A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions

Fluorescent protein (FP) has enabled the analysis of biomolecular interactions in living cells, and bimolecular fluorescence complementation (BiFC) represents one of the newly developed imaging technologies to directly visualize protein–protein interactions in living cells. Although 10 different FPs that cover a broad range of spectra have been demonstrated to support BiFC, only Cerulean (cyan FP variant), Citrine and Venus (yellow FP variants)-based BiFC systems can be used under 37 °C physiological temperature. The sensitivity of two mRFP-based red BiFC systems to higher temperatures (i.e., 37 °C) limits their applications in most mammalian cell-based studies. Here we report that mLumin, a newly isolated far-red fluorescent protein variant of mKate with an emission maximum of 621 nm, enables BiFC analysis of protein–protein interactions at 37 °C in living mammalian cells. Furthermore, the combination of mLumin with Cerulean- and Venus-based BiFC systems allows for simultaneous visualization of three pairs of protein–protein interactions in the same cell. The mLumin-based BiFC system will facilitate simultaneous visualization of multiple protein–protein interactions in living cells and offer the potential to visualize protein–protein interactions in living animals.     

Antibacterial activity of silkworm hemolymph after hemopoietic organ being injured with radiosurgery

Abstract:  To study the effect of hemopoietic organs damage on hemocyte function and antibacterial activity of hemolymph, silkworm ( Bombyx mori ) larvae were locally irradiated with carbon ion beams (¹²C⁵⁺, 100 Gy), live and death ratio of hemocytes and antibacterial activity of hemolymph were investigated. For unirradiated controls, the ratio of died hemocytes hardly changed at the fifth instar, but for locally irradiated silkworms, with growth died hemocytes and low-functional hemocytes increased clearly, and reached an extremely significant level at the later stage of the fifth instar. For irradiated individuals, the phenolxidase activities and sterilization effect of hemolymph were clearly lower than those of controls. So it is considered that after irradiating hemopoietic organs with heavy ion beams, not only the number of hemocytes decreased but the function of hemocytes also dropped, and they at last lead to a decline in immunity.     

The interface behavior and biocatalytic activity of superoxide dismutase at carbon nanotube

The interface behavior and biocatalytic activity of superoxide dismutase (SOD) were studied at carbon nanotube (CNT) surface with cyclic voltammetry (CV). The results show that SOD participates in a rapid exchange with CNT and the process is a single-electron-single-proton process. The electron-transfer coefficient was calculated to be 0.52, the electron-transfer rate constant was 1.4s(-1) and the average surface coverage was measured to be 6.93 x 10(-11)+/-4.2 x 10(-12) mol cm(-2). The bioactive measurements show that SOD keeps its bioactivity at the CNT surface. SOD's remarkable ability to catalyze the dismutation of the superoxide anion (O (2)(-)) has been demonstrated.     

Root Morphology and Zn2+ Uptake Kinetics of the Zn Hyperaccumulator of Sedum alfredii Hance

Root morphology and Zn2 uptake kinetics of the hyperaccumulating ecotype (HE) and nonhyperaccumulating ecotype (NHE) of Sedum alfredii Hance were investigated using hydroponic methods and the radiotracer flux technique. The results indicate that root length, root surface area, and root volume of NHE decreased significantly with increasing Zn2 concentration in growth media, whereas the root growth of HE was not adversely affected, and was even promoted, by 500 μmol/L Zn2 . The concentrations of Zn2 in both ecotypes of S. alfredii were positively correlated with root length, root surface area and root volumes, but no such correlation was found for root diameter. The uptake kinetics for 65Zn2 in roots of both ecotypes of S. alfredii were characterized by a rapid linear phase during the first 6 h and a slower linear phase during the subsequent period of investigation. The concentration-dependent uptake kinetics of the two ecotypes of S. alfredii could be characterized by the Michaelis-Menten equation, with the Vmax for 65Zn2 influx being threefold greater in HE compared with NHE, indicating that enhanced absorption into the root was one of the mechanisms involved in Zn hyperaccumulation. A significantly larger Vmax value suggested that there was a higher density of Zn transporters per unit membrane area in HE roots.     

造血器官机能障碍后家蚕血淋巴中蛋白质成分的变化

为了研究家蚕Bombyx mori造血器官机能障碍后其血淋巴中蛋白质成分的变化,利用重离子射线局部照射家蚕幼虫的造血器官,检测了照射后家蚕血淋巴中的蛋白质成分及注射大肠杆菌后在体内诱导出现的应急蛋白量的变化。结果表明,照射蚕血淋巴中的蛋白质含量与对照蚕之间没有明显的差异。但在成分分析时发现,5龄起蚕血淋巴中70 kD附近的3条蛋白质谱带比对照蚕的浓度要高,随着个体的发育两者的浓度都上升;5龄后期则相反,对照蚕的浓度比照射蚕高;脂肪体中贮藏蛋白质的含量具有相似的变化趋势。用家蚕贮藏蛋白质SP-1及SP-2的抗血清进行免疫印迹反应的结果显示：70 kD附近的3条蛋白质谱带的最上面的一条为贮藏蛋白质SP-1,下面的二条为贮藏蛋白质SP-2;同时照射蚕血淋巴中分子量约为24 kD的蛋白质成分也发生变化,5龄前期的浓度比对照蚕低,5龄第3天几乎检测不到;全体照射与造血器官局部照射蚕之间的结果相似。照射蚕注射大肠杆菌后在体内诱导出现的应急蛋白量明显比对照蚕要少。由此认为家蚕幼虫造血器官与血淋巴中的蛋白质成分有关,造血器官的机能障碍、血球的数量减少可影响脂肪体中蛋白质的合成,从而使存在于血淋巴中的蛋白质成分发生变化。     

