Abstract:Transient outward potassium current (I_A), highly expressing in hippocampal dendrites, has characteristics of rapid activation and inactivation following appropriate voltage depolarization. I_A is the predominant outward potassium current at the subthreshold range of the action potential and during early phase of repolarization. IA current plays an important role in modulating synaptic input and affecting action potential back-propagation by reducing the speed of depolarization and delaying the action-potential initiation, suggesting its important roles in regulating the electric signal integration and synaptic plasticity. I_A currents have been associated with many diseases, such as seizure.

Abstract:RNA interference mediated by short interfering RNA is widely used to study functional genes and also being developed for therapeutic applications. However, recent study demonstrated that siRNA might activate innate immune system and induce huge production of inflammatory cytokines in mammals, and also randomly inhibit expression of undesired genes. Designing highly effective siRNAs or modifying the siRNA to retain or enhance the silence efficiency and meanwhile abolish the off-target effects associated with immunostimulation then become the key techniques in application of siRNAs as safe and effective therapeutic agents.

Abstract:The T help cell 1(Th1) and Th2 cell classification have provided the framework for understanding CD4⁺ T cell biology and the interplay between innate and adaptive immunity for almost two decades. Recent studies have defined a previously unknown subset of the CD4⁺ T cell effectors, the Th17 lineage. The uncover on the differentiation of IL-17-producing effector T cells from naive T cell precursors provides insights into mechanisms by which signals from cells of the innate immune system guide alternative pathways of Th1, Th2 or Th17 development. Th17 lineage has an important role in autoimmune and inflammatory diseases. This promises to change our understanding on the immune regulation, immune pathogenesis and host defense. The identification, differentiation and immune function of the new subsets of Th cells, Th17 cells will be reviewed.

Abstract:L1 cell adhesion molecular is a type Ⅰ membrane glycoprotein of immunoglobulin superfamily. L1 is expressed mainly in nervous system and plays important roles in nervous system development, learning and memory. L1 cytoplasmic domain (L1CD) is important for L1 function by mediating signaling. To investigate the molecular mechanisms of signaling mediated by L1CD, yeast two-hybrid assay was carried out to screen the human brain cDNA library using L1CD as the bait. After sequence analysis and BLAST, several candidates were identified. One candidate is PAX6 transcription factor. L1CD and PAX6 cDNA were cloned in expression vectors and cotransfected into COS-7 cells. The interaction between L1CD and PAX6 was verified by co-immunoprecipitation assay. These preliminary results suggest that L1CD may be involved in transcription regulation.

Abstract:STGC3, a novel tumor related gene, was cloned recently. The previous studies indicated that STGC3 can inhibit the proliferation of CNE2 cell line in vitro. To examine the effect of STGC3 on the tumorigenicity of CNE2 cell line and explore its mechanism in nude mice. The Tet/pTRE/CNE2-STGC3 cell line was planted under the front leg skin of nude mice and induced by doxycycline (Dox). The mRNA and protein level of STGC3 in transplanted tumor tissues were detected with RT-PCR and Western Blotting. The apoptosis ratio of the tumor cell was analyzed with flow cytometry. STGC3, Bcl-2 and Bax proteins were examined by immunohistochemistry method. The results indicated that high level of STGC3 expression can inhibit tumorigenicity of CNE2 cell line in nude mice. Tumor grew slowly, later and smaller. Cell apoptotic percentage increased. Bcl-2 protein expression was down-regulated and Bax protein expression was up-regulated in Tet/pTRE/CNE2-STGC3 cell line (P < 0.01). The data indicated that STGC3 had a role as a tumor suppressor gene in vivo, which was in line with the results in vitro.

Abstract:LPLUNC1 is a newly cloned gene and was found down-expressed in 71% NPC biopsies but high-expressed in normal adult and fetal nasopharyngeal epithelial tissue, which indicates that it may play an important role in the tumorigenesis of nasopharyngeal carcinoma. The object of the research is to study the effect of LPLUNC1 on nasopharyngeal carcinoma cells. The full length of LPLUNC1 cDNA was cloned into the pcDNA3.1(+) vector, then the pcDNA3.1(+)-LPLUNC1 plasmid was stably transfected into the NPC cell line HNE1 cells using lipofectamine. After being selected with G418 and detected with RT-PCR and Northern-blot, the stable transfectants of LPLUNC1 over-expression were constructed. Cell growth curve, MTT, BrdU labeling, flow cytometry, soft agar and nude mice were performed to study the effects of LPLUNC1 over-expression on HNE1 cells. The results showed that HNE1 cells with over-expression of LPLUNC1 grew slower significantly than that of the control. MTT and BrdU labeling assay showed that LPLUNC1 inhibited the HNE1 cell proliferation. And LPLUNC1 gene can delay G1-S phase cell cycle progression of HNE1 cells. Furthermore, LPLUNC1 was found that it decreased the HNE1 clonogenicity in soft agar and inhibited the tumor formation in nude mice obviously. In conclusion, LPLUNC1 can suppress the malignant phenotype of HNE1 and plays an important role in inhibiting the tumorigenesis and development of NPC.

