Abstract:Prion diseases are thought to arise through misfolding of the cellular protein PrP, which can exist in both cellular, PrP^C, and pathological, PrP^Sc, forms. According to the “protein only” hypothesis, disease results from infection with the misfolded prion form of the protein, or by inherited mutations in the PrP gene which apparently increase the propensity of the protein to misfold. The result is one of a number of devastating neurological diseases, which are inevitably fatal and are characteristed by spongiform changes in the brain. Hence prion diseases are also known as transmissable spongiform encephalopathies (TSEs). New advances in prion research were reviewed focusing on the structural characteristics of the PrP protein. Putative mechanisms for the conversion between PrP^C and PrP^Sc, and the factors thought to influence this change, are described. Progress in determining the physiological function of the PrP protein and prospects for diagnosis and treatment are discussed.

Abstract:Nociceptin was discovered at the end of 1995. It is the endogenous ligand of opioid receptor like 1 (ORL1 or LC 132) receptor. It plays an important role in pain modulation, cardiovascular system, ion channel, dependence and tolerance, learning and memory, etc. It is becoming a new research field to investigate the structure-activity relationship of ORL1 receptor and its ligands in recent years. Now, the studies on NC fragments, agonists, partial agonists, and antagonists that were found in the course of investigating the structure-activity relationship of nociceptin are reviewed.

Abstract:Discrimination between self and non-self is the basic function of innate immunity that is found in both vertebrates and invertebrates. A critical first step in any immune response is the recognition of an invading organism as foreign. In the innate immune systems of both vertebrates and invertebrates, such recognition, termed pattern recognition, is mediated by a group of proteins, known as pattern recognition proteins or receptors. Different pattern recognition proteins recognize and bind to molecules (pathogen associated molecular patterns, PAMPs) present on the surface of microorganisms but absent from animals. Binding of pattern recognition proteins to PAMPs triggers responses such as phagocytosis, nodule formation, encapsulation, activation of proteinase cascades, melanization and synthesis of antimicrobial peptides. The characterizations and functions of several classes of pattern recognition receptors involved in immune responses of the invertebrate, including peptidoglycan recognition proteins, thioester-containing proteins, Gram negative binding proteins, scavenger receptors, C-type lectins, galectins, Toll-like receptors and hemolins were reviewed.

Abstract:Expression vectors are critical to high efficiency expression of foreign proteins. Thus a vector containing human elongation factor 1α subunit promoter (P_EF-1α) for transcription of genes of interests, and mouse dihydrofolate reductase (dhfr) gene under control of SV40 promoter (P_SV40) for clonal selection and amplification was first constructed . The vector was named pED5. The expression efficiency difference between pED5 and pCdhfr1, a vector utilizing CMV enhancer/promoter (P_CMV-IE) for foreign protein production, was analyzed using human interferon-β (IFN-β) gene and human secreted alkaline phosphatase (SEAP) gene as reporters. When analyzed in transient expression, pED5 showed a little more protein produciton than pCdhfr1. However, in continuous expression, when serum concentration was lessened to slow down cell proliferation, pED5 expressed 3.1 times more reporter proteins than pCdhfr1, which implied that P_EF-1α was less affected by cell cycle status in contrast to P_CMV-IE, making pED5 a good expression vector for foreign protein production.To further enhance protein productivity of expression vectors, two artificial transcription activating domains, AH and VP2, were linked to cI repressor protein of phage λ through a soft linker, respectively, and thus two artificial transcription activators were created. The O_R2-O_R1 sequence of phage λ was inserted into P_EF-1α about 200 bp before TATA box. The artificial transcription activators can bind to O_R2-O_R1 sequence, and are thus conscripted near to the TATA box of P_CMV-IE, activating gene transcription with artificial activating domain AH or VP2. All the components mentioned above were integrated into pED5, producing two vectors: pER-AH and pER-VP2. The two vectors were tested for their efficiency of expressing IFN-β and SEAP reporter genes in comparison with pERFRT, a vector similar to pER-AH and pER-VP2 except for lacking of artificial transcription activators. To abolish transcription efficiency affected by copy number or integration site in chromosome, all the vectors were integrated into FRT site by co-transfecting with an Flp recombinase producing plasmid. The FRT site was pre-inserted into CHO cell chromosome by Invitrogen Corporation. It was showed that pER-AH was only a little more efficient than pERFRT, however, pER-VP2 was 2.7 times more efficient than pERFRT in expressing reporter genes.

