Herpesviruses have evolved a unique mechanism for nuclear egress of nascent progeny nucleocapsids: the nucleocapsids bud through the inner nuclear membrane into the perinuclear space between the inner and outer nuclear membranes (primary envelopment), and enveloped nucleocapsids then fuse with the outer nuclear membrane to release nucleocapsids into the cytoplasm (de-envelopment). We have shown that the herpes simplex virus 1 (HSV-1) major virion structural protein UL47 (or VP13/VP14) is a novel regulator for HSV-1 nuclear egress. In particular, we demonstrated the following: (i) UL47 formed a complex(es) with HSV-1 proteins UL34, UL31, and/or Us3, which have all been reported to be critical for viral nuclear egress, and these viral proteins colocalized at the nuclear membrane in HSV-1-infected cells; (ii) the UL47-null mutation considerably reduced primary enveloped virions in the perinuclear space although capsids accumulated in the nucleus; and (iii) UL47 was detected in primary enveloped virions in the perinuclear space by immunoelectron microscopy. These results suggested that UL47 promoted HSV-1 primary envelopment, probably by interacting with the critical HSV-1 regulators for viral nuclear egress and by modulating their functions.
The influenza A virus possesses an eight-segmented, negative-sense, single-stranded RNA genome (vRNA). Each vRNA segment binds to multiple copies of viral nucleoproteins and a small number of heterotrimeric polymerase complexes to form a rod-like ribonucleoprotein complex (RNP), which is essential for the transcription and replication of the vRNAs. However, how the RNPs are organized within the progeny virion is not fully understood. Here, by focusing on polymerase complexes, we analyzed the fine structure of purified RNPs and their configuration within virions by using various electron microscopies (EM). We confirmed that the individual RNPs possess a single polymerase complex at one end of the rod-like structure and that, as determined using immune EM, some RNPs are incorporated into budding virions with their polymerase-binding ends at the budding tip, whereas others align with their polymerase-binding ends at the bottom of the virion. These data further our understanding of influenza virus virion morphogenesis.
RNAi-mediated suppression of the endogenous storage proteins in MucoRice-CTB-RNAi seeds affects not only the levels of overexpressed CTB and RAG2 allergen, but also the localization of CTB and RAG2. A purification-free rice-based oral cholera vaccine (MucoRice-CTB) was previously developed by our laboratories using a cholera toxin B-subunit (CTB) overexpression system. Recently, an advanced version of MucoRice-CTB was developed (MucoRice-CTB-RNAi) through the use of RNAi to suppress the production of the endogenous storage proteins 13-kDa prolamin and glutelin, so as to increase CTB expression. The level of the ?-amylase/trypsin inhibitor-like protein RAG2 (a major rice allergen) was reduced in MucoRice-CTB-RNAi seeds in comparison with wild-type (WT) rice. To investigate whether RNAi-mediated suppression of storage proteins affects the localization of overexpressed CTB and major rice allergens, we generated an RNAi line without CTB (MucoRice-RNAi) and investigated gene expression, and protein production and localization of two storage proteins, CTB, and five major allergens in MucoRice-CTB, MucoRice-CTB-RNAi, MucoRice-RNAi, and WT rice. In all lines, glyoxalase I was detected in the cytoplasm, and 52- and 63-kDa globulin-like proteins were found in the aleurone particles. In WT, RAG2 and 19-kDa globulin were localized mainly in protein bodies II (PB-II) of the endosperm cells. Knockdown of glutelin A led to a partial destruction of PB-II and was accompanied by RAG2 relocation to the plasma membrane/cell wall and cytoplasm. In MucoRice-CTB, CTB was localized in the cytoplasm and PB-II. In MucoRice-CTB-RNAi, CTB was produced at a level six times that in MucoRice-CTB and was localized, similar to RAG2, in the plasma membrane/cell wall and cytoplasm. Our findings indicate that the relocation of CTB in MucoRice-CTB-RNAi may contribute to down-regulation of RAG2.
Rotavirus-induced diarrhea is a life-threatening disease in immunocompromised individuals and in children in developing countries. We have developed a system for prophylaxis and therapy against rotavirus disease using transgenic rice expressing the neutralizing variable domain of a rotavirus-specific llama heavy-chain antibody fragment (MucoRice-ARP1). MucoRice-ARP1 was produced at high levels in rice seeds using an overexpression system and RNAi technology to suppress the production of major rice endogenous storage proteins. Orally administered MucoRice-ARP1 markedly decreased the viral load in immunocompetent and immunodeficient mice. The antibody retained in vitro neutralizing activity after long-term storage (>1 yr) and boiling and conferred protection in mice even after heat treatment at 94°C for 30 minutes. High-yield, water-soluble, and purification-free MucoRice-ARP1 thus forms the basis for orally administered prophylaxis and therapy against rotavirus infections.
