Hepatitis B virus DNA-negative dane particles lack core protein but contain a 22-kDa precore protein without C-terminal arginine-rich domain

DNA-negative Dane particles have been observed in hepatitis B virus (HBV)-infected sera. The capsids of the empty particles are thought to be composed of core protein but have not been studied in detail. In the present study, the protein composition of the particles was examined using new enzyme immunoassays for the HBV core antigen (HBcAg) and for the HBV precore/core proteins (core-related antigens, HBcrAg). HBcrAg were abundant in fractions slightly less dense than HBcAg and HBV DNA. Three times more Dane-like particles were observed in the HBcrAg-rich fraction than in the HBV DNA-rich fraction by electron microscopy. Western blots and mass spectrometry identified the HBcrAg as a 22-kDa precore protein (p22cr) containing the uncleaved signal peptide and lacking the arginine-rich domain that is involved in binding the RNA pregenome or the DNA genome. In sera from 30 HBV-infected patients, HBcAg represented only a median 10.5% of the precore/core proteins in enveloped particles. These data suggest that most of the Dane particles lack viral DNA and core capsid but contain p22cr. This study provides a model for the formation of the DNA-negative Dane particles. The precore proteins, which lack the arginine-rich nucleotide-binding domain, form viral RNA/DNA-negative capsid-like particles and are enveloped and released as empty particles.     

Visualization of spatiotemporal activation of Notch signaling: live monitoring and significance in neural development

Notch signaling plays various key roles in cell fate determination during CNS development in a context-dependent fashion. However, its precise physiological role and the localization of its target cells remain unclear. To address this issue, we developed a new reporter system for assessing the RBP-J-mediated activation of Notch signaling target genes in living cells and tissues using a fluorescent protein Venus. Our reporter system revealed that Notch signaling is selectively activated in neurosphere-initiating multipotent neural stem cells in vitro and in radial glia in the embryonic forebrain in vivo. Furthermore, the activation of Notch signaling occurs during gliogenesis and is required in the early stage of astroglial development. Consistent with these findings, the persistent activation of Notch signaling inhibits the differentiation of GFAP-positive astrocytes. Thus, the development of our RBP-J-dependent live reporter system, which is activated upon Notch activation, together with a stage-dependent gain-of-function analysis allowed us to gain further insight into the complexity of Notch signaling in mammalian CNS development.     

Influence of glycosylation on the efficacy of an Env-based vaccine against simian immunodeficiency virus SIVmac239 in a macaque AIDS model

The envelope glycoprotein (Env) of human immunodeficiency viruses (HIVs) and simian immunodeficiency viruses (SIVs) is heavily glycosylated, and this feature has been speculated to be a reason for the insufficient immune control of these viruses by their hosts. In a macaque AIDS model, we demonstrated that quintuple deglycosylation in Env altered a pathogenic virus, SIVmac239, into a novel attenuated mutant virus (delta5G). In delta5G-infected animals, strong protective immunity against SIVmac239 was elicited. These HIV and SIV studies suggested that an understanding of the role of glycosylation is critical in defining not only the virological properties but also the immunogenicity of Env, suggesting that glycosylation in Env could be modified for the development of effective vaccines. To examine the effect of deglycosylation, we constructed prime-boost vaccines consisting of Env from SIVmac239 and delta5G and compared their immunogenicities and vaccine efficacies by challenge infection with SIVmac239. Vaccination-induced immune responses differed between the two vaccine groups. Both Env-specific cellular and humoral responses were higher in wild-type (wt)-Env-immunized animals than in delta5G Env-immunized animals. Following the challenge, viral loads in SIVmac239 Env (wt-Env)-immunized animals were significantly lower than in vector controls, with controlled viral replication in the chronic phase. Unexpectedly, viral loads in delta5G Env-immunized animals were indistinguishable from those in vector controls. This study demonstrated that the prime-boost Env vaccine was effective against homologous SIVmac239 challenge. Changes in glycosylation affected both cell-mediated and humoral immune responses and vaccine efficacy.     

