HPV58 E6体外降解p53蛋白作用的研究

通过PCR方法从有乳头瘤病毒58型（HPV58）全基因克隆中扩增出HPV58E6基因片段，克隆至真核表达质粒pcDNA3的T7启动子下游，并在体外转录成mRNA后，在源自网织红细胞的翻译缓冲液中成功表达了含有生物素标记的HPV58E6蛋白，并在体外成功发现HPV58E6能够降解p53蛋白，从而推断结合并降解p53蛋白是其致癌作用的关键环节，这为日后在细胞内对其致癌机理行进一步研究以及针对其进行治疗奠定下坚实的基础。     

牛乳头瘤病毒E6蛋白与p73蛋白的相互作用

为进一步探讨和阐明E6蛋白的致癌机制以及充分认识新发现的抑癌基因p73的作用，在体外通过免疫共沉淀法研究p73和牛乳头瘤病毒1型E6蛋白（BPI－1E6）的相互结合情况，后将表达p73和BPV－1E6的质粒导入SAOS－2细胞内，进一步研究细胞内E6蛋白对p73蛋白的诱导调亡和转录活化功能等生物学活性的影响。结果发现，BPV－1E6与p73蛋白结合较弱，且未检测到E6能诱导p73蛋白的降解。在SAOS－2细胞内，BPV－1E6蛋白确实能部分抑制p73蛋白的诱导细胞凋亡的功能，并且p73蛋白的转录激活作用也有影响，但这种影响作用颇弱。     

E6-p53和E7-pRb模式在人乳头瘤病毒致癌过程中的作用

在全球范围特别是发展中国家，宫颈癌的发病率居女性恶性肿瘤的第二位。目前认为人乳头瘤病毒（HPV）是宫颈癌的主要致病因子，而早期基因E6、E7是HPV致癌机制研究中的重点。体内体外实验均表明E6、E7是细胞转化所必需的，尤其是在高危型HPV感染机体时，E6结合并灭活肿瘤抑制因子p53、E7结合并降解抑制蛋白pRb，最终导致细胞无限分裂而不发生凋亡。E6、E7分别与抑癌蛋白p53、pRb相互作用的模式为人乳头瘤病毒致癌机制的进一步研究提供了基本的理论基础。     

HPV与宫颈癌关系及疫苗研究进展

流行病学和病原学研究表明,人乳头瘤病毒（HV）感染是妇女发生宫颈癌重要的原因之一.HPV是无包膜的小型双链环状DNA病毒,不同基因型病毒对细胞的转化能力不同,其中HPV-16、18与子宫颈癌关系最密切.HPV诱发官颈癌的主要机制,是其E6和E7蛋白基因在宫颈细胞中的表达增加,产生的E6和E7蛋白两个癌蛋白分别与抑癌蛋白p53和pRb结合而诱导后两者降解.HPV疫苗包括预防性疫苗和治疗性疫苗两大类.本文对HPV感染与宫颈癌发生的关系、疫苗研究进展进行了系统的阐述.     

p53与抗肿瘤免疫

p53是迄今发现的与肿瘤发生、发展关系最为密切的抑癌基因,正常的p53蛋白通过诱导细胞周期阻滞和细胞凋亡等途径对细胞增殖发挥负调控作用,而在肿瘤细胞中异常表达的p53,尤其是突变型p53蛋白可以诱导机体产生细胞和体液免疫反应,导致对肿瘤细胞的免疫耐受或排斥。本文中对p53的功能、p53在抗肿瘤等免疫反应中的作用,及其在肿瘤治疗中的应用做一综述。     

西多福韦对人宫颈癌细胞CaSki内人乳头瘤病毒16抑制作用研究

目的从抗病毒角度探讨西多福韦对宫颈癌细胞 CaSki内人乳头瘤病毒16（HPV16）的抑制作用及对细胞周期的影响。方法用 MTT法检测西多福韦对细胞的毒性;用实时定量 PCR法检测其对病毒 E6、E7 mRNA水平的影响;用 Western blot方法检测其对病毒蛋白 E6、E7和细胞抑癌蛋白 p53、pRb表达水平的影响;用流式细胞法检测其对宫颈癌细胞周期的影响。结果西多福韦对宫颈癌细胞毒性较正常细胞大。可使HPV16阳性宫颈癌细胞 CaSki内 E6、E7 mRNA和蛋白水平降低,最大抑制率分别为（33.38±8.00）%、（28.32±2.73）%和98.92%、97.46%;可以使 p53、pRb蛋白水平升高,最大浓度时可以上调12.06和3.53倍;对 HPV16阴性宫颈癌细胞 C-33A p53蛋白表达无影响,但可提高 pRb蛋白水平;可导致 CaSki和 C-33A细胞发生 S期阻滞,最高浓度组细胞相对对照组 S期分别增加22.83%和67.64%。结论西多福韦可以在对细胞无毒的浓度下,抑制宫颈癌细胞内的 HPV16,诱导宫颈癌细胞发生 S期阻滞。     