Abstract:The MUC1 protein is expressed as a stable heterodimer from a single polypeptide, which was cleaved into two subunits in endoplasmic reticulum. It localizes at the cell membrane as an α/β-complex, tethered by the β-subunit transmembrane domain. Previous studies implicated that the three amino acids of the transmembrane domain adjacent to the cytoplasmic domain in MUC1 β-subunit are the residues Cys-Gln-Cys (CQC). Therein, site-directed mutagenesis of the CQC motif was performed and the cell lines were established. These cell lines include HCT116/MUC1, HCT116/MUC1(CQC→AQA), HCT116/MUC1(△CQCRRK), HCT116/MUC1C-ter(CQC→AQA), which can express wild type or mutant MUC1 on the cell surface，or its cytoplasmic domain. The effects of CQC→AQA mutation or CQCRRK deletion were investigated in vitro and in vivo. Compared with wild type MUC1, the mutants depressed soft agar colony formation and showed abrogated tumorigenicity in nude mice. These findings implicate that CQCRRK motif mediate the formation of MUC1 protein complex. As a result of this research, disruption of MUC1-C-terminal subunit-associated dimerization by mutation of CQC→AQA might represent a novel therapeutic approach for tumor.

Abstract:Tissue factor pathway inhibitor (TFPI) plays a vitally important role in the blood coagulation system. Recent studies showed that TFPI could also inhibit the proliferation of vascular smooth muscle cells. Studies has suggested that TFPI may find wide applications for the therapy of various diseases such as sepsis, DIC, vascular stenosis, atherosclersis, and miocardial infarction, etc. However, the rapid clearance of TFPI from the circulation remains to be one of major drawbacks to its clinical application. The aim of the present study is to prolong the half-life of recombinant TFPI while maintaining its biological activities by reducing the interaction between TFPI and cell surface, which is mediated by the negative charges on the membrane of the cells and the positive charge on the molecule of TFPI. For this purpose, three rTFPI mutants, m₀TFPI, m₁TFPI, and m₂TFPI, were generated by introducing mutations in the DNA sequences coding for the C-terminal amino acid sequences. The clearance rate of the experimental group (three rTFPI mutants) and the control group (wild-type TFPI, namely mTFPI) in rats was investigated. The inhibitory function on FXa and the heparion binding capacity of the TFPI mutants were also studied. It was shown that at 10 min after intravenous injection, (59.06 ± 13.54) % of the injected mTFPI remained in the circulation, while (71.14 ± 13.04) % of the m₀TFPI, (77.99 ± 2.53) % of the m₁TFPI, and (82.95 ±11.36) % of the m₂TFPI were still in the circulation. The relative half-life of m₀TFPI, m₁TFPI and m₂TFPI were prolonged 0.5, 0.9, and more than 1 times respectively in comparison with that of mTFPI, while their FXa inhibiting activity and heparin-binding capacity were little changed.

Abstract:The microRNAs (miRNAs) are a kind of single strand small noncoding RNAs(approximately 22nt) with probable roles in the silencing gene expressions. To date, a large number of miRNAs have been identified in several organisms, but the function of the vast majority of these molecules remains to be determined. To study their functions, a library of vectors expressing miRNAs, including more than 170 kinds of human miRNAs were developed. The ability of some miRNA vectors to express miRNAs was validated with Northern blot and dual luciferase assays. The results indicated that these vectors could express pre-miRNAs and mature miRNAs in HEK-293 cells. Furthermore, they could suppress the expression of luciferase tagged with a sequence complementary to corresponding miRNAs. All these data show that these miRNA expression vectors can drive functional miRNA expression, and they can be used to screen functional miRNAs.

