Effects of DNA binding of the zinc finger and linkers for domain fusion on the catalytic activity of sequence-specific chimeric recombinases determined by a facile fluorescent system

Artificial zinc finger proteins (ZFPs) consist of Cys(2)-His(2)-type modules composed of ～30 amino acids with a ββα structure that coordinates a zinc ion. ZFPs that recognize specific DNA target sequences can substitute for the binding domains of enzymes that act on DNA to create designer enzymes with programmable sequence specificity. The most studied of these engineered enzymes are zinc finger nucleases (ZFNs). ZFNs have been widely used to model organisms and are currently in human clinical trials with an aim of therapeutic gene editing. Difficulties with ZFNs arise from unpredictable mutations caused by nonhomologous end joining and off-target DNA cleavage and mutagenesis. A more recent strategy that aims to address the shortcomings of ZFNs involves zinc finger recombinases (ZFRs). A thorough understanding of ZFRs and methods for their modification promises powerful new tools for gene manipulation in model organisms as well as in gene therapy. In an effort to design efficient and specific ZFRs, the effects of the DNA binding affinity of the zinc finger domains and the linker sequence between ZFPs and recombinase catalytic domains have been assessed. A plasmid system containing ZFR target sites was constructed for evaluation of catalytic activities of ZFRs with variable linker lengths and numbers of zinc finger modules. Recombination efficiencies were evaluated by restriction enzyme analysis of isolated plasmids after reaction in Escherichia coli and changes in EGFP fluorescence in mammalian cells. The results provide information relevant to the design of ZFRs that will be useful for sequence-specific genome modification.     

Association of zinc ion release and oxidative stress induced by intratracheal instillation of ZnO nanoparticles to rat lung

Zinc oxide (ZnO) nanoparticles are one of the important industrial nanoparticles. The production of ZnO nanoparticles is increasing every year. On the other hand, it is known that ZnO nanoparticles have strong cytotoxicity. In vitro studies using culture cells revealed that ZnO nanoparticles induce severe oxidative stress. However, the in vivo influence of ZnO nanoparticles is still unclear. In the present study, rat lung was exposed to ZnO nanoparticles by intratracheal instillation, and the influences of ZnO nanoparticles to the lung in the acute phase, particularly oxidative stress, were examined. Additionally, in vitro cellular influences of ZnO nanoparticles were examined using lung carcinoma A549 cells and compared to in vivo examinations. The ZnO nanoparticles used in this study released zinc ion in both dispersions. In the in vivo examinations, ZnO dispersion induced strong oxidative stress in the lung in the acute phase. The oxidative stress induced by the ZnO nanoparticles was stronger than that of a ZnCl(2) solution. Intratracheal instillation of ZnO nanoparticles induced an increase of lipid peroxide, HO-1 and alpha-tocopherol in the lung. The ZnO nanoparticles also induced strong oxidative stress and cell death in culture cells. Intracellular zinc level and reactive oxygen species were increased. These results suggest that ZnO nanoparticles induce oxidative stress in the lung in the acute phase. Intracellular ROS level had a high correlation with intracellular Zn(2+) level. ZnO nanoparticles will stay in the lung and continually release zinc ion, and thus stronger oxidative stress is induced.     

A proteomics approach to the cell-surface interactome using the enzyme-mediated activation of radical sources reaction

We previously reported a simple method to analyze the interaction of cell-surface molecules in living cells. This method termed enzyme-mediated activation of radical sources (EMARS) is featured by radical formation of the labeling reagent by horseradish peroxidase (HRP). Herein, we propose an approach to the cell-surface molecular interactome by using combination of this EMARS reaction and MS-based proteomics techniques. In the current study, we employed a novel labeling reagent, fluorescein-conjugated arylazide. The fluorescein-tagged proteins resulting from the EMARS reaction were directly detected in the electrophoresis gels with a fluorescence image analyzer. These products were also purified and concentrated by immunoaffinity chromatography with anti-fluorescein antibody-immobilized resins. The purified fluorescein-tagged proteins were subsequently subjected to an MS-based proteomics analysis. Analysis using HRP-conjugated cholera toxin subunit B, which recognizes a lipid raft marker, ganglioside GM1, revealed 30 membrane and secreted proteins that were candidates for the cell-surface molecules coclustering with GM1. The proposed approach will provide a clue to study functional molecular interactions in a variety of biological events on the cell surface.     

Fibroblast growth factor-2 is an important factor that maintains cellular immaturity and contributes to aggressiveness of osteosarcoma

Osteosarcoma is the most frequent, nonhematopoietic, primary malignant tumor of bone. Histopathologically, osteosarcoma is characterized by complex mixtures of different cell types with bone formation. The role of environmental factors in the formation of such a complicated tissue structure as osteosarcoma remains to be elucidated. Here, a newly established murine osteosarcoma model was used to clarify the roles of environmental factors such as fibroblast growth factor-2 (Fgf2) or leukemia-inhibitory factor (Lif) in the maintenance of osteosarcoma cells in an immature state. These factors were highly expressed in tumor environmental stromal cells, rather than in osteosarcoma cells, and they potently suppressed osteogenic differentiation of osteosarcoma cells in vitro and in vivo. Further investigation revealed that the hyperactivation of extracellular signal-regulated kinase (Erk)1/2 induced by these factors affected in the process of osteosarcoma differentiation. In addition, Fgf2 enhanced both proliferation and migratory activity of osteosarcoma cells and modulated the sensitivity of cells to an anticancer drug. The results of the present study suggest that the histology of osteosarcoma tumors which consist of immature tumor cells and pathologic bone formations could be generated dependent on the distribution of such environmental factors. The combined blockade of the signaling pathways of several growth factors, including Fgf2, might be useful in controlling the aggressiveness of osteosarcoma.     

