低稳定性绿色荧光蛋白EGFP-PEST的载体构建及表达验证

目的:构建含有蛋白降解基序PEST序列的增强型绿色荧光蛋白(EGFP)融合基因表达载体pQCXIP-EGFPPEST-N1,并检测其对蛋白稳定性的调节。方法:以NFATx6-mPro-EGFP-PEST-YES为模板,PCR扩增EGFP-PEST序列,克隆入逆转录病毒载体pQCXIP-EGFP-N1构建pQCXIP-EGFP-PEST-N1,病毒包装后感染293FT细胞获得稳定细胞系;用蛋白酶体抑制剂MG-132处理细胞,Western印迹检测EGFP的表达变化。结果:构建获得逆转录病毒载体pQCXIP-EGFP-PEST-N1,感染293FT细胞后,与对照组相比,EGFP表达显著降低,并且该降低可被MG-132处理逆转。结论:构建了EGFP-PEST蛋白的表达载体,PEST可以介导EGFP蛋白发生蛋白酶体依赖的降解。为实现基因编辑效果的可视化筛选奠定了基础。     

GAPDH降解途径在鱼藤酮诱导多巴胺能神经元损伤中的作用

目的探讨GAPDH降解途径在鱼藤酮(rotenone)诱导的多巴胺能神经元损伤中的作用。方法用鱼藤酮诱导多巴胺能神经细胞SH-SY5Y建立帕金森病(Parkinson’s disease,PD)细胞模型,并分别加入蛋白酶体和自噬抑制剂和激活剂,显微镜观察细胞形态,MTT法检测细胞活性,Western blot检测GAPDH的表达及降解。结果鱼藤酮、蛋白酶体和自噬途径抑制剂可导致细胞活性下降,各药物组细胞生长受抑制;在鱼藤酮诱导的SH-SY5Y细胞内GAPDH水平上调,加入蛋白酶体和自噬途径抑制剂能够进一步上调GAPDH水平,而加入两个降解途径的激活剂则显著抑制鱼藤酮对GAPDH水平的上调作用。结论蛋白酶体和自噬溶酶体途径功能异常在GAPDH代谢中发挥重要作用,两者均参与GAPDH的降解。激活这两条降解途径可增加过表达的GAPDH的清除,可能成为PD治疗潜在的新靶点。     

抑制或激活泛素-蛋白酶体途径对大鼠肺泡巨噬细胞内质网应激的影响

目的:研究肺泡巨噬细胞(NR8383)不同蛋白酶体激活程度对内质网应激的影响。方法:构建UbG76V-GFP融合蛋白,将含有UbG76V-GFP的质粒导入NR8383细胞,筛选出可稳定表达UbG76V-GFP的细胞系,通过蛋白酶体抑制剂(MG132)、蛋白酶体激活剂(阿霉素)干预蛋白酶体活性。荧光显微镜观察不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时蛋白酶体活性,Western blot及PCR技术检测不同蛋白酶体活性下大鼠肺泡巨噬细胞在缺氧复氧2 h、4 h、6 h时泛素化蛋白及内质网应激相关基因的表达。结果:在缺氧复氧2 h、4 h、6 h这3个时间点,加入MG132组大鼠肺泡巨噬细胞绿色荧光及泛素化蛋白(Ubiquitin)表达明显降低(P0.05),而PCR及Western blot示内质网应激基因BIP(免疫球蛋白结合蛋白)、XBP-1(X-盒结合蛋白)和CHOP(C/EBP同源蛋白)平均扩增量及蛋白表达量明显增加(P0.05);加入阿霉素组大鼠肺泡巨噬细胞在缺氧复氧2 h、4h、6 h表现出相反的实验结果,绿色荧光及Ubiquitin蛋白相对表达均明显增加(P0.05),而PCR及Western blot示内质网应激基因BIP、XBP-1和CHOP平均扩增量及蛋白表达量明显增加(P0.05)。结论:本实验结果表明活细胞泛素-蛋白酶体活性程度与内质网应激存在紧密联系,外源性增强泛素蛋白酶体活性会抑制内质网应激,外源性减弱泛素蛋白酶体活性会增强内质网应激。     

