Abstract:Universality as one of the fundarmental features for genetic codes has been known since its proposal. But some extraodinary conditions have been discovered recently. Mitochondria use one set of genetic codes for different meaning and some nonsense codes which should have the meaning to stop translation may redefined. UGA may be redefined as selenocysteine. Results of recent research showed that some amber codens are translated as a sense coden for the 22nd natural amino acid, pyrrolysine.

Abstract:nogo is a recently discovered gene. It mainly encodes three proteins: Nogo-A, Nogo-B and Nogo-C. To date, it has been proved to be associated with axonal outgrowth inhibition in mature central nervous system(CNS) and apoptosis-inducing. Nogo receptor is a glycosylphosphatidylinositol(GPI)-anchored protein. To investigate the Nogo and Nogo receptor is important for understanding of neurite outgrowth inhibition following CNS injury and cancer.

Abstract:The aminoacyl-tRNA synthetases (AARS) are very important during the protein biosynthesis, which can make the gene sequence be accurately translated into the protein sequence by the specific recognition between AARS and tRNA/amino acids. However, the recognition between AARS and tRNA/amino acids can be either specific or compatible, which is not only related with evolution of AARS structures, but also with the different evolutionary stage of life organisms containing AARS. AARS could undergo a evolutionary process from “ambiguous specificity” (multiple specificities )to “accurate specificity” (single specificity).

Abstract:The INK4a/ARF gene locus on human chromosome 9p21, is one of the most frequent targets of inactivating mutations in the human tumors. Containing two different promoters, INK4a/ARF can encode two distinct proteins in alternative reading frames, p16^INK4a and p14^ARF (the mouse homologues is called p19^ARF). p16 is a recognized tumor suppressor that induces a G1 cell cycle arrest by inhibiting the phosphorylation of pRb by CDK4/6. While ARF inhibits oncoprotein MDM2, resulting in the stabilization of p53. These activities of ARF promote p53-mediated G1 and G2/M cell cycle arrests or apoptosis. So just like p16, the ARF protein also acts as a tumor suppressor.

Abstract:Extreme enzymes have super biological stability. They demonstrate biological activity at extreme temperatures, pH, pressure and ionic intensities. Thus, extreme enzymes provide good opportunities for biological catalysis and transformation. The discovery of the new extreme species, the confirmation of the genome sequence and the application of gene engineering technology have expedited the discovery and preparation of the new enzymes. Protein engineering and directed evolution further alter the enzymatic activity and peculiarity of the extreme enzymes, which fosters the industrial application of the extreme enzymes. Research on extreme enzymes increases comprehension of the mechanism of enzymatic stability and enriches the theory of molecular evolution.

Abstract:The widespread use of gene knockout technology promotes the course of the research of gene function in post-genomic era greatly. Recently, the research of p38α knockout mice, which is one member of MAPKs, is very typical in filtering noise from critical signals in signal transduction and translating phenotype into gene function. Looking back on the research of p38α knockout mice showed: the reasonable use of gene knockout technology, based on carefully designed and analyzed mutant mice, is very important for defining the function in vivo of important signal molecular and pushing forward the research of function gene.

Abstract:In the past decade,systematic evolution of ligands by exponential enrichment (SELEX) technology has been under development,and is a general approach for identification of aptamers or oligonucleotide ligands that bind with high-affinity and specificity to a wide rang of selected molecules on their versatility. The SELEX protocal is a process of synthesis random oligonucleotide sequents library pool, select special aptamers,amplify and iterative in vitro.Combinatorial modify groups libraries offer the convience in the SELEX process,making the screening process fast, easy and high-throughput.Here the current designs and application of modified nucleotides for in SELEX process are reviewed, and expected to further the utility of this method in both practical and theoretical terms.

