TMEM16A钙激活氯离子通道在Fisher大鼠甲状腺滤泡上皮细胞的表达及其电生理特性研究

 目的：探讨跨膜蛋白16A（TMEM16A）钙激活氯离子通道在Fisher大鼠甲状腺滤泡上皮（FRT）细胞的表达及其电生理特性。方法：构建pUB6/V5-TMEM16A真核表达载体。脂质体方法转染TMEM16A至FRT细胞,同时优化脂质体和载体的量和比例,获得最佳转染效率和表达效果,杀稻瘟菌素（blasticidin）进行抗生素筛选,获取稳定表达TMEM16A的FRT细胞株。RT-PCR和免疫荧光检测TMEM16A于FRT细胞的表达情况;倒置荧光显微镜下观察TMEM16A在FRT细胞中的表达和定位;应用全细胞膜片钳技术和卤族元素敏感的荧光蛋白YFP-H148Q/I152L检测TMEM16A钙激活氯离子通道的功能。结果：BamHⅠ和XbaⅠ双酶切的琼脂糖凝胶电泳和测序结果表明目的基因TMEM16A成功克隆到真核表达载体pUB6/V5中;RT-PCR和免疫荧光实验结果表明经杀稻瘟菌素筛选后的FRT细胞在mRNA和蛋白水平表达TMEM16A,倒置显微镜下观察结果表明TMEM16A在FRT细胞膜上有表达;应用全细胞膜片钳技术和卤族元素敏感的荧光蛋白YFP-H148Q/I152L证实稳定表达于FRT细胞的TMEM16A具有经典的钙激活氯离子通道特性。结论：FRT细胞可高效表达TMEM16A。TMEM16A是钙激活氯离子通道的分子基础。     

基于钙激活氯离子通道检测胞质内第二信使Ca2+

目的 建立一种基于钙激活氯离子通道（CaCC）可敏感检测胞质内第二信使Ca²⁺的细胞模型。 方法 构建氯离子通道蛋白1（ANO1）和YFP-H148Q/I152 L真核表达载体,应用脂质体转染法构建共表达ANO1和YFP-H148Q/I152 L的FRT细胞,倒置荧光显微镜观察其表达情况,流式细胞仪检测细胞纯度;应用膜片钳技术研究CaCC生理特性;荧光淬灭动力学实验验证细胞模型的有效性;荧光淬灭动力学实验验证细胞模型可筛选CaCC调节剂;荧光探针法检测加入CaCC激活剂后胞质内的Ca²⁺浓度。 结果 倒置荧光显微镜下观察到ANO1表达在细胞膜上,YFP-H148Q/I152 L表达于胞质中;该模型具有经典的钙激活氯离子通道的生理特性;成功构建共表达ANO1和YFP-H148Q/I152 L的FRT细胞模型;该模型可筛选CaCC调节剂,荧光变化斜率值与CaCC调节剂浓度成剂量依赖关系;荧光变化斜率值可反映胞质内Ca²⁺浓度,该模型可敏感检测胞质内Ca²⁺浓度。 结论 此细胞模型可以高效敏感检测胞质内第二信使Ca²⁺浓度,为Ca²⁺信号相关靶点的研究提供了一种简便快捷的方法。     

稳定表达UT-A2的FRT细胞系的建立与鉴定

目的：建立稳定表达尿素通道蛋白A2（UT—A2）的FRT细胞系，为寻找UT—A2抑制剂提供细胞模型。方法：通过真核表达质粒-脂质体介导的方法，将UT—A2 cDNA与真核表达载体pUB6／V5连接后的重组质粒转染入FRT细胞，经稳定筛选建立稳定表达UT—A2的FRT细胞系。功能检测实验将细胞分为对照组和稳定转染组，用2mol／L尿素负荷试验检测稳定表达UT—A2的FRT细胞膜的尿素通透性。结果：经BSD筛选21d后得到稳定表达UT—A2的细胞株；Western blotting证实UT—A2蛋白稳定表达；免疫荧光分析结果提示UT—A2的质膜定位。2mol／L尿素试验证实了该细胞系具有明显尿素通透性。结论：在非肾脏上皮细胞获得了稳定质膜定位表达UT—A2的FRT细胞株，该细胞株可用于UT—A2抑制剂的筛选。     

