Abstract:筛选特异神经元表达的Gal4转基因品系对于果蝇神经系统及其功能的研究极其重要。GAL4蛋白是酵母中的一类转录因子，它能够与特定的上游激活序列（Upstream Active Sequences, UAS）结合，并驱动UAS下游基因的表达。果蝇的转座子P因子（P-element）可以在其自身产生的转座酶的作用下，在果蝇的基因组内移动。通过剔除P因子内转座酶基因，使其成为转基因的载体。在外源转座酶的帮助下将带有一个弱启动子的Gal4基因随机插入到果蝇的基因组中，如果插入的位置正好在某个增强子的作用范围内，就会驱动Gal4基因的表达，获得具有特定表达模式的Gal4转基因品系，这就是“增强子陷阱”（enhancer-trap）技术。通过转基因载体同样可以将UAS及其下游目的基因（如绿色荧光蛋白GFP）一起转入果蝇的基因组中，从而获得UAS-GFP转基因品系。当Gal4转基因品系与UAS-GFP转基因品系的果蝇杂交后，在其子代中产生的GAL4蛋白与UAS特异性结合，驱动UAS下游的GFP表达，从而标记出Gal4转基因品系的表达模式。经过大规模的筛选就可以得到大量具有特定表达模式的Gal4转基因品系。由于已有的Gal4品系本身不能产生转座酶，保证了已有Gal4品系的稳定遗传。如果人为地为已有的Gal4品系提供一次转座酶，就可以引起P因子的再次转座，从而可能产生具有特定表达模式的新Gal4品系。本期发表的《用“增强子陷阱” 技术构造并筛选果蝇UAS/Gal4系统中Gal4新品系及脑基因表达图谱数据库的开发》（见xx-xx页），报道了清华大学钟毅实验室经过开展大规模的遗传筛选，找到了一批目前国外Gal4基因库中还没有发现的具有特异神经元表达的Gal4品系，将极大地拓展果蝇脑结构与功能的研究。根据Gal4品系的表达模式，他们开发了在果蝇脑中特异表达的图谱数据库，将会方便快速查找实验所需的特定表达的Gal4转基因品系。这项工作不仅扩大了国际上果蝇Gal4品系资源，也为建立我国特有果蝇信息与资源平台打下了良好的基础。 果蝇作为一种经典的模式生物，其Gal4/UAS系统被比喻为瑞士军刀（Swiss Army Knife），是探索生命科学问题的强有力的工具。凭借Gal4/UAS系统，果蝇已经被应用于生命领域的各个学科中。如神经科学的研究中，需要研究各种基因、突触、神经元以及神经元网络在各种神经活动中的功能。有了大量特异神经元表达的Gal4转基因品系，就使得上述研究得心应手。如将UAS转基因品系中的目的基因换成要研究的基因，与特定表达模式的Gal4品系杂交，可以研究该基因在相关部位的功能。如果将UAS转基因品系中的目的基因换成各种功能阻断分子等，与各种特定表达的Gal4品系杂交，就可以准确研究果蝇脑中从单个神经元、神经元群到神经回路的功能，从而最终实现果蝇脑功能的精确定位。 目前国际上已经建立了多个果蝇的信息与资源平台，如美国印第安纳大学的布卢明顿果蝇种系中心（Bloomington Drosophila Stocks Center），日本京都工业技术大学的果蝇遗传资源中心（Drosophila Genetics Resource Center，DGRC）以及奥地利维也纳果蝇RNAi中心（Vienna Drosophila Resource Center, VDRC）等等。这些果蝇的信息与资源平台为各国科研人员进行信息交流、获得果蝇品系、促进科研发展起到了非常重要的作用。由于建立果蝇信息与资源库的成本较高，既需要大量工作人员进行长期的筛选工作，又需要稳定的资助来维持果蝇资源与数据库的运转。钟毅实验室克服了各种困难，建立了具有自己特色的Gal4转基因品系库和相关的数据库，并且还在不断的扩大和完善已有的资源与信息，极大地推动了国内果蝇信息与资源平台的建设。另一方面，随着国内果蝇实验室的不断发展与壮大，科研水平的日益提高，新的基因突变体和转基因品系不断增多，如何有效的利用国内的果蝇资源，如何推动国内果蝇信息与资源的共享，还需要国内果蝇实验室的不断努力。另外，建立和维持果蝇品系库需要大量人员和财力的支撑，我们也希望国内的果蝇实验室在国家相关部门的大力支持下，通过多种形式建立和完善各种果蝇的信息与资源平台，推动国内科研持续稳定地发展。