Abstract:Baculoviruses are host specific for insects. The recent researches indicated that baculoviruses could entry into different mammalian cells, but do not replicate. The baculoviruses have been successfully used to deliver foreign DNA into mammalian cells in vitro and in vivo and efficiently mediated the expression of interest genes. These results indicated that baculovirus could be developed as potential gene therapy vectors. The recent development and prospects on using baculoviruses as target gene delivered vectors in gene therapy is reviewed.

Abstract:HIF-1 (hypoxia-inducible factor-1), a transcription factor involved in homeostasis of oxygen concentration, upregulates gene expression in processes of glycolysis, cell proliferation and apoptosis, angiogenesis, and contributes to mammalian organism hypoxia adaptation, embryo development, various ischemic diseases and tumors. The regulation of HIF-1 activity is the focus of hypoxia responsive genes expression. The regulation occurs dominantly at the two divergent signaling pathways rooting from Ras, transactivation mediated by Ras/Raf/MEK pathway and PI(3)K/Akt dependent stabilization of HIF-1alpha protein, cooperatively but independently regulates the activity of HIF.

Abstract:Although genomic research has opened up more new avenues for therapeutic interventions based on genetic therapy, it will not be possible to realize the full potential of these therapies until the issue of gene delivery has been resolved. With the development of nanotechnology, the systemic and cellular mechanisms of nanoparticulate carriers for gene delivery have been gradually elucidated. Research and applications of long-circulating and target-specific nanoparticulate carriers maybe help to allevate the gene delivery bottleneck, and realize the efficient, targeted and safe gene delivery.

Abstract:The transcription factor, activating protein-1(AP-1) plays an important role in the regulation of cell proliferation, cell survival and apoptosis. c-Jun is the major component of AP-1. The activity of c-Jun is up-regulated and down-regulated at three main levels: transcriptional control, posttranslational regulation (major by phosphorylation) and the modulation by interacting proteins. Eight sites of c-Jun can be phosphorylated by kinases such as JNK1, GSK3, CKII and Abl. Moreover, c-Jun is regulated through interacting with bZIP transcriptional factors, coactivators and other proteins via its N-terminal transcription-activation domain and C-terminal DNA-binding domain. Other molecules can regulate the AP-1 activity in a coactivator-dependent manner. So the regulatory mechanism of AP-1 activity is complicated.

Abstract:Recent studies suggest that polypeptide signals, such as systemin, RALF, PSK, ENOD40, SCR, CLV3, regulate plant growth and development process as well as plant responses to the environment. Most of the receptors of the polypeptide signals in plants are identified and the procession, release and signal transduction of the polypeptides show high similarity to those in animals and yeast. The possible roles of polypeptide signals in plants and the future prospects in this area are discussed.

Abstract:Cloning of bovine, pig and monkey etc. has been successful by nuclear transfer since “dolly” was born, but the overall efficiency of cloning is very low(typically between 0% and 3%), and many cloned animals display abnormalities in some degrees. Recent studies showed that the reprogramming of the genome that occurs during normal development is aberrant in cloned embryos, especially demethylation is inefficient. Progress in reprogramming of genome of early cloned embryos and the somatic nucleus remodeling in the recipient cytoplasm is reviewed in order to offer some clues to resolve the two important problems in cloning.

Abstract:Mutations in the adenomatous polyposis coli(APC) gene are responsible for familial adenomatous polyposis coli(FAP) and sporadic colorectal tumours. APC gene encodes a protein with multiple function domains and different phosphorylation states. APC is involved in regulating cell adherin,migration,prolification,through its interation with multiple proteins.APC binds to microtubles with its C terminal region directly and indirectly, but APC' middle region could also bind to microtubles,but the mechanism is still unclear.For further studying the interactions of APC and other proteins, using the middle fragment of APC(1 500 bp～4 800 bp)as bait, through yeast two-hybrid technology screen the human fetal brain cDNA library, got a novel APC binding protein SMAP/KAP3,and then generated the SMAP/KAP3 antiserum and the flowing coimmunoprecipitation and coimmunofluoresce staining identificated the interaction of APC and SMAP/KAP3.This suggested that the APC utilize the SMAP/KAP3-KIF3A-KIF3B as a motor protein move along the microtubles.

