TSG101基因研究进展

人TSG101基因是近年来发现的一个新的抑瘤基因候选,定位于11p15.1-15.2,其编码产物TSG101蛋白N端区与泛素连接催化区同源。近年来的研究表明,TSG101基因与多种肿瘤密切相关,其产物TSG101蛋白具有多种重要功能,由于其参与HIV病毒的感染过程,使得研制和开发针对HIV病毒的基因药物来治疗艾滋病成为可能。     

TSG101在病毒出芽过程中的作用及其机制

TSG101基因是新发现的抑癌基因候选者,定位于人类 11号染色体 p1511-p1512,其编码产物TSG101蛋白N端区域与泛素结合酶(UBC)同源。近年来研究发现,TSG101基因具有多种重要的功能,与多种病毒出芽密切相关,所以TSG101可作为一个新的抗病毒靶点。本文主要从TSG101在多种病毒（HIV、IAV、MARV、ASV等）出芽过程中扮演的角色,TSG101与多种蛋白(泛素、Nedd4、ARMMs、Tom1、Gag、VP40、NP等)的相互作用进而辅助病毒出芽的机制,以及TSG101抑制剂的研究等方面进行阐述。     

一个新鼻咽癌抑瘤候选基因的克隆及其功能初步分析

从cDNA 代表差异分析法（cDNA representational difference analysis, cDNA RDA）分离的新cDNA片段入手,进一步采用RT-PCR验证,其中发现AF152605片段在74%鼻咽癌(nasopharyngeal carcinoma,NPC)活检组织中表达下调和缺失,DNA印迹显示其代表一转录本为2.1 kb的基因,结合生物信息学,运用文库筛选克隆该基因,命名为NAG4基因,GenBank登录号AF179285,定位于6q22.1～22.33,至少含有两个外显子,并在第一外显子的上游有TATA盒样序列,编码一个508个氨基酸组成的、分子质量为57.4 ku的蛋白质;功能预测NAG4基因产物与小鼠溴区蛋白BP75有84%同源,是含有多个磷酸化位点和溴区结构域的核内转录因子;突变分析表明NAG4基因在HeLa细胞株中发生整码突变.以上结果说明NAG4基因是鼻咽癌抑瘤基因的良好候选者,其表达的下调参与了鼻咽癌的发生.     

下调TSG101基因对人急性髓性白血病细胞的调节作用

构建肿瘤易感基因101(TSG101)基因的小干扰RNA载体并将其转导入HL-60细胞,获得稳定转染的阳性克隆后,应用RT-PCR和Western印迹进行鉴定;MTT法和流式细胞仪检测细胞转染前后生长速度和细胞周期的变化;DNA梯带(ladder)法和流式细胞仪检测细胞对顺铂诱导的凋亡的变化;Western印迹检测耐药相关蛋白P-gp和MRP的表达变化。结果表明,成功构建了TSG101的小干扰RNA载体;经蛋白质水平检测证实成功建立了稳定的TSG101低表达的白血病细胞模型;转染TSG101小干扰RNA后的细胞生长速度显著减慢,出现G1期阻滞;对顺铂的敏感性明显增强,形成DNA条带,且低表达P-gp。因此,下调TSG101基因能抑制HL-60细胞生长,增加细胞对化疗药物的敏感性,并提示该基因具有进行白血病基因治疗的可行性。     

与泛肽途径可能相关的新基因UBAP1的克隆和表达分析

在先前确定了鼻咽癌9p21-22区域的一个最小共同缺失区内的基础上,为了筛选和克隆鼻咽癌相关的修选抑瘤基因,应用EST介导的定位候选克隆策略,用RT－PCR及Northern杂交检测了22个表达序列标签(expressed sequence tag,EST）在鼻咽癌细胞株HNE－1和原代培养的正常鼻咽上皮细胞中的表达水平,发现其中一个EST w56112在鼻咽癌细胞株HNE－1中的表达显著下调,RNA印迹显示其代表一转录本为2.7kb的基因。进一步运用cDNA测序和RACE方法克隆了该EST代表的基因全长cDNA,Genbank登录呈AF222043,同时结合生物信息学方法克了该基因在小鼠中的同源基因,Genbank登录AF275549。该基因cDNA全长2.7kb,编码由502个氨基酸组成的、分子质量为55kD的蛋白质。数据库分析显示该基因编码的蛋白质羧基段含有两个重要的泛肽相关结构域（UBA domain）,属于泛肽相关蛋白家族的一个新成员,因此征得国际人类基因组命名委员会同意,将其命名为UBAP1基因。运用Northern杂交和 RT-PCR方法检测发现UBAP1基因在所检测的人和小鼠的组织中广泛表达。采用RT－PCR和直接测序的方法,未能发现UBAP1基因编码区在鼻咽癌细胞株HNE－1和10例鼻咽癌活检标本中存在突变。UBAP1基因作为一个泛肽相关蛋白家族的新成员,有可能参与泛肽信号途径;结合其在9p的定位信息及在鼻咽癌中的表达下调。有等对UBAP1基因进行为精细的突变分析,以进一步研究其表达下调参与鼻咽癌发生发展的可能机制。     

