端粒酶RNA基因在白细胞中表达的研究

用RT－PCR的方法钓取端粒酶RNA（hTR）基因的cDNA，将hTR基因克隆到逆转录病毒载体（pLNCX）构建哺乳动物细胞表达质粒，然后经脂质体介导转染人体正常的外周血白细胞中表达。结果表明端粒酶RNA基因的表达，不能激活或重建白细胞的端粒酶活性，流式细胞技术分析结果显示，外源hTR基因的表达不能延长白细胞的寿命，反而抑制白细胞的增殖并促进其凋亡，这可能是因为外源hTR基因的表达一定程度上阻碍了细胞内端粒酶RNA同端粒酶催化亚基的结合，同时也阻碍了端粒酶RNA模板区与端粒DNA之间的结合，从而影响端粒酶活性，抑制了细胞的增殖。     

反义端粒酶RNA对SMMC7721细胞P16、P21表达的增强作用

目的 观察反义端粒酶RNA对SMMC7721细胞周期的影响,检测P16和P21的表达,探讨端粒酶与细胞期调控间的关系及反义端粒酶RNA在肝癌基因治疗中的意义。方法 经脂质体介导,将反义端粒RNA真核表达载体pBBS212－hTR导入SMMC7721细胞中。细胞克隆转移扩大培养后,采用流式细胞仪、TRAP－PCR－ELISA及免疫组化技术,结合图像分析,在检测SMMC7721／pBBS212－hTR细胞端粒酶活性的同时,检测P16和P21的表达。结果 与对照组相比较,反义端粒酶RNA在抑制SMMC7721／pBBS212－hTR细胞端粒酶活性的同时,检测P16和P21的表达。结果 与对照组相比较,反义端粒酶RNA在抑制SMMC7721细胞端粒酶活性的同时,P16和P21蛋白的表达水平显著增高。结论 转染反义端粒酶RNA在封闭细胞端粒活性表达的同时,伴发P21与P16表达水平的升高。本实验研究为探讨肝癌发病机制及治疗提供了一定的依据。     

端粒酶RNA反义核酸降低鼻咽癌细胞p53蛋白表达水平

目的:研究端粒酶活性抑制后鼻咽癌细胞p53蛋白表达水平的变化。方法:脂质体介导端粒酶反义寡核苷酸转染鼻咽癌CNE1和CNE2Z细胞株,MTT法检测细胞增殖能力,流式细胞术检测细胞凋亡,免疫组化法检测端粒酶逆转录酶(hTRT)和p53蛋白表达水平。结果:端粒酶RNA反义寡核苷酸可抑制CNE1和CNE2Z细胞的增殖并呈剂量依赖性,流式细胞仪检测出现凋亡峰,反义寡核苷酸组hTRT和p53蛋白表达水平显著低于其它处理组及空白对照组(P＜0.01)。结论:端粒酶RNA反义寡核苷酸抑制鼻咽癌细胞端粒酶活性后可降低p53蛋白表达水平。     

表达反义c-myc RNA的重组逆转录病毒载体对人胰腺癌细胞恶性表型的逆转作用

为研究表达反义c－mycRxA的逆转录病毒载体抑制靶基因的表达和翻译，使胰腺癌细胞恶性表型逆转的机理，利用PXTI逆转录病毒载体构建了一个能表达反义c－mycRNA的质粒，经病毒包装细胞PA317包装后，使之成为具有感染力的重组病毒。用该病毒感染人胰腺癌细胞系PC－2细胞，经G418筛选得到稳定的转化细胞系。Northernblot杂交证实包装细胞PA317及转化的PC－2细胞中均有病毒的高表达，体外合成的单链RNA探针杂交结果表明转化PC－2细胞中有高表达的反义c－mycRNA，而内源性c－mycRNA的表达明显受抑；Westernblot分析发现c－myc癌基因产物P62蛋白的表达显著下降。反义c－mycRNA使人胰腺癌细胞生长速率、~3H-胸腺嘧啶掺入、软琼脂集落形成能力以及探鼠致瘤能力等均明显下降。实验结果表明：表达反义c－mycRNA的逆转录病毒载体能有效地抑制靶基因的表达和翻译，使胰腺癌细胞恶性表型部分逆转。     

hTERT-逆转录病毒载体构建及其介导的脐血间质干细胞基因转移研究

目的：构建hTERT-逆转录病毒载体，向脐血间质干细胞（MSCs）中导入hTERT基因，观察该基因对细胞端粒酶活性的影响，能否延长细胞的寿命以及转基因细胞的分化能力。方法: PCR法扩增出hTERT全部片段，定向克隆入pLNCX2载体。将病毒质粒导入包装细胞内，筛选阳性产毒细胞，测定病毒滴度。取病毒上清感染脐血MSCs，筛选转基因细胞。检测转染前后hTERT在mRNA水平的表达，端粒酶活性的变化，hTERT转入后对细胞增殖的影响。用5-氮杂胞苷将转基因细胞向心肌方向诱导，检测心肌特异抗体的表达。结果:用BglⅡ和NotⅠ双酶切构建质粒，证明PLNCX2-hTERT构建成功，转基因细胞的hTERT基因在mRNA水平得到表达，细胞端粒酶阳性。生长曲线显示转基因细胞增殖速度快于未转染细胞。向心肌细胞诱导后，心肌特异性抗体 α-sarcomeric actin和connexin-43均有表达。结论:在逆转录病毒介导下，外源性hTERT基因转入脐血MSCs后得到表达，激活细胞端粒酶的活性，有效延长了细胞的寿命，不影响其分化功能。     

