人类抗中心体自身抗体的研究

用间接免疫荧光法以小鼠腹水癌细胞为底物从硬皮病病人的自家抗血清中筛选出几个抗中心体的抗血清。因为中心体是化学构分复杂的细胞结构,而自家抗体又是多克隆的,故本文只用其中一个抗中心体抗血清作进一步研究。为了定位其抗原,同时也采用了Ｌ９２９培养细胞为底物。发现这抗血清结合于微管,有丝分裂期的纺锤体,中心体以及其它一些核结构。同时也用此抗血清在细胞裂解液的免疫印迹膜上检出了分子时与微管蛋白相同的主要条带,     

绿豆根尖细胞微管骨架有丝分裂时相发育变化的研究

提纯猪脑微管蛋白,制备兔抗微管蛋白抗血清,以此抗体与羊抗兔ｌｇＧ－ＦＩＴＣ因清,对绿豆根尖细胞进行间接免疫荧光标记和荧光显微镜检,得到了绿豆根尖细胞有丝分裂微管骨架周期发育变化的时相,如：早前期带,纺棰体微管,成膜体微管等,结果证明了双子叶植物具有与单子叶植物相似的细胞分裂微管周期时相,表明了微管架周期时相变化在高等植物中具有普遍性和共同变化的规律,讨论了微管骨架时相发育变化与染色有丝分裂行为的关     

慈姑根尖细胞微管骨架的免疫荧光观察

分别用考马斯亮蓝染色和间接免疫荧光标记,并运用荧光倒置显微镜和激光共聚焦显微镜,对慈姑根尖固定后酶解获得的去壁细胞和细胞团块以及根尖细胞分裂周期中微管骨架列阵进行详细观察,以探索高等植物微管周期的普遍性。结果表明:慈姑根尖固定后酶解可获得大量结构完整的去壁细胞与细胞团块;考马斯亮蓝染色观察可见,慈姑根尖细胞中丰富的蛋白物质以及处于不同分裂期的细胞核染色体;免疫荧光观察可见,慈姑根尖细胞周期中微管骨架保存较好,主要有周质微管、早前期带微管、纺缍体微管和成膜体微管4种循序变化的排列方式,构成了高等水生植物分裂细胞中典型的微管周期。实验结果证明,高等水生植物与陆生植物微管周期具有相似性,为植物微管周期概念提供了新的实例。     

核微管组织中心的研究

本文利用间接免疫荧光手段对间期核微管组织中心的性质进行了初步探讨。当分离核与6S微管蛋白保温后,用抗管蛋白抗体进行间接免疫荧光染色,在群体中只有极少数核上有微管生长。Triton X-100或溶血卵磷脂去膜核的实验证实能长微管的核是核膜缺失或破损核。用秋水仙素解聚核上长出的微管,可见核内荧光亮点数多于染色体数,说明核内微管组织中心数目多于着丝粒数,很可能包括着丝粒和一部分染色质颗粒。将秋水仙素和6S微管蛋白混合后与去膜核保温,结果6S微管蛋白与核内微管组织中心有亲和力。本文就这些结果进行了讨论。     

利用自身免疫病人血清鉴定新的中心体蛋白质组分

与其他亚细胞结构相比, 中心体由于其分离和纯化方面的困难使其蛋白质组学研究一直处于滞后状态. 通过免疫荧光证明特异性识别中心体的6名自身免疫病人的自发抗血清被用来鉴定它们相应的抗原即中心体蛋白. 利用细胞全蛋白的Western blot检测血清后, 从Western blot膜上每条单独条带上洗脱结合的抗体, 并确定产生中心体特异性荧光染色的抗体洗脱来源. 通过免疫沉淀获得并通过质谱鉴定该抗体相应的抗原蛋白, 共鉴定出6种中心体蛋白, 包括2种已知的中心体蛋白和4种未知定位或未见中心体定位报道的蛋白. 这些蛋白涉及细胞周期调控、信号转导通路、分子伴侣和代谢酶类, 反映了中心体功能的多样性.     

中心粒及其功能

在动物细胞中,普遍存在一种叫做中心体（ｃｅｎｔｒｏｓｏｍｅ）的结构。在细胞有丝分裂中,纺锤体微管在中心体的位置生长形成,在间期细胞中的大多数微管其负极也在中心体区由负极至正极向四周辐射,因此一般认为,中心体起到微管组织中心的作用。电镜观察显示,中心体...     

