Improved synaptic and cognitive function in aged 3 × Tg-AD mice with reduced amyloid-ß after immunotherapy with a novel recombinant 6Aß15-TF chimeric vaccine.

Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder impairing memory and cognition. In this study, we describe the immunogenicity and protective efficacy of the novel recombinant 6Aß15-TF chimeric antigen as a subunit protein vaccine for AD. Recombinant 6Aß15-TF chimeric vaccine induced strong Aß-specific humoral immune responses without Aß-specific T cell immunity in C57/BL6 and 3 × Tg-AD mice at different ages. As an early immunotherapy model for AD, this vaccine induced high titers of long-lasting anti-Aß42 antibodies in aged 3 × Tg-AD mice, which led to improve behavioral performance and markedly reduced the levels of insoluble and soluble Aß and Aß oligomers. In agreement with these findings, immunotherapy with 6Aß15-TF prevented the Aß-induced decrease of presynaptic and postsynaptic proteins in aged 3 × Tg-AD mice. Our results suggest that this novel and highly immunogenic recombinant 6Aß15-TF chimeric vaccine provides neuroprotection in AD mice and can be considered an effective AD candidate vaccine.

The highly pathogenic H5N1 influenza A virus down-regulated several cellular MicroRNAs which target viral genome.

Higher and prolonged viral replication is critical for the increased pathogenesis of the highly pathogenic avian influenza (HPAI) subtype of H5N1 influenza A virus (IAV) over the lowly pathogenic H1N1 IAV strain. Recent studies highlighted the considerable roles of cellular miRNAs in host defence against viral infection. In this report, using a 3'UTR reporter system, we identified several putative miRNA target sites buried in the H5N1 virus genome. We found two miRNAs, miR-584-5p and miR-1249, that matched with the PB2 binding sequence. Moreover, we showed that these miRNAs dramatically down-regulated PB2 expression, and inhibited replication of H5N1 and H1N1 IAVs in A549 cells. Intriguingly, these miRNAs expression was differently regulated in A549 cells infected with the H5N1 and H1N1 viruses. Furthermore, transfection of miR-1249 inhibitor enhanced the PB2 expression and promoted the replication of H5N1 and H1N1 IAVs. These results suggest that H5N1 virus may have evolved a mechanism to escape host-mediated inhibition of viral replication through down-regulation of cellular miRNAs, which target its viral genome.

Analysis of Soluble protein complexes in Shigella flexneri reveals the influence of temperature on the amount of lipopolysaccharide.

Shigella flexneri, which is closely related to Escherichia coli, is the most common cause of the endemic form of shigellosis. In this study, 53 homomultimeric protein complexes and nine heteromultimeric protein complexes from S. flexneri 2a strain 2457T were separated and identified. Among these, three potential homomultimeric protein complexes had not been previously described. One complex, thought to be composed of 12 PhoN1 subunits, is a periplasmic protein with an unknown physiological role encoded on the virulence plasmid of S. flexneri. The abundance of the protein complexes was compared following growth at 37 or 30°C, and the abundance of three protein complexes (PyrB-PyrI, GlmS, and MglB) related to the synthesis of lipopolysaccharides (LPS) appeared to be temperature-dependent. Many studies have shown that LPS is essential to the virulence of S. flexneri. Here, we report the influence of temperature on the amount of LPS.

Intracellular delivery of artificial transcription factors fused to the protein transduction domain of HIV-1 Tat.

Protein transduction domains (PTDs), such as the TAT peptide derived from HIV Tat protein, may transduce macromolecules into cells. In the present study, the TAT peptide-fused artificial transcription factors (ATFs) were generated by fusion of the N-terminal TAT peptide with SV40 promoter-targeted three-fingered C2H2 zinc finger proteins and the KRAB transcriptional repression domain. The fusion proteins were then expressed in an E .coli system and purified by Ni-NTA affinity chromatography. The purified fusion proteins were tested on mammalian cell lines CHO DG44 and L929. TAT-ATF-S, which contains the zinc fingers that bind to the SV40 promoter with high specificity, exhibited the desired transcriptional repression activity to the reported genes, indicating the successful cellular delivery and desired conformation of TAT-ATF-S. Our study has provided a new strategy for intracellular ATF delivery.

The extremely high level expression of human serum albumin in the milk of transgenic mice.

