Abstract:Extracellular signal-regulated kinase3 (ERK3) is distinguished from other ERK family members especially in its molecular biological characteristics including the big intron between exons in its gene structure, the serine189 mono-phosphorylated site and C-terminal extention of its kinase structure. The specially activating phosphorylation site of serine189 indicates that all MEKs, which phosphorylate serine/threonine double phosphorylation sites of MAPKs, are unable to activate ERK3. The C-terminal extension involves both subcellular localization of ERK3 and binding to intact cyclin D3, which can profoundly affect cell cycle regulation. According to update reports, ERK3 signal pathway in the regulation of cell cycle might be as follows: Ras→B-Raf→ERK3kinase→ERK3→decrease of CDK compounds of G1-phase→increase of the inhibiting factor (retinoblastoma protein) of S-phase→blockage of S-phase of cell cycle→cell differentiation entry while cell proliferation arrest. Moreover, the activation of ERK3 signaling pathway is also associated with cell differentiation, embryonic development, insulin secretion and cancer diseases.

Abstract:Calmodulin (CaM) is ubiquitous, multifunctional calcium (Ca²⁺) sensor that exists in all eukaryotes. As it has no enzymatic activity, CaM transmits the Ca²⁺ signal by interacting with CaM-binding proteins (CaMBPs) to function in cellular regulation. In recent years, it has been found that CaM not only signals in the presence of Ca²⁺, it can also directly bind to target proteins as ApoCaM. Ca²⁺-independent CaM-binding proteins (ApoCaMBPs) is also an important way to elucidate the mechanism of CaM functions. Recent progresses in studies on animal and plant ApoCaMBPs were summarized. ApoCaM differs from Ca²⁺-CaM in its tertiary structure. It binds target proteins differently, utilizing different binding motifs such as the IQ motif, noncontiguous binding sites and others. The ApoCaMBPs are a diverse group of proteins including enzymes, transcription activators, as well as cytoskeletal and other membrane proteins, including receptors and ion channels. The overall picture that emerges is that CaM cycles between its Ca²⁺-bound and Ca²⁺-free states and in each state binds to different proteins and performs essential functions. Although much of the research focus has been on the roles of Ca²⁺-CaM binding proteins, the roles of ApoCaMBPs are equally vital but less well understood. Researches on ApoCaM and its binding proteins will make us understand the variety of CaM signaling pathway.

Abstract:Usage of the fruit and bark of a Melia-family plant as a digestive tract-parasiticide and agricultural insecticide was recorded about two thousant years ago in ancient China. Toosendanin (TSN), a triterpenoid, is an effectual ingredient extracted from the plant. Studies have demonstrated that TSN selectively affects neurotransmitter release, effectively antagonizes botulism, induces cell differentiation and apoptosis and inhibits proliferation of various human cancer cells, inhibits feeding and dovelopment in insects and modifies K⁺- and Ca²⁺-channel activity. The research data to demonstrate that TSN inhibits K⁺-channel and facilitates L-type Ca²⁺-channel are summarized, and the mechanism of action of TSN is discussed.

Abstract:Nasopharynx epithelial cells are constantly exposed to both commensal and pathogenic micro-organisms. After being stimulated by some microbial, the nasopharynx epithelial cells secrete a lot of inflammatory factors that lead to the inflammation of nasopharynx and further result in the nasopharyngeal carcinoma and other disorders. LPS is the most important commensal or pathogenic bacteria constituents. It is related to the pathogenesis of a variety of disease. It has been shown that the 5-8F cells could bind with the FITC-labeling LPS for its expression of CD14, TLR4 and MD2 with flow cytometry and RT-PCR assay. With immunofluoresense, Western-blot and luciferase reporter system assay, it is indicated that LPS activated the TLR4 signaling pathway in 5-8F cells. Phospho-NFκB p65 expression was increased and entered into the nuclear in 5-8F cells with LPS inducement. Furthermore, after LPS stimulation, TNF-α promoter activity and the relevant amount of TNF-α being produced were increased in 5-8F cells. In conclusion, 5-8F cells expressed CD14, TLR4 and MD2 that are crucial for LPS binding. When nasopharnyx epithelial cells were induced by LPS, they did respond to LPS via TLR4 signaling pathways and activated NFκB signaling pathway, which can further lead to nasopharnyx inflammation and other nasopharnyx disorders.

