Abstract:With the completion of human genome sequencing, the next challenge is to understand the function of each gene. RNA interference (RNAi) library can be used in large scale loss-of-function and phenotype screening. Although RNAi libraries have been proved to be a powerful tool of functional genetic screening in model organisms such as Caenorhabditis elegans, their use in mammalian cells has been hampered by toxic effect induced by long dsRNA. However, since 2003, several RNAi libraries that can be used in mammalian cells have been established and used for functional genetic screening. As a simple, effective, large scale and high-throughput screening technique in functional genomics, RNAi library can be used in functional genetic screening, drug target discovery and validation, disease gene discovery and many other areas.

Abstract:With the development of the technique of nuclear transplantation, nuclear-transfered embryonic stem cell line of cattle, mice and human-rabbit inter-species now have produced. Although experimented therapeutic cloning in animal showed potentially clinical applications in human being, research of human nuclear-transfered embryonic stem cell is confronted with many problems, such as low efficiency of production of nuclear-transfered embryonic stem cell lines, the limited derivation of oocytes, dispute of ethic and politics, therapeutic security and etc. In the long run, the efforts should be focused on the development of cloning efficiency and the solution of ethical and politics concerns with scientific and idealistic progress. Nuclear-transfered embryonic stem cells will meet the high expectations of human for rejuvenation of the aging or diseased body.

Abstract:RNA mainly existed as the single-stranded molecule in organisms is the genetic information carrier, and plays an important role in the transfer of genetic information. In addition, RNA as a nucleic acid enzyme regulates the cellular metabolism. There are three types of RNA molecules that could travel in plants systemically, including the virus RNA, mobile signals of RNA silencing and endogenous RNA. The current advances of systematic transport of RNA and their functions in plant gene expression are reviewed.

Abstract:Conserved in many eukaryotic cells, RNA silencing is a mechanism that suppresses gene expression through RNA-mediated sequence-specific interactions. In plants, RNA silencing plays an important role against virus infection. A counter-defensive strategy in plant viruses has evolved by encoding suppressor proteins to overcome RNA silencing. Up to date, over twenty suppressors encoded by plant, animal and human viruses had been identified. A hot topic in virology has been focused on the identification of suppressors of RNA silencing and the mechanism for inducing silencing. The discovery and identification of viral suppressors of RNA silencing, possible mechanisms involved in RNA silencing suppression and its relationship with symptom formation of viral diseases, and suppressors of animal viruses were reviewed. The applications of these suppressors in plant biological and biotechnological research were also discussed.

Abstract:Smu.260 encodes a putative protein of 200 residues in Streptococcus mutans, a primary pathogen for human dental caries. Smu.260 was cloned into expression vector pET28a and expressed in good amount from the E.coli strain BL21 (DE3). Smu.260 protein was purified to homogeneity in a two-step procedure of Ni²⁺ chelating and size exclusion chromatography. The purified protein exists in two forms, a dimer form about 46 ku with yellow color and a tetramer form without apparent color. Crystals were obtained from the dimer protein by hanging-drop vapor-diffusion method. The crystals diffracted to about 2.3 Amstrong resolution and belong to orthorhombic space group P2₁2₁2₁ with cell dimensions of a=89.88 Amstrong, b=90.91 Amstrong, c=105.17 Amstrong. The asymmetric unit is expected to contain two dimers with solvent content of 53%.

Abstract:In order to establish an animal model of atherosclerosis in minipigs and investigate the change of ATP binding cassette transporter A1(ABCA1) expression in atherosclerotic minipigs, Chinese minipigs were fed a normal control diet (CD) or a high fat/high cholesterol diet (HFHC) and carotid overstretch ballon injury for 12 months. Plasma total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG) were determined by commercially enzymatic methods. ABCA1 mRNA and protein level were determined by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot and immunohistochemistry, respectively. At the end of 12 months, plasma total cholesterol, HDL cholesterol and triglyceride in atherosclerotic minipigs were increased. It is obvious that atherosclerotic plaques and lipid stripes in aonta, iliaca anteria and coronary artery of atherosclerotic minipigs ATP binding cassette transporter A1 expression in liver, aorta and small intestine of atherosclerotic minipigs was upregulated. HFHC may induce atherosclerosis and upregulation of ATP binding cassette transporter A1 expression in atherosclerotic minipigs.

