Abstract:Zebrafish, as a new model of animal, has been gradually accepted and widely used in biology research due to its special characteristics. Besides its convenience used in development research, the application in ethology is more and more extensively. Because of the transparency before 2 days post-fertilization, the eye size nearly half of the brain and its conspicuous circadian rhythm, zebrafish has been significantly applied in vision research. The organs of olfactory and auditory are both visible on the surface, such structure makes it very easy to detect the olfactory and auditory function by means of behavior experiments. Observation of motion is very convenience as zebrafish's propensity is active. It is also used more and more in social biology research. The behavior test of zebrafish is a simple and effective method to analyze the integrative neuronal function in vivo while some experimental models have been established which reviewed as follows.

Abstract:Recently, advances in high-throughput experimental technologies enable an ever-increasing amount of data on protein interaction networks available. These data provide new insights into the evolutionary processes of protein interaction networks. The researches associated with analyzing such data from an evolutionary perspective was reviewed at five different levels: from proteins to protein interactions, motifs, modules and the whole network. Two aspects were focused on: 1) the constraints of the network organization on protein evolution, 2) origins and evolution of the topological features of protein interaction networks which are different from those of random networks. In addition, the enlightenments from the former studies were presented and the development trends in this field were discussed.

Abstract:Many investigations have demonstrated that microRNA (miRNA) is not only involved in the modulation of nerve cell growth and physiological activity, but also responsible for dysfunctions in synaptogenesis or synaptic plasticity, neurodegenerative diseases, tumorgenesis in the nervous system, as well as in cerebrovascular disorders. With the intensive researches in miRNA, it is possible gradually to explain the related pathogenic mechanisms of some major diseases in the nervous system.

Abstract:Dynamic fucosylation of glycoprotein especially 80 ku which bound to UEA and LCA during the course of rat hepatocarcinogenesis was investigated. In patient hepatocellular carcinoma, more UEA- and LCA-bound proteins were also observed in patients with high metastatic potential than those with low metastatic potential. Fucosylated glycans constitute important adhesion molecules such as Lewis antigens. A differential expression pattern of Lewis antigens was further confirmed on various metastasis potential hepatocellular carcinoma cells (HCC). High metastatic hepatocellular carcinoma cell line (HMCC97H) expressed much more Lewis x and b than low metastasis HMCC97L cells. Moreover, surface Lewis x, or b expression level declined significantly after the cells were treated by retinoic acid. Not only in experimental metastasis foci, but in HCC as well, both α1,3/1,2 and α1,6 fucosyltransferase activities were quite high. After retinoic acid treatment, α1,3/1,2 fucosyltransferase activities were significantly inhibited, Lewis x on epidermal growth factor receptor reduced, and the EGFR was less phosphorylated. These results suggested that fucosylated glycans such as Lewis x played an important role in HCC development and metastasis.

Abstract:Apoptosis-inducing factor (AIF), normally confined to mitochondria, can move from mitochondria to nuclei, participate in the execution of cell death. The mechanism by which AIF induces cell apoptosis is unclear. In order to analyze proteins which may interact with AIF, interceptive AIF gene (mitochondrial localization sequence, MLS, was deleted) which was amplified from the total mRNA extracted from hepatoma HepG2 cells by reverse transcriptase polymerase chain reaction(RT-PCR), was inserted into pcDNA3.1 plasmid.The recombinant plasmids constructed successfully were then transfected into HepG2 cells and expressions of interceptive AIF were detected by RT-PCR and Western blot. Immunoprecipitation (IP) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to isolate and identify proteins which can interact with interceptive AIF respectively. Screened out proteins which may interact with AIF were then confirmed by CO-IP and Western blot. The result showed that AIF may interact with β-actin.

Abstract:Uridine diphosphate (UDP)-GalNAc∶polypeptide N-acetylgalactosaminyltransferase (ppGalNAcT) catalyzes the initial step in mucin type O-glycosylation in the Golgi apparatus. Here generation and characterization of a polyclonal antibody to human ppGalNAcT2 were described. The subcellular location of ppGalNAcT2 in SGC7901 cell line was investigated using Western blot analysis of fractionated cell extracts and confocal microscopy with this antibody and two Golgi markers: Golgi SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) of 28 ku (GS28) and trans-Golgi network (TGN) 38, markers for the cis- and trans-Golgi apparatus, respectively. Morphometric analyses indicated that ～60% of the ppGalNAcT2 signal colocalized with the GS28, while ～36% of the cis-Golgi marker colocalized with the ppGalNAcT2. Approximately 34% of the ppGalNAcT2 signal colocalized with the TGN38, whereas 38% of the trans-Golgi marker colocalized with the ppGalNAcT2. The results provide unequivocal evidence for the location of ppGalNAcT2 within the Golgi apparatus, and further highlight the importance of this organelle in the initiation of O-linked glycosylation.

