皮层蛋白介导斑马鱼(Danio rerio)原肠化细胞运动

在胚胎发育过程中, 细胞运动对指导原肠期胚胎细胞的时空定位并决定其发育命运具有核心作用, 然而活体状态下原肠化过程中细胞运动的调控机制目前并不清楚. 微丝结合蛋白皮层蛋白(cortactin)是微丝核化过程的重要调控分子, 它通过激活微丝相关蛋白2/3复合物(Arp2/3 complex)促进微丝在细胞前导缘区域迅速组装, 从而直接作用于细胞运动. 为阐明斑马鱼(Danio rerio)原肠化细胞运动的分子调控机制, 本研究首先检测了皮层蛋白在斑马鱼胚胎发育过程的表达水平. Western blotting分析证明皮层蛋白在斑马鱼原肠期胚胎中大量表达; 整装胚胎抗体染色结果表明在斑马鱼原肠化过程中, 皮层蛋白主要分布于胚胎背侧胚盾区域的细胞中, 在发生活跃运动的上皮层细胞和下皮层细胞中含量较高;在亚细胞水平, 皮层蛋白和Arp2/3复合物共同定位于运动的皮层区域, 并在细胞连接处也有大量分布. 此外, 研究还发现皮层蛋白在发育中的中枢神经系统中表达量较高. 本研究结果首次表明皮层蛋白和Arp2/3复合物介导的微丝聚合参予了斑马鱼原肠化细胞运动, 并在中枢神经系统发育中扮演重要角色.     

一例硬皮病人自发抗核仁抗体的免疫荧光及其与银染的比较研究

从硬皮病人血清中筛选出一例含有自发抗核仁抗体的血清,利用这个血清对核仁抗原的性质及其在细胞中随分裂周期不同产生的分布变化做了初步研究,并把结果与核仁嗜银蛋白做了比较。间接免疫荧光染色及细胞化学分析表明,这种核仁抗原的性质是蛋白质,其分布与嗜银蛋白相似,在间期,抗原呈颗粒状簇集在核仁中,而在分裂中期,抗原颗粒与染色体NORs部位接合,但有证据指出,这种抗原蛋白与核仁嗜银蛋白有所不同,同时还发现,经长时间秋水仙素处理诱导产生微核化的多核细胞中尽管微核的数目远多于细胞中NORs的数目,免疫荧光染色和银染都显示出每个微核中类核仁小体的存在。这说明(1)类核仁小体也是由核仁物质构成;(2)某些类核仁小体的产生可能与NORs无关。对这个现象的意义进行了讨论。     

热激蛋白HSP70在肿瘤免疫反应中作用的研究进展

热激蛋白（heat shock proteins,HSPs)是细胞受应激原刺激后诱导产生的一组应激蛋白,在进化上高度保守,热激蛋白的功能有很多,充当分子伴侣,参与蛋白质折叠和转运,最近研究表明HSP70还在肿瘤免疫反应过程中起协同作用,充当呈递载体,将抗原肽呈递于APC表面引起CTL毒性杀死感染细胞和肿瘤细胞,在肿瘤的免疫治疗中作为免疫疫苗的载体有很好的前景。     

白芷细胞外钙调素结合蛋白(ECBP21)免疫组化及金标定位分析

ECBP21是我们从白芷悬浮培养细胞外纯化及cDNA克隆的一种钙调素结合蛋白(CaMBP),亦是植物中首次报道的细胞外CaMBP。本实验以大肠杆菌表达的重组ECBP21蛋白为抗原,制备了高效价特异性抗体;随后,利用免疫组化及金标定位技术,研究了ECBP21蛋白组织特异性分布及亚细胞定位。免疫组化结果表明:ECBP21在白芷各组织中均有分布,但在叶、花、花序轴中较多,而在根中较少,并且ECBP21在细胞中多分布于细胞壁区域;免疫胶体金电镜定位结果显示:在白芷花序轴细胞中金颗粒主要分布在细胞壁,表明ECBP21蛋白主要定位于细胞壁区域,从而首次为细胞外CaMBP(ECBP21)的胞外存在提供了直观证据,并为进一步研究其在植物生长发育中的功能提供了初步信息。     

