Abstract:Protein synthesis is generally known as consisting of three steps: initiation, elongation, and termination. Much less known is the fourth step: disassembly of the posttermination ribosomal complex and recycling of the machinery necessary for the next round of translation. In bacteria, after reaching the end of a protein-coding sequence, the ribosome binds release factor RF-1 or RF-2 in response to stop codon in the ribosomal A site, activating hydrolysis of the polypeptide chain from peptidyl-tRNA. Release factor RF-3 then catalyzes dissociation of RF-1 or RF-2, leaving a posttermination complex consisting of the 70S ribosome, mRNA, and deacylated tRNA in the P site. How the posttermination complex is disassembled for next round of protein synthesis is a very basic process. The possible mechanisms of posttermination complex disassembly during protein synthesis was summarized: the forth step of protein synthesis is catalyzed by the concerted action of ribosome recycling factor (RRF) and elongation factor G (EF-G).

Abstract:Pyrrolysine, known as the 22nd amino acid, is found in Methanosarcina barkeri (M.barkeri) methylamine methyltransferases. It comes from the sense decoding of the UAG amber stop codon. It has specific pyrrolysyl-tRNA synthetase and tRNA^Pyl. tRNA^Pyl has noncanonical secondary structure. M.barkeri has two routes: direct and indirect routes to synthesize pyrrolysyl-tRNA^Pyl. The special structure in the mRNA and other unknown mechanisms may control the decoding of UAG as pyrrolysine or termination signal. Pyrrolysine was compared with the 21st amino acid: selenocysteine.

Abstract:The estrogen-related receptor ERR, belonging to the nuclear receptor superfamily, is the first orphan nuclear receptor to be identified. ERR comprise ERRα, ERRβ and ERRγ. ERRs participate in estrogen signal pathway in various patterns and they share common transcriptional target genes with estrogen receptor in muscles and breast. Analysis of ERR expression in human breast cancer has proposed ERRα and ERRγ as prognostic markers of this cancer. ERR also play an pivotal role in the metabolism pathway. The natural ligand of ERRs have not been identified till now, so identification of modulators (positive or negative) of ERR activities would be highly useful in understanding of estrogen-related pathologies, such as human osteoporosis, breast cancer and diabetes.

Abstract:Connexins form gap junctions that mediate the transfer of ions, metabolites, and second messengers between contacting cells. Connexin31(Cx31) is an important member of connexin β family. Mutations in Cx31 are associated with erythrokeratodermia variabilis(EKV) ， hearing impairment and peripheral neuropathy. The pathological mechanism for Cx31 mutants in these diseases remains unknown. Assembly, intracellular transport, plaque assembly and stability and channel conductivity of Cx31 are finely regulated and likely involve proteins that interact with Cx31. However, little is known about the Cx31 interaction proteins. And then the differential immunogenic peptide was synthesized, the purified peptide was coupled to the hemocyanin from keyhole limpets(KLH). The peptide-KLH solution was mixed with freund's complete or incomplete adjuvant and immunized rabbit to prepare specific polyclonal antibody against the topology of connexin31. A proteomics approach was used to screen Cx31 binding proteins using HT1080 cells stably expressing a myc-tagged Cx31. Immunoprecipitation followed by peptide sequence analysis identified association of actin and annexin Ⅱ . Interaction between annexin Ⅱ and Cx31 is further confirmed by coimmunoprecipitation, and immuno-colocalization. This result would be useful to study the function of connexin31 further.

Abstract:Laryngeal carcinoma related gene LCRG1, cloned by the laboratory using mRNA differential display, has the suppressive function to none expression LCRG1 Hep-2 cell line. Bioinformatics analysis using software showed LCRG1 may play function in cellular signal transduction. In order to further elucidate the function of LCRG1, RT-PCR and colony efficiency were used to identify whether LCRG1 expressed and had the tumor suppressive function in incubated Hep-2/LCRG1 cell lines. The results suggested LCRG1 was expressed in Hep-2/LCRG1 cell lines and had the significant suppressive proliferation ability. Hence, the total proteins of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines were separated by immobilized pH gradient(IPG)-based two-dimensional gel electrophoresis(2DGE), coupled with anti-tyrosine phosphorylated antibody immunoblotting and matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS) identifying tyrosine-phosphorylated proteins. The well-resolved, reproducible 2DGE patterns of Hep-2/LCRG1 and Hep-2/pcDNA3.1(+) cell lines were established and 13 differential tyrosine-phosphorylated proteins were identified using immunoblotting, analysis software and MALDI-TOF-MS methods. These proteins were involved in the signal transduction and cell cycle. So it was speculated that LCRG1 may be involved in the processes of cellular proliferation, metabolic pathways and apoptosis etc. and play tumor suppressive functions through regulating the phosphorylation/ dephosphorylation status of these proteins. These data will be helpful to elucidate the molecular mechanism of LCRG1 tumor suppressive function.