Abstract:Soluble peroxidase in pericarp of litchi (Litchi chinensis sonn. Cv. Heiye) fruit was extracted by phosphate buffer and purified by ammonium sulfate precipitation, ion exchange chromatography using DEAE Sephadex A-50 column, and gel filtration using Sephadex G-100 column. The specific activity of the purified enzyme increased 65.70 fold over the crude extract with 45.66% recovery. The effects of pH and temperature on activity of peroxidase(POD) were assayed. The K_m for H₂O₂ and 4-methylcatechol were determined by Lineweaver-Burk plots. Several compounds including phonelic compounds were used as the substrates of the enzyme for specificity study. The effects of various inhibitors on this peroxidsae were also assayed.

Abstract:The major biochemical process of apoptosis involves the activation of a group of proteases (caspases) and the selective cleavage of a set of intracellular proteins that leads to the collapse of cell survival mechanism. Among the caspases identified, caspase-3 stands out because it is commonly activated by numerous death signals and cleaves a growing number of cellular components. In order to reveal potential targets of caspase-3 in primate neural tissue, an alternative cell free system based on adult monkey brain was established to reproduce the downstream part of apoptotic program, initiated by the addition of granzyme B. Through Western blot analysis, caspase-3 was found to become mature in a two-step manner and its activity was exhibited by the cleavage of the synthetic substrate, Ac-DEVD-pNA. Investigations on native proteins in the brain extract showed that poly (ADP-ribose) polymerase (PARP) was cleaved to an 85 ku fragment, suggestive of caspase-3 activity. And more intriguingly, a neuronal apoptosis inhibitory protein (NAIP)-immunoreactive fragment with molecular mass of approximately 40 ku was detected in granzyme B-treated brain extract and its production was not blocked by the caspase-3 inhibitor, Ac-DEVD-CHO. According to the substrate specificity of granzyme B and the size of cleavage product, putative cleavage site may be located immediately after the third DIR domain of NAIP. These data suggest that cleavage events involved in apoptosis can be reproduced in matured primate brain extract and NAIP is likely to be the target of granzyme B, but not of caspase-3, during apoptosis.

Abstract:Human leukocyte antigen-G(HLA-G) is a nonclassical major histocompatibility complex Ⅰ (MHC Ⅰ) molecule. As the ligand of NK inhibitor receptors, it can transmit the inhibitory signal and inhibit the cytotoxicity of NK cells. In order to study the effect of HLA-G1 on the recognition of effector-target cells, laser scanning confocal microscopy (LSCM) and flow cytometry were used to analyse the expression and function of full-length HLA-G1 and the real-time change of ［Ca²⁺］_i (free intracellular Ca²⁺ concentration) in the target cells. Results showed that full-length HLA-G1 was expressed in the cytoplasm and on the cytomembrane of K562, JAR and CHO cells. HLA-G1 partially inhibited the cytotoxicity of NK92 in a 4h-⁵¹Cr-release assay. The ［Ca²⁺］_i in the CHO and GFP-CHO (which expresses the green fluorescence protein) obviously rised after the addition of activated NK92. The expression of HLA-G1 inhibited this kind of rising. These results demonstrated that the rising of ［Ca²⁺］_i in target cells is necessary for the effective cell cytotoxicity. The immune inhibition function of HLA-G1 is possible closely related to the inhibition of ［Ca²⁺］_i in the target cells.

Abstract:In order to study the mechanism of hhlim gene transcriptional regulation, a series of deleted fragments of 5′ flanking region extending from +16 to －2 537 bp were subcloned into the pGL3-Basic vector respectively to identify the specific functional regions by detecting the luciferase activities. The results indicated that there was a silencer in the distal region of －2 537～－1 537 bp and an enhancer in proximal fragment of －253～－157 bp, respectively. Electrophoretic mobility shift assay showed that the patterns of shifted bands were different between the nuclear protein from differentiated C2C12 myotubes and undifferentiated C2C12 myoblasts when they bound to the region including the region of －253～－157 bp of hhlim gene. In addition, the results also showed that ET-1 and bFGF could not only significantly induce the hhlim gene expression in C2C12 cells but also activate the luciferase gene transcription promoted by －253～－157 bp regulatory region. It was suggested that hhlim gene was regulated by ET-1 and bFGF.

