单颗粒冷冻电镜揭开G蛋白偶联受体结构生物学研究的新篇章

G蛋白偶联受体（GPCR）是生物体内一类庞大而又多样的细胞膜蛋白。GPCR可以应细胞外信号发生构象变化,进而结合不同效应蛋白激活下游多种信号转导通路,调控众多生命活动过程,参与几乎所有病理过程的发生和发展。近年来,冷冻电镜技术在研究生物大分子结构方面取得了突飞猛进的发展,基于冷冻电镜的GPCR信号转导复合物的高分辨率三维结构不断出现。本文阐述了GPCR和G蛋白复合物相互作用的共性结构特征——受体第六个跨膜螺旋的构象变化,也展示了GPCR识别不同G蛋白亚型选择性的结构基础。单颗粒冷冻电镜提供了更加高效地鉴定受体与配体相互作用分子机制的方法,为GPCR信号通路的机制研究以及基于结构的药物理性设计提供了重要信息。     

引起成人腹泻的新轮状病毒在原代人胚肾细胞上的培养和传代

目的 探讨用细胞培养系统在体外培养新轮状病毒（RV）。方法 含新RV的病人粪便标本经高浓度胰蛋白酶（100μg/ml)37℃作用1h,接种于原代人胚肾（PHEK）细胞,37℃吸附1h后,补加无血清含胰蛋白酶（100μg/ml）的DMEM,37℃转鼓培养3d,收获细胞培养液,冻融1次,作为下一代培养的接种物,培养传代。用聚丙烯酰胺凝胶电泳、免疫荧光（IF）、电镜（EM）、免疫电镜（IEM）等方法对细胞培养液进行病毒核酸、抗原及形态检查。结果 从10份粪便标本中培养分离到1株病毒,定名为J19。该病毒在PHEK细胞上稳定培养传代（28代）,其病毒核酸（dsRNA）电泳图型与初始用于接种细胞的粪便标本中的dsRNA电泳图形完全一致,而与A、B、C组RV dsRNA电泳图型明显不同。J19株感染的细胞与病人恢复期血清呈IF阳性反应,与成人腹泻轮状病毒（ADRTV）腹泻病人恢复期血清、兔抗A组RV和兔抗ADRV血清均为IF阻性;EM和IEM在该病毒的细胞培养液中观察到,在形态上与初始接种物中病毒一样的轮状病毒颗粒,而且可被新RV腹泻病人恢复期血清凝集,结论 成功地从含新RV的成人腹泻粪便标本上分离培养出1株轮状病毒－－J19,并能在细胞稳定传代。J19株不属于A、B、C组RV,而是一种新RV。关于新RV在细胞上培养传代,本文是第一次报告。     

轮状病毒肠炎病原的快速诊断

轮状病毒(RV)是秋冬季婴幼儿腹泻的主要病原。目前可用电镜、ELISA、核酸电泳、乳胶凝集等技术从患儿粪便中检出病毒。其中以ELISA法应用最广、也较敏感,且试剂盒易得。我们采用ELISA(即A组轮状病毒单克隆抗体快速一步法)检测粪便RV抗原。该方法是一种诊断轮状病毒肠炎病原的快速、简便方法。现将结果报告如下:     

轮状病毒及轮状病毒单克隆抗体

轮状病毒(Rotavirus,RV)是引起人和动物急性胃肠炎的主要病毒,遍及全世界。主要感染对象为婴幼儿,也可侵袭成人。据WHO统计,在病毒性腹泻中,RV感染占30～65％,仅发展中国家,由于RY感染每年就引起500万人死亡。1978年,我国用免疫电镜(IEM)技术证明也有婴幼儿轮状病毒流行,八十年代     

婴幼儿轮状病毒肠炎治疗新进展

1973年澳大利亚学者Bishop在电镜下于急性腹泻患儿十二指肠黏膜活检标本中,发现上皮细胞内存在大量球形病毒颗粒,根据其类似车轮状形态,1975年命名为"轮状病毒"(Rota Virus,RV).     

