Abstract:Nobel Prize 2009 in Physiology or Medicine is awarded to three American scientists, Elizabeth H. Blackburn, Carol W. Greider and Jack W. Szostak, for the discovery of “how the chromosomes are protected by telomeres and the enzyme telomerase”. Telomere is the specific structure at the ends of the chromosomes and protects it from fusion and degradation. Telomerase synthesizes telomere DNA to maintain the telomere length. Studies suggest that telomere length and telomerase activity is directly associated with cell life and the genesis of many diseases. With the progress of study, how to control the telomere length and telomerase activity is helpful to shed light on the studies in “cancer, inherited diseases and senescence”, and will stimulate the development of potential new therapies.

Abstract:The 2009 Nobel Prize in Chemistry was awarded to Venkatraman Ramakrishnan, Thomas A. Steitz, and Ada E. Yonath "for studies of the structure and function of the ribosome". The atomic-resolution ribosome structures solved by the laureates revealed the structural basis of the mechanisms of peptide syntheses by ribosome. They demystified several long standing functions of ribosome, such as the high fidelity of the translation process and the wobble effect. Their work also paved ways for the development of new antibiotics, just when public health is faced with increasing threat from drug-resistant pathogenic bacteria.

Abstract:Tumor metastasis, the main characteristic of malignance tumor, is the primary cause of death for most cancer patients. The initiation of tumor metastasis involves complex signaling pathway within tumor cell and microenvironment, mediating primary tumor metastasis, invasion, survival and arrest in the blood circulation, and progressive growth at the distant site. The most research on the mechanism of tumor metastais will help understand the metastasis process, and identify promising molecular targets for cancer clinical diagnosis and treatment.

Abstract:Epigenetics refers to non-coding sequence changes such as DNA methylation, histone modifications, chromosome remodeling and non-coding RNA regulation. Histone modifications include acetylation, phosphorylation, methylation, ubiquitination and ADP ribosylation. The combinations of different histone modifications, known as "histone code", are dynamic during development and differentiation and play important roles in the regulation of gene expressions in spatial-temporal manners. The modification on a particular residue in a histone affects not only the modifications at different residues in its own protein but also other histones. The histone modifications is a complicated network and the regulation remains elusive.

Abstract:The RD-1 locus has been considered crucial in the pathogenesis of M. tuberculosis, the RD-1 locus is 9.5 kb and spanning open reading frames Rv3871 to Rv3879c encoding 9 different proteins separately. The RD-1 locus is missing in all bacillus Calmette-Guerin (BCG) strains, and is one of the key virulence factor in M. tuberculosis. The RD-1 locus participates in a new secreting system named ESX-1, which can facilitate the secretion of some special proteins. The two important proteins encoded by the RD-1 locus named CFP-10 and ESAT-6 can form a tight 1∶1 complex, and has been shown to be coordinately secreted and lead to a strong T cell response, which suggests that these two proteins may act as ideal target antigens in diagnosis and prevention of tuberculosis (TB).

Abstract:Angiogenesis is of great importance to a variety of normal physiological processes and pathological disorders. It is tightly regulated by many mechanisms, among which vascular endothelial growth factor (VEGF) is one of the most potent promoters. VEGF binds and activates its specific receptor tyrosine kinases, especially vascular endothelial growth factor receptor-2(VEGFR-2). VEGFR-2 mediates the key functional and biochemical effects of VEGF in endothelial cells including proliferation, migration, survival, and permeability. Following its binding to VEGF, VEGFR-2 dimerizes and undergoes autophosphorylation on tyrosine residues within its cytoplasmic portion. This creates docking sites for adapter molecules to be recruited through their Src homology domain-2 (SH2). These adapter molecules can then initiate the activation of downstream signaling cascades. Further down-stream effector molecules are activated, and regulate the biological effects of endothelial cells. It is also foound that VEGF/VEGFR-2 signaling pathway may negatively regulate the function of human monocyte-derived mature dendritic cells (DCs) as well as the maturation of immature-DCs. Advances in the understanding of the VEGF/VEGFR-2 signaling pathway may contribute to the discovery of kinds of pharmaceutical agents.