Abstract:Extensive studies have indicated that synaptic plasticity of neurons, including functional and structural plasticity, is intimately related to learning and memory. Recently, a long-term potentiation (LTP) induced by learning was successfully recorded in hippocampal neurons of the trained rats, which lost their retention memory if the late LTP was blocked by a kinase inhibitor. These results show that LTP may be a molecular mechanism underlying memory. Therefore, further studies on synaptic plasticity in the mammalian brain are of significance to revealing molecular mechanisms underlying learning and memory. Furthermore, abnormal morphology, shrinkage and reduced density of dendritic spines and defects in LTP were observed in brains of the patients suffering from mental retardation and neurodegenerative diseases; many mutant genes found from these patients encode component proteins of signal transduction for neuronal plasticity. These studies on synaptic plasticity would certainly promote making the effective prevention and treatment procedures for mental and neurodegenerative diseases. Advances in synaptic plasticity studies and looks into the future of this research field are reviewed.

Abstract:EBP50(ERM-binding phosphoprotein-50)，a multifunctional adapter protein with 358 amino acids and two PDZ domains, regulates cell growth and migration. Lines of evidences indicate that it is a potential cancer suppressor protein. Loss of heterozygosity (LOH) and intragenic mutation of the ebp50 gene have been found in both primary breast tumors and breast cancer cell lines. EBP50 suppresses the breast cancer cell proliferation via its interaction with many tumor suppressor protein including PTEN, SYK, MERLIN, etc. Here the molecular structure of EBP50, signal pathway regulated by EBP50, and the relationship between breast cancer development and EBP50 are discussed.

Abstract:The PHD finger is a Zn-binding domain found in all eukaryotic genomes, and typically show a C4HC3 signature. Notably, many if not all PHD fingers are found in nuclear proteins whose functions are associated with the regulation of transcription, cell cycle and apoptosis. Increasingly evidences suggest that the PHD finger has multiple functions, including the protein-protein interaction, especially interact with nucleosomes. The pattern and combination of histone modifications, for example, methylation, acetylation, phosphorylation and ubiquitination etc, have been believed to be an important regulator of gene expression and state of the chromatin, which have raised the histone code hypothesis. With the feature of specific recognizing methylated histone, the PHD finger may functions as an important reader of the histone code.

Abstract:According to the report from World Health Organization (WHO), about two billion people worldwide have been infected with hepatitis B virus (HBV), of which 350 million people with chronic HBV infection. Now China has 130 million HBV carriers and 30 million patients suffered from hepatitis B. About 20%～40% of patients after years of inflammatory damage may develop into liver cirrhosis and liver cancer. Even today, however, people still have not found a specific drug that can completely cure chronic hepatitis B. Since the phenomenon of RNA interference (RNAi) was discovered, people have been constantly trying to apply it to the anti-viral drug research and development. Findings show that RNAi can indeed inhibit the replication of HBV. However, the efficiency of gene silencing varies considerably due to the different sequences of RNAi. In addition, the safety and effectiveness of RNAi-based antiviral drugs tested on humans still need to be further investigated. And the "off target" phenomenon of RNAi is another problem that must be resolved before its clinical trial.

Abstract:p27^kip1 is an important negatively regulator of cell cycle progression and plays a central role in the pathogenesis of a member of tumors including breast cancer. In breast cancer cells, the level of p27^kip1 expression usually decreases during tumor development and progression, in addition, cytoplasm　mislocalization of p27^kip1 has been reported, but less is known about the exact molecular mechanisms. Studies have indicated that phosphorylation is the key regulation way, several signal transduction pathways are involved in the regulation of the expression and distribution of p27^kip1. To further understand the mechanism, the disparity of the interacting protein profiling between tumor cells and normal cells must be identified first. Including cyclins, cyclin-depend kinases, CRM1, jab1, SKP2, p27^kip1 has various interacting molecules. There are also several interacting molecules especially for breast cancer cells. It seems that different protein profiling cause the different expression and intracellular distribution in different cell cycle phase. So, disparity of the p27^kip1 protein profiling may be the main mechanism of its down-expression and mislocalization in breast cancer cells.

