Site-directed mutation position of two nuclear factors.

<p>Site-directed mutation position of two nuclear factors.</p

Effects of site-directed mutation in transcriptional factors LEF-1 and AP-2α on transcriptional activation of the human CTNNAL1 luciferase reporter.

<p>Shown is transcriptional activation of the human CTNNAL1 luciferase reporter in BEC. Data represent the means of relative luciferase activities (normalized to Renilla reniformis) from at least 5 independent transient transfection experiments each performed in triplicate. (^*P<0.05 versus pGL3/CTNNAL1 under resting condition; ^#P<0.05 versus pGL3/CTNNAL1 under ozone-stressed condition).</p

Expression of CTNNAL1 mRNA in ozone stressed mouse lung.

<p>(A) It was shown by in situ hybridization that the structure of lung tissues was damaged after being stressed with ozone. Goblet cell hyperplasia and bronchial epithelial denudation were seen in the 2^nd and 4^th day of ozone stress. The expression of CTNNAL1 mRNA was increased with the ozone stress (×200, bar = 100 µm). Arrows indicate airway epithelium damage. (B) Real time PCR showed the time course of CTNNAL1 expression in mice lung tissue. The result showed that the expression of CTNNAL1 mRNA was increased with the ozone stress, peaked on the 2nd day, and then decreased. (n = 5, *P<0.05).</p

Screen of transcription factors binding to the human CTNNAL1 promoter region.

<p>(A) Depicted is the 400 bp DNA sequence of the human CTNNAL1 promoter (−552∼−152 from ATG) which was divided equally to probes 1–8 used for EMSA. Underlines represent possible nuclear-factor-binding sites in corresponding probes. The shaded residues represent the mutated sites in different mutated probes. (B) DNA-protein complexes binding to the human CTNNAL1 promoter were demonstrated in probes 1, 2, 3, 5, and 8. (C) EMSA competiton test of probe 1, 2, 3, 5, and 8. Competition with unlabeled primer significantly reduced the entire five probe's binding intensity. (D) The nuclear factors in the retarded bands were verified using the assay of mutated probes binding with extracts. 9 mutations according to putative binding sites search by TESS were designed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031158#pone-0031158-g003" target="_blank">Figure 3A</a>. The valid mutations (specific binding bands was be competed out by unlabelled and 100-fold excess mutation probes) were 1.2, 2.1 (representing the LEF-1 site), 3.1 (representing the AP-2 site) and 8.2 (representing the CREB site).</p

Time course of human CTNNAL1 mRNA expression and DNA-protein binding activities of AP-2α and LEF-1.

<p>(A) The time course of AP-2α and LEF-1 DNA binding to CTNNAL1 promoter sequence under ozone stress was determined by EMSA. Expression of human CTNNAL1 mRNA was assayed by real-time PCR. Data represent means ± SD of 4 independent experiments.</p

Oligonucleotide primers used for real-time PCR analysis.

<p>Oligonucleotide primers used for real-time PCR analysis.</p

Supershift analyses were performed using nuclear proteins from HBEC that were incubated with specific antibodies against LEF-1, AP-2α, and CREB.

<p>The supershifted complex detected with each antibody was indicated by “▴”. Note that each of the LEF-1, AP-2α, and CREB antibodies caused a marked decrease in the intensity of the complex (marked by “•”) formed by the interaction of antibody and nuclear extract.</p

The expression of CTNNAL1 on cultured human BEC was increased significantly after ozone stress.

<p>(A) CTNNAL1mRNA expression on BEC assayed by in situ hybridization (DAB staining, ×200). (B) CTNNAL1mRNA expression on BEC assayed by real time PCR (n = 4, *<i>P<</i>0.05). (C) CTNNAL1 protein expression on BEC assayed by flow cytometry detection.</p

Analysis on the Relevance of Asthma Susceptibility with the Alteration of Integrin β 4 Expression

<div><p>Accumulated research has suggested the importance of the adhesion molecules modulation as therapeutic approach for bronchial asthma. Adhesion molecules expression alteration contributes to the pathogenesis of asthma. In order to probe the roles of expression imbalance of adhesion molecules in asthma pathogenesis, expression profiling of adhesion molecules was performed using cDNA microarray assay. The results showed that the expression pattern of adhesion molecules was altered in peripheral blood leucocytes of asthma patients. In this study, we focused on one of the abnormally expressed molecule, integrin β4, which was down-regulated in all asthma patients, to analyze the relevance of asthma susceptibility with the alteration of integrin β4 expressions. Real time PCR was used to verify the down-regulation of integrin β4 in additional 38 asthma patients. Next, the 5′flanking region of integrin β4 DNA were amplified, sequenced and site-directed mutagenesis technology in correspondent variation sites were carried out. Among 4 variation sites found in 5′ flanking region of integrin β4, 3 were related to asthma susceptibility: -nt1029 G/A, -nt 1051 G/A, and -nt 1164 G/C. A reduction of human integrin β4 promoter activity was observed at mutants of these sites. This study demonstrates that various adhesion molecules in asthma patients are abnormally expressed. Mutations in 5′ flanking region result in reduced integrin β4 expression, which is related to increased risk of asthma.</p></div

Group characteristics.

<p>Forced expiratory volume in one second (FEV1) is expressed in (%), which is defined as FEV1% of the patient divided by the average FEV1% in the population for any person of similar age, sex and body composition. Normal value is approximately 86%.</p