Abstract:In previous study, the 150-ku oxygen-regulated protein (ORP150) was identified as a candidate glycoprotein related to hepatocellular carcinoma. In order to further validate the expression level of ORP150 in hepatocellular carcinoma, protein expression was determined by Western blot and cell immunochemistry, and messenger RNA (mRNA) expression was detected by quantitative real-time polymerase chain reaction. The effect of ORP150 on apoptosis and invasive potential of hepatocellular carcinoma cells was evaluated using the small interference RNA (siRNA) technique. Both the protein and mRNA expression levels of ORP150 were significantly upregulated in hepatocellular carcinoma cell lines compared with a non-tumor human liver cell line. After transfection with the specific siRNA of ORP150, significantly greater apoptosis of hepatocellular carcinoma cells was induced compared with untransfected cells. However, no significant effect on invasive potential was found. Overexpression of ORP150 was associated with hepatocellular carcinoma, and ORP150 might promote the proliferation of hepatocellular carcinoma cells by inhibiting apoptosis. ORP150 could be a potential therapeutic target for hepatocellular carcinoma.

Abstract:Human ribosomal gene (hrDNA) targeting vectors ( 10～14 kb) constructed by the group are novel non-viral vectors which could specifically integrate into the ribosomal loci and characterized by their security and stable expression of therapeutic genes. However, the low transfection efficiency handicapped their clinical application. Although nuclear localization signals (NLS) could facilitate the nuclear entry of non-viral vectors and improve the transfection efficiency, the expression of therapeutic gene varied dramatically with the coupling methods and the type of chemistry used. The hrDNA vectors were conjugated by succinimidyl-[4-(psoralen-8-yloxy)]butyrate (SPB) with Simian Virus 40 NLS (SV 40 NLS) peptide through electrostatic interaction efficiently, which could protect the plasmid DNA(pDNA) from degradation of DNase. The polyethylenimine (PEI), which is an economical and low toxic polymer and wildly used in vivo gene therapy, was employed to transfect the primary human dermal fibroblasts (HDF). When conjugated with NLS peptide, the 12 kb hrDNA emerged in the nucleus within 60 min under the view of confocal microscopy. The GFP fluorescence analysis by flow cytometry showed that the transfection efficiency was increased to 4～5 folds. In conclusion, an effective procedure was developed to improve the non-viral transfection efficiency and promise the preclinical trial of hrDNA vectors.

Abstract:In order to validate and estimate the capability of BMP9 to induce osteogenic differentiation of multipotent stem cells, three multipotent stem cells (C3H10, MEFs and BMSC) were used as target cells, and BMP9 was introduced into these cells by using recombinant adenoviruses assay, the effect of BMP9 on osteogenic differentiation of multipotent stem cells was demonstrated by using luciferase reporter assay, alkaline phosphatase(ALP) quantitative assay, calcium deposition assay, real time PCR, animal experiment and histological staining assay. The results demonstrated that BMP9 can induce ALP expression of C3H10, MEFs and BMSC by a dose dependent manner. BMP9 can also stimulate calcium deposition of C3H10 and MEFs in vitro, the osteogenic markers (ALP, Runx2, osteopontin, osteocalcin) were increased after stimulated by BMP9. BMP9 can activate canonical TGFβ-Smad pathway, and promote the expression of osteogenic master gene Runx2. The animal experiment and histological staining assay show that BMP9 can induce ectopic bone formation in naked mice. To sum up, BMP9 is a more powerful cell factor to induce osteogenic differentiation of multipotent stem cells.