Abstract:“Enhancer trap” is an important technique to build and explore Drosophila brain gene expression pattern and its database. Large genome-wide screen for specific expression pattern in adult Drosophila brains by enhancer trap method provides fly tools for further understanding neural basis of learning and memory. Enhancer trap method was utilized to generate Drosophila P[Gal4] mutant library. Each mutant in library was crossed with GFP reporter flies (UAS-eGFP); heads of the adult off-springs were dissected to obtain the whole brain which were examined under the epifluorescence microscope and expression pattern of each mutant were further analyzed. JavaScript was utilized to establish an expression pattern library. A total of 2 677 mutant lines of Drosophila were obtained by P-element induced mutagenesis and subjected to expression pattern analysis. Most of mutated genes express in adult fly. 368 mutated genes specific express in the mushroom body, which was reported to involve in Drosophila olfactory learning and memory, and some others specific express in other neuron circuitries. Drosophila mutant libraries and genome-wide expression pattern database was built by enhancer trap method, which as a tool will be utilized to understand the function of genes and particular neural circuitries in Drosophila in the future.

Abstract:The incorporation of site-specific recombination systems can help to overcome bottlenecks in livestock transgenic technology. For evaluating the efficiency of Cre/lox mediated DNA recombination in embryos and somatic cells, a working model was established using rat mammary carcinoma cell line SHZ-88, aimed at creation of and use repeatedly of selected “friendly loci” in transgenic livestock. An integration vector pTE-lox2272-DsRed-loxP-GFP-loxP, which red fluorescence gene DsRed served as the first target gene and green fluorescence gene GFP as marker gene, was constructed for introduction of acceptor loci in genome. At the same time a replacement vector pT-lox2272-neo-loxP in which Neo coding sequence served as the second target gene was also constructed for replacing DsRed gene. Transgenic cell clones were produced by electroporating SHZ-88 cell with the integration vector. Cells from three transgenic clones selected randomly were further amplified and were then co-electroporated with the replacement vector as well as cre gene. Analysis of the expression patterns of DsRed and GFP indicated that among the 1 070 cell colonies the efficiency on marker GFP deletion was 91.1% and the efficiency on gene replacement was 29.3%. Molecular analysis by PCR and Southern blotting confirmed that the color patterns as expressed by cell colonies could represent the actual molecular events. This working model mediated by Cre/lox system should be useful for the improvement of the present animal transgenic technology.

Abstract:The previous studies have showed that neutrophil gelatinase-associated lipocalin (NGAL) was overexpressed in the progress of human esophageal epithelial cells and novel 12-O-tetradecanoyl phorbol-13- acetate (TPA) response elements (TRE) might be located on the -152～-60 position of the 5′ flanking region of the promoter. However, the nucleic proteins bound to the elements have not been identified. In present study, the TRE binding-proteins from EC109 cells following TPA induction were purified by oligonucleotide trapping DNA affinity chromatograph, further separated by SDS-PAGE and stained with silver staining. And then the protein bands were subsequently identified by MALDI-TOF-MS and the resulting spectra were searched to NCBI protein data bank through Mascot. C19, KIAA1949, TDRD1, RXRβ, FAM54A, KLF15, KLF10 and YY-1 eight proteins were identified according to their function, localization and molecular mass. Finally, RT-PCR was performed to determine the TPA responsiveness of these nucleic proteins. Results showed that C19, KIAA1949, TDRD1, RXRβ and KLF15 proteins have the obvious TPA responsiveness in the transcription level. The results suggest these proteins might respond the stimulation of TPA and regulate the expression of NGAL in EC109 cells.

Abstract:To study the biocompatibility of the titanium dental implant surfaces coated with nacre in vitro, osteoblast-like cells ( MC3T3E1) growth on the substrates coated with nacre were compared with that growth on the substrates coated with hydroxyapatite (HA) or nothing and the cells of the blank. MC3T3E1 were incubated on the respective surface for 3days, 5days and 7 days. Cell morphology was estimated by inverted phase contrast microscope and scanning electron microscope (SEM) and cell proliferation was measured by flowcytometry. The King's chromometry was used for measuring the activity of ALP in the cell lysate and the expression of TGF-β1 was detected by Western blotting. The results showed that MC3T3E1 cultured on the nacre-coated surface were more spreaded and plumper than the controls on the 3days, 5days and 7days of culture. Meanwhile, the test group showed a superiority to control groups and blank with respects to the cell proliferation index[(35.9±2.5)%, (69.7±3.3)% , (58.2±2.6)%], ALP activity [(6.123±2.917), (17.486±1.986), (23.987±1.372) U/g] and TGF-β1 level. In conclusion, the surface coated nacre can promote the proliferation and differentiation of osteoblast, which the mechanism partly may be the increasing expression of TGF-β1.