HPV与宫颈癌关系及疫苗研究进展

流行病学和病原学研究表明,人乳头瘤病毒(HV)感染是妇女发生宫颈癌重要的原因之一.HPV是无包膜的小型双链环状DNA病毒,不同基因型病毒对细胞的转化能力不同,其中HPV-16、18与子宫颈癌关系最密切.HPV诱发官颈癌的主要机制,是其E6和E7蛋白基因在宫颈细胞中的表达增加,产生的E6和E7蛋白两个癌蛋白分别与抑癌蛋白p53和pRb结合而诱导后两者降解.HPV疫苗包括预防性疫苗和治疗性疫苗两大类.本文对HPV感染与宫颈癌发生的关系、疫苗研究进展进行了系统的阐述.     

siRNA抑制HPV18 E6 基因对HeLA细胞凋亡的影响

目的 研究特异小干扰RNA (small interfering RNA, siRNA)对宫颈癌HeLa细胞中人乳头瘤病毒（human papillomavirus, HPV）18型 E6基因的抑制及其对细胞凋亡的影响。方法 针对HPV18 E6 基因设计siRNA序列，经PCR方法体外扩增，得到含有U6启动子以及siRNA序列的PCR产物，利用LipofectamineTM2000脂质体转染HeLa细胞，在U6启动子的作用下于细胞内转录siRNA。针对转染后不同时间点采用四唑盐（MTT）比色法测定细胞活力，流式细胞仪PI染色法检测细胞凋亡率，RT-PCR测定HPV18 E6 mRNA变化。结果 转染siRNA后细胞活力受到显著抑制（P＜0.05），光镜下出现明显的凋亡形态，72h 的凋亡率达到55.8%。RT-PCR结果显示，细胞转染24、48和72h后HPV18 E6 mRNA分别减少了57％、78％和40%，而siRNA阴性对照与未转染细胞相比差异不显著。结论 siRNA可特异有效的干扰宫颈癌HeLa 细胞内HPV18 E6基因的表达，从而可诱导肿瘤细胞凋亡。     

p73基因与细胞凋亡

p73是经典抑癌基因p53的类似物。经研究证实，p73具有两面性。它可表达为两种相互独立却又密切相关的蛋白质即TApT3和DNp73。它们在细胞凋亡与肿瘤发生的过程中起着彼此对立以相互影响的作用。同时在细胞凋亡的过程中，p73的功能又受到E2F-1、C-Abl、C-Myc、P300、PKCδ等各种因素的影响。本文现就p73在细胞凋亡中的作用以及其影响因素作一综述。     

p53功能调节的新进展

p53的结构和功能异常与肿瘤的发生密切相关。研究表明 ,p53的磷酸化和 或乙酰化可直接影响p53的稳定性及活性 ,多种蛋白包括癌基因蛋白 (如Mdm2 ,p3 3 ING1,E1a ,c Myc等 )亦通过不同途径影响p53的功能。了解p53功能的调节将助于进一步认识其在肿瘤发生发展中的作用。     

Renal dysplasia and asplenia in two sibs

A family is reported in which two sibs, one male and the other female, both died within 24 hours of birth with enlarged polycystic kidneys. Postmortem histology in the second child showed gross renal dysplasia. In both children the pancreas was enlarged, nodular and cystic but the liver appeared macroscopically normal. In the second child, histological examination confirmed pancreatic fibrosis with cystic dilation of ducts, but showed portal fibrosis with bile duct proliferation in the liver.
This combination of findings is very reminiscent of those in a girl and her brother reported by Ivemark et al. (1959). The children reported here also showed absence or hypoplasia of the spleen, cardiac anomalies and other features of the Ivemark syndrome (Ivemark 1955), a quite different, usually sporadic, congenital disorder. It is suggested that the children described here have a distinct lethal congenital disorder, probably inherited in an autosomal recessive manner.     

