Abstract:Transmenbrance serine proteases 6(TMPRSS6) is a recently identified transmembrane serine protease, this protein regulates the expression of hepcidin in the process of iron metabolism, thereby affecting the body of the iron steady. The discovery of TMPRSS6 is not only very important to the understanding of mechanism of iron metabolism but also reveals the etiology of iron metabolism-related diseases, it provides a new way of thinking about how to treat these diseases.

Abstract:Emerging and re-emerging human viral pathogens, such as severe acute respiratory syndrome (SARS), which emerged in 2003, and the recently emerged swine-origin H1N1 influenza virus, which causes global pandemics, have had a worldwide impact and therefore represent a serious threat to human health. Viruses as the obligate parasites strictly depend on host cells for replication and, throughout co-evolution with hosts, viruses have developed strategies to evade and subvert the host antiviral innate immune response. A wide variety of RNA viruses have been reported to encode proteins that inhibit host innate immune responses. Papain-like protease (PLP) of human coronavirus is a novel viral-encoded deubiquitinase and is an IFN antagonist for inhibition of host antiviral innate immune response through disruption of ERIS (also called MITA/STING)-mediated signaling. The novel mechanisms by which human coronavirus inhibits host IFN response and new findings that papain-like protease (PLP) of coronavirus is an IFN antagonist which targets specific components of the IFN induction pathway were introduced.

Abstract:Source memory refers to memory for the source of information (e.g., memory for how information is acquired or memory for the modality thorough which information is acquired). Emotional stimuli refers to those stimuli that can arouse positive or negative emotions in individuals. With regard to the effect of emotional stimuli on source memory, the research findings have been controversial. Integrating the relevant research findings, the attention narrowing theory and the priority binding theory were described, and the current experimental studies were summarized that respectively demonstrate the enhancement, reduction and null effect of emotional stimuli on source memory. Finally the limitations of the current research and the directions for future research were pointed out.

Abstract:Mitofusin-2 (Mfn2) is a highly conserved transmembrane GTPase which plays a critical role in mitochondrial fusion process. Recent data have been demonstrated that Mfn2 is involved in the regulation of several crucial cellular pathways beyond fusion, including mitochondrial metabolism, cellular signaling cascade, apoptosis and proliferation. With multiple functions and complex mechanisms, Mfn2 might play potential role in the applications in modern medicine. The structure and basic biological function of Mfn2 were summarized, furthermore, the dysfunction of Mfn2 in certain diseases and its therapeutic value were also discussed.

Abstract:Equine infectious anemia virus (EIAV) vaccine is the first successfully applied lentiviral vaccine, but its mechanism on inducing protective immunity is not clear. Previous studies found that the EIAV vaccine strain EIAV_FDDV12 transmembrane protein (gp45) had a high-frequent translational terminating mutation at the site of 261W, resulting in a truncation of 154 amino acid residues at the C-terminus. To explore the biological meaning of the gp45 truncation, a gp45-truncated molecular clone was constructed by using an infectious clone of EIAV_FDDV12 as the backbone. Replications features of the gp45-truncated EIAV and its prototype virus were analyzed and compared in cultivated monocyte-derived macrophages (MDM) of equine and donkey and fetal donkey dermal cells (FDD). Results showed that the replication capacity of the gp45-truncated EIAV in equine and donkey MDMs was significantly decreased compared to the untruncated gp45 EIAV(P < 0.01), especially in the horse macrophages. In contrast, the truncated EIAV replicated significantly faster than the untruncated EIAV in FDD cells (P < 0.01). In addition, the reduced replication of the gp45-truncated EIAV in equine MDM led to a significant decline of cytotoxicity of the host cells when compared with the gp45 untruncated EIAV (P < 0.05). These results suggest that the truncation of the 154-residue C-terminus of the EIAV gp45 glycoprotein was an adaptation to the attenuation of the EIAV vaccine strain in FDD cells. This truncated mutation reduces the replication of the vaccine strain in macrophages, the primary in vivo target cell of EIAV, which leads to a further attenuation of the vaccine.

