丙型肝炎病毒结构蛋白E1在昆虫细胞中的表达及定位

本文在大肠杆菌中表达了与GST融合无跨膜区的丙型肝炎病毒(Hepatitis C Virus,HCV)E1蛋白,并通过免疫兔制备了兔抗E1的抗血清。然后利用Bac-to-Bac杆状病毒表达系统构建了含有HCV结构蛋白E1基因的重组杆状病毒vAcHCVE1。通过Western blot分析,E1蛋白在Sf9细胞中表达分子量大小为30kDa大于预测的20kDa,表明存在翻译后修饰如糖基化等。通过Confocal显微镜观察当感染48h后E1蛋白定位在细胞质和细胞膜上。     

Psychological stress induces chemoresistance in breast cancer by upregulating mdr1

Psychological distress reduces the efficacy of chemotherapy in breast cancer patients. The mechanism may be related to the altered neuronal or hormonal secretions during stress. Here, we reported that adrenaline, a hormone mediating the biological activities of stress, upregulates mdr1 gene expression in MCF-7 breast cancer cells via alpha(2)-adrenergic receptors in a dose-dependent manner. Mdr1 upregulation can be specifically inhibited by pretreatment with mdr1-siRNA. Consequently, adrenergic stimulation enhances the pump function of P-glycoprotein and confers resistance of MCF-7 cells to paclitaxel. In vivo, restraint stress increases mdr1 gene expression in the MCF-7 cancers that are inoculated subcutaneously into the SCID mice and provokes resistance to doxorubicin in the implanted tumors. The effect can be blocked by injection of yohimbine, an alpha(2)-adrenergic inhibitor, but not by metyrapone, a corticosterone synthesis blocker. Therefore, we conclude that breast cancers may develop resistance against chemotherapeutic drugs under psychological distress by over-expressing mdr1 via adrenergic stimulation.     

青鱼生长激素的重组表达及其多克隆抗体的制备

以含有的青鱼生长激素编码区cDNA的重组质粒pbcGHc为模板,高保真PCR扩增青鱼生长激素（GH）成熟肽cDNA序列,定向插入原核表达载体pET-28a,构建青鱼GH原核表达质粒pET-bcGH。将pET-bcGH转化大肠杆菌BL21(DE3),IPTG诱导青鱼GH基因在大肠杆菌中的融合表达,SDS-PAGE凝胶电泳结果显示一条23 kDa的诱导表达重组青鱼GH带。以草鱼GH多克隆抗体为一抗,Western Blot证明,该重组青鱼GH具有免疫学活性。将经过亲和层析、透析纯化后的重组青鱼GH作为抗原,采用改进的方法对家兔进行皮下免疫注射,获得青鱼GH多克隆抗血清。以该多抗为一抗,Western Blot 可以检测出4 ng的抗原量;并且在青鱼垂体组织抽提液中和血清中检测到一种能与该抗血清作用的大小为21 kDa的蛋白质。这些结果表明本研究得到的青鱼GH多克隆抗血清具有较好的免疫特性。     

地塞米松与硫酸镁对大鼠小肠缺血再灌注损伤的作用及机制

目的研究地塞米松和硫酸镁对大鼠小肠缺血再灌注（I/R）损伤的保护作用,并初步探讨其机制。方法制作小肠I/R模型,实验分为假手术阴性对照组、I/R组、硫酸镁治疗组、地塞米松治疗组、地塞米松和硫酸镁联合治疗组,比较五组血浆二胺氧化酶（DAO）、丙二醛（MDA）的含量,同时比较小肠的病理切片观察治疗效果。结果①I/R组小肠组织病理变化明显,血浆DAO、MDA比假手术阴性对照组显著升高;②硫酸镁治疗组和地塞米松治疗组小肠病理变化减轻,血浆DAO、MDA比I/R组显著降低,且两组无显著差别;③硫酸镁和地塞米松合用组的血浆MDA比I/R组显著升高,但是小肠病理变化和I/R组相比无明显区别,血浆DAO也和I/R组无明显差别。结论硫酸镁,地塞米松分别对大鼠小肠缺血再灌注有保护作用而二者合用却无明显的保护作用。     

重组人粒细胞-巨噬细胞集落刺激因子包涵体提取、复性和纯化的研究

由基因工程大肠杆菌表达的重组人粒细胞－巨噬细胞集落刺激因子（ｒｈＧＭ－ＣＳＦ）以包涵体的形式存在于细胞中,通过破菌、洗涤获得包涵体,再经过溶解、凝胶过滤、复性、疏水和离子交换柱导析得到了均一的产品,经高压液相和ＳＤＳ－ＰＡＧＥ电泳测定纯度均大于９８％,ｒｈＧＭ－ＣＳＦ的比活为３．２×１０＾７ＩＵ／ｍｇ,纯化获得的ｒｈＧＭ－ＣＳＦ为一酸性蛋白,等电点约为５．２,ＮＨ２－末端有２０个氨基酸序列测定结果     

Subcellular Distribution and Chemical Forms of Cadmium in the Medicine Food Homology Plant <i>Platycodon grandiflorum</i> (Jacq.) A.DC.

