9 research outputs found

    Proposed mechanism of HCV NS3 and adenovirus E1A in the Notch-mediated transcription.

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    <p>The HCV NS3 protein activates both the SRCAP and p400 functions for the Notch-mediated transcription, whereas adenovirus E1A protein activates only the p400 function. Since E1A disrupts the interaction of p300 and p400, enhancement of SRCAP-mediated transcriptional activation of Notch target genes by NS3 is inhibited by the competition with the E1A function. The NS3 function for p400-mediated transcriptional activation is not competitive with the E1A function. Thus an additional expression of NS3 enhances the E1A function for increments in the p400-mediated transcriptional activation.</p

    Identification of SRCAP as a host factor which is targeted by HCV NS3.

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    <p>(A, B) HEK293 cells were transfected with the expression vectors for HA-tagged NS3 and FLAG-tagged SRCAP (A), or the expression vectors for HA-tagged SRCAP and FLAG-tagged NS3 (B). After 48 hrs, the cells were harvested and the whole cell lysates were subjected to the immunoprecipitation assay using anti-FLAG M2 agarose (Sigma) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020718#s4" target="_blank">Materials and Methods</a>. The asterisk (*) indicates the bands which are considered to be degradation products of exogenously expressed SRCAP. (C) HEK293 cells were transfected with expression vectors for FLAG-tagged NS3 and SRCAP proteins. After 24 hrs, the cells were harvested, and were fractionated into nuclear and post-nuclear fractions. Anti-histon H1 and anti-actin antibodies were used as a nuclear control and a loading control, respectively.</p

    Specific primer sets used in this study.

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    Chondroitin sulfate (CS) is a glycosaminoglycan, and CS derived from various animal species is used in drugs and food supplements to alleviate arthralgia. The CS is a high molecular weight compound, and hydrolysis of CS by intestinal microbiota is thought to be required for absorption in mammalians. Chondroitin sulfate oligosaccharides (Oligo-CS) are produced by hydrolysis with subcritical water from CS isolated from a species of skate, Raja pulchra for the improvement of bioavailability. The present study conducted in vitro experiments using murine cell lines, to compare the biological activities of Oligo-CS and high molecular weight CS composed with the similar disaccharide isomer units of D-glucuronic acid and N-acetyl-D-glucosamine (CS-C). The results show that Oligo-CS inhibits osteoclast differentiation of RAW264 cells significantly at lower concentrations than in CS. The cell viability of a myoblast cell line, C2C12 cells, was increased when the cells were grown in a differentiated medium for myotubes with Oligo-CS, where there were no effects on the cell viability in CS. These results suggest that in vitro Oligo-CS exhibits stronger bioactivity than high-molecular weight CS.</div

    Marker gene mRNA expressions in C2C12 cells grown in adipocyte differentiation medium.

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    (A) C2C12 cells were grown in adipocyte differentiation medium for a total of 5 days as described in the Materials and Methods. The concentrations of accumulated lipids were measured by the Oil Red O staining method. (B-G) Marker gene mRNA expressions in the C2C12 cells grown in adipocyte differentiation medium were monitored by real-time RT-PCR analysis using the specific primer set for each of the genes. The data indicate relative expressions compared to cells grown in growth medium after normalization with GAPDH mRNA expression. Error bars indicate standard deviations (n = 3). Asterisks (*: p p < 0.001) indicate that the difference is statistically significant between two groups.</p

    Stimulation with AP-PG effectively induces TRAIL mRNA in a human macrophage-like cell line, Mono Mac 6 cells.

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    <p>(A, B) 6 × 10<sup>5</sup> of Mono Mac 6 cells cultured in a 35 mm culture dish were stimulated with AP-PG at a concentration of 100 μg/ml. After 0, 6, 24, 48, and 72 hours, the cells were harvested, and the total RNAs isolated from the cells were subjected to real-time RT-PCR using a specific primer sets for TRAIL (A) and TNF-α (B) genes. (C) Mono Mac 6 cells were stimulated with 100 μg/ml of AP-PG for 21 hours, and then the cells were treated with 10 μg/ml of Brefeldin A (Bref. A). After an additional 3 hour incubation period, the whole cell extracts prepared from the cells were subjected to Western blotting analysis using a specific antibody for the TRAIL protein. An anti-β-actin antibody was used for loading the controls. (D, E) THP-1 cells (6×10<sup>5</sup> cells in a 35 mm culture dish) differentiated into macrophages using PMA were simulated with 100 μg/ml of AP-PG for 0, 3, 6, 12, and 24 hours, and then the total RNAs isolated from the cells were subjected to real-time RT-PCR analysis using specific primer sets for TRAIL (D) or TNF-α (E) genes. (F) 1 × 10<sup>5</sup> of normal human monocytes were differentiated into macrophages, and then stimulated with 100 μg/ml of AP-PG. After 0, 3, 6, 12, and 24 hours, the cells were harvested, and the total RNAs isolated from the cells were subjected to real-time RT-PCR analysis using a specific primer sets for TRAIL mRNA. Data represent relative expression amounts compared with that of control cells after normalizing with the GAPDH mRNA expression. The values in the graphs indicate the means, and error bars indicate standard deviations calculated from three independent experiments.</p

    Expression of RIG-I and MDA5 is upregulated in the RAW264.7 cells, after AP-BG stimulation.

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    <p>(A–C) RAW264.7 cells were stimulated with AP-BG at the concentration of 100 µg/ml. At the time point indicated in the figure, the cells were harvested, and the total RNAs isolated from the cells were subjected to real-time RT-PCR analysis using specific primer sets for retinoic acid-inducible gene-I (RIG-I; A), melanoma differentiation-associated protein 5 (MDA5; B), and interferon-β promoter stimulator 1 (IPS-1; C). The data represent relative mRNA expression compared with the expression level of the initial time point, and the calculated values for each time point were normalized with the expression level of G3PDH mRNA. Error bars indicate standard deviations which were calculated by three independent experiments. (D, E) THP-1 cells which were differentiated into macrophages using 100 nM of phorbol 12-myristate 13-acetate (PMA), were stimulated with 100 µg/ml of AP-BG. After 6 hrs, the cells were harvested, and the total RNAs isolated from the cells were subjected to the real-time RT-PCR analysis using specific primer sets for RIG-I (D) and MDA5 (E). (F) RAW264.7 cells were stimulated with AP-BG (100 µg/ml) or PBS for 6 hrs, and then the PR8 strain of influenza A virus was infected to the cells (MOI = 10). After the incubation for the periods indicated in the figure, the virus titers in the cultured medium were measured by plaque assay. Single asterisk (*) and double asterisk (**) indicate significant differences between AP-BG-treated cells and control cells with P<0.05 and P<0.01, respectively.</p
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