Abstract:In order to study the effect of IFN-γ on ATP binding cassette transporter A1(ABCA1) expression and cholesterol efflux in THP-1 macrophage-derived foam cells, after exposure of the cultured THP-1 macrophage-derived foam cells to IFN-γ for different time, cholesterol efflux and ABCA1 mRNA and protein level were determined by FJ-2107P type liquid scintillator and reverse trancriptase-polymerase chain reaction(RT-PCR) and Western blot, respectively and the mean ABCA1 fluorescence intensity on THP-1 macrophage-derived foam cells was detected by flow cytometry. It was demonstrated that exposure of the cultured THP-1 macrophage-derived foam cells to IFN-γ for different time results in decreasing cholesterol efflux, the expression of ABCA1 mRNA and protein and the mean ABCA1 fluorescence intensity in THP-1 macrophage-derived foam cells in a time-dependent manner. Exposure of the cultured THP-1 macrophage-derived foam cells to IFN-γ for different time results in increasing total cholesterol, free cholesterol and cholesterol ester in THP-1 macrophage-derived foam cells in a time-dependent manner. It can be concluded that IFN-γ decreases cholesterol efflux and the expression of ABCA1 in THP-1 macrophage-derived foam cells.

Abstract:A new lectin extracted from lablab can maintain the multipotential of hematopoietic stem/progenitor cells in vitro for a long time in addition to its hemagglutination property. This new lectin FRIL (Flt3-rectptor interacting lectin) was purified from Dolichos lablab, its biological speciality was identified and its receptors were located on hematopoietic stem/progenitor cells. When CD34⁺ cells were cultured in the presence of this lectin or its counterpart FL, within the 28 days of culture, FRIL can keep more cells in G0/G1 phase (beyond 80%) and 1.5 fold colony forming cells more than FL does after 14-day's culture. So, it is concluded that FRIL maintain HSC/HPCs self-renewal property by halting its cell cycles.

Abstract:A glucocorticoid receptor(GR) interacting protein JAB1 was isolated from human marrow cDNA library by two-hybrid screening in yeast using the GR ligand-binding domains (GR-LBD) as bait. To further demonstrate the interaction between GR and JAB1 and the effect of JAB1 on GR, PCR was performed to amplify GR-LBD fragments and it was cloned into the bait vector pGBKT7 to create the plasmid pGBKT7-GR LBD. The plasmid was used as bait to screen a cDNA library constructed in the pACT2 vector. Transformants were plated on SD/－Ade/－His/－Leu/－Trp. Yeast colonies were assayed for α-galactosidase activity. The positive colonies were sequenced in which JAB1 cDNA fragments were cloned into the vector pGEX-4T-1 for GST pull analysis. A GRE-driven reporter gene was used for CAT activity assay. The results were as follows: 42 clones turned blue in the yeast α-galactosidase assay and contained pACT2 sequence in which 5 clones corresponded to a fragment of JAB1 as shown by DNA sequencing. Yeast two-hybrid and GST pull down assay verified that JAB1 interacted with GR-LBD. GR, a GRE-driven reporter gene cotransfected with JAB1, strongly potentiated the transactivation properties of GR. The studies suggest that JAB1 interacts with GR-LBD expressed in COS7 cells in vitro and it potentiates the transactivation properties of GR.

Abstract:Using a bioinformatical approach, a hybrid gene encoding the Fc-binding domains of both SPA and SPG (1 269 bp) was designed and synthesized. The sequence was designed by substitution of high-usage codons for low-usage ones of Escherichia coli. A total of 64 oligonucleotides were assembled in a single-step, in which T4 DNA ligase ligated DNA fragments from a pool of overlapping oligonucleotides. The synthetic gene was cloned to pSK vector. After sequencing and remending, the gene was expressed using the expression vector pET-28a-c(+). A band of 47 ku was detected with Western blot analysis. The protein so called MproteinAG has been covalently conjugated to the carboxyl-terminated magnetic particles. With the MproteinAG magnetic particles,IgG from blood serium of rabbit, pig,dog,mouse, human, macaque, rat, cat, sheep, cattle, horse can be purificated in one hour.

Abstract:The deduced amino acid sequence of the VER2 gene was analyzed in a computer. A peptide corresponding to 204～215 amino acids in VER2 sequence was synthesized by chemical method and purified using HPLC. The polypeptide was conjugated with BSA and the covalent complex was used as antigen to immunize rabbits. The antibody was then affinity purified before being used to probe VER2 protein in Paraplast sections. The immunolocalization results detected by the peptide antibody were consistent with that of the full length VER2 protein antibody. It is concluded that antibody prepared with synthetic peptide is credible and sensitive enough for immune labeling of proteins in paraplast sections. It is a sensitive and effective approach to combine preparation of antibody from synthetic peptide with paraplast section production for protein immunocytochemistry study.