Abstract:Diallyl trisulfide (DATS) is known to have an anticancer effect on human cancer cells. However, the exact mechanisms of this anticancer activity remain unclear. To explore the effect of DATS on human hepatocellular carcinoma HepG2 cell apoptosis. HepG2 cells were either treated with 50 μmol/L, 100 μmol/L of DATS for 0, 6, 12，24，48 and 72 h, respectively. The mitochondrial membrane potential was visualized by the JC-1 fluorescence staining. The cytochrome c in HepG2 cells was detected by Western blotting. The activity of caspase-3 in HepG2 cells was measured with ELISA. Diallyl trisulfide induces HepG2 cell apoptosis. The apoptosis percentage in HepG2 cells is up to 60.33% and 93.67%, respectively, when the cell was treated with 50 μmol/L or 100 μmol/L of DATS for 48 h. Diallyl trisulfide degrades the mitochondrial membrane potential and lowers the cytochrome c in mitochondria while increases the cytochrome c in the cytoplasma that mitochondria was deleted. Diallyl trisulfide activates caspase-3 kinase in HepG2 cells. These results indicate that diallyl trisulfide induces apoptosis by decreasing mitochondrial membrane potential, facilitating cytochrome c release from mitochondria into cytoplasm, and activating caspase-3 kinase in human hepatocellular carcinoma HepG2 cell.

Abstract:Anthrax toxin is one of the two virulence factors of Bacillus anthracis and it is a binary bacterium toxin composed of protective antigen (PA), lethal factor (LF) and edema toxin (EF). Among of them, LF is the enzyme moiety lead to the death of the cell. To investigate key function amino acid of LF and study the lethal mechanism of lethal factor, random mutagenesis was carried out. The mutation library of the 774 bp fragment of lethal factor from site 1 550 to 2 324 bp was constructed using Diversify^TM PCR Random Mutagenesis Kit (Clontech) with some modifiication. Transformants of lef mutation library were induced by 0.2 mmol/L IPTG at 28℃ for 4 h and then T7 phage were added into the culture, incubating for further 2～3 h to lyse the bacterium. The lysis was centrifuged at 12 000 r/min for 8 min and the supernatant which contained the target protein was collected. To determine the activity of the mutants, the cell cytotoxicity assay was carried out. RAW 264.7 microphage cells were seeded into 96-well plates, 24 h ahead of experiments. When assaying, the medium was removed and 100 μl DMEM medium containing 1.0 mg/L of purified recombinant and 10 μl of the lysis supernatant was added into each well. Then the plates were incubated at 37℃ for 4 h. The cell viability was determined by using the alamarBlueTM (AbD Serotec) dye according to the manufacturer’s direction. The mutants whose activities had decreased significantly were sequenced and further purified by Ni-histidine tag affinity chromatography. The cell cytotoxicity and competition binding assay were demonstrated to analyse the character of the mutants. Five mutants which had low biological activity determined through the cell cytotoxicity assay were obtained. Furthermore, two mutant proteins with single-site mutation, K518E and L519C, were purified for the first time. The cytotoxicity assay showed that the two mutant proteins lost activity significantly, that means, Lys518 and Leu519 played an important role in the function of lethal factor. The competition binding assay showed K518E endowed with the character of competition inhibition of wild type LF, whereas L519C doesn’t have the character. Probably, mutant K518E change Lys into Glu carrying negative charge and affects the catalytic groove of LF which is negative charge. Therefore, Lys 518 maybe has an important role to stabilize the conformation of LF and combine the substrate.

