Gel mobility shift assay using the <i>OsDXS3</i> upstream region.

<p>(A) DNA probes used in GMSA. Closed triangles indicate TGACGT sequences. (B) GMSA was performed using purified recombinant GST-fused OsTGAP1 (GST-OsTGAP1) protein and ³²P-labelled DNA probes containing the TGACGT sequences in the <i>OsDXS3</i> promoter. WT: wild-type probe, m1–m3: mutated probes.</p

Overview of the results of ChIP-seq.

<p>(A) Distribution of OsTGAP1-binding regions in the rice genome. The upstream region includes the binding regions within 2 kbp of the transcription start site. The gene region includes the binding regions that are located between the transcription start site and the transcription termination site. The downstream region includes the binding regions within 2 kbp of the transcription termination site. The remaining binding regions were assigned to intergenic regions. (B) Distribution of OsTGAP1-binding regions in the upstream and downstream regions. Red lines indicate the average of number of OsTGAP1-binding regions. Statistical analysis was performed for each 100-bp region, and significantly enriched regions were indicated by asterisks (<i>P</i><0.01 the binomial test and the Bonferroni correction). (C) Overrepresented motifs in the OsTGAP1-binding regions analysed in the untreated and elicitor-treated conditions using Partek Genomics Suite based on Gibbs motif sampler <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105823#pone.0105823-Neuwald1" target="_blank">[37]</a>. (D) Distribution of TGACGT sequences around the OsTGAP1-binding regions.</p

OsTGAP1-binding regions in the <i>OsDXS3</i> upstream region.

<p>(A) The mapped ChIP-seq reads in untreated (E–, grey) and elicitor-treated (E+, black) conditions were visualized using Partek Genomics Suite. Two biological replicates were performed. Black bars indicate the positions of TGACGT sequences. The gene structure of <i>OsDXS3</i> is shown in the bottom row. Open and closed squares indicate untranslated and coding regions, respectively. Lines indicate introns. (B) ChIP-PCR was performed using ChIP DNA immunoprecipitated by the anti-OsTGAP1 antibody and normal rabbit IgG. Values indicate the ratio of the amount of DNA in the ChIP DNA to the amount in the ‘Input’ control (n = 3); bars indicate the standard deviation of the mean.</p

Expression profiles and functional classification of the OsTGAP1 target genes.

<p>(A and B) Venn diagrams showing the overlap of genes that possess an OsTGAP1-binding regions within the 400-bp upstream region with the upregulated or downregulated genes in OsTGAP1-overexpressing rice cells. (C and D) Hierarchical clustering of the OsTGAP1 target genes using the cosine correlation and average linkage methods. WT and OX represent wild-type rice cells and OsTGAP1-overexpressing rice cells, respectively. Each column represents the mean of three biological replicates at each time point shown above the heat map. Colours represent induction (red) and repression (green), as indicated by the colour bar. The values of the heat maps are relative to those in the WT cells 0 h after the elicitor treatment. (E) Functional classification of the OsTGAP1 target genes. Categories are as follows: biosynthetic enzyme, defence response, transcription factor, protein kinase, transporter, other function, and unknown function.</p

Transactivation assay using a 250-bp upstream region of <i>OsDXS3.</i>

<p>(A) Nucleotide sequence of the 250-bp upstream region of the ATG translation start site of <i>OsDXS3</i>. Closed triangles indicate TGACGT sequences. The TGACGT sequences in the nucleotide sequence are also indicated by bold characters. The putative TATA box is underlined. Transcription start sites on RAP-DB are indicated by black arrows. (B) Transactivation assay using a 250-bp fragment of the <i>OsDXS3</i> promoter with GUS or OsTGAP1 effector plasmid (0.1 µg per bombardment). Values indicate the relative luciferase (LUC) activities (firefly LUC/<i>Renilla</i> LUC) after 24 h incubation of rice cells with or without chitin elicitor treatment (n = 3); bars indicate the standard error of the mean. Statistically different data groups are indicated by different letters (<i>P</i><0.01 by one-way ANOVA with a Tukey’s <i>post hoc</i> test). (C) Transactivation assay using mutated or deleted <i>OsDXS3</i> promoters with GUS or OsTGAP1 effector plasmids (0.1 µg per bombardment). Values indicate the relative LUC activities (firefly LUC/<i>Renilla</i> LUC) after 24 h incubation of rice cells without chitin elicitor treatment (n = 12); bars indicate the standard error of the mean. Statistically different data groups are indicated by different letters (<i>P</i><0.01 by one-way ANOVA with a Tukey’s post <i>hoc test</i>).</p