钙激活氯通道ANO1在小鼠心肌细胞的表达及其功能鉴定

目的:探讨钙激活氯通道蛋白anoctamin 1(ANO1)在小鼠原代培养心肌细胞中的表达及其功能特性。方法:采用胰酶与胶原酶共同消化,联合2次差速贴壁法获得C57BL/6小鼠原代心肌细胞;并用免疫荧光染色法检测α-横纹肌肌动蛋白,以鉴定心肌细胞纯度;应用RT-PCR检测小鼠心肌细胞ANO1 mRNA的表达;免疫印迹检测ANO1蛋白在小鼠心肌细胞的表达情况;应用荧光淬灭动力学实验检测ANO1钙激活氯通道的功能特性。结果:RT-PCR结果表明原代培养的小鼠心肌细胞表达ANO1 mRNA。免疫印迹实验结果显示原代培养的小鼠心肌细胞表达ANO1蛋白。荧光淬灭动力学实验证实表达于小鼠心肌细胞的ANO1具有钙激活氯通道典型的阴离子转运功能特性。结论:ANO1在小鼠心肌细胞中有明确表达,并具有钙激活氯离子通道特性,提示ANO1是钙激活氯通道的分子基础。     

乙醇激活的鼻咽癌细胞氯电流的分子机制

 目的：研究乙醇对低分化鼻咽癌CNE-2Z细胞氯通道的影响并探讨其分子机制。方法：MTT检测乙醇对细胞生长的影响;全细胞膜片钳技术记录乙醇对氯电流的影响;应用氯通道阻断剂分析该电流的特性;siRNA干扰技术分析乙醇敏感氯通道的分子本质。结果：在等渗灌流液中记录到微弱的背景氯电流,随灌流液中乙醇浓度（0.17~170 mmol/L）的升高,所激活的氯电流呈倒U型曲线状,17 mmol/L的乙醇激活电流达到峰值,电流呈较明显的外向优势,能被高渗液及氯通道阻断剂5-nitro-2-(3-phenylpropylamino)benzoic acid（NPPB）所阻断;干扰ClC-3氯通道基因表达后乙醇激活的氯电流明显减小。结论：乙醇可能通过激活CNE-2Z细胞的ClC-3氯通道,诱导氯电流的产生。     

原代培养猪冠脉平滑肌细胞的电压依赖和钙激活的钾通道

作者应用改良的细胞膜片钳制技术对原代培养的猪冠状动脉平滑肌细胞的电压依赖性和钙激活的钾通道的特性进行了研究。整个实验均在计算机的控制下进行,各种膜片中引导出的电流,通过膜片钳制放大器后,均输入至计算机进行采样、存贮和分析。结果显示:①这种通道活动呈明显的电压依赖性,随膜的去极化而激活,电流-电压关系     

蟾蜍灵激活低分化鼻咽癌细胞氯通道

目的: 研究蟾蜍灵(bufalin)对低分化鼻咽癌(CNE-2Z)细胞氯通道的激活作用以及通道的特性。方法: 采用全细胞膜片钳技术记录蟾蜍灵激活CNE-2Z细胞膜电流并分析其电流特征。结果: 细胞外灌流1 μmol/L 的蟾蜍灵可诱发CNE-2Z细胞产生一个氯电流,该电流潜伏期较长,为(12.1 ± 6.4)min, 其翻转电位接近氯离子平衡电位。该电流具有较明显的外向优势,没有明显的时间依赖性失活和电压依赖性失活。氯通道阻断剂他莫昔芬(tamoxifen)可完全抑制该电流, 细胞外灌流高渗液也可完全抑制该电流。结论: 蟾蜍灵可以激活CNE-2Z细胞氯通道产生氯电流,与容积激活性氯电流相比,该电流的潜伏期较长,并且有更明显的外向优势。     

顺铂激活的低分化鼻咽癌细胞氯电流

目的： 研究抗癌药顺铂对低分化鼻咽癌细胞（CNE-2Z）氯通道的激活作用以及该电流的特性。方法：采用膜片钳技术记录顺铂激活的CNE-2Z细胞全细胞电流;离子置换法等方法分析通道的特性。结果：细胞外灌流5 μmol/L的顺铂诱发CNE-2Z细胞产生一个有明显外向优势的电流,电流的翻转电位为(-6.69 ± 0.51) mV,接近氯离子平衡电位(-0.9 mV)。该电流的激活依赖于细胞内ATP。顺铂激活的细胞膜氯通道对阴离子的通透性顺序为：I-≥Br->Cl->gluconate。氯通道阻断剂tamoxifen完全抑制该电流。结论：抗癌药顺铂可以激活一个电流特征与容积激活性氯通道相似的氯通道。     