Abstract:In order to explore the role of Krupperl-like factor 4(KLF4) in regulating the expression of IL-6 induced by lipopolysaccharide (LPS). mRNA and protein levels of KLF4 in various periods were determined by RT-PCR and Western blot. A full-length cDNA or antisense oligonucleotides of KLF4 was transfected into RAW264.7 macrophages. The expression and release of IL-6 were analyzed by RT-PCR and ELISA. The transcription and DNA binding activities of KLF4 to the IL-6 promoter were detected by the luciferase reporter and EMSA. The results showed that treatment of RAW264.7 macrophages with LPS resulted in increase in KLF4 and IL-6 protein levels; KLF4 overexpression decreased the expression and release of IL-6, while KLF4 inhibition increased the expression of IL-6. The results from luciferase reporter gene assay demonstrated that KLF4 could inhibit the transcriptional activity of IL-6 gene. The results from EMSA demonstrated that KLF4 could not bind to the KLF4 binding sites on the promoter of IL-6 gene. These data suggested that LPS can stimulated the expression of KLF4 and release of IL-6 in RAW264.7 macrophages; KLF4 can inhibited the release of IL-6 in LPS-stimulated RAW264.7 macrophages; KLF4 can inhibited the transcription activity of IL-6 promoter in RAW264.7 macrophages; this effect of KLF4 is not through binding the IL-6 promoter directly.

Abstract:In order to investigate the messenger ribonucleic acid(mRNA) and protein expression status of ANXA1 gene in nasopharyngeal carcinoma cell lines, so as to explore the correlation between ANXA1 methylation status and gene expression, four nasopharyngeal carcinoma (NPC) cell lines, including CNE1, CNE2, 5-8F, 6-10B, and immortalized non-neoplastic human nasopharyngeal epithelial cell line NP69 were cultured in vitro for research. Methylation status of ANXA1 gene was detected by methylation specific polymerase chain reaction (MSP), while mRNA expression level were also evaluated by reverse transcriptional polymerase chain reaction (RT-PCR). Subsequently, different end concentration (0 μmol/L as control, 0.1 μmol/L, 1 μmol/L, 5 μmol/L,10 μmol/L) of 5-aza-2′-deoxycytidine(5-aza-2dC) were added into culture medium of NPC cell lines for 72 hours′ de-methylation treatment, then methylation status of ANXA1 gene were detected by MSP and mRNA expression were evaluated by RT-PCR. In addition, ANXA1 protein expression was detected by Western-blotting. Without de-methylation treatment, ANXA1 gene was methylated in all the four NPC cell lines but NP69, and the methylation extent is correlated with differentiation state and metastasis potential of the cells. mRNA expression was lower in all of the four NPC cell lines without de-methylation treatment compared with NP69, and the expression level was correlated with gene methylation level. 5-aza-2dC de-methylation treatment reversed ANXA1 methylation status, and increased the expression levels of mRNA and protein in all the four NPC cell lines. In summary, the current research verified down-regulated ANXA1 gene expression in NPC cell lines from both mRNA and protein level, the expression down-regulation was mainly caused by gene methylation, and 5-aza-2dC de-methylation treatment restored the down-regulated expression of ANXA1 in NPC cell lines to the level in the non-neoplastic cell line NP69.

Abstract:Insulin resistance in insulin sensitive organ results in metabolic disorder such as hyperglycemia, hyperinsulinemia and hyper triglyceridemia which are common features of type 2 diabetes. Insulin resistance in liver cells mainly causes impaired glycogen synthesis, failed to suppress glucose production which is the major contribution to hyperglycemia. FGF-21 as a new metabolic regulator can control fasting blood glucose. The mechanism of FGF-21 effects on regulating plasma glucose has little to known. In order to establish an in vitro insulin resistant model of liver cells and evaluate the effects and mechanism of FGF-21 on glucose metabolism in the cell model, HepG2 cells were incubated with 10^-7 mol/L insulin for 24 h to build insulin-resistant cell model. To evaluate the cells for insulin resistance, the cells were stimulated with fresh insulin for 24 h and the glucose uptake by these cells was carried out. The insulin-resistant cells were treated with different concentrations of FGF-21 for 24 h and insulin-treated cells were used as a control. The glucose uptake by the cells was detected by the method of glucose oxidizes/peroxides (GOD-POD); the synergy between insulin and FGF-21 was evaluated. The mRNA expression of GLUT1 in the insulin-resistant cells was detected by the real-time PCR. Glycogen synthesis of the cells was examined by the anthrone method. The results showed that HepG2 cells treated with 10^-7 mol/L insulin for 24 h became resistant to insulin and the insulin resistance status was maintained for 48 h without change of cell morphology. FGF-21 could stimulate glucose consumption of the insulin-resistant model in a dose-dependent manner. The glucose consumption and glycogen synthesis of the insulin-resistant model were significantly improved by FGF-21 treatment. FGF-21 showed strong synergy with insulin in glucose uptake and glycogen synthesis of the model cells. While the cells became resistant to insulin, FGF-21 could increase the mRNA expression of GLUT1. Thus, It is concluded that FGF-21 stimulates glucose uptake in insulin resistant HepG2 cells through GLUT1 expression, stimulates glycogen synthesis and improves the glucose metabolism in the insulin resistant liver cell model.