Highly topographic organization of neural circuits exists for the regulation of various brain functions in corticobasal ganglia circuits. Although neural circuit-specific refinement during synapse development is essential for the execution of particular neural functions, the molecular and cellular mechanisms for synapse refinement are largely unknown. Here, we show that protocadherin 17 (PCDH17), one of the nonclustered ?2-protocadherin family members, is enriched along corticobasal ganglia synapses in a zone-specific manner during synaptogenesis and regulates presynaptic assembly in these synapses. PCDH17 deficiency in mice causes facilitated presynaptic vesicle accumulation and enhanced synaptic transmission efficacy in corticobasal ganglia circuits. Furthermore, PCDH17(-/-) mice exhibit antidepressant-like phenotypes that are known to be regulated by corticobasal ganglia circuits. Our findings demonstrate a critical role for PCDH17 in the synaptic development of specific corticobasal ganglia circuits and suggest the involvement of PCDH17 in such circuits in depressive behaviors.
Autophagy is a cellular self-catabolic process wherein organelles, macromolecules, and invading microbes are sequestered in autophagosomes that fuse with lysosomes. In this study, we uncover the role of nitric oxide (NO) as a signaling molecule for autophagy induction via its downstream mediator, 8-nitroguanosine 3,5-cyclic monophosphate (8-nitro-cGMP). We found that 8-nitro-cGMP-induced autophagy is mediated by Lys63-linked polyubiquitination and that endogenous 8-nitro-cGMP promotes autophagic exclusion of invading group A Streptococcus (GAS) from cells. 8-nitro-cGMP can modify Cys residues by S-guanylation of proteins. We showed that intracellular GAS is modified with S-guanylation extensively in autophagosomes-like vacuoles, suggesting the role of S-guanylation as a marker for selective autophagic degradation. This finding is supported by the fact that S-guanylated bacteria were selectively marked with polyubiquitin, a known molecular tag for selective transport to autophagosomes. These results collectively indicate that 8-nitro-cGMP plays a crucial role in cytoprotection during bacterial infections or inflammations via autophagy upregulation.
The purpose of this study was to test the hypothesis that obese diabetic mice exhibit marked skin fragility, which is caused by increased oxidative stress and increased matrix metalloproteinase (MMP) gene expression in the subcutaneous adipose tissue. Scanning electron microscopy of skin samples from Tsumura-Suzuki obese diabetic (TSOD) mice revealed thinner collagen bundles, and decreased density and convolution of the collagen fibres. Furthermore, skin tensile strength measurements confirmed that the dorsal skin of TSOD mice was more fragile to tensile force than that of non-obese mice. The mRNA expressions of heme oxygenase 1 (Hmox1), a marker of oxidative stress, Mmp2 and Mmp14 were increased in the adipose tissue of TSOD mice. Antioxidant experiments were subsequently performed to determine whether the changes in collagen fibres and skin fragility were caused by oxidative stress. Strikingly, oral administration of the antioxidant dl-?-tocopherol acetate (vitamin E) decreased Hmox1, Mmp2 and Mmp14 mRNA expressions, and improved the skin tensile strength and structure of collagen fibres in TSOD mice. These findings suggest that the skin fragility in TSOD mice is associated with dermal collagen damage and weakened tensile strength, and that oxidative stress and MMP overexpression in the subcutaneous adipose tissue may, at least in part, affect dermal fragility via a paracrine pathway. These observations may contribute to novel clinical interventions, such as dietary supplementation with antioxidants or application of skin cream containing antioxidants, which may overcome skin fragility in obese patients with diabetes.
The formation and refinement of synaptic connections are key steps of neural development to establish elaborate brain networks. To investigate the functional role of protein tyrosine phosphatase (PTP) ?, we employed an olfactory sensory neuron (OSN)-specific gene manipulation system in combination with in vivo imaging of transparent zebrafish embryos. Knockdown of PTP? enhanced the accumulation of synaptic vesicles in the axon terminals of OSNs. The exaggerated accumulation of synaptic vesicles was restored to the normal level by the OSN-specific expression of PTP?, indicating that presynaptic PTP? is responsible for the regulation of synaptic vesicle accumulation. Consistently, transient expression of a dominant-negative form of PTP? in OSNs enhanced the accumulation of synaptic vesicles. The exaggerated accumulation of synaptic vesicles was reproduced in transgenic zebrafish lines carrying an OSN-specific expression vector of the dominant-negative PTP?. By electron microscopic analysis of the transgenic line, we found the significant increase of the number of OSN-mitral cell synapses in the central zone of the olfactory bulb. The density of docked vesicles at the active zone was also increased significantly. Our results suggest that presynaptic PTP? controls the number of OSN-mitral cell synapses by suppressing their excessive increase.
