Abstract:Complex diseases are the any ones that there is no classical mendelian dominant or recessive inheritance model for a single locus. And cancers are a kind of common complex diseases. Based on the linkage analysis and association analysis, many genetic mapping approaches of complex diseases had been grown up such as functional cloning, candidate cloning, positional cloning, positional candidate cloning and systems biology approach and so on. Among them, systems biology approach is in the ascendant now. Because it integrates all of the informations from DNA to proteins, it gives a better commentary of the complicated gene- regulatory network. This advantage makes it become one of the most potential methods in the 21 century. Nearly one hundred of cancers had been mapped so far. Furthermore, the genetic mappings of complex diseases still shoulder heavy responsibility.

Abstract:Bromodomain is an evolutionally conserved domain and is identified in proteins strongly implicated in signal-dependent gene transcription regulation. BRD7, a novel bromodomain gene, has been cloned by cDNA RDA (cDNA Representational Difference Analysis) in 1999. The GenBank accession number is AF152604 or AF152605. eMotif analysis revealed that BRD7 protein contains a conserved bromodomain and several important phosphorylation sites. Multiple sequence alignment program showed high ratio identity between BRD7 protein, protein Celtix-1 and musculus bromodomain-containing protein BP75. Over expression of BRD7 in Nasopharyngeal carcinoma (NPC) cells can inhibit cell proliferation and cell cycle progression from G1 to S phase, and can partly inhibit the aberrant growth of NPC cells. In order to further address the signal pathway and the possible functions of BRD7 gene, function analysis of BRD7 gene was performed through three different cellular physiology levels including up- and down-stream, interplay issues of BRD7 gene.

Abstract:Heparanase is the only mammalian endo-β-D-glucuronidase known to cleave heparan sulfate (HS) components of proteoglycans(PGs) at limited intra-chain sites. Heparanase release in response to an inflammatory stimulus or in relation to tumor metastasis alters the composition and structural integrity of extracellular matrix and basement membrane. The enzymatic degradation of HS by heparanase is, therefore, involved in range of biological phenomena, from pregnancy, morphogenesis and development to inflammation, vascularization, and cancer progression. In normal physiological processes, expression of the active enzyme is tightly regulated by promoter methylation, mRNA splicing, transcription factors, proteolytic processing and inflammatory cytokines. The recent findings about the strict regulation of heparanase from the expression of the gene to processing of the protein product to yield the active enzyme are reviewed.

Abstract:In order to establish an in vitro attachment culture method for obtaining highly purified neural progenitor cells (HPCs) from the hippocampus of adult Wistar rats and to identify functional L-type calcium channels in those cells, the adult rat hippocampal tissue was dispersed into a single cell suspension, and the dissociated cells were cultured in serum-free DMEM/F12 medium containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), N2 and B27 supplement. After six passages, the proportion of nestin-positive cells reached to 99.9%. Following fourteen-days culture in differentiation medium, neuron-like and astrocyte-like cells were observed, which expressed β-tubulin Ⅲ (Tuj1) and glial fibrillary acidic protein (GFAP), respectively. Immunofluorescent double labeling and Western blot showed an expression of Cav1.2α1C and Cav1.3α1D subunits in HPCs, and functional L-type calcium channels were confirmed by confocal microscopic Ca2+ imaging. Moreover, L-type calcium channel currents were recorded in those cells by using whole-cell patch clamp techniques. The results indicated that adult rat HPCs express functional L-type calcium channels.

Abstract:Studies have shown that host actin is essential for baculovirus replication and assemblage. In order to further elucidate the function of actin in the late and very late infection stages, a recombinant virus vAc-ph/70GA were constructed using Bac-to-Bac system, in which eGFP-actin fusion gene under control of hsp70 promoter and polyhedrin gene (ph) with its’own promoter were inserted. vAc-ph, which only contains ph, was also constructed as control. After vAc-ph/70GA infected Sf9 cells, actin was expressed persistently. However, there were no polyhedra, while the control virus vAc-ph did produce polyhedra. Further analysis such as SDS-PAGE and RT-PCR did not detect the transcription and expression of polyhedrin after vAc-ph/70GA infected Sf9 cells, indicating that the late expression of actin inhibit polyhedra formation. Expression of actin, however, did not change the viral infectivity. It can be concluded that the persistently expression of actin during late stage of baculovirus infection inhibit the polyhedrin expression, and then the formation of polyhedra. Here, some results were shown by electro microscopy, SDS-PAGE and RT-PCR with vAc-ph/70GA.