The recombinant production of human serum albumin has been challenging due to the low unit price and huge amount needed, for the commercial production of rhSA at an economically feasible level, It will be well worth the effort to exploit new method for the extremely high level expression of rhSA. To this end, here a hybrid gene locus strategy was employed, a 37 Kb mWAP-hSA hybrid gene locus was constructed and used as mammary gland specific expression vector, in which the 3 Kb genomic coding sequence in the 24 Kb mouse whey acidic protein (mWAP) gene locus was substituted by the 16 Kb genomic coding sequence of human serum albumin (hSA), exactly from the start codon to the end codon. Corresponding transgenic mice were generated and rhSA was secreted into the milk at an extremely high level of 11.9 g/L. Our transgenic mice carrying the mWAP-hSA hybrid gene locus represent a model system for the cost-effective production of human serum albumin.

The proteome of Shigella flexneri 2a 2457T grown at 30 and 37 degrees C.

To upgrade the proteome reference map of Shigella flexneri 2a 2457T, the protein expression profiles of log phase and stationary phase cells grown at 30 and 37 degrees C were thoroughly analyzed using multiple overlapping narrow pH range (between pH 4.0 and 11.0) two-dimensional gel electrophoresis. A total of 723 spots representing 574 protein entries were identified by MALDI-TOF/TOF MS, including the majority of known key virulence factors. 64 hypothetical proteins and six misannotated proteins were also experimentally identified. A comparison between the four proteome maps showed that most of the virulence-related proteins were up-regulated at 37 degrees C, and the differences were more notable in stationary phase cells, suggesting that the expressions of these virulence factors were not only controlled by temperature but also controlled by the nutrients available in the environment. The expression patterns of some virulence-related genes under the four different conditions suggested that they might also be regulated at the post-transcriptional level. A further significant finding was that the expression of the protein ArgT was dramatically up-regulated at 30 degrees C. The results of semiquantitative RT-PCR analysis showed that expression of argT was not regulated at the transcriptional level. Therefore, we carried out a series of experiments to uncover the mechanism regulating ArgT levels and found that the differential expression of ArgT was due to its degradation by a periplasmic protease, HtrA, whose activity, but not its synthesis, was affected by temperature. The cleavage site in ArgT was between position 160 (Val) and position 161 (Ala). These results may provide useful insights for understanding the physiology and pathogenesis of S. flexneri.

The interaction between the PARP10 protein and the NS1 protein of H5N1 AIV and its effect on virus replication.

BACKGROUND: During the process that AIV infect hosts, the NS1 protein can act on hosts, change corresponding signal pathways, promote the translation of virus proteins and result in virus replication. RESULTS: In our study, we found that PARP domain and Glu-rich region of PARP10 interacted with NS1, and the presence of NS1 could induce PARP10 migrate from cytoplasm to nucleus. NS1 high expression could reduce the endogenous PARP10 expression. Cell cycle analysis showed that with inhibited PARP10 expression, NS1 could induce cell arrest in G2-M stage, and the percentage of cells in G2-M stage rise from the previous 10%-45%, consistent with the cell proliferation result. Plague forming unit measurement showed that inhibited PARP10 expression could help virus replication. CONCLUSIONS: In a word, our results showed that NS1 acts on host cells and PARP10 plays a regulating role in virus replication.

The NS1 protein of influenza A virus interacts with heat shock protein Hsp90 in human alveolar basal epithelial cells: implication for virus-induced apoptosis.

BACKGROUND: Our previous study showed that the NS1 protein of highly pathogenic avian influenza A virus H5N1 induced caspase-dependent apoptosis in human alveolar basal epithelial cells (A549), supporting its function as a proapoptotic factor during viral infection, but the mechanism is still unknown. RESULTS: To characterize the mechanism of NS1-induced apoptosis, we used a two-hybrid system to isolate the potential NS1-interacting partners in A549 cells. We found that heat shock protein 90 (Hsp90) was able to interact with the NS1 proteins derived from both H5N1 and H3N2 viruses, which was verified by co-immunoprecitation assays. Significantly, the NS1 expression in the A549 cells dramatically weakened the interaction between Apaf-1 and Hsp90 but enhanced its interaction with cytochrome c (Cyt c), suggesting that the competitive binding of NS1 to Hsp90 might promote the Apaf-1 to associate with Cyt c and thus facilitate the activation of caspase 9 and caspase 3. CONCLUSIONS: The present results demonstrate that NS1 protein of Influenza A Virus interacts with heat hock protein Hsp90 and meidates the apoptosis induced by influenza A virus through the caspase cascade.

Hepatitis B virus X protein regulates the mEZH2 promoter via the E2F1-binding site in AML12 cells.