Schizophrenia in a North Swedish geographical isolate, 1900–1977. Epidemiology, genetics and biochemistry

Over 200 schizophrenic patients belonging to three major and interrelated pedigree complexes have been investigated over the past 30 years in a North Swedish geographically isolated population, presently numbering about 6,000. An intensive investigation of a number of biochemical correlates and genetic markers in a few selected families belonging to one of the major pedigrees has indicated new strategies for the current research program.
Schizophrenia, as defined operationally, is significantly associated with decreased activities of two enzymes (1) blood platelet monoamine oxidase, (2) plasma dopamine-β-hydroxylase, and (3) with the genetic marker Gc² (group specific antigen). Both enzymes are subject to genetic variation. A positive score for linkage between schizophrenia and low plasma DBH activity has been calculated, but, so far, available data are insufficient for discrimination between linkage and partial contribution of genetically controlled low plasma DBH to the pathogenesis of the disease. Alternatively, both mechanisms could be involved.
As a model for continued research, schizophrenia is explained as based on a double dominant-recessive genotype (Aabb), representing a vulnerability which in about 50 % of cases develops into clinical schizophrenia. It is suggested that the dominant mutation (A) operates on or affects MAO activity, and that the recessive genotype (bb) is instrumental in low variates of DBH activity and very likely such variates within the normal range of physiological variation. Moreover, it is suggested that the combined effects of MAO- and DBH-reduced efficiency on the metabolism of e.g. dopamine could be an essential pathogenic mechanism for the schizophrenic illness which is segregating in this population.     

The dominant diseases of modernized societies as omega-3 essential fatty acid deficiency syndrome: Substrate beriberi

About 1900, modern food selection and processing caused widespread $\text{epidemics}$ of the B vitamin deficiency diseases of beriberi and pellagra which, for genetic reasons, often expressed as different diseases ranging from bowel and heart disease to dermatoses and psychoses. But the B vitamins merely help convert essential fatty acids (EFA) into the prostaglandin (PG) tissue regulators and it now turns out that, through hydrogenation, milling and selection of w3-poor southern foods, we have also been systematically depleting, by as much as 90%, a newly discovered trace Nordic EFA (w3) of special importance to primates and sole precursor of the PG3(4) series, even as a concurrent fiber deficiency increases body demand for EFA. Since substrate EFA is processed by many B vitamin catalysts, an EFA deficiency will mimic a $\text{panh}$ypovitaminosis B, i.e., a mixture of $\text{substrate}$ beriberi and $\text{substrate}$ pellagra resembling vitamin beriberi and pellagra but exhibiting as even more diverse $\text{endemic}$ disease. This would consitute a second stage of the Modern Malnutrition and explain why some workers now hold the dominant diseases of modermized societies to be new, nutritionally based, pellagraform yet lipid-related and to range, once again, from heart disease to psychosis. It is an assumption that our dominant diseases are unrelated to each other or are merely revealed by our diagnostic acumen and therapeutic success; and that hydrogenating millions of tons of food oils annually, to destroy the rancidity producing w3-EFA, is safe for $\text{primates}$. Extensive beriberiform disease is reported here in 32 typical cases taken from medical practice which responds strikingly to linseed oil supplements (60% w3-EFA) in confirmation of identical results in Capuchins.     

What will studies of Fulani individuals naturally exposed to malaria teach us about protective immunity to malaria?

There are an estimated over 200 million yearly cases of malaria worldwide. Despite concerted international effort to combat the disease, it still causes approximately half a million deaths every year, the majority of which are young children with Plasmodium falciparum infection in sub-Saharan Africa. Successes are largely attributed to malaria prevention strategies, such as insecticide-treated mosquito nets and indoor spraying, as well as improved access to existing treatments. One important hurdle to new approaches for the treatment and prevention of malaria is our limited understanding of the biology of Plasmodium infection and its complex interaction with the immune system of its human host. Therefore, the elimination of malaria in Africa not only relies on existing tools to reduce malaria burden, but also requires fundamental research to develop innovative approaches. Here, we summarize our discoveries from investigations of ethnic groups of West Africa who have different susceptibility to malaria.     

