Abstract:Signal transduction is vitally important for development and cell survival of all animals. Most of signaling processes involve phosphorylation and dephosphorylation of amino acid residues in proteins. The kinases and phosphatases involved in signaling processes are regulated by different mechanisms. So far, the studies on protein phosphorylation almost exclusively limited to protein serine/threonine and tyrosine phosphorylation, phosphorylation of histidine has only been sparsely reported. However, phosphorylation of histidine residues has been extensively studied in prokaryotes. It is estimated that histidine phosphorylation may account for 6% of total protein phosphorylation in eukaryotes, 10- to-100-fold more than phosphotyrosine, though less abundant than phosphoserine. Although the presence of phosphohistidine in vertebrate protein was described as early as in the 1960s, accumulated knowledge in vertebrates so far is still limited to O-phosphates. The protein phosphatases were introduced, and the knowledge on the key mechanisms of the bacterial two-component system was summarized. Most importantly, novel mechanisms of phosphorylation and dephosphorylation of histidine residues are described. Finally, the recent studies about histidine phosphatases are discussed.

Abstract:Antibacterial peptides (ABP) are a new type of antimicrobial substance. It is widely distributed in many living organisms, from the lowest virus and germs to the higher propagation. The research foregone mostly focused on the mechanism of ABP against the cell membrane, and three modes have been put forward. Recent researches indicate that many antibacterial peptides can penetrate into the bacterial cell effectively. They act on biologic molecules inside bacterium directly without disruption of the membrane. According to different structure of antibacterial peptides, there are several mechanisms to penetrating through the membrane. By interacting with nucleic acids, proteins and signal transmissions, antibacterial peptides can finally kill and wound bacterium.

Abstract:The one-trial passive avoidance task (OTPAT) and sickness-conditioned learning task (SCLT) models are widely used in studies on the mechanisms of learning and memory in day-old chicks and a numerous progress has been achieved. Previous findings have shown that the intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) are principal structures involved in memory formation in the chick brain. In light of studies on the relevant molecular mechanisms, pharmacological experiments have discovered a number of medicines which may affect the memory formation at its different stages. As an example, noradrenalin may enhance and modulate the long-term memory. Since the principal structures and their functions in the avian brain are, to some extents, comparable to those in the mammal, the above-mentioned studies on chicks may provide important clues for exploring the learning and memory in human brain.

Abstract:To screen the differential expression genes of nasopharyngeal carcinoma(NPC), the cDNA microarray technique was employed to analyze the changes of gene expressions with GenMAPP. Total RNA was extracted from NPC and normal nasopharyngeal epithelium tissue respectively. Reverse transcription was performed and cDNA probe was random-prime labeled with 33P-dATP and hybridized with the cDNA microarray membrane containing 16 150 genes and ESTs. The hybridized result was confirmed by RT-PCR analysis. The results suggested that 339 genes were differentially expressed over two fold, in which 160 genes were up-regulated and 179 genes were down-regulated in NPC. These genes were involved in cell proliferation, gene transcription, apoptosis, signal transduction, DNA damage and repair, tumor differentiation and metastasis, cell cycle, and so on. Therefore, the data suggested that some genes play a critical role in NPC, and provided an important clue to elucidate the mechanism of these genes.

Abstract:Taq pol, MMLV- RT and human pol β are polymerases lack of 3′-5′ exonuclease activity. The Taq polymerase binding with DNA template-primer (T-P) with 1, 2, or 3 mismatched base/bases at the terminus of primer was studied by surface plasmon resonance (SPR)　biosensor, comparing with the binding with full matched DNA T-P. The experiment showed that the affinity of Taq pol binding with DNA T-P decreases when the number of mismatched bases increased, indicating that Taq pol preferred to binding with matched DNA T-P. With “incorrect” dNMP, the kinetics of Taq pol binding with matched DNA T-P could be analyzed by 1∶1 Langmuir model while the kinetics of MMLV RT－ binding with matched DNA T-P might involve conformation change. Conformation change model fitted well with Taq pol and MMLV－ RT-DNA binding curves in the presence of “correct”dNMP. The　binding affinity was 20 times and 64 times higher than that without dNMP, respectively, indicating “correct” dNMP induced the conformation change of polymerse-DNA complex and enhanced the binding tightness. In the presence of dNMP, the kinetics of pol β binding with DNA T-P changed significantly and pol β grasped DNA closely.

