Abstract:Proteomics is an emerging research area that focuses on the repertoires of entire set of proteins of the cells and the activities of global cellular proteins, thus providing a new view on cell activities. A brief review of the background and current progress of proteomics is given.

Abstract:Ribosome is a molecular machine based on RNA,which provides the workshop and tools to synthesize all of the proteins in cells.Its sophisticated structure enables crystallographers regarding it as the Mount Everest for a long time.Recently ,the breakthrough has made for ribosome structure studies.For the first time,several proteins of known three-dimensional structure,and many regions of double-stranded rRNA,have been located in highly complex electron-density maps of ribosomal subunits. And the delicate structure of subunit interface and complicated tRNA,mRNA and ribosome interactions have been revealed.

Abstract:Cre site-specific DNA recombinase system has been developed as a novel powerful tools for manipulating DNA both in vitro and in vivo. It has been used in transgenic mice to induce site-specific DNA recombination leading to gene expression or deletion/mutation not only in a tissue-specific or at a certain stage of development, by combining with inducible systems for controlling Cre expression or function, but also in temporally and spatially manner. These recombination-based strategies are likely to have a profound impact on study of gene function and the generation of animal models of human diseases.

Abstract:Recently the green fluorescent protein (GFP) from the jellyfish Aequorea victoria has emerged. It has been widely studied and used in biochemistry and cell biology. The protein emits green light (λ_max＝508 nm) when excited with ultraviolet (UV) or blue light (λ_max＝395 nm, minor peak at 479 nm). It is extremely useful as a marker for monitoring gene expression and as a tag in studying protein localization in organisms, intact cells and organelle. Mutagenesis and engineering of GFP fused into chimeric proteins are opening new prospect in physiological indicators, biosensors, photo-chemistry and production of luminescent fiber.

Abstract:Lipid-transfer proteins(LTPs) are a group of basic, small (9 ku) proteins which transfer lipids between biomembranes by in vitro assays. So they are thought to participate in the lipid transferring during biomembrane synthesis. Their purification, structure, gene expression and biological functions have been studied in various monocotyledonous and dicotyledonous plants. The latest studies found that they are secreted and located in the cell wall, and that it is suggested that plant LTPs are possibly related with cutin formation, defense reactions against phytopathogens and plant adaptation to various environmental stresses.

Abstract:The function of water-stress-inducible gene products and signal transduction in water-stress response was mainly introduced. Genes induced during water-stress conditions are thought to function not only in protecting cells from water deficit but also in the regulation of genes for signal transduction in water-stress response.At least four independent signal transduction pathways exist between the initial dehydration signal and gene expression,two are ABA independent and two are ABA dependent,one of the ABA-dependent pathways requires protein biosynthesis,one of the ABA-independent pathways overlaps with that of the cold response.

Abstract:With the development of biochip techniques, several new types of biochip, such as bioelectronic chip, gel element microarray chip, drug controlled-release chip, capillary eletrophoretic or electrochromatographic chip, PCR chip and biosensor chip, had sprung up. These biochips, which were different from typical molecular microarrays such as DNA chip, were based on the microarray of various structures, and have successfully applied to analyze DNA mutations, polymorphisms and sequences, to separate mixtures and monitor biomolecular interactions. Because analyses on these chips have many advantages such as quick detection, high efficiency, little sample consumed and low cost, they will become novel tools in the field of life science and medicine.

Abstract:Vision research is very important to reveal the mystery of brain. Functional magnetic resonance imaging(fMRI) is a method for measuring hemodynamic responses to changes in neural activity in the brain. As the activity in the human brain can be observed by fMRI non-invasively with spatial resolution of a few millimeters and temporal resolution of less than a second, fMRI has become an important approach to human brain research since the 1990s. The recent application of fMRI to visual studies has begun to elucidate how the human visual system is anatomically and functionally organized. There is much more work to do to investigate the neural mechanism of the higher-level functions such as mind、attention and memory.

Abstract:Baculovirus expression system(BES) is one of the most important expression systems. Baculovirus also has potential ability as pesticide. DNA replication is the central step in its life cycle. Recent advances of the genes related to DNA replication were discussed.

Abstract:Plasma high density lipoprotein(HDL) can be oxidized in vitro and in vivo as can low density lipoprotein(LDL), which causes many changes in HDL properties, such as peroxidation of polyunsaturated fatty acids, hydrolysis of phosphatidylcholine, apolipoprotein aggregation or degradation. HDL oxidative modification in vivo might be induced by arterial endothelial cells, macrophages and blood polymorphonuclears, monocytes. Ox-HDL might metabolize through scavenger receptors but not normal HDL receptors. Ox-HDL has many atherogenic roles. Vitamin E, C supplementation can inhibit HDL oxidation and may prevent the atherosclerosis.