Abstract:The mammalian target of rapamycin (mTOR) is a serine-threonine kinase downstream in the PI3K-Akt pathway, which belongs to the phosphatidylinositol kinase-related kinase (PIKK) family. The mTOR palys an important role in cell cycle regulation and protein synthesis. mTOR signaling pathway is evolutionarily conserved, which can integrate and converge a wide range of signals, including intracellular and extracellular nutrients, growth factors, energy and stress conditions, and has a crucial role in vertebrate growth control. The upstream regulation and downstream effects mediated by mTOR form a network of critical growth signaling pathways. Hence, dysregulation of components within mTOR signaling network result in impaired internal environment conditions, and first meiotic division can also be influenced if occurring during starfish and sea urchin oocyte maturation. However, its functions in mammalian meiosis are unclear. The localization and function of mTOR during mouse oocyte meiotic maturation were investigated with the oocytes of Kunming mouse. Location of mTOR was examined in Germinal vesicle (GV) stage, Germinal vesicle breakdown (GVBD) stage and second metaphase (MⅡ) stage during mouse oocyte maturation by immunofluorescence technology. The rate of GVBD and polar body extrusion (PB1) during mouse oocytes maturation was also detected by treatment with different concentration rapamycin (0.5 g/L, 2.5 g/L, 3.3 g/L, 5.0 g/L, 10 g/L ). Immunofluorescent staining showed that mTOR mainly is located on nuclear membrane during GV stage, distributed with the chromosome after GVBD, and distributed with the spindle apparatus during MⅡ stage. The oocyte maturation was inhibited by treatment with rapamycin, and the inhibitory action was concentration dependent, when the drug concentration reached to 3.3 g/L, the disparity was significant (P < 0.05). Meanwhile, the location and morphous of mTOR were changed: under treatment with 3.3 g/L rapamycin, mTOR protein expression became obviously weaker and distributed more in intranuclear in the stage of GV; and didn't distribute with the chromosome after GVBD, the morphous of chromosome was changed. The mTOR can not be detected in MⅡ stage, and the chromosome arrangment was irregular. The results suggest that the expression and distribution of mTOR shows stage specificity during mouse oocyte maturation, rapamycin had an effect on mouse first meiotic division, including chromosome arrangment and spindle formation, demonstrating that a rapamycin-sensitive pathways is involved in this mechinism. mTOR plays an important role during procedure of GVBD and first polar body extrusion, which could regulate maturation process of mouse oocyte through the changes of its expression and localization.