Abstract:Human hepatocarcinoma cells, HepG2 were cultured onto biodegradable polyglycolic acid (PGA) polymer scaffolds, which were cultured in a rotating cell culture system (RCCS) to form a three-dimensional (3D) multicellular culture in vitro. The RCCS can simulate microgravity effects with low shear stress and well exchanging for gas. Then the growth characteristics and some mechanism of the cells in RCCS were detected by scanning electron microscopy (SEM), transmission electron microscopy (TEM), RT-PCR and flow cytometry (FCM). The results indicate that the cells grew well with polyhedron morphology and lots of microvilli, mitochondria and tight junctions in this system, which means that this system is useful for cells to form 3D structure to mimic cell status in vivo. The expression of some cell adhesion molecules (CAMs) were changed markedly, which are closely associated with cancer invasion and metastasis. The characters of increased expression of integrin β1(CD29), CD44, intercellular adhesion molecule-1(CD54) and depressed expression of E-cadherin presumably show that the HepG2 cells cultured in RCCS could recur some characters of primary liver cancer in vivo, the capacity of invasion and metastasis. It is necessary for acquiring perfect and external results to select an appropriate research model for studying in vitro. This 3D culture in vitro under simulated microgravity can provide a useful and reasonable model for oncology, anticancer drugs research and other research.

Abstract:MicroRNAs(miRNAs) are short non-coding RNAs that play important regulatory roles in both animals and plants. While the first miRNAs were discovered using experimental methods, experimental miRNA identification remains technically challenging and incomplete. Hence, computational approaches are a natural choice to complement experimental approaches to miRNA gene identification. A de novo miRNA precursor prediction method was proposed. In constructing the recognition model, both primary sequence and secondary structure were combined into an input sequence through encoding, and the input space was mapped into a feature space via k-gram method. After applying feature selection, those selected features was used to construct SVM-based models for the recognition of miRNA precursors. In the mean time, the method was compared with the HMM learning method. Experimental results show that the method outperforms HMM. The reason is that microRNAs are so short that it is not easy for HMM model to capture the signals for differentiating the genuine microRNAs from those pseudo-microRNA genes. From features selected, it was found that they are mostly come from the primary and secondary structure of microRNAs. This phenomenon may tell us to put more efforts in the microRNAs themselves in designing computational method before we fully understand the transcription mechanism of microRNA biologically.

Abstract:To study whether the late-acting co-stimulatory molecules ICOS can suppress the apoptosis and sustain the survival and proliferation of T cells through the survivin pathway, ICOS signals deficient T-cells were infected with adenovirus carried survivin gene, other T-cells were given ICOS co-stimulatory signals, then infected with adenovirus carried dominant-negative mutant survivin gene. Apoptosis and proliferation were determined by TUNEL and CCK-8 respectively. The results show that engagement of ICOS signal increased the expression level of survivin significantly. Survivin can sustain co-stimulatory deficient T cells survival and suppress the apoptosis. Mutant survivin inhibits ICOS signal positive T cells survival and increase its apoptosis. Late-acting co-stimulatory molecules ICOS can suppress the apoptosis and sustain the survival of T cells through the survivin pathway.

Abstract:Learning a new behavior is an exploration process that involves the formation and the modulation of association set between stimuli and neuronal networks' responses. To construct one appropriate learning model at networking level, on multi-electrode array, the closed-loop low frequency (1 Hz) pair stimulation was used to train the cultured hippocampal neuronal network and induce the learning behavior at networking level. Once the hippocampal neuronal network was trained successfully, the early postsynaptic responses were increased evidently, the response/stimulus ratio was increased and the response time decreased, furthermore, selective. These results indicate that the formation and the modulation of association between stimuli and neuronal networks' responses have been set, which means the learning behavior at networking level has occurred.