Negatively stained influenza virions sometimes show irregular morphology and are often referred to as pleomorphic. However, this irregular morphology has not been visualized when ultrathin-section transmission and scanning electron microscopies are used. This study focused on the effects of ultracentrifugation on influenza A virion morphology, as negative staining often involves ultracentrifugation to concentrate or purify virions. The morphologies of unfixed, glutaraldehyde-fixed and osmium tetroxide-fixed virions were quantitatively compared before and after ultracentrifugation, and it was found that, without chemical fixation, approximately 30% of virions were altered from oval to irregular shapes following ultracentrifugation. By contrast, most glutaraldehyde-fixed virions remained uniformly elliptical, even after ultracentrifugation. When a virus with an 11 aa deletion at the C terminus of its M2 cytoplasmic tail was ultracentrifuged, its morphology was appreciably deformed compared with that of the wild-type virus. These results demonstrate that the native morphology of influenza A virions is regular but is disrupted by ultracentrifugation, and that the cytoplasmic tail of M2 is important for virion integrity.
The impact of quorum sensing (QS) in in vivo models of infection has been widely investigated, but there are no descriptions for ischemic wound infection. To explore the role of QS in Pseudomonas aeruginosa in the establishment of ischemic wound infection, we challenged a pressure ulcer model in rats with the PAO-1, PAO-1 derivatives ?lasI?rhlI and ?lasR?rhlR strains, which cannot induce the virulence factor under QS control, thus the reduced tissue destruction was expended in these mutant strains. However unexpectedly, on postwounding day 3, the inflammatory responses in the three groups were similarly severe and the numbers of bacteria in tissue samples did not differ among the three strains. Biofilm formation was immature in QS-deficient strains, defined by the absence of dense bacterial aggregates and extracellular polymeric substance, which was confirmed by scanning electron microscopy. The Pseudomonas aeruginosa QS signal, acylated homoserine lactone, was only quantified from wound samples in the PAO-1 group. The swimming and twitching motilities were significantly enhanced in the ?lasR?rhlR group compared with the PAO-1 group in vitro. A significantly larger wound area was correlated with the bacterial motility. The inflammation in the early phase of bacterial challenge to wounds with immature biofilm formation in the QS-deficient strains indicated that the role of QS was more crucial for the chronic phase than for the acute phase of infection. The present findings indicate a difference in the importance of QS in ischemic wound infections compared with other infection models.
In this study, we demonstrated a new airway Ag sampling site by analyzing tissue sections of the murine nasal passages. We revealed the presence of respiratory M cells, which had the ability to take up OVA and recombinant Salmonella typhimurium expressing GFP, in the turbinates covered with single-layer epithelium. These M cells were also capable of taking up respiratory pathogen group A Streptococcus after nasal challenge. Inhibitor of DNA binding/differentiation 2 (Id2)-deficient mice, which are deficient in lymphoid tissues, including nasopharynx-associated lymphoid tissue, had a similar frequency of M cell clusters in their nasal epithelia to that of their littermates, Id2(+/-) mice. The titers of Ag-specific Abs were as high in Id2(-/-) mice as in Id2(+/-) mice after nasal immunization with recombinant Salmonella-ToxC or group A Streptococcus, indicating that respiratory M cells were capable of sampling inhaled bacterial Ag to initiate an Ag-specific immune response. Taken together, these findings suggest that respiratory M cells act as a nasopharynx-associated lymphoid tissue-independent alternative gateway for Ag sampling and subsequent induction of Ag-specific immune responses in the upper respiratory tract.
When Ebola virus nucleoprotein (NP) is expressed in mammalian cells, it assembles into helical structures. Here, the recombinant NP helix purified from cells expressing NP was characterized biochemically and morphologically. We found that the recombinant NP helix is associated with non-viral RNA, which is not protected from RNase digestion and that the morphology of the helix changes depending on the environmental salt concentration. The N-terminal 450 aa residues of NP are sufficient for these properties. However, digestion of the NP-associated RNA eliminates the plasticity of the helix, suggesting that this RNA is an essential structural component of the helix, binding to individual NP molecules via the N-terminal 450 aa. These findings enhance our knowledge of Ebola virus assembly and understanding of the Ebola virus life cycle.
