Abstract:Neuronatin(Nnat) is a recently cloned brain-specific gene that is selectively expressed during brain development. Human Nnat gene spans 3 973 bases and contains three exons and two introns. Nnat mRNA has two different splice isoforms, α and β, the former encoding a protein of 81 amino acids and the latter 54 amino acids. These two isoforms share the same open reading frame, but the α-form contained an additional 81 bp sequence inserted in the middle of the coding region. The human Nnat is a single copy gene located at chromosome 20q11.2～12, while the rat gene is located on the distal region of chromosome 2, 2H1. Nnat is an imprinted gene and expressed from the paternal allele, while the maternal allele is methylated and silenced. As Nnat is highly expressed in the central nervous system from mid-gestation through early postnatal development and down-regulated in adulthood and senescence, Nnat may be involved in brain development and differentiation. The expression is also detected in the adult brain stem, suggesting roles in neuroplasticity. The deduced protein sequence contains a hydrophobic N-terminal and a hydrophilic C-terminal, and appears to code for a transmembrane protein. The biological function of Nnat remains to be elucidated.

Abstract:Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) play pivotal roles in the degradation and remodeling of the extracellular matrix (ECM). The cooperation and balanced expressions of MMPs and TIMPs are essential issues for the cyclic growth, differentiation, maintenance, and degradation of the tissues during physiologic and pathologic processes. Transforming growth factor-β (TGF-β) could perform its biological effect on ECM by regulating the gene expressions of MMPs and TIMPs. The different modulating effects of TGF-β on MMPs and TIMPs in different cell types are due to the activating of Smad pathway, MAPK signaling pathway or inducing the formation of AP-1 complex by extracellular TGF-β signal.

Abstract:The human immunodeficiency viruses(HIV) cause the destruction of CD4 lymphocytes, resulting in the development of acquired immunodeficiency syndrome (AIDS). The entry of HIV into host cells is mainly mediated by the fusion of the viral and cellular membranes, which involves the interactions of a series of biomacromolecules, such as gp120，gp41，CD4 and CCR5. The deep understanding of the crystal structures, the surfactant details of their interaction, and the change of conformation during interaction of the macromolecules provides a new idea for the anti HIV drugs. At present some new drugs have been found arising from this idea.

Abstract:Alterations in chromatin structure by histone post-translational modifications appear to play a central role in the regulation of gene transcription. Histone modifications consist of methylation, acetylation, phosphorylation and ubiquityination. Among them histone acetylation is of critical importance. The level of histone acetylation depends on the activity of two families of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). HATs, which is frequently part of multisubunit coactivator complexes, lead to the relaxation of chromatin structure and transcriptional activation, while HDACs tend to associate with multisubunit corepressor complexes, resulting in chromatin condensation and transcriptional repression of specific target genes. Chromosomal translocations are often associated with acute leukemias, and a significant number of translocations involve genes encoding HATs and HDACs. On the other hand, some histone acetylation-modifying enzymes have been located within chromosomal regions that are particularly prone to chromosomal breaks. The recent achievements in studies aimed at elucidating the biological roles of histone acetylation modifying enzymes and their potential impacts on the molecular changes involved in the development of cancers are reviewed.

Abstract:Several topics in gene therapy of diabetes mellitus are described as follows: 1) Insulin-producing plasmid or cell is a plausible target of gene therapy of diaetes mellitus. Development in this research includes construction of single-chain insulin analogue (SIA), which possesses biologically active insulin activity without enzymatic conversion, and new evaluation of K cells which can be engineered to secrete insulin and represent a viable mode of therapy for diabetes. 2)Immunotherapy of diabetes is now focusing on induction of tolerance to beta cell antigens using target cytokins or monoclonal anti-T-cell antibodies. Some have reached the clinical arena. 3)Very rencently, PDX-1 was found to be an important factor to diabetes. Study on mice got to reveal the therapeutical effect of PDX-1.

Abstract:Skeleton is mainly composed of two different tissues: bone and cartilage. Recent studies show that Cbfa1 is not only a key regulator controlling bone formation and growth, but also important in cartilage maturation. And it may also be involved in osteoclastogenesis and cartilage vascular invasion.