Although Platycodon grandiflorum (Jacq.) A.DC. is a renowned medicine food homology plant, reports of excessive cadmium (Cd) levels are common, which affects its safety for clinical use and food consumption. To enable its Cd levels to be regulated or reduced, it is necessary to first elucidate the mechanism of Cd uptake and accumulation in the plant, in addition to its detoxification mechanisms. This present study used inductively couple plasma-mass-spectrometry to analyze the subcellular distribution and chemical forms of Cd in different tissues of P. grandiflorum. The experimental results showed that Cd was mainly accumulated in the roots [predominantly in the cell wall (50.96%–61.42%)], and it was found primarily in hypomobile and hypotoxic forms. The proportion of Cd in the soluble fraction increased after Cd exposure, and the proportion of insoluble phosphate Cd and oxalate Cd increased in roots and leaves, with a higher increase in oxalate Cd. Therefore, it is likely that root retention mechanisms, cell wall deposition, vacuole sequestration, and the formation of low mobility and low toxicity forms are tolerance strategies for Cd detoxification used by P. grandiflorum. The results of this study provide a theoretical grounding for the study of Cd accumulation and detoxification mechanisms in P. grandiflorum, and they can be used as a reference for developing Cd limits and standards for other medicine food homology plants.     

Revealing the community and metabolic potential of active methanotrophs by targeted metagenomics in the Zoige wetland of the Tibetan Plateau

The Zoige wetland of the Tibetan Plateau is one of the largest alpine wetlands in the world and a major emission source of methane. Methane oxidation by methanotrophs can counteract the global warming effect of methane released in the wetlands. Understanding methanotroph activity, diversity and metabolism at the molecular level can guide the isolation of the uncultured microorganisms and inform strategy-making decisions and policies to counteract global warming in this unique ecosystem. Here we applied DNA stable isotope probing using ¹³C-labelled methane to label the genomes of active methanotrophs, examine the methane oxidation potential and recover metagenome-assembled genomes (MAGs) of active methanotrophs. We found that gammaproteobacteria of type I methanotrophs are responsible for methane oxidation in the wetland. We recovered two phylogenetically novel methanotroph MAGs distantly related to extant Methylobacter and Methylovulum. They belong to type I methanotrophs of gammaproteobacteria, contain both mxaF and xoxF types of methanol dehydrogenase coding genes, and participate in methane oxidation via H₄MPT and RuMP pathways. Overall, the community structure of active methanotrophs and their methanotrophic pathways revealed by DNA-SIP metagenomics and retrieved methanotroph MAGs highlight the importance of methanotrophs in suppressing methane emission in the wetland under the scenario of global warming.     

Degradation of WTAP blocks antiviral responses by reducing the m6A levels of IRF3 and IFNAR1 mRNA

N ⁶‐methyladenosine (m⁶A) is a chemical modification present in multiple RNA species and is most abundant in mRNAs. Studies on m⁶A reveal its comprehensive roles in almost every aspect of mRNA metabolism, as well as in a variety of physiological processes. Although some recent discoveries indicate that m⁶A can affect the life cycles of numerous viruses as well as the cellular antiviral immune response, the roles of m⁶A modification in type I interferon (IFN‐I) signaling are still largely unknown. Here, we reveal that WT1‐associated protein (WTAP), one of the m⁶A “writers”, is degraded via the ubiquitination‐proteasome pathway upon activation of IFN‐I signaling. With the degradation of WTAP, the m⁶A levels of IFN‐regulatory factor 3 (IRF3) and interferon alpha/beta receptor subunit 1 (IFNAR1) mRNAs are reduced, leading to translational suppression of IRF3 and instability of IFNAR1 mRNA. Thus, the WTAP‐IRF3/IFNAR1 axis may serve as negative feedback pathway to fine‐tune the activation of IFN‐I signaling, which highlights the roles of m⁶A in the antiviral response by dictating the fate of mRNAs associated with IFN‐I signaling.