原生动物贻贝棘尾虫微管胞器的荧光标记与显示

采用FLUTAX直接荧光标记和抗α微管蛋白抗体的间接免疫荧光标记显示,原生动物贻贝棘尾虫(Stylonychia mytilus)细胞微管胞器由口围带、波动膜、额腹横棘毛、左右缘棘毛、背纤毛等纤毛器微管骨架、纤毛器基部附属微管和其他皮层微管骨架组成。纤毛器微管骨架和基部附属微管按皮层纤毛模式定位;皮层左、右侧微管带和领肋壁微管等其他皮层微管构成细胞特定位置的皮层微管骨架,并可能为具有背腹分化的腹毛目纤毛虫所特有,对维持细胞背腹面的形态、支持附近纤毛器(如左、右缘棘毛)的运动起作用。本文较完整地阐述了其细胞骨架的三维构形,对于深入了解纤毛虫细胞微管骨架的结构和分布特征,进一步揭示微管类胞器的功能是有意义的。     

培养的正常细胞和肿瘤细胞内微管变化的研究:1.正常细胞内微管的免疫荧光细胞化学观察

本工作用我们自制的兔抗管蛋白血清经过间接免疫荧光染色检查,效价达到1:32,显示出我国小儿包皮成纤维细胞(正常二倍体细胞)内微管的特异分布。我们观察到在间期细胞内的微管(CMTC)系由核附近的微管组织中心(MTOC)发出到达细胞的边沿,或终止于质膜下方,或弯曲沿细胞表面平行分布;胞质内的微管纤维有的紧绕核周,有的伸入细胞突起内与主轴方向平行排列。当细胞进入分裂期(M期)时,不仅CMTC解聚,同时有丝分裂器纺锤体微管出现,细胞从扁平形状变成圆的外形。当有丝分裂完成后,纺锤体微管荧光消失,CMTC又逐渐代替而出现,细胞也恢复成为扁平形。CMTC在早G_1期开始出现,此时的子细胞之间由于极间微管束的残迹而呈现出荧光染色的阳性反应,是为中体。用秋水仙胺(0.06微克/毫升)在37℃下处理培养细胞,2小时后,CMTC解聚,变为弥散于细胞质内的管蛋白荧光,细胞外形变成更近似圆形或不规则的外形。洗去秋水仙胺,细胞在37℃的新鲜培养基内保温1.6小时后,CMTC又可复现,同时细胞外形又恢复到处理前的成纤维细胞形状。细胞在低温(0—4℃)处理1小时后,CMTC消失;当细胞再放回到37℃下保温16分钟后,CMTC开始恢复,30分钟后完全恢复。本实验用未免疫的同一家兔血清做对照染色,结果为阴性。本工作改用冷氯仿:甲醇(2:1)液固定细胞,在染色和洗涤时采用非离子性去污剂Triton X-100处理法,微管的荧光染色效果好,背景的非特异荧光减少。     

粗茎鳞毛蕨原叶体细胞有丝分裂过程中微管列阵的变化

应用Steedman‘s wax切片法,间接免疫荧光标记技术和激光共聚焦扫描显微镜技术研究了粗茎鳞毛蕨（Dryopteris crassirhizoma Nakai)原叶体大液泡化细胞和分生组织细胞有丝分裂过程中微管列阵的变化。结果显示：应用高浓度的多聚甲醛（8%）可以很好地保持大液泡化细胞的结构和微管的抗原性。结果也显示Steedman‘s wax切片法和间接免疫荧光标记技术的优点;（1）避免在微管标记过程中酶解细胞壁;（2）在乙醇脱水过程中样品中叶绿素的自发荧光被减到最小;（3）能够详细观察到有丝分裂过程中微管骨架的变化。因此,这种方法可以被广泛用来调查简单植物体和复杂植物体中细胞的有丝分裂过程以及发育过程中微管骨架的变化。     