Abstract:hhLIM, a member of LIM protein family, has two LIM domains and plays an important role in gene regulation, cytoskeleton organization and cell hypertrophy. To understand the functional importance of hhLIM in cytoskeleton organization and muscle hypertrophy, hhLIM and its mutants of two LIM domains were constructed and function of each LIM domain and its interaction with actin were studied. GST pull down assay and immunofluorescence assays showed that LIM domain 2 at the C-terminus of hhLIM is critical for its interaction with actin. The mutant of the LIM domain 2 in which two important Cys are replaced by Ser lost the capacity of hhLIM to interact with actin. Mutation of the LIM domain 1 at the N-terminus of hhLIM impaired the capacity of hhLIM to interact with actin. F-actin cross-linking assay identified that hhLIM could make F-actin to cross-link into bundles by interaction between LIM domain 2 and actin. In conclusion, LIM domain 2 at the C-terminus of hhLIM plays a central role in F-actin polymerization and cytoskeleton stabilization, whereas the first LIM domain is essential for the nuclear localization of hhLIM.

Abstract:The rapid development of silicon microelectrode arrays provides an ideal means for the study of spatio-temporal features of neuronal activity in the brain. The stability of the linear silicon electrode array (LSEA) in recording neuronal potentials and its validity in recording unit activity are investigated. The experimental results showed that during the recording of field potentials in the hippocampal CA1 region of anesthetized rats, upward and downward movements of the recording probe for a distance of 200 μm did not affect the orthordromic and antidromic evoked potentials significantly. The data indicated that the probe movements caused very small damage to the neurons, and the recording was stable. The contact sites that located in the pyramidal cell layer acquired CA1 neuronal unit activity validly. Different types of unit activity from independent neurons were easily distinguished in epochs of recording from a same recording site. These results demonstrated the features of the LSEA, including the facility of probe manipulation, the stability of recording and the abundance of data acquirement. The data will be helpful to the researchers involved in the application of microelectrode array for neuroscience researches.

Abstract:In order to present clues to find safer and more efficient ciliary neurotrophic factor(CNTF), computer molecular modeling system was used to gain the three-dimension structure of CNTFRα.Then through analysing the CNTF/CNTFRα crucial sites to its bioactivity combined with the homologous sequences comparison and CNTF molecular characters, two CNTF mutants were designed. The mutants' bioactivity were tested by the experiment of TF-1 proliferation. The two mutants' bioactivity reached 10⁶ U/mg level after expression and purification and the bioactivity of A is higher than that of B. The results proved primarily that the design was reasonable and provided premise and basement for CNTF's development and application.

Abstract:Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear hormone receptors belonging to the steroid receptor superfamily. Three PPAR isoforms, PPARα, PPARδ (also known as PPARβ) and PPARγ have been found in the mouse. They can activate expression of many genes, including those involved in lipidmetabolism. PPARδ is ubiquitously expressed, but the level of expression differs markedly between different cell types. PPARδ is expressed in skeletal muscle at 10- and 50-fold higher levels compared with PPARα and PPARγ, respectively. A role for PPARδ in skeletal muscle is to increase the genes expression with relation to oxidative metabolism. In order to determine the molecular mechanisms governing PPARδ gene expression in muscle, a 2 kb 5′ flanking region was cloned and analyzed. The DNA fragment is able to transcribe GFP in COS7 cells. Dual luciferase assay is used to quantify promoter activity. Deletion analysis of the 2 kb PPARδ promoter fragment in COS7 and NIH 3T3 cells shows that the proximal promoter sequence, nt －197 to +120, confers basal transcriptional activity of the mouse PPARδ gene. Computational analysis of putative cis-acting elements located within the ～2.0 kb mouse PPARδ 5′-flanking sequence was performed using the TRANSFAC database and MatInspector software and 4 potential MEF2A binding sites were found. And there is a potential binding site sharing 100% identity with positive element of MEF2A in the proximal promoter (nt －261). Co-transfection experiments of the PPARδ promoter reporter and pMEF2A expression plasmid (pMEF2A) showed that MEF2A significantly enhanced transcription activity of PPARδ promoter in NIH 3T3. Moreover, the enhancive effect depended on the concentration of plasmid pMEF2A transfected into cells. The results suggested that MEF2A may enhance transcription activity of the PPAR promoter in muscle cells.

