WRKY proteins, a big family of transcription factors, are involved in regulation diverse developmental and physiological processes in plants. Here, a novel WRKY gene, OsWRKY52, was isolated from a rice cDNA library. This gene included an open reading frame of 1 719 bp in length, and the deduced polypeptide contained 572 amino acids,sharing 54% identity with a WRKY1 protein from Avena sativa. Expression of OsWRKY52 gene was induced rapidly by Magnaporthe grisea in the incompatible interaction with rice plant. OsWRKY52 protein, expressed prokaryotically bound specifically to W box cis elements derived from the promoter of a rice PR1a. Transcriptional activation assay was performed by a yeast one- hybrid method. Regions of transactivation were identified to be the N-terminal serine- and threonine-rich islands and the C-terminal acidic domain of OsWRKY52. These results suggest that OsWRKY52, as a transcription activator, may be involved in defense responses against Magnaporthe grisea in rice plants.

Data analysis poses a significant challenge to the large-scale proteomics studies. Based on the structured and controlled vocabularies-Gene Ontology (GO), and the GO annotation from related databases, a strategy composed of several programs and local databases is developed to identify the functional distribution and the significantly enriched functional categories of the proteomic expression profile. It would be helpful for understanding the overall functions of these identified proteins and supply the fundamental information for further bioinformatics exploration. This strategy has been successfully used in the Human Fetal Liver (HFL) proteomic research, which is available online at http://www.hupo.org.cn/GOfact/.

Template direct dye-terminator incorporation with fluorescence-polarization (TDI-FP assay) is a technology for genotyping single nucleotide polymorphisms (SNPs). To apply this method in analyses of A647G variation in human papillomavirus (HPV) 16 E7 gene from HPV 16-positive cervical tissues, a total of 91 and 49 HPV 16-positive DNA samples obtained from women with cervical cancer and normal/inflamed cervices living in Shaanxi in northwest China were subjected to the partial E7 gene PCR with nucleotide (nt) 647 in the products. Then, the oligonucleotide probe designed to anneal immediately to nt 647 was hybridized to the template within the PCR amplicons, and extended specifically by TAMRA-ddTTP or R110-ddCTP directed by the base at nt 647. The increasing FP values were read and the base at nt 647 was identified. The prevalence of nt 647 A→G was 35.71% (50/140). The variation 647G detected in 42.86% (39/91) of women with cervical cancer was significantly higher than 22.45% (11/49) detected in those with normal/inflamed cervices (x2 = 5.778, P = 0.016). The odds ratio (OR) between these two groups was 2.59 (95% confidence interval=l.17～5.71). The results demonstrate that TDI-FP method can be potentially applied in analysis of interest point mutations in HPVs. The incidence and risk implication of HPV 16 A647G variant infection in Shaanxi, China, displays significant geographic difference from other areas. The HPV 16 with E7 gene A647G point mutation appears to have a higher risk for invasive cervical cancer in women living in Shaanxi.

MutL and MutS or their homologues are two crucial proteins of DNA mismatch repair (MMR) system. A new method was described for observation of the interaction between MutS and MutL which is based on the fusion gene/fusion protein technique. Three fusion proteins, MutL-GFP fusion (Trx-His6-GFP-(Ser-Gly)6-MutL), MutL-Strep tag Ⅱ fusion (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) and MutS fusion (Trx-His6-(Ser-Gly)6-MutS), were constructed and expressed in E. coli AD494 (DE3). Interaction assay between MutS and MutL was performed in a 96-well microtiter plate.MutS fusion protein was immobilized on the wells and provided a surface for the interaction between MutS and MutL.Results showed that only after binding of MutS to the mismatched DNA, there was an interaction between MutS and MutL.The binding events could be indicated by GFP signal or the signal generated from alkaline phosphatase and its substrate. In addition, the method based on fusion molecular system also serve as a model for studies on the interactions among other proteins or biomolecules.

The manipulation and direct mechanical measurement of single DNA molecule could give much information about its elastic properties. Single stranded DNA (ssDNA) of 100 bases was adsorbed onto flat gold surface fabricated by depositing gold onto mica surface. Atomic force microscope (AFM) was used to observe the surface topography with different ssDNA concentration. Then the ssDNAs were stretched by AFM tip and in 50% cases ssDNAs could be stretched.Various kinds of force curves have been observed due to the different interaction between AFM tip and ssDNAs.

