人过敏毒素C5a反义肽与其正义肽的相互作用研究

目的　证实过敏毒素C5a与其反义肽分子间是否确实存在选择性相互识别作用 ,进一步以反义肽分子设计的思路 ,寻找过敏毒素C5a拮抗反义多肽的依据。方法　选择人过敏毒素C5a与其受体 (C5aR)相互作用的两个功能区域 ,按照反义肽分子设计的模型 ,分别设计合成其对应的反义肽 ,并利用生物分子相互作用分析 (BIA)技术 ,对C5a正、反义间相互作用进行分析。结果　在设计合成的 4条反义肽中 ,有一条反义多肽与全长C5a以及其对应的正义肽分子间均存在选择性相互作用 ,其解离平衡常数值分别为 6 .6 2× 10 - 6 M、4 .5 0 8× 10 - 6 M。结论　过敏毒素C5a及其反义肽分子间确实存在选择性相互作用 ,从反义肽的思路寻找C5a生物学效应的拮抗多肽 ,进行新药的设计和开发是可能的。     

光学生物传感器在研究正、反义肽间选择性相互作用中的应用

目的　观察生物分子间相互作用动力学、亲和力及特异性 ,研究C5a与其反义肽的相互作用规律。方法　应用生物分子相互作用实时分析系统IAsysPlus,以反义肽R4为固定相 ,L2和rhC5a为流动相 ,采用FASTplot软件对选择结果优化的反应模式 ,计算各动力学参数值。结果　反义肽R4与配体rhC5a相互结合的KD=6 .6 2× 1 0 -6mol,直线在Y轴上的截距(解离常数 )Kdiss为 0 .0 2 2 5s-1 ;与L2的KD=4 .5 0 8× 1 0 -6mol。结论　生物传感器技术可以用于正义 反义肽之间相互作用的研究中 ,且反义肽R4可与其正义肽、与rhC5a发生特异性相互作用     

研究C5a反义肽的理论价值和实际意义

存在于蛋白质中某些相邻片段的微小表面之间的疏水作用力可以稳定天然蛋白质的构象 ,它也是小肽与蛋白质特异性结合过程中的主要驱动力。在正义DNA链上编码亲水性氨基酸残基的密码子 ,其同一阅读框内所对应的反义DNA链上大多会编码疏水性氨基酸 ,反之亦然。Mekler Biro Blalock模型 (MBB)认为正义 反义肽的相互作用是根据Kyte&Doolittle的疏水 亲水复合指数的疏水性反互补机制 (hydrophobicanticomplementarity)而实现的。在离体和在体实验中 ,都存在有正义 反义多肽发生相互作用的现象 ,如 :一氧化氮合成酶 (nitricoxidesynthase ,NOS)的钙调蛋白结构域的反义肽 ,0 .0 1～ 1 .0mmol L的浓度就能够抑制结构型和诱导型NOS ,其IC(50 )值分别为 98mmol L和 56mmol L ;内皮素受体 (endothelinreceptor ,ETR A)一个片段的反义肽ETR P1 f可抑制内皮素诱导的颈动脉和股动脉血管收缩 ,1 .0 μmol LETR P f反义肽将明显抑制ET 1的功能活性。 0 .2 5μmol L的人C5a反义肽可以保护小鼠免受rh C5a的攻击 ,单独使用rh C5a会使小鼠发生中毒反应 ,血细胞减少     

用生物传感器测定重组人α2a-干扰素单克隆抗体的亲和力及抗原识别表位

目的 :测定抗重组人α2a 干扰素单克隆抗体的抗原识别表位及其亲和常数。方法 :借助于生物传感器技术进行抗原抗体相互作用的动力学分析。结果 :抗体的亲和常数介于 10 -5～ 10 -7之间 ,5株抗体识别干扰素分子上 4种不同的抗原表位。结论 :生物传感器在研究抗原抗体相互作用中确为一个理想的工具 ,适合于抗原抗体结合与解离的动力学分析及预测表位的构像关系     

