Complete structure of the hemagglutinin gene from the human influenza A/Victoria/3/75 (H3N2) strain as determined from cloned DNA

The complete sequence of a hemagglutinin (HA) gene of a recent human influenza A strain, A/Victoria/3/75, is 1768 nucleotides long and contains the information for 567 amino acids. It codes for a signal peptide of 16 amino acids, the HA1 chain of the mature hemagglutinin of 329 amino acids, a connecting region between HA1 and HA2 consisting of a single arginine residue and the HA2 portion of 221 amiino acids. The sequence is compared with the hemagglutinin of two members of other subtypes, the human H2 strain A/Jap/305/57 and the avian Hav1 strain A/FPV/Rostock/34, and with one of the same H3 subtype, A/Memphis/3/72. To align the HA1 chain of different major subtypes several deletions/insertions of single amino acids must be invoked, but two more extensive differences are found at both ends, one leading to an extension of the amino terminal sequence of HA1 and the other (four residues) occurring in the region processed away between HA1 and HA2. Comparison of the HA1 of two H3 strains suggests that drift probably depends on single base mutations, some of which change antigenic determinants. The HA2 region, which apparently is not involved in the immune response, is highly conserved even between different subtypes, and single base substitutions account for all the observed diversity. A hydrophobic segment of 24 residues is present in the same position close to the carboxyl terminus of HA2 in both Victoria and FPV, and presumably functions in implantation into the lipid bilayer. The many conserved features not only in HA2 but also in HA1 suggest a rather rigid architecture for the whole hemagglutinin molecule.     

Proteomic analysis of up-regulated proteins in human promonocyte cells expressing severe acute respiratory syndrome coronavirus 3C-like protease

The pathogenesis of severe acute respiratory syndrome coronavirus (SARS CoV) is an important issue for treatment and prevention of SARS. Previously, SARS CoV 3C-like protease (3CLpro) has been demonstrated to induce apoptosis via the activation of caspase-3 and caspase-9 (Lin, C. W., Lin, K. H., Hsieh, T. H., Shiu, S. Y. et al., FEMS Immunol. Med. Microbiol. 2006, 46, 375-380). In this study, proteome analysis of the human promonocyte HL-CZ cells expressing SARS CoV 3CLpro was performed using 2-DE and nanoscale capillary LC/ESI quadrupole-TOF MS. Functional classification of identified up-regulated proteins indicated that protein metabolism and modification, particularly in the ubiquitin proteasome pathway, was the main biological process occurring in SARS CoV 3CLpro-expressing cells. Thirty-six percent of identified up-regulated proteins were located in the mitochondria, including apoptosis-inducing factor, ATP synthase beta chain and cytochrome c oxidase. Interestingly, heat shock cognate 71-kDa protein (HSP70), which antagonizes apoptosis-inducing factor was shown to down-regulate and had a 5.29-fold decrease. In addition, confocal image analysis has shown release of mitochondrial apoptogenic apoptosis-inducing factor and cytochrome c into the cytosol. Our results revealed that SARS CoV 3CLpro could be considered to induce mitochondrial-mediated apoptosis. The study provides system-level insights into the interaction of SARS CoV 3CLpro with host cells, which will be helpful in elucidating the molecular basis of SARS CoV pathogenesis.     

Quantitative phosphoproteomic analysis reveals γ‐bisabolene inducing p53‐mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation

γ‐Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti‐proliferative activities against human oral squamous cell carcinoma (OSCC). γ‐Bisabolene activated caspases‐3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9‐22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ‐bisabolene was identified using TiO2‐PDMS plate and LC‐MS/MS, then confirmed using Western blotting and real‐time RT‐PCR assays. Phosphoproteome profiling revealed that γ‐bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein–protein interaction network analysis proposed the involvement of PP1‐HDAC2‐p53 and ERK1/2‐p53 pathways in γ‐bisabolene‐induced apoptosis. Subsequent assays indicated γ‐bisabolene eliciting p53 acetylation that enhanced the expression of p53‐regulated apoptotic genes. PP1 inhibitor‐2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ‐bisabolene‐treated Ca9‐22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ‐bisabolene‐induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1‐HDAC2‐p53 and ERK1/2‐p53 pathways in mitochondria‐mediated apoptosis of γ‐bisabolene‐treated cells. This study demonstrated γ‐bisabolene displaying potent anti‐proliferative and apoptosis‐inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ‐bisabolene‐induced apoptosis. The novel insight could be useful for developing anti‐cancer drugs.     

Oxidative stress induces lipid-raft-mediated activation of Src homology 2 domain-containing protein-tyrosine phosphatase 2 in astrocytes

Several protein phosphatases are involved in neuroprotection in response to ischemic brain injury. Here, we report that reactive oxygen species (ROS)-mediated oxidative stress promotes phosphorylation of endogenous SHP-2 through lipid rafts in rat primary astrocytes. SHP-2 was transiently phosphorylated during hypoxia/reoxygenation, an effect abrogated by a ROS scavenger and an NADPH oxidase inhibitor. Additionally, exogenous treatment with hydrogen peroxide (H₂O₂) triggered SHP-2 phosphorylation in a time- and dose-dependent manner and led to its translocation into lipid rafts. H₂O₂-mediated SHP-2 phosphorylation and translocation were inhibited by filipin III and methyl-β-cyclodextrin (MCD), lipid-raft-disrupting agents. In the presence of H₂O₂, SHP-2 formed a complex with STAT-3 and reduced the steady-state STAT-3 phosphorylation level. Interestingly, the effect of H₂O₂ on SHP-2 phosphorylation was cell-type specific. Remarkably, SHP-2 phosphorylation was induced strongly by H₂O₂ in astrocytes, but barely detectable in microglia. Our results collectively indicate that SHP-2 is activated by ROS-mediated oxidative stress in astrocytes and functions as a component of the raft-mediated signaling pathway that acts through dephosphorylation and inactivation of other phosphotyrosine proteins, such as STAT-3.     

