Abstract:Different types of neurons are regularly dispersed on retina. For finding the mechanism of eye and retina formation, it is important to know how this regular pattern is formed during embryonic stage. It was claimed that multiple genes regulate the development of eye and retina. These genes regulate the development of tissues and differentiation of cells in different parts of the visual system during embryo development. Also, it was found that different tissues and differentiated cells cohere with each other both temporally and spatially to form the eye.

Abstract:Pituitary adenylate cyclase-activating polypeptide (PACAP) which belongs to the secretin/glucagon/VIP family has been originally isolated from the sheep hypothalamus on the basis of its ability to stimulate cAMP formation in culture rat anterior pituitary cells. Post-translational processing of the PACAP precursor generates two biologically active molecular forms, PACAP-38 and PACAP-27. The primary structure of PACAP has been remarkably conserved during evolution. The sequence of PACAP-27 exhibits substantial similarities with those of vasoactive intestinal polypeptide (VIP), glucagon and secretin. The gene encoding the PACAP precursor is widely expressed in brain and various peripheral organs, notably in endocrine glands, gastro-intestinal，uro-genital tracts and respiratory system. In vivo and in vitro studies have shown that PACAP exhibits multiple activities especially a trophic activity during ontogenesis, notably in the adrenal medulla and the central nervous system. The biological effects of PACAP are mediated through three distinct receptor subtypes which exhibit differential affinities for PACAP and VIP. The PAC1 receptor, which shows high selectivity for PACAP, is coupled to several transduction systems. In contrast, VPAC1 and VPAC2, which bind with the same affinity for PACAP and VIP, are mainly coupled to the adenylyl cyclase pathway. In conclusion, PACAP is neuropeptide, and it functions as a hypothalamic hormone, neurohormone, neuromodulator, vasodilator, neurotransmitter or trophic factor in the brain and the various organs.

Abstract:The cell's sense direction is closely related with the proteins that contain PH (pleckstrin homology) domain. PH domain has been found in about 60 proteins, many of which could activate the sequent events of signal transduction via combining with the related binding sites on the surface of chematactic cells. The characteristics of this combining are: a.rapid and transient; b.It is only related to the concentration gradient of surroundings outside cells, which is the base of spatial model; c.The distribution of the binding sites on the cell membrane changes when the researchers altered the position of the chemoattractant. This is the base of temporal model. To deeply investigate the effects of all kinds of proteins which contain PH domain on cell's sense of direction will greatly promote the research of this field, and hence, has great theoretical significance.

Abstract:In two models for DNA replication and transcription, traditional one known as sliding model postulates that proteins involving replication and transcription track on the DNA template as a locomotive. In factory model proposed recently, those proteins are immobilized on nuclear structure, to pull the template. Growing evidence from biochemistry, biophysics, and cell biology suggests that the factory model is an actual fact in vivo.

Abstract:Anandamide (N-arachidonoylethanolamide), an arachidonic acid derivative, is an endogenous ligand for cannabinoid receptors, which are members of the G protein (Gi)-coupled receptors family. Ananamide is mainly found in central nervous system, immune system and uterus etc and mimics most of the effects of (－)Δ⁹-tetrahydrocannabinoid ［(－)Δ⁹-THC］, a psycoactive derivative of marijuana. Fatty-acid amide hydrolase (FAAH), which is involved in hydrolyzing anandamide to arachidonic acid and ethanolamide, may quickly regulate level of anandamide in vivo.

Abstract:DNA computer is a new research field which combines both the computer science and molecular biology. DNA computer is proposed to solve a class of hard problems of mathematical complexity by using a set of DNA sequences encoding all candidate solutions to the computational problem of interest and find out the correct answers by serial manipulations of biochemical reactions. DNA computer is exactly a biomolecular computer which stores a vast quantity of information with high density. DNA computer, by means of its huge parallel computation and brute force search strategy, can solve the NP complete problems with polynomial time. The recent advances and principle of DNA computer are introduced. The future development and the bioinformatical significance of DNA computer are also analyzed and discussed.

Abstract:Tensegrity structure is comprised of compression-resistant elements and a set of continuous tensile elements that are interconnected with each other. The stability of such system depends on maintenance of tensional integrity inside the structure, or what has come to be termed “tensegrity”. According to studies on biology, cell structures are assembled on the basis of tensegrity mechanism. Tensegrity of cytoskeleton can affect cell shape and function. Furthermore, some basic rules of mechanochemical transduction in cells can be well explained using tensegrity theory.