肿瘤发生与相关基因突变的关联

肿瘤的发生常与其相关基因的突变有关,这些突变会引起编码基因的蛋白质的结构或数量的改变,从而导致其相关基因功能的丧失。突变在基因上发生的位置及类型的不同也会对基因的表达造成不同的影响。本文介绍了存在于肿瘤相关基因编码区及非编码区的突变与肿瘤发生发展的相互关系。本文还就肿瘤相关基因的突变类型阐述肿瘤的靶向治疗,提供了癌症治疗及预防的新思路。     

泛素特异性蛋白酶46的分子克隆及基因突变对其功能的影响

该文探讨了行为绝望相关基因—泛素特异性蛋白酶46(usp46)基因的生物学功能及编码区错义突变对其功能的影响。从人类食管癌细胞株TE-1中提取总RNA,RT-PCR扩增获得人类usp46基因,q RT-PCR检测小鼠各组织中该基因的m RNA相对表达水平;运用去泛素化酶活性检测体系,将USP46与模型底物表达质粒共转化,研究USP46的活性;采用定点突变法构建错义突变V89I,用上述方法检测突变对酶活性的影响。结果发现,人类usp46基因具有1 101 bp的开放阅读框,编码366个氨基酸;序列比对显示,其在各个物种中高度保守;在小鼠脾脏中m RNA表达量较高,而骨骼肌中表达较少;人类的USP46蛋白具有去泛素化酶活性,V89I突变后,其活性显著下降,仅为野生型的37.67%±2.52%(P0.05)。结果表明,usp46基因在小鼠各组织中的m RNA表达水平存在差异;人类USP46蛋白具有去泛素化酶活性,而且其编码区V89I错义突变后,其活性显著下降,为抑郁症等神经精神性疾病的病因学研究提供了新的线索。     

已建株鼻咽癌细胞的PLUNC基因启动子区序列多态性分析

目的检测人类标准鼻咽癌细胞中是否存在已知的PLUNC基因启动子-437bp-＋87bp区域的单核苷酸多态性（SNP）。以便进一步探索SNP与鼻咽癌的关系。方法采用PCR产物直接测序的方法,对7株体外培养的鼻咽癌细胞基因组DNA的PLUNC基因启动子区进行序列分析。结果发现7株PLUNC基因的启动子区皆存在已知的3个SNP位点（1888、2128和N2）和未知一个突变位点（N1）,其测观杂合度分别为85．7％、100％、100％和28．6％。其中3个已知SNP位点在筛查的细胞株中均存在T-C的突变,而且SUNE-1鼻咽癌细胞株的1888位点基因型为突变纯合子CC型。结论体外培养的标准鼻咽癌细胞株中存在已知的3个SNP位点（1888、2128和N2）的突变现象,且突变率为100％;1888位点鼻咽癌易患型（CC型）已在体外稳定建株;首次发现启动子-195bp区域N1突变位点。     

HIV-1感染对细胞内宿主因子TSG101及与Alix表达的影响

探讨HIV-1感染宿主细胞后对其宿主蛋白肿瘤易感基因101蛋白(Tumor Susceptibility Gene 101,TSG101)及ALG-2相互作用蛋白X(ALG-2-interacting protein X,Alix)表达的影响。以HIV-1感染性克隆病毒pNL4-3感染TZM-bl PM1、Jurkat细胞株和人外周血单个核细胞(PBMCs),感染24h后收获细胞提取总RNA,逆转录PCR检测在RNA水平各因子的表达差异;感染48h后收获细胞提取总蛋白,Western-blot检测各因子在蛋白水平的表达差异。结果显示:HIV-1感染对原代PBMC与细胞系表达Alix与TSG101影响显著不同,细胞系主要表现为下调,而原代PBMC主要表现为TSG101上调;细胞系中的下调又细分为Jurkat细胞的Alix与TSG101的双下调、TZM-bl细胞的Alix单下调以及PM1细胞无影响三种情况。HIV-1感染对细胞宿主分子TSG101及Alix在RNA和蛋白水平的表达均有影响,这种影响因细胞的不同而有差异。HIV-1感染调节Alix与TSG101的机制生物学意义尚有待于进一步阐明。     

MutPrimerDesign:用于人类基因编码区域突变位点的引物设计程序

位于基因编码区的DNA突变与基因的功能密切相关。在已知人类基因编码区的突变位点时,如何在基因组上设计引物验证该突变是一个重要的问题。本文利用Python语言开发了引物设计程序MutPrimerDesign。MutPrimerDesign通过解析人类基因组序列数据库以及基因注释信息,转换基因编码区坐标为基因组坐标,并调用Primer3的python程序包接口,可批量自动化完成基因突变位点的引物及探针序列设计。MutPrimerDesign使用简便,可识别多种数据库的基因名称,并能够修改引物常规参数,实现引物的快速调整。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

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.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.