Abstract:As a new identified help T cell lineage different from Th1 and Th2 cells, Th17 cell has been found played important roles in the pathogenesis of autoimmunity and inflammatory disease. To further identify their roles, the differentiation and regulation of Th17 cells has been widely explored recently. Now it has been confirmed that TGF-beta, combined with IL-6 or IL-21, play critical roles in the differentiation of Th17 cells. While IL-23 mainly contribute in promoting the secretion of IL-17 and maintaining the function of Th17 cells. Corresponding with the Th1,Th2, and Treg cells, which has special transcription factors T-bet、GATA3、Foxp3 respectively, now it has been confirmed that ROR-γt(retinoid-related orphan receptors-γt) is the special transcription factor which specially regulate the differentiation of Th17 cells. Th17 cells function through their secreted pro-inflammatory cytokines, including IL-17A, IL-17F, IL-21, IL-22, IL-6, TNF-α. Among them IL-21，which act as a autocrine cytokine of Th17 cells, play critical roles in promoting the differentiation of Th17 cells while inhibiting the differentiation and function of Th1 and Treg cells. On the other hand, IL-2, which is obligatory for the growth of Th1,Th2,Treg and CD8⁺T cells, now has been found negatively regulate the differentiation of Th17 cells. In all, differentiation of Th17 and Treg,Th1 cells are exactly regulated in vivo, in which TGF-beta played critical roles. As both Th1 and Th17 cells participate in the pathogenesis of autoimmunity and inflammatory diseases, are they play synergistic roles or function at different time point or location? How TGF-β regulate Th17 and Treg cells？Can Th17 cells be used as a target for immune tolerance induction? All above questions will certainly be of continuing interest.

Abstract:NKR and TLR are most important receptor superfamilies in innate immunity and act as first line of host defense against infection. Those receptors exert peculiar recognition mechanisms to sense danger signals and distinguish infectious nonself from noninfectious self. More importantly, they coordinate and regulate each other and therefore play major roles in initiation of innate immunity and also help to direct adaptive immune responses. The importance of recognition and interaction of those receptors are highlighted. The precise mechanisms can be harnessed to aid the rational design of therapy against infection, inflammation, cancer or autoimmune diseases.

Abstract:Inflammation is a basic way in which the body reacts to infection, irritation or other injury. The key clinical feature of inflammation is redness, warmth, swelling and pain. A series of immune cells and cytokines are involved in the complicated and interrelated events during inflammation to work together to defend the body. Innate immune cells, including phagocytes, NK cells and dentritic cells, are the main effector cells in initiating an inflammatory response. Adaptive immune cells, for example, T cells which take part in the battle at the later phase of an inflammatory response could also temper the initial inflammatory responses during acute infection. On one hand, hosts rely on inflammatory responses to eliminate the pathogen, control the infection within the local site, and induce the adaptive immune response. However, on the other hand, over-reactive and chronic inflammation can also lead to some diseases. Consequently, the study of the mechanism of inflammation might lead to new treatment for patients with inflammation-related diseases.

Abstract:During lymphocyte development antigen receptor genes undergo V(D)J recombination to obtain antigen binding specificity and diversity. This process is not only controlled by genetic factors, such as tissue- and stage- specificity of RAG-1/2 protein, germline transcriptional activity and ACEs, but also regulated at epigenetic level. The chromatin accessibility of recombinase is associated with the chromatin configuration around the targeted gene segments. Thus, activation of V(D)J recombination requires the recruitment of remodeling complexes for changing the accessibility in the localized chromatin. Moreover, docking of remodeling complexes, which serve for creating active chromatin environment, relies on certain patterns of chromatin modification. Some recent findings regarding epigenetic regulation mechanisms in V(D)J recombination, such as CpG methylation, histone modification, nucleosome remodeling and nuclear topology were reviewed.

Abstract:Novel oncogene with kinase-domain (NOK) can activate multiple mitogenic signaling pathways including the janus kinases (JAK) and signal transducer and activator of transcription proteins (STAT). It was showed that NOK specifically and physically interacted with STAT3 in human embryo kidney 293T (HEK293T) cells. In addition, NOK could directly interact with most of the STAT3 subdomains except coiled-coil and C-terminal domains. Removing ectodomain and transmembrane domain of NOK markedly enhanced its intermolecular interaction with STAT3. Also, NOK could co-immunoprecipitate with JAK2 in vivo. Importantly, co-expression of NOK and JAK2 produced a synergistic effect on NOK-mediated STAT3 activation, while inactivating the kinase domain of JAK2 completely prevented this synergistic effect. Overall, the results indicated that NOK might complex with both STAT3 and JAK2 and activate STAT3 signaling by a JAK2-dependent mechanism.