ONYX-015 and H101 are E1B 55-kDa protein-deficient replicating C group adenoviruses that are currently in clinical trials as antitumor agents. However, their application in cancer gene therapy is limited by the native tropism of C group adenoviruses. This is in part due to low expression of the C group adenovirus receptor (coxsackievirus-adenovirus receptor, CAR) on malignant tumors. An H101-based chimeric virus vector containing sequences encoding the Ad35 fiber domain instead of the Ad5 fiber (H101-F35) was constructed. This modification allowed infection of tumor cells through CD46, a membrane protein over-expressed on tumors. The CAR and CD46 RNA expression was evaluated by RT-PCR method. H101-F35 conferred a stronger cytocidal effect than H101 and ONYX-015 in tumor cell lines that lacked CAR expression (MDA-MB-435 and MCF-7), while the cytocidal effect of H101-35, H101 and ONYX-015 was similar in high-level CAR expressing cancer cell lines (A549, NCI-H446, Hep3B, LNCaP, ZR-75-30 and Bcap-37). In an MDA-MB-435 xenograft mouse tumor model, tumor growth in mice receiving H101-F35 was significantly inhibited compared with mice injected with H101. These results suggest that the chimeric oncolytic adenovirus H101-F35 vector might be a useful candidate for gene therapy of cancer.

Radish phospholipid hydroperoxide glutathione peroxidase (RsPHGPx) was identified as a mitochondrion-targeting PHGPx in previous work. To determine its cleavage site of the targeting peptide, the immunoaffinity chromatography (IAC) purification approach was carried out to isolate the native RsPHGPx protein.Polyclonal antibodies directed against recombinant RsPHGPx were raised in rabbit. Monospecific anti-RsPHGPx antibodies were isolated by means of affinity chromatography using the recombinant RsPHGPx as affinity ligand, and employed in assembling an IAC column. A single-step, highly specific and easy-to-use protocol was developed for purification of the active RsPHGPx protein through the assembled IAC column. Using this approach, a specific protein of the expected molecular size was obtained from the mitochondrial fraction of radish seedlings. Western blot analysis showed that it could be specifically recognized by anti-RsPHGPx antibodies, and an enzyme activity assay indicated that it exhibited significant PHGPx activity, suggesting that the purified protein should be the desired native RsPHGPx. These results will lead to clarification of the targeting peptide and the active mature protein of RsPHGPx and will be helpful to further probe the intracellular localization mechanism and biological fun ction of this plant PHGPx.

Oligonucleotide microarray technology is a powerful data-mining platform and has been widely applied in biosciences. To improve the performance of assays on the oligonucleotide microarray, the factors that influence the hybridization effects such as surface chemistry, probe size, spacer length, hybridization conditions etc were intensely studied and optimized. However, it is a key problem with DNA microarrays how to generate higher fluorescent signals to improve the detection sensitivity. Two types of multiply labeled primers, termed multiply labeled linear primer and multiply labeled branched primer, were used to enhance the fluorescent signal obtained from two-dimensional DNA microarrays.The signal was intensified by increasing the number of fluorophores labeled on the target DNA segment. It was indicated that the detection limit (minimum template amoumt for detection) of the multiply labeled primers is about 1% of that of the singly labeled primer. Multiple labeling is an effective signal amplification method to increase the detection sensitivity of the probes in a miniaturized array format.

The canonical Wnt/β-catenin signaling pathway plays critical roles in both embryonic development and tumorigenesis. Central to the pathway is the turnover of β-catenin, a protein that functions in both cell adhesion and transcription. In the absence of a Wnt signal, free cytosolic β-catenin is phosphorylated by a large protein complex called the "β-catenin destruction complex" that targets β-catenin for degradation by an ubiquitin ligase/proteasome system. In the presence of a Wnt signal, the binding of Wnt to its receptor Frizzled and co-receptor LRP leads to the inhibition of β-catenin phosphorylation in the β-catenin destruction complex through an unknown mechanism. Inhibition of the β-catenin destruction complex leads to the accumulation of nuclear β-catenin, which in turn forms a complex with Tcf and BCL9. Recent studies have provided important clues regarding the molecular mechanism of the β-catenin destruction complex as well as an explanation for how β-catenin switches between its roles in cell adhesion and transcription.

Calcium oscillation may regulate gene transcription in a frequency-decoding manner during agonist stimulation,which provides an indicator of transcription level in cells. To determine whether persistent exposure to hypoxia may sensitize or blunt cell response to histamine, the effects of 24 h subacute mild hypoxia on histamine-stimulated calcium oscillation frequency were examined in pulmonary artery endothelial cells (PAECs). The results are: (1) 24 h subacute mild hypoxia significantly increased the histamine-stimulated calcium oscillation frequency in PAECs. The averaged frequency of calcium oscillation in posthypoxic PAECs was significantly higher than that in normoxic ones. (2) NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI, 10 μmol/L), abolished histamine-stimulated calcium oscillations both in normoxic and posthypoxic PAECs. (3) Xanthine oxidase inhibitor, oxypurinol (100 μmol/L), did not affect the calcium oscillation kequency in normoxic PAECs. However, it significantly decreased the elevation of calcium oscillation frequency in posthypoxic PAECs. These results demonstrated that, during pulmonary disease related to persistent hypoxia,PAECs become more sensitive to histamine. During histamine stimulation, NADPH oxidase plays a critical role in generating calcium oscillations, while xanthine oxidase may contribute to, at least in part, the increase of calcium oscillation frequency in posthypoxic PAECs.