轮状病毒的提纯和理化性质

用PEG6000沉淀和密度梯度离心从组织培养病毒感染液和粪便标本中浓缩提纯了人和牛轮状病毒,这有助于低浓度大体积标本的检出。对纯化病毒作了电镜、浮力密度、结构蛋白和紫外吸收等理化性质的研究。     

轮状病毒感染对乳鼠肠道菌群及小肠屏障功能的影响

目的 探讨轮状病毒感染后乳鼠肠道菌群结构、小肠上皮细胞紧密连接蛋白及小肠通透性的改变。方法 构建轮状病毒感染乳鼠腹泻模型,收集结肠和直肠混合肠道内容物,利用16S rDNA测序技术检测乳鼠感染轮状病毒后肠道菌群结构及组成;采用实时荧光定量PCR检测轮状病毒感染后乳鼠粪便中病毒拷贝数及乳鼠肠道细胞紧密连接蛋白1 (TJP-1)、紧密连接蛋白4 (claudin-4)、链接黏附分子（F11R）基因的表达。采用FITC-葡聚糖检测乳鼠肠道上皮细胞的渗透性。结果 连续灌胃4 d后轮状病毒感染（RV）组乳鼠出现黄色水样粪便。16S rDNA测序结果显示,RV组与对照（NC）组的菌群分类组成有显著差异,筛选出10个显著差异菌属。NC组的优势菌属为Lactobacillus、Paenibacillus、Brevundimonas、Parabacteroides、Bacteroides和Alloprevotella,而RV组的富集菌属为Enterococcus和Shigella。实时荧光定量PCR结果显示,与NC组相比,RV组claudin-4及F11R基因表达水平显著升高（P <0.001）,...     

婴幼儿轮状病毒肠炎治疗新进展

1973年澳大利亚学者Bishop在电镜下于急性腹泻患儿十二指肠黏膜活检标本中,发现上皮细胞内存在大量球形病毒颗粒,根据其类似车轮状形态,1975年命名为“轮状病毒”(Rota Virus,RV)。该病毒是引起婴幼儿腹泻的主要病原之一,在发展中国家是引起婴幼儿死亡的主要原因之一。我国的资     

乳鼠流行性腹泻研究概况

乳鼠流行性腹泻(EDIM)是开放饲养小鼠的乳鼠常见疾病,早在1947年,Cheever等首次系统地研究了实验乳鼠所发生的流行性腹泻。其特点为2周龄乳鼠发生急性腹泻,患病率高,传染性强,但死亡率相对较低。1957年Kraft认为本病致病原是病毒。1963年又经Adams用电镜观察证实,起名为乳鼠流行性腹泻病毒。病毒形态与引起牛、猴、猪和羊等腹泻的轮状病毒(Rotavirus,RV)相似,以后证明它与这些RV有血清学关系,定名为小鼠轮状病毒(MRV)。由于其基因组由双股RNA组成,归为呼肠孤病毒科,轮状病毒属。最近,有资料表明,我国也有许多品系乳鼠发生由MRV引起的EDIM。一、病原学MRV形态为典型RV。完整MRV直径75—80nm,缺乏外膜的不完整MRV直径约为65nm左右。它在56℃或60℃半小时内仍具感染性,纯化MRV可在     

脂筏依赖性内吞途径对轮状病毒感染细胞表面NHE3蛋白的调控作用

目的 轮状病毒(RV)可引起儿童急性腹泻症状,但机制均尚不明确.研究表达NHE3的Caco-2细胞经轮状病毒感染后表面NHE3蛋白的改变及其机制.方法 表达HA-NHE3的单层极化Caco-2细胞分别加入等体积无血清培养液、RV病毒液(1×104TCID50/mL)、脂筏破坏剂MβCD(10 mmol/L)和MβCD+...     

Recent advances in the identification of hepatitis viruses.