蛋白酶体抑制剂MG132的浓度对人白血病细胞K562凋亡的影响

探讨蛋白酶体抑制剂MG132 在诱导人白血病K562细胞凋亡过程中作用.分别以不同浓度的蛋白酶体抑制剂MG132 处理人白血病细胞K562,通过MTT法检测K562细胞活力,应用Annexin Ⅴ和PI 双染的细胞流式法检测K562细胞凋亡率和细胞内活性氧(ROS) 水平,应用酶标仪法检测K562细胞内Caspase- 3活性变化的情况.结果表明,随着MG132浓度的增加,各个指标与对照组比较差异均有显著性(P<0.05):K562细胞增殖明显受到抑制;细胞凋亡率明显增加,且当MG132浓度为900 nmol/L时,细胞凋亡率达36.5 %;同时,ROS 水平和caspase- 3活性明显升高.因次,蛋白酶体抑制剂MG132可显著抑制人白血病细胞K562增殖并促进其凋亡.     

蛋白酶体抑制剂MG132诱导K562和HeLa细胞凋亡程度存在明显差异

蛋白酶体抑制剂MG132诱导人白血病细胞K562和宫颈癌细胞HeLa凋亡,用3个不同浓度的蛋白酶体抑制剂MG132处理人白血病细胞K562和宫颈癌细胞HeLa,通过MTT检测、annexin Ⅴ/ PI 双染法、流式细胞术、酶标仪和Western 印迹分别检测MG132对K562细胞和HeLa细胞的生长效应、细胞凋亡率、细胞内活性氧(ROS)水平和caspase-3活性变化的影响.蛋白酶体抑制剂MG132诱导K562细胞凋亡明显,对HeLa细胞诱导凋亡不明显.结果表明,蛋白酶体抑制剂MG132特异性诱导不同肿瘤细胞凋亡的程度存在明显差异.     

NIRF泛素化p53的作用过程中NIRF与p53结合域的测定

NIRF(Np95/ICBP90-like RING finger protein)是2002年发现的一种核蛋白,其功能涉及细胞增殖调节、蛋白多聚泛素化降解、细胞癌变进程控制等领域．已有研究报道,NIRF能与p53相互作用, NIRF本身也是一个高度调节蛋白,在细胞正常的生理状态下发挥泛素化E3连接酶的作用,结合p53并将其降解,但NIRF与p53结合的蛋白结合域目前尚不清楚．本文研究证明,NIRF能与p53结合成复合体参与泛素化蛋白降解途径,并测定出NIRF与p53结合的区域．为了检测NIRF的蛋白结合域,将空载体和NIRF缺失突变体质粒分别转染于HEK293细胞,蛋白表达水平通过Western印迹用两种抗体分别检测. 结果显示,所有的突变体都能在细胞中表达,并且两种抗体检测结果完全一致. 同时,免疫共沉淀技术用于进一步分析实验结果. 由于泛素化蛋白通常伴随蛋白酶体通路介导的降解,免疫共沉淀的蛋白纯化过程中用蛋白酶体抑制剂MG-132以抑制蛋白降解. 本研究结果显示,NIRF 通过PHD区域与p53形成复合体. 该复合体可能参与蛋白分选、蛋白降解、DNA修复以及细胞凋亡等一系列重要的细胞活动,从而形成与细胞增殖相关的新的信号通路,在肿瘤的发生发展中可能发挥某种程度的作用．     

藤黄新酸抑制肝癌细胞生长的机制研究

在肿瘤细胞中蛋白酶体活性的抑制可以导致细胞凋亡和周期阻滞.藤黄新酸(TH2)是从中药藤黄中提取的一个新的(口山)酮类衍生物.研究结果显示,TH2可以抑制人肝癌Bel-7402细胞的增殖,诱导细胞产生凋亡,且呈浓度和时间依赖性.同时,10μmol/L的TH2与细胞作用24h后,可以导致早期凋亡标志性蛋白PARP发生裂解.在体外采用特异性荧光底物检测TH2对蛋白酶体活性的影响,发现该化合物能够抑制蛋白酶体的糜乳蛋白酶样、胰蛋白酶样活性和谷氨酰后水解活性.抑癌基因p53是细胞内的蛋白酶体降解底物,TH2可使p53蛋白的降解受到阻滞,表达增加.由此可见,TH2具有抑制人肝癌Bel-7402细胞增殖、诱导细胞凋亡的作用,可能的分子机制与其抑制细胞内蛋白酶体活性、导致p53蛋白降解受阻有关.     