'Very sore nights and days': the child's experience of illness in early modern England, c.1580-1720

Sick children were ubiquitous in early modern England, and yet they have received very little attention from historians. Taking the elusive perspective of the child, this article explores the physical, emotional, and spiritual experience of illness in England between approximately 1580 and 1720. What was it like being ill and suffering pain? How did the young respond emotionally to the anticipation of death? It is argued that children’s experiences were characterised by profound ambivalence: illness could be terrifying and distressing, but also a source of emotional and spiritual fulfilment and joy. This interpretation challenges the common assumption amongst medical historians that the experiences of early modern patients were utterly miserable. It also sheds light on children’s emotional feelings for their parents, a subject often overlooked in the historiography of childhood. The primary sources used in this article include diaries, autobiographies, letters, the biographies of pious children, printed possession cases, doctors’ casebooks, and theological treatises concerning the afterlife.     

Guidelines for the Validation and Use of Immunoassays for Determination of Introduced Proteins in Biotechnology Enhanced Crops and Derived Food Ingredients

Recent advancements in agricultural biotechnology have created a need for analytical techniques to determine introduced proteins in crops enhanced through modern biotechnology techniques. These proteins are expressed in plant tissues and may be present in food ingredients. Immunoassays are ideally suited for protein detection and may be used as both quantitative and threshold methods. Microplate ELISA and lateral flow devices are two of the most commonly used immunoassay formats for agricultural biotechnology applications. This paper provides general background information and a discussion of criteria for the validation and application of immunochemical methods to the analysis of proteins introduced into plants and food ingredients using biotechnology methods. It is the result of a collaborative effort of members of the Analytical Environmental Immunochemical Consortium. This collaborative effort represents the combined expertise of several organizations to reach consensus on establishing guidelines for the validation and use of immunoassays. Further, the paper offers developers and users a consistent approach to adopting the technology as well as aid in producing accurate and meaningful results.     

HLA in North Colombia Chimila Amerindians

HLA-A,-B,-C,-DRB1 and -DQB1 alleles have been studied in Chimila Amerindians from Sabana de San Angel (North Colombian Coast) by using high resolution molecular typing. A frequent extended haplotype was found:HLA-A*24:02-B*51:10-C*15:02-BRB1*04:07-DQB1*03:02 (28.7%) which has also been described in Amerinndian Mayos Mexican population (Mexico, California Gulf, Pacific Ocean). Other haplotypes had already been found in Amerindians from Mexico (Pacific and Atlantic Coast), Peru (highlands and Amazon Basin), Bolivia and North USA. A geographic pattern according to HLA allele or haplotype frequencies is lacking in Amerindians, as already known. Also, five new extended haplotypes were found in Chimila Amerindians. Their HLA-A*24:02 high frequencies characteristic is shared with aboriginal populations of Taiwan; also, HLA-C*01:02 high frequencies are found in New Zealand Maoris, New Caledonians and Kimberly Aborigines from Australia. Finally, this study may show a model of evolutionary factors acting and rising one HLA allele frequency (-A*24:02), but not in others that belong to the same or different HLA loci.     

Ultracryotomy: development and application (with examples from the yeast cytology) (author's transl)

The preparation steps usually necessary for obtaining ultrathin frozen sections of biological material (chemical prefixation, enclosing, cryoprotective treatment, freezing, sectioning, and post-staining the sections for transmission electron microscopy) are submitted to a critical analysis. The application of cryo-ultramicrotomy, in particularly for cytochemical purposes, is reviewed. Fundamental considerations of chemical prefixation and poststaining are supported by examples from yeast cytology. Furthermore, the efficiency of the cryo-ultramicrotomy (electron optical resolution of ultrastructural details) is demonstrated on yeast cells and protoplasts.     

The universal muscular chamber. A basic unit of the genitourinary, cardiovascular, gastro-intestinal, skeletal, cutaneous, respiratory and other systems, endocameral hypertension; and spasm, the key to their idiopathic diseases and arthropathies

Starting with the integument, we see many organs are contractile sacs or multiples thereof, which tubes or bags constitute the major part of the entire body. Recognition of this basic unit and its characteristics sheds new light, individually and collectively, on many disorders previously considered unrelated. Muscular tears and perforations develop in the walls of these chambers, being no way peculiar to those organs, wherein, hydrochloric acid occurs. So, it is not necessary to explain the absence of excessive acid from patients who exhibit holes in the gastric, uterine, aortic, duodenal, rectal, pulmonary, retina, and other walls. Muscle, not acid is the great common factor relating idiopathic disorders in the gastrointestinal tract to each other and to similar diseases in other systems. When the units are linked together, the lesions tend to appear as arthropathies, i.e. at the joints. Rephrasing common-place observations, frees us from conventional, conceptual cul-de-sacs. An observation is only as good as its interpretation, so all possibilities must be considered, otherwise, we will remain blinded by our misconceptions.