Abstract:Membrane fusion between the virus envelope and host cells is the first step of the enveloped virus entry into the host cells. This process involves the interaction of viral envelope proteins and their cellular receptors (proteins or sialic aids), which leads to the conformational changes of the envelope proteins. Avian paramyxovirus-2 (APMV-2) has the hemagglutinin- neuraminidase (HN) glycoprotein in which there are the stalk and globular head regions, and the fusion (F) glycoprotein in which there are the heptad repeat 1 (HR1) and heptad repeat 2 (HR2) and heptad repeat 3 (HR3) regions. To construct and express the correlative genes with membrane fusion of APMV-2 ， the relative sequences basing on the published sequences of avian paramyxovirus-1 (APMV-1) and using BLAST bio-software were ensured, then constructed genes by PCR and cloned genes into the BamHⅠ-XhoⅠ restriction sites of the GST fusion expression vector pGEX-6P-I, in which there is a rhinovirus 3C protease cleavage site for the fusion protein. E. coli strain BL21 (DE3) was transformed with the recombinant GST fusion plasmids. The supernatants lysed by sonication and clarified by centrifugation were passed over Glutathione-Sepharose 4B column for purifying, respectively. The GST fusion proteins were then cleaved by GST-fusion rhinovirus 3C protease and then were purified by the affinity chromatography. The LearnCoil-VMF and ExPASy bio-softwares were used for predict and analysis the structure and function of five peptides. And the results of Gel-filtration and circular dichroism (CD) showed that the HR1 and HR2 form a six-helix structure.

Abstract:Hypertension is a major problem worldwide. There is much evidence to suggest that reactive oxygen species (ROS) radical may play a role in the development of organ damage associated with cardiovascular disease and hypertension. ( － )Epicatechin, a member of tea catechins belonging to flavonoid group, is known to be a potent anti-oxidant. The study has been undertaken to evaluate the effect of ( － )epicatechin on markers of oxidative stress: reduced glutathione (GSH) and membrane sulfhydryl ( — SH) groups in erythrocytes from hypertensive patients. The effect of ( － )epicatechin was also compared with a known anti-oxidant L-ascorbic acid. The erythrocyte intracellular GSH content and membrane — SH group content were significantly (P<0.01) decreased in hypertensive subjects. In vitro incubation with ( － )epicatechin caused an increase in GSH and — SH content, the effect was more pronounced in hypertensive erythrocytes. Similar results were obtained with L-ascorbic acid. The observed decrease in the level of GSH and — SH groups in hypertension is an indicator of oxidative stress condition. Observation of an increase in red cell GSH content and the protection of membrane — SH group oxidation by ( － )epicatechin in hypertensive subjects is a convincing reason to suggest that high dietary intake of foods rich in catechins may help to reduce oxidative stress and concomitant free radical damage in hypertensive patients.

Abstract:Whether protein could adopt multiple conformations coexisting in solution is disputable. In a previous report, the conformation heterogeneity of apoazurin mutant M121L had been identified. The thermal unfolding of wild type apoazurin from Pseudomonas aeruginosa is re-investigated with differential scanning calorimetry (DSC) and circular dichroism (CD) methods. The results show that unfolding in the pH range from 4 to 9 is associated with two heat capacity maxima. The low temperature transitions are reversible at all pH conditions used, while the high temperature transitions are irreversible. The two unfolding transitions were analyzed by the two-interchangeable-conformation model with the fraction for the first transition (N1) from 64% at pH 4.0 to 55% at pH 9.0. Temperature induced unfolding monitored at 219 nm shows also two separate transitions. The ratio of the signal changes is consistent with the fractions obtained from the corresponding DSC measurements. These results provide further support for the hypothesis that at least two conformations of apoazurin coexists in solution.