Abstract:The Wnt signalling pathway plays an important role in regulation of haematopoietic stem cells (HSCs) self-renewal. Purified Wnt3a could remarkably enhance expansion of HSCs. A transgenic bone marrow stromal cells were established by using adenoviral vector mediated Wnt3a gene transfection. The effect of transgenic stromal cells as feeder layer on expansion of CD34+ cells was studied. The group of Wnt3a transfected BM stromal cells with cytokines displays the best expansion effect on of human umbilical cord blood CD34+ hematopoietic stem/progenitor cells in four groups. In vitro, expansion of CD34+ cells, when cocultured with Wnt3a modified stromal cells in the presence of cytokines, was significantly enhanced: CFC by (1.55±0.06) fold; CFC(mix) by (1.95±0.26) fold; HPP-CFC by (1.45±0.40) fold; LTC-IC by (3.83±0.86) fold ,compared with nontransgenic stromal cells with cytokines group. These results strongly suggest that Wnt3a modified BM stromal cells may be a suitable feeder layer for expansion of haematopoietic stem/progenitor cells in vitro.

Abstract:In oder to study effect of BRD7 gene on NPC cell line CNE1, BRD7 was introduced into CNE1 cells by liposome transfection. BRD7 transfected cells were resulted in a declined growth curve. To account for the mechanism of this gene function on CNE1, two-dimensional polyacrylamide gel eletrophoresis(2-D PAGE) and MALDI-TOF were performed. After image analysis and MALDI-TOF identification, 19 differential expression proteins were identified. These proteins included BCCIP(BRCA2 and CDKN1A (p21(Waf1/Cip1)), FHL2(four and a half LIM domains 2), Chloride channel regulatory protein, Hin-1 (high-in-normal-1), WISP-1 (connective tissue growth factor related protein), SREC-4(scavenger receptor expressed by endothelial cells-2), folate receptor, which involved in transcription regulation, adherence and so on. The study extended the research field of BRD7 and reinforced the evidences that BRD7 act as a NPC-related candidate suppressor gene.

Abstract:HER2/neu is an attractive target for tumor therapy since its overexpression in a number of tumors. In order to enhance the cytotoxicity of anti-HER2/neu antibody, and the specificity of TNF-α for tumor cell, a fusion gene of antiHER2-hTNF-α was constructed. The recombinant fusion protein was produced in E.coli, and their refolding has done through affinity chromatography on a His-column. The ELISA assay showed that antiHER2-hTNF-α specifically bound to HER2/neu expressing SKOV-3 and MCF-7 cells, but it did not recognize the HER2/neu negative cells A375. In vitro study, it was found that antiHER2-hTNF-α inhibited the proliferation of SKOV-3 and MCF-7 cells, whereas it did not effect on the A375. These studies suggest that the recombinant antiHER2-hTNF-α immunotoxin has a potential application in HER2/neu expressing tumor therapy.

Abstract:Using methods based upon the PCR, two DNA sequences which are identical to two JH genes(accession No.AY158087 ， AY149283) from one Holstein bovine genomic DNA have been cloned, the results demonstrate that all the two JH genes(accession No.AY158087 ， AY149283) are present in the same Holstein bovine genomic DNA. Two kinds of IgM cDNA was cloned from bovine spleen total RNA. Sequencing the cDNA clones show that the last exon of JH gene (accession No.AY149283) JH6 is a functional gene encoding part of CDR3 and whole FR4 region of bovine IgM. The sequences of two IgM cDNA clones indicate each constant region is identical to one of the two Cμ genes(accession No.AY230207 ， U63637), there are greater diversities in the IgM CH1 domain than the other CH domains. PCR products of JH-Cμ gene suggested that each Cμ gene (accession No.AY230207 ， U63637) is in series with the same JH gene (accession No. AY149283) in the bovine germline genes, they are all functional genes in the process of bovine IgM generation.

Abstract:TGIF ( TG interacting factor) is an inhibitor of TGF-βsignaling pathway. However, TGF-β can overcome the cell growth in the early stage of tumorigenesis, but promote the invasion and metastasis of neoplasms in the later stage. However its role in carcinogenesis is still unknown. After gastric carcinoma cell line, SGC-7901, was stablely transfected with plasmid PcDNA3.1-TGIF, the effect of TGIF on it was investigated via MTT method, flow cytometry, plate clone formation, nude mice tumorigeneity and the expressions of MMP2, MMP9 and VEGF proteins were detected in tumors originated from inoculated TGIF transfected, PcDNA3.1 transfected and SGC-7901 cells via immunohistochemistry. The contents of VEGF, active MMP2 and MMP9 in supernants of three cells were examined via ELISA and zymograph respectively. TGIF has no effect on the proliferation, cell cycle distribution and plating efficacy of SGC-7901 cells. In vivo experiment, tumors originated from TGIF transfected cells have no embolism formed inside whereas tumors originated from control cells have obvious embolisms. The expression levels of MMP9 and VEGF in tumors from TGIF transfectant cells are less than that from appropriate control cells, whereas MMP2 has no detectable difference among the three cells. The concentrations of VEGF in supernants of TGIF transfectant, PcDNA3.1 transfectant and SGC-7901 cells were (635±20.3) ng/L, (780±25.4) ng/L and (791±23.9) ng/L respectively. The content of VEGF in TGIF transfectant cells was significantly lower than that in control cells (P < 0.01). The contents of active MMP9 protein in supernants of TGIF transfectant cells were also significantly lower than that in control cells. Although TGIF can partially resist TGF-β mediated growth inhibition, it can not worsen the biological behaviors of gastric cancer cells. Inversely, TGIF can downregulate the expressions of VEGF and MMP9 and then mitigate their metastasis.