Abstract:A soluble Jagged-1/Fc chimera protein (Jagged-1/Fc) was directly used to induce the differentiation of lymphonode cells in mice into CD4⁺CD25⁺ regulatory T cells in vitro. A fluorescein-labeled monoclonal antibody staining combined with flow cytometry was applied to detect the effect of different doses of Jagged-1/Fc on the differentiation of the lymphonode cells into CD4⁺CD25⁺ T cells at different time, and to measure intracellular cytokine changes of the T cells induced by Jaggde1/Fc. The level of TGF-β1, IL-4 and IL-10 secreted by the T cells that were induced by Jagged-1/Fc was determined by ELISA. The results showed that over 500.0 μg/L of Jagged-1/Fc led to the obvious enhancement of the proportion of CD4⁺CD25⁺ T cells within the day 4 to 6 of induction, which was abrogated with an anti-Jagged-1/Fc monoclonal antibody. This induction action of Jagged-1/Fc on CD4⁺CD25⁺ T cells was also inhibited by the block of a Notch signal pathway with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). The level of IL-4 and IL-10 in the supernatant of T cell culture and their intracellular level were elevated by the induction of Jagged-1/Fc，and the level of TGF-β1 in the supernatant was not altered. These findings suggest that a soluble Jagged-1/Fc chimera protein may induce the differentiation of mouse lymphonode cells into CD4⁺CD25⁺ regulatory T cells in vitro.

Abstract:Laryngeal carcinoma related gene 1(LCRG1) is a candidate tumor suppressor gene of Laryngeal carcinoma. To further investigate its transcriptional regulation, the transcriptional start sites for LCRG1 gene have been identified by 5′ RACE (rapid amplification of cDNA ends) based on the bioinformation analysis of LCRG1. Then eleven luciferase expression vectors which contained potential human LCRG1 gene promoter were constructed. Luciferase reporter assay indicated that LCRG1 promoter region was mainly located in －169～＋127 region nearby the major transcriptional start site. These results suggested that the region (－169～＋127) includes an essential promoter for human LCRG1 gene transcription.

Abstract:In order to identify virulence factors of the pathogen, the outer membrane proteins of virulent and avirulent strains of Riemerella anatipestifer were compared by a proteome analysis. Three protein spots differentially expressed between the two strains were observed by 2-DE gels, and were further analyzed using in gel tryptic digestion and peptide mass fingerprinting. Three proteins were identified. W1 was Hsp20, W2 and W3 were transposon. Although the exact role of these proteins has not been characterized, the exclusive expression in virulent strain may indicate that they play an important role in the pathogenesis of Riemerella anatipestifer infection. Although only two virulence factors identified, it opens a path to the further analysis of virulence factors of Riemerella anatipestifer.

Abstract:The adhesion of leukocytes to vascular endothelium is crucial for the generation of inflammatory responses. The selectins and β₂-integrin (Mac-1) play a major role in the process. Recently, it was reported that RO-heparin can inhibit selectin-mediated leukocyte adhesion. The effect of RO-heparin on the Mac-1-mediated neutrophils adhesion were further tested. The results showed that RO-heparin could effectively inhibit neutrophils binding to ICAM-1, adhering to COS-7 cells expressing human ICAM-1, and adhering to human umbilical vein endothelial cells (HUVECs) under static and flow conditions. The findings suggest that the effect of RO-heparin on leukocyte adhesion is mainly due to its inhibition on the interaction between selectins or Mac-1 and their ligands and that RO-heparin might be useful in preventing inflammation diseases.