Abstract:Gravity is the most fundamental biomechanical stimulus for posture change. Pressure of blood flow is one of primary indicators to evaluate cardiovascular performance. Up to now, the underlying mechanism of effects of posture change on cardiovascular system is still unclear. A 3D FSI mathematical model with posture change was presented. By applying the body force terms to the fluid equation and the vessel wall equation, the model could be used to study posture change and the effects of gravity on the pressure of blood flow quantitively. Under different inlet-outlet pressure difference(IODP) and different postures such as horizontal, upright and upside-down one, the effects of gravity were simulated. In horizontal position, the pressure distributions of blood flow transformed from 2D(two-dimensional) axis-symmetry without gravity to 3D asymmetry with gravity under small IODP. With IODP increasing, gravity had less effects on pressure distribution and extreme value. As IODP reached 10 665.6 Pa(80 mmHg) and 2 666.4 Pa(20 mmHg) respectively, this effect was observed to be constant. Similar results were obtained from 3D fluid-only model. In either upright or upside-down position, 2D axis-symmetric pressure distribution was observed with and without gravity, yet the position, in which extreme pressure appeared, was different in upright position from that in upside-down one. Finally, the effect intensity of gravity in upright or upside-down position was more than twice as much as that in horizontal one. The results indicate that introducing body force term into the fluid and solid equations to present a novel model, which was based on hemodynamics, will provide a new way to study posture change. Effects of gravity on pressure distribution and extreme value changed with different postures and IODP. If IODP is small, ignoring effect of gravity and postures so as to simplify the hemodynamics model to 2D axis-symmetric one, the conclusion should be drawn with caution.

Abstract:To perform an evaluation of the usefulness of lectin microarray approach for the identification of characteristic glycan profilings related to different aggressive and metastatic potentials between liver cell lines. At first, CHO and its glycosyltransferase-defective mutants Lec1 were chose to validate the feasibility of the lectin microarray system, then the difference of the glycan profiling on cell surface of L02 (normal control), Hep3B (no metastasis) and HCCLM3 (high metastatic potential) cell lines was characterized by lectin microarray. Comparing with L02, Hep3B showed increased affinity for PHA-L, ConA, AAL, MPL and decreased signals for WGA. HCCLM3 presented elevated signals for LCA, MAL-Ⅰ, MAL-Ⅱ, WGA, PHA-E and decreased signals for RCA-I with contrast to Hep3B. The results of fluoresceinated lectin staining of cells with biotinylated LCA, WGA, PHA-E and RCA-I support some observation from array. Lectin microarray was an applicable and useful tool in identifying the glycan profiling changes that accompany the biological processes.

Abstract:In order to investigate the inhibiting effect of RNAi on the proliferation of Bombyx mori nucleopolyhedrovirus (BmNPV), BmN cells were transfected with transgenic vector pigA3-LEF-Neo containing lef-1 dsRNA expression cassette, to produce stable transformation cell line screened with G418(750～800 mg/L). Virus infection test showed that (1) the infection ratio of the stable transformation cells was lower than that of normal cells by 53%, (2) the quantity of polyhedron from transformation cells were 2/3 of that of normal cells and (3) the number of liberation virus from cell culture′s supernatant decreased more than 90%, suggesting that the virus′ proliferation in the transformation cells was depressed significantly. The result underlying the transcriptional level of the lef-1 gene in the transformation cells was only 2/5～3/5 of that in the normal cells suggested that the transcribed lef-1 dsRNA in transformation cells inhibited the expression of the lef-1 gene. Inverse PCR was used to locate the site of exogenous DNA fragment insertion in genome, of which the result showed that in the transformation cells, exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for piggyBac element transposition.

Abstract:Using gene fusion technology, polypeptide fusion tags can be engineered into target proteins at the genetic level. The resultant recombinant proteins may possess the biochemical properties of the imported fusion tags. Therefore, it is possible to take advantage of fusion tags to improve and evaluate protein expression, to detect and track protein targets, and to purify and characterize proteins. However, it is necessary to eliminate any influence of the fusion tag in structural characterization experiments or in isolating pharmaceutical proteins. Scientists must therefore remove fusion tags prior to structural and functional analyses when fusion tags are suspected of interfering with the biological activity of a protein or influencing its behavior. The fusion tag can be removed by several methods including harsh chemical treatment, mild enzymatic cleavage by endoprotease or exoprotease, and intein-mediated self-cleavage. Here the literature is reviewed in relation to principles, applications, and approaches of each method.