培养的正常细胞和肿瘤细胞内微管变化的研究——Ⅱ.肿瘤细胞内微管的免疫荧光细胞化学观察

本实验用管蛋白抗体间接免疫荧光细胞化学方法,观察了我国建株的人胃低分化粘液腺癌MGc 80-3,人胃腺癌SGC-7901,人鼻咽癌上皮样细胞CNE,人食管癌上皮细胞ECa-109,人肺鳞癌LTEP-78,人啼腺癌LTEP-a_1,人肺小细胞癌LTEP-p七株癌细胞和HeLa细胞,小鼠S_(180)-V肉瘤细胞的微管形态。与人的正常包皮成纤维细胞和食管上皮细胞内精细的CMTC结构对比,肿瘤细胞间期的胞质微管普遍有减少或缺如的现象。参考Brin-kley对微管免疫荧光染色图形的分型方法,我们将观察的各种微管染色图形归纳为四种类型,比较各种细胞群体内微管类型的分布。肿瘤细胞群体内多数为微管缺如型和稀疏型,未见典型的丰满型,而正常细胞群体内都是丰满型。同时,肿瘤细胞的MTOC区面积明显增大。分裂期的肿瘤细胞内,有丝分裂器纺锤体微管荧光形态与正常细胞的没有差别。本文对肿瘤细胞间期胞质微管减少和缺如以及MTOC区明显增大的现象及其可能的意义进行了讨论,认为这是癌变机制研究中值得深入探讨的重要课题之一。     

Electrophoretic and immunologic characterization of proteins of merozoites of Eimeria acervulina, E. maxima, E. necatrix, and E. tenella.

Merozoites of Eimeria acervulina, Eimeria maxima, Eimeria necatrix, and Eimeria tenella were compared by gel electrophoresis, western-blotting with chicken antiserum, indirect fluorescent antibody reactions, and antiserum neutralization. Merozoites from the 4 species had dissimilar patterns of proteins and antigens in soluble and membrane fractions. Coomassie blue staining of SDS-PAGE gels revealed 16-22 protein bands depending on the species of merozoite but only 3 bands per species in the membrane fractions. Homologous and heterologous antisera recognized 5-12 soluble fraction bands and 3-7 membrane fraction bands on immunoperoxidase-stained western blots, depending on the species. When antisera from infected chickens were used in an indirect fluorescent antibody reaction, the merozoites of E. tenella and E. necatrix had a strong reaction with homologous and heterologous antisera. Merozoites of E. acervulina and E. maxima reacted with homologous antisera but had a weak or no reaction with heterologous antisera. Chicken antiserum against E. tenella had no effect on the viability of E. tenella merozoites when they were inoculated into chicken embryos.     

Fibroblast-like cells from embryonic chick cornea, heart, and skin are antigenically distinct.

Populations of fibroblast-like cells from 14 day embryonic chick cornea, heart, and skin were grown in vitro as primary cultures and found to be antigenically distinct from one another. Corneal fibroblasts were obtained by dissection, whereas heart and skin fibroblast-like cells were separated from nonfibroblastic cell types by their rapid adhesion to substrata. Cultured cells were used as antigens in rabbits. Antisera were first absorbed against homogenates of embryonic chicks from which the homologous tissue was removed. Each such 1° absorbed antiserum then was absorbed against homogenates of the two respective heterologous fibroblast-like cell populations (2° and 3° absorptions). Resulting 3° absorbed antisera were tested for specificity by immunodiffusion, immune agglutination, immune cytotoxicity (trypan blue uptake and ⁵¹Cr release), and indirect immunofluorescence. Each 3° antiserum was judged tissue specific when it reacted only with the fibroblast-like cells of its own tissue, i.e., the homologous population. Unabsorbed antisera reacted with both homologous and heterologous fibroblast-like cells, as did 1° absorbed antisera. Absorption of 1° antisera with homogenates of the two heterologous fibroblast-like populations removed antibodies against the heterologous populations without significantly reducing the 3° antiserum titer against the homologous fibroblast cell type. Moreover, absorption of 1° antisera with each of the two heterologous fibroblast-like populations removed antibodies not removed by the other. Thus, the fibroblast-like cells from cornea, heart, and skin are antigenically different from one another in vitro. The stable antigenic differences detected may have arisen during the differentiation of these cells in vivo. Some of the tissue-specific antigens detected must occur on the cell surface.     

