Abstract:During the translation process, aminoacyl tRNA enters the ribosome, decoding the codon on the mRNA and brings the mRNA moving forward towards the 5′ direction of mRNA, until the de-acylated tRNA leaves the ribosome, it moves through the ribosome in one direction. Recently, with the finding and identification of the highly conserved protein LepA, a new kind of tRNA movement inside the ribosome, namely the back-transloation of the tRNAs and mRNA in the direction of mRNA 3′ is discovered. With the in-depth research, the physiological meaning behind the back-translocation for the translational efficiency and fidelity has been studied.

Abstract:Functions of the 3′untranslated regions (3′UTR) of eukaryotic mRNAs are complicated. They can control the stability and intracellular localization of mRNAs, and direct the translation of special amino acids. Mutations in the 3′UTR of some mRNA can cause serious diseases, and recent studies showed that the 3′UTR of some eukaryotic mRNAs possess tumor-suppression function.

Abstract:The ubiquitously expressed family of Id(inhibitor of differentiation) helix-loop-helix(HLH) proteins function as dominant negative regulators of basic HLH (bHLH) transcriptional regulators. In eukaryotic organisms, Id proteins have been implicated in development, cell differentiation, proliferation, angiogenesis, invasion and migration. Recent studies revealed that Id proteins are not only significantly correlated with cancer progression and prognosis, but also exploited as a tumor therapeutic target. The roles of Id proteins in tumorigenesis were reviewed and the opportunities of Id proteins in cancer targeted therapy were discussed.

Abstract:The toxin-antitoxin (TA) system consists of a pair of co-expressed genes. The upstream gene encodes an unstable antitoxin protein, and the downstream gene encodes a stable toxin. The toxin-antitoxin system was originally found on the low-copy plasmid to maintenance the plasmid stability. Recently the TA loci have been widely identified on the chromosomes of bacteria, including the some pathogens. The TA systems play an important role in the bacterial growth control or the bacterial programmed cell death under starvation and other stress conditions. The toxin in different TA system has different cellular targets. CcdB toxin in the ccdAB system interacts with the catalytic GyrA subunit of gyrase to inhibit the DNA replication. RelE toxin in the relBE system assists the RNA cleavage at the ribosome A site with a high coden specificity. PemK toxin in the pemIK system and MazF toxin in the mazEF system are identified as endoribonucleases, which cleave the cellular mRNA in a sequence-specific manner to interfere with mRNA function and inhibit the protein synthesis. This review summarizes the mechanism of the toxin in TA system and evaluates the applications of TA system in the future.

Abstract:With the rapid developing of nanotechnology, the use of nanomaterials in medical imaging, disease diagnoses, drug delivery, cancer treatment, gene therapy, and basic research have also progressed quickly. The beneficial uses of nanomaterials may cause human exposure through inhalation, ingestion, skin uptake, and intravenous injection. When nanomaterials interact with biological systems, they may generate adverse biological effects. The possible toxic effects of these nanomaterials are unknown until now. The positive applications in biomedicine as well as the negative biological effects of nanomaterials on cardiovascular, respiratory and other systems are highlighted, the possible mechanisms by which nanoparticles result in cardiovascular and pulmonary morbidity are also discussed, and the possible interaction routes and sequences is farther reviewed. Finally, the perspective of risk assessment of nanomaterials in the future is summarized.

Abstract:15 SARS-CoV N Protein Interacting Protein (NPIP) were selected from host cells using Yeast Two-hybrid system (Y2H). These are Angiogenin, acyglycerol kinase, cytochrome oxydase subunit I, CXC chemokine ligand 16, epidermal growth factor receptor pathway substrate 15, glutathione S-transferase kappa 1,integrin beta 1, jun oncogene, NIMA (never in mitosis gene a)-related kinase 10, protein tyrosine kinase 2 beta,homo sapiens SH3KBP1 binding protein 1 and ubiquitin specific peptidase 53. With the aid of immunological co-precipitation (CO-IP), it was confirmed that chemokine CXCL16 was the interactor with SARS-CoV N protein in host cells.