Abstract:To elucidate biological functions of different protein Annexin1 discovered from hepatocellular carcimomar (HCC) cell lines by comparative proteomics approach. The different expression level of Annexin1 among HCC cell lines was further validated using other approachs including RT-PCR, Western blot and cell immunochemistry. The pcDNA3.1(+) AS Annexin1 expression plasmid was constructed, and then transfected into high metastatic cell lines MHCC97H to observe the alteration of biological functions of MHCC97H (motility, invasion, extracellular matrix metalloproteinase). All data from validation tests confirmed the overexpression of Annexin1 in high metastatic HCC cell lines with the comparison of non-metastatic one. Inhibition of the expression of Annexin1 in MHCC97H was successfully detected by RT-PCR after transfected with anti-sense RNA. According to analysis sequence followed as MHCC97H/pcDNA3.1(+) AS Annexin1, MHCC97H/ pcDNA3.1(+) and MHCC97H. Cell motility assay in vitro showed the average amount of invading cell per field were 11.13±3.31, 18.88±2.03, 21.86±3.39 respectively. The average amount of invading cell per field in cell invasion assay were 16.43±2.23, 16.4±1.57, 16.86±1.52 respectively.The Average clone formation ration of MHCC97H transfected was (14.33±0.46)%,(19.35±0.49)%, (20.25±0.35)%. The amount of apoptosis cell transfected by FCM analysis was 22.2%, 6.44%,6.97%. The number of transfected cell in each phase during cell cycles displayed G0-G1 phase 79.5%/ 76.34%/ 80.5%, S phase 13.26%/ 14.4%/9.69%, G2-M phase 7.25%/ 9.26%/ 9.81%. The concentration of MMP9 in serum free culture media by quantitative assay (ABC-ELISA) were 26.37 μg/L, 28.00 μg/L, 31.90 μg/L and MMP2 were 29.79 μg/L, 26.37 μg/L, 26.53 μg/L. The activities of MMPs in serum free culture media were detected by Gelatin zymography. No significant changes were found between target sample and control. Analysis of the results mentioned, some biological properties (including motility, clone formation potential) of MHCC97H transfected with pcDNA3.1(+) AS Annexin1 were all down-regulation, and yet the amount of apoptosis cell transfected were increased. No significant changes in cell invasion, cell cycle, MMPs were discovered. All the data suggested that Annexin1 may involved in HCC metastasis by alteration of cell apoptosis and of cell motility property.

Abstract:Tau is one of the major microtubule-associated proteins in neuron. The abnormal hyperphosphorylation of tau is believed to be critical for pathogenesis of Alzheimer disease. The autopsied human brain tissue is commonly available with longer than 2~3 h of postmortem delay. Therefore, understanding whether and how postmortem delay affects tau phosphorylation is very important for studies of the function of tau and the role of tau phosphorylation in AD pathogenesis. Tau phosphorylation in normal rat brain and its change during postmortem delay were measured by using site-specific and phosphorylation dependent anti-tau antibodies. It was found that tau in normal rat brain was phosphorylated with different extents at Thr181, Ser199, Ser202, Thr205, Thr212, Ser214, Thr217, Ser396, and Ser404, but not at Ser262, Ser409, and Ser422. Tau was rapidly dephosphorylated at all of the sites measured in 3 h after death and the dephosphorylation continued till 6 h postmortem delay although total tau level did not change during this period of time. The activities of both tau kinase and tau phosphatase in rat brain were found decreased upon temperatures, and the kinase activity was more sensitive to temperature than phosphatases activity at 25℃. The rapid dephosphorylation of tau after animal death was due to the relative higher activity of tau phosphatase in the brain.