Abstract:Apoptosis inducing factor (AIF) is a mitochondrial intermembrane space protein ubiquitously expressed in various kinds of cells. When death stimuli present, AIF is released from mitochondria to the cytoplasm and then translocated to the nucleus, inducing peripheral chromatin condensation and large-scale fragmentation of DNA (～50 kb). The full-length AIF gene was amplified by RT-PCR firstly, then its N-terminal mitochondrial localization sequence (MLS) was deleted, being replaced with PE transmembrane domain, and then the recombinant gene was inserted into the pIRES2 -EGFP eukaryotic expression vector. After transfection into HeLa cells with lipofectamine, the expression of these recombinant AIF gene and their effects on HeLa cell growth were detected by fluorescent microscopy, confocal microscopy, and electron microscopy analyses. The result proved that the expression of the recombinant human AIF gene could induce HeLa cell death, which provided new strategy for killing cancer cells.

Abstract:Helicobacter pylori infection is the major etiological factor of chronic active gastritis and most peptic ulcer disease，and is closely associated with gastric cancers such as adenocarcinoma and MALT lymphoma. Since the Hp adhesin conservative region(AB) is outer membrane protein and porin type component, while these two kinds of protein are the excellent immunogen candidates of vaccination. The gene ab was amplified by PCR and inserted into the prokaryotie expression vector pET-22b（+）and expressed in the BL21（DE3）E.coli strain. DNA sequenced showed one open reading frame of 588 bp,which encoded polypeptides of 195 amino acids. SDS-PAGE and scan analysis show the AB molecular mass is 22.5 ku and recombinant protein amounted to 29% of the total bacterial protein. The AB purity amounted to 96% through affinity chromatography. Western blot analysis of AB confirmed it could be specially recognized by serum from rabbit immunized with AlpA.Acquire of AB established foundation for further studying the molecular adhesin, prevention and cure immunity mechanism of the adhesin.

Abstract:To explore the cerebral cortex mechanism of visceral nociceptive sensation and its characteristics on the cell level with intracellular recording techniques，spontaneous biological electric activities and evoked responses of neurons in anterior cingulate gyrus（ACG） to stimulating ipsilateral greater splanchnic nerve（GSN） in 18 cats were investigated. Among 312 neurons，82 were visceral nociceptive neurons（VNNs），which mainly having five patterns of spontaneous biological electric activities. According to the characteristics of the evoked responses，VNNs were classified into specific visceral nociceptive neurons （SVNNs，92.68％） and non-specific visceral nociceptive neurons（NSVNNs，7.32％）. Modes of the evoked responses could be excitatory（65.86％），inhibitory（17.07％），or mixed ones（17.07％）.The results suggest that ACG may be one of the representative areas of the ipsilateral GSN afferent pathway，and there exist two kinds of VNNs in ACG，which may be differently involved in pain modulation. The results may provide new experimental data for specific theory of pain.