Abstract:To investigate the mRNA expression profile of novel candidate of tumor suppressor gene NGX6 in the several common types of cancer, analyze the correlation between NGX6 mRNA expression and its clinicopathological and to evaluate the validity that NGX6 mRNA act as molecular marker for tumor metastasis and prognosis. Multi-tumor tissue and nasopharyngeal cancer tissue microarrays were constructed previously and tissue microarrays combined with in situ hybridization were used to detect the expression of NGX6 mRNA in the human several common types of cancer. Results showed that expression of NGX6 mRNA in the nasopharyngeal cancer (NPC), lung cancer, gastric cancer and colon-rectal cancer was significantly lower than that in their non-cancer normal tissue (P < 0.05, P < 0.01). Expression level of NGX6 mRNA was evidently lower in the NPC, laryngeal cancer, lung caner and colon-rectal cancer with lymph node metastasis (P < 0.05, P < 0.01). Expression of NGX6 mRNA in the NPC, lung caner and colon-rectal cancer had significant correlation with their clinical stages, which NGX6 mRNA was significantly lower in the clinical stage T2, T3 or T4 that than their clinical stage T1(P < 0.05, P < 0.01). The results suggested that the NPC, lung caner, gastric cancer and colon-rectal cancer had evidently lower expression of NGX6 mRNA, NGX6 mRNA might be used as molecular markers for invasion, metastasis and prognosis of NPC, lung caner and colon-rectal cancer.

Abstract:Besides differentiating into multiple mesenchymal tissues, bone marrow mesenchymal stem cells(BMMSCs) can also develop into hepatocyte-like cells in vitro and in vivo. CTLA4Ig-gene modified BMMSCs have a strong immunosuppressive function cultured in both normal and hepatic differentiation medium and this gene modification improves the biological function of BMMSCs. Recombinant adenovirus containing CTLA4Ig gene was constructed, and transferred into rat BMMSCs in vitro. The transfected BMMSCs, cultured in hepatic differentiation medium containing HGF for 14 days, could express hepatocyte-specific markers including AFP, Alb and CK18, and these differentiated cells from BMMSCs also had the capabilities of glycogen deposition and ICG uptake and excretion, which are hepatocyte-specific functions. Expression of CTLA4Ig in the transfected BMMSCs cultured in the normal growth medium or the differentiation medium was confirmed by RT-PCR, Western blot and fluorescent immunocytochemistry, and the expression at 14 d was weaker than that at 7 d. In the model of MLR, the transfected BMMSCs could suppress immune response, and the suppression was statistically stronger than that of BMMSCs at the same number. Even if cultured in hepatic differentiation medium, the transfected BMMSCs also had the same immunosuppression function. In rat liver transplantation model, the CTLA4Ig-gene modified BMMSCs infused into liver graft after operation could prolong the survival time of the recipients. Adenovirus-mediated CTLA4Ig gene transfer into BMMSCs can not change the potential of BMMSCs to differentiate into hepatocytes, and on the other hand, this gene transfer can improve the immunosuppressive function of BMMSCs, which can broaden the applying space of BMMSCs in the clinical therapy for liver diseases.

Abstract:Flavones and flavonols belong to flavonoids that have anti-cancer activities. In order to explore molecular mechanism and inhibitory effects of flavones and flavonols on human esophageal carcinoma cells, the inhibition of proliferation and the induction of G2/M cell cycle arrest in KYSE-510 cells and OE33 cells treated with three flavones (luteolin, apigenin, chrysin) and three flavonols (quercetin, kaempferol, myricetin) were analyzed by MTT array and flow cytometry. Among these compounds, luteolin and quercetin were the most active flavonoid to inhibit the proliferation of KYSE-510 cells and OE33 cells, respectively. The genes related to cell cycle control were analyzed by gene chip, after KYSE-510 cells and OE33 cells were treated by luteolin and quercetin, respectively. The results were shown that the expression of p21^waf1 was induced and the expression of cyclin B1 was suppressed in KYSE-510 cells, and that the expression of GADD45β and 14-3-3σ were induced and the expression of cyclin B1 was suppressed in OE33 cells. These results were verified by real-time RT-PCR and Western-blot. The comparative effects of all six compounds on the regulation of these gene expressions at the mRNA and protein levels were also analyzed by real-time RT-PCR and Western-blot. The results were shown that p21^waf1, GADD45β, 14-3-3σ and cyclin B1 were the target genes which mediated the effects of flavones and flavonols on induction of cell cycle arrest in KYSE-510 cells and OE33 cells.