Abstract:The transcriptional repressor RE1 silencer transcription factor (NRSF/REST) is an important factor that restricts some neuronal traits in neurons. It has been confirmed that the insulin gene was regulated by NRSF via NRSE. Here, the relationship between NRSF and insulin gene was investigated by means of RNA interference (RNAi). The expression of NRSF was down-regulated by lentiviral RNA interference vector, and the interference efficiency is 56% (n = 6, P < 0.01). When the expression of NRSF was down-regulated, genes regulated by NRSF, such as SCG10, BDNF, pax4 and insulin, began to express in HeLa cells. The human insulin promoter activity was up-regulated 2.4-fold after RNA interference (n = 3, P < 0.01), confirmed by dual-luciferase assay.

Abstract:In order to investigate the effect of sequence-specific small interfering RNA on suppressing cyclin D1 expression and proliferation and cell cycle and expression of G1 phase regulators of fibroblasts derived from keloid, the plasmid expression vector of siRNA targeted against cyclin D1 was constructed and transfected into fibroblasts with Lipofectamine^TM 2000. The changes of cyclin D1 expression were detected by fluorescent quantitative PCR(FQ-PCR), semi-quantitative RT-PCR. The effect of sequence- specific small interfering RNA in suppressing the proliferation of fibroblasts was detected by MTT assay. Flow cytometry were used for evaluation of cell cycle. The expression of cyclin D1, CDK4, pRb and P16 was detected by immunohistochemical method. The results showed that: (1) The sequence- specific siRNA effectively suppressed cyclin D1 expression at both mRNA levels with inhibition rate of 63.68% and 92.83% (P < 0.01). (2) Significantly inhibited the proliferation of fibroblasts, and changed cell cycle in percentage of G0/G1 phase cells was increased compared with the controls groups in fibroblasts(P < 0.05). (3) 72 h after transfection, the expression of cyclin D1, CDK4 and pRb decreased, and the expression of P16 increased. It can be concluded that the plasmid expression vector for the RNAi against cyclin D1 constructed in the study can effectively and specifically suppress cyclin D1 expression, and progression of G1/S is effected by G1 phase related regulatory protein, and suppresses proliferation of fibroblasts derived from keloid.

Abstract:Aerenchyma formation has been described in depth in a number of species at a histological level. But large gaps remain in our understanding of its regulation as a developmental process. It is attempted to analyse essential mineral elements like K, Mg, Cu, Zn, Ca and P in the cell wall of aerenchyma cells in petioles of S. trifolia at five different developmental stages by CSEM-EDX technique. At early stage, K and Cl concentrations in cell wall were high up to 36% and 4.3% of dry weight, respectively. It supported the hypotheses that aerenchyma spaces are filled with liquid at early developmental stages of aerenchyma in S. trifolia petiole. Mg concentration was high at stage 2, up to 0.86% of dry weight. Zinc and Cu were detected only at rapid expansion stages, during which the concentrations were up to 1.5% and 2.5%, respectively. Calcium was detected in the cell wall only at mature stages, the concentration was high up to 1.3% of dry weight at stages 4 and 5. These results confirmed that the element concentration of aerenchyma cell wall undergoes dynamic changes during different developmental stages, and a low Ca with high Zn and Cu concentration are needed for cell expansion. Copper and Zn deposition in the cell wall showed a significant positive linear correlation, suggesting that these two elements share same or similar uptake and transport mechanism in plants.

Abstract:MiRP1 is encoded by KCNE2, which contains a single membrane-spanning domain. Inherited mutations in KCNE2 have been identified in patients with long QT syndrome (LQT6). However the mechanism by which KCNE2 mutations increase the risk of arrhythmias in patients remains unclear. Previous work has reported that MiRP1 plays an important role in regulating transient outward current (I_to) function and maintaining the ventricular electrical stability. The role of two kinds of long QT syndrome (LQT6)-associated mutations in KCNE2, I57T and V65M in regulation of Kv4.3, the major α subunit of I_to was investigated in COS-7 cells to elucidate the mechanism for pro-arrhythmia induced by these two inherited mutations. The results demonstrate that coexpression of KCNE2 with Kv4.3 slowed the rate of activation and inactivation, and caused a positive shift of voltage-dependence of inactivation of Kv4.3 channel. Moreover, KCNE2 accelerated recovery of Kv4.3 channel from inactivation. This is in agreement with previous study． In comparison with the effect of wild type KCNE2, I57T exerted no significant effect on Kv4.3 gating kinetics and recovery property. The phenotype of I57T and Kv4.3 coexpressed channel was similar to that of Kv4.3 expressed alone. In contrast, V65M enhanced all the effects of KCNE2 on Kv4.3, including slower activation and inactivation, more positive shift of voltage-dependence of inactivation and faster recovery from inactivation versus Kv4.3+KCNE2. Furthermore, V65M dramatically decreased Kv4.3 current amplitude. These results suggest that I57T causes “loss of function” while V65M results in “gain of function” of KCNE2. It is therefore proposed that KCNE2 is an important modulator of I_to. LQT6-associated mutations in KCNE2 may perturb the contribution of I_to to electrical stability in the heart through changing KCNE2 regulation of Kv4.3, predisposing the heart to arrhythmia under certain conditions.

