白芷细胞外钙调素结合蛋白（ECBP21）免疫组化及金标定位分析

ECBP21是我们从白芷悬浮培养细胞外纯化及cDNA克隆的一种钙调素结合蛋白(CaMBP),亦是植物中首次报道的细胞外CaMBP。本实验以大肠杆菌表达的重组ECBP21蛋白为抗原,制备了高效价特异性抗体;随后,利用免疫组化及金标定位技术,研究了ECBP21蛋白组织特异性分布及亚细胞定位。免疫组化结果表明：ECBP21在白芷各组织中均有分布,但在叶、花、花序轴中较多,而在根中较少,并且ECBP21在细胞中多分布于细胞壁区域;免疫胶体金电镜定位结果显示：在白芷花序轴细胞中金颗粒主要分布在细胞壁,表明ECBP21蛋白主要定位于细胞壁区域,从而首次为细胞外CaMBP(ECBP21)的胞外存在提供了直观证据,并为进一步研究其在植物生长发育中的功能提供了初步信息。     

A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems. Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2). However, slow maturation is still a big obstacle to the use of GFP variants for visualization. These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles. Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications. Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-). For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired. Here we describe the development of an improved version of YFP named "Venus". Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation. As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl-. We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells. Its secretion was readily monitored by measuring release of fluorescence into the medium. The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices. With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before.     

Repeated pre-training sleep restriction in adolescent rats impaired spatial performance

Sleep and Biological Rhythms - Chronic sleep deprivation (SD) is an overwhelming problem in young students. Firstly, we investigated whether different levels of pre-training SD had effects on...     

A novel protein MAJN binds to Jak3 and inhibits apoptosis induced by IL-2 deprival

To find a possible signal interacting with the Jak3 N-terminal, we screened the human peripheral blood cDNA library through both a two-hybrid system and a tyrosine-phosphorylation-modified two-hybrid system using the N-terminal of Jak3 as bait. Results showed that one new homologue of myosin heavy chain, designated MAJN (molecule associated with Jak3 N-terminal), could bind to Jak3 in a tyrosine-phosphorylation-independent manner. The interaction between Jak3 and MAJN was further confirmed by immunoprecipitation in BAF-B03 beta cells. To investigate the function of MAJN, we have constructed the BAF-B03 beta/MAJN cell line that stably expresses MAJN and found that overexpression of MAJN can partially inhibit the apoptosis induced by interleukin-2 deprival. Further studies are needed to elucidate how MAJN executes its function to antagonize BAF-B03beta cell death in the absence of IL-2.     

Gene Systems Network Inferred from Expression Profiles in Hepatocellular Carcinogenesis by Graphical Gaussian Model

Hepatocellular carcinoma (HCC) in a liver with advanced-stage chronic hepatitis C (CHC) is induced by hepatitis C virus, which chronically infects about 170 million people worldwide. To elucidate the associations between gene groups in hepatocellular carcinogenesis, we analyzed the profiles of the genes characteristically expressed in the CHC and HCC cell stages by a statistical method for inferring the network between gene systems based on the graphical Gaussian model. A systematic evaluation of the inferred network in terms of the biological knowledge revealed that the inferred network was strongly involved in the known gene-gene interactions with high significance Open image in new window , and that the clusters characterized by different cancer-related responses were associated with those of the gene groups related to metabolic pathways and morphological events. Although some relationships in the network remain to be interpreted, the analyses revealed a snapshot of the orchestrated expression of cancer-related groups and some pathways related with metabolisms and morphological events in hepatocellular carcinogenesis, and thus provide possible clues on the disease mechanism and insights that address the gap between molecular and clinical assessments.     

A chimeric mouse-human antibody that retains specificity for HIV gp120 and mediates the lysis of HIV-infected cells

Murine mAb BAT123, which was made against the envelope glycoprotein gp120 of HTLV-IIIB strain of HIV type 1 (HIV-1), is capable of neutralizing HTLV-IIIB in vitro. It also inhibits the fusion between uninfected CD4+ cells and HIV-1-infected cells to form syncytia. As a step to explore the potential utility of the anti-HIV antibody in vivo, we have constructed a mouse-human chimeric antibody by rDNA techniques. The chimeric antibody, which bears the variable domains of mouse antibody BAT123 and constant domains Cr1 and C kappa of human Ig retains the Ag specificity of BAT123 as determined by its reactivity with HIV-1-infected H9 cells, gp120 in Western blot analysis, and the oligopeptide recognized by BAT123. The antiviral activities of the chimeric antibody in neutralizing HIV-1 infection as well as inhibiting the syncytia formation are also found identical to those of the parent murine antibody. Moreover, in the presence of human blood mononuclear cells, the chimeric antibody but not BAT123 (mouse IgG1) induces antibody-dependent cellular cytotoxicity. The findings point to the potential usefulness of the chimeric antibody in treating patients infected with HIV-1.     

Complete mitochondrial genome of Tubulipora flabellaris (Bryozoa: Stenolaemata): the first representative from the class Stenolaemata with unique gene order

Mitochondrial genomes play a significant role in the reconstruction of phylogenetic relationships within metazoans. There are still many controversies concerning the phylogenetic position of the phylum Bryozoa. In this research, we have finished the complete mitochondrial genome of one bryozoan (Tubulipora flabellaris), which is the first representative from the class Stenolaemata. The complete mitochondrial genome of T. flabellaris is 13,763 bp in length and contains 36 genes, which lacks the atp8 gene in contrast to the typical metazoan mitochondrial genomes. Gene arrangement comparisons indicate that the mitochondrial genome of T. flabellaris has unique gene order when compared with other metazoans. The four known bryozoans complete mitochondrial genomes also have very different gene arrangements, indicates that bryozoan mitochondrial genomes have experienced drastic rearrangements. To investigate the phylogenetic relationship of Bryozoa, phylogenetic analyses based on amino acid sequences of 11 protein coding genes (excluding atp6 and atp8) from 26 metazoan complete mitochondrial genomes were made utilizing Maximum Likelihood (ML) and Bayesian methods, respectively. The results indicate the monopoly of Lophotrochozoa and a close relationship between Chaetognatha and Bryozoa. However, more evidences are needed to clarify the relationship between two groups. Lophophorate appeared to be polyphyletic according to our analyses. Meanwhile, neither analysis supports close relationship between Branchiopod and Phoronida. Four bryozoans form a clade and the relationship among them is T. flabellaris + (F. hispida + (B. neritina + W. subtorquata)), which is in coincidence with traditional classification system.