Comparative studies of antigens of erythrocytic and exoerythrocytic merozoites of Plasmodium lophurae: characterization of a surface glycoprotein from exoerythrocytic merozoites

Rabbits were immunized with merozoite-enriched preparations of erythrocytic and exoerythrocytic Plasmodium lophurae. The antisera were used to compare antigens of the two types of merozoites. The indirect immunofluorescent antibody test showed the presence of common antigens. The growth of exoerythrocytic parasites was inhibited by the homologous antiserum and to a lesser extent by the antiserum prepared against erythrocytic forms. Cultures of exoerythrocytic parasites as well as their normal host cells were labeled metabolically with 35S-methionine, tritiated proline and glucosamine. Nonidet P-40 extracts of labeled merozoite-enriched preparations, infected cells, and normal cells were immunoprecipitated with the two types of antisera and the immunoprecipitates were analyzed on polyacrylamide gels. The results showed that erythrocytic and exoerythrocytic merozoites have several common proteins. A major difference was a glycoprotein with an approximate molecular weight of 110,000 daltons. This glycoprotein was associated with the surface of exoerythrocytic merozoites and was not recognized by antibodies prepared against erythrocytic forms.     

Lipopolysaccharides as Determinants of Serological Variability in Pseudomonas corrugata

The variation in biochemical and serological features of 128 isolates of Pseudomonas corrugata has been studied with 56 isolates from Spain and 72 isolates from other countries. Isolates were analyzed with common diagnostic tests and with the AP150CHE system. Variability among isolates for some standard tests usually listed as positive or negative for this species, such as arginine dihydrolase and gelatin hydrolysis, lipase and lecithinase activities, pigment production, and wrinkled colony morphology, was observed. Three antisera were raised against the type strain and two Spanish isolates from tomato and pepper plants. Serological reactions were studied by indirect immunofluorescence and indirect enzyme-linked immunosorbent assay. Eighty-three isolates reacted with a single antiserum, 6 reacted with two antisera, and none reacted with three antisera. Thirty-nine isolates did not react with any of the three antisera. These results suggest that serology will not be a useful method for routine diagnosis of P. corrugata unless common antigens can be identified. Electrophoresis and immunoelectrotransfer were used to study the antigens involved. Each antiserum reacted with whole-cell lysates, giving two common bands for P. corrugata isolates and other Pseudomonas species and a ladder-like pattern characteristic of lipopolysaccharides (LPS). Common bands were not observed after proteinase K treatment. More than 10 LPS patterns were distinguished in 98 isolates after silver staining of polyacrylamide gels. There was no correlation between the geographical origin or host of the isolates and the LPS patterns. A correlation between LPS groups and serological reaction was observed.     

Development, Characterization, and Use of Monoclonal and Polyclonal Antibodies against the Myxosporean, Ceratomyxa shasta

Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.     

Immunological properties of gap junction protein from mouse liver

Hepatic gap junctions were purified as plaques from BALB/c mice and separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS). Antisera were raised in rabbits and rats against gap junction plaques as well as protein bands of the following apparent molecular weights: 44K to 49K ("dimer" proteins), 26K, and 21K. Using an enzyme immunoassay, we found that the reactivities of the different antisera towards gap junction plaques decreased in the following order: anti-plaque antisera, anti-26K antisera, anti-"dimer" protein antisera, and anti-21K antisera. The gap junction protein bands separated by SDS-polyacrylamide gel electrophoresis were transferred by blotting onto nitrocellulose paper and the immunological cross-reactivities were compared: the anti-26K antisera reated with the dimer protein bands and the 26K band but did not cross-react with the 21K protein band. The rabbit anti-21K antiserum reacted weakly with the 21K protein. The missing immunological cross-reaction of the 26K and the 21K protein band can be most easily explained if both proteins were independent of each other. No inhibition of metabolic cooperation between fibroblastoid mouse 3T6 cells was observed in the presence of Fab fragments prepared from rabbit antiplaque antiserum or from rabbit anti 26K antiserum. When the total proteins of plasma membranes from mouse liver were separated by SDS-polyacrylamide electrophoresis, only the 26K protein reacted with rabbit anti 26K antiserum. This result opens the possibility for direct quantitation of gap junction protein in tissues and cell fractions.     

Localization of Z-protein in isolated Z-disk sheets of chicken leg muscle

Immunoblotting studies with antisera against Z-protein, desmin, and alpha-actinin showed that Z-protein is clearly distinguishable from desmin and alpha-actinin. Z-protein is not a proteolytic product of another protein but is an intrinsic component of chicken breast muscle myofibrils. In these experiments, an SDS extract of intact muscle was first electrophoresed in a polyacrylamide gel, and then proteins were transferred to a nitrocellulose paper sheet. Detection of each protein on the sheet was made possible by the application of the indirect immunofluorescence technique with the respective antiserum. Immunofluorescence microscope studies using these antisera revealed that Z-protein has the same distribution as alpha-actinin in isolated Z- disk sheets. Anti-Z-protein antiserum and anti-alpha-actinin antiserum stained the interior of Z-disks. On the other hand, antiserum against desmin stained the periphery of Z-disks in isolated Z-disk sheets.     

