Abstract:Programed cell death 4 (Pdcd4) is a newly discovered tumor suppressor gene，which was found to inhibit oncogenesis by suppressing transcription and translation related genes. Expression of Pdcd4 has been found loss or low in several types of human tumors. It is reported that 5’CpG island methylation is the predominant cause of Pdcd4 mRNA silencing in gliomas. MicroRNA-21 regulates Pdcd4 in post-transcription by binding Pdcd4, and the target site is at the Pdcd4 mRNA 3’-UTR region. The mean Pdcd4 protein levels in cells is correlated with the antitumor activity of some drugs, upregulation of Pdcd4 expression may increase cytotoxicity of certain antitumor drugs and downregulation of Pdcd4 expression may reduce cytotoxicity of certain antitumor drugs. Some antitumor drug may affect the expression of Pdcd4. Pdcd4 interferes with the acetylation of p53. Phosphorylation of Pdcd4 by Akt/PKB (protein kinase B) causes nuclear translocation of Pdcd4 and a decreased ability to function as an inhibitor of AP-1( activator protein-1)-mediated transcription. Phosphorylated Pdcd4 by protein kinase S6K1 can be degraded via the ubiquitin pathway by SCF^βTRCP ligase.

Abstract:Monoclonal antibodies(mAbs) have the advantages of high specificity and stable expression, while avian IgY antibodies provide a series of advantages such as less interference reaction with non-specific proteins in immunoassays, and the immune system of birds reacts stronger with IgY production against highly conserved mammalian antigens than the mammalian immune system. Monoclonal IgY(mIgY) is a new antibody development concept to combine the both advantages of IgY and mAbs. It was reviewed the current research situation and improvement of mIgY, mainly focused on the theoretic background, techniques involved, research cases and application prospects of mIgY.

Abstract:Essential hypertension (EH) is a complex disease caused by interaction of genetic and environmental factors. Increasing evidences suggest that hypomethylation of certain genes is involved in pathogenesis of EH. Alteration in methylation status affects expression of genes encoding 11β-hydroxysteroid dehydrogenase-2 (11β-HSD-2), endothelin converting enzyme-1 (ECE-1) and angiotensinⅡ receptor type 1b (AT1b) and hence results in hyperactivation of renin-angiotensin-aldosterone system and reduced renal sodium retention. Genomic hypomethylation can be induced by homocystine (Hcy). Study of metabolizing enzyme and receptor gene methylation regulation and its relation to antihypertensive effects will help understand the pathogenesis of EH and provide evidence for improving clinical treatment of EH.

Abstract:Alzheimer's disease (AD) is a neurodegenerative disease. Recent years, AD has been found closely related to cell apoptosis. It is reported that the synthesis of excessive tumor necrosis factor-α (TNF-α) has been widely considered as a potential inducer of apoptosis contributing to neurodegenerative disease such as AD. However, the molecular mechanism of TNF-α-mediated apoptosis in neuron remains unclear. The signaling pathways involved in TNF-α-induced apoptosis in living differentiated PC12 cells were investigated by using confocal microscope and FRET (fluorescence resonance energy transfer) technique for the first time. Experimental results show that the TNF-α induced apoptosis in differentiated PC12 cells through "extrinsic" or death receptor-initiated pathway, and the "intrinsic" or mitochondrial pathway. NF-κB can inhibit mitochondrial pathway apoptosis through up-regulation of Bcl-x_L by TNF-α induced. Further results show that Bim_L displaces Bcl-x_L in the mitochondria and promotes Bax translocation during TNF-α-induced apoptosis. Furthermore, SP600125 (specific inhibitor of JNK) can inhibit the Bax translocation to mitochondria. Finally, Bax is found to translocate to mitochondria in Naive PC12 cells with co-expressing of GFP-BimL and YFP-Bax. The research demonstrates the important role of Bim_L, and reveals that Bim_L activate Bax indirectly during TNF-α-induced apoptosis.