Abstract:In order to study the effect of the cellular repressor of E1A-stimulated genes (CREG) on differentiation and migration of human vascular smooth muscle cells (VSMCs)-HITASY, the full length human sense and antisense-CREG cDNA retroviral vectors, pLNCX₂( ＋ )/CREG and pLXSN( － )/CREG, were constructed. Western blot and immunoflourescence analysis showed that the expression of CREG and SM α -actin increased in HITASY after infection with pLNCX₂( ＋ ) /CREG. The migration of HITASY infected with pLNCX2( ＋ )/CREG obviously enhanced compared with that of normal HITASY and pLXSN( － )/CREG cells showed by scrape-wounding and time-lapse analysis. Moreover, CREG over-expression increased the secretion of MMPs in HITASY tested by Western blot. Gelatin SDS-PAGE zymography analysis revealed that the activities of MMPs also increased in HITASY infected with pLNCX₂( ＋ )/CREG . On the other hand, the opposite effects were observed when CREG expression decreased by using antisense pLXSN( － )/CREG . These results suggest that CREG may be able to induce the VSMCs differetiation and promote the VSMCs migration in the meantime.

Abstract:Toll-like receptor 2 is an important pattern recognition molecule of innate immune, which could recognize diverse pathogens and their products. Using the eukaryotic expression TLR-2 extracellular fragment as target to screen peptide mimics to ligand of TLR-2 from Ph.D.-12 phage display peptide library, 20 of positive phage clones were sequenced , which shared a very conservative sequence and was named as P12-1. Biotinylated peptide P12-1 could bind with different form of TLR-2 extracellular fragment, and also stimulate THP-1/CD14 cells to secrete TNFα. These results indicated that P12-1 could mimic the structure and activity of ligand of TLR-2.

Abstract:The single mutant BR_E204Q and tri-mutant BR_{I119T/T121S/A126T} of bacteriorhodpsin were constructed by directed-mutation method. The M412 Flash-dynamics spectrum indicates that the life of M intermediate of the single mutant BR_E204Q is 7.10 ms and the life of M intermediate of the tri-mutant BR_{I119T/T121S/A126T} is 8.23 ms, whose lives of M intermediate are longer than that of the wild-type BR with life of the M intermediate 6.23 ms. The result shows that the tri-mutant has the longest M intermediate life which exceeds wild-type BR by thirty-two percent. The Proton-pump functional research of the two mutants illustrates that their proton-pump functions decresae compare with that of wild-type BR respectively and the proton-pump function of the tri-mutant BR_{I119T/T121S/A126T} is the weakest one.

Abstract:Protein A and protein L are bacterial cell wall proteins of importance in pathogenesis which have different crystal structures and bind different sites of host immunoglobulin (Ig). A pair of primers containing SacⅠ sequence were synthesized to amplify A,B,C,D domain of protein A and B3 domain of protein L by PCR respectively. After digestion with restriction enzyme SacⅠ , these PCR prepared Ig-binding domains were ligated randomly with each other to come into being a combinatorial molecular library. The library was displayed on phage surface by cloning into SacⅠ site of phagemid pCANTAB5S. The capacity of the phage library were calculated as 3.4×107 clones,and the titer was 6.2×1010 TU/ml. The sequence analysis showed that the displayed DNA fragments in the library comprise of various Ig-binding domains ligated in random. After three or four rounds affinity selection with human Ig, 36 positive clones were sequenced at random to analyze structure of the recombinant molecules. The sequence analysis showed 3 kinds of new molecular structures existed in the selected molecules which were totally different from its natural molecules. The characteristic structure of (MDPL-MDPA)n which consists of the repetition of mono-domain of protein A(MDPA) and mono-domain of protein L (MDPL) existed predominantly in 32 of 36 positive clones. The effort to proceed molecular evolution study of Ig-binding domains combinatorial molecular library by phage display not only provides potent approach for research involved in the relationship between structure and function of Ig-binding molecules, but also a basis for Ig-binding molecules rebuilding.

Abstract:Fusion protein IIn-UK was constructed by fusing ScFv specific against human fibrin with low molecular urokinase with linker (G₄S)₃, and this fusion protein was a potential targeting thrombolytic agent. But the fusion protein leaned to be proteolysed while expressed in CHO cells. To overcome this problem, four new linkers were selected from linkers database by using the program at http://ibivu.cs.vu.nl /programs/ linkerdbwww, and four IIn-UK fusion genes were reconstructed by replacing linkers. And other four fusion genes were reconstructed by changing reletive position of two moities and replacing the linker. The degree of proteolysis of eight reconstructed IIn-linker-UK or UK-linker-IIn fusion proteins were analysed with Western blot by using anti-urokinase antibody, the results showed that all eight constructed fusion proteins were degraded partly while expressed in CHO cells. So three new IIn-linker UK fusion genes were constructed by using a new linker coming from pro-urokinase and removing one or two cleavage site of proteolytic enzyme around the linker. The Western blot showed that IIn-UK fusion protein removed two protyolitic enzyme cleavage sites had good anti-proteolysis ability in COS7 cell and CHO cell. Thus it laid a foundation for preparation of IIn-UK fusion protein in CHO cells and further research of targeting thrombolytic agent.