Resulting directly from the discovery of virus-related antigens, rapid progress has marked the last decade of viral hepatitis research. The hepatitis B virion has been tentatively identified as a DNA virus with an endogenous DNA polymerase, and new serological markers for type B hepatitis have been discovered. Hepatitis A antigen has been identified on a virus-like particle thought to be the hepatitis A virion. Progressively more sophisticated assays for hepatitis antigens and antibodies have been applied to the study of viral hepatitis epidemiology and biochemical-biophysical characterization of the agents. Most recently, knowledge learned from such studies has been exploited to develop a prototype non-infectious but immunogenic hepatitis B vaccine using hepatitis B surface antigen (HBsAg) purified in large quantities from chronic HBsAg carriers. Especially exciting is the prospect, suggested by serological studies of viral hepatitis, that hepatitis viruses besides hepatitis A and B viruses will be identified.     

Human Herpes Virus Type 2 （ HSV2 ）, Human Cytomegalovirus （HCMV） in the Male Genital Tract and Fertilization

The possibility of infection of the human male genital tract by human herpes virus type 2 ( HSV 2) or human cytomegalovirus ( HCMV ) is well established and their sexual transmission has been the object of many studies. Moreover, medically assisted procreation, which helps in numerous fertility problems, raises the question of new viral risks linked to the application of these new technologies. In this review, we shall consider current knowledge in terms of the presence of HSV2 and HCMV in the different parts of the genital tract of immunocompetent or immunodepressed men. We shall also consider the possibility of viral transmission by the sexual act or by the various techniques used in medically assisted procreation. We shall describe studies in human beings and in animals.     

Post-infectious disease syndrome

Many post-infectious syndromes have been recognized in the last 50 years, some following viral infections and others closely related to bacterial disease. The occurrence of prolonged fatigue following an apparent viral illness of varying severity is also well documented. The lack of a recognizable precipitating cause and the tendency for epidemic fatigue to occur among hospital staff led many to believe that the illness may be psychogenic in origin. However, there is serological evidence that some cases may follow enterovirus infections or occasionally delayed convalescence from infectious mononucleosis. Much interesting work is currently in progress relating fatigue to persisting immunological abnormalities, and the development of molecular immunology makes this a most exciting field of research. This paper reviews the evidence for and against a definitive post-viral fatigue syndrome and examines the results of research carried out in the last 50 years.     

病毒感染与特发性血小板减少性紫癜

特发性血小板减少性紫癜(ITP)是一种免疫机制参与而使血小板生成减少和破坏增多的疾病。病毒感染是其主要病因之一。近年来,随着免疫学和分子生物学技术的发展,研究发现病毒感染引起血小板减少的发病机制是多方面的,包括直接作用于巨核细胞和血小板、免疫学机制和多因素联合作用等。病毒感染引起ITP的主要发病机制为机体对病毒感染后的天然免疫反应,或因病毒感染改变了血小板抗原,导致自身抗体产生,使巨核细胞发育成熟障碍。     

Rotavirus infection in young children in the highlands of Papua New Guinea

Rotavirus infections were detected by electron microscopy examinations in 54 of 66 children (82%) with acute gastroenteritis which necessitated admission to hospital during April to July, 1979, in the Highlands of Papua New Guinea. Longitudinal epidemiological studies may confirm rotavirus infections to be more important aetiolgical agents of childhood gastroenteritis in this region than in many other countries studied to date.     

The coronary arteries in active viral cardiomyopathies

These studies reveal that in mice, Coxsackie B₄ and EMC viruses produce not only myocardial disease but extensive disease of the myocardial capillaries, coronary arteries, aorta and mesenteric arteries and veins and other arteries and veins. These lesions impair luminal calibre and, in turn, lead to myocardial ischaemia. This ischaemia contributes further to the myocardial damage. It is postulated that viruses initiate arteriosclerosis in man. A 19-year-old man who died of Coxsackie B₄ viral cardiomyopathy had changes in the aorta noted on light and electron microscopy which are compatible with atherosclerosis. The relationship of these aortic changes to the viral infection can only be opened to speculation. Nevertheless, all of these studies establish a new concept of the possible initiating cause of arteriosclerosis, a common and extremely important disease of man.     