Abstract:Erythropoietin (EPO) plays an important role in modulating proliferation and differentiation of erythrocytes. The fetal liver stromal cell lines(FLSCs) expressing EPO has been established steadily by lentiviral system. The EPO gene was cloned from human fetal liver by RT-PCR. The EPO recombinant lentiviral plasmid was steadily transfected into FLSCs. The efficiency of virus transfection was identified by expression of enhanced green fluorescence protein(eGFP) analyzed by fluorescence microscope, then the high eGFP espression FLSCs were sorted by fluorescence-activated cell sorting (FACS) according to strong eGFP expression. Analysis of strong eGFP expression was detected by RT-PCR and ELISA. The EPO expression at mRNA level of strong eGFP expression FLSCs are 5.63 and 5.71-fold for the FLSCs no transfected and the FLSCs transfected by the control lentivirus. And at protein level, the content of EPO expression is 263 U/L. Then the supernatant from the EPO transfected FLSCs could induce the CD34+ cell differentiated into hematopoietic cell, especially erythrocytes. This would provide an alternative for cell therapy and blood cell transfusion.

Abstract:Theoretical and computational studies have suggested that the visual cortex processes natural sensory information with characterized pattern that is termed as "sparse coding", which means that each individual neuron rarely fires intensely (lifetime sparseness), and meanwhile, only a small subset of neurons within a large population are activated in response to a given instantaneous stimulus (population sparseness). Temporal and spatial patterns of the chicken retinal ganglion cells′(RGCs) activities in response to time-varying natural images (movies) as well as pseudorandom white-noise checker-board flickering sequence (control) were analyzed. The sparseness indices of the RGCs′ response over lifetime and across population were calculated, the detailed temporal and spatial characteristics underlying such sparseness were also investigated. The results show that the lifetime sparseness and the population sparseness were both more profound for the neuronal responses evoked by natural stimuli as compared to that elicited by checker-board flickering. Further analysis shows that there were more action potentials fired in "burst" form in response to natural stimuli. Coincident bursts of adjacent neurons were prevalent in response to both kinds of stimulation, but occurred more frequently during natural movies stimulation. These results suggest that the RGCs encode natural sensory inputs efficiently. In this scheme, individual neuron fires at a low rate to save metabolic energy, while dynamically grouped small subsets of neurons are activated with adjacent neurons firing concertedly to transmit information to the postsynaptic neurons efficiently.

Abstract:The entry of glucose from blood into the brain through the blood-brain barrier relies on glucose transporters (GLUT). GLUT1 is the major GLUT in the blood-brain barrier and one of the molecular markers of the endothelial cells of brain capillaries. It has been demonstrated that GLUT1 expression is increased after acute cerebral ischemia in animal models. GLUT1 level in brain tissue from 7 cases of chronic ischemic cerebrovascular diseases (ICVD) were determined and compared with that from 11 cases of the age- and postmortem interval- matched controls. It was found that GLUT1 level was decreased markedly in ICVD, and this decrease appeared to result from a down-regulation of hypoxia-inducible factor-1α(HIF-1α). It was also found that the level of protein O-GlcNAcylation was not altered in the ICVD brain. These results shed new light into the molecular mechanism of ICVD.

Abstract:The huge datasets produced from high-throughput microarray technology can elucidate unknown mechanisms of gene regulation in biological systems. Because biological processes are dynamic, it is relevant to focus on certain condition-specific gene regulatory sub-networks. The cell cycle is a basic cellular process, thus, identifying cell cycle specific regulatory sub-networks in yeast will provide a basis for understanding the cell cycle and may be important in other cellular conditions. With a gene expression differential equation model (GEDEM), dynamic cell cycle-related regulatory relationships were indentified from a static regulatory network. Compared to cell cycle-related regulatory interactions previously published, this method identified more true regulatory relationships and show higher performance than other methods. On larger datasets, the GEDEM identified regulatory sub-networks with high sensitivity and specificity. Further analysis on combinatorial regulation revealed that condition-specific regulatory sub-networks exhibited more significant correlations between transcription factors than previously implied in static network analyses, which infer that the condition-specific sub-networks are closer to reality than static network. Additionally, the GEDEM identified more potential co-regulatory transcriptional factors in the cell cycle.