Abstract:Molecular motors are the proteins that translate the free energy/electrochemical gradient into mechanical work. They are generally divided into two classes: one is linear motor, including myosin, kinesin, dynesin, RNA polymerase and DNA helicase, the other is rotary motor, such as F₁-ATPase and the bacterial flagellar motor. The molecular motors play an important role in the cell transport,DNA replication, transcription, and ATP synthesis.

Abstract:A cDNA encoding a basic phospholipase A₂ (A.aBPLA₂) from Agkistrodon acutus was inserted into a bacterial expression vector pBLMVL2 and effectively expressed in E.coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column superose^TM12. The enzymatic activity of the expressed A.aBPLA₂ is close to those of denatured-refolded native acidic phospholipase A₂ from Agkistrodon halys Pallas, A.aBPLA₂ has the same hemolytic activity as denatured-refolded basic phospholipase A₂ from Agkistrodon halys Pallas, but its inhibiting effect on platelet aggregation is negligible. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A₂ are discussed.

Abstract:The extracellular hemicellulase from Penicillium was immobilized on chitosan by glutaraldehyde.The results indicated that the immobilized-hemicellulase prepared by 0.5 g chitosan crosslinking with 4% glutaralhyde and then combining with 2.5 mg protein showed higher enzyme activity and better activity recoveries(45.6%).The optimum pH of soluble enzyme and immobilized enzyme were pH 4.6 and pH 3.6 respectively.The optimum temperature of soluble enzyme was 55℃, whereas immobilized showed high activity in 60～75℃.Thermal stability of immobilized enzyme was better than soluble enzyme at 65℃.The apparent K_m′ of the immobilized enzyme was 3.58×10^－2 g/L and the K_m of soluble enzyme was 5.0×10^－2 g/L with hemicellulose as the substrate.

Abstract:A molecular beacon probe was used in the detection of prawn white spot bacilliform virus (WSBV). The probe possesses a stem and loop structure. The sequence of loop is complementary to the WSBV DNA, and the stem is formed by two complementary arm sequences which are unrelated to the WSBV DNA. The probe underwent a fluorogenic conformational change as it hybridized to the WSBV DNA. The probe could not affect the amplification , and was specific and sensitive when used in PCR. It was showed that the fluorescent intensities increased as the cycles of PCR and the number of target DNA copy increased.

Abstract:Magnetic fileld was made of Fe₃O₄ particles and soybean licithin which was only 20 nm in diameter. The heat effect was studied under 10 kW, 100 kHz magnetic field. The temperature change was measured by a glass thermometer (±0.1℃) in agarose gel and distilled water with different Fe₃O₄ content respectively. The effect of magnetic intensity was discussed. This was an experimental basis for hyperthermia treatment tumors. The results indicated that the tempereature raised quickly as the increase of Fe₃O₄ concentrations and the magnetic field intensity. The tempereature finally remained constant due to the balance of heat generation and heat transfer to the surroundings. When Fe₃O₄ concentration were 1 g/L and 2 g/L, the balance temperature were respectively 41.9℃ and 47.5℃ as the magnetic field was 2.78×10⁴ A/m. In 1 g/L Fe₃O₄ concentration, the magnetic field intensity were 2.78×10⁴ A/m and 1.11×10⁴ A/m, the temperature plateaus were at 41.9℃ and 30.7℃ respectively. The heat transfer faster in water than in agarose gel.

Abstract:Two three-dimensional structure models of the 21nt oligodeoxyribonucleotides, CP1 (G3TG-2TGT2G5TG2TGT) and CP3 (TGTG2TG5T2-GTG2TG3), were constructed by InsightII (MSI) software in IRIS Indigo2 (SGI) workstation using the crystal structure of TAT triplex formation as the template. The initial structures subsequently were minimized by molecular mechanics. The final structures were believed as the dominant conformation. The results showed that the energy of CP1 is lower than that of CP3, and the former is more stable than the latter. Moreover, the results further proved that the 21nt oligodeoxyribo-nucleotide CP1 stably combines with the core promoter (Cp) fragment of hepatitis B virus (HBV) to form a triplex DNA, and CP1 specifically inhibits a specific cellular factor (DNA binding protein) binding to Cp fragment. These results indicated that specific repression of gene transcription of HBV DNA might be possible by triplex-formation DNA.

Abstract:Mitogen activated protein kinase (MAPK) is a group of important protein kinases involved in phosphorylation cascade in the mitogen-initiated signal transduction pathways.NGF from Agkistrodon halys has been used to investigate its effects on MAPK and MAPKK of PC12 cells. The results showed that this kind of NGF increased MAPK and MAPKK expression and MAPK activity. The above increases were NGF concentration dependent in the range of 25～100 μg/L. The use of PKC inhibitor H-7 indicated that the increased expressions of MAPK and MAPKK expression were PKC-dependent.