Abstract:The roles of fucosylated glycoproteins and oligosaccharides in hepatoma cells were studied by means of fucosylation analysis. It was noted that the protein bands between 23 ku and 40 ku which bound to ulex europaeus agglutinin (UEA) and lens culinaris agglutinin (LCA) reduced significantly until 20 weeks when the liver mass formed, but the band at 80 ku became denser week after week during the course of rat hepatocarcinogenesis induced by N-nitrosodiethylamine. In comparison to the hepatoma cells with low metastatic potential, high metastatic hepatoma cells were found more protein bands binding to UEA and LCA within a broad range of molecular mass. Fucosyltransferase activities were furthermore investigated in the metastatic tissues of hepatoma. It was observed that the activity of 1,6 fucosyltransferase in metastatic liver mass was significantly higher than those of liver tissues without metastasis. Therefore, the fucosylated glycoproteins were isolated and directly used for the treatment of 7721-k3 hepatoma cells. Interestingly the glycoprotein isolated by aleuria aurantia lectin (AAL) and LCA chromatography significantly inhibited the migration of 7721-k3 hepatoma cells. Glycopeptides digested from the glycoproteins above still have the same inhibitory effects on 7721-k3 cells or even stronger. Results by a series of lectin binding analysis showed that these oligosaccharides had a strong affinity to concanavalin A and bound to L-type and E-type phaseolus vulgaris agglutinin as well. This evidence suggested that the fucosylated oligosaccharides might change into high mannose type in hepatocarcinogenesis and played an important role in hepatoma cell migration and metastasis.

Abstract:The distribution of hexa-structures in secondary structure sequences of different classes of proteins has been found. Based on this, two methods for the recognition of the structural class of a protein are proposed. The first is the method of Mahalanobis distance which is based on the frequencies of tri-structures in secondary structure sequence. The second is the method of diversity measure which is based on the frequencies of hexa-structures that are regarded as the source of diversity. The prediction has been done in a set of 1 130 proteins of four classes, namely α-class, β-class, α/β-class and multi-domain protein. The successful rates for two recognitions are about 81% and 83% respectively. The method introduced here also gives an approach to predict the compact structural domain of proteins.

Abstract:Human lung cancer cells (ASTC-a-1) were infected with EGFP plasmid vectors, and incubated in RPMI 1640 culture medium supplemented with 15% FCS and 800 mg/L G418. Strong fluorescence of five cell clones were observed after several generations. The flow cytometry was used to determine the GFP expression stability, the results showed that there was significant difference between 2#(3#) and 4#(5#) (P＜0.01). The nude mouse, 6 weeks of age, was injected s.c. with a single dose of 3×10⁶ infected tumor cells. The GFP fluorescence was directly exited by 488 nm argon ion laser and recorded by digital camera with 530 nm long pass filter.

Abstract:Human neuroglobin (NGB) is a newly-discovered gene functioned specifically in the oxygen supply and oxygen consumption in the brain. However, the full-length cDNA sequence of NGB was not reported yet. Using the in silicon sequence elongation technique, 5′-RACE and 3′-RACE technique, the full-length cDNA sequence of human neuroglobin was successfully obtained (GenBank accession number AF422797). The cDNA sequence of human NGB is 1 909 bp in size and capable of encoding a protein of 151 amino acids. The 5′-untranslated region is 375 bp, while the 3′-untranslated region is 1 078 bp including 27 bp of poly(A) sequence. Results demonstrated that the RACE technique coupled with electronic sequence elongation technique is useful to obtain unknown sequence of 5′-terminal or 3′-terminal part of a new gene.

Abstract:In order to obtain a quality subtractive cDNA library of age-related cataract which contains more large fragments from small amounts of sample, an improved subtractive hybridization which using magnetic beads isolation and biotin labelling was used to obtain the subtractive cDNA. Then the large fragments of the subtractive cDNA were amplified by using selective PCR method, so the subtrative cDNA library of age-related cataract was successfully constructed. 22 clones of the library were selected at random and were identified by the restriction enzymes digestion. 7 fragments were larger than 1 000 bp,which is 31.8% of the totals, 15 fragments were larger than 750 bp, which is 68.2% of the totals. The cDNA fragments are larger and can be used in further study. A subtractive cDNA library with high quality and more large fragments can be constructed rapidly and effectively by improved subtractive hybridization and selective PCR method from small amounts of sample.