Abstract:PRL-3 is a key gene related to metastasis of colorectal carcinoma. However, it is known little about the possible regulatory mechanisms of PRL-3 gene expression. There were three possible promoter regions predicted by TRED, a promoter prediction software，which were all located in the upstream regions of PRL-3 gene. One of PRL-3 gene candidate promoters was located in the region of about -1kb upstream proximal to 5′ UTR of PRL-3 gene. Many possible transcription factor binding sites such as Snail, n-MYC, ARNT, E74A, NF-kappaB, NRF-2 and AML-1 were predicted in the region by Consite, a promoter analysis web system. Interestingly, a 5′ CACCTG 3′ core sequence and other related sequences of snail binding sites were found in promoter region of PRL-3 genes. Two PRL-3 gene promoters between －699 to 299 nt and between －642 to －383 nt were cloned into pGL3 vector with luciferase report gene. Both of them had promoter activities in four different cell lines including colorectal carcinoma cell lines SW480 and SW620, nasopharyngeal carcinoma cell line CNE2 and human embryo kidney cell line 293A. Interestingly, the luciferase activities of the short DNA fragmentations with Snail binding site′s core sequence 5′ CACCTG 3′ were higher than that of the longer one. PRL-3 promoter obtaining the 5′ CACCTG 3′ core sequence of Snail binding sites, was validated to bind to snail by chromatin immunoprecipitation (CHIP) analysis and electrophoretic mobility shift assay (EMSA) in SW480 cells. The data suggested that Snail was involved in regulation of PRL-3.

Abstract:Nitric oxide (NO) reacts rapidly with superoxide anion and produces speroxynitrite (ONOO^－), which is a highly reactive free radical that has been shown to mediate much of the toxicity of NO. ONOO^－ has also been implicated as playing a role in the pathology of various brain disorders. The aim of the study was to investigate the actions of ONOO^－ on voltage-gated sodium currents and its effect on membrane excitability in hippocampal CA1 neurons. SIN-1, which leads to the simultaneous generation of superoxide anion and NO, and then forms the highly reactive species ONOO^－, induced a dose-dependent inhibition in amplitudes of transient potassium currents (I_Na). SIN-1(500 μmol/L) delayed the recovery of I_Na from inactivity, but did not have a marked effect on the activation and inactivation parameters of I_Na. However, co-treamtent with SIN-1 and Urate (100 mmol/L), an ONOO^－ scavenger, had no effect on I_Na currents. SIN-1 (500 μmol/L) led to the decrease in the amplitude and firing rate of action potential. Pretreatment with adenylate cyclase inhibitor MDL-12, 330A (25 μmol/L) and NEM (50 μmol/L) did not block the effects of ONOO^－. On the other hand, the responses to ONOO^－ were inhibited by guanylate cyclase inhibitor ODQ (10 μmol/L). The effects of ONOO^－ on hippocampal neurons were mediated through activation of cGMP-I_Na-AP (action potential) signaling cascades and independent from PKA and S-nitrosylation pathway, which maybe one of underlying mechanisms likely related to neurotoxicity of ONOO^－.

Abstract:Human ADAM-19 is a newly identified member of ADAM family, and is highly expressed in human placenta. But its functions in human trophoblast cells invasion have yet to be elucidated. A well-accepted human trophoblast cell model, NPC cell line, was used as in vitro model to investigate the influence of hADAM-19 on cell invasion in human trophoblasts. The regulation of hADAM-19 expression by transforming growth factor β1 (TGF-β1) was investigated by RT-PCR and Western blotting. It was shown that over-expression of hADAM-19 in NPC cells led to down-regulation of MMP-9, MMP-2 mRNA expression and decrease in cell invasion. In NPC cells, the expression of hADAM-19 was significantly up-regulated by TGF-β1 in a dose-dependent manner. The data indicated that there existed paracrine regulation of ADAM-19 in human placenta. ADAM-19 was involved in invasion-inhibiting regulation of human trophoblast cells, and it may function, at least in part, through coordination with various MMPs.

