Abstract:Aminoacyl-tRNA synthetases (ARS) are the oldest enzymes in the evolution. The amino acids are transferred to the cognate tRNAs by the aminoacyl-tRNA synthetases, and then involved in the synthesis of the protein. This process is very important to keep the stability and variety of the lives. With the coming of the post-genome research, one of the main objectives becomes to illustrate the structures and the function of the aminoacyl-tRNA synthetases. Based on the data from the structure biology and the bioimformatics, the multi-ARS complex seemed to be the main form in the eukaryote. In addition, mammalian ARSs consist of a sophisticated macromolecular network with the auxiliary proteins or the elongation factors. The recent discoveries show that aminoacyl-tRNA synthetases not only are the most important enzymes to the synthesis of the protein, but also are involved in the diverse cellular processes, such as regulation on the transcription and translation level, RNA splicing and trafficking, apoptosis, angiogenesis and inflammation.

Abstract:Rho is a member of the Rho family of small GTPase, and acts as a molecular switch in some signal transduction pathway. Rho can induce growth cones collapse and inhibit axonal outgrowth by regulating the actin cytoskeleton. Recent studies have indicated that three myelin-derived inhibitors including Nogo-A,MAG and OMgp all can inhibit axonal regeneration by activating the Rho-mediated signal transduction pathway.

Abstract:The results of many researches show that 3′untranslated region plays a very important role in the life cycle of single strand plus RNA virus. The high structures in 3′UTR such as stem-loop, pseudoknot and tRNA-like structure are also generally believed to be partly involved into the transcription, replication and translation of plus-strand RNA virus. The common methods and the last results about 3′UTR of this kind of virus are reviewed.

Abstract:FRAP and FLIP are two techniques that are usually applied to study the dynamics of protein in living cells. In recent years, studies of protein dynamics by these two techniques showed: some proteins are mobile, and they rapidly associate and dissociate with their respective compartments in the nucleus of living cells. Furthermore, protein movement is mainly independent of energy, which indicates that proteins may use a passive mechanism of movement. Additionally, covalent modifications have effect on the mobility of some proteins. Protein dynamics in the karyon is important to the nuclear architecture and gene regulation, however, the detail mechanism is still unknown and expected to further study.

Abstract:Previous study has shown that melanoma cell adhesion molecule (MCAM/CD146) is highly expressed by invasive trophoblasts in normal pregnancy, but it is absent in pregnancy disorder pre-eclampsia in which the invasion ability of trophoblast is seriously impaired. The mechanism that CD146 affects the trophoblast invasion was investigated. Immunofluorescence staining revealed that CD146 was selectively expressed on invasive intermediate trophoblasts, but was not expressed on non-invasive cytotrophoblasts and syncytiotrophoblasts. Functional studies showed that the anti-CD146 antibody significantly inhibited the migration of trophoblasts and the activity of matrix metalloproteinases secreted by trophoblastic cells, which are two critical factors for trophoblast invasion. The results demonstrate that adhesion molecule CD146 plays a critical role in trophoblast invasion, which will provide a valuable molecular model for the study of trophoblast and tumor cell invasion.

Abstract:The molecular genesis of a tumor is a multi-step and complex process in which many factors are involved such as oncogenes, tumor suppressor genes and MHC-Ⅰ etc. Metastasis is the essential character causing mortality from such tumors and little is currently known about mechanisms underlying cancer metastasis. As a step toward understanding the complex differences between high metastatic and low metastatic cells, metastasis-associated gene expression patterns and proteomes were separated and identified by suppression subtraction hybridization and comparative proteomics analysis between high metastatic cell AccM and low metastatic cell Acc2. Although extensive similarity was noted between the expression profiles of the two cell lines, twenty-eight genes highly expressed were obtained in tester, low metastatic cells, compared with their low expression in driver, high metastatic cells while six genes in reverse hybridization. These function of genes are related to cell proliferation, differentiation or cell metabolism while several novel EST sequences which function are unknown. Two novel genes were ACC metastasis-associated RNH ACC metastasis-associated suspected protein. Meanwhile, ten proteins were randomly selected which are expressed different qualitatively or quantitatively and identified by mass fingerprinting (MALDI-TOF-MS).These proteins are stratifin, symplekin, B7, TPMsk1 tropomyosin isoforms, serologically defined breast cancer antigen NY-BR-20 etc. Intriguingly, most identified candidate proteins have been shown to be somehow associated with distinct aspects of tumor metastasis such as cell communication protein, motility, adhesion, invasion and tumor immunity associated protein, etc. These results suggest that attaining the ability to metastasize is related to the changed expression of these genes or protein. These data provide insight into the extent of expression differences underlying metastasis-related genes that may prove useful as diagnostic or prognostic markers.