Abstract:The optical intrinsic signal imaging (OISI) at 550nm was applied to examine the parietal cortex of focal cerebral ischemia rats with the left middle cerebral artery occlusion (MCAO). A series of spontaneous spreading depression (SD) waves (10.3±4.6 times) were observed during the next 4 h after MCAO. In the earlier 2 h, the SD waves usually spread across the whole left parietal cortex, however, the optical signals showed significant regional differences. During the later 2 h, the waves were restricted in the medial parietal cortex, and the propagated area decreased reversely with the increasing times of SD waves. The baseline intensity in the lateral area increased steadily, up to (8.4±1.2) % at the end of 4 h. By TTC staining, the lateral area proved to infract focus. The results suggested that the optical imaging supplied an effective method to identify the ischemic penumbra and monitor its dynamic evolution.

Abstract:Ca2+/H+ antiporters play important roles in plant nutrition and signal transduction. A novel Ca2+/H+ antiporters gene OsCAX3 was identified from rice. Sequence analysis revealed that OsCAX3 has 11 transmembrane domains, and an acid motif consisting of 17 amino acids between sixth and seventh transmembrane domains. When expressed in yeast, OsCAX3 complemented the mutant growing deficiency at high Ca2+ concentrations, and its N-terminal(1~26 amino acid) partially suppressed its ability to transport Ca2+. RT-PCR analysis showed that OsCAX3 mRNA was induced by exogenous calcium. OsCAX3 was predicted to localize in plant plasma membrane by PSORT prediction, and this was further confirmed by transiently expressing OsCAX3-GFP fusion protein in mesophyll cell protoplast of Arabidopsis. All these findings strongly suggested that OsCAX3 is a Ca2+/H+ antiporter located in plasma membrane.

Abstract:In order to develop a novel, simple and effective relative quantitative method using fluorescence real-time RT-PCR, the formulas were derived from the standard curve for quantifying relative expression of mRNA. The formulas show that the relative expression index is only associated with CT value and the slope of standard curve. RNA (DNA) standard for absolute quantitative standard curve is obtained generally by cloning and transcription in vitro. When input copy numbers of serial diluted RNA standards were increased or decreased n-fold (n > 0) proportionally, the slope of standard curve remains invariable, suggesting that the slope is independent of actual copy numbers of RNA standard. Therefore, anyone of tested RNA (cDNA) samples could be used after serial dilution as RNA standards to obtain slope. Compared with absolute quantitative method, the present one appears to have a more excellent consistency (difference less than 4%) in the relative expression indices with absolute quantitative method, than traditional relative method 2-ΔΔCT (difference more than 5%). The results show that the method described here is simple, precise and cost-effective for relative quantifying gene expression.

Abstract:A bicistronic expression vector, p3.1-IRES-CD34, has been constructed to facilitate the selection and screening of transfected cells by magnetic cell sorting (MACS). In this vector, an engineered variant of CD34, deleted CD34 (dCD34), was chosen as the marker gene and the internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) was employed to allow simultaneous expression of the inserted 5′-end heterologous gene and the CD34 marker gene. To test the utility of the vector, the enhanced green fluorescent protein (EGFP) gene was inserted into the multiple cloning site (MCS) of the vector. Transfected cells were selected by magnetic cell sorting (MACS). The results demonstrated that the transfected cells can be enriched to high purity (>95%). The vector p3.1-IRES-CD34 would provide a rapid (2~3 days for transient transfected cells and 10 ~ 15 days for stable transfected cells) and efficient method for the selection of transfected cells.

Abstract:Human salivary gland cells (HSG) were irradiated with carbon ion beams supplied by the Heavy Ion Medical Accelerator in Chiba (HIMAC), Japan at a low dose rate of 0.5 Gy/h. Clonogenic survival effects of HSG cells after exposure to carbon beams with three different dose-averaged LETs were obtained by means of standard colony-forming assay. Compared to the previous surviving data acquired for carbon beams with similar dose-averaged LETs at therapeutic dose rates (1~5 Gy/min), HSG cells show an obvious dose-rate sparing effect. To derive relative biological effectiveness (RBE) of the carbon beams for HSG cells under the same dose-rate condition, the dose surviving effect of HSG cells irradiated with 60Co γ-rays was measured as well at the dose rate of 0.5 Gy/h. The RBE values obtained at the low dose rate are totally reduced in contrast with those determined at the preceding therapeutic dose rates. The dose-rate sparing effect and the reduction in RBE at the low dose rate imply that the radiation-induced damage by high-LET carbon ion beams at low dose rates could be repaired to a great extent. Based on the important findings, the essential reason of cell lethality by radiation was discussed.