Up-Regulation of Imp3 Confers In Vivo Tumorigenicity on Murine Osteosarcoma Cells

Osteosarcoma is a high-grade malignant bone tumor that manifests ingravescent clinical behavior. The intrinsic events that confer malignant properties on osteosarcoma cells have remained unclear, however. We previously established two lines of mouse osteosarcoma cells: AX cells, which are able to form tumors in syngeneic mice, and AXT cells, which were derived from such tumors and acquired an increased tumorigenic capacity during tumor development. We have now identified Igf2 mRNA-binding protein3 (Imp3) as a key molecule responsible for this increased tumorigenicity of AXT cells in vivo. Imp3 is consistently up-regulated in tumors formed by AX cells, and its expression in these cells was found to confer malignant properties such as anchorage-independent growth, loss of contact inhibition, and escape from anoikis in vitro. The expression level of Imp3 also appeared directly related to tumorigenic ability in vivo which is the critical determination for tumor-initiating cells. The effect of Imp3 on tumorigenicity of osteosarcoma cells did not appear to be mediated through Igf2-dependent mechanism. Our results implicate Imp3 as a key regulator of stem-like tumorigenic characteristics in osteosarcoma cells and as a potential therapeutic target for this malignancy.     

Histochemical analyses of hepatic architecture of the hagfish with special attention to periportal biliary structures

The hagfish liver was histochemically examined with special attention to biliary structures around the portal veins. Hepatocytes were organized into tubular structures surrounded by sinusoids. Biliary ductule structures, which resemble the ductal plates transiently appearing in mammalian liver development, were observed around the portal veins, but they did not appear around central veins. Thus, the hagfish liver demonstrates the same basic structure as the mammalian liver; that is, a vascular system from portal to central veins via sinusoids, and portal triad structures consisting of portal veins, hepatic arteries, and intrahepatic bile ducts. The epithelial cells of the ductal platelike structures strongly expressed cytokeratin, had some lectin-binding sites, and were delineated by the basal lamina, which was reactive for periodic acid-Schiff (PAS) staining and Iectin histochemistry. The lumina of the ductal plate-like structures were comparatively small and heterogeneous in diameter around the portal veins, suggesting that the biliary structures may not be efficient for bile secretion. The epithelial cells of the gall bladder had a simple columnar shape and were a PAS-positive cytoplasm. Those of bile ducts near the hilus, including extrahepatic and hepatic ducts, were simple columnar or cuboidal cells, and had large lumina. The cytoplasm in these cells was PAS-positive. These phenotypes with the expression of lectin-binding sites were clearly different from those of the ductal plate-like structures in the liver proper, suggesting that the extrahepatic and intrahepatic biliary structures may have different developmental origins.     

Effect of single-site charge-reversal mutations on the catalytic properties of yeast cytochrome c peroxidase: mutations near the high-affinity cytochrome c binding site

Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) have been constructed to determine the effect of localized charge on the catalytic properties of the enzyme. The mutations are located on the front face of CcP, near the cytochrome c binding site identified in the crystallographic structure of the yeast cytochrome c-CcP complex [Pelletier, H., and Kraut, J. (1992) Science 258, 1748-1755]. The mutants are characterized by absorption spectroscopy and hydrogen peroxide reactivity at both pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1-ferrocytochrome c(C102T) as a substrate at pH 7.5. Some of the charge-reversal mutations cause detectable changes in the absorption spectrum, especially at pH 7.5, reflecting changes in the equilibrium between penta- and hexacoordinate heme species in the enzyme. An increase in the amount of hexacoordinate heme in the mutant enzymes correlates with an increase in the fraction of enzyme that does not react with hydrogen peroxide. Steady-state velocity measurements indicate that five of the 15 mutations cause large increases in the Michaelis constant (R31E, D34K, D37K, E118K, and E290K). These data support the hypothesis that the cytochrome c-CcP complex observed in the crystal is the dominant catalytically active complex in solution.     

热带季节雨林多花白头树年内径向生长动态及其对环境因子的响应

为了解热带地区树木的季节性生长动态和规律,在西双版纳热带季节雨林利用高精度生长仪和微树芯法对落叶树种多花白头树的径向生长季节动态进行监测。结合木质部非结构性碳水化合物和环境因子的监测,分析其形成层活动和径向季节动态的生理生态驱动因子。结果表明: 在2020年,生长仪的监测显示,多花白头树于5月底(儒略日DOY:149.3±7.2)开始生长,8月底(DOY:241.0±14.7)生长结束,年生长量为3.12 mm,最大生长速率为0.04 mm·d^-1。而微树芯法显示,扩大细胞3月9日(DOY:69.2±6.2)开始出现,9月19日(DOY:262.8±2.8)细胞加厚结束,木质部生长量为1.76 mm,最大生长速率为0.009 mm·d^-1。多花白头树径向日生长量与生长季的降水、相对湿度、日最低气温、深度为20 cm的土壤含水量和温度呈显著正相关,而与日最高气温、水汽压亏缺、最大风速和水汽压呈显著负相关。多花白头树边材淀粉含量和可溶性糖含量均在生长季开始之前保持较高水平,淀粉含量在3月底达到最低,而可溶性糖含量5月中旬达到最低,随着生长季的结束淀粉和可溶性糖的含量分别在10月中旬和12月底达到最高。