Abstract:To investigate action mechanism of the expression of rVBMDMP gene regulated by hTERT promotor inhibited cell growth of lung cancer, the Core domain of hTERT promotor was obtained by PCR and its function activity was detected. Then, retroviral vector of pLNSX/hTERT/rVBMDMP was recombined, and lung cancer A549 cells were infected by the collected vetrovirus. Further, it was observed that expression of rVBMDMP gene regulated by hTERT promotor influenced cellular morphous, cell growth, cell apoptosis and expression of Caspase-3 protein. Moreover, it was observated that expression of rVBMDMP gene regulated by hTERT promotor inhibited the growth of transplanted tumor in the nude mice, the cellular apoptosis of tumor tissue and the expression of Caspase-3 protein. The results showed: a. The Core domain of hTERT promotor can regulate the expression of rVBMDMP gene (n=3, P < 0.05); b. Expression of rVBMDMP gene regulated by hTERT promotor was abled to inhibit the growth of lung cancer A549 cells and transplanted tumor in nude mice (n=6, P < 0.05); c. The expression of rVBMDMP gene promoted the apoptosis of lung cancer A549 cells and the expression of Caspase-3 protein. These results suggested that expression of rVBMDMP gene regulated by hTERT promotor inhibited the growth of lung cancer A549 cells because it enhanced expression level of Caspase-3 protein to increase cell apoptosis.

Abstract:T7 endonucleaseⅠ(T7EⅠ) is a multiple functional endonuclease from bacteriophage T7, which can act as a reslovase to bind and resolve recombination intermediate Holliday Junction (HJ) specifically, act as a nicking enzyme to nick double-stranded DNA randomly, act as a specific endonuclease to recognize and cleave nicked sites and single-base mismatched sites on duplex DNA. Vector pET21a-MBP-T7EⅠ(E20K) and pET28a-T7EⅠ(E65K) were constructed, and MBP and His6 tagged T7EⅠ mutants MBP-T7EⅠ(E20K) and His6-T7EⅠ(E65K) were expressed and purified respectively. Two T7EⅠ mutants exist as homodimers, two domains of each mutant are inactive. One MBP-T7EⅠ(E20K) subunit and one His6-T7EⅠ(E65K) subunit can form a heterodimer, which have one active domain and one inactive domain (so named T7EⅠ-SAD). T7EⅠ-SAD was obtained by two approaches: The first concerns co-expression of two plasmids in E. coli strains ER2566 and purification of T7EⅠ-　SAD by amylose and Ni-NTA affinity columns. The second concerns co-denaturing and co-refolding of MBP-T7EⅠ(E20K) and His6-T7EⅠ(E65K) in equal molar and isolation of heterodimer T7EⅠ-SAD by double affinity tagging. Using the cruciform structure-containing plasmid pUC(AT) and general closed circular plasmid pUC19 as substrates, the resolving, random nicking and nicked sites cleavage activity of T7EⅠ-SAD were analyzed. T7EⅠ- SAD can recognize cruciform structure, but it loses the capability to introduce two nicked sites simultaneously. In buffer with Mg²⁺, the specific nicking activity of T7EⅠ-SAD to pUC(AT) and random nicking activity to pUC19 are close to that of wide-type enzyme. In buffer with Mn²⁺, the random nicking activity of T7EⅠ-SAD is higher than wide-type T7EⅠ significantly. Results of stepwise denaturing and elution experiment showed that the subunits of T7EⅠ-SAD can disassociate in 5～6 mol/L urea or 1.75～2.0 mol/L guanidine hydrochloride. Gel-shift assay showed T7EⅠ-SAD can binding to four-way junction substrate Junction3 specifically. This study provides an efficient strategy to prepare cytotoxic protein, it provides a sample intuitive approach to probe the stability of protein dimerization too.