Abstract:Children Salter's typeⅢ and Ⅳ growth plate injuries always induce the skeletal deformity because of bony bridge formation. However, the underlying cellular and molecular changes of the remaining cartilage adjacent to the injury site are still unclear. The purpose of this investigation was to understand the molecular mechanisms of bony bridge formation. The in vivo cellular and molecular changes in the adjacent cartilage were studied in the rat growth plate injury models. Consisted with the histological changes, both Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay and in situ hybridization experiment using Col2a1 probe showed there was the sub-injury cartilage region adjacent to the original injury site. Despite the sub-injury region remained normal cartilage structure and Collagen typeⅩ in the extracellular matrix, the chondrocytes within this region showed the dislocation with the cartilage lacunas, and the strand breaks of cleaved DNA in TUNEL assay. That these chondrocytes didn't express Col2a1 mRNA further confirmed they were dead cells. Along with the degradation of sub-injury cartilage, some fibroblast-like cells presented to the cartilaginous region between the sub-injury region and uninjured cartilage. In situ hybridization experiment for Patched 1 (Ptch1), indicator of Indian Hedgehog (IHH) signaling, indicated these fibroblast-like cells could respond to Hh signaling. These results suggest that the bony bridge formation involves series of changes of chondrocytes and Ihh signaling may be involved in the formation of the transient perichondrium-like structure between the sub-injury cartilage and normal cartilage, and partially contribute to the bony bridge formation. Investigating the underlying cellular and molecular changes after the transphyseal injury will contribute us to explore a prevention treatment in the future clinic.

Abstract:The effects of glutamate transporters on synaptic plasticity in rat models of pilocarpine-induced status epilepticus were investigated. Male Wista rats ((304.06±13.79) g) were randomly divided into 5 groups, short-term seizures (SE) and its control (SC), long-term seizures (LE) and its control(LC), normal control (Sham) groups. Epilepsy rat models were induced by injection of pilocarpine(25 mg/kg, i.d.). Glutamate transporter inhibitor, DL-threo-benzyloxyaspartate (TBOA, 7.5 nmol，1 μl) was microinjected into right side of hippocampus after 14 days of initial status epilepticus in SE and LE groups. The same volumes of artificial cerebrospinal fluid were injected into same side of hippocampus in SC and LC groups. Electroencephalographys (EEG) were detected in SE and SC groups after 2 h of drug injection. Long term potential (LTP) at perforant pathway and dentate gyrus(PP-DG) and EEG were recorded in LE and LC groups after two weeks of drug injection. Example of Fluoro-Jade-B staining in the rat brain was made at the end of electrophysiological experiment. The results showed that there was a significant decrease in theta band power of EEG in SE group compared with that of SC group (P < 0.05). There was no significant difference in theta band power of EEG between LE and LC groups(P > 0.05). The slope of excitatory postsynaptic potential (EPSP) was significantly increased in LE group compared with that of LC group (P < 0.01). Fluoro-Jade-B showed more neuronal degeneration in LE group compared with that of LC group. The results suggested that TBOA induced damage of glutamate transporters and enhanced the neurotoxicity of status epilepticus, which contributed the synaptic plasticity in status epilepticus rats.

Abstract:Epitope prediction of a new gene wx2 in Toxoplasma gondii was analyzed by bioinformatics. The epitope encoding fragments W2b and W2a were amplified from new gene by PCR, respectively. The single-epitope vaccines pcDNA3-W2b, pcDNA3-W2a and double-epitope vaccine pcDNA3-W2b2a were successfully constructed. Mice in group pcDNA3-W2b, pcDNA3-W2a, pcDNA3-W2b2a and two control groups, pcDNA3 and NS, were injected intramuscularly with pcDNA3-W2b、pcDNA3-W2a and pcDNA3-W2b2a epitope vaccines, respectively. As controls, mice were inoculated with NS or empty plasmid pcDNA3. The induced immune responses were tested by ELISA detecting IgG, and flow cytometry sorting the subsets of T lymphocyte. All mice were challenged with highly virulent RH tachyzoites to observe the survival time. Results display that the level of IgG in sera of mice inoculated with pcDNA3-W2b2a was significantly higher than those in pcDNA3 and NS control group, CD4⁺T/CD8⁺ T cell proportion of immunized mice was significantly lower than those in control group. After challenged with highly virulent tachyzoites, the mean survival time of immunized mice in pcDNA3-W2b2a group was significantly longer than groups pcDNA3-W2b, pcDNA3-W2a and control group, indicating that the epitope vaccine from new gene wx2 can induce protective immunity in mice．and the protective efficacy of pcDNA3-W2b2a double epitope vaccine elicits better immunoprotectivity than pcDNA3-W2b,pcDNA3-W2a single-epitope vaccine.