Histone lysine methyltransferase EZH2 has been reported to be frequently overexpressed in hepatocellular carcinoma (HCC) tissues and associated with hepatocarcinogenesis. However, the exact mechanism of EZH2 up-regulation in HCC has not been determined. In this study, we used murine hepatocyte AML12 cells to investigate the role of hepatitis B virus X protein (HBx) in regulating the expression of mEZH2. Western blot analysis demonstrated that the expression level of mEZH2 protein in AML12 cells was up-regulated by HBx in a dose-dependent manner. To further investigate the mechanism of mEZH2 overexpression, the 2500 bp regulatory sequence upstream from the first exon of the mEZH2 gene was amplified from AML12 genomic DNA and constructed into a luciferase reporter plasmid. The luciferase activity of the mEZH2 promoter significantly increased in AML12 cells co-transfected with HBx plasmid, and deleting the -486/-214 promoter region decreased HBx-induced mEZH2 promoter activation by nearly 50%. The -486/-214 region was then analyzed in the TRANSFAC 6.0 database and a typical E2F1-binding site was found. Mutation of this E2F1-binding site or knockdown of E2F1 expression by RNAi led to a dramatic decrease in HBx-induced activation of the mEZH2 promoter and mEZH2 overexpression in AML12 cells. These results provide evidence that HBx up-regulates mEZH2 expression by transactivating the mEZH2 promoter through E2F1 transcription factor, thereby providing new epigenetic evidence for the carcinogenic effect of HBx.

Global analysis of a plasmid-cured Shigella flexneri strain: new insights into the interaction between the chromosome and a virulence plasmid.

Shigella flexneri is an important human pathogen that causes dysentery, and remains a significant threat to public health, particularly in developing countries. The virulence of this pathogen is dependent on an acquired virulence plasmid. To investigate the crosstalk between the bacterial chromosome and the exogenous virulence plasmid, a virulence plasmid-cured strain was constructed using plasmid incompatibility. The global patterns of gene expression of this strain compared with the wild-type strain were analyzed using 2-DE combined with MALDI-TOF MS. Most known virulence factors of S. flexneri were identified in the 2-DE gels. Interestingly, the expression of the glycerol 3-phosphate (glp) regulon-encoded proteins was increased when the virulence plasmid was absent. Microarray analysis confirmed that regulation occurred at the transcriptional level. Purification and identification of DNA binding proteins with affinity for the regulatory region of the glp genes revealed that regulation mediated by the virulence plasmid to control the expression of the glp regulon might in turn be mediated by protein GlpR. To our knowledge, this is the first study analyzing the interaction between a pathogen chromosome and a virulence plasmid at the proteomic level.

Development and evaluation of candidate vaccine and antitoxin against botulinum neurotoxin serotype F.

To produce a vaccine suitable for human use, a recombinant non His-tagged isoform of the Hc domain of botulinum neurotoxin serotype F (rFHc) was expressed in Escherichia coli and purified by sequential chromatography. The rFHc was evaluated as a subunit vaccine candidate in mouse model of botulism. A dose-response was observed in both antibody titer and protective efficacy with increasing dosage of rFHc and number of vaccinations. These findings suggest that the rFHc is an effective botulism vaccine candidate. Further, we developed a new antitoxin against botulinum neurotoxin serotype F (BoNT/F) by purifying F(ab')(2) fragments from pepsin digested serum IgGs of horses inoculated with rFHc. The protective effect of the F(ab')(2) antitoxin against BoNT/F was determined both in vitro and in vivo. The results showed that the F(ab')(2) antitoxin could prevent botulism in mice challenged with BoNT/F and effectively delayed progression of paralysis from botulism in the therapeutic setting. Thus, our results provide valuable experimental data for this new antitoxin as a potential candidate for treatment of botulism caused by BoNT/F.

Highly pathogenic avian influenza A virus H5N1 NS1 protein induces caspase-dependent apoptosis in human alveolar basal epithelial cells.

BACKGROUND: It is widely considered that the multifunctional NS1 protein of influenza A viruses contributes significantly disease pathogenesis by modulating a number of virus and host-cell processes, but it is highly controversial whether this non-structural protein is a proapoptotic or antiapoptotic factor in infected cells. RESULTS: NS1 protein of influenza A/chicken/Jilin/2003 virus, a highly pathogenic H5N1 strain, could induce apoptosis in the carcinomic human alveolar basal epithelial cells (A549) by electron microscopic and flow cytometric analyses. NS1 protein-triggered apoptosis in A549 cells is via caspase-dependent pathway. CONCLUSIONS: Influenza A virus NS1 protein serves as a strong inducer of apoptosis in infected human respiratory epithelial cells and plays a critical role in disease pathogenesis.

