Abstract: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.

Abstract:Parkin is a causative gene of autosomal recessive juvenile parkinsonism. Now it is believed that Parkin functions as an E3 ubiquitin protein ligase which involves in protein's ubiquitination. Parkin defect makes its substrates accumulate, which eventually leads to dopaminergic neuron selective death. More and more evidence shows that Parkin can protect neurons from various neuron toxic stimulations and may participate in the formation of Lewy body, therefore Parkin may be important in the pathogenesis of sporadic Parkinson's disease.

Abstract:The Notch pathway is a wildly utilized, evolutionarily conserved regulatory signaling pathway that plays a central role in the fate decisions of multipotent precursors including common lymphoid precursors, which will undergo either T or B cell differentiation. Notch signaling participates in the process of lymphocyte development. It can promote formation of Tαβ cells, induce development of regulatory T cells from na?ve T cells, and block CD4+ T cell to differentiate to Th1 cell. It can increase the numbers of MZB cells. On the basis of structure of Notch and the new progress on Notch, the role of Notch signaling in the process of lymphocyte development and its molecular mechanisms have been reviewed.

Abstract:Nicastrin is a novel typeⅠ transmembrane glycoprotein, which has been shown to be a critical component of γ-secretase complex. It has been well established that nicastrin is essential for the stability and trafficking of the other γ-secretase components. Furthermore, nicastrin plays an important role in Aβ peptide generation of Alzheimer's disease and Notch signaling pathway.

Abstract:In order to study the effect of UBAP1 gene on UBAP1 down-regulated nasopharyngeal carcinoma (NPC) cell line HNE1, the pcDNA3.1(+)/UBAP1 mammalian expression recombination was constructed and transfected into HNE1 cells. G418 was used to obtain the neomycin-resistant transformants of which indicated the vector was present in the HNE1 cells. The expression of UBAP1 gene was detected by RT-PCR and dot blot. The cytobiologic characterizations of the transfected HNE1 cells were probed by population double time (PDT), xenograft of nude mice and cell cycle analysis. The results showed that the PDT of HNE1 cells with expression of UBAP1 was longer than that of vector transfected HNE1 cells and untrasfected HNE1cells, and the most UBAP1 transfected cells went into phase G0-G1 compared with the two other cells. It also presented inhibiting the tumor formation in nude mice. Thus, these data suggested that UBAP1 gene plays an important role in the cell growth of HNE1, and might be a good candidate for tumor suppressor gene correlated with NPC.

Abstract: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.

Abstract:Obtained by homologous recombination of replacing part coding region and common promotor region of cathepsin (v-cath) and chitinase (ChiA) genes with polyhedrin gene, the recombinant baculovirus BmNPVpolh+ CP－ChiA－GMCSF+, in which the v-cath and ChiA genes were inactivated, could express hGM-CSF gene and polyhedrin gene simultaneously. It was revealed that the inactivation of v-cath and ChiA genes had no significant effect on viral growth or polyhedron production in BmN cells, and the cells infected with the recombinant baculovirus BmNPVpolh+CP－ChiA－GMCSF+ survived 2 days more than those infected by BmNPV and BmNPV-hGMCSF, the expression level of foreign gene could be obviously improved. The B.mori larva infected with BmNPVpolh＋CP－ChiA－GMCSF＋ retained a healthy, normal white epidermis color, in contrast to the typical discoloration of the larval epidermis seen after death in wild-type BmNPV-infected larvae, whose cuticle becomes soft and easily punctured.

Abstract:It has been demonstrated that there are differences between introns of highly-transcribed genes and those of lowly-transcribed genes in sequence length, position preference and oligonucleotide usage. Further observation showed a similar phenomenon: the lengths of upstream intergenic sequences of highly-transcribed genes are generally longer than those of lowly-transcribed genes. Based on the statistical comparative analysis on the occurrence frequencies of oligonucleotides in the upstream intergenic regions of the two sets of genes, some potential sites were extracted in the upstream regions of highly-transcribed genes which are likely to enhance the transcription of genes. These regulatory elements turned out to be also over-represented by comparing the upstream sequences of highly-transcribed genes to all non-coding sequences of yeast genes. Most of these elements are agreement with transcription factor binding sites obtained from experimental analyses. And these elements are G,C rich, this seems to be supplement to those potential sites in introns extracted before, which are A,T rich, in base composition. Such sequence structures of highly-transcribed genes are favorable to the transcription of genes.

Abstract:When either massive damage is inflicted on the liver or liver regeneration after damage is compromised by a variety of toxins and carcinogens, the hepatocyte progenitor/stem cells which called “oval cells” is activated and differentiate into a variety of cell lineages including hepatocytes and biliary epitheliums. Those models used in the study of rodents' oval cells are obviously not suitable for the study on human hepatic oval cells. A new hepatic oval cells produced model suitable for human need be developed. Mouse embryonic stem cells (ES cells) were cultured and induced to develop into embryonic bodies. The induced group and the control group were set up then. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) was added to the culture medium of the induced group. The markers such as A6 antigen, which is expressed by hepatic oval cell, are detected by means of immunocytochemistry. As the descendants of mouse ES cells, hepatic oval cells sorted by FACS were detected by RT-PCR and transmission electron microscopy. The hepatic oval cells sorted by FACS were further cultured and tested the ability of bipotential differentiation by ICC and RT-PCR. It was firstly confirmed that the midterm phase of hepatic oval cells which are bipotential does occur during the course of the directional induction and differentiation of mouse ES cells into hepatic parenchyma cells. The differentiation ratios of hepatic oval cells from the induced group are significantly higher than that from the control group, and the maximal ratio from the induced group could be about 6.11%. HGF and EGF could promote the proliferation of hepatic oval cells derived from ES cells. About 4.59% of the cells sorted by FACS are Sca-1+/CD34+, and about 90.81% of the Sca-1+/CD34+ cells are A6 positive. Highly purified A6+/ Sca-1+/CD34+ hepatic oval cells derived from ES cells could be obtained by FACS. A new hepatic oval cells produced model suitable for human was developed.