Abstract:In order to screen and characterize aptamers against hepatitis C virus(HCV) NS3 helicase, an 81bp single stranded DNA (ssDNA) random library was subjected to 8 rounds of selection against HCV NS3 helicase by SELEX method. The selected aptamers were cloned and sequenced.The primary sequences of the aptamers were analyzed by Clustal W, and the affinities of aptamers to HCV NS3 helicase were determined. After 8 rounds selection, the percentage of the ssDNA pool bound to HCV NS3 helicase from 0.45% inceased to 29.5%. The primary sequences of the aptamers were divided into 5 families with 4 conserved sequences. The affinity of aptamer H2 to HCV NS3 helicase was the highest, with K_d values as low as 140 nmol/L. Aptamer H2 (10 μmol/L) inhibited approximately 44% of the helicase activity of HCV NS3 in vitro. The results suggest that aptamer against HCV NS3 helicase have been identified by means of SELEX methods from an 81bp single stranded DNA random library.

Abstract:The relationship between the concentration of intracellular ATP and the glycolytic flux in Torulopsis glabrata was studied by adding oxidative phosphorylation inhibitors (rotenone, antimycin A and oligomycin). When 10 mg/L rotenone and antimycin A were added to the cell cultures, the concentrations of intracellular ATP were approximately 43% and 27.7% less than that of the control, respectively. The specific activity of phosphofructokinase, one of the rate limiting enzymes of the glycolytic pathway, increased by a factors of 3.4 and 2.3, in comparison of the control respectively. With the specific activity of phosphofructokinase increased, the rate of glucose consumed increased by a factor of 3.6 and 2.4 compared with the control, and the rate of pyruvate produced increased by 17% and 8.5% respectively. The specific activities of hexokinase and pyruvate kinase were not affected by the addition of rotenone or antimycin A. Furthermore, the concentration of intracellular ATP decreased by 64.3% upon addition of 0.05 mg/L oligomycin to the cell culture, and the growth of Torulopsis glabrata was ceased when 0.4 mg/L oligomycin was added to the culture broth at 24 h. Both the rate of glucose consumed and the rate of pyruvate produced were enhanced with increasing the concentration of oligomycin ( ＜ 0.6 mg/L) in the cell cultures. As a result, the activity of phosphofructokinase (r²=0.9971), the rate of glucose consumed (r²=0.9967) and the rate of pyruvate produced (r²=0.965) were enhanced by a decrease in the energy level of the cell (the concentration of intracellular ATP). Increase of the rate of glucose consumed rooted in elevation of the specific activity of phosphofructokinase (r²=0.9958) and pyruvate kinase (r²=0.8706). These results are the first answer to the fundamental question of what controls the flux through glycolysis in Torulopsis glabrata.

Abstract:To investigate whether there exits the dose- and time- dependent effect of RNA interference(RNAi) when the introduced extraneous reporter gene was suppressed by RNAi in mammalian cell lines.The expression vectors carrying reporter component were cotransfected with the plasmids coding short hairpin RNAs(shRNAs) into HEK293H cell using lipofectamine 2000 reagent, and the consequent inhibitory effect mediated by RNA interference was observed. After transfection, the transient expression of shRNAs could specifically inhibit the extraneous reporter in mammalian cell.The expression of mRNA and protein of enhanced green fluorescent protein(EGFP)gene was determined at 12, 24, 48, 60, 72, 96 h after transfection in HEK293 cell. The results showed that the decrease of EGFP mRNA or protein level was not obvious at 12 h, but gradually became more evident during from 24 to 48 h.The decrease achieved the maximal degree during from 48 to 72 h, then became weakened and restored subsequently. It indicated that the effect of RNA interference underwent a tendency of from weak to strong, then from strong to weak, and ultimately disappeared gradually. The efficiency of inhibition caused by RNAi was related to the dose of RNA interfering vector within a confined limit in HEK293H cell cotransfected with a series of dose-proportional vectors as pd1EGFP and psh-d1EGFP, whereas it nearly kept constant when the dose of interfering plasmid was sufficient to depress the expression of extraneous genes. Simultaneously, the variation of luciferase activity also displayed the similar effect when its expression was suppressed by RNAi in HEK293H or HeLa cell.. It concluded that vector-based RNA interference took on time- and dose- dependent effect in mammalian cell, which provides certain theoretical reference or valuable clue to the utility of RNAi.