Abstract:Intracellular antibody refers to the recombinant antibody expressed intracellularly.Single-chain Fv fragment,one common form of intracellular antibody with high affinity,can be expressed in transfected nonlymphocytes and targets to a particular cellular compartment to interfere the activity of some macromolecular substances or the process of their secretion.Intracellular antibody has been proved to be capable of inhibiting growth factor receptors,inactivating oncoproteins and inhibiting HIV-1 replication. Intracellular antibody technique is a novel gene therapy approach with potentiality in medical application.

Abstract:During long period infection of HIV, plenty of reactive oxygen species are accumulated in patients, and then oxidative stress forms. Oxidative stress mediated by reactive oxygen species can activate a transcription factor, NFκB which in turn stimulates HIV gene expression.Besides, oxidative stress causes the disturbance of biological functions of infected patients'bodies, such as DNA damage, loss of Ca²⁺ homostasis, incurable destruction of enzyme system, block of energy production and so on, It is still at experimental stage to use antioxidants in AIDS therapy.

Abstract:The recent theoretical researches on the conductivity in proteins were reviewed. The investigations of the past decades were briefly introduced and commented. It was discussed that the approximations and numerical methods on the calculations on the electronic structures and theoretical conductivity of an entire molecule of proteins. The basic characteristics of the electronic structures and theoretical conductivity of proteins were concluded from the calculated results. Finally, an electronic channel model on the trans-membrane signal transformation by insulin and its receptor was presented from those results of insulins.

Abstract:Bacteriorhodopsin (BR) is the only protein in the purple membrane (PM) from Halobacterium halobium. Wild-type BR contains a single-chain polypeptide of 248 aminoacid residues and a retinal attached to Lys216 of the peptide via a schiff base, BR has the function of proton pump, Upon illumination, BR goes through a photocycle which is considered to be correlated with its proton pumping process. The good progress has been made in the studay of the structure and function of BR, but the path of photocycle and mechanism of proton pump are still not clear. The progress of the structure, photocycle and proton pump of BR in recent years are briefly introduced. And the four models of the path of photocycle which are in great argument are discussed in detail.

Abstract:The insulin protein engineering is quickly progressing. The recent progesses in the studies of fast-acting, slow-acting and high potent-acting insulins, including their molecular design, biological activity and clinical prospect, which were obtained by means of protein engineering are introduced. The progesses in the receptor binding sites of insulin and the intereaction of insulin with its receptor is also discussed.

Abstract:Superoxide dismutases (SODs) are metal-containing enzymes that catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide. Three classes of SODs,Fe-SOD, Mn-SOD and CuZn-SOD, have been distinguished according to their bound metals.The Fe-SOD is essentially a prokaryotic enzyme,while the CuZn-SOD is essentially an eukaryotic enzyme. The Mn-SOD is present in both prokaryotes and eukaryotes. The Fe- and MnSODs appear to highly similar in the primary structure, three-dimensional structure and other physiochemical properties, but have no resemblance to CuZn-SOD. The Fe- and Mn-SOD can be traced to a common ancestor, whereas CuZn-SOD has evolved independently at a later time.

Abstract:DNA damage is the cause of gene mutation and contributes to cell transformation.The cellular response to DNA damage is complex in its components and regulation. First, DNA repair need cell cycle arrest to avoid mutation accmulating in generated cell. Second, there is strong connection between repair and transcription. Third, nucleotide excision repair and mismatch repair are important pathway of mutation avoidance. If any event mentioned above was altered, mutations would accumulate, and the chance of tumor developing would increase. The relationship between DNA repair,transcription and the cell cycle, and the cell transformation were discussed.

Abstract:In the research of cell cycle regulation, the knowledge of the functions of cyclinCDKs in the cell cycle regulation has been made a signifcant progress. Recently, the isolation and studies of the family of CDK inhibitor proteins, p16 and p21, imply that cyclins and CDIs form a very complicated cascad regulation network in the regulation of the activity of CDKs,through which they impose control upon the proliferation and differentiation of cell, are related to the formation and development of cancer.

Abstract:The methods and progress of knowledge-based protein three-dimensional structure prediction are mainly introduced. To date, the methods of knowledge-based structure prediction can be grouped into two categories. One is homologous protein modeling, which is relative mature and can give reliable results, but it is restricted to modeling of the target sequence that shares a high sequence identity with thehomologous proteins. The other method, inverse protein folding, can be empolyed to predict the structure of protein with limited sequence homology, which mainly includes 3D Profile and potential-based functions, and gives the topology of the target protein.