Abstract:Genome of Arabidopsis has a kind of genes encoding proteins with ARM repeat domains and some of these proteins are known to play important roles in plant development and responses to hormone. An Arabidopsis mutant lfr with a distinct phenotype was got in leaf and flower development. The gene is predicted to encode a protein with ARM repeat domains. In order to study its function and molecular mechanism, recombination expression plasmid pGEX-2TGST∶LFR was constructed and transformed into the host bacteria strain Rosetta. Then IPTG was used to induce the recombinant protein expression in engineering strain. The expression products were detected by 12% SDS-PAGE. The GST∶LFR fusion protein was existed in soluble form with a relative molecular mass 77 ku, which is fit with the molecular mass supposed from gene coding frame. After purification by GST-tag affinity chromatography and electroelution, the fusion protein was used as antigen to prepare polyclonal antiserum in rabbits. After the fifth injection of antigen, the antiserum was obtained and further purified by decreased nonspecific bacteria and GST-tag antibody with method of immuno-precipitation. Western blot analysis showed that the purified antiserum, raised against the recombination LFR protein in rabbits, could react to the recombinant protein expressed in Rosetta specifically. And then the nuclear proteins of Arabidopsis wild type and mutant were extracted and separated by SDS-PAGE. Western blot assays revealed that there was a protein band, with a relative molecular mass 50 ku, indicating that antiserum could react to the native protein expressed in Arabidopsis specifically.

Abstract:Higher plant photosystem Ⅱ (PS Ⅱ) generates superoxide anion radicals (O₂^·) under strong illumination, in which the O₂^· plays a pivotal role in reactive oxygen species (ROS) metabolism during higher plant photosynthesis. The photosystem of Chlamydomonas reinhardtii is highly similar to that of higher plants. The generation of O₂^· in C. reinhardtii thylakoid membranes and PS Ⅱ particles has been firstly proved by means of spin trapping-ESR technique and NBT-mediated spectral assay. Referring to the spin trapping-ESR evidences in spinach, the mechanisms of O₂^· generation both in C. reinhardtii and spinach look identical. Compared to higher plant, C. reinhardtii has several irreplaceable advantages: simpler structure; clearer genetic background and genomic sequencing has been finished; easy to introduce mutations in photosystem. As a result, the study concerning the O₂^· generation in C. reinhardtii introduces a new promising model for further investigation of ROS metabolism in higher plant photosynthesis.

Abstract:The application of the protein design method based on the HNP model and the relative entropy theory is discussed for four structural classes of real proteins, and the results are compared with that of the HP model. Testing on 190 proteins shows that this method is generally effective for the different structural classes of proteins. Further studies show that the success rate of this method on regular secondary structures is higher than that on the random coil. Additionally, the success rate for different types of amino acids is also analyzed. It is found that the success rate on the hydrophilic residues is higher than those of the other two types. Furthermore, the success rate of this method on the conserved residues is higher than the non-conserved residues. The reasons resulting in the difference of the success rate on different systems were also analyzed. All analyses mentioned above make the foundation for the development and the application of this method in the future.

Abstract:Previous study indicated that the expression of a LRR receptor-like protein kinase OsRLK in root tips of rice could be induced by salt stress. In order to study the functions of OsRLK, the extracellular fragment of OsRLK gene was obtained through RT-PCR. The target fragment was subcloned into pET29a, and the recombinant plasmid pET29a-RLK was transformed into E. coli BL21(DE3). The target fragment over-expressed in host strain, and the expression level was about 30% of the total cellular protein. After separated by SDS-PAGE, the target band was excised from the gel, and was used as an antigen to raise the antibody in New Zealand rabbits. After separation and purification of antiserum, the anti-OsRLK polyclonal antibody with the titers of 1∶20 000 was successfully prepared. Western blot analysis showed that the antibody could specifically recognize the expressed fragment in E. coli and the OsRLK protein in root tips from rice. In addition, for the first time, the OsRLK was confirmed as a salt stress responsive protein.

Abstract:Histone acetyltransferases (HATs) are involved in the regulation of gene transcription in eukaryotic cells and their inhibitors could be a promising class of drugs due to their ability to modulate transcription and exert antiviral, anti-inflammatory as well as antioxidant effects. Nonradioactive spectrophotometric HAT assay is an alternative method to the widespread radioactive assay but suffers from drawbacks as lack of sensitivity and accuracy. A simple, non-radioactive fluorescent assay that measures the production of CoASH was established by its facile reaction with O-phthalaldehyde and 2-amino-ethanol. This method gives much higher accuracy compared to spectrophotometric assay, and allows screening of various compounds with potential HAT inhibition. The novel assay should be a valuable tool in transcriptional research and especially drug discovery.