Abstract:The remodeling of chromatin is a key step in controlling and regulating the temporal expression of genes. In the senescent human diploid fibroblast, there is a specific heterochromatic structure accumulating in the nucleus in the form of punctate foci, which is termed as senescence-associated heterochromatic foci (SAHF). The histone H3 methylated on lysine 9 (K9M-H3) and Heterochromatin Protein 1(HP1) are the marker proteins of SAHF. During the process of SAHF formation, many factors such as p16^INK4a/Rb pathway and HMGA proteins play a very important role. Recent studies have shown that SAHF may suppress the expression of some E2F-target genes, thereby making the cell keep in a stable senescent state. The discovery of SAHF has provided a new biomarker for the research of cellular senescence, and it also gives us a molecular explanation for the stability of the senescent state.

Abstract:Transcriptomics studies the variety, structure, function and regulation of all transcripts in a given cell and in a given time. It provides a novel procedure for revealing the molecular mechanism and regulatory network of different development stages of nasopharyngeal carcinoma. The progress of isolation, identification and function study of tumor susceptibility/suppressor gene, gene transcription profiling and transcription regulatory networks have been introduced.

Abstract:Endoplasmic reticulam is an imprtant organelle in cells. Normal functions of the ER could be impaired and that causes endoplasmic reticulam stress (ERS). ERS activates unfolded protein response (UPR), including immediate stoppage of new protein synthesis, up-regulation of ER chaperones and folding enzymes, and inducement of ER-associated degradation as a self-protective mechanism and induces rescue or adaptive response. If stress is prolonged and functions of the ER are severely impaired, to protect the organism by eliminating the damaged cells, apoptotic signals are generated through several mechanisms including: induction of C/EBP homologous protein CHOP, IRE-1-mediated activation of ASK1/JNK, cleavage and activation of procaspase-12 and Bcl-2-regulated Ca²⁺ release from the ER.

Abstract:Bidirectional signal transduction is a newly elucidated mechanism in intercellular communication. The bidirectional signal transduction mediated by the Eph-ephrin is an important representative in this field. The Eph family receptor tyrosine kinases and their membrane-bound ligands, the ephrins, play pivotal roles in the development of nervous system, angiogenesis, etc. The signal transduction into cells by Eph receptors is the forward signal, whereas the signal transduction by ephrins is the reverse signal. Based on their molecular structures, the ephrins can be divided into two subclasses, i.e. ephrinA and ephrinB. The ephrinBs are transmembrane proteins, which can activate FAK, JNK and Wnt signal transduction pathways through phosphotyrosine-dependent signaling and PDZ-binding motif-dependent signaling. The ephrinAs are glycosylphosphotidylinositol (GPI) anchored proteins, which can also mediate reverse signal transduction.

Abstract:Previous work showed that there was high ratio of Mycoplasma hyorhinis infection in human gastric carcinoma. P37 protein is a membrane lipoprotein of Mycoplasma hyorhinis. It was reported that P37 inhibited cell adhesion and induced tumor invasiveness. To investigate the function of P37 in cancer development, the p37 gene was subcloned into shuttle vector of pAdTrack-CMV. Linearized pAdTrack-CMV-p37 was co-transformed into BJ5183 cells with adenoviral genomic plasmid pAdEasy-1. The identified recombinant DNA was transfected into 293 cells to package adenovirus. From the supernatant and cell lysis, the presence of recombinant adenovirus P37 was proved by PCR. And then BICR cells with this recombinant adenovirus of P37 were successfully infected. RT-PCR and Western blot demonstrated the transcription and expression of P37 in infected BICR cells. And the soluble P37 protein can be secreted into the culture supernatant of infected BICR cells. In the migration assay in vitro, the number of migration BICR cells infected with Ad-p37 was 52.03% more than that of control. The construction of recombinant adenovirus provided a good tool for studying P37 function and its molecular mechanism, which will be further used for in vivo migration and invasion experiments.