CHO细胞表达重组猪干扰素-γ及其抗病毒活性

为利用中国仓鼠卵巢(Chinese hamster ovarian,CHO)细胞表达系统制备重组猪干扰素-γ(recombinant porcine interferon gamma,r Po IFN-γ),并分析其体外抗病毒活性,本研究首先构建r Po IFN-γ真核表达质粒pc DNA3.1-Po IFN-γ,转染悬浮培养的CHO细胞,进行上清分泌表达,利用亲和层析纯化目的蛋白,并进行SDS-PAGE和Western blotting鉴定;通过CCK-8实验分析r Po IFN-γ对细胞的毒性,用VSV/PK-15系统检测其抗病毒活性效价;最后分析r Po IFN-γ对塞内卡病毒A (Seneca virus A,SVA)的抗病毒活性及其对细胞内干扰素刺激基因(interferon-stimulated genes,ISGs)和细胞因子的诱导作用。结果显示,本研究利用CHO悬浮细胞表达系统成功制备了纯化的r Po IFN-γ,该r Po IFN-γ对细胞无毒性,VSV/PK-15系统检测其活性效价为5.59×107 U/mg;此外,r Po IFN-γ可诱导细胞内多种ISGs和细胞...     

蛋白酶体抑制剂MG132对青扦花粉萌发和花粉管生长的影响

泛素/蛋白酶体系统(UPP)是真核细胞内蛋白质选择性降解的主要途径,而蛋白酶体是UPP中蛋白质降解的场所。本文应用细胞学、统计学方法以及FTIR技术研究了蛋白酶体抑制剂MG132对青扦(Peceawilsonii)花粉萌发、花粉管生长的影响。结果表明:MG132显著抑制青扦花粉萌发和花粉管生长,并导致花粉管形态异常,主要表现为花粉管亚顶端出现液泡化,并且液泡随着培养时间的延长而扩大到整个花粉管,花粉管濒临死亡;而DMSO以及非蛋白酶体抑制剂E-64不产生类似结果;半薄切片结果表明,MG132处理后不仅花粉管细胞质发生液泡化,生殖细胞也发生液泡化;FTIR分析进一步表明,MG132处理后,花粉管顶端的细胞壁蛋白和果胶质含量大幅度下降。上述结果表明:MG132通过抑制蛋白酶体活性显著影响青扦花粉萌发及花粉管生长;UPP在青扦花粉萌发、花粉管极性生长模式的建立和维持过程中起重要作用;抑制蛋白酶体活性将导致青扦花粉管的程序性死亡。     

hK-Fc融合蛋白的改良、表达及其生物活性的分析

为了延长人激肽释放酶(hK)的血清半衰期,提高分泌蛋白的产率,制备了重组激肽释放酶-IgG1 Fc融合蛋白(hK'-Fc)。采用PCR扩增hK基因和IgG1的Fc序列,用鼠源信号肽序列替换hK基因原有的信号肽序列,构建改良型融合蛋白hK'-Fc以及天然型融合蛋白hK-Fc的表达载体,转染中国仓鼠卵巢细胞(CHO)细胞,筛选稳定分泌融合蛋白的细胞株,通过Western blotting鉴定信号肽改造效果,利用Protein A+G亲合层析柱纯化融合蛋白,酶学实验检测融合蛋白的体外活性。结果表明:成功构建了pcDNA-hK'-Fc以及pcDNA-hK-Fc重组表达载体;获得了稳定表达融合蛋白的细胞株,产量达11mg/L以上;信号肽改造后融合蛋白的分泌效率提高约5～10倍;融合蛋白能水解其特异性的底物S-2266,具有生物学活性。本研究为进一步探讨融合蛋白的体内半衰期打下了坚实基础,也为研制治疗脑梗塞疗效更好的第二代hK蛋白和其他药用蛋白的改良提供新的线索。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.