Abstract:Calcium oscillation may regulate gene transcription in a frequency-decoding manner during agonist stimulation, which provides an indicator of transcription level in cells. To determine whether persistent exposure to hypoxia may sensitize or blunt cell response to histamine, the effects of 24 h subacute mild hypoxia on histamine-stimulated calcium oscillation frequency were examined in pulmonary artery endothelial cells (PAECs). The results are: (1) 24 h subacute mild hypoxia significantly increased the histamine-stimulated calcium oscillation frequency in PAECs. The averaged frequency of calcium oscillation in posthypoxic PAECs was significantly higher than that in normoxic ones. (2) NADPH oxidase inhibitor, diphenylene iodonium chloride (DPI, 10 μmol/L), abolished histamine-stimulated calcium oscillations both in normoxic and posthypoxic PAECs. (3) Xanthine oxidase inhibitor, oxypurinol (100 μmol/L), did not affect the calcium oscillation frequency in normoxic PAECs. However, it significantly decreased the elevation of calcium oscillation frequency in posthypoxic PAECs. These results demonstrated that, during pulmonary disease related to persistent hypoxia, PAECs become more sensitive to histamine. During histamine stimulation, NADPH oxidase plays a critical role in generating calcium oscillations, while xanthine oxidase may contribute to, at least in part, the increase of calcium oscillation frequency in posthypoxic PAECs.

Abstract:To investigate the regulatory function of chaperone interacting protein CHIP in TGF-β signal pathway, a tetracycline induced CHIP expression stable cell line (Mv1Lu-Tet off-CHIP) was generated. In this cell line model, it was observed that overexpression of CHIP dramatically reduced the endougenous Smad2/3 protein level. Luciferase reporter analysis showed that CHIP inhibited the Smads induced transcription activity. Furthermore, Western blot results indicated that CHIP could downregulate gene expression of JunB, a quick response gene induced by TGF-β. These results provided new evidences that CHIP might be a novel signal protein negatively regulating TGF-β signal pathway.

Abstract:The effect of the thickness of ultrathin sections on the topographical contrast in the images of atomic force microscopy (AFM) had been studied. Three different cell lines, Tca8113, C6 and ECV-304 were treated with conventional TEM fixation and embedment techniques. Cut by ultramicrotomy and collected with mica pieces, thin sections with different thickness were imaged by AFM on both sides. The images observed from the lower surfaces showed that the cell regions were always concave compared to the epoxy resin regions, and the concavity was increased with the increase of the ultrathin section thickness. Interestingly, the images from upper surfaces showed a peculiar convexness in the cell regions when the thickness is small, and achieved a maximum when the thickness was about 80 nm, and finally became concave when the thickness was larger than 120 nm. Statistic analysis showed that this trend was a general phenomenon. The relevant mechanism has been discussed.

Abstract:An antimicrobial polypeptide was isolated and purified from the acid soluble proteins of human LAK cells. Its N-terminal amino sequence was identical to HMGN2 (high mobility group nucleosomal binding domain 2). Mass spectrum identification and Western blotting analysis also indicated its individual character of HMGN2. The antimicrobial assay showed that MICs of the recombinant HMGN2 against E.coli ML-35p (an ampiciline-resistance strain), Pseudomonas aeruginosa ATCC 27853, and Candida albicans ATCC 10231 were 12.5, 25, and 100 mg/L respectively. In contrast, the recombinant holo-HMG-17 was inactive against Staphylococcus aureus ATCC 25923. The immunocytochemistry staining, ELISA, and Western blotting revealed that HMGN2 was present in the cytoplasm of mononuclear leukocytes and released to the extracellular environment when stimulated with IL-2. The results indicated that HMGN2 was a new effector molecule of human LAK cells.

Abstract:Human embryonic intestinal tissues were digested by collagenase, epithelial cells were cultured, glucagon-like peptide 1(1~37) was adapted to induce human embryonic intestinal epithelial cells (hEIECs) to differentiate, and hEIECs without GLP-1 induction were set as control. hEIECs were successfully isolated and cultured, they were stained by cytokeratin 18 and cytokeratin19, the marker for intestinal epithelial cells; they were also stained by somatostatin and glucagon, the hormones secreted by endocrinal cells from pancreas, but they were negative for insulin. After GLP-1(1~37) induction for 6 days, insulin-positive cells could be identified in intestinal epithelial cells by immunocytochemistry and RT-PCR showed that these cells expressed pancreatic duodenal homeobox-1 (PDX-1), glucose transporter-2 (GLUT-2) and insulin. No insulin-positive cells were identified in the control (no GLP-1(1~37) induction), RT-PCR showed that the control expressed PDX-1 and GLUT-2, but not insulin. The findings suggest that GLP-1(1~37) could induce insulin production in developing human embryonic intestinal epithelial cells in vitro, and GLP-1(1~37) may represent a new therapeutic approach to diabetes mellitus.