Abstract:Photodynamic therapy (PDT) has been widely used as an anticancer treatment in the clinic. However, the damage to the vessel induced by PDT, one of the most important aspects to tumour therapy, is not clearly understood. In order to study the effect of vascular damage in vivo during PDT, the chicken chorioallantoic membrane (CAM) tumor model by using stable high GFP-expressing human adenoid cystic carcinoma cell strain (ACC-M-GFP) is built for the experiment. The fluorescence imaging was used to show the location and the size of the tumor and treatment region. The dynamic parameter change of blood with different photodynamic doses in PDT was monitored in real time by laser speckle contrast imaging (LSCI) technique. The experimental results show that: the photodynamic effects are observably different at different photodynamic doses. It proved that: firstly the GFP-expressing tumor CAM model is extremely suitable for studying the photodynamic effect on the vessels, because of its convenience to locate the tumor and treatment region on the CAM. Secondly, with the aid of the LSCI technique, it is able to perform real-time monitoring of vessel structure and flow velocity change to identify the perfusion variation in PDT and to additionally investigate the damage to the vessels around the tumor induced by PDT.

Abstract:Monokine induced by interferon-γ (MIG, CXCL9) is a CXC chemokine which chemoattracts lymphocytes or neutrophils. Recent studies suggest that some members of CXC family, such as PF-4 and IL-8, CRG-2 and MIG also possess angiogenic or angiostatic properties. Recombinant mouse MIG protein were produced and purified and test its biological effects on endothelial cells. Mouse MIG cDNA was PCR amplified from mouse brain and cloned into pFASTBAC 1 vector in Bac-To-Bac baculovirus expression system. The recombinant mouse MIG Bacmid was introduced into High-Five (Tn-5B1-4) insect cells. After 48~72 h of incubation, the supernatant was collected and the MIG protein was purified by S-Sepharose. The yield of MIG was 10 mg/L and the purity is 90%. The MIG protein migrated as several bands on SDS-PAGE with the main band at 14.4 ku and was further confirmed by Western blot analysis. Functional assays demonstrated that purified mouse MIG protein not only elicited influx of calcium in mouse cytotoxic T cell lines but also inhibited both mouse endothelial cell migration and proliferation in a dose dependent manner. Thus, mouse MIG protein is not only a potent chemoattrant for lymphocytes but also an anti-angiogenic factor, which has important implications for angiogenesis-related diseases, such as tumor. It is the highest yield of recombinant mouse protein production described in the literature so far. The availability of large amount of purified active mouse MIG protein produced from baculovirus expression system has paved the way for the further studies of its in vivo angiostatic functions in mice.

Abstract:Based on flow chamber technique and using carrier microspheres as force magnifiers, an investigation of the interaction force between surface immobilized ligand and objective molecule was carried out. Human immunoglobulin G (IgG) and goat anti-human IgG (anti-IgG) were employed as model ligand and model objective molecule respectively. The parameters of the flow field were designed based on Plane Poiseuille Flow and the design was validated by a numerical simulation. Using bovine serum albumin (BSA) as negative control, it was concluded that the adhesion force between the microspheres and the chip surface came from the specific interaction between the ligand and the objective molecule. And this conclusion was confirmed by an anti-IgG deactivation comparison. It was found that the adhesion force between the spheres and the chip surface was affected by the ligand surface concentration. The wall shear rate at which 95% microspheres were removed from the chip surface was set as the critical value, and the relationship between the critical shear rate and the ligand surface concentration was obtained. A mechanical analysis model considering both the ligand surface concentration and the difference of molecular bonds' position was proposed, which finally gave the result that the average interaction force between a single pair of ligand and objective molecule was about 342pN.

Abstract:HCV NS3 protein plays an important role in the carcinogenesis of HCV related HCC. But the mechanism remains to be determined. The cell lines and transgenic mice used in previous studies were rarely based on HCV natural infection process, so the results acquired may need to be further evaluated. Under such background, the eukaryotic plasmid expressing HCV NS3 protein (pcDNA3.1/NS3 ) was constructed. Through transfecting the human hepatocyte line QSG7701 with pcDNA3.1/NS3, the research system expressing HCV NS3 protein was constructed successfully. On this basis, the effects of HCV NS3 on cell proliferation, the phosphorylation of MAP kinases and activity of transcription factors AP-1, NF-κB and STAT3 were investigated. The results showed that HCV NS3 protein could enhance proliferation, up-regulate phosphorylation of Erks and the activity of transcription factors AP-1, NF-κB and STAT3 in human hepatocytes. It is speculated that, HCV NS3 protein up-regulates ERKs/AP-1 activities is an important mechanism by which HCV NS3 protein enhances cell proliferation, its up-regulation of activity of transcription factors NF-κB and STAT3 might be relevant to the acute hepatitis associated with HCV infection.