Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-7

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p>g region of with respect to the start site of transcription; the y axis indicates the percentage of the subclones that were methylated

Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-5

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p>showing MsrA (26 kDa) and Sp1 (105 kDa) protein levels

Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-1

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p>uence of the band from the RACE -2 primer reaction is reported. The upstream sequence from the fragment cloned from the band is in upper case and the arrow indicates the transcription start site (+ 1). The open reading frame codifying for MsrB1 is in lower case and the start codon is in boldface type

Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-3

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p> was designed P-203 Sp1EMut. Luciferase reporter activity was assessed following transfection into MCF7 cells. Luciferase activity was inhibited by approximately 80% by mutation of Sp1E site. () the sequences spanning Sp1-binding sites (Sp1C and Sp1E) contained in the P-296 WT construct were subjected to site-directed mutagenesis. The constructs obtained were designated as P-296 Sp1CMut and P-296 Sp1EMut. In addition a double mutant containing both mutations was indicated as P-296 Sp1C/EMut. The mutated constructs were transiently expressed in MCF7 cells for luciferase assays. Luciferase activity was inhibited by approximately 30% by mutation of Sp1C site; 24% by mutation of Sp1E site and by approximately 76% by mutation of both Sp1 sites (Sp1C/Sp1E). The results are expressed as mean ± S.D. of at least five different experiments, in duplicate for each construct. Statistically significant differences compared to the appropriate WT construct are indicated by *P < 0.005 and **P < 0.002

Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-0

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p>t levels in MCF7 and MDA-MB231 cells were analysed using Chemi Doc System (Bio-Rad) and normalized by GAPDH signal intensities of the corresponding lanes. The data are reported as mean ± S.D. of three independent experiments; * P < 0.001. Immunoblotting of MCF7 and MDA-MB231 extracts with a mouse monoclonal anti-MsrB1. The MsrB1 protein (13 kDa) levels in MCF7 and MDA-MB231 cells were analysed using Chemi Doc System (Bio-Rad) and normalized by β-actin (42 kDa) signal intensities of the corresponding lanes. Values are reported as mean ± S.D. of at least five different immunoblotting experiments; ** P < 0.005

Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter-4

<p><b>Copyright information:</b></p><p>Taken from "Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter"</p><p>http://www.biomedcentral.com/1471-2199/8/39</p><p>BMC Molecular Biology 2007;8():39-39.</p><p>Published online 22 May 2007</p><p>PMCID:PMC1885803.</p><p></p>complexes. The control + lane was DNA that had been sonicated and pre-cleared with protein G beads. The control-lanes were processed according to the protocol, but did not have any Ab added to the samples. PCR were performed on isolated DNA using primers encompassing MsrB1 promoter region -169 to +76. A schematic map of the amplified DNA fragment (245 bp) containing Sp1 binding motifs and TSS position is illustrated as well

<i>Stenotrophomonas maltophilia</i> Virulence and Specific Variations in Trace Elements during Acute Lung Infection: Implications in Cystic Fibrosis

<div><p>Metal ions are necessary for the proper functioning of the immune system, and, therefore, they might have a significant influence on the interaction between bacteria and host. Ionic dyshomeostasis has been recently observed also in cystic fibrosis (CF) patients, whose respiratory tract is frequently colonized by <i>Stenotrophomonas maltophilia</i>. For the first time, here we used an inductively mass spectrometry method to perform a spatial and temporal analysis of the pattern of changes in a broad range of major trace elements in response to pulmonary infection by <i>S. maltophilia</i>. To this, DBA/2 mouse lungs were comparatively infected by a CF strain and by an environmental one. Our results showed that pulmonary ionomic profile was significantly affected during infection. Infected mice showed increased lung levels of Mg, P, S, K, Zn, Se, and Rb. To the contrary, Mn, Fe, Co, and Cu levels resulted significantly decreased. Changes of element concentrations were correlated with pulmonary bacterial load and markers of inflammation, and occurred mostly on day 3 post-exposure, when severity of infection culminated. Interestingly, CF strain – significantly more virulent than the environmental one in our murine model - provoked a more significant impact in perturbing pulmonary metal homeostasis. Particularly, exposure to CF strain exclusively increased P and K levels, while decreased Fe and Mn ones. Overall, our data clearly indicate that <i>S. maltophilia</i> modulates pulmonary metal balance in a concerted and virulence-dependent manner highlighting the potential role of the element dyshomeostasis during the progression of <i>S. maltophilia</i> infection, probably exacerbating the harmful effects of the loss of CF transmembrane conductance regulator function. Further investigations are required to understand the biological significance of these alterations and to confirm they are specifically caused by <i>S. maltophilia</i>.</p></div

Trace elements, bacterial load, and cytokine levels observed in lung tissue of <i>S. maltophilia</i>-infected DBA/2N mice.