Abstract:To study biological activity and mechanism of two modified anti-tumor peptide of tumstatin, the nucleotide sequences of 19peptide (amino acid185～203) and 21peptide based on modified T7 peptide (amio acid 75～95) of tumstatin were designed, artificially synthesized and inserted into the fusion protein vector pTYB2. After transformed and expressed in E. coli BL-21 (DE3) by means of fusion protein, the soluble 19peptide and 21peptide were obtained from one step chitin affinity chromatograph. By such experiment as MTT assay, cell growth curve, TUNEL assay，flow cytometry，the effect of the H22 ascitic fluid transfevent hepatoma of mice and histopathological slice, the biological activity of 19peptide and21peptide were studied. Experiments in vitro and in vivo identified that 19peptide could inhibit tumor cell proliferation, promote tumor cell apoptosis and stop tumor cell in G0/G1 cycle. It also could inhibit human umbilical vein endothelial cell proliferation to some extend. The anti-tumor activity of 19peptide mainly relied on affecting tumor cell directly and partly on inhibiting vascularization.21peptide inhibited proliferation of human umbilical vein endothelial cell much better than that of tumor cell. It almost could not promote endothelial cell and tumor cell apoptosis.21peptide mainly exhibited indirect anti-tumor activity by anti-angiogenesis. The combination of 19peptide and 21peptide obviously inhibited endothelial cell and tumor cell proliferation and promoted them apoptosis. A combination application could markedly enhance anti-tumor activity, make up the defect of 19peptide, 21peptide alone and bring about synergism. It would be an efficient method for tumor therapy in future, which will lay foundation on its mechanism of action research and clinical tumor therapy.

Abstract:The inhibitory effects of growth-inhibitor on the growth of Isochrysis galbana, malondialdehyde (MDA) content and activities of antioxidant enzymes (superoxide dismutase and peroxidase) were studied. And the inhibition effects of 4 species of antioxidants on this damage to its cells were also investigated. The results showed that 0.10 mg/L of GI marked decreased cell densities, activities of superoxide dismutase (SOD) and peroxidase (POD), and MDA content increased. With increase of GI concentrations, cell densities and activities of SOD and POD decreased sharply, and MDA content increased obviously. When concentration of GI was 0.30 mg/L, the cell densities, activities of SOD and POD decreased to 0.05, 0.56 and 0.59 folds of the control, respectively, and MDA content increased to as higher as 2.2 folds of that of the control set. L-Ascorbic acid, citrazinic acid, lenediamiaetetraacetic acid disodium salt (Na₂EDTA) and 3-tert-butyl-4-hydroxyanisole could decrease MDA content obviously, and marked increased cell densities and activities of SOD and POD. When 4 species of antioxidants were added to the medium, the cell densities, and activities of SOD and POD increased to as high as 1.38～1.90, 1.49～2.12 and 1.55～2.13 folds of that of the control set, respectively, and MDA content decreased by 57.7%～87.9%. It was indicated that the excess active oxygens were produced under growth-inhibitor stress and active oxygen participated in the damage of growth-inhibitor to Isochrysis galbana; the L-ascorbic acid, citrazinic acid, lenediamiaetetraacetic acid disodium salt and 3-tert-butyl-4-hydroxyanisole could effectively inhibit membrane lipid peroxidation and further alleviated algal damage.

Abstract:MRF4 is one of muscle regulatory factors and plays critical roles during skeletal muscle development. The muscle development is important for the fish growth which is an important economic factor for the fish culture. To analyze the function of MRF4 in fish, the founder MRF4 antibody was prepared. The flounder MRF4 was cloned, ligated into prokaryotic expression vector pET-30b and expressed in strain E. coli BL21 (DE3). The recombinant flounder MRF4 fusion protein was soluble and purified with cobalt IMAC resins. To prepare MRF4 polyclonal antibodies, rabbits were immunized with the soluble protein and the increasing level of antibodies was determined by Western blot. Also, the endogenous flounder MRF4 was recognized by the anti-serum. The result further proved the existence of the anti-MRF4 antibody in the anti-serum, which will be useful for studies on the function of flounder MRF4.