Abstract:DNA mismatch repair system exists in nearly all originisms. They have different components and mechanisms from prokaryote E.coli to eukaryote and human. Defects in human mismatch repair genes cause genome instability and hereditary nonpolyposis colon cancer (HNPCC) and others tumors. MutS in E.coli MMR system can specifically recognize mispaired or unpaired base and has been developed into powerful tools for detecting gene mutations and polymorphisms.

Abstract:The advances of research on the pathogenicity and pandemicity of Vibrio cholerae in recent years are reviewed. The gene ctxAB encoding cholera toxin CT is not a native component of Vibrio cholerae genome, while it originally comes from a bacteriophage CTXΦ. CTXΦ can specifically infect Vibrio cholerae and integrate into its genome, forming a prophage; while it can also secrete CTXΦ under the induction of the environmental factors.RS1 and CTX gene elements not only closely located in chromosome position, but also closely related functionally. RS1 element can also transform into a filamentous phage RS1Φ, secrete and transfer horizontally. VPI comes from VPIΦ,which can transmit between Vibrio cholerae strains, and it is the bridge for Vibrio cholerae to acquire CTX gene element. Two gene regions VSP-Ⅰ and VSP-Ⅱ closely related to pandemicity of the Vibrio cholerae were identified recently. The relationship between pathogenicity and pandemicity is discussed.

Abstract:Sleeping Beauty (SB), a member of the Tc1/mariner superfamily of transposable elements, is the only active DNA transposon system from vertebrate. The research progresses of SB in recent years are reviewed and some modifications are proposed to improve the performance of SB as a genetic manipulating system.

Abstract:The decapeptide gonadotropin-releasing hormone（GnRH）is a kind of regulative neuropeptide synthesized by GnRH-producing cell bodies in hypothalamus and is an important information molecule among nervous, immune and neuroendocrine systems. GnRH plays a pivotal role in the regulation of reproduction. In recent years, GnRH-analogues have been one of the most widely applied hormonal medicine. The research progresses of GnRH were reviewed, including structure, localization and distribution of GnRH and its receptor. A series of evidences on GnRH regulating reproduction activity at different levels such as pituitary and gonad, factors affecting secretion of GnRH, as well as prospects of studies on GnRH are also introduced.

Abstract:Lipid rafts are liquid-ordered membrane microdomains with a unique protein and lipid composition found on the plasma membrane. Caveolae, a type of lipid rafts, is characterized by high levels of cholesterol, sphingolipids and proteins, and is identified by the presence of the protein caveolin. The structure and component of lipid rafts is used of reciprocity and comformational change between proteins. Lipid rafts are associated with signal transduction and cellar proteins movement. The disfunction of lipid rafts is related to some diseases such as infection, heart disease, cancer, muscular dystrophy and prion protein diseases.

Abstract:The β-subunit of human chorionic gonadotropin (hCGβ) is secreted by trophoblastic cells during normal pregnancy. In addition, hCGβ is also synthesized by many tumor and cancer cells, and it has been one of target molecules for active immunotherapy to prevent and treat hCGβ dependent tumors and cancers. The full coding region of hCGβ was isolated by the RT-PCR method, and was inserted into PCR3.1 eukaryotic expression vector to construct the recombinant PCR3.1-hCGβ DNA vaccine successfully. Using HeLa cells transient expression system, the capability of hCGβ expression of PCR3.1-hCGβ was confirmed in vitro, and the expressed hCGβ protein was mainly with intracellular state. With 20 μg PCR3.1-hCGβ plasmid DNA through intramuscular inoculation after bupivacaine-HCl inducing, the immunized BALB/c mice could express hCGβ antigen to induce both intensive hCGβ-specific humoral immune responses and CTL responses, and the antibody titer reached high to 1∶8000. Moreover, both two types of immune responses elicited by PCR3.1-hCGβ could attack HeLa cells to induce apoptosis in vitro. The results indicated that the immune responses induced by PCR3.1-hCGβ DNA vaccine have antitumor effects in vitro, and would be helpful to detect the antitumor effects of PCR3.1-hCGβ DNA vaccine in vivo.