Abstract:The proteomics definition, investigation method and its application in cancer research were simply introduced. Proteomic research is to reveal the function of genes from an integrated, kinetic and quantitative view at the global protein level, which is an important component of post-genome project. Cancer is a kind of complex disease involved by multi-genes. Proteomic research will be helpful to discover the mechanism of cancer development, to find special malignant tumor markers and targets of drug treatment.

Abstract:Mitochondria are involved not only in energy metabolism but also in free radical metabolism. Superoxide anion can be generated through a way of electron leak of respiratory chain and the reactive oxygen species (ROS) can be formed in the further reactions of O₂^-_· in mitochondria. The role of mitochondria in anti-oxidant functions and cell apoptosis is discussed in terms of electron leak of respiratory chain, uncoupling of oxidative phosphorelation, mitochondrial pore, Box- or/and PTP-mediated release of cytochrome c from mitochondria and so on. The signaling act of ROS is emphasized in the regulation of cell apoptosis.

Abstract:monocarboxylate transporter (MCT) are vital transmembrane transporters involved in multiple cellular functions including the regulation of intracellular pH and lactate transport. At least eight isoforms of MCT have been cloned and characterized to date, they constitute a new gene family of mammal transporters. These isoforms have the differences in substrate and inhibitor specificities and tissue distribution. Thus it may provide a new way of diagonosis and treating for dieases such as cancer by investigating on the structure and function and regulational mechanism of MCT.

Abstract:Gene disruption by homologous recombination is a powerful tool for investigating gene function in yeast. Since 1980’s, it has been developed a lot. PCR-mediated gene disruption technique makes the manipulation easier and it can be used to precisely delete genes in yeast. Multi-gene disruption technique can delete several genes successively in the same yeast strain. After the completion of the yeast Saccharomyces cerevisiae genome sequencing, the gene disruption technique for systematic analysis meets the need of the functional genomics in yeast. It also enlighten the study on human functional genomics.

Abstract:Circadian rhythms describe biological phenomena that oscillate with an 24 hour cycle. These rhythms include blood pressure, body temperature, hormone level, the number of immune cells in blood, and the sleep-wake cycle. The aim is to introduce common genes between species that are responsible for determining the circadian behavior, especially some transcription factors that serve to regulate many circadian rhythm genes. And the common molecular mechanism of biological clocks between fly and human will be introduced.

Abstract:Since the first intein was found, more and more attention were paid on it. It not only enrichs the content of the process that the gene transfers its information but also can be used in protein purification. The recent advance in the sequence characteristic, transfer, evolution and the mechanism of splicing of intein was summarized.

Abstract:Regulation of gene expression is one of the most important responses of cells to DNA damage induced by radiation. A novel expressed sequence tag (EST) fragment had been cloned from human embryo lung cells induced by 50cGy radiation and named RIG1. To clone the full-length cDNA of RIG1, a non-cloned cDNA library of human embryo lung cells induced by low dose irradiation had been established. This library was used as template in enchanced nest RACE PCR and biotin-labeled probe was used for further purification. The 3′ flanking sequence of this EST was cloned and sequenced with this set of technology. It was illuminated by homology analysis that this 3′ flanking sequence and the original EST are well aligned with a BAC clone of 20^th chromosome and the predicted exons' sequence of this chromosome is well consistence with the real EST. Thus the RIG1 can be roughly located in 20^th chromosome. By use of the exons' sequence predicted from chromosome sequence by GENSCAN, full-length of RIG1 gene has been cloned. Chromosome location of RIG1 gene is further determined by this successful verification of Bioinformatics prediction by experiment. By the same step, genome sequence of RIG1 has been determined. Therefore,by the combined use of Bioinformatics analysis，the full-length cDNA sequence and genome sequence of RIG1 gene are obtained and the predicted protein sequence is determined.

Abstract:In order to study the expression and the feasibility of scaled production of neuropeptide in the routine expression system such as E.coli with the pituitary adenylate cyclase activating polypeptide(PACAP) as an example, the following experiments were carried out. First, on the basis of the reported amino acid sequence of PACAP, DNA sequence of PACAP was deduced and six partially complementary oligonucleotide fragments were designed. The coding region of PACAP was obtained by renaturing the DNA fragments and ligation and identified by DNA sequencing. The coding region of PACAP was cloned into plasmid pGEX-4T-3 and transformed into E.coli BL21(DE3). An expression strain BLPACAP was selected. SDS-PAGE analysis revealed that the GST-PACAP fusion protein was highly expressed and accumulated to about 30% of the total bacterial proteins. By affinity chromatography, up to 90% GST-PACAP was purified by one step from bacterial lysate. The purified protein could promote neurite outgrowth of PC12 cells and the survival of spinal cord neurons.