Abstract:ku80 gene of radiosensitive cell line SX-9 was cloned by RT-PCR.Compared cDNA of the radiosensitive cell line with its parental cell line, it was found that there were two mutation sites in ku80 gene which could interact with DSB DNA and Ku70.It was confirmed by EMSA and Southwestern blot that the DSB end binding activity of Ku80 in the nucleus extract of the radiosensitive cell decreased greatly，which infered that the radiosensitivity of the cell line is caused by dysfunction of its Ku80 protein.

Abstract:A study by the technique of electron spin resonance (ESR) has been made on the interaction between the liposomes containing unsaturated fatty acid and cancer cell membrane, and probed into that they have may be biological significance in damaged and inhibited cancer cells. Experimental results showed following: the effect of the oleic heterogeneous liposomes causes that the powerful fixed effect of the spin probe is weakened that the weak fixed effect of the spin probe is strengthened, and that the free direction of the spin probe movement is increased. The effect of linoleic heterogeneous liposomes causes that the powerful fixed effect of the spin probe is strengthened, and the weak fixed effect of the spin probe was weakened on the membrane of mammary tumor, and that the free direction of the spin probe movement is impeded. The effect of ricinoleic heterogeneous liposomes is the same with linoleic heterogeneous liposomes on the neoplasm cell. The result indicates that the unsaturated fatty acid can react with mercaptan groups and change the conformations of membrane proteins of tumor cells.

Abstract:To construct a cDNA subtractive library of human renal cell carcinoma (RCC) with technique called suppression subtractive hybridization. The library only contains the differently expressing cDNAs between RCC and normal kidney. Poly(A)⁺ RNA were isolated from cell lines of RCC and normal kidney respectively.Moreover, single-strand cDNAs and double-strand cDNAs were synthesized in turn. After enzyme restriction,cDNAs between 400～600 bp were obtained. RCC cDNAs then were divided into two groups and ligated to the specific adaptor l and adaptor 2 respectively .After RCC cDNAs hybridized with normal kidney cDNA twice and underwent two times of nested PCR,then with arms of T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with the E.coli strain Top 10F′. Human RCC subtrctive library with high subtractive efficiency was set up sucessfully. The amplified library contains 6 500 positive clones.Random analysis of 350 clones with enzyme restriction shows that all plasmids in the clones contain 400～600 bp inserts. The constructed cDNA subtractive library of human RCC is a highly efficient one and lays solid foundation for screening and cloning new and specific oncogenes or tumor suppressor genes of RCC.

Abstract:In order to isolate the related genes in the development of systemic lupus erythematosus (SLE) in BXSB mice, bone marrow cells were seperated from the BXSB and C57-BL-6 mouse, poly(A) RNA was extracted, the cDNAs were synthesized by reverse transcription, and the differentially expressed genes were cloned by cDNA-representational difference analysis(RDA). Three novel genes were isolated, their accession numbers in GenBank are AF060113, AF060111, AF060110; at the same time, some genes that have been reported to be related to the development of SLE such as endogenous retrovirus and Line -1 reverse transcriptase were found. This can provide a novel method to do further research about autoimmune diseases.

Abstract:A self-cleaving ribozyme was comprised of a hammerhead ribozyme and its target sequence located the downstream of the hammerhead ribozyme. The self-cleaving ribozyme gene was synthesized, amplified and cloned into the Bluescript SK plasmid. The construct harboring 10 copies of the self-cleaving ribozyme gene was obtained through successively cloning for four times. The result of polyacrylamid denatured gel electrophoresis showed: the multimeric ribozymes caused self-cleaving during the transcription reaction in vitro and formed RNA step ladders from 70 nt to 706 nt, which indicates that the self-cleavage ribozyme transcripts can be used as RNA molecular mass markers.

Abstract:PCR is now one of the key procedure in the field of molecular biology, but too many things affect the PCR result. Sometimes, a lot of PCR optimization experiments must be done before satisfactory results are obtained. So an optimization project with minimum times of test is needed. Uniform Design is what can meet this requirement. For 244 bp fragment in human apo E gene, the optimization of the ampified conditions included concentration of Mg²⁺, concentration of DMSO, denature time, annealing temperature, elongation time, number of cycles based on Uniform Design was reported. The results show that a set of PCR conditions with specific, high yield amplified product can be obtained for purified template or simple-treated template by 6～10 times of optimization experiment. The Uniform Design is useful for the optimization of PCR conditions.