Abstract:hhlim was a new heart-related gene cloned from human embryonic heart whose product participated in transcriptional regulation and cell development as a kind of transcriptional factor. Over expression of hhlim gene using recombinant plasmid was sufficient to induce a greater than 1.5 fold increase in C2C12 muscle cell area compared with that untransfered with hhlim. RT-PCR and Western blot testified that transfection of hhlim into the C2C12 muscle cells could induce α-actin over expression and trigger the expression of embryonic related gene-BNP, which is related to the cell hypertrophy. hhlim-green fluorescence fusion protein in hhlim-pEGFP-C3 transfected C2C12 muscle cells was recorded with fluorescence microscopy during C2C12 muscle cell differentiation induced by horse serum. The data showed that the distribution change of green fluorescence of hhlim-GFP fusion protein from cytoplasm to nucleus was observed. Co-immunoprecipitation proved that hhlim protein exists in a manner that associates with α-actin in cytoplasm.

Abstract:The senescent phenotype of human embryo lung diploid fibroblasts was obtained by four treatments of early passage cells with 50 μmol/L H₂O₂ for 30 min every 2 days. After total mRNA extraction from the two kinds of cells, the young cells and H₂O₂-induced premature senesence cells，the cDNA of two kinds of cells were labled by Cy3 and Cy5 respectively were hybridized with microarray containing 4 096 human genes. The gene expression changes were identified by GnePix 4000B and GenePix 3.0. 123 genes changed their expression significantly, which involved in progress of cell cycle,metabolism and protein processing, formation and modification of cytoskeleton and extracellular matrix and signal transduction, and the most interesting finding was that secretory function seemed to be enhanced in premature senescence of fibroblasts.

Abstract:To screen NF-κB antagonistic drugs and research singal transduction pathway related to NF-κB, two vectors, p4κB-d2EGFP containing destabilized enhanced green fluorescent protein(d2EGFP) reporter gene and p4κB-EGFP with EGFP gene, were constructed on the base of 4 copies of NF-κB cis-element κB as enhancer, SV40 as basic promoter and neo^r gene as selective gene. The time and dose effects of d2EGFP and EGFP induced by p65 protein showed that p4κB-d2EGFP was the better NF-κB-responsive GFP reporter gene system because it is more sensitive to detect the changes of gene transcription regulation after p65 vector transiently contransfected with the two vectors respectively. The NF-κB-responsive d2EGFP clonal cell line named HEK-d2EGFP was established after p4κB-d2EGFP stablely transfected into HEK 293 cells. With the cotransfection of NF-κB transcription factor decoy (TFD) and p65 vector into HEK-d2EGFP cells, the results showed the groups of 1 mg/L and 2 mg/L TFD could antagonized the d2EGFP expression induced by p65 protein significantly. It was demonstrated that the NF-κB-responsive d2EGFP reporter system could report and detect NF-κB activation accurately and dynamically.

Abstract:SynaptotagminⅠ(sytⅠ) is an abundant integral membrane protein of synaptic vesicle and the C2AB domain is the important functional domain in its cytoplasmic part. Recent studies show that C2AB prefers to interact with plasmic membranes of neuron cells in vivo and it is believed that such interaction is closely related to the sytⅠ physiological function as a Ca²⁺ sensor in the Ca²⁺-regulated neurotransmitter release, but the mechanism of the interaction is not clearly understood. Monolayers at an air/water interface combined CD and fluorescence experiments were used to study the characteristic of interaction between C2AB and a phospholipid membrane. The results in the monolayer experiment showed that C2AB domain preferred insertion into the negatively charged phosphatidylserine monolayer and Ca²⁺ ions were required for the interaction. Electrostatic force was mostly responsible for the insertion of C2AB into PS monolayers. Further CD and fluorescence experiments showed that the secondary structure of C2AB domain in the presence or absence of PS/PC liposome had some relatively small change. The experiments provide useful information concerning the important role of sytⅠ as a Ca²⁺ sensor in the fusion of secretary vesicles to the plasma membrane, and better understanding the mechanisms of membrane fusion in exocytosis.