Abstract:A host-plasmid lethal balancing system was constructed based on asd gene in an avirulent strain of S.flexneri to express coli surface antigen 6 of enterotoxigenic Escherichia coli. The results of Western-blotting demonstrated that avirulant strain of S.flexneri FWL01 expressed CS6 steadily. Immunofluorescence analysis showed that S.flexneri FWL01 carrying the plasmid pZLG6 can be excited fluorescence on its surface. Antibodies against CS6 and LPS of Shigella can be detected in sera of mice immunized with recombinant bacteria either orogastrically (o.g.) or intranasally(i.n.); simultaneously sIgA against CS6 can also be detected in the intestine. This is helpful for constructing multivalent recombinant vaccine for prevention of bacterial diarrhea.

Abstract:Mismatches during amplification result in some mutations in the PCR products. The simulation indicates that the rate of mutation is quite low and has no influence on the consequent sequencing analysis when template DNA molecules are abundant, but it is not the case when the PCR amplification is performed with single or few molecules as templates. The error rates of single molecule PCR under different conditions showed that it is essential to select different polymerases according to different experimental purposes.

Abstract:Avian influenza (AI) was caused by A avian influenza virus in chicken all over the world. One 1.6 kb DNA fragment was amplified by RT-PCR from the RNA of AIV A/Chicken/Henan/1/1999(H9N2) and proved to be HA gene by sequencing. The HA gene expression plasmid pVAX-H9 was constructed by inserting the fragment into the pVAX1 vector. 3-week-old SPF chickens were inoculated with 50 μg DNA of plasmid pVAX-H9 by electroporation, 5 weeks later, all chickens were challenged with 100×EID50 of AIV A/Chicken/Henan/1/1999(H9N2), 1 week post challenge all birds were sampled by cloacal swabbing to isolate virus. The virus isolation was negative in all vaccinated birds and positive in all control birds. The HI titers reached to 10lg2 in vaccinated group, in contrast to 2lg2～4lg2 in control group post challenge respectively. These results showed that the plasmid pVAX-H9 designed as DNA vaccine would be able to elicit a firm protective immune response against AIV infection.

Abstract:The Logistic model is improved for the description of severe acute respiratory syndrome (SARS), which is infecting all over the world especially in China. The model simulates the situations of SARS in different countries and in different provinces of China. The results reveal the asymmetrical phenomenon of the infection of SARS. The conditions that cause the infectious disease in natural circumstance and the factors that added by human for the control of SARS are considered in the model. The different measures, which are used to control the disease, can lead to different results. The phenomenon of super-spread events (SSEs) is also discussed.

Abstract:A new simple and effective approach completely based on the physical theory is proposed for protein design. Compared with the similar works, the algorithm saves a vast deal of trouble in exploring sequence space. The method is an improvement over previous works. The design procedure was tested on three lattice models in two dimensions and the successful results have been obtained. The method can be readily implemented for three-dimensional off-lattice models of real proteins.

Abstract:ARF can induce cell cycle arrest or apoptosis. By using the A375 cell overexpressing p14^ARF as a model, the molecular machanism of cell cycle arrest by ARF was studied. It was indicated that overexpressing p14^ARF resulted in G1-phase and G2-phase arrest. In the A375 cells overexpressing p14^ARF, the level of p53, p21^cip1 and p27^{kip1 was upregulated, but the protein lever of p-ERK1/2, Cyclin D1 and Cyclin E was inhibited. The results indicated that p14ARF induced the cell cycle arrest of A375 not only in a p53-dependent way, but also in coopration with the ERK signal transduction pathway.}

Abstract:The PCR production of vascular endothelial growth factor (VEGF) was subcloned into a T-vector, and the recombined plasmids were then transformed into competent cell JM109. Eight white clones were selected randomly, and the recombined plasmids were extracted and mixed as template. Three selected primers were adapted and two rounds of PCR were performed to introduce mutations. The production of those were confirmed by enzyme digestion, PCR amplification and sequencing. The method is proved to be a simple，quick and easy way to introduce site-directed mutagenesis, and make latter work easy to do.

Abstract:Semiconductor quantum dots（QDs）are inorganic nanocrystals consisted of a cadmium selenide (CdSe) core which was wrapped in an outer shell of zinc sulfide (ZnS). In comparison with common organic dyes, this class of fluorescent labels is 20 times as bright, 100 times as stable against photobleaching. The emission wavelength of QDs was controlled by the size of the core and each single-color of QDs has narrow symmetrical emission peak. These advantages of the optical properties make them broad applications in medical diagnosis，high-speed screening drugs and high-throughput analysis of genes and proteins. Based on the stability and biocompatibility of the QDs, it is possible to make movies of long-term interaction of biological molecules in a living cell by tagging each biomolecule with different color of QDs.