An evaluation of herpes simplex virus antigenic markers in the study of established and developing cervical neoplasia

Previous reports from our laboratory have shown that antiserum to "pure" AG-e, a type-common HSV antigen, specifically stains atypical cervical cells in indirect immunofluorescence. These observations have been confirmed and extended. Antisera were prepared against the two protein components of pure AG-e, designated ICP 12 (M. W. = 140,000) and ICP 14 (M. W. = 130,000), and were purified to radiochemical homogeneity by SDS-acrylamide gel electrophoresis. The antisera reacted as well as antiserum to pure AG-e in immunofluorescence with HSV-2-(G)-infected cells, and their reactivity was adsorbed with pelleted HSV-2 (G) virions. Unlike antiserum to pure AG-e, the antisera to ICP 12 and ICP 14 were nonreactive in immunodiffusion, and only antiserum to ICP 12 showed complement fixation with soluble viral antigenic mixtures. Antisera to pure AG-e, ICP 12 and ICP 14 specifically stained exfoliated cervical cells from patients with herpetic cervicitis and atypical cells from patients with atypia, carcinoma in situ (CIS) or invasive cancer. However, both the number of patients with a positive response and the number of staining atypical cells were greater with antiserum to pure AG-e than with antisera to ICP 12 or ICP 14, suggesting that AG-e is a superior marker. Cells staining with antiserum to pure AG-e, individually identified, were classified as atypia (mild to marked), CIS or cancer. The ability of the antiserum to pure AG-e to identify atypical cervical cells was compared to cytopathologic screening in a blind study of 26 patients. A good correlation (80% to 93.8%) was observed, indicating that pure AG-e is a sensitive and specific marker for the identification of atypical cells.     

Relationship between unicellular cyanobacteria established by indirect immunofluorescence of intact cells

Antisera prepared against intact, viable cells were used to show the applicability of a serological approach to detect relationships between unicellular cyanobacteria. Antisera were raised against eight unicellular cyanobacteria and two chlorophycean unicellular organisms. The staining reactivity of each antiserum was tested by the fixed indirect immunofluorescence assay against the different organisms, and each organism was tested for its reactivity with all of the different antisera. Absorption of antisera with the appropriate heterologous antigens was used to further characterize the relationship betweenAnacystis nidulans andSynechococcus cedrorum, and also the relationship between two subcultures of an isolate distinguished by morphological features. Absorption of antiserum was also used for the removal of antibodies to contaminating bacteria. The approaches used are suggested as a useful tool for determining relationships between unicellular cyanobacteria.     

The identification of mammalian centrosomal antigens using human autoimmune anticentrosome antisera.

Human autoimmune sera were screened for the presence of anticentrosome autoantibodies. Two high titer sera were identified that reacted with HeLa, CHO, and PtK2 centrosomes by immunofluorescence, although the fluorescent patterns that were obtained using the two antisera were separate and distinct. Serum obtained from patient IJ contained antibodies that reacted with epitopes present only in mitotic centrosomes; staining of interphase centrosomes was never detected uing IJ antiserum. Immunoblot analysis demonstrated that antibodies present in IJ antiserum reacted with a 190 kD spindle pole antigen. Immunofluorescent staining of cultured mammalian cells demonstrated that antibodies present in serum obtained from patient SPJ reacted with both interphase and mitotic centrosomes. Characterization of SPJ antiserum by immunoblotting demonstrated that antibodies present in the SPJ serum recognized proteins of Mrs of 39, 185, and 220 kD, although the possibility that the 185 kD polypeptide was a proteolytic breakdown product of the 220 kD protein has not been eliminated. Neither antiserum was able to inhibit microtubule nucleation from centrosomes in a lysed cell system in which pure 6S tubulin was added to permeabilized cells following pretreatment of the cells with either SPJ or IJ antiserum. These antisera should be useful probes for studying the biochemistry of the mammalian centrosome.