人端粒酶催化亚单位hTERT基因重组腺病毒载体的构建与鉴定

为构建人端粒酶催化亚单位(hTERT)重组腺病毒载体,采用PCR法从pCI-neo-hTERT中钓取hTERT全长cDNA,定向克隆到pADTrack-CMV穿梭质粒。通过分步转化把pADEasy-1和重组穿梭质粒(pADTrack-hTERT)转化入BJ5183菌进行同源重组。hTERT重组腺病毒骨架质粒(pAD-hTERT)转染HEK293细胞,荧光显微镜观察绿色荧光蛋白(GFP)的表达,收获重组腺病毒(AD-hTERT)提取DNA行PCR鉴定并扩增。经PCR、酶切和测序鉴定证实hTERT的cDNA正确插入穿梭质粒且与pADEasy-1正确同源重组;PCR鉴定及报告基因分析说明hTERT重组腺病毒包装正确且具有较好的感染活力。结果表明本研究已成功构建了hTERT重组腺病毒,为hTERT的神经保护作用研究奠定了基础。     

短发夹RNA对人端粒酶催化亚基表达的影响

目的:研究短发夹RNA(shRNAs)对人端粒酶催化亚基(hTERT)表达的影响。方法:将编码针对hTERT的shRNA的寡聚核苷酸克隆入哺乳细胞shRNA表达载体pUC18U6形成pUC18U6ht,然后以脂质体法转染入HepG2细胞。被pUC18U6和表达抗绿色荧光蛋白shRNA的pUC18U6GFPsir转染的HepG2细胞作为对照。转染细胞的hTERTmRNA以实时定量RT-PCR定量测定。结果:与对照相比,shRNA可使hTERTmRNA的水平降低49%(P<0.05)。结论:shRNAs抑制了hTERT的表达。     

人端粒酶反转录酶启动子调控的肿瘤特异性载体的构建及表达

 目的: 构建由人端粒酶反转录酶(hTERT)启动子调控的具有肿瘤细胞特异性表达能力的荧光蛋白质粒。方法: 利用PCR技术克隆hTERT 基因启动子,利用分子生物学方法以该启动子替换pEGFP-C3质粒的CMV启动子,构建重组质粒（hTERTp-EGFP）。用脂质体转染法将hTERTp-EGFP质粒分别转染至肿瘤细胞和正常细胞, 观察此两种细胞内绿色荧光蛋白的表达差异。结果: 成功构建重组质粒hTERTp-EGFP,转染结果显示该质粒在肿瘤细胞中表达,而在正常细胞中无明显表达。结论: 重组质粒hTERTp-EGFP具有肿瘤特异性表达能力。     

反义hTERT基因对白血病细胞端粒酶表达及活性的抑制作用

目的探讨反义hTERT基因体外对白血病细胞端粒酶基因表达及其活性的抑制作用。方法采用基因重组技术设计并构建了针对端粒酶活性蛋白催化亚单位mRNA的5’端序列的反义pcDNA-hTERT真核表达载体,通过SuperFect(QIAGEN)将其瞬时转染人早幼粒白血病细胞株Hk及人红白血病细胞株K562,并运用流式细胞术测定其细胞凋亡与细胞周期的变化,同时运用实时荧光定量逆转录-聚合酶链反应方法对其端粒酶蛋白催化亚单位基因的表达水平进行了检测,端粒酶活性的检测则采用了非放射性TRAP-银染法。结果成功地构建了反义pcDNA-hTERT真核表达载体,并将其转染至K562和HL60白血病细胞株。结果与空载体组、空白组相比,反义pcDNA-hTERT组体外显示出明显有效地抑制端粒酶的基因表达及其活性的作用;在细胞凋亡与细胞周期方面,反义组能引起细胞周期左移,增殖分裂能力降低,同时伴有细胞凋亡数的增加。结论研究构建的反义hTERT基因体外确实能明显有效地下调白血病细胞端粒酶的基因表达及其活性,在抗肿瘤基因治疗中具有特异的靶向性和潜在应用的广谱性。     

互补于hTERT关键区段的反义RNA抑制肝癌细胞

目的研究针对人类端粒酶催化亚单位(hTERT)调控区C-MYC结合位点的反义RNA抑制肝癌细胞生长。方法细菌内同源重组构建反义RNA腺病毒(rAd-asmycb),转染HepG2.2.15肝癌细胞,流式细胞术检测细胞凋亡,透射电镜下观察凋亡细胞形态,聚合酶链反应-酶联免疫分析(PCR-ELISA)、逆转录-PCR(RT-PCR)分别检测细胞端粒酶活性及mRNA水平上hTERT表达。结果反义RNA抑制肝癌细胞生长,细胞凋亡率为40.7%,显示特征性凋亡细胞形态,能够显著降低细胞端粒酶活性,在mRNA水平上抑制hTERT表达。结论hTERT调控区C-MYC结合位点可能是肝癌基因治疗的有效靶位。     

AML患者端粒酶逆转录酶mRNA的表达与端粒酶活性的检测

目的:建立一种实时荧光RT-PCR方法,定量检测急性髓细胞白血病(AML)患者外周血单个核细胞端粒酶逆转录酶(hTERT)mRNA的表达水平,观察其与AML发生及复发的关系,探讨hTERT mRNA的表达水平与端粒酶活性的相关关系。方法:采用实时荧光RT-PCR与Lightcycler荧光PCR仪定量检测hTERT mRNA的表达水平,采用PCR-ELISA检测端粒酶活性。结果:①AML首治患者、复发患者、完全缓解期患者以及健康体检者NhTERT分别为299.2±292.8、550.1±441.3、14.0±9.2和12.3±6.7,与健康体检者和完全缓解期患者比较,AML首治患者、复发患者hTERT mRNA表达水平显著升高,而且AML复发患者hTERT mRNA的表达水平显著高于首治患者;②AML首治患者、AML复发患者、AML完全缓解期患者以及健康体检者端粒酶的活性分别为32.8%±24.3%4、8.6%±31.4%、7.4%±5.1%和7.6%±3.6%,AML首治患者、复发患者端粒酶活性显著升高,而且AML复发患者端粒酶活性显著高于首治患者;③hTERT mRNA表达水平与端粒酶的活性呈现良好的相关性,相关系数为0.78。结论:hTERT mRNA表达水平与端粒酶活性的上调是AML发生与复发的一个重要因素,且hTERT mRNA表达水平与端粒酶活性呈良好的正相关。     