敲低IK1钾通道抑制ClC-3氯通道的表达和功能

 目的: 探讨ClC-3氯通道是否为IK1钾通道的调节靶点,重点研究鼻咽癌细胞IK1钾通道对ClC-3氯通道功能及蛋白表达的影响。方法: 采用siRNA转染技术抑制低分化鼻咽癌上皮细胞(CNE-2Z) IK1 基因的表达;real-time PCR技术检测ClC-3 mRNA的表达;Western blot检测ClC-3的蛋白表达;细胞免疫荧光结合激光共聚焦显微镜技术检测ClC-3和IK1蛋白在细胞内分布;全细胞膜片钳记录细胞氯电流。结果: IK1 siRNA可以成功转染CNE-2Z细胞,有效抑制鼻咽癌细胞IK1钾离子通道的表达;用IK1 siRNA抑制鼻咽癌细胞IK1钾离子通道的表达后, ClC-3的mRNA表达上调而ClC-3蛋白却表达减少:在低分化鼻咽癌上皮细胞,低渗刺激可激活氯通道,产生一个较大的氯电流,在成功转染IK1 siRNA的细胞,此氯电流明显减弱。结论: 敲低IK1钾离子通道可抑制ClC-3氯离子通道的表达和功能。     

CⅡTA基因编码区不同单倍型cDNA的功能研究

目的 研究MHCⅡ类反式激活因子(cⅡTA)基因编码区非同义单核苷酸多态性(SNP)位点C19170G(Leu45Val)和C30799G(Ala500Gly)构成的4种不同单倍型cDNA的功能.方法 将4种不同单倍型的真核表达载体和卒载体分别转染至HeLa细胞.用RT-PCR和间接细胞免疫荧光技术检测未经转染的HeLa细胞、转染4种真核表达载体及卒载体的HeLa细胞cⅡTA mRNA与3种HLAⅡ类分子(HLA-DR、DP、DQ)的表达,并用流式细胞技术对其表达的3种HLAⅡ类蛋白进行定量分析.结果 未经转染和转染空载体的HeLa细胞均尤CⅡTA mRNA和3种HLAⅡ类分子的表达,而转染4种不同单倍型真核表达载体的HeLa细胞均出现CⅡTA mRNA表达,并表达3种HLAⅡ类分子.证实了转染4种不同单倍型真核表达载体后的HeLa细胞3种HLAⅡ类分子表达水平差异无统计学意义(P均>0.05).结论 中国人CⅡTA基因编码区这两个SNP位点的多态性(2个位点氨基酸的改变)不影响CⅡTA反式激活HLAⅡ类基因表达的能力.     

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 universal muscular chain reaction,muscle spasm,Torsions, ruptures and extravasations. Chameleons of pathology and some manifestations of simple muscular disorders

Most bodily functions require the coordinated actions of complementary and supplementary paired muscle groups. Where this essential muscular cooperation is lacking, hollow organs may burst and others become literally screwed up, giving rise to many similar spastic diseases such as Torticollis, Twisted ovarian cyst, Torsion of the Testis, Volvulus of the intestines, Varicose Veins, Megacolon, Aortamegaly, Scoliosis, Erb's Palsy, Peyronie's Disease, Main-en-Griffe, Undescended Foot (Pes Cavus), Talipes, Strabismus. Spasm is “panenepidemic” and unclassified examples of Torsion Dystonia and Dyskinesia really are as common as debt and taxes.     

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.     