Abstract:To obtain information of glycan changes on cell surface in hepatocarcinoma progress, a high throughout lectin microarray was established to detect glycans on cell surface based on the principle of lectin to glycan binding affinity. Cells extracted from normal or model mice tissues and H22 cell lines were labeled with fluorescence and caught by lectins through the distinctive binding specificities, bound cells were directly detected by a laser fluorescence scanner to obtain the glycome profile of the cell surface according to the distinctive binding specificities of lectins on microarray and the appearance of bound cells were observed under the microscope. The optimal blocking buffer, optimal incubation time and temperature, optimal cell concentration were studied and specificity of lectin microarray was validated through the mannose blocking assay, flow cytometry and diffenrent blood type erythrocyte. High level of diversity of glycoprofiling was present between normal and hepatocarcinoma mice, only PSA, DSL, STL, NPL captured cells in normal group, all lectin captured cells except LTL and DBA in hepatocarcinoma group , the result show that Sia, GluNAc, GalNAc, Man and Gal increased on hepatocarcinoma cell surface. These results imply that these carbohydrates and correlate glycoprotein may play key roles in occurrence and development in liver canceration. The lectin microarray established a stable, real-time and throughout method and provides a novel strategy for study profiling changes of the cell surface glycome on tumor metastasis.

Abstract:In order to explore the function of PCNA gene on the proliferation and apoptosis of hepatocelluar carcinoma HepG2 cells, to provide the experimental evidence and a new tool to further explore the function of PCNA gene and the feasibility of its gene therapy, the eukaryotic expression vector targeting PCNA was constructed. According to the sequence screened in previous work, PCNA siRNA was converted into cDNA coding expression of shRNA (small hairpin RNA). The cDNA was synthesized and inserted into plasmid pSilencer2.0-U6 to construction of eukaryotic expression vector of siRNA specific for targeting PCNA gene, the negative plasmid pShPNA was also constructed. The plasmids were identified by sequence analysis. The plasmid was transiently transfected into hepatocelluar carcinoma HepG2 cells for 48 h, proliferation and the clone formation of HepG2 cells were detected by CCK-8 and clone formation assay respectively. The percentage of hypodiploid cells and early apoptotic cells was detected by flow cytometry. The morphology was examined by fluorescence microscope after Hoechst 33258 staining. Compared with control group and negative control group, PCNA mRNA level was reduced by pShPCNA detected by RT-PCR. The proliferation and clone formation of HepG2 cells treated with pShPCNA for 48 h were significantly inhibited (P < 0.01). The migration was also attenuated when PCNA was knocked down by shRNA, and flow cytometry analysis showed an increase of the percentage of G0/G1 phase cells, along with a decrease of cell population in the S phase. Percentages of hypodiploid cells and early apoptosis rates were significantly higher in treatment groups than those in control and negative control group (P < 0.01). Mitochondrial membrane potential was reduced in pShPCNA group detected by JC-1 fluorescent staining. Apoptotic morphology such as cell shrinkage, nuclear condensation, nuclear fragmentation, chromatin condensation and apoptotic bodies were also observed by staining with Hoechst33258 under fluorescence microscope. The study indicated the eukaryotic expression vector, PCNA shRNA has been successfully constructed, and effectively inhibits proliferation and induces apoptosis and arrested HepG2 cells in G0/G1 phage.

Abstract:To explore the functional role of TRAIL on the proliferation，apoptosis of mouse decidual stromal cells (DSC) artificially induced in vitro, a TRAIL gene expressing eukaryotic vector and a short interfering RNA(siRNA) expressing vector were constructed. 72h after transfecting with each vector, semi-quantitative reverse transcription polymerase chain reaction (semi-qRT-PCR) and Western blotting were applied to detect the expression levels of TRAIL mRNA and protein, respectively in DSC. Besides, MTT assay was performed to investigate the cell growth activity and proliferation capacity, and flow cytometry was used to examine cell cycle distribution and apoptosis rate of DSC. Restriction endonuclease digestion and sequencing of nucleotide acid confirmed the correct construction of both over-expression and interfering vectors. The combined results from TRAIL overexpression and interfering in DSC demonstrated that TRAIL is capable of markedly inhibiting DSC proliferation by blocking most of cells at G0/G1 phase, meanwhile the apoptosis rate of DSC was notably increased.