Abstract:The Arabidopsis thaliana glutathione S-transferases zeta class (AtGSTZ) is a multi-functional enzyme, which plays important role in cellular metabolism and environmental purification. Error-prone PCR and cycles of DNA shuffling were used to construct a mutagenesis library of AtGSTZ. The screening of the resultant libraries was carried out by a pH indicator dye-based colorimetric assay. Nine mutants which enhanced the dichloroacetic acid dechlorination activity were obtained. Among them, NN23 contained 25 amino acid substitutions with the activity improving 120%, whereas NN20 contained 24 amino acid substitutions with the activity improving 102%. EC1 contained 2 amino acid substitutions with the activity improving 47%. The rest 6 mutants contained one amino acid substitution with their activity increasing from 9% to 60%. The enzymatic characterization showed that all the evolved enzymes increased their catalytic efficiencies towards dichloroacetic acid and binding affinity towards glutathione whereas some of them increased the renaturability. However there is no obvious change in their thermostability. Based on these data, functional residues related to catalysis and refolding of AtGSTZ were discussed.

Abstract:RT-PCR assays have been used to analyze expression patterns of atherosclerosis related genes in aorta from 1 to 3-month and liver from 14-day to 3-month old of atherosclerotic model, young apoE deficient (apoE^-/-) mice in normal chow diet. The level of plasma lipids was also analyzed. Combined with the pathomorphological characteristics of the aortic root, the sequential expression characteristics of atherosclerosis related genes in the early stage of the atherosclerosis were studied. ApoE^-/- mice compared with WT mice, the genes detected in the aorta, the expressions of VCAM-1, IκB and TGF-β in 1-month old of the apoE^-/- mice were prominently up-regulated; while the expression of PDGF-α and CD36 were significantly up-regulated in 2-month old, the mRNA levels of TNF-α and MMP2 in 3-month old mice were prominently up-regulated, and IL-1β was significantly up-regulated from 1 to 3-month old. The genes detected in the liver, the mRNA level of CRP was significantly up-regulated at 3-month old of the apoE^-/- mice. The mRNA level of NF-κB had no significant change at different ages in the aorta and liver in the two types of mice. Serum TC, TG and LDL-C levels in different ages of apoE^-/- mice were significantly higher than those of WT mice. It appeared lipid deposition in the intima of aortic root in 2-month old apoE^-/- mice. With the increasing of age, the lesions became more serious. The results implied that the sequential and differential expression of genes related to atherosclerosis formed a complex regulatory network which NF-κB is the core, and the genes may interact with each other and play important roles in the early stages of atherosclerosis of young apoE^-/- mice.

Abstract:The inverted terminal repeat (ITR) is the only cis element of AAV genome essential for rAAV rescue, replication and packaging. It is prone to mutation or loss when it is latent in host cell or in plasmid. Plasmids with different ITR types were cloned to compare the influence of ITR types on the AAV packaging and infectivity. The vector plasmids were transformed the competent SURE cells to get different colonies. The ITR types of plasmids were screened by digestion with SmaⅠ. AAV vector plasmid pScGFPud has two ITRs at both ends of AAV genome and plasmid pScGFPu has only one ITR at upstream end of AAV genome. When the two plasmids were co-transfected 293 cells to prepare rAAVs, 1.08×10¹³ viral particles (AAV1-GFPud) were produced from 20-dishes of 293 cells cotransfected with plasmid pScGFPud, 4.28×10¹² viral particles (AAV1-GFPu) were produced from 20-dishes of 293 cells cotransfected with plasmid pScGFPu. Virus AAV1-GFPud infected 293, HeLa and NCI H446 cells more efficiently than did virus AAV1-GFPu. This suggests that defect ITRs in AAV genome is deleterious to AAV packaging and AAV infectivity and vector with complete ITRs is favorable to the yield and activity of rAAV.

Abstract:Hepatitis B virus surface antigen with mutation on site 145 may cause omission in detection of hepatitis B virus with HBsAg diagnostic reagent. Monoclonal antibody against this mutation was obtained through hybridoma technique. The sub-class of the monoclonal antibody was determined to be IgG1(κ type, the titer is 1∶9×10⁴), and the monoclonal antibody has no cross-reaction to other hepatitis virus. The method of AC-ELISA which was established with monoclonal antibody and polyclonal antibody showed high analysing susceptibility for HBV mutant samples.