A novel human monoclonal antibody potently neutralizes human adenovirus serotype 7 by primarily targeting the adenovirus hexon protein.

Human adenovirus serotype 7 (HAdV-7), belonging to species B, has caused severe lower respiratory tract diseases and even deaths recently. However, no adenovirus vaccine or therapeutic is available thus far. In this study, a HAdV-7-specific human monoclonal antibody (HMAb), 3-3E, isolated from single plasma cells obtained from the peripheral blood mononuclear cells of HAdV-7-infected patients showed potent HAdV-7 neutralization activity. The results showed HMAb 3-3E only binds to the hexon protein of intact HAdV-7 or the recombinant hexon protein and it does not bind to other intact virion particles. This could mean the antibody recognizes a conformational epitope of the hexon protein. Further, HMAb 3-3E potently neutralized HAdV-7 in vitro at low concentrations. In vivo studies showed HMAb 3-3E protected from HAdV-7 infection in a murine model. Therefore, HMAb 3-3E is promising as a safe and effective prophylactic and therapeutic treatment for HAdV-7 infection.

A mWAP-hLF hybrid gene locus gave extremely high level expression of human lactoferrin in the milk of transgenic mice.

The production of pharmaceuticals by current mammary gland bioreactor techniques is limited by the low expression level of foreign proteins. We describe here a novel method that solves this problem. A successive three-step gap-repair strategy was developed to replace the genomic coding sequence in mouse whey acidic protein (mWAP) gene locus with that of human lactoferrin (hLF) precisely from the start code to the end code. A 50-kb mWAP-hLF hybrid gene locus was constructed, and corresponding transgenic mice were generated. An extremely high-level expression of rhLF in the milk was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot. The expression level ranged from 16.7 to 29.8 g/l among five transgenic lines, as indicated by the ELISA assay. Importantly, the expressed rhLF maintained the same antibacterial activity as the native hLF. Our strategy can very likely also be used for the efficient expression of other valuable pharmaceutical proteins.

Interaction of influenza virus NS1 protein with growth arrest-specific protein 8.

NS1 protein is the only non-structural protein encoded by the influenza A virus, and it contributes significantly to disease pathogenesis by modulating many virus and host cell processes. A two-hybrid screen for proteins that interact with NS1 from influenza A yielded growth arrest-specific protein 8. Gas8 associated with NS1 in vitro and in vivo. Deletion analysis revealed that the N-terminal 260 amino acids of Gas8 were able to interact with NS1, and neither the RNA-binding domain nor the effector domain of NS1 was sufficient for the NS1 interaction. We also found that actin, myosin, and drebrin interact with Gas8. NS1 and beta-actin proteins could be co-immunoprecipitated from extracts of transfected cells. Furthermore, actin and Gas8 co-localized at the plasma membrane. These results are discussed in relation to the possible functions of Gas8 protein and their relevance in influenza virus release.

PC-1/PrLZ contributes to malignant progression in prostate cancer.

PC-1/PrLZ gene overexpression has been identified to be associated with prostate cancer progression. Previous studies have revealed that PC-1 possesses transforming activity and confers malignant phenotypes to mouse NIH3T3 cells. However, the functional relevance of PC-1 expression changes during prostate cancer development and progression remains to be evaluated. In this study, gain-of-function and loss-of-function analyses in LNCaP and C4-2 cells, respectively, were implemented. Experimental data showed that PC-1 expression was in positive correlation with prostate cancer cell growth and anchor-independent colony formation in vitro, as well as tumorigenicity in athymic BALB/c mice. Moreover, PC-1 expression was also found to promote androgen-independent progression and androgen antagonist Casodex resistance in prostate cancer cells. These results indicate that PC-1 contributes to androgen-independent progression and malignant phenotypes in prostate cancer cells. Furthermore, molecular evidence revealed that PC-1 expression stimulated Akt/protein kinase B signaling pathway, which has been implicated to play important roles in promoting androgen refractory progression in prostate cancer. Increased PC-1 levels in C4-2 cells may represent an adaptive response in prostate cancer, mediating androgen-independent growth and malignant progression. Inhibiting PC-1 expression may represent a novel therapeutic strategy to delay prostate cancer progression.

Cloning and functional analysis of 5'-upstream region of the Pokemon gene.