Abstract:Calmodulin (CaM) is a ubiquitous, multifunctional calcium (Ca²⁺) sensor that exists in all eukaryotes . Calmodulin-binding proteins (CaMBPs) play important roles in various signal pathway of calmodulin. The finding of new CaMBPs will be useful for illustrating mechanism of CaM implicating in plant growth and development. Yeast two-hybrid system is an effective method for studying protein-protein interactions in vivo. In the study of plant signal transduction, many important signal transduction molecules were obtained by this system. Here, Arabidopsis flower were used as the material. The Yeast two-hybrid library of Arabidopsis flower was constructed by co-transforming yeast strain AH109 with ds cDNA, pGADT₇-rec and pGBKT₇-ACaM2. A positive clone was identified. DNA sequencing and analysis indicated that this cDNA clone encodes calmodulin-binding protein AtIQD26. The AtIQD26 protein contains a plant-specific domain of 67 conserved amino acid residues, referred to as the IQ67 domain(IQD), which is characterized by a unique and repetitive arrangement of three different CaM recruitment motifs, known as the IQ, 1-5-10, and 1-8-14 motifs. Yeast two-hybrid analysis and gel overlay experiments demonstrated that AtIQD26 interacted with CaM both in the presence or absence of Ca²⁺. A striking feature of AtIQD26 is the high isoelectric point (～10.6) and frequency of serine residues (～10%). To uncover potential roles for AtIQD26, a series of expression vectors were constructed about it, and the relative transgenic works were finished. Using these transgenic lines, the tissue expression and the subcellular localization of AtIQD26 were studied. Fusion GFP reporter showed that AtIQD26 was located at the nucleus and near cytoplasm membrane. GUS histochemical assay showed that AtIQD26 had characteristic of universal tissue expression, especially in the renascent tissue. Interaction of AtIQD26 with CaM and the presence of predicted CaM binding sites in it suggest that AtIQD26 is a new member of CaM targets. The basic isoelectric point and its potential nuclear localization suggest that AtIQD26 links Ca²⁺ signaling pathways to the regulation of gene expression. Expression similarity indicates that AtIQD26 combined with CaM to regulate plant development and growth.

Abstract:Cardiomyocytes hypoxia resulting from ischemia is a major pathological factor in ischemic heart disease(IHD), and diabetes is one of the most common complications in IHD. myocardial damages aggravate and prognosis is worse in patients with coronary heart disease and diabetes, which is probably concerned with the reduction of hypoxia-inducible factor 1 (HIF-1) expression. But the mechanisms of HIF-1 signal transduction system in diabetes are not clear. In recent years, many studies have indicated that increasing expression and activity of HIF-1α in IHD can promote neovascularization and cardiomyocytes survival, decrease ischemical reperfusion injury and myocardial infarction (MI) size, and increase myocardial function. Therefore regulating expression and activity of HIF-1α becomes a new way to treat IHD. Myocardial infarction models were made in GK diabetes rats through the ligation of left anterior descending coronary artery (LADCA). By using immunohistochemistry staining and RT-PCR methods, results suggested that HIF-1α expression decreased and myocardial infarction size increased in GK diabetes rats combined with MI. When treated with cobalt chloride(CoCl₂), blood glucose level decreased, HIF-1α expression increased and myocardial infarction size reduced in GK diabetes rats, which may provide a new insight on treatment of coronary heart disease combined with diabetes by regulating HIF-1 signal transduction system.

Abstract:Rab GTPases serve as master regulators of vesicular membrane transport on both the exo- and endocytic pathways. Though there are many reports on Rab proteins, the function of these small proteins still remain in speculation. And no report has ever clarified the character of human Rab26. Here it was reported that a novel Rab protein Rab26 is membranous organelle related and involved in endocytosis of HeLa cells. By using RT-PCR method a novel Rab26 cDNA full-length cDNA of Rab26 that is 1656 bp was identified.The cDNA sequence that at 1197 is ‘A’ other than ‘G’, while ‘C’ at 956 substitutes for ‘T’, and has ‘GCC’ insertion at 48 to 50 compared with published sequences. The complete open reading frame (ORF) is 771 bp in length encoding 256-residue protein with a calculated molecular mass of 27.9 ku (GenBank accession No.AY646153), rather than a shorter one with 190-amino acid residue as reported previously. GFP labeled full-length Rab26 expression showed that Rab26 was mainly sublocated in membranous organelles and could enhance endocytosis which means could took PE labeled protein as an endocytic tracer. RT-PCR analysis showed Rab26 was detected to express in several kinds of adenocarcinoma cell lines such as Acc2, AccM, SPC-A1 and HeLa cell lines, which indicated that Rab26 expression might be associated with some carcinomas.