Trx1 in nuclei stimulates dexamethasone-induced death in thymocytes from immature mice.

<p>Western blot shows NLS-hTrx1 expression in thymocytes of Tg mice (A) and in transfected HeLa cell lysates used as NLS-hTrx1 protein control. Isolated thymocytes from Tg (6 mice) and WT (6 mice) were incubated with dexamethasone (Dex, 0, 1, 10 µM) for 4 h. Quantification of surviving cells in each group was measured 4 h after Dex treatment by WST-1 assay and confirmed by trypan blue cell counting. (Data are shown as means ± SE, 3 independent experiments, * p<0.05).</p

NLS-hTrx1 transgenic mice have increased NF-κB activation and proinflammatory cytokine (TNF-α and IL-6) production after influenza viral infection.

<p>Lung tissues obtained from Tg and WT before (3 mice each for Tg and WT) and 3-d post infection (6 mice for Tg, 5 mice for WT) were examined for mRNA of cytokines (A) and NF-κB activity (B). mRNA levels of TNF-α and IL-6 were analyzed and quantified by real-time PCR. * p<0.05 for 3-d post infection in Tg compared to 3 d post infection in WT and 0 d in Tg. B, NF-κB activity was examined by EMSA using same tissues analyzed for cytokines shown in A. Bands indicated as NF-κB bound DNA probe show activity of NF-κB. Densitometry values shown as fold difference were obtained from measuring relative intensities compared to that in WT before infection (lane 2). [lanes 1–6 are as follows; 1, NF-κB probe alone; 2, WT before infection; 3, Tg before infection; 4, WT 3 days post infection; 5, Tg 3-d post infection; 6, Tg 3-d post infection (cold NF-κB probe incubation followed by labeled NF-κB probe incubation)]. C, NF-κB activity (NF-κB Luc) was examined by expression of NLS-hTrx1 WT or dominant negative mutant of Trx1, NLS-hTrx1 C35S by transient cotransfection with NF-κB luciferase and β-galactosidase. Quantified luciferase activity as a measure of NF-κB activity was normalized by β-galactosidase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018918#pone.0018918-Go4" target="_blank">[37]</a>. Western blot confirms expression of NLS-hTrx1 WT and NLS-hTrx1 C35S (C, top) and β-actin (C, middle) for equal protein loading in HeLa cells 2 d after transfection. Results of luciferase activity in bar graph are shown as means ± SE (n = 8, * p<0.05).</p

Changes in GSH/GSSG redox potential (E_hGSSG) in Tg and WT following H1N1 influenza virus infection.

<p>The same lung samples described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018918#pone-0018918-g005" target="_blank">Fig. 5</a> were examined for GSH and GSSG concentrations by HPLC, and E_hGSSG was calculated using the Nernst equation <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018918#pone.0018918-Jones2" target="_blank">[60]</a>. No differences in concentrations or E_hGSSG oxidation were observed in lung tissues of Tg or WT following infection, but more oxidation of plasma E_hGSSG was observed in Tg compared to WT. Three mice for each Tg and WT without infection were analyzed as control (0 in A). Plasma GSH, GSSG, and E_hGSSG in Tg and WT before and 3 d post infection are shown in B. * p<0.05.</p

Effect of influenza virus infection on (A) body weight and (B) survival in NLS-hTrx1 Tg mice compared to WT littermates.

<p>WT (n = 18) and Tg mice (n = 17) were infected with 2009 H1N1 influenza A/California/04/2009 virus (1× LD50) as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018918#pone.0018918-Quan1" target="_blank">[38]</a>. Body weight and survival rates (WT; 7/18, Tg; 2/17) were monitored daily and presented as percentages based on day 0.</p

mRNA levels of NLS-hTrx1 in different tissues.

<p>Different organs harvested from Tg and littermate WT mice were analyzed to examine mRNA abundance of nuclear compartment-targeted hTrx1 and β-actin. Each cDNA converted from purified tissue RNA (1 µg) by reverse transcription was analyzed by real-time PCR. Quantified values (picomole) are represented as mean ± SE for 4 Tg and 4 WT mice. Symbols are as follows; H, heart; K, kidney; Li, liver; Lu, lung; SM, skeletal muscle; SI, small intestine; Sp, spleen; Th, thymus.</p

Nuclear localization of NLS-hTrx1 in Tg mouse.

<p>Cells isolated from kidneys of Tg and WT were analyzed to examine localization by immunofluorescence (A) and western blotting (B). A, Nuclear compartmentalized expression of NLS-hTrx1 was visualized by myc antibody followed by Cy3 (orange, Tg). As a control, actin distribution in all area of cells was obtained by Alexa Fluor 488 phalloidin, shown for both Tg and WT (green). Subcellular fractions including cytoplasm and nuclei were obtained from kidney tissues of Tg and WT. Western blotting using each fraction confirmed nuclear localization of NLS-hTrx1 in Tg. Western blot analysis of lamin and GAPDH were used as markers to verify purity of nuclear and cytoplasmic fractions, respectively.</p

Increased endogenous mouse Trx1 level in nuclei by infection with influenza H1N1 virus.

<p>WT mice infected with influenza H1N1 virus (A/California/04/2009) at day 3 post infection (day 3) or uninfected WT control mice (day 0) were examined for nuclear Trx1. Cytoplasm (Cyto) and nuclear (Nuc) fractions of lung tissues (A) and immune cells isolated from lungs were examined for Trx1 level by western blot analysis. The same blot was probed with a lamin A/C antibody as a nuclear protein marker. Results are representatives of 3 analyses.</p

A scheme for stimulation of inflammatory response in NLS-hTrx1 Tg by infection.

<p>Virus infection and other inflammatory stimuli affect cytoplasmic redox state, e.g. oxidation of E_hGSSG. Cytoplamic oxidation results in phosphorylation and degradation of I-κBα, which then translocates NF-κB to nucleus. Upon its translocation, p50 subunit (Cys62) reduced by nuclear localized Trx1 together with Ref-1 and Prx stimulates its DNA binding activity followed by subsequent gene expression, e.g. TNF-α and IL-6. Increased inflammatory cytokines further activate NF-κB as feedback stimulation.</p

Correction: Metabolic Characterization of the Common Marmoset (<i>Callithrix jacchus</i>)

<p>Correction: Metabolic Characterization of the Common Marmoset (<i>Callithrix jacchus</i>)</p

Metabolites higher in female than male marmosets.

<p>Female and male marmosets were grouped according to age as younger (y, 2 to 7 y) and older (o, 8 to 15 y). Following ANOVA, post-hoc tests showed differences between groups as indicated by a, female versus male in younger marmosets; b, female versus male in older marmosets. Group sizes were: females, 12 y, 12 o: males 13 y, 13 o.</p