Abstract:One major problem to successful treatment of cancer is the development of resistance by tumor cells to multiple chemotherapeutic drugs, a phenomenon named multidrug resistance (MDR). Searching for the novel chemotherapeutical agents is one of the important strategies for overcoming MDR. By using a cytotoxicity assay, flow cytometry analysis, Western-blotting and RT-PCR, a drug (Taxol, TAX) resistant human nasopharyngeal carcinoma KB cell line (KB/TAX) was established by addition of the drug to the cell cultures gradually, then a novel N-sugar substituted thalidomide analogue (STA-35) was investigated for its reversal effect on MDR of KB/TAX cells and possible mechanism. The results showed that KB/TAX cells were resistant to several chemotherapeutical agents, and the relative resistance to TAX was 73.1. Compared with parental KB cells, the function and protein expression of P-glycoprotein (P-gp), as well as mdr1 gene in the KB/TAX cells were remarkable reduced. Moreover, both KB and KB/TAX cells were sensitive to STA-35, the relative resistance to TAX on KB/TAX cells was decreased by the addition of STA-35. Furthermore, STA-35(5～20 μmol/L)was capable to reduced the activity of P-gp by increasing the accumulation of rhodamine 123, decreasing P-gp expression in KB/TAX cells in a dose dependent manner, but had no effect on the mdr1 gene expression. These results suggest a potential action of STA-35 as MDR reversing agent, and one of the possible mechanisms could be the suppression of P-gp function and protein expression.

Abstract:A H5N2 subtype avian influenza virus isolated from goose belongs to highly pathogenic avian influenza virus, and the intravenous pathogenicity indexes (IVPI) =2.99. But ducks are not sensitive to this isolated influenza virus. The virus can infect mouse but only replicates in lung and has no pathogenicity. HA and NA gene of this isolated strain share 99.4% and 99.8% nucleotide sequence identity to the HA gene of A/chicken/Hubei/ 489/2004 (H5N1) and the NA gene of A/chicken/Jilin/53/01(H9N2), and share 99.3% and 99.6% amino acid sequence identity to the HA protein of A/chicken/Hubei/489/2004(H5N1), A/swan/Guangxi/307/2004(H5N1), A/wild duck/ Guangdong/314/2004(H5N1), A/chicken/Henan/210/2004(H5N1) and the NA protein of A/chicken/ Jilin/53/01(H9N2). There are several continuous basic amino acids (-RRRKKR-) at the cleavage site of HA protein. Phylogenetic trees analysis of HA and NA gene suggests that the isolated influenza virus probably originated from the reassortment of H5N1 and H9N2 subtype influenza virus.

Abstract:The primary neural stem cells were isolated from SD rat and formed the neuropheres, the neuropheres were passaged and planted on the dish coated with 0.1% gelatin, the colony was picked up under the microscope, then dispersed and cultured, to obtain the clone proliferated from one cell, passaging and picking up the cells 5～6 times at least. The NSC and its differentiated cells were identified with the marker genes respectively. The results showed that the neural stem cells were isolated from the SD rat embryos and the real clone were obtained by picking up the cells again and again, and then cultured in the form of monolayer. The marker genes of the neural stem cells and its differentiated cells could be detected at last. It will provide the rat model the resource of the cells for the treatment and the basic research for the morphology standard.

Abstract:Proteins are the major molecules performing life activities, and their spatial and temporal expression profiles in organisms are very important for understanding their accurate functions. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a unique antioxidant enzyme that directly reduces lipid hydroperoxides in biomembranes and plays a vital role in defense against membrane peroxidation damage. The protein expression profiles of rice PHGPx (OsPHGPx) were investigated in different rice tissues and under various stress treatments by using Western blot analysis. The results showed that in mature rice plants, OsPHGPx was mainly distributed in leaves, especially flag leaves, and in rice seedlings, OsPHGPx was detected in shoots and leaves. Moreover, OsPHGPx expression in rice seedlings could be markedly induced by H₂O₂ and NaCl, but weakly influenced by several plant hormones. Time- and dose-dependent effects were observed in both H₂O₂ and NaCl treatments, and the strongest induction was observed when rice seedlings were treated with 0.5 mmol/L H₂O₂ for 12 h or 500 mmol/L NaCl for 24 h. Additionally, dimethylthiourea, a H₂O₂ trap, inhibited H₂O₂-enhanced expression of OsPHGPx, but did not impair the enhanced effect of NaCl, implying that NaCl-induced OsPHGPx expression was not mediated by H₂O₂. These results will contribute greatly to further study the exact physiological function of OsPHGPx in rice.

Abstract:Rat synoviocytes were cultured in vitro and the changing of cellular cytoskeleton induced by PMA was investigated with atomic force microscopy. PMA is usually used to stimulate PKC signal transduction pathway in synoviocytes and to simulate process of inflammation. The results presented here show that synoviocyte cytoskeleton has changed significantly after PKC activation by using the imaging of atomic force microscopy. The results also indicate that the activation of PKC might play crucial role in cytoskeleton changing of synoviocytes，which, to some extent, provides some experiments and ideas on understanding the mechanism underlying rheumatoid arthritis (RA).