Effect of Adiponectin on Kidney Crystal Formation in Metabolic Syndrome Model Mice via Inhibition of Inflammation and Apoptosis

<div><p>The aims of the present study were to elucidate a possible mechanism of kidney crystal formation by using a metabolic syndrome (MetS) mouse model and to assess the effectiveness of adiponectin treatment for the prevention of kidney crystals. Further, we performed genome-wide expression analyses for investigating novel genetic environmental changes. Wild-type (+/+) mice showed no kidney crystal formation, whereas ob/ob mice showed crystal depositions in their renal tubules. However, this deposition was remarkably reduced by adiponectin. Expression analysis of genes associated with MetS-related kidney crystal formation identified 259 genes that were >2.0-fold up-regulated and 243 genes that were <0.5-fold down-regulated. Gene Ontology (GO) analyses revealed that the up-regulated genes belonged to the categories of immunoreaction, inflammation, and adhesion molecules and that the down-regulated genes belonged to the categories of oxidative stress and lipid metabolism. Expression analysis of adiponectin-induced genes related to crystal prevention revealed that the numbers of up- and down-regulated genes were 154 and 190, respectively. GO analyses indicated that the up-regulated genes belonged to the categories of cellular and mitochondrial repair, whereas the down-regulated genes belonged to the categories of immune and inflammatory reactions and apoptosis. The results of this study provide compelling evidence that the mechanism of kidney crystal formation in the MetS environment involves the progression of an inflammation and immunoresponse, including oxidative stress and adhesion reactions in renal tissues. This is the first report to prove the preventive effect of adiponectin treatment for kidney crystal formation by renoprotective activities and inhibition of inflammation and apoptosis.</p></div

Additional file 1: Figure S1. of Bacillus subtilis 5′-nucleotidases with various functions and substrate specificities

Genetic organization of the altered chromosomal loci; ΔycsE::spc(A), ΔyktC::cat(B), ΔyqeG::kan(C), and amyE::(Pspac-yqeG lacI erm)(D). Genes and primers are indicated schematically by thick arrows and arrowheads, respectively. (PDF 144 kb

Confirmation of selected genes detected by microarray analysis.

<p>A–F. Among the selected genes with significant expression changes in the microarray analysis, the expression of several characteristic genes belonging to 6 categories based on GO analysis was evaluated by immunohistochemical staining. Magnification is ×20 (in box: ×400). 6 categories are as follows. A. Inflammation and immune-related gene group: LYZ1, Lysozyme 1.CD44, CD44 antigen; MHC-class 2, major histocompatibility complex-class2. B. Apoptosis-related gene group: STAT3, signal transducer and activator of transcription 3; AURKA, aurora kinase A, Thymidine kinase 1, C. Cell repair and proliferation-related gene group: MCM5, minichromosome maintenance deficient 5. D. Adhesion and fibrosis-related gene group: Fn, Fibronectin. E. Oxidative stress-related gene group: 8OHdG, 8-Hydroxydeoxyguanosine. F: transporter-related gene group; SLC12A1, solute carrier family 12, member 1.</p

TUNEL staining and the number of TUNEL-positive cells.

<p>Data are indicated as the mean ± SD. *, p<0.05 between groups at the same time. **, p<0.01 between groups at the same time point. †, p<0.01 compared with the same group at day 6.</p

Confirmation that selected genes detected by microarray analysis showed differential expression between different treatment groups.

<p>The expression of several characteristic genes belonging to 5 categories based on GO analysis was evaluated by quantitative PCR. Expression levels are expressed relative to <i>Actb</i>. Data are indicated as the mean ± SE. *, p<0.05 between the groups at the same time point. †, p<0.01 compared with the same group at day 0. 5 categories are as follows. A. Inflammation and immune-related gene group: <i>Lcn2</i>, lipocalin2; <i>Cd44</i>, CD44 antigen; <i>Lyz1</i>, lysozyme1. B. Apoptosis-related gene group: <i>Stat3</i>, signal transducer and activator of transcription 3; <i>Aurka</i>, aurora kinase A. C. Cell repair and proliferation-related gene group: <i>Mcm5</i>, minichromosome maintenance deficient 5. D. Adhesion and fibrosis-related gene group: <i>Vcam1</i>, vascular cell adhesion molecule 1; <i>Col3a1</i>, collagen, type III, alpha 1. E. Transporter-related gene group: <i>Slc12a1</i>, solute carrier family 12, member 1; <i>Slc7a13</i>, solute carrier family 7, member 13.</p