HIV-1假病毒的研究进展

假病毒是一种病毒的核酸由另一种病毒编码的包膜蛋白所包被,从而形成的具有外源性病毒囊膜,而基因组保持着逆转录病毒本身基因组特征的病毒.假病毒因其丧失了病毒的自我复制能力及其安全系数高等优点,已经被广泛应用于包括人类免疫缺陷病毒(HIV)在内的多项研究中.HIV假病毒通过识别膜蛋白基因的功能有助于研究病毒侵入过程、病毒调节基因功能以及评估中和抗体效价.     

单纯疱疹病毒潜伏和激活机制研究进展

单纯疱疹病毒（HSV，包括HSV-1和HSV-2）是引起多种疾病的重要病原，通常这些疾病具有反复发作和无法根治的特点。HSV在外周黏膜组织表面进行增殖式感染后，可进入感觉神经元并建立无复制的潜伏感染。潜伏的病毒还可以通过激活的方式重新进入复制周期，并回到外周组织进行复发感染。这种既能通过潜伏逃避宿主免疫攻击又能在激活过程中传播的能力是该病毒高超的生存策略，也是当前任何药物无法彻底消灭该病毒的根本原因。虽然HSV潜伏与激活的研究众多，且不断有新的研究进展，但由于潜伏和激活过程本身的复杂性和研究模型的局限性，其中很多具体过程相关机制依然不明确，甚至常有争议。本文重点梳理HSV-1潜伏和激活的主要研究成果，并讨论其发展趋势。     

基因-病毒载体的研制及其抗肿瘤作用的初步研究

目的：研究一种结合肿瘤基因治疗与病毒治疗优势的新型肿瘤治疗载体系统,即基因－病毒载体系统。方法：利用病毒重组技术将抗癌基因插入肿瘤细胞特异性的增殖病毒的病毒基因组中,通过细胞病理作用、荧光显微镜、免疫酶链技术及电镜等技术分别观察病毒的杀伤效应、报告基因绿色荧光蛋白、抗癌基因小鼠白细胞介素12表达量及病毒复制情况。结果构建了一种新型基因－病毒载体系统,该载体系统腺病毒E1b55000蛋白缺失,保留了腺病毒E1a蛋白。该载体系统具有肿瘤增殖病毒ONYX－015的相似功能,即它可在肿瘤细胞内复制及增殖,而在正常细胞内不能复制及增殖,从而特异性杀灭肿瘤细胞。该载体系统还可携带各种抗癌基因以进一步提高抗肿瘤的疗效。应该该载体系统携带该报告基因荧光蛋白可使绿色荧光蛋白在肿瘤细胞内高效表达,其表达量明显高于传统基因治疗的腺病毒载体系,而在正常细胞内低表达,表达量与传统腺病毒载体系统相似或更低。应用该载体系统携带抗癌基因小鼠白细胞介素12,也产生类似结果。电镜也证实该载体系统携带抗肿瘤基因小鼠白细胞介素12可在肿瘤细胞株中复制及增殖。结论：基因－病毒载体将抗癌基因插入肿瘤增殖病毒基因组,可数百倍乃至上万部提高抗癌基因的表达量,进一步提高抗肿瘤的疗效。     

Chronic obstructive pulmonary disease: the clinical management of an acute exacerbation

Exacerbations of chronic obstructive pulmonary disease impose a considerable burden of morbidity, mortality, and health care cost. Management guidelines outlining best practice, based largely on consensus expert opinion, were produced by a number of organisations during the last decade. Current interest in the field is high. This has resulted in the publication of many further studies which have extended our understanding of the pathology involved and provided, for the first time, an evidence base for many of the therapeutic options. In this review we aim to bring the non-specialist reader up to date with current management principles and the evidence underlying such interventions.