Abstract:Single components and multicomponent of R-phycoerythrin (R-PE ), Cphycocyanin (C-PC ) and allophycocyanin(APC) LB multilayers were prepared. The three phycobiliproteins in the LB films were orderly oriented as in the natural phycobilisomes. The absorption and fluorescence spectra remainedsimilarly to these in their aqueous solutions. The phenomenon of excitation energy transfer among R-PE, C-PC and APC in their stacking LB multilayers were observed through steady-state fluorescence spectral measurements and spectral deconvolutions. From the quenching degree of R-PE fluorescence, the efficiency of energy transfer from R-PE through C-PC to APC in their stacking LB multilayers was calculated to be 51%.

Abstract:Investigation into effects of conformation variation on optical absorption of phycobilins is important in photosynthesis research. It is found that the electronic excited-state of phycobilin exhibits a quasi-Boltzmann distribution due to inhomogeneous random variation of its con formation, and that the envelope of the vibronic absorption transition band can be described as convolution with the inhomogeneous random distribution of con formation variation. Inhomogeneous random distribution of phycobilin conformation variation results in asymmetric broadening of vibronic absorption transition band.

Abstract:Assayed by gel retardation, it is shown that IL-2 treatment on CTLL-2 causes formation of a DNA-binding factor that strongly recognizes a DNA sequence termed gammainterferon activation site (GAS). This DNA binding factor is named as IL-2 nuclear-activated factor (IL-2-NAF). The activation of IL-2-NAF is rapid and does not require protein synthesis. The level of IL-2-NAF activity increases within minutes of IL-2 stimulation and reaches maximum at 1 hour, then begins to decrease. Activation of IL-2-NAF is sensitive to an inhibitor for tyrosine protein kinasc (TPK)，but not to those specific for PKA or PKC. Moreover, protein tyrosine phosphatase(PTPase) or antibodies specific for phosphotyrosine(Anti-P-Tyr) can block the formation of IL-2-NAF-DNA complex. This demonstrates that activation of IL-2-NAF requires tyrosine phosphorylation. IL-4 or y-IFN treatment of CTLL-2 causes activation of common DNA-binding factors that recognize SIE(sis-inducible element)，but seems to have no effect on any DNA-binding factor that recognizes GAS. Thus, IL-2, IL-4 and γ-IFN induce their signals to nucleus by different path-ways in CTLL-2. The data also show that treatment of Hut-102 cell with IL-2 or IL-4 can weakly activate a DNA-binding factor specifically to recognize GAS.

Abstract:Procedures for the determination of the spatial distribution and the dynamic changes of intracellular Ca²⁺ signaling in single intact living cells are described using laser scanning con focal microscopy (LSCM) and the highly fluorescent Ca²⁺ -sensitive dye, fluo-3/AM. The duration, dye concentration, and requirement for Pluronic are determined empirically for the loading conditions. It is especially important for the proper intracellular dye concentration to provide adequate signal strength for detection while not to disturb normal intracellular physiology. For example, in C57BL/6J macrophages, it was found that cells cultured on glass cover slips were incubated for 1 h at 37℃ with 6 μmol/L fluo-3/AM, resulting in excellent LSCM imaging of intracellular Ca²⁺.The similar results have been successfully obtained by this method for other types of cells such as vascular smooth muscle cells, Chinese hamstar oocytes. It may provide an available, visual experimental approach for investigation into the dynamic changes of Ca²⁺ signal and their relationship to transmembrane Ca²⁺ gradient in living cells under the physiological and pathological conditions.

Abstract:The effects of T-2 toxin on the activities of B-type, L-type and T-type Ca²⁺ channels were recorded on cultured ventricular myocardiocytes of neonatal Wistar rats by cell-attached patch clamp technique. T-2 toxin at dose of 10 mg/L significantly inhibited the activities of the three types of Ca²⁺ channels. The open time of Ca²⁺ channels was shortened and the openstate probability was decreased, whereas there were no significant variations in the amplitude of ionic current flowing through the Ca²⁺ channels. Compared with verapamil and Bay K 8644, the effects of T-2 toxin on the Ca²⁺ channels were similar to the blockade effect of verapamil. It was suggested that the cytotoxic action of T-2 toxin may be due to the damages of cell membrane .

Abstract:The following algorithms of RNA secondary structure prediction: the maximum base pairing, Zuker's minimum free energy, the optimal stacking of belieal regions, the random stacking of belieal regions and all possible combinations and the RNA secondary structure drawing based on primary belieal regions have been used to construct RNA secondary structure prediction system named Rnafold. In addition, the former four methods are compared through 20 tRNA sequences in the two aspects of free energy and cloverleaf structure. The statistical difference of free energy was analysed by t-test. From the point of view of cloveleaf structure, the method of randorrT stacking is the best, the second are the optimal stacking and Zuker's algo-rithms and the worst is the maximum base pairing. Finally, the difference between two methods of minimum free energy was analysed.