Hypo- und Hypermagnesämie aus kardiologischer Sicht

Zusammenfassung Eine Reihe pathologischer Zustände bedingen Magnesiummangel. Zustände mit Hypermagnesämie sind ebenfalls bekannt, doch wesentlich seltener. Für den Kardiologen beachtenswert ist, daß unter Therapie mit bestimmten Diuretica bei Herzinsuffizienz, bei Herzinfarkt, Kardiomyopathie, Digitalisintoxikation und bestimmten Herzrhythmusstörungen Hypomagnesämie beobachtet wurde. Leider kann in der klinischen Routine nur ein extracelluläres Magnesiumdefizit durch Serumbestimmungen gemessen werden; über Magnesiummangel einzelner Organe kann nichts ausgesagt werden. Hinweise für Magnesiummangel geben aber neben der Messung des Serumspiegels Anamnese, klinischer Befund, bestimmte EKG-Veränderungen wie auch evtl. Hypokalämie, ein Zustand, bei dem sich oft — besonders bei Aldosteronismus — parallele Veränderungen zeigten.Tierexperimente deuten darauf hin, daß infarktähnliche Läsionen unter Magnesiummangel entstehen, doch ob Herzinfarkt beim Menschen durch Magnesiummangel ausgelöst werden kann, ist noch ungeklärt. In Leichenherzen zeigte sich im Infarktgebiet neben Calciumakkumulation signifikanter Magnesiumverlust, wobei unklar blieb, ob sich Ursache oder Folge des Infarktes widerspiegelten. Falls ein ursächlicher Zusammenhang besteht, ist er im Myokardstoffwechsel selbst zu suchen, wie bei der Alkoholkardiomyopathie, wo myokardialer Magnesiummangel zumindest als pathogenetischer Teilfaktor anerkannt wird. Andererseits versucht man aber auch Beziehungen zwischen Atherosklerose, Blutgerinnung und Hypomagnesämie herzustellen, in der Meinung, daß Magnesiummangel auch über den coronaren Pathomechanismus des Herzinfarktes wirken könnte. Sicher scheint, daß gewisse EKG-Veränderungen und Herzrhythmusstörungen durch einen irritierten Magnesiumhaushalt bedingt sein können, da sie bei Gabe bzw. Entzug von Magnesium verschwinden. Daß Magnesiummangel die Glykosidtoleranz verringert, wird tierexperimentell bestätigt. Unter Hypomagnesämie bewirkt Acetylstrophanthidin eher und länger Rhythmusstörungen als ohne, außerdem lassen diese sich durch Magnesiumgaben eliminieren. Da in gewissen Fällen spontane und digitalisinduzierte Herzrythmusstörungen durch Magnesiuminjektionen beseitigt wurden, scheint Magnesium als Therapeuticum angebracht. Einsatz verschiedener Magnesiumsalze bei Angina pectoris, degenerativen Herzerkrankungen und Herzinsuffizienz ohne geprüften und offensichtlich gestörten Magnesiumhaushalt ist fragwürdig, weil keine eindeutigen klinischen Erfolgsbeweise vorliegen. Immerhin mag es aber larvierte, durch Serumbestimmungen nicht erfaßbare Mangelzustände geben. Allgemein erscheint es aus kardiologischer Sicht ratsam, den Magnesiumhaushalt zu überwachen und in entsprechenden Fällen auszugleichen, um möglichen Myokardläsionen oder fatalen Herzrhythmusstörungen entgegenzuwirken.     

Environmental risk factors and their role in the management of atopic dermatitis

Introduction: The etiology of atopic dermatitis (AD) is multifactorial with interaction between genetics, immune and environmental factors.

Areas covered: We review the role of prenatal exposures, irritants and pruritogens, pathogens, climate factors, including temperature, humidity, ultraviolet radiation, outdoor and indoor air pollutants, tobacco smoke exposure, water hardness, urban vs. rural living, diet, breastfeeding, probiotics and prebiotics on AD.

Expert commentary: The increased global prevalence of AD cannot be attributed to genetics alone, suggesting that evolving environmental exposures may trigger and/or flare disease in predisposed individuals. There is a complex interplay between different environmental factors, including individual use of personal care products and exposure to climate, pollution, food and other exogenous factors. Understanding these complex risk factors is crucial to developing targeted interventions to prevent the disease in millions. Moreover, patients require counseling on optimal regimens for minimization of exposure to irritants and pruritogens and other harmful exposures.     

'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 genes in Amerindians from Mexico San Vicente Tancuayalab Teenek/Huastecos

Huastecos or Teenek Amerindians are presently living at North East Mexico (San Luis Potosi State). They have probably one of the most ancient culture of Mexico and Central America together with Mayas and Olmec groups with which also show close relationships. Proximity to Atlantic Ocean/Mexican Gulf originated that Spaniards had very early contact with them at about 1519 CE or before. In the present paper we have aimed to study HLA gene profile which may be useful for HLA and disease epidemiology and transplant programs in Teeneks. HLA-DRB1*04:07, -DRB1*14:06 and -DRB1*04:11 have been found in high frequency like in other Amerindian groups. High frequency typical Amerindians HLA extended haplotypes have been found, such as A*02-B*35-DRB1*04:07-DQB1*03:02; A*68-B*39-DRB1*04:07-DQB1*03:02 and A*02-B*39-DRB1*04:07-DQB1*03:02; also new haplotypes have been described, like A*02-B*52-DRB1*04:11-DQB1*03:02, A*68-B*35-DRB1*14:02-DQB1*03:01 and A*68-B*40-DRB1*16:02-DQB1*03:01. Genetic proximity is observed not only to linguistically close Mayans, but also to Mazatecans, Mixtecans and Zapotecans, who speak an altogether different languages; it shows once more that genes and languages do not correlate. This population was greatly diminished after European contact between 1500 and 1600 years CE; in fact, North and South America First Inhabitants population was brought from 80 down to 8 million people because of diseases (i.e.: measles, smallpox or influenza), slavery and war.     

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.