Abstract:In order to study single viral gene functions in the context of genome and analysis interactions between one gene with another, the HEK293/p2089 stable cell line was established by transfecting the plasmid of DNA (p2089) into EBV-negative 293 cells and selecting for resistance against hygromycin. The plasmid p2089, which was kindly provided by Prof.Hammerschmidt, contained the whole EBV genome of wild-type B95-8. Two eukaryotic expression vectors (pcDNA3.1(+)/BZLF1 and pcDNA3.1(+)/BALF4) were constructed and then transiently cotransfected into the HEK293/p2089 stable cells, so as to induct EBV lytic replication and product recombinant EBV particles visualized through GFP-expressing. To estimate the EBV production, Raji cells were incubated with supernatants from the induced 293 cells carrying p2089 DNA, as revealed by indirect visualization of the Raji cells. GFP-positive cells were evaluated by inverted fluorescence microscope or FACS analysis. The different virus supernatants were quantified with the help of “green Raji units” per ml as an absolute number of infectious particles. This technique makes it possible for the reconstitution of viral progeny or mutants by transfection of BAC plasmid into eukaryotic cells, and any genetic modification in E. coli, thereby facilitating the analysis of viral gene functions in the context of genome. This new technique has provided a useful tool for the study of pathogenesis mechanism of EBV, especially for that of cancer-associated.

Abstract:In order to investigate the expression and roles of SelS in human umbilical vein endothelial cells (HUVECs, ECV₃₀₄ cells), total RNAs were extracted with TRIzol from adipose tissues of human, then the 1 102 bp fragment of SelS was amplified by RT-PCR. After the expected 1 102 bp PCR fragment was purified, SelS was cloned into pMD18-T vector. Purification, RT-PCR, restriction endonuclease analysis and DNA sequencing were performed to confirm the results. pMD18-SelS was incubated in presence of XhoⅠ/ClaⅠ and then ligated into pLNCX2 vector corresponding restriction sites. RT-PCR and DNA sequencing were performed to confirm a 1 102 bp SelS gene fragment was successfully inserted into pLNCX2. The ECV₃₀₄ cells were stably infected with pLNCX2-SelS or pLNCX2 alone and positive clone was obtained by G418 selection. After transient transfection, ECV₃₀₄ cells expressed pLNCX2 was verified by neo^r gene detection and the expression of recombinant SelS in the ECV₃₀₄ cells was 1.76-fold higher than the endogenous level by RT-PCR. After the cultured ECV₃₀₄ cells was treated with hydrogen peroxide (H₂O₂, 100, 200, 300, 400 μmol/L) for 24 h, cell viability was measured by MTT assay and the intracellular maleic dialdehyde(MDA)was detected by TBA method. The results showed that ECV₃₀₄ cells were injured by H₂O₂, overexpressing SelS increased the cell viability apparently and inhibited the production of MDA induced by H₂O₂. So overexpressing SelS protects ECV₃₀₄ cells from injuring by H₂O₂, and SelS may have the antioxidant protection effective on endothelial cells.

Abstract:Using conventional electrophysiological technique, the plasticity of characteristic frequency (CF) of neurons in rat auditory cortex (AC) at postnatal age of two, three, four, five, six and eight week was investigated by determining CF and frequency tuning curve (FTC) shifts of neurons. It was found that when the frequency difference between conditioned stimulus frequency (CSF) and CF of neuron was in 1.0 kHz with conditioned stimulation, conditioned stimulus can induce CF shift toward higher frequency side (HFS), lower frequency side (LFS) and both sides (BLH) of neuron's FTC. The proportion of CF shift becomes lower and the proportion of non CF shift becomes higher with the increasing of the age. Neurons with bigger Q₁₀-dB value and symmetry index >0 of the FTC, increasing of the proportion of CF shift toward higher frequency side (HFS) of neuron's FTC is much higher. There is significant correlation between the time course and the age of rat, the older is the age, the longer is the shift time (P < 0.05) and the recovery time (P < 0.05) of neurons. The results suggest that plasticity of characteristic frequency in rat auditory cortical neurons is correlated with the postnatal age. The findings provided important data to the study of the mechanisms for the developmental plasticity of central nervous system.

Abstract:The effect of hydrazine on blue membrane was investigated by the UV/VIS absorption spectrum technique and the flash photolysis technique, the results show that: hydrazine can convert blue membrane to purple membrane and the photocycle returns, but the rate of decay of photcycle intermediate(M₄₁₂) quickens, this phenomenon is not seen when metal cations are added to blue omembrane solution(the rate of decay of photcycle intermediate slowers). At the same time , the effect of pH and temperature on the interaction between hydrazine and blue membrane was investigated. When hydrazine was added to blue membrane solution, the sensitivity of the reaction is pH and temperature dependent. Over the pH range(2～4.8), the sensitivity of the reaction lowers with the increase of the acidity. Over the temperature range(20～40℃), the sensitivity of the reaction lowers with the increase of the temperature.