Nitrogen-containing bisphosphonates are pyrophosphate analogs that have long been the preferred prescription for treating osteoporosis. Although these drugs are considered inhibitors of prenylation and are believed to exert their effects on bone resorption by disrupting the signaling pathways downstream of prenylated small GTPases, this explanation seems to be insufficient. Because other classes of prenylation inhibitors have recently emerged as potential antiviral therapeutic agents, we first investigated here the effects of bisphosphonates on simian virus 40 and adenovirus infections and, to our surprise, found that viral infections are suppressed by bisphosphonates through a prenylation-independent pathway. By in-house affinity-capture techniques, dynamin-2 was identified as a new molecular target of bisphosphonates. We present evidence that certain bisphosphonates block endocytosis of adenovirus and a model substrate by inhibiting GTPase activity of dynamin-2. Hence, this study has uncovered a previously unknown mechanism of action of bisphosphonates and offers potential novel use for these drugs.
Bone regenerative medicine via tissue engineering is expected to be an alternative treatment for conventional autogenous bone graft, as it is less invasive. One of the best triads for bone engineering is bone marrow stromal cells, calcium phosphate ceramics, and bone morphogenetic protein (BMP). However, the optimal mixing conditions for BMP-induced osteoblasts and ceramic granules remain unclear. Therefore, we investigated the effect of the mixing conditions for cell scaffolds on the bone-forming potential. The cells were mixed with beta-tricalcium phosphate (beta-TCP) granules followed by osteoblast induction with recombinant human BMP-2 (rhBMP-2) (first mixture), or were first induced with rhBMP-2 on plastic dishes and then mixed with the beta-TCP granules (last mixture) just prior to the operation. Both the first and last mixtures were transplanted into nude mice subcutaneously, with the amount of bone formation analyzed histomorphometrically. In addition, cell numbers and alkaline phosphatase (ALP) activity before transplantation was determined in both the mixtures. In vitro analyses revealed that cell numbers were greater in the last mixture, whereas ALP activity was greater in the first mixture. In vivo analyses revealed that the first mixture was much more osteogenic than the last mixture with respect to new bone formation and osteocalcin synthesis. These data suggest that cell-scaffold mixing conditions have a significant influence on the bone-forming capacity via bone engineering and that first mixture might be the optimal condition for rhBMP-2-induction of human osteoblasts.
The eye is protected by the ocular immunosurveillance system. We show that tear duct-associated lymphoid tissue (TALT) is located in the mouse lacrimal sac and shares immunological characteristics with mucosa-associated lymphoid tissues (MALTs), including the presence of M cells and immunocompetent cells for antigen uptake and subsequent generation of mucosal immune responses against ocularly encountered antigens and bacteria such as Pseudomonas aeruginosa. Initiation of TALT genesis began postnatally; it occurred even in germ-free conditions and was independent of signaling through organogenesis regulators, including inhibitor of DNA binding/differentiation 2, retinoic acid-related orphan receptor gammat, lymphotoxin (LT) alpha1beta2-LTbetaR, and lymphoid chemokines (CCL19, CCL21, and CXCL13). Thus, TALT shares immunological features with MALT but has a distinct tissue genesis mechanism and plays a key role in ocular immunity.
Us3 is a serine/threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). We recently identified serine at Us3 position 147 (Ser-147) as a physiological phosphorylation site of Us3 (A. Kato, M. Tanaka, M. Yamamoto, R. Asai, T. Sata, Y. Nishiyama, and Y. Kawaguchi, J. Virol. 82:6172-6189, 2008). In the present study, we investigated the effects of phosphorylation of Us3 Ser-147 on regulation of Us3 catalytic activity in infected cells and on HSV-1 pathogenesis. Our results were as follows. (i) Only a small fraction of Us3 purified from infected cells was phosphorylated at Ser-147. (ii) Us3 phosphorylated at Ser-147 purified from infected cells had significantly higher kinase activity than Us3 not phosphorylated at Ser-147. (iii) Phosphorylation of Us3 Ser-147 in infected cells was dependent on Us3 kinase activity. (iv) Replacement of Us3 Ser-147 by alanine significantly reduced viral replication in the mouse cornea and the development of herpes stromal keratitis and periocular skin disease in mice. These results indicated that Us3 catalytic activity is tightly regulated by autophosphorylation of Ser-147 in infected cells and that regulation of Us3 activity by autophosphorylation appeared to play a critical role in viral replication in vivo and in HSV-1 pathogenesis.
To determine the extent to which hepatic stellate cell (HSC) activation contributes to liver fibrosis, it was found necessary to develop an alternative structural and functional stellate cell marker for in situ studies. Although several HSC markers have been reported, none of those are associated with particular HSC functions.