Abstract:Double-stranded RNA (dsRNA) can induce sequence-specific inhibition of gene function, this phenomenon is termed RNA interference (RNAi). An effective RNAi method of silkworm (Bombyx mori) embryo by introduction of double-stranded RNA corresponding to the silkworm Bmwh3 gene into the preblastoderm eggs of wild-type has been established based on the previous research. The results showed that the RNAi was successful when injected Bmwh3 dsRNA into the preblastoderm eggs within 8h after laid of wide-type silkworm and the concentration of the Bmwh3 dsRNA must be high to 2.0 g/L. It can also induce the other w3 mutant phenotype——translucent larvae when injected Bmwh3dsRNA into the third day of developed eggs. A conclusion can be primarily drawn that the Bmwh3 is not only involved in the transport of the precursors of the ommochrome and pteridine pigments to the eye and egg，but also involved in the transport of the precursors of the pigments to the larvae skin.

Abstract:The secretion of proteins depends on signal peptide. Gene trap is a powerful tool for functional gene identification and characterization. Classical gene trap employs geo as the reporter, and gives no discrimination on the types of trapped genes. A new reporter gene named peo was obtained by in turn fusing Exotoxin A of Pseudomonas aeruginosa, 2A sequence, transmembrane region of IL-2 receptor and neomycin phosphotransferase genes, Peo is able to report genes encoding secretory proteins specifically. To prove peo's specificity for secretory proteins, three plasmid vectors, representing three types of sequences possibly trapped by gene trap vector during screening, were constructed based on peo reporter. After transfecting HeLa cells with these plasmids, followed by G418 selection and PLAP staining, it is shown that the new vector could distinguish secretory proteins from others, and the gene trap vector taking peo as the reporter can trap secretory protein genes efficiently.

Abstract:Hepatopoietin (HPO) has diverse functions in the regulation of cell growth and differentiation. Its high level expression in testis suggests that HPO may have important role for testicular physiological functions. Using HPO as “bait”, a yeast two-hybrid library screen of human testis was performed. By screening and selecting the positive colonies, retesting interactions in yeast, amplifying the AD/library inserts, sequencing and sequence comparing, four HPO-interacting proteins were identified: NADH dehydrogenase 1, Na⁺/K⁺ ATPase beta-3 subunit (ATP1β3), phospholipase C delta 1 and epididymal secretory protein. The significance of identification of these proteins may provide mechanistic insight into the biologic role of HPO in testis.

Abstract:Tubulin is one superfamily of proteins. α- and β-tubulins are the major proteins of microtubules, while γ-tubulin plays a critical role in the nucleation of microtubules. Western blotting and immunofluorescent confocal microscopy were used to study the assembly and transformation of γ-tubulin during meiotic maturation, fertilization and parthenogenetic activation of pig oocytes. γ-Tubulin exists in the porcine oocytes and its quantity remains stable during meiotic maturation. It is located in the region where there is microtubule assembly, especially on the spindle poles at metaphase and middle plate or midbody of elongating spindle at anaphase and telophase. After in vitro fertilization and parthenogenetic activation, γ-tubulin concentrated around the male and female chromatin and pronuclei. It is also found to be localized in sperm acrosomal cap and the neck. During early cleavage, γ-tubulin distributes around the blastomere nuclei. The results suggest that γ-tubulin is a major regulator of microtubule assembly in porcine eggs and that both oocyte and sperm contribute centrosome materials to fertilized eggs.

Abstract:In order to unravel the mechanism of inhibition of the cell proliferation induced by overexpression of PEMT2, the cell clone transfected with pemt2-cDNA was constructed and the effect of overexpression of PEMT2 on PI3K/Akt signaling pathway was investigated by the methods of RT-PCR, immunocytochemical and flow cytometry. The results show that the overexpression of PEMT2 could inhibit the expression of PI3K and Akt, and induce cell apoptosis. The results indicate that the down-regulating of PI3K/Akt signaling pathway could, at least partly, account for the inhibition of hepatoma cell proliferation induced by overexpression of PEMT2.