Abstract:The plasmid pXJ-41-p15, which contains the full length DNA coding for p15 was introduced into human melanoma cell line A375 in which p15 was deleted by DNA recombination and transfection. Using G418, the positive clones were selected. And the cell model overexpressing p15 was constructed successfully through the analysis of PCR and Western blot. It is showed that the expression of p15 was further enhanced after the cells overexpressing p15 were treated with PMA for 72 hours. In contrast of the control cells, the PKC activity was further declined in the cells overexpressing p15 after treated with PMA. At the same time, the growth rate of cell was decreased more significantly and approximate 30% apoptotic cells were found. The expression of Caspase-3(P20) was increased in the apoptotic cells. It is indicated that CKI p15 was related to PKC signal transduction in the regulation of cell proliferation and apoptosis. They may be involved in the apoptotic pathway including Caspase3, thus inducing the apoptosis of cells.

Abstract:In order to obtain the bifunctional chimeric molecule of single-chain urokinase-type plasminogen activator (scu-PA) which can inhibit platelet aggregation, PRGDWR peptide was inserted into the site between Gly¹¹⁸ and Leu¹¹⁹ (called insertion mutant B, InB). The recombinant gene of InB was expressed by Pichia pastoris. The secreted protein was purified by metal chelate affinity and strong cation exchange chromatography. The amidolytic ability of mutant InB is 5 900 IU/mg, the kinetic constants is: K_m,plg^InB=56.8 μmol·L^-1，k_cat,plg^InB=0.33 s^-1. The kinetic constants of plasminogen activation reaction is: K_m,plg^InB=0.397 μmol·L^-1，k_cat,plg^InB=0.0164 s^-1. Fibrin inhibit the catalytiv ability of InB during plasminogen activation, the influence factor is 0.463(means InB remain 46.3% of the catalytic ability when fibrin was involved in the reaction system). The mutant not only has almost the same catalytic ability as wild type scu-PA, but also has strong ability of anti-platelet aggregation(compared with scu-PA), IC₅₀ of InB is 12.7 μmol·L^-1.

Abstract:Because the influence of fibrin on the reaction of plasminogen activation by various plasminogen activators is different, the kinetic constant of the reaction of plasminogen activation catalyzed by InB with and without fibrin were detected. The result is: K_m^fibrin＝4.2 μmol·L^-1，greater than the normal K_m=0.379 μmol·L^-1; k_cat^fibrin＝0.107 s^-1，greater than the normal k_cat=0.0165 s^-1. The results suggest that existence of fibrin in the reaction system of plasminogen activation depress the affinity between InB and plasminogen, but accelerates the hydrolysis of plasminogen by InB. The count up effect is inhibition.

Abstract:P₅₃ gene (exon7～8) mutatins and p53 proteins and HPV 6，11，16，18-DNA were examined in 49 cervical carcinoma by immunohistochemistry, polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) in order to investigate their role and mutual relation and clinical significance in the oncogenesis of cervical carcinoma. The results showed that first, p53 proteins positive rate was 48.98％, and not outstandingly related to the differentiation and the invasive degree of cervical carcinoma(P＞0.05); the defects of P₅₃ gene (exon7～8) were not found but P₅₃ (exon7～8) mutations were detected in 7 of 49(14.29％) cervical carcinoma; then, HPV16-DNA positive rate was much higher than HPV6,11,18-DNA positive rate respectively(P＜0.001),and the different HPV-DNA was simultaneously tested in one cervical carcinoma; last, not all cases of P₅₃ mutations had p53 proteins positive, but the cases of P₅₃ mutations and p53 proteins negative certainly had HPV infections, and HPV positive cases were much more than its negative one in the cases of p53 proteins positive(P＜0.001). These results proved that the oncogenesis of cervical carcinoma is mainly associated with HPV16 infections, and second related to P₅₃ (exon7～8) mutations. p53 proteins positive results from P₅₃ mutations or/and HPV infections in cervical carcinoma.