Abstract:FAAP (focal adhesion associated protein), encoded by murine D10Wsu52e gene, is a member of function-unknown protein family, UPF0027. These protein family homologues are conserved and ubiquitously expressed in various species, organs and cells. But the functions are quite unclear yet. Here, FAAP were demonstrated to deposited in the cytoplasmic fractions and the plasma membrane fractions by ultra-speed centrifugation. The expression levels of FAAP are significantly correlative with 67 ku Laminin receptor (LR) in HeLa cells. Moreover, FAAP, similar with LR, could effectively restrain the cultured cells attachment. Together, these results suggest the effects of FAAP on cell attachment.

Abstract:Elucidating the complex molecular regulatory mechanisms underlying hepatic differentiation at early stage contributes both to fully harness embryonic stem cells in liver regeneration, and to understand the differentiation-related liver diseases. Pluripotent embryonic stem cells can be induced into hepatocytes in vitro. mESC-D3, maintained in adherent monolayer culture condition, were induced to differentiate along hepatic lineage with addition of some factors to the medium at different time. The factors included FGF, HGF, OSM and so on. The differentiated cells showed a hepatocyte-like morphology, expressed hepatic marker genes and have also produced and stored glycogen by phase contrast and transmission electron microscopy, reverse transcription-polymerase chain reaction, immunocytochemistry, and periodic acid-Shiff staining respectively. Stem cell differentiation-related microarray was used to analyze the differential gene expression profiling during hepatic differentiation of mESC-D3 at early stage. Quantitative PCR was performed to verify the microarray data. Microarray analysis presented 48 genes expressed differentially (2 fold), including 20 genes up-regulated and 28 genes down-regulated. Further bioinformatics analysis showed the majority of these genes were extracellular matrix, intercellular junction and FGF, BMP, Notch and Wnt signaling pathways molecules, which suggests these alterations may be closely associated with the hepatic differentiation of embryonic stem cells at early stage.

Abstract:To study the role that the novel nongenomic membranous estrogen receptor GPR30 plays in the hippocampal synaptic plasticity, the developmental profile and subcellular localization of GPR30 in the hippocampus of postnatal female rats was examined by nickel-intensified immunohistochemistry and immunoelectronic microscopy. Results showed that the immunoreactivity of the GPR30 was predominantly localized in the neurons of the hippocampal pyramidal layer of the CAs and grannual layer of the dentate gyrus, and an increased profile with postnatal development was also noticed. GPR30 immunoreactivity was first detected at P7 in the CA2, at P14 it was detected in the CA1, CA2 and dentate gyrus, hereafter it was detected in the CA1, CA2, CA3 and dentate gyrus with higher expression in the adults (P60). Under light microscopy GPR30 immunopositive materials were found in the cytoplasm of the neurons, while under electronic microscopy they were localized in the membranous structure of the neuronal cytoplasm, predominantly the rough endoplasmic reticulum. The above results demonstrated that GPR30 is a membranous estrogen receptor, showing an increased expression profile in the hippocampus of postnatal female rats, it may mediate the rapid, non-genomic effect of estrogen on the morphology and function of the hippocampus, such as morphological maturation, synaptic plasticity, learning and memory.

Abstract:Balb/c mice were immunized four times by the purified fusion CA protein which was emulsified with freund′s adjuvant. Then, cell fusion was conducted according to standard procedure. Positive hybridoma clones were screened by indirect ELISA and Western blot. Three hybridoma clones that stably secreted specific monoclonal antibody against JSRV-CA were developed. Meanwhile, JSRV ca gene was divided into four overlapping fragments and expressed in E. coli BL21 respectively. Pepscan technology was employed to screen the antigen epitope through the expressed fusion proteins were detected respectively with three McAbs by Western blot. Then three liner epitopes recognized by three McAbs were preliminary identified, and the functional affinities of anti-CA McAbs were assessed with non-competitive ELISA method. All this may be helpful in understanding molecular properties of JSRV-CA and may be useful for pathogenic diagnosis and vaccine design.

Abstract:RNA secondary structure predicting is a classical problem in bioinformatics and the optimal algorithms based on minimal free energy (MFE) criterion are the widely used methods. However, pseudoknots render the problem of computing the RNA MFE structure with pseudoknot becomes a NP-hard problem. A heuristic algorithm——StemFind to predict RNA secondary structure with pseudoknot was presented. The algorithm regard stem as the basic search unit, adopting heuristic search strategy, and search the most possible RNA secondary structure in stem combination space. The StemFind algorithm to a large number of test sets was applied. Performance evaluation demonstrates that StemFind not only outperforms the well-known optimal and heuristic algorithms in overall sensitivity and specificity but also requires significantly less time than the optimal algorithm.