Co-transfection and tandem transfection of HEK293A cells for overexpression and RNAi experiments

pIRES2-EGFP was employed and a non-target shRNA expressing plasmid was constructed to simulate overexpression and RNAi (RNA interference) experiments. Transfection of pIRES2-EGFP into HEK293A cells by cationic lipids VigoFect demonstrated that transfection efficiency increased in a dose-dependent manner with amount of DNA plasmid used, and optimal transfection time and cell density should be identified to reach a compromise of higher transfection efficiency and lower toxicity. Co-transfection experiments indicated that the two co-transfected plasmids were equivalently delivered into the same cells, and the co-transfection efficiency was rarely affected by cell density and proportion of the two plasmids. However, plasmid-receipted cells seemed indisposed to accept plasmid again during the second transfection, and very low co-transfection efficiency was observed in tandem transfection.     

Root Morphology and Zn^2＋ Uptake Kinetics of the Zn Hyperaccumulator of Sedum alfredii Hance

Root morphology and Zn^2＋ uptake kinetics of the hyperaccumulating ecotype （HE） and nonhyperaccumulating ecotype （NHE） of Sedum alfredii Hance were investigated using hydroponic methods and the radiotracer flux technique. The results indicate that root length, root surface area, and root volume of NHE decreased significantly with increasing Zn^2＋ concentration in growth media, whereas the root growth of HE was not adversely affected, and was even promoted, by 500μmol/L Zn^2＋. The concentrations of Zn^2＋ in both ecotypes of S. alfredii were positively correlated with root length, root surface area and root volumes, but no such correlation was found for root diameter. The uptake kinetics for ^65Zn^2＋ in roots of both ecotypes of S. alfredii were characterized by a rapid linear phase during the first 6 h and a slower linear phase during the subsequent period of investigation. The concentration-dependent uptake kinetics of the two ecotypes of S. alfredii could be characterized by the Michaelis-Menten equation, with the Vmax for ^65Zn^2＋ influx being threefold greater in HE compared with NHE, indicating that enhanced absorption into the root was one of the mechanisms involved in Zn hyperaccumulation. A significantly larger Vmax value suggested that there was a higher density of Zn transporters per unit membrane area in HE roots.     

人参皂甙Rg1、肉桂酸和丹参酮IIA组合对成骨肉瘤MG-63细胞增殖与相关基因表达的影响

研究中药有效成分人参皂甙Rg1、肉桂酸和丹参酮IIA组合对人成骨肉瘤MG-63细胞增殖抑制和相关基因表达影响,探索其对肿瘤细胞的生物学效应.以33 μg/ml人参皂甙Rg1、296.32 μgml肉桂酸和0.3 μg/ml丹参酮IIA的组合(简称RCT)处理人成骨肉瘤MG-63细胞,以肿瘤细胞分化诱导物HMBA处理MG-63细胞为平行对照,用流式细胞仪、免疫细胞化学检测及光镜观察系统研究RCT组合对MG-63细胞的作用.生长曲线及细胞周期检测显示RCT组合可显著抑制MG-63细胞的增殖,细胞生长抑制率达72.37%,细胞周期阻滞于G0/G1期;免疫细胞化学检测显示RCT组合处理后MG-63细胞的癌基因c-fos、c-myc表达下调,抑癌基因p27、Rb表达上调.RCT组合对MG-63细胞增殖及相关基因表达的影响与分化诱导物六亚甲基双乙酰胺(HMBA)处理组相似.     

载脂蛋白M

载脂蛋白M(apoM)是一类在血液中主要与高密度脂蛋白（HDL）结合的载脂蛋白,呈组织特异性表达且有着众多生物学功能.体内外多种因素可从转录或转录后水平对其表达进行调控:肝细胞核因子-1α,4α（HNF-1α,4α）、肝受体同系物-1（LRH-1）、叉头框转录因子a2（Foxa2）、血小板活化因子（PAF）等可上调其表达;肝X受体（LXR）、维甲酸X受体（RXR）、法尼酯X受体（FXR）、小异源二聚体-1(SHP-1)以及绝大多数细胞因子可下调其表达,具体调节机制复杂. 结构上,apoM含有一个特征性的疏水性信号肽,可结合1 磷酸鞘氨醇（S1P）等小的生物活性脂,以此介导多项生命活动. 功能上,apoM能促进preβ-HDL的生成,并提高其一系列抗动脉粥样硬化的生物活性,如胆固醇逆向转运、抗炎、以及低密度脂蛋白（LDL）的抗氧化等.在一些糖尿病病人体内,apoM的含量也显著降低,而apoM含量的提高可以降低血糖含量,增加胰岛素分泌以及改善胰岛素抵抗,不少学者将其视为该病发生发展的一项预测指标.本文就近年来对apoM的生物学特性,特别是其表达调控机制和功能的研究进展进行综述.