构建人端粒酶催化亚单位基因慢病毒表达载体转染人骨髓基质细胞

背景：骨髓基质细胞不表达端粒酶,因而在体外传代扩增过程中端粒长度逐渐缩短,导致细胞衰老,这是限制其用于细胞治疗应用的一个重要因素。 目的：构建人端粒酶催化亚单位基因慢病毒表达载体,探讨以慢病毒介导的人端粒酶催化亚单位基因修饰人骨髓基质细胞的可行性。 方法：以pReceiver-M02-hTERT质粒为模板PCR扩增获得目的基因。用BP重组系统将目的片段重组到载体pDONR221上。然后使用LR重组系统将目的序列重组到载体pLenti6.3/V5-DEST上。将重组载体与包装质粒充分混合,利用脂质体共转染293FT细胞获得慢病毒颗粒。 结果与结论：成功构建人端粒酶催化亚单位慢病毒表达载体,病毒的平均物理滴度为1.07×10¹² LP/L。以之转染人骨髓基质细胞,目的基因表达水平有显著提升,细胞正确表达人端粒酶反转录酶蛋白。说明以慢病毒介导的人端粒酶催化亚单位基因修饰人骨髓基质细胞能强化细胞表达人端粒酶催化亚单位。     

反转录酶活性检测实时荧光定量PCR方法的建立

目的 在传统产物增强性反转录酶(PERT)活性检测方法基础上建立检测反转录酶活性的实时荧光定量PCR方法.方法 以噬菌体MS2 RNA为模板,设计、合成引物和探针,并分别以连续10倍倍比稀释的AMV反转录酶(1×10-1～1×10-9 U/μl)标准品催化体外反转录反应合成相应cDNA.采用实时荧光定量PCR法检测底物cDNA模板量,并获得相应的PCR荧光值曲线,分析AMV反转录酶的相对活性.同时应用传统PERT方法对cDNA进行扩增,测定该方法的灵敏度.结果 将AMV反转录酶标准品连续10倍倍比稀释后催化反转录反应,实时荧光定量PCR分析所得的反转录产物cDNA,结果得到与理论值完全相符合的PCR标准荧光值曲线;定量分析结果显示,浓度为1×10-2～1×10-8U/μl的AMV反转录酶均可得到相应的荧光值.不同稀释度的AMV反转录酶,经传统PERT方法扩增后,结果显示浓度为1×10-3～1×10-7 U/μl的AMV反转录酶均可见大小为112 bp的阳性条带,检测灵敏度达到1×10-7U/μl.结论 成功建立了基于实时荧光定量PCR技术的检测反转录酶活性的新方法,实验操作简单,具有高精确性和无污染性,为生物制品外来污染尤其是反转录病毒污染的检测提供了参考指标.     

反转录酶活性检测实时荧光定量PCR方法的建立

目的在传统产物增强性反转录酶（PERT）活性检测方法基础上建立检测反转录酶活性的实时荧光定量PCR方法。方法以噬菌体MS2RNA为模板,设计、合成引物和探针,并分别以连续10倍倍比稀释的AMV反转录酶（1×10-1～1×10-9U/μl）标准品催化体外反转录反应合成相应cDNA。采用实时荧光定量PCR法检测底物cDNA模板量,并获得相应的PCR荧光值曲线,分析AMV反转录酶的相对活性。同时应用传统PERT方法对cDNA进行扩增,测定该方法的灵敏度。结果将AMV反转录酶标准品连续10倍倍比稀释后催化反转录反应,实时荧光定量PCR分析所得的反转录产物cDNA,结果得到与理论值完全相符合的PCR标准荧光值曲线;定量分析结果显示,浓度为1×10-2～1×10-8U/μl的AMV反转录酶均可得到相应的荧光值。不同稀释度的AMV反转录酶,经传统PERT方法扩增后,结果显示浓度为1×10-3～1×10-7U/μl的AMV反转录酶均可见大小为112bp的阳性条带,检测灵敏度达到1×10-7U/μl。结论成功建立了基于实时荧光定量PCR技术的检测反转录酶活性的新方法,实验操作简单,具有高精确性和无污染性,为生物制品外来污染尤其是反转录病毒污染的检测提供了参考指标。     

端粒、端粒酶与细胞衰老及永生化

正常细胞体外培养时,表现为有限生长特性,经一定的细胞倍增次数后,失去了对促分裂因子刺激的反应,不可逆地失去增殖能力而停止分裂,细胞开始衰老的历程.目前认为染色体末端(端粒)的缺失会使细胞逐渐失去增殖能力,导致细胞的衰老和死亡.人端粒酶催化亚单位(hTERT)可以激活端粒酶的活性,延长染色体末端DNA,维持基因组的稳定.端粒、端粒酶、hTERT的发现为细胞衰老的研究提供了新的思路,同时也应用于永生化细胞系的建立,特别是在组织工程种子细胞生物性能研究和细胞库的建立中将发挥重要作用.     

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.