Abstract:CD72 is a B cell specific receptor that exists in multiple alternative splicing forms. Eight novel alternative splicing forms of CD72 were identified from the spleenocytes of BALB/C mice. Two very unique intron sequences were found in those alternative splicing forms. One kind of splicing variants retained the intron1 in the mRNA. This intron can be translated into 32 amino acid residues without changing the reading frame of the whole proteins. Another kind of splicing variants used an alternative 3' splice site in intron 3(3'AS) which led to premature termination of its encoded protein. The differential expression of the CD72 splicing variants were compared in BALB/C and NZB/W mice that were at different stage of systematic lupus erythematosis(SLE) disease development. It was found that 1) splicing forms containing 3'AS was rare in all samples examinated; 2) splicing forms containing two ITIM domains and transmembrane domains were more abundant in BALB/C mice than in NZB/W mice, even in some cases the two ITIM domains were separated by the intron 1; 3) a shorter splicing form with both exon2 and exon3 missing was expressed highly in terminally diseased NZB/W mice.These results suggested an important role of CD72 alternative splicing forms in B cell receptor signaling and in SLE.

Abstract:Epidemiologic studies suggest that intake of excess all-trans-retinoic acid (RA) during embryogenesis induces various developmental defects and the central nervous system (CNS) represents a major site of the teratogenic action of RA. It is therefore important to understand which parameters are affected early by excessive RA in order to devise and improve protective nutritional strategies. The modulations of beta-catenin and caspase-3 levels were investigated in the KM mouse embryo following maternal treatment with a single oral dose of 30mg/kg body weight of RA during the neurula period. In addition, retinoic receptors (RARs) are key transcription factors regulating gene expression in response to RA-activated signals. So the experiment was designed to evaluate whether the alterations in protein expression of RAR alpha and beta during the time of neural tube closure were induced by excessive RA. Maternal intake of excess RA induced early downregulation of RAR alpha and beta, beta-catenin and caspase-3 expression, which was followed by an increase in their expressive levels in the neural tube tissue of mouse embryos. This finding suggests that the alterations in the expression profile of RAR alpha and beta, beta-catenin and caspase-3 may be implicated in the teratogenesis induced by excess RA in KM mouse embryo.

Abstract:In order to obtain MicroRNA (miRNAs) expression profiles of human hematopoietic stem cells (HSCs), and to preliminarily investigate functions of HSC-correlative miRNAs, by using miniMACS magnetic beads and fluorescence-activated cell sorting (FACS), isolated hematopoietic stem cells (HSCs) were isolated from human umbilical cord blood and performed total RNA extraction. Next, using a microarray, miRNA gene expression profiles of HSCs were obtained. CFC assays were performed to research on differentiation-promoting function of miR-520h, enriched in HSCs. Results showed that CD34⁺ hematopoietic cells and HSCs were successfully isolated from human umbilical cord blood, and 31 HSCs-correlative miRNAs were screened by microarrray. Among them, 22 were low in HSCs, and 9 were high. The result of real time RT-PCR confirmed high expression of miR-520h in HSCs. CFC assays showed that miR-520h promotes differentiation of HSCs into progenitor cells. In conclusion, human HSCs have a set of specific miRNAs that contribute to regulation of HSCs functions, which pave the way for exploring the roles of miRNAs in development of hematopoiectic system.