N-Glycosylation profiling of turtle egg yolk: expression of galabiose structure

To understand the roles of species-specific carbohydrates, systematic studies of interspecific glycan analyses are imperative. An extensive series of glycomics studies on approximately 180 kinds of bird eggs have demonstrated that 60-70% of the birds, which are closely related in phylogeny, express the α-Galp-(1→4)-Galp structure on their egg glycoproteins. This prompted us to investigate the glycosylation profiles of eggs from an evolutionarily related organism, a sea turtle (reptilian). We performed N-glycosylation profiling of turtle egg yolk by using HPLC mapping in conjunction with mass spectrometric methods and thereby demonstrated that the α-Galp-(1→4)-Galp groups are displayed on approximately 38% of total N-glycans. Our findings suggest that the ability to express the galabiose structure was acquired at an early stage of diversification in amniotes.     

Astrocytes in injury states rapidly produce anti-inflammatory factors and attenuate microglial inflammatory responses

Microglia are known to be a primary inflammatory cell type in the brain. However, microglial inflammatory responses are attenuated in the injured brain compared to those in cultured pure microglia. In the present study, we found that astrocytes challenged by oxygen-glucose deprivation (OGD) or H(2) O(2) released soluble factor(s) and attenuated microglial inflammatory responses. Conditioned medium prepared from astrocytes treated with OGD (OGD-ACM) or H(2) O(2) (H(2) O(2) -ACM) significantly reduced the levels of interferon-γ (IFN-γ)-induced microglial inflammatory mediators, including inducible nitric oxide synthase, at both the mRNA and protein levels. The anti-inflammatory effect of astrocytes appeared very rapidly (within 5min), but was not closely correlated with the extent of astrocyte damage. Both OGD-ACM and H(2) O(2) -ACM inhibited STAT nuclear signaling, as evidenced by a reduction in both STAT-1/3 binding to the IFN-γ-activated site and IFN-γ-activated site promoter activity. However, both phosphorylation and nuclear translocation of STAT-1/3 was unchanged in IFN-γ-treated microglia. The active component(s) in OGD-ACM were smaller than 3kDa, and displayed anti-inflammatory effects independent of protein synthesis. These results suggest that, in the injured brain, astrocytes may act as a controller to rapidly suppress microglial activation.     

Regulation of Weibel-Palade Body Exocytosis by α-Synuclein in Endothelial Cells

α-Synuclein is a small presynaptic protein implicated in the pathogenesis of Parkinson disease. Nevertheless, its physiological roles and mechanisms remain incompletely understood. α-Synuclein is not only expressed in neurons but also in the vascular endothelium, which contains intracellular granules called Weibel-Palade bodies (WPBs) that contain a number of chemokines, adhesive molecules, and inflammatory cytokines. This study explored whether the exocytosis of WPB is regulated by α-synuclein. Phorbol 12-myristate 13-acetate-, thrombin-, or forskolin-induced von Willebrand factor release or translocation of P-selectin from endothelial cells were inhibited by α- and β-synuclein but not γ-synuclein. Three point mutants (A30P, A53T, and E46K) found in familial Parkinson disease also inhibited WPB exocytosis similar to that of wild-type α-synuclein. Furthermore, the negative regulation of WPB exocytosis required the N terminus or the nonamyloid β-component of Alzheimer disease amyloid region of α-synuclein, but not the C-terminal acidic tail, and α-synuclein affected WPB exocytosis through interference with RalA activation by enhancing the interaction of RalGDS-β-arrestin complexes. Immuno-EM analysis revealed that α-synuclein was localized close to WPBs. These findings imply that α-synuclein plays as a negative regulator in WPB exocytosis in endothelial cells.     

Screening of protective antigens of Japanese encephalitis virus by DNA immunization: a comparative study with conventional viral vaccines

In this study, we evaluated the relative role of the structural and nonstructural proteins of the Japanese encephalitis virus (JEV) in inducing protective immunities and compared the results with those induced by the inactivated JEV vaccine. Several inbred and outbred mouse strains immunized with a plasmid (pE) encoding the JEV envelope protein elicited a high level of protection against a lethal JEV challenge similar to that achieved by the inactivated vaccine, whereas all the other genes tested, including those encoding the capsid protein and the nonstructural proteins NS1-2A, NS3, and NS5, were ineffective. Moreover, plasmid pE delivered by intramuscular or gene gun injections produced much stronger and longer-lasting JEV envelope-specific antibody responses than immunization of mice with the inactivated JEV vaccine did. Interestingly, intramuscular immunization of plasmid pE generated high-avidity antienvelope antibodies predominated by the immunoglobulin G2a (IgG2a) isotype similar to a sublethal live virus immunization, while gene gun DNA immunization and inactivated JEV vaccination produced antienvelope antibodies of significantly lower avidity accompanied by a higher IgG1-to-IgG2a ratio. Taken together, these results demonstrate that the JEV envelope protein represents the most critical antigen in providing protective immunity.