<p>A–C) Score and loading plots obtained by Principal Component Analysis. These plots show PC1 vs PC2summarizing 56%, 76%, and 59% of the variation among lung samples collected on days 1 (A), 3 (B), and 7 (C) p.e. to <i>S. maltophilia</i> (Sm111 CF strain, red circles; C39 environmental strain, green triangles) or PBS only (controls, black rhombi). The amounts of variance explained by each PC are shown in parentheses. D–E) Correlation maps. The heatmaps show the unsupervised hierarchical clustering of lung elements, bacterial counts in lung homogenate (CFU), and cytokines assessed using Spearman's correlation in response to <i>S. maltophilia</i> infection by Sm111 CF (D) or C39 (E) strain. Direct and inverse correlations are shown in red and green, respectively. Correlation and cluster analysis were performed using MetaboAnalyst statistical analysis module <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088769#pone.0088769-Xia1" target="_blank">[29]</a>.</p

Assessment of lung damage in DBA/2 lung following infection with <i>S. maltophilia</i>.

<p>Lung damage was macroscopically and histologically assessed in DBA/2 mice (n = 18) on days 1, 3, and 7 p.e. to <i>S. maltophilia</i> (Sm111 CF strain, red line; C39 environmental strain, green line), or PBS only (control; black line). A–C) Macroscopic examination. A) Photographs of control and infected mouse lungs are representative for lung damage observed at each time point (days 1, 3, and 7 p.e.) in 8 mice per group. C) Lung damage was assessed by using “four-point scoring system” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088769#pone.0088769-Johansen1" target="_blank">[24]</a>, and results are shown as median values. B–D) Histological assessment. B) Lung sections were stained with hematoxylin-eosin and ten fields/lung were observed at low (10×), and high (63×) magnification. For each group, only one representative microscopic field was reported. Since there were no significant differences in the degree of inflammation at any of the time point when evaluating the left and the right lung separately, the data were pooled and considered representative for both lungs. Mean percentage of tissue area characterized by the presence of neutrophilic infiltrate: 60% (Sm111-infected mice) vs 20% (C39-infected mice) (p<0.001, chi-square test.). D) Lung damage was quantified by using the “five-point scoring system” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088769#pone.0088769-Dubin1" target="_blank">[25]</a>, and results are shown as median histopatological index. *** p<0.001, ** p<0.01, * p<0.05 vs ctrl; °° p<0.01, ° p<0.05 Sm111 vs C39; Kruskall-Wallis followed by Dunn's multiple comparison <i>post-hoc</i> test.</p

MG-related protein damage and expression of MG-targeting detoxification system in hippocampi of mice undergoing 2- or 4-mo moderate treadmill running.

<p>Immunoreactivity levels against arg-pyrimidine and protein expression of glyoxalase 1 (GLO1) in hippocampi of mature CD-1 female mice undergoing a two- or four-month moderate and regular treadmill-based exercise program (E2 or E4, respectively); age-matched sedentary animals (S2, S4) were used as controls (n = 12 per group). As shown in panel a, an age-dependent increase in the levels of MG-damaged protein was detected (S4 vs S2), and an increasing trend in MG-related protein damage was observed after 2 months of running (E2 vs S2). The immunoreactivity against arg-pyrimidine decreased after 4 months of regular exercise (E4 vs S4). Dot-blots signals were normalized against the Comassie Brilliant Blue-based total protein staining. In the panel a, right section, representative dot immunoblots of three independent experiments were reported. As reported in panel b, an age-dependent increase in the GLO1 protein expression levels was detected (S4 vs S2). 2-mo exercise increased GLO1 protein levels (E2 vs S2), while the expression levels of GLO1 decreased after four months of regular running (E4 vs S4). Immunosignals were normalized against the housekeeping β-actin. Representative western immunoblots of three independent experiments were reported in panel b. Values were given as means ± std. dev. The level of statistical significance was computed by using two-way ANOVA and post-hoc Newman-Keuls test: *** P<0.001; ** P<0.01. Experiments were performed in triplicate.</p