Abstract:Nanometer Fe₃O₄ particle was synthesized by coprecipitation, magnetic polymeric microspheres- magnetic chitosan microspheres, containing a magnetic core and a polymer shell, were prepared by the suspension cross-linking technique. Novel affinity magnetic microspheres were obtained after immobilizing heparin onto the nanoparticles. The size, morphology, structure and magnetic response properties were studied by means of SEM, FT-IR and XRD and so on. The novel affinity magnetic microspheres′ properties for thrombin were examined by its adsorption and purification, and compared with the traditional means of column chromatography—DEAE-Sepharose Fast Flow. The results showed the affinity magnetic microspheres had a spherical appearance, narrow distribution of diameter, the particle diameter is about 50 nm, and one-step purification of thrombin using it the specific activity was 1 879.7 U/mg, recovery rate was 85% and purification fold was 11.057. Whereas, purification of thrombin by column chromatography, the product exhibits an activity of 909.84 U/mg, recovery rate of 72% and purification fold of 5.33. In conclusion, chitosan nanometer affinity magnetic microspheres was prepared and magnetic separation techniques was applied for purification of thrombin, the results is better than that from traditional methods and these results have some references values in the production and subsequent study on the research and development.

Abstract:Heat shock response rises when the organisms in the environmental stimulate. During heat shock, a lot of proteins which is so called heat shock protein that do not express in normal state largely express. These proteins′ expression is regulated by a family of transcription factor. These proteins were called heat shock transcriptional factors (HSFs). Among these HSFs, HSF1 is the most important protein. And HSFs are also regulated by other proteins, such as heat shock factor binding protein1 (HSBP1). HSBP1 interact with the trimer formation of HSF1 to convert the HSF1 from its active trimer state to inert monomer state. In order to do the further functional investigation, the gene of HSBP1 was cloned by PCR using the virus cDNAs as templates and expressed in Escherichia coli BL21 (DE3). Furthermore, the expressed HSBP1 protein was purified and crystallized. The distinct crystal form was obtained by the hanging-drop vapor-diffusion process to carry on screening and the optimization to the crystallization condition. The crystals belong to R3 space group with the cell parameter a = b =35.2Å, c= 233.3Å.

Abstract:To investigate the role played by mutations of LDHC gene in male infertility, 100 male patients with unexplained infertility were screened by activity staining of LDH-C₄ on sperm smears and those with lowered or no LDH-C₄ activity in their sperms were subject to DNA analysis. The encoding exons were PCR amplified and the PCR products were screened by DHPLC for mutations. The PCR products with abnormal elution peaks were subject to DNA sequencing. In 14 infertile male patients with lowered LDH-C4 activity in sperms, it was identified a heterozygous T > A mutation in base 115 of exon 5 of LDHC gene(GenBank accession number: GU479375), changing codon 178 (TTG, encoding leucine) to a stop codon (TAG). This nonsense mutation (L178X) was predicted to result in a severely truncated protein. The mutation and the heterozygosity were further confirmed by T-cloning. To the best of the knowledge, this is the first mutation found in human LDHC gene, substantiating that mutations in LDHC gene might cause male infertility.

Abstract:Myostatin is a TGF-β superfamily member that negatively regulates the growth of the skeletal muscle mass. Remarkable muscle increase was observed in myostatin-knockout mice. Injection and electroporation of myostatin-targeting shRNA into rat tibialis anterior resulted in an increase in its weight, fiber size, and MHCⅡ expression. Two siRNAs targeting mouse myostatin were identified to block mouse myostatin expression upon co-transfection with a myostatin-expressing plasmid into HEK293 cell culture. These siRNAs were cloned into shRNA expression vectors and transferred into C2C12 myoblasts. ShRNA-positive cells were screened by neomycin selection and flow cytometry. By using real-time PCR, it was determined that the endogenous myostatin mRNA expression decreased by 10.2% and 35.5% in Mst-shRNA1-treated and Mst-shRNA2-treated C2C12 myoblasts, respectively. Western blot analysis indicated that the myostatin protein expression level decreased by 29.3% and 64.7%, respectively, in the two groups. it was also demonstrated that downstream MyoD pathway was affected by myostatin blockade, as evidenced by the 24.4% and 40.4% upregulation of MyoD expression in shRNA-treated cells. The results indicate that myostatin-targeting siRNA produced endogenously could efficiently downregulate myostatin expression. This RNAi-based method of increasing muscle mass could provide an alternative strategy to gene knockout methods for genetic breeding and may be useful in improving the economic properties of livestock.