Abstract:The earthworm fibrinolytic enzymes (EFE) were separated by affinity chromatography using soybean trypsin inhibitor as a matrix. The enzymes were further separated and purified into 12 components after DEAE-32 chromatography and preparative electrophoresis. The pI of these components gradually decreased from pH 4.0 according to electrophoresis mobility from higher to lower on PAGE. The molecular weights were in the range of 22～34 ku. 6.5 and 7 were glycoproteins proved by staining with the shiff reagent and thymol/sulfuric acid. The fibrinolytic activity of 7 was highest as determined using chromzym UK and chromzym PL as specific substrates.

Abstract:To screen and identify the mimotopes for lipopolysaccharide(LPS) epitope, a random phage displayed dodecapeptide library was screened with a monoclonal antibody 2B4 specifically against LPS conservative epitope. The positive clones were identified by phage ELISA and competitive inhibition assay by either S.typhi T8-61 LPS or E.coli O111:B4 LPS. After three rounds of biopanning,the clones binding with 2B4 antibody were well enriched with positive rate of 80%. The bindings between 12 of positive phage clones and screening antibody were competitively inhibited by the two kinds of LPS,indicating that the positive clones have similar epitope with LPS. The positive peptide sequences were deduced from the corresponding DNA sequences. There were identical sequences among them. The sequences were GPPQWFFSQPQL （5/12，41.7%），LPQYFWNTATTA （3/12，25%），FPQNHWNVPWAT（2/12，16.6%），HSQSFWNAPLAM and AHPWTHGYFPPL （1/12，8.3%） respectively. The results demonstrate that the peptides screened with 2B4 antibody are mimotopes for LPS conservative epitope.

Abstract:Four methods were compared to purify F_o from porcine heart mitochondria. The best results were obtained by the following method: after removing F₁-ATPase with NaBr incubation from submitochondrial F_oF₁-ATPase, F_o was solubilized with CHAPS and purified by sucrose density gradient centrifugation. SDS-PAGE with silver staining showed about 85% purity of the isolated F_o and 9 different subunits including b, OSCP, d, a, e, F₆, IF₁, A6L and c. The purified F_o was then incorporated into asolectin liposomes, the reconstituted F_o showed higher H⁺ translocation activity and after F_o was reconstituted with F₁-ATPase, the resulted F_oF₁-ATPase complex exhibited high ATP hydrolysis activity and high sensitivity to oligomycin. The results provide evidence for successful purification, reconstitution of F_o with high H⁺ translocation activity and its relationship with phospholipids.

Abstract:The synchronized HeLa cells were used to study the effect of protein kinase A(PKA) inhibitor on the progression of S phase. Synchronized cells in S phase were obtained by the method of TdR double block through ³H-TdR incorporation assay. The PKA inhibitor typeⅢ obviously increased the level of ³H-TdR incorporation of S phase in HeLa cells. In contrast with control, the activity of thymidine kinase (TK) in S phase increased, too. It indicated that PKA played an inhibitory role in S phase progression of HeLa cells. With the method of Western blotting, the PKA inhibitor typeⅢ enhanced the level of CyclinA and PCNA, inhibited the expression of p21, which is a negative regulator of cell cycle, but had no effect on the expression of CDK2. The results showed that PKA could negatively regulate the S phase progression by affecting the level of CyclinA, PCNA and influencing the expression of p21 protein. This may be one of the molecular mechanisms which is involved in the negative regulation of S phase progression by PKA in HeLa cells.

Abstract:To obtain active hFKBP52 protein for screening novel neurotrophic drugs. Semi-nested and overlap PCR and affinity chromatography were used. hFKBP52 gene was cloned successfully from human fetal brain cDNA library, and then highly expressed (about 30%) as fusion protein in pET28a(+) vector system. The recombinant protein was purified as one band on SDS-PAGE. The purified hFKBP52 showed peptidyl-prolyl cis-trans isomerase (PPIase) activity, similar to the wild type.

Abstract:The Epstein-Barr virus latent membrane protein 1 (LMP1) oncoprotein causes multiple cellular changes, including activation of the NF-κB transcription factor. To elucidate its possible mechanism, the interaction between LMP1 and the tumor necrosis factor receptor associated factor (TRAF) molecules was detected by the immunoprecipitation-Western blotting assay. Results showed that LMP1 was co-precipitated with TRAF1,2,3 in the LMP1-HNE2 cell line. In the meantime, κB reporter gene analysis revealed that over expression of TRAF1 or TRAF2 augmented LMP1-mediated NF-κB activation from LMP1, suprisingly, overexpression of either TRAF3 or an dominant negative TRAF3 inhibited the NF-κB activation, indicating that TRAF1 or TRAF2 is a positive modulator of LMP1-mediated NF-κB activation, whereas,TRAF3 is a negative modulator. Rather both CTAR1 (carboxy-terminal activating region 1) and CTAR2 domains of LMP1 can independently activate NF-κB by interacting with TRAF proteins. These data indicate that LMP1 interacts TRAF1,2,3 which are important for LMP1-mediated NF-κB activation, and further suggest that signaling from TRAFs may be involved in the progression to malignancy in cells of epithelial origin such as nasopharyngeal carcinoma (NPC).