Abstract:Hepatitis B virus X-interacting protein (HBXIP), a cofactor of survivin, previously had been found to promote cell proliferation. In order to demonstrate the molecular mechanismsthe effect of HBXIP on transcriptional activity of NF-κB was investigated. The eukaryotic expression vector of HBXIP (pcDNA3-hbxip), or the RNA interference vector of HBXIP (pSilencer-hbxip) and NF-κB luciferase reporter (pNF-κB-Luc) were co-transfected into H7402 hepatoma cells. The pRL-TK vector was used as an internal control for normalization of luciferase activity, and the luciferase activity was determined by using the dual luciferase reporter assay system. The luciferase assay indicated that the over-expression of HBXIP could stimulated NF-κB transcriptional activity in H7402 hepatoma cells. RNA interference (RNAi) targeting HBXIP mRNA resulted in the opposite effects. Statistically no significant difference was observed between the control cells and pcDNA3 empty vector-transfected cells or pSilencer-control transfected cells. The phosphorylation level of IκBα, an inhibitor of NF-κB, was increased by Western blot analysis when over-expression of HBXIP in H7402 cells. The p65/NF-κB level in nucleus extraction from H7402 cells was examined by Western blot analysis after transfection. The data indicated that over-expression of HBXIP in H7402 cells was able to increase the levels of p65/NF-κB in the nucleus. However, down-regulation of HBXIP mRNA in the cells by RNAi resulted in the opposite results. In conclusion, HBXIP is involved in cell proliferation by regulating NF-κB signal pathway.

Abstract:Human herpesvirus 7 (HHV-7) infection is dependent on the functions of structural glycoproteins at multiple stages of the viral life cycle. These proteins mediate the initial attachment and fusion events that occur between the viral envelope and a host cell membrane, as well as cell to cell spread of the virus. To characterize the HHV-7 glycoproteins that can mediate cell fusion, a cell-based fusion assay was used. 293T cells expressing the HHV-7 glycoproteins of interest along with a luciferase reporter gene under the control of the T7 promoter were cocultivated with SupT1 cells transfected with T7 RNA polymerase. HHV-7 glycoproteins gB, gH, gL and gO can mediate the fusion of 293T cells with SupT1 cells, and the fusion can be inhibited by anti-CD4 mAbs. Thus, the coexpression of HHV-7 gB, gO, gH and gL is sufficient and necessary for HHV-7 induced membrane fusion, and one of these glycoproteins or protein complex formed by these glycoproteins might be the ligand(s) of CD4 molecule.

Abstract:DNA recombinase Cre can recognize LoxP sites and result in recombination of DNA molecules. Recombination between two directly oriented LoxP sites excises the inserted DNA. And recombination between two circular DNA molecules, which contains a directly oriented LoxP site each one , generates a cointegrate. Based on these traits of Cre recombinase, a gene-transfer and gene sub-cloning system was constructed. gfp gene was excised from gene-donor vector pTLG and transferred directly to gene-receiving vector pET-LoxP mediated by Cre recombinase, completing the construction of pET-gfp very quickly and simply. Afterwards, gfp gene was expressed in Escherichia coli BL21(DE3) and generated visible green fluorescence colonies. It extremely simplified traditional courses of vector construction to only a reaction mediated by recombinase. Recombination ratios affected by circular gene-donor vector pTLG and linear pTLG were quantified and compared with. A good reference was provided for gene easy cloning or sub-cloning mediated by Cre recombinase.

Abstract:BAC-FISH (bacterial artificial chromosome and fluorescence in situ hybridization) is a powerful tool for molecular cytogenetics such as chromosome identification and physical mapping in plant. Due to the large proportion of repetitive sequence occurring in some plant species，especially the polyploidy species, the application of BAC-FISH was hindered severely. Molecular markers from a high-density genetic map of tetraploid cotton were used to screen BAC libraries. Protocols of BAC-FISH in cotton mitotic and meiotic cells were introduced by some modifications such as root tip preparation, slide denaturation and post-hybridization washing according to previous protocols. The dual-color, dual-probe and repeated FISH were also introduced by applying them to physical mapping a random BAC clone on the corresponding chromosome.