1,25-二羟基维生素D3协同转染反义端粒酶RNA诱导肝癌细胞凋亡的实验研究

目的观察在抑制肝癌细胞端粒酶活性表达情况下,1,25-二羟基维生素D3(1,25-VD3)对肝癌细胞凋亡的影响。方法经脂质体介导,将反义端粒酶RNA真核表达载体pBBS212/hTR导入维生素D3受体(VDR)阳性的肝癌细胞株SMMC7721细胞中培养。细胞克隆转移扩大培养,将其分为对照组、药物组、转染组和转染药物组。添加1,25-VD3,分别作用于转染药物组、药物组,用脱氧核糖核苷酸末端转移酶介导的缺口末端标记法(TUNEL)和电子显微镜检测肝癌细胞凋亡。结果转染组和对照组细胞的端粒酶活性分别为0.686和1.685,说明反义端粒酶RNA可显著降低肝癌细胞端粒酶活性。对照组、药物组、转染组和转染药物组细胞凋亡率分别为3.4%、12.2%、8.8%、23.6%;差异有显著统计学意义(P<0.01)。超微结构检查也证实存在凋亡发生。1,25-VD3或转染反义端粒酶RNA对肝癌细胞有促细胞凋亡作用,两者协同对肝癌细胞凋亡作用显著增强。结论转染反义端粒酶RNA,可降低肝癌细胞端粒酶活性,可显著增强1,25-VD3对肝癌细胞的促凋亡作用,并且对细胞的增殖也存在明显的抑制作用。     

乙型肝炎病毒C基因重组逆转录病毒的构建和在真核细胞的表达

目的　构建含HBVC基因的重组逆转录病毒以用于慢性乙型肝炎的免疫治疗。方法　用PCR法扩增HBV的C基因片段 ,定向插入逆转录病毒质粒载体pLXSN中 ,重组质粒用脂质体转染包装细胞PT67,G418筛选抗性细胞 ,用抗性细胞培养上清液中的重组病毒感染NIH3T3、EL4和鼠骨髓来源的树突状细胞 (DC) ,用PCR检测目的基因的插入 ,用间接免疫荧光和流式细胞仪检测目的基因的表达 ,基因修饰的DC免疫C57BL 6鼠观察其诱导细胞免疫的能力。结果　含HBVC基因重组质粒载体经PCR、酶切和序列分析鉴定为阳性 ,转染PT67后经G418筛选获得抗性细胞 ,抗性细胞培养上清液中含有重组逆转录病毒 ,病毒效价达 3× 10 5CFU ml,感染NIH3T3和EL4细胞后PCR鉴定含目的基因 ,间接免疫荧光、流式细胞仪鉴定有HBcAg表达 ,基因修饰后的DC免疫C57BL 6鼠能诱导特异性CTL应答。结论　构建的HBVC基因重组逆转录病毒可将目的基因转移至真核细胞并得到有效表达     

丙型肝炎病毒核心蛋白在HepG2细胞中的表达及其对端粒酶活性的影响

目的 建立HCV核心蛋白细胞表达模型，并探讨其对细胞端粒酶活性的影响。方法 用PCR法扩增出HCV核心基因cDNA，将其插入真核表达载体pBK-CMV的HindⅢ和BamHⅠ位点间，构建重组质粒pBK-HCVc。再将重组质粒pBK-HCVc和空载体分别导入肝癌细胞株HepG2中，G418筛选，RT－PCR、免疫组化和蛋白印迹鉴定HCV核心蛋白表达。PCR－ELISA法检测端粒酶活性。结果 构建的pBK-HCVc质粒在HepG2细胞中有稳定表达。表达HCV核心蛋白的细胞HepG2-C的端粒酶活性较转染空载体的细胞HepG2-CMV明显升高。结论 HCV核心蛋白上调了端粒酶活性，可能是HCV诱发肝细胞癌的一种途径。     

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.     

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.     

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.     

'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.     

Der Einfluß von Nahrungsmitteln und Rauchen auf die klinisch-chemische Diagnostik von Phäochromozytom,Neuroblastom und Karzinoid-Syndrom

Zusammenfassung Der Einfluß von verschiedenen Nahrungsmitteln auf Methoden zur Bestimmung von Adrenalin (AD), Noradrenalin (NA), Vanillinmandelsäure (VMS), Metanephrinen (MN), Homovanillinsäure (HVS) und 5-Hydroxyindolessigsäure (5-HIE) im 24 h-Harn zur Diagnose des Phäochromozytoms bzw. Karzinoid-Syndroms wurde untersucht. Die in die Untersuchung einbezogenen Nahrungsmittel waren: Tee, Kaffee, Mandeln, Ananas, Käse, Walnüsse, Vanillepudding, Bananen, Tomaten und Milchschokolade. Außerdem wurde der Einfluß des Zigarettenrauchens auf die Bestimmung von AD, NA, VMS und MN untersucht.Walnüsse führten zu einer starken Erhöhung der 5-HIE-Ausscheidung. Bananen erhöhten die Ausscheidung von AD, NA, VMS, MN und 5-HIE. Kaffee und Ananas bewirkten eine geringe Zunahme der MN-Werte. Rauchen von 20–30 Zigaretten/Tag beeinflußte keine der vier Variablen.Wenn die beschriebenen Methoden benutzt werden, sollte lediglich auf den Verzehr von Bananen und Walnüssen vor und während der Harnsammelperioden verzichtet werden, da die oberen Normgrenzen im Harn überschritten werden könnten. Ein Verzicht auf Kaffee und Ananas in normalen Mengen ist nicht erforderlich. Es besteht kein Anlaß, weiterhin die bisherigen umfangreichen Restriktionen der übrigen Nahrungsmittel beizubehalten.