Pokemon, the POK erythroid myeloid ontogenic factor, not only regulates the expression of many genes, but also plays an important role in cell tumorigenesis. To investigate the molecular mechanism regulating expression of the Pokemon gene in humans, its 5'-upstream region was cloned and analyzed. Transient analysis revealed that the Pokemon promoter is constitutive. Deletion analysis and a DNA decoy assay indicated that the NEG-U and NEG-D elements were involved in negative regulation of the Pokemon promoter, whereas the POS-D element was mainly responsible for its strong activity. Electrophoretic mobility shift assays suggested that the NEG-U, NEG-D and POS-D elements were specifically bound by the nuclear extract from A549 cells in vitro. Mutation analysis demonstrated that cooperation of the NEG-U and NEG-D elements led to negative regulation of the Pokemon promoter. Moreover, the NEG-U and NEG-D elements needed to be an appropriate distance apart in the Pokemon promoter in order to cooperate. Taken together, our results elucidate the mechanism underlying the regulation of Pokemon gene transcription, and also define a novel regulatory sequence that may be used to decrease expression of the Pokemon gene in cancer gene therapy.

Effect of O-superfamily conotoxin SO3 on synchronized spontaneous calcium spikes in cultured hippocampal networks.

SO3 belongs to the O-superfamily of conotoxins and is known to have analgesic effects in experimental animals. In order to explore the mechanism of its potential pharmacological actions, the effect of SO3 on synchronized spontaneous calcium spikes was examined in cultured hippocampal networks by calcium imaging. Spontaneous oscillations of intracellular concentrations of calcium (Ca(2+)) in the form of waves and spikes are found in cultured hippocampal networks. Exposure to increasing concentrations of SO3 resulted in a progressive decrease in synchronized spontaneous calcium spikes. The higher concentrations (0.1 micromol/L and 1 micromol/L) of SO3 showed the strongest inhibition. The rank order of inhibition was 1 micromol/L > 0.1 micromol/L > 10 micromol/L > 0.01 micromol/L. This action of SO3 in reducing synchronized calcium spikes suggests a possible application for therapeutic treatment of epilepsy.

miR-21-3p Regulates Influenza A Virus Replication by Targeting Histone Deacetylase-8.

Influenza A virus (IAV) is responsible for severe morbidity and mortality in animals and humans worldwide. miRNAs are a class of small noncoding single-stranded RNA molecules that can negatively regulate gene expression and play important roles in virus-host interaction. However, the roles of miRNAs in IAV infection are still not fully understood. Here, we profiled the cellular miRNAs of A549 cells infected with A/goose/Jilin/hb/2003 (H5N1) and a comparison A/Beijing/501/2009 (H1N1). miRNA microarray and quantitative PCR analysis showed that several miRNAs were differentially expressed in A549 cells during IAV infection. Subsequently, we demonstrated that IAV replication was essential for the regulation of these miRNAs, and bioinformatic analysis revealed that the targets of these miRNAs affected biological processes relevant to IAV replication. Specifically, miR-21-3p was found to be down-regulated in IAV-infected A549 cells and selected for further detailed analysis. Target prediction and functional study illustrated that miR-21-3p repressed the expression of HDAC8 by targeting its 3'UTR. Furthermore, we confirmed miR-21-3p could promote virus replication, which was similar to the result of knocking down HDAC8, indicating that miR-21-3p promoted IAV replication by suppressing HDAC8 expression. Altogether, our results suggest a potential host defense against IAV through down-regulation of miR-21-3p.

Potent Neutralization Ability of a Human Monoclonal Antibody Against Serotype 1 Dengue Virus.

The incidence of dengue virus (DENV) infections has been escalating in tropical and subtropical countries, but there are still no effective therapeutic options. In the present study, a DENV-1-specific human monoclonal antibody (HMAb), 1G5, isolated from single plasma cells obtained from the peripheral blood mononuclear cells of dengue patients was found to have potent neutralization activity against serotype 1 DENV (DENV-1). Its neutralization activity against DENV-2 was not as strong, and it was almost absent for DENV-3 and DENV-4. The results showed that HMAb 1G5 only binds to the envelop protein of intact DENV-1 or the envelop protein under unheated and non-reducing conditions, and that it does not bind to recombinant envelope protein. This could mean that the antibody recognizes a conformational epitope of the envelope protein. Further, the findings showed that HMAb 1G5 potently neutralizes DENV-1 in both the pre- and post-attachment phases of the virus at low concentrations. In vivo studies showed that HMAb 1G5 provides protection from DENV-1 infection in a murine model. In addition, antibody-dependent enhancement that occurs at lower doses of the antibody was completely abrogated by the introduction of Leu-to-Ala mutations (1G5-LALA) or deletion of nine amino acids (1G5-9del) in the Fc region. Therefore, HMAb 1G5 shows promise as a safe and effective agent for prophylactic and therapeutic treatment of DENV-1 infection.