Abstract:A novel method was developed to prepare an ExonucleaseⅢ-partially-digesting RNA as a competitive reference standard (RNA-CRS) of human stem cell factor (hSCF) gene in quantitative RT-PCR: complete hSCF cDNA was already amplified from HepG2 cells using RT-PCR and cloned into pGEM-T vector. After the recombinant pGEMSCF was treated with Exonuclease Ⅲ and S1 nuclease at a favorable condition to make a limited deletion in hSCF cDNA, the recombinant pGEMSCF mimic was constructed successfully and transcribed in vitro to obtain the RNA-CRS. The hSCF RNA-CRS with a 110 bp deletion from base 499 to 608 in hSCF cDNA was identified by DNA sequencing and it is suitable to be used as a reliable RNA-CRS for the quantitation of the transcriptional expression level of recombinant hSCF in eukaryotic cells by quantitative RT-PCR.

Abstract:In order to further identify expressing difference of cDNA fragments isolated from cDNA representational difference analysis (cDNA RDA) in nasopharyngeal carcinoma(NPC) biopsies and to clone those deregulated genes related with NPC, reverse transcription polymerase chain reaction (RT-PCR) was used to identify the differentially expressing of cDNA fragment, among which AF152605 was found to be down-regulated in sporadic NPC samples (74%). As it were shown by Northern blot, AF152605 is highly expressed in heart,brain, skeletal muscle and placenta whose transcription size is 2.1 kb. Combining bio-informatics, the gene (GenBank accession number AF179285) named as NAG4, was cloned by using library screening, which locates at 6p22～6p23.3. Comparative analysis shows that the NAG4 gene has at least two exons and a “TATAA BOX” sequence lies at upstream of the first exon. And the NAG4 gene encodes a protein proposed of 508 amino acid with predicted molecular mass of 57.4 ku, Blast homology searches reveal that NAG4 protein, containing a bromodomain and several important phosphorylation sites, takes on high similarity to musculus bromodimain-containing protein BP75. Moreover, condon mutation of NAG4 gene was found in HeLa cell line. So it is further demonstrated that NAG4 gene is a good candidate of putative tumor suppressor genes associated with NPC, whose down-expression may be involved in the development of NPC.

Abstract:As a specific angiogenesis inhibitor, angiostatin can inhibit new vascular formation, and it has potential clinical practical value. Human plasminogen was purified from human plasma by affinity chromatograph, and then in situ digestion of plasminogen by elastase was carried out to produce angiostatin fragments. After washing, angiostain was eluted specifically by 0.2 mol/L 6-aminocaproic acid solution. This improvement made this method be simpler, rapider and more efficient. In vitro and in vivo experiments indicated that this purified protein has potent antiangiogenic activity.

Abstract:After the UMP-ADH-Sephsrose 4B column was made as the affinity manterial,human erythrocyte pyrimidine-5′-nucleotidase(P5′N EC 3.1.3.5.) was purified from the blood of normal subjects by a combination of DEAE-cellulose chromatography、ammonium sulfate fractionation、ion-exchange and affinity chromatography. The results show that the P5′N can adhere to the UMP-ADH-Sephsrose 4B column tightly with high specificity. Polyacrylamide electrophoresis of the purified material shows one strong protein band. The enzyme has pI of 5.2 and a relative molecular mass of 28 000 by polyacrylamide electrophoresis.The anyibody was obtained after the rabbits were immunized with the purified enzyme. It might play an important role in clinical study on hereditary or acquired erythrocyte pyrimidine-5′-nucleotidase dificiency.

Abstract:In western blotting, the electrophoretic transfer effect of the proteins can be affected by sodium dodecyl sulfonate (SDS) in the transfer buffer. Using three transfer buffer with or without SDS in both semidry and tank transfer systems, the Bcl-2 protein levels were tested and the spectrum of the protein blots were compared. The addition of SDS in buffer reduced the binding of Bcl-2 protein with PVDF membrane, either in semidry or tank electric transfer. The transfer efficiency of both semidry and tank systems were also compared. The semidry electric transfer markedly minimized the transfer times, but the transfer result were a little poorer than the tank transfer system.

Abstract:Ultra-weak chemiluminescence analytical technology were used to research of oncology. The luminescence intensity of serum and urine of bone tumor patients and normal persons were measured by BPCL ultra-weak luminescence analyzer. The results showed that the luminescence intensities of serum and urine of bone tumor patients were higher than those of the normal subjects (P＜0.05). The urine luminescence intensity of bone tumor patients significantly decreased after operation (P＜0.05). The luminescence from blood and different organs of nude mice were measured. The results showed that the luminescence intensities from different organs increased greatly after inoculation.