Abstract:The gene smu.776 encodes a possible S-adenosylmethionine-dependent methyltransferase of 385 residues in Streptococcus mutans, a primary pathogen for human dental caries. The DNA fragment of smu.776 was cloned into pET28a and expressed in good amount from the E. coli strain BL21 (DE3). Smu.776 protein was purified to homogeneity in a two-step procedure of Ni²⁺ chelating and size exclusion chromatography. Crystals were obtained by hanging-drop vapor diffusion method and diffracted to 2.0 Å resolution. The crystal belongs to orthorhombic space group C2 with cell dimension of a=168.47 Å, b= 50.66 Å, c=53.96 Å, β=104.22°. The asymmetric unit is expected to contain one molecule with solvent content of 51.3%.

Abstract:Staphylococcus epidermidis is the common pathogen of nosocomial infection. SarA (staphylococcal accessory regulator A) is a global factor regulating expression of most virulence genes in S. epidermidis. It is reported that SarA regulates the transcriptions of lipA encoding lipase, zinC encoding membrane- associated zinc metalloprotease and atlE encoding autolysin. SarA exhibiting an up-regulation on lipA and zinC, and down-regulation on atlE were demonstrated by reverse-transcription PCR and reporter gene lacZ analysis respectively. The results demonstrate that SarA regulated directly the expressions of lipA, zinC and atlE by protein-DNA binding to the promoter region of AT-rich sequence. The consensus sequence of binding sites in atlE, lipA and zinC promoter regions of S.epidermidis RP62A was forecasted by using program Omiga 2.0. The results suggest that SarA could be treated as the target in the prevention of Staphylococcus epidermidis infection.

Abstract:Protein kinase CK2 is an attractive target for anti-neoplastic and antiviral drugs and the inhibitors of CK2 have clinical therapeutic potential. The effect of recombinant human CK2 holoenzyme activity and cellular CK2 activity by digitoflavone was assayed by detecting incorporation of ³²P of [γ-³²P]ATP into the substrate. The mRNA expression of CK2α, α' and β subunits were detected by multiplex RT-PCR. CK2 kinetic analysis was carried out by using the Lineweaver-Burk plot. Digitoflavone was shown to inhibit strongly the activity of recombinant human CK2 holoenzyme in vitro (IC₅₀=0.86 μmol/L) and endogenous CK2 in HL-60 cells, which was more effective than the positive control 4Br-2-azabenzimidazole (TBB). After being treated for 2 h with digitoflavone, the mRNA expression of CK2α and α' subunit decreased, while β subunit was not changed fundamentally. Kinetic studies of digitoflavone on recombinant human CK2 showed that digitoflavone acted as an inhibitor of competitive with ATP and mixed types with casein. These results indicating that digitoflavone is a potent endogenous inhibitor of protein kinase CK2.

Abstract:A glycomic method was used to screen the aberrantly α1-6 fucosylated glycoproteins related to HCC metastasis and analyze the alteration of CK8 both in its expression level and its glycan parts associated with metastatic ability. Based on the approach, 2-DE coupled with lectin affinity blot, lectin affinity precipitation followed by MALDI-TOF-MS/MS, the lens culinaris agglutinin (LCA) affinity glycoprotein profiles from MHCC97-L and MHCC97-H cells, two higher metastatic HCC cell lines, were obtained, in which a differentially displayed protein spot was indicated when compared with Hep3B, in the region within 55～60 ku in molecular mass and 4～6 in isoelectric point. The identification result was CK8 by MALDI-TOF-MS/MS. To confirm the relation between increased core-fucosylation of CK8 and HCC metastasis, LCA affinity precipitation was used to extract the α1-6 fucosylated glycoproteins, followed by Western blot. And it was found that CK8 was highly fucosylated in both MHCC97-L and MHCC97-H cells compared to Hep3B. Immunofluorescence analysis and Western blot were used to detect its intracellular localization and its protein expression levels, indicating that CK8 distributed in cytoplasm and increased protein expressions in MHCC97-L and MHCC97-H cell lines. And further lectin binding studies found that CK8 has a high affinity for Con A in both MHCC97-H and Hep3B cells, indicating that CK8 was a glycoprotein with high-mannose type N-glycans. But the amount of the lectin RCA-1 binding to CK8 was greater in MHCC97-H than Hep3B, suggesting that CK8 contained the increased terminal galactose residues β-1, 4-linked to GlcNAc in MHCC97-H. All the results suggested that the increase of CK8 in its protein expression level, core-fucosylation and terminal gal β1,4 GlcNAc disaccharides might be related to HCC metastatic ability.