Abstract:Proteome research has become a new hot spot in the post-genome era. High-resolution two-dimensional gel electrophoresis (2-DE), which provides the most comprehensive analysis system of the whole proteome, was highly improved in recent years. With the development of computer techniques, the powerful and user-friendly image analysis systems appeared to help high-throughput, large-scale proteomic studies. Using new generation two-dimensional image analysis software, ImageMaster 2D Elite, the 2D gels of proteins extracted from cultured Schwann’s cells were processed. The analysis procedure, including image acquirement, spot detection, match, background subtraction, pI/M_r calibration, analysis results report and database query, were reported and discussed.

Abstract:An improvement of chemiluminescent system for determination of peroxynitrite anion (ONOO^－) has been made. In this system, the effect of some antioxidants for scavenging ONOO^－ was tested. The constitute of this system and the program of starting were followed as: The ozone (O₃) was bubbled through a glass-frit into 10 ml 0.01 mol/L solution of sodium azide in CB(pH 10.5) to generate ONOO^－. The 800 μl ozonized solution of azide was injected into a glass tude in situ which contains 100 μl sample and 100 μl luminol solutions to initiate chemiluminescence (CL). The pluses / 6 seconds (CP6S) were determined immediately and continually for 10～30 times. A certain CL intensity (CP6S) was chosen as evaluation index to compare the activity of antioxidants. This chemiluminescent system is sensible, simple and stable. The determination limit was 8.74 μmol/L ONOO^－. The linear rang was 8.74～74.04 μmol/L ONOO^－. The intra batch and inter batch variation coefficient (CV%) of the analysis were 3.35%(n=10) and 5.52%(n=10) respectively. It was tested that Vit.C, teapolyphenol, procyanidin and thiourea all have effects on scavenging ONOO^－.

Abstract:A new and efficient reaction system has been set up, in which the MseⅠ primers were fluorescent labeled for auto-sequencer. PCR reagents and primers and adapters of MseⅠ and EcoRⅠ, which were synthesized, the AFLP protocol has been modified, and reaction and electrophoresis conditions were optimized, the results obtained can be comparable to that of AFLP fluorescent labeled AFLP kits with less cost.

Abstract:The α chain of hemoglobin of 615 mouse was isolated and purified on CM-Celullose-23 colomn chromatography. The N-terminal amino acid of the α chain was valine determined with DABITC/PITC method.The amino acid composition was determined and it was different from the parent(C₅₇BL)in literature on the number of leucine residue,histine residue and valine residue.An undissoluble ‘core’ and dissoluble peptides were found when the α chain of 615 mouse was hydrolysised by trypsin and it was found that the eighth amino acid residue from N-terminal of one particular peptide fragment mutated from valine (C₅₇BL) to leucine.

Abstract:More and more DNA sequences have been obtained since the start-up of human genome project. Powerful system is badly needed for data mining on these DNA sequences. Based on a personal computer and Linux operating system, the Phred/Phrap/Consed software and Blast software were used to construct a platform for batch analysis of the sequences, including identifying raw DNA sequence from chromatogram file, vector sequence removing, contig analysis (sequence assembly), repeat sequence identifying and sequence similarity analysis. Result demonstrated that this robust platform could accelerate data analysis for large-scale DNA sequencing.

Abstract:Sepharose 6B was activated by epichlorohydrin in the strong base condition, and then reacted with solution of iminodiacetic sodium. The arms of IDA were conjuncted to the activated Sepharose 6B. Then the products were reacted with the solution of NiSO₄. The arms of IDA were chelated with Ni²⁺,and the chelating resin―Ni²⁺-IDA could be prepared. The physicochemical indexes and performance in purifying protein of the expressing product were assayed with atomic absorption method and purifying aimed protein-human B lymphocyte stimulator(hBLyS) from the expressing products in E.coli. The results indicated that the performance of made gel is very good, and its price is less than 1/10 of that of commodity gel.