Herpes simplex virus 1 (HSV-1) enters cells either via fusion of the virion envelope and host cell plasma membrane or via endocytosis, depending on the cell type. In the study reported here, we investigated a viral entry pathway dependent on the paired immunoglobulin-like type 2 receptor alpha (PILRalpha), a recently identified entry coreceptor for HSV-1 that associates with viral envelope glycoprotein B (gB). Experiments using inhibitors of endocytic pathways and ultrastructural analyses of Chinese hamster ovary (CHO) cells transduced with PILRalpha showed that HSV-1 entry into these cells was via virus-cell fusion at the cell surface. Together with earlier observations that HSV-1 uptake into normal CHO cells and those transduced with a receptor for HSV-1 envelope gD is mediated by endocytosis, these results indicated that expression of PILRalpha produced an alternative HSV-1 entry pathway in CHO cells. We also showed that human and murine PILRalpha were able to mediate entry of pseudorabies virus, a porcine alphaherpesvirus, but not of HSV-2. These results indicated that viral entry via PILRalpha appears to be conserved but that there is a PILRalpha preference among alphaherpesviruses.
Sepsis is a major clinical challenge and septic encephalopathy is its nasty complication. The pathogenesis and underlying mechanisms of septic encephalopathy are not well understood. This study sought to fully characterize sepsis-associated biochemical and histopathological changes in brains of mice after cecal ligation and puncture, regarded as a highly clinically relevant animal model of polymicrobial sepsis. Real-time PCR analysis showed that gene expression levels of proinflammatory cytokines, including tumor necrosis factor-? and interleukin-1?, were significantly up-regulated in brain tissues from septic mice, but to a much lesser extent when compared with those in peripheral tissues such as lungs. Blood-brain barrier (BBB) permeability was significantly increased in septic mice, as determined by the measurement of sodium fluorescein and Evans blue content. Sepsis resulted in increases in NADPH oxidase activity and expression of p47(phox) and p67(phox) and up-regulation of inducible nitric oxide (NO) synthase in brains, indicating that superoxide, produced by NADPH oxidase, reacts with NO to form peroxynitrite, that maybe lead to the loss of BBB integrity. Light and electron microscopic examination of septic mouse brain showed serious neuronal degeneration, as indicated by hyperchromatic, shrunken, pyknotic, and electron-dense neurons. These histopathological changes were prevented by treatment with the free radical scavenger edaravone. Together, these results suggest that sepsis can lead to rapid neurodegenerative changes in brains via free radical species production and possibly subsequent injury to the BBB. We may also provide a potentially useful therapeutic tool for treating septic encephalopathy.
Polypyrimidine tract-binding protein (PTB) is a well-characterized RNA-binding protein and known to be preferentially expressed in neural stem cells (NSCs) in the central nervous system; however, its role in NSCs in the developing brain remains unclear. To explore the role of PTB in embryonic NSCs in vivo, Nestin-Cre-mediated conditional Ptb knockout mice were generated for this study. In the mutant forebrain, despite the depletion of PTB protein, neither abnormal neurogenesis nor flagrant morphological abnormalities were observed at embryonic day 14.5 (E14.5). Nevertheless, by 10 weeks, nearly all mutant mice succumbed to hydrocephalus (HC), which was caused by a lack of the ependymal cell layer in the dorsal cortex. Upon further analysis, a gradual loss of adherens junctions (AJs) was observed in the ventricular zone (VZ) of the dorsal telencephalon in the mutant brains, beginning at E14.5. In the AJs-deficient VZ, impaired interkinetic nuclear migration and precocious differentiation of NSCs were observed after E14.5. These findings demonstrated that PTB depletion in the dorsal telencephalon is causally involved in the development of HC and that PTB is important for the maintenance of AJs in the NSCs of the dorsal telencephalon.
The influenza A virus genome consists of eight single-stranded negative-sense RNA (vRNA) segments. Although genome segmentation provides advantages such as genetic reassortment, which contributes to the emergence of novel strains with pandemic potential, it complicates the genome packaging of progeny virions. Here we elucidate, using electron tomography, the three-dimensional structure of ribonucleoprotein complexes (RNPs) within progeny virions. Each virion is packed with eight well-organized RNPs that possess rod-like structures of different lengths. Multiple interactions are found among the RNPs. The position of the eight RNPs is not consistent among virions, but a pattern suggests the existence of a specific mechanism for assembly of these RNPs. Analyses of budding progeny virions suggest two independent roles for the viral spike proteins: RNP association on the plasma membrane and the subsequent formation of the virion shell. Our data provide further insights into the mechanisms responsible for segmented-genome packaging into virions.
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