Abstract:Sirtuin type 1 (SIRTl), a novel regulatory factor of adipocyte and myocyte, interacts with target the forkhead box O family 1 (FoxOl) to modulate cell proliferation and differentiation, aging, apotosis and metabolism. The effect of nicotinamide (NAM, inhibitor of SIRT1) on the apoptosis of bovine preadipocytes was investigated with Acridine Orange (AO) staining, the flow cytometry detection and quantitative real-time PCR (qPCR). The results showed that NAM inhibited the differentiation of bovine subcutaneous preadipocytes (BSP) and bovine intramuscular preadipocytes (BIP), and their apoptosis rates were higher than control. SIRT1 influenced the apoptosis of bovine preadipocytes by regulating FoxO1 and adipocyte marker genes. The different mechanisms of SIRT1 and its related genes exist in BSP and BIP.

Abstract:The mechano growth factor (MGF) is originated from alternative splicing of Igf-1 gene, which is mainly expressed in stretched osteoblasts. The main purpose of this study is to examine the effects of MGF and E peptide (the C-terminal 24 amino acids peptide in the E domain of MGF, MGF-Ct24E) on differentiation of osteoblast MC3T3-E1 cells. The results indicate that the MGF and MGF-Ct24E activated the extracellular signal-regulated kinase 1/2(Erk1/2) and affected the expression of osteoblast-associated genes, including the reduced expression of alkaline phosphatase (ALP) and type 1 collagen (Col1), the increased product of osteopontin (OPN) and the decreased nuclear levels of activated Cbfa-1. These effects could be abolished by the blockade of Erk1/2 activation by inhibitor PD98059. In addition, the MGF could preferably activate the PI3K-Akt pathway, which is essential for the differentiation of osteoblasts, so the osteoblast has higher expression of ALP, type 1 Col1, and the formation of bone mineralization nodule under the treatment of MGF than that under the treatment of MGF-Ct24E. These advantages could be inhibited after blockading of Akt by inhibitor LY294002. It was concluded that the MGF-Ct24E could inhibit osteoblasts differentiation through the activated Erk1/2 cascade, while the MGF can induce differentiation through the activated PI3K-Akt. The MGF consists of MGF-Ct24E and IGF-1, which could activate Erk1/2 and PI3K-Akt respectively. Therefore, the MGF possess double effects on differentiation of osteoblasts, which presented as that inhibitory effect at the early differentiation, and promoting effect at the later differentiation.

Abstract:The investigation of electromagnetic fields (EMF) in human health risk assessment include epidemiology, human and animals, cellular system, biochemistry and molecular biology, and biophysical mechanisms. The causal chain describes a series of sequential steps to lead to disease outcome for EMF to cause biological effect. To complete the first step, or say primary effect, the physical reaction between EMF with bio-molecules must come into being chemical reaction in organism, and then bring biological reaction. To research relationship of radical, energy and cytosolic calcium has significance to know primary effect of EMF. The effect of exposure to 48 h at 0.1mT, 0.5mT and 1.0mT power frequency EMF on rat-hippocampal neurons was investigated by evaluating the reactive oxygen species (ROS) and cytosolic Ca²⁺ concentration. The results show that the reactive oxygen species (ROS) and cytosolic Ca²⁺ concentration at 0.1mT, 0.5mT and 1.0 mT EMF have be all signification increaser (P < 0.01) than control (sham exposure), ROS at 0.1mT and 0.5mT and Ca²⁺ at 0.1mT have be no difference (P > 0.05) than Trolox + EMF, ROS at 1.0 mT and Ca²⁺ at 0.5mT and 1.0 mT have be signification increaser (P < 0.01) than Tolox + EMF. It is known that EMF may enhance ROS formation in neurons, and there is positive correlation between ROS level and cytosolic Ca²⁺ concentration.