B7反义肽抑制同种异基因移植排斥反应的研究

目的 :探讨B7反义肽竞争抑制CD2 8 B7共刺激通路、抑制移植排斥反应的作用 ,为该反义肽在器官移植方面的应用提供实验依据。方法 :固相合成法合成含有MYPPPYmotif的B7反义肽N’ EFMYPPPYLD C’ ,纯度 95 % ,分子量 (MW)为12 71 6 ;用灭活的C5 7(H 2 d)脾细胞和体外培养新生C5 7心肌细胞作为刺激细胞 ,以适当浓度的B7反义肽处理刺激细胞后 ,再与BALB C(H 2 d)脾细胞作为应答细胞进行混合培养 ,观察此反义肽在体外抑制淋巴细胞增殖的效果 ;用此反义肽预封闭新生C5 7小鼠心肌后 ,移植到BALB C小鼠耳后 ,观察此反义肽在体内抑制移植排斥反应、延长心肌移植存活的情况。结果 :B7反义肽能抑制前述两种淋巴细胞增殖反应 ,抑制率分别为 38 4 %和 36 6 % ;并呈现出明显的剂量依赖关系 ,此反义肽预处理过的新生C5 7小鼠心肌 ,移植到BALB C小鼠 ,可较对照组平均延长 5 4天 (n =6 ,P <0 0 0 1)。结论 :人工合成的B7反义肽在体外可以通过抑制CD2 8 B7共刺激通路 ,抑制淋巴细胞的增殖 ,并可用于延长心肌移植物存活 ,为进一步研究其在移植免疫方面的应用提供了有益的启示。     

人C5a B细胞表位的设计与其单克隆抗体的结合

我们在分子设计的基础上 ,采用B细胞优势表位预测并结合实测的方法 ,设计合成人C5a的B细胞表位多肽 ,以此为抗原按常规方法免疫BALB c小鼠 ,制作表位多肽单克隆抗体 ,在传统ELISA鉴定的基础上 ,利用生物传感器分析方法 ,分别研究单克隆抗体与表位多肽和全长C5a分子的结合动力学规律。材料和方法软件 :GOLDKEY核酸蛋白质分析软件 (军事医学科学院吴加金等 )和PC Gene软件系统 (BairochA等编制 ,UniversityofGeneva ,Switzerland)。生物相互作用实时分析系统IAsysPlus(美国 ) ;CMD(羧甲基葡聚糖生物传感片 ,Thermo公司 )。主要…     

生物传感器对抗戊型肝炎病毒单克隆抗体部分特性的研究

目的　应用生物传感器对新制备的抗戊型肝炎病毒单克隆抗体 (mAb)的亲和力、Ig亚类 (型 )及抗原结合的动力学进行研究。方法　用HEVORF2区基因工程重组蛋白NE2 ,免疫BALB/c小鼠 ,经杂交瘤技术制备mAb ,采用ELISA、Westernblot和生物传感器鉴定其有关特性。结果获得 12株可稳定分泌抗NE2mAb的杂交瘤细胞系。用ELISA及生物传感器等鉴定各株mAb ,分别为IgM和IgG1、IgG2a,轻链均为κ型。其中在用ELISA法对mAb3F5亚类鉴定过程中 ,发现其与HRP GAMIgG2a和HRP GAMIgM均有反应 ,生物传感器鉴定其为IgM。Westernblot验证各株mAb的特异性 ,同时各株mAb对于不同聚合形式的NE2蛋白的反应性有一定的差别。应用生物传感器测定了 4株mAb的KD 值 ,mAb 8C11为 4 .36× 10 7,mAb8H3为 1.5 3× 10 5,mAb 13D8为 1.2 1× 10 6,mAb 16D7为8.0 3× 10 7。从生物传感器测得的各株mAb与NE2的结合、解离过程中发现 ,mAb 8H3与NE2可快速结合、快速解离 ,其它 3株mAb结合稳定。结论　经多种方法鉴定 ,所获得的 12株杂交瘤细胞分泌的抗体 ,为抗HEVORF2区段的特异性抗体     