Abstract:Two novel antibacterial peptides were isolated and characterized from the earthworm Eisenia fetida. The antibacterial peptides were purified to homogeneity by means of cation-exchange chromatography and reverse FPLC and named F-1 and F-2. By means of ESI-MS, F-1 and F-2 were determined to be two peptides with the relative molecular mass of 535.27 and 519.27. By means of MS/MS, the amino acid sequence of F-1 and F-2 were determined to be Ac-Ala-Met-Val-Ser-Ser and Ac-Ala-Met-Val-Gly-Thr respectively. F-1 and F-2 exibited an antibacterial activity not only to bacteria but also to fungi. Minimal inhibitory concentration(MIC) of F-1 and F-2 against several Gram-positive and Gram-negative bacteria were determined by incubating approximately 10⁴～10⁵ CFU/ml of cell with serial dilutions of peptide in a 96-well microtiler plate. The MIC of F-1 and F-2 against Enterococcus gallinarum, Pseudomonas pyocyanea, Acinetobacter baumanii, Klebsiella terrigena1 were 11.4 mg/L and 12.85 mg/L. The MIC of F-1 and F-2 against Enterococcus faecalis was 22.8 mg/L and 25.68 mg/L.

Abstract:Silk sericin protein powder can be produced from the sericin solution by the processing of degumming in the high temperature and high-pressure conditions, purifying, condensing and spray drying. Molecular mass of the sericin protein is high up to 200 ku. Taking on white color and about 10 μm average granularity, the powder of the protein is hot water soluble. L-Asparaginase was immobilized on the natural silk sericin powder by cross-linked with glutaraldehyde. The enzyme activity and dynamical properties of the immobilized L-asparaginase were described. It was found that the immobilized enzyme is very stable, and the stability to heat increased in comparison with free enzyme, and this immobilized enzyme has preferable stability of operation. The resistance to trypsin hydrolysis was also improved greatly compared to that of soluble enzyme.

Abstract:The 3α-hydroxysteroid dehydrogenase gene (3α-hsd)from Comamonas testosteroni was amplified by PCR from the genomic DNA of Comamonas testosteroni. It was cut out from PCR product by the digestion of the restriction enzymes NdeⅠ/BamHⅠ and was cloned to plasmid pET-15b again. The recombinant plasmids pET-15b with proper orientation were identified by the analysis of restriction enzymes and DNA sequencing. Then the host bacteria containing recombinant plasmids pET-15b with proper orientation grew with IPTG induction. The total cell lysate of the host bacteria was extracted for SDS-PAGE and the determination of its enzymatic activity. Afterwards the recombinant protein with an N-terminal His tag sequence was purified by affinity chromatography on a Ni²⁺-Sepharose column. The recombinant plasmid pET-15b with proper orientation containing the 3α-hsd gene was selected and the fusion protein was overexpressed in the host bacteria of E.coli BL21(DE3) pLysS. The enzymatic activity of the crude extracts was up to 2.45×10⁵ U/L.The fusion protein could be purified in one step using metal chelate chromatography to homogeneity as judged by SDS-PAGE. And the recovery rate was about 68%. The above work has laid a necessary foundation for the construction of a enzymatic cycling method to determine the serum total bile acids.

Abstract:Three scientists were awarded the 2002 Nobel Prize in Physiology of Medicine for discoveries concerning the genetic regulation of organ development and programmed cell death. The laureates have established the nematode Caenorhabditis elegans as an experimental model, mapped the invariant cell lineage, identified key genes regulating organ development and programmed cell death and shown that corresponding genes exist in higher species, including man. These discoveries are of primary importance for understanding the pathogensis of various of human diseases.

Abstract:The progress of science is in part attributed to the development of new methodologies. The application of mass spectrometry (MS) and nuclear magnetic resonance (NMR) in the study of biological macromolecules is such a representative example, which is the subject of this year's Nobel prize award in chemistry shared by three scientists. The works include the development of soft desorption ionization methods for mass spectrometric analysis of biological macromolecules and the development of NMR for determining the three-dimensional structure of biological macromolecules in solution. These developments revolutionized the analytical methods for biomolecules such as proteins and facilitate the study of biological macromolecules so much enough to have deep effects on the whole life sciences.