Abstract:In order to investigate if minimally modified low-density lipoprotein (MM-LDL) could induce apoptosis in human umbilical vein endothelial cells (HUVECs) and the potential role of cytosolic phospholipase A₂ (cPLA₂) in this process. Cell viability was determined by MTT assay, cell apoptosis was assessed by phase-contrast microscopy, fluorescence microscopy and flow cytometry (FCM), PLA₂ activity was determined by measuring the release of ³H-arachidonic acid (³H-AA) from prelabeled cells, phosphorylation of cPLA₂ was analyzed using Western blot, changes of ［Ca²⁺］_i in single cell were monitored by laser scanning confocal microscope (LSCM). The results showed that MM-LDL (100～300 mg/L) induced decrease in cell viability in a dose- and time- dependent manner. After 48 h exposure to 300 mg/L MM-LDL, apoptosis with cell shrinkage, chromatin condensation and nuclear fragmentation were observed. FCM assay showed that the apoptotic ratio rose with an increase in concentration of MM-LDL. MM-LDL caused a rapid elevation of ［Ca²⁺］_i in HUVECs partly through calcium influx. Incubation with MM-LDL induced calcium-dependent cPLA₂ activation companied with its phosphorylation. Inhibition of cPLA₂ activity with 5 mmol/L EGTA or 15 μmol/L AACOCF₃ reduced MM-LDL-induced apoptosis by 41.8% and 33.6%, respectively. Addition of exogenous AA (50 μmol/L) reversed AACOCF₃-induced reduction of apoptosis. It was demonstrated that MM-LDL induced apoptosis in HUVECs. cPLA₂ activated by increase in intracellular Ca²⁺ was involved in the signal pathway.

Abstract:Effort was made to screen expressed genes of a white-rot fungus Trametes gallica by using cDNA microarray from Phanerochaete chrysosporium, a well known model strain used for studying lignin-degrading systems. The results showed that there were only 172 positive clones detected on the microarray and 95.9% of the clones had a ratio of Cy-5/Cy-3 from 0.5 to 2.0. However, only 3 and 4 clones with the Cy-5/Cy-3 ratio over 2.0 and lower than 0.5 respectively, were found, which are corresponding to 5- and 12-day cultures of T.gallica. One hundred and twenty-two clones were randomly selected and sequenced, of which 118 clones could be perfectly located in the genome of P.chrysosporium. The result indicates that there is obvious sequence difference between probes from T.gallica and inserts on the chip from P.chrysosporium, exhibiting far relative between these two fungi. Two interesting clones have been obtained from homology comparison, one corresponding to a fragment of peroxidase lpoB gene of P.chrysosporium and the other encoding a sort of heat shock protein.

Abstract:Two-dimensional electrophoresis (2-DE) was employed to compare the global protein patterns between human macrophage cell line U937 infected and that uninfected with Mycobacterium tuberculosis (MTB) isoniazid (INH) resistant strains, and 32 protein spots were found to express differentially. Five protein spots which remarkably increased in U937 infected with MTB INH-resistant strains were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from peptide mass fingerprinting were used in protein database search. Five protein spots in gel were identified as HSP105_β, inhibitor of apoptosis protein-1, phosphoglycerate mutase 1, cathepsin B, desmocollin 3. These data provide insight into the changed global protein patterns of the U937 while in stasis shortly after infection and may prove useful for further study in the interaction between MTB INH-resistant strains and host.

Abstract:In order to reveal the events during the course of killing target cells of immunotoxins, two forms of Pseudomonas exotoxin A based immunotoxins against carcinoembryonic antigen (CEA) were constructed and named CEA(Fv)/PE38/KDEL and PE35/CEA(Fv)/KDEL. Antigen binding activity of the immunotoxins were assessed by flow cytometry, internalization of immunotoxins was detected by optical confocal microscopy and flow cytometry quantitatively. Cell apoptosis and necrosis were analyzed by staining the cells with FITC-annexin V/PI. The MTT method was used to assay the cytotoxicity of the immunotoxins. The biological functions of the two forms of immunotoxins in the different functional phases were compared. And the interaction between different functional phases was analyzed. The results revealed the whole functional process from the cell binding to the cytotoxicity of the immunotoxins, which provided more details about the biological functions of immunotoxins and could be valuable in the mechanism research and the optimization of immunotoxins.

Abstract:With the rapid development of the genetically modified plant, more and more genetically modified plant food has been pouring into the market. It has been paid much attention to detection of GMP under the controversy of GMP safety. Electrochemiluminescence method is the chemiluminescent reaction of species that are generated electrochemically at the surface of an electrode. It is a high efficiency and accurate detection method. Electrochemiluminescence PCR method makes electrochemiluminescence technique, PCR technique and two nucleic acid probes hybridization technique band together for the first time. In this method, whether plant containing GM component by detection of CaMV(Cauliflower mosaic virus)35S promoter are distinguished. The results show thus, it is indicate that, the method can detect genetically modified plant exactly. The method is simple, reliable and correctly for analysis of GMP.

Abstract:Proteins are core executors of biological activity inside cells, therefore, construction of protein-protein interaction network is so important to interpret protein function and decipher the secret of various cellular phenomena. Tandem affinity purification (TAP) is recent advance in technology made over past few years now enables the study of protein-protein interaction in vivo and the knowledge gathered from such methods will decipher the protein complexes network. Now, it is also a powerful tool available for proteomics research. This new technology will continue to be developed as it is now became extraordinary valuable to study protein-protein interaction.