小鼠抗人C5aR短肽（9—30）单克隆抗体制备及鉴定

目的获得有生物功能的小鼠抗人 C5 a R短肽单克隆抗体。方法对 C5 a受体 (CD88)二级结构和 B细胞表位进行分析研究 ,采用 Fmoc方案固相合成 C5 a R N-端第 9～ 30位氨基酸残基的 2 2 -肽 ,以此为抗原免疫 Balb/ c小鼠。结果建立了 1株小鼠抗人 C5 a R短肽杂交瘤细胞系 E3,其平均染色体数目为 10 2条 ,所分泌抗体为 Ig G1,κ型 ,腹水抗体效价为 1×10 - 4 ～ 1× 10 - 6 ,可识别 U 937、人脐静脉内皮细胞 (VEC)和 PMN等表达 C5 a R细胞。 E3单抗的亲和常数 Ka=2 .5× 10 5 ,其结合表位为 C5 a R第 15～ 2 1位氨基酸基序 D1 5 DKDTL2 0 D。结论 B细胞表位多肽具有免疫原性 ,可制作单克隆抗体 ,为 C5 a-C5 a R相互作用的研究以及 AL I、ARDS等 C5 a相关疾病的研究提供实验材料。     

从噬菌体随机肽库中筛选拮抗IL-5的抑制多肽

目的　通过噬菌体随机肽库筛选与白细胞介素 5 (IL 5 )高亲和力结合的相关多肽 ,观察这些多肽是否能够抑制IL 5的生物功能。方法　以重组人IL 5作为筛选分子 ,应用M13噬菌体PⅢ呈现随机 7肽库进行筛选 ,经ELISA进一步鉴定噬菌体克隆与IL 5的亲和力。阳性噬菌体克隆呈现多肽和人工合成多肽对IL 5介导的嗜酸细胞 (Eos)存活的影响。结果　经过 3轮淘筛后 ,经鉴定得到9个阳性噬菌体克隆能与IL 5呈高亲和力结合 ,其中 3个具有抑制IL 5介导的Eos存活延长作用。选择抑制作用最强的呈现多肽进行人工合成 ,发现其中一个合成多肽能够诱导Eos凋亡 ,抑制IL 5介导的存活延长作用 ,但作用强度不如噬菌体呈现多肽。结论　通过噬菌体肽库能够筛选到抑制IL 5功能的相关多肽 ,为进一步研究抗IL 5的分子药物奠定了基础。     

NF-κB p65亚基DNA结合域相互作用多肽的筛选及功能鉴定

目的以NF-κB p65亚基DNA结合域为诱饵蛋白,筛选能够与其发生相互作用的多肽,并鉴定这些多肽对NF-κB DNA结合活性的抑制效应。方法首先利用PCR搭桥扩增方法获取NF-κB p65亚基DNA结合域的基因片段,后将其克隆到酵母双杂交pGBKT7DNA-BD载体上,与GAL4DNA-BD融合,形成GAL4DNA-BD/p65BD融合蛋白;再以GAL4DNA-BD/p65BD融合蛋白为诱饵蛋白行酵母双杂交实验,自16肽随机肽库中筛选与NF-κB p65亚基DNA结合域相互作用的多肽;最后选择性人工合成筛选获得的多肽,并进行EMSA实验检测相互作用多肽对NF-κB DNA结合活性的抑制效应。结果扩增获得了NF-κB p65亚基DNA结合域的基因片段,并成功构建pGBKT7DNA-BD/p65BD载体。自1.28×107个酵母转化子中最终筛选获得5个阳性克隆,测序结果和同源氨基酸序列比对表明5个阳性多肽为p65BD新型相互作用多肽,其中4条多肽拥有一个共同的基序。EMSA实验结果表明合成的两条多肽对NF-κB的DNA结合活性均具有一定的抑制效应。结论经初筛、复选和假阳性鉴定,自16肽库中获得5个能与NF-κB p65亚基发生相互作用的新型多肽,并已证实其中两条多肽对NF-κB的DNA结合活性具有抑制效应。     

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.