Abstract:The evolution of phoA gene fragment distant from the Asp101-Ser102-Ala103 encoding region to increase the catalytic activity of EAP with a single mutant D101S as parent was directed. Through two cycles of error prone PCR, coupled with a sensitive screening method, an evolved variant 4-186 was obtained. Its catalytic activity was 3-fold higher than that of D101S parent and 35-fold more active than wild-type EAP. The kinetic analysis indicated that the evolved enzyme exhibits a higher substrate binding ability and a higher catalytic efficiency than the D101S parent enzyme. DNA sequence revealed that 4-186 contains two amino acid substitutions, K167R and S374C, both of which locate neither the substrate-binding sites nor the metal-binding sites of EAP.

Abstract:LIM domain protein KyoT interacts with transcription factor RBP-J and modulates Notch signaling pathway. It was previously found that KyoT was specifically expressed in lung, kidney and testis of mice. To investigate the level of the expression of KyoT in mice embryogenesis and the distribution of the expression the 17 dpc (days of gestation), Northern blot and immunohistochemical SABC methods were used in the experiments. Northern blot showed that KyoT was expressed in almost all stages of embryos of mice and the level was highest in the 17 dpc. Moreover, Northern blot and immunohistochemical SABC showed that the mRNA and protein of KyoT were expressed at the high level in lung, kidney and muscle of 17 dpc mice. These results suggest that KyoT was expressed in mice embryogenesis and the distribution of the expression of KyoT in 17 dpc. was similar to that in adult mice and located in lung, kidney and muscle etc.

Abstract:To observe whether the re-expression of NAG7 gene could affect the gene expression of HNE1 cells, the cDNA microarray technique was employed to analyze the changes of gene expressions. 250 μg total RNA was extracted and 1 μg polyA mRNA was isolated. Reverse transcription was performed and cDNA probe was random-prime labeled with ³³P-dATP and hybridized with the cDNA microarray membrane containing 16 150 genes and ESTs. The hybridized result was confirmed by Northern blot analysis. FLA-3000A Plate scanner and Array Gauge software were used to screen and analyze the expressions of each gene. The background was eliminated and the differences of signal intensity of matched spots over 2 times were set as a marker to identify the differential expression genes. The results suggested that 179 genes were differential expressed, in which 91 genes were up-regulated and 88 genes were down-regulated in NAG7 re-expressed cells. These genes were involved in gene transcription, regulation, proliferation, metabolism, apoptosis, and so on. Northern blot result testified that growth arrest specific protein 1 (gas 1) expressed up-regulated. Especially, the previous results of proteomic research also found that the protein of gas 1 expressed up-regulated. Therefore, the data suggested that gas 1 gene plays a critical role in NAG7 re-expressed HNE1 cells, and provides an important clue to elucidate the mechanism of NAG7 gene.

Abstract:Alzheimer's disease (AD) is neuropathologically characterized by the presence of extracellular amyloid plaques and intracellular neurofibrillary tangles. The core of senile plaque is amyloid β-protein (Aβ)， which comes from its precursor —— amyloid β-protein precursor (APP). The pQE-APP_28～123 plasmids was constructed by gene recombination. The APP_28～123 protein was expressed in Escherichia coli and then purified. The purified products were examined for GM1 binding abilities by Western blotting. The effects of GM1 on conformation of APP N-terminus were detected by fluorescence and circular dichroism(CD) techniques. APP_28～123 protein could bind with GM1.The intrinsic fluorescence intensity of APP_28～123 protein in PC/GM1 vesicles or GM1 solution remarkably increased and the fluorescence peak value blue shifted 20 nm. CD results showed that the major secondary structure of APP_28～123 in PBS buffer was α-helix. When APP_28～123 incubated with PC/GM1 vesicles or GM1 solution，the α-helix content increased markedly. These results suggested that GM1 might affect the physiological function of APP and change APP span-membrane process and interfere APP trafficking and internalization by anchoring this molecule on the membrane， which may provide much more substrate for γ-secretase and enhance Aβ generation on the cells.