Abstract:Smu_195c is a protein with 86 amino acids in Streptococcus mutans, a primary pathogen for human dental caries. The specific function of Smu_195c is still unknown and there are no conserved domains in it. In order to find out its function, the gene encodes Smu_195c was cloned and expressed in E. coli as N-terminally 6*His tagged recombinant protein. Two crystal forms were obtained by the hanging drop method. Form Ⅰ belongs to space group P6₁22 or P6₅2 with the unit cell parameters a = b = 62.93 Å, c = 90.63 Å, γ=120° and form Ⅱ belongs to the space group P4₁2₁2 or P4₃2₁2 with the unit cell parameters a = b= 57.97 Å, c = 103.51 Å. Crystals from the protein with His-tag belong to form Ⅰ, however, crystals from the protein without His-tag belong to both.

Abstract:Basic fibroblast growth factor (bFGF or FGF-2) plays an important role in modulating proliferation, migration, differentiation and survival of various cells, such as fibroblasts, endothelial cells, smooth muscle cells and neural cells. The fetal liver stromal cell lines (FLSCs) expressing bFGF stably has been established by lentiviral system. The FLSCs were isolated from aborted fetus with informed consent, the growth character was identified by MTT method and the surface marker analyzed by flow cytometry. The cells could maintain normal karyotype after passaged for 35 generations. The coding region of 18 ku bFGF was cloned from fetal bone marrow mesenchymal stem cells (BMSCs) by RT-PCR. The bFGF recombinant lentiviral plasmid was steadily transfected into FLSCs. The low and high bFGF-expression FLSCs that transfected by the recombinant lentivirus were sorted by fluorescence-activated cell sorting (FACS) according to weak and strong eGFP expression. Analysis of weak and strong eGFP expression transfected FLSCs by RT-PCR and Western-blot demonstrated the stable expression of bFGF after 15 passages. The bFGF expression at mRNA level of weak and strong eGFP expression FLSCs are 2.33 and 6.19-fold for the FLSCs transfected by the control lentivirus, and then at the protein level are 1.76 and 5.05-fold. hES cells can be matained for 20 passages using the bFGF/FLSCs as feeder layers supplemented with a little or no additional recombinant bFGF. The establishment of bFGF/FLSCs could be acted as feeder cells for sustainment of human embryonic stem cell in vitro by supplying an eligible microenvirnment free of animal feeder layers.

Abstract:IκB kinase-α (IKKα) is required in lymphoid organogesis and mammary gland development. It has been reported that expression of IKKα is up-regulated in human cancers. IKKα-knockdown mouse model was established in order to study of its physiological function and its role in tumorigenesis. IKKα-knockdown lentiviral vectors were injected into the subzonal space between the cytoplasmic membrane and the zona pellucida. Transgenic mice were generated with the average positive rate of 15% and the IKKα gene knockdown in this mouse model was characterized. IKKα mRNA levels were markedly lower in blood samples collected from transgenic mice as compared to wild type mice. This IKKα-knockdown transgenic mouse model proves to be a useful tool to study physiological function of IKKα gene in vivo. Further phenotype analysis is under the way.

Abstract:Xenopus Paraxial Protocadherin (PAPC), which was initially identified in a screen for genes present in the Spemann organizer of Xenopus embryos, is required for gastrulation, somitogenesis and otic vesicle formation. In order to investigate its function in various developmental events, an antibody was prepared which could specifically recognize Xenopus PAPC. Glutathione S transferase (GST) expression system was used to express the fusion protein GST-PAPC. Rabbits were immunized with GST-PAPC fusion protein and the anti-PAPC polyclonal antibody was obtained. In dilution of 1∶3 000 the antibody recognized a specific band in Western blotting analysis of FL-PAPC transfected HEK 293T cells lysates, which could be specifically blocked by pre-adsorption of prokaryotic expressed GST-PAPC fusion protein. Furthermore, by using immunofluorescence analysis the polyclonal antibody recognized membrane-bound PAPC in FL-PAPC transfected 293T cells and Xenopus animal cap cells. By Western blotting analysis, the endogenous 150 ku PAPC protein was detected in Xenopus embryos using the anti-PAPC antibody. Take together it could be concluded that a polyclonal antibody specifically against Xenopus PAPC was developed.