Comparative simulations of aquaporin family: AQP1, AQPZ, AQP0 and GlpF

Molecular dynamics simulations were performed for four members of the aquaporin family (AQP1, AQPZ, AQP0, and GlpF) in the explicit membrane environment. The single-channel water permeability, pf, was evaluated to be GlpF approximately AQPZ > AQP1 > AQP0, while their relative pore sizes were GlpF > AQP1 > AQPZ > AQP0. This relation between pf and pore size indicates that water permeability was determined not only by the channel radius, but also another competing factor. Analysis of water dynamics revealed that this factor was the single-file nature of water transport.     

Regulation of spinophilin Ser94 phosphorylation in neostriatal neurons involves both DARPP-32-dependent and independent pathways

Spinophilin is a protein phosphatase-1 (PP-1)- and actin-binding protein that is enriched in dendritic spines. Phosphorylation of the actin-binding domain of rat spinophilin at one or more sites by protein kinase A (PKA) inhibits actin binding. Here, we investigated the regulation of mouse spinophilin that contains only a single PKA-site (Ser94) within its actin-binding domain. In vitro phosphorylation of Ser94 resulted in the dissociation of spinophilin from actin filaments. In mouse neostriatal slices, phospho-Ser94 (p-Ser94) was dephosphorylated mainly by PP-1 and also by PP-2A. Activation of dopamine D1 receptors in striatonigral medium spiny neurons, and of adenosine A 2A receptors in striatopallidal medium spiny neurons increased, whereas activation of dopamine D2 receptors in striatopallidal neurons decreased, spinophilin Ser94 phosphorylation. In neostriatal slices from DARPP-32 (dopamine- and cAMP-regulated phosphoprotein of 32 kDa) knockout mice, the effects of D1, D2 and A 2A receptors were largely attenuated. Activation of NMDA receptors decreased Ser94 phosphorylation in a PP-2A-dependent, but DARPP-32-independent, manner. These results suggest that PKA-dependent phosphorylation of spinophilin at Ser94 in both striatonigral and striatopallidal neurons requires synergistic contributions from the PKA and DARPP-32/PP-1 pathways. In addition, PP-2A plays a role in Ser94 dephosphorylation in response to activation of both D2 and NMDA receptors.     

The minimum endpoint variance trajectory depends on the profile of the signal-dependent noise

It has been proposed that the central nervous system determines reaching movement trajectories so as to minimize the positional variance of the endpoint in the presence of signal-dependent noise. The hypothesis well reproduces the empirical movement trajectories for noise to the control signal whose variance is proportional to the second power of the amplitude of the control signal. However, empirical studies do not necessarily exhibit such a simple signal-noise relationship. The studies exhibit a wide distribution of estimates of the value of the exponent. This discrepancy raises the question of how the minimum endpoint variance trajectory depends on the value of the exponent. To address this question, we calculated minimum endpoint variance trajectories in simulations in which the value of the exponent was varied from 0 to 3. We found that the optimal trajectories differed according to the value of the exponent, and the profiles of optimal trajectories gradually diverged from the empirical ones as the value approached 0, though this change was not remarkable for larger values. Moreover, the optimal trajectories failed to replicate Fitts' law when the value was not equal to 2. These results suggest that the acceptability of the minimum endpoint variance theory depends on the value of the exponent in our motor system.     

Ammonia-induced apoptosis is accelerated at higher pH in gastric surface mucous cells

Gastric luminal ammonia produced by Helicobacter pylori has been shown to cause gastric mucosal injury. This study was conducted to examine the mechanisms by which gastric luminal ammonia causes apoptosis of gastric epithelial cells. Monolayers of GSM06 cells, developed from murine gastric surface mucous cells, were cultured in the absence or presence of 10-30 mM NH(4)Cl at ambient pH of 5.0, 6.0, and 7.0. In the presence of luminal NH(4)Cl, GSM06 cells showed 1) cell shrinkage and nuclear chromatin condensation, 2) DNA fragmentation into oligonucleosomes, 3) leakage of cytochrome c into cytosolic fraction without affecting bax expression, and 4) increases in activity of caspases-3 and -9. These changes were accentuated when the cells were cultured at pH 7.0. In the absence of NH(4)Cl, none of these changes was detected at any pH examined. These results suggest that gastric luminal ammonia, at concentrations detected in H. pylori-infected subjects, induces apoptosis of gastric epithelial cells by release of cytochrome c from mitochondria, followed by activation of caspases-9 and -3, especially at higher ambient pH.