Abstract: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.

Abstract:Bioinformatics and gene chip are two new technologies in the field of life science. Bioinformatics is very important to the data analysis of gene chip. The expression and regulation of gene have a profound influence on progression and metastasis of malignant tumors. In the present report, the oligonucleotide microarray technique was used to identify gene expression profiling and screen differential expression genes in breast cancer with a special emphasis on metastasis factors. Then a linear differential model is employed to construct rough genetic regulatory network of gene related to breast cancer metastasis. An available gene chip system was used to analyze surgical samples, including both breast cancer primary tissue and metastasis tissue, collected from 30 patients in different clinical staging. 27 differential expression genes were identified. 14 of the total are up-regulation genes whose Ratio is large for 3, and the rest are down-regulation gene whose Ratio is small for 0.33. In search of putative metastasis and tumor progression factors with Bioinformatics method, linear differential model, a linear model estimated -by Lest Squares Method, was used to construct rough regulatory network of gene related to breast cancer metastasis. With the analysis of the rough regulatory network constructed by the linear differential model, the reliability of regulatory network and abnormality of signal transmission pathways caused by multiple gene abnormal expression, which maybe correlate to breast cancer metastasis, were manifested.

Abstract: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.

Abstract: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 tagⅡ 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.

Abstract:Based on the characteristics of base distribution in promoter and non-promoter region the method of Increment of Diversity with Quadratic Discriminant analysis (IDQD) was used to predict the pol Ⅱ promoter in human genome. The prediction has attained accuracy higher than 90%. The transcription start sites have also been predicted successfully with sensitivity 86% and specificity 91%, better than other top softwares currently published.

Abstract:A therapeutic gene———p27kip1 gene were encapsulated into poly (lactic-co-glycolic acid) (PLGA) NPs by an emulsification/solvent evaporation technique, and NP size distribution was assessed by submicro laser defractometer. The particle morphology was observed by scanning electron microscopy. The diameter of p27kip1gene-NPs was around 288.9 nm with very narrow size distribution, and p27kip1 Nps showed good spherical shape with smooth uniform surface. The p27kip1 loading in NPs was about 3%. Encapsulation efficiency of gene was about 86%. In vitro gene release from the NPs was performed in TE buffer at 37℃ under rotation (130 r/min) utilizing double-chamber diffusion cells on a shake stander, the result showed that gene release from the NPs lasted above two weeks. P27kip1 gene NPs was transduced in smooth muscle cell in vitro and cell cycle was tested by flowcytometry. Vein grafting model was established in 120 rats by transplanting internal branch of jugular vein to carotid artery. The rats were randomly divided into three groups: 1)The p27kip1 gene NPs treated grafting group; 2) Control group (treated with empty NPs without p27kip1 gene); 3) Shame grafting group. The grafted veins were harvested at 3d, 7d, 14d and 28d respectively after the operation. Intimal hyperplasic (IH) was observed by morphologic evaluation. The expression of PCNA, E2F were detected by immunohistochemistry and analyzed by computer digitizing system. The p27kip1 gene transfection mediated by NPs complex enabled to produce protein expression of p27kip1 gene (P < 0.05) and significantly inhibits hyperplasia of the grafted vein especially for 7~28 days in p27 group (P < 0.01). Immunohistochemical analysis of PCNA indicated decreased positive cell in the p27kip1 group compared with the control group at 7~28 d (P < 0.01). The expression of E2F was decreased in p27 group at 7~14 d (P < 0.01). There is no significant difference between control group and grafting group in expression of E2F and PCNA. The p27kip1 gene NPs can prevent IH and SMC proliferation, NPs is an effective gene transfecting carrier.

Abstract: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=1.17~5.71). 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.