Abstract:Five purified preparations of ribosome-inactivating proteins (RIPs ), trichosanthin, momorcharin, saporin, luffin and gelonin were shown to exhibit ribonuclease activity, under PH 5.0, with celery 4.5 S RNA as substrates. The autoradiography showed that different RIPS had different base specificities on RNA molecules.

Abstract:The E gene fragment was amplified from RNA isolated the Chinese dengue-2 virus by RT-PCR method. It was 1.29 kb in length. This unmodified E fragment was directly inserted into the EcoRV-cut T-tailed pBluescript ⅡKS⁺ vector DNA. The positive recombinant colonies were identified by the PCR and the restriction endonuclease digesting method. Then nucleotide sequence analysis of the inserted E gene fragment was conducted directly by using the common M₁₃ primer. The result of the restriction endonuclease and sequence analysis confirmed that the 120 by of 5’-teminal nucleotides sequence of the amplified E gene fragment was the same sequence as reported.

Abstract:The purification of cardiac troponin T(cTnT) from human left vertricular muscle has been developed. The 5 mg cTnT were obtained from 100 g cardiac muscle by homogenization, 70℃ treatment, imidazol-HCl dialysis, and DEAE-cellulose chromatography. The purity of total cTnT was 97.6 %. The cTnT was used to immunize Balb/C mice by intraspleenic injection and five hybridoma cell lines (G₃, G₈, G₁₀, A₅, A₇), which secreted stably monoclonal antibody against human cTnT, were obtained by cell fusion, identification, cloning, four lines were IgM, one line IgG by immunological analysis. The number of hybridomas chromosomes were 92～110, the McAb titers from ascitic fluid were about 3.2×10^-6～1. 6×10^-7.

Abstract:Chicken liver dihydrofolate reductase purified by Aga rose-methotrexate (MTX-Agarose) column has been analyzed. The boundsubstrates, dihydrofolate or folate, were removed by flat-bed isoelectric focusing electrophoresis. The effect of the bound-substrates on studying the conforrnation of the enzyme was discussed, and the wrong conclusion in the literature obtained with the enzyme in the presence of bound-substrates was pointed out.

Abstract:The human IL-2 cDNA was cleaved from pHIG53 and inserted in the intermediate vector pSP-72 which has the restriction sites comptible with clone site of the expression vector pDOR-neo, then IL-2 cDNA was directionally inserted into the retroviral vector pDOR-neo to construct a eukaryotic expression vector. The vector was trans feeted into human osteosarcoma cell line by lipofectin. The IL-2 was bioassayed in the culture of tumor cell after selection of G418. The production of IL-2 was 50～800 U per 1×10⁵ cells every 24h, which makes it possible to carry out osteosarcoma gene therapy.

Abstract:A strategy based on polymerase chain reaction (PCR) and a phagedisplay system for the rapid method for constructing and screening functional single chain antibody genes was reported. Total RNA from a hybridoma (C50) producing an antibody that reacts with carcinoembryonic antigen (CEA) was used as a template to make the first strand of a cDNA, lightand heavy-chain variable-region genes were separately amplified by PCR. Then whole single chain antibody gene was synthesized and amplified by using two sets of DNA primers that(1) hybridized to the ends of the light- and heavy-chain variable regions, ( 2 ) encoded a Linker peptide, and(3)contained appropriate restriction enzyme sites for cloning. After 30 cycles of PCR, the DNA fragments containing sequences encoding the light- and heavy-chain variable regions were cloned into pCANTAB 5 phagemid containing gene Ⅲ of the phage. Clones encoding single-chain antibody was express on the surface of the phage as a fusion with gene Ⅲ protein. Recombinant phage expressing anti-CEA single chain antibody which retained its antigen-binding capability was screened by affinity selection and enriched. By using this approach it should be possible to rapidly clone and screen the functional variable region sequences of many different antibodies from hybridoma RNA.

Abstract:Plasma glutamine was measured, following ultrafiltration by high performance liquid chromatography with a mean recovery of 96.75%, linearity of the measurement was in the range of 130 to 1300 μmol/L (r= 0.9906). Normal values in healthy adults was (694.97 ±102.31 ) μmol/L in male and (623.79±99.27) μmol/L in female with a significant difference (P＜0.05 ). The method is simple,efficient in performance and can be applied in the study of bowel structure and function and in routine measuements in nutritional support.

Abstract:Traditional method of constructing recombinant baculovirus was modified: Transfer only recombinant plasmid DNA which was purified by PEG precipitation into insect cells first,then attack these cells with low dose vinous 12～23 hours later. This method is simple, convinient, time saving, economical and has a high ratio of recombinant viruses. It is suitable for general laboratories.