Abstract:The dynamics character of H1 peptide in aqueous solution at different temperatures has been investigated through temperature replica exchange molecular dynamics (T-REMD) simulations with GROMOS 43A1 force field. The two independent T-REMD simulations were completed from initial conformations α-helix and β-sheet structures, respectively. Each replica was run for 300 ns. 36 replicas have been simulated and the total MD simulation time of all replicas was 21.6 ?滋s, the convergence of conformation sampling was verified by simulations starting from β-sheet structure. The structural character of H1 peptide at each temperature (300K, 330K, 350K and 370K) was assessed from the parameters such as the distributions of backbone dihedral angles, the number of native hydrogen bond, formation of β-turn and the favorite conformation at different temperatures. The simulations sampled conformation cluster close to β-sheet structure as demonstrate by the 0.05 nm Cα RMSD, and this cluster contains 39%, 23%, 13% and 11% conformations in total at 300K, 330K, 350K and 370K respectively. The results indicate that: GROMOS 43A1 force field has precision of the description of the conformational equilibriums, however, efforts have been made to refine the treatments of hydrogen bond, and the comparative study of H1 peptide at different temperatures can afford information for refine molecular force field.

Abstract:The light-chain actin-binding protein caldesmon (l-CaD) stabilizes microfilaments and stress fibers in cells, it also can dissociate from the actin filament by phosphorylation. Curiously, in many tumor and transformed cells CaD is down-regulated, but in metastatic cancer lines, the expression of l-CaD is very high, in order to explore the role of l-CaD in metastatic cancer cell mobility, transwell migration assays and contractility measurements at cellular levels by traction microscopy were performed using metastatic human breast cancer cell lines the MDA MB-231.By over-expression of wild-type or mutated CaD, including A1234 (unphosphorylatable，both PAK- and ERK-sites converted to Ala) and D1234 (phosphorylation mimics，both PAK- and ERK-sites converted to Asp) in MDA MB-231 cells, l-CaD phosphorylation in cells was disturbed to detect how the l-CaD phosphorylation mediate cancer cell migration. Afterwards, by siRNA, the l-CaD expression in MDA MB-231cells was knocked down and the migration activities was compared with the non-metastatic human breast cancer cell line MCF-7. The result showed that the A1234 mutant cells exhibited most robust traction force and the wild-type CaD transfected cells also showed much stronger traction force than control cells, whereas the same transfection resulted in most severely hampered migration in both types of cells. These A1234 and wild-type expressing cells also exhibited enhanced stress fibers and delayed trypsin-induced detachment from substratum. Upon the inhibition of the CaD expression, the l-CaD knock-down cells lost the stress fiber structures and exhibited significant decrease in migration activities which was similar to the non-metastatic cancer cell line MCF-7. Moreover, because of the disruption of the cytoskeleton by l-CaD knock-down, the contractility of the l-CaD knock-down cells was decreased and much easier to detach from the substratum. Taken together, l-CaD phosphorylation is an important pathway that mediate the migration activity of the metastatic cancer cells, the high l-CaD expression level with the efficient phosphorylation cycling is a critical factor that maintains the high migration activities of the metastatic cancer cells. The findings thus not only shed lights on the mechanism by which CaD regulates cell motility, but also provide new insights into the nature of metastasis of cancer cells.

Abstract:To prepare Citrinin(CIT)-protein antigen, CIT was conjugated with bovine serum albumin (BSA) by 1,4-butanediol diglycidyl ether. HPLC, UV and IR absorption suggested that CIT was correlated with the carrier protein, and the molar ratio of CIT to BSA was 8.16. The polyclonal antibodies (PcAb) against CIT were produced in serum of immunized BALB/C mice with CIT-BSA, and the titer of antibody reached to 1.1×10⁵ by indirect enzyme-linked immunoassay. The indirect competitive ELISA showed that the detection limit of CIT was 10 μg/L, with a good linearity ranging 10～250 μg/L, and IC₅₀ was 100 μg/L. Immunogenicity of antigens prepared by different methods was analyzed. The result showed that the epitope was the carboxyl group at C₇. This work would be helpful for establishing the technology and developing the kit to determine CIT-contaminated samples by ELISA.

Abstract:Multiplex PCR was used in many biological fields. Primer design and amplification conditions are critical for enhancing the efficiency of multiplex PCR. In order to improve the multiplex PCR assay, five house-keeping genes of mouse were selected to design primers for multiplex PCR analysis by using the MPprimer program, followed with optimizing the conditions for PCR reactions. Result showed that MPprimer is a valuable tool for multiplex PCR primer design. In addition, by optimizing the conditions for multiplex PCR such as the annealing temperature and extension time, the efficiency of multiplex PCR assay could be significantly improved. This work can be used to advance large-scale gene expression analysis in the post-genome era.