白芷细胞外钙调素结合蛋白（ECBP21）免疫组化及金标定位分析

ECBP21是我们从白芷悬浮培养细胞外纯化及cDNA克隆的一种钙调素结合蛋白(CaMBP),亦是植物中首次报道的细胞外CaMBP。本实验以大肠杆菌表达的重组ECBP21蛋白为抗原,制备了高效价特异性抗体;随后,利用免疫组化及金标定位技术,研究了ECBP21蛋白组织特异性分布及亚细胞定位。免疫组化结果表明：ECBP21在白芷各组织中均有分布,但在叶、花、花序轴中较多,而在根中较少,并且ECBP21在细胞中多分布于细胞壁区域;免疫胶体金电镜定位结果显示：在白芷花序轴细胞中金颗粒主要分布在细胞壁,表明ECBP21蛋白主要定位于细胞壁区域,从而首次为细胞外CaMBP(ECBP21)的胞外存在提供了直观证据,并为进一步研究其在植物生长发育中的功能提供了初步信息。     

克氏原螯虾消化道内分泌细胞的鉴别与定位

目的 对克氏原螯虾（Procambarus clarkii）消化道的嗜银细胞及5种内分泌细胞进行鉴别与定位。方法应用整块组织Grimelius银染法和过氧化物酶标记的链霉亲和素（SP法）免疫组织化学技术结合生物统计学分析。结果嗜银细胞在克氏原螫虾消化道除幽门胃外的各段均有分布,位于消化道上皮细胞间及结缔组织中。五羟色胺（5-HT）细胞在除幽门胃外的消化道各段均有分布。生长抑素（SS）细胞仅在食道和后肠中有分布。胃泌素（Gas）细胞分布于除幽门胃和中肠外的消化道各段。胰高血糖素（Glu）细胞在除幽门胃外的整个消化道均有分布,在食道和贲门胃中最多。胰多肽（PP）细胞仅在肠道中有较多分布。结论克氏原螫虾消化道中存在多种内分泌细胞,它们的分布情况与其它甲壳动物存在一定的共性,然而也有其一定的特异性。     

鲤、青鱼肠粘膜内分泌细胞的免疫组织化学鉴别和定位

使用过氧化物酶——抗过氧化物酶(PAP)的免疫组织化学染色技术,用10种哺乳动物激素培育出的抗血清对鲤、青鱼肠道粘膜中内分泌细胞的鉴別表明,它们的肠道粘膜上皮中存在有胃泌素、P物质、牛胰多肽、亮氨酸脑啡肽、胰高血糖素样免疫反应物、抑胃多肽等6种免疫活性內分泌细胞;而五羟色胺、胆囊收缩素和神经降压素没有免疫活性反应。在鲤的肠粘膜中存在生长抑素免疫活性内分泌细胞;青鱼中则未见到这种细胞。两种鱼的各种免疫活性內分泌细胞多数在前肠的分布密度较大;但青鱼肠粘膜中P物质和亮氨酸脑啡肽两种免疫活性内分泌细胞却在直肠中分布最多;胰高血糖素样免疫活性内分泌细胞在中肠分布最多。P物质和胃泌素免疫活性內分泌细胞大多数分布于肠褶顶部;其他各种免疫活性內分泌细胞则主要分布于肠褶的中、底部。本文比较了鲤、青鱼的肠内分泌细胞在各肠段中的分布密度,并对其形态学及分布特点进行描述和讨论。     

大鳞副泥鳅肠道黏液细胞及消化酶活性分布特征研究

采用阿利新兰-碘酸雪夫氏反应(AB-PAS)染色法及酶学方法研究了大鳞副泥鳅成熟个体肠道各段黏液细胞分布及消化酶活性。结果表明, 大鳞副泥鳅肠道黏液细胞分为Ⅰ、Ⅱ、Ⅲ和Ⅳ 4种类型。前肠至后肠, 黏液细胞数量逐渐减少。前肠主要分布Ⅲ和Ⅳ混合型黏液细胞, 后肠则以Ⅱ和Ⅳ型酸性黏液细胞为主。肠道胰蛋白酶活性显著高于淀粉酶和脂肪酶。且后肠消化酶活性显著低于前肠和中肠。根据黏液细胞及消化酶活性分布特点, 表明大鳞副泥鳅属于杂食性鱼类, 前肠为其主要的消化吸收场所, 后肠中性黏液细胞的数量较少以及消化酶活性较低, 表明其对食物的消化吸收功能较弱, 与其为辅助呼吸功能的特点相关。     

无花粉蛋白在体内定位的放射自显影研究

天花粉蛋白是从中草药发掘出来的一个植物蛋白性的中期引产和抗增生性滋养层细胞疾病的药物。在临床应用中已累积了大量经验。关于作用机理的研究也进行了各方面的工作(上海实验生物研究所第二研究室,1976)。在小白鼠以间接荧光标记抗体法,追踪了给药后不同时间,“纯制天花粉”抗原分布的动态。结果证明,天花粉蛋白的原     

嗜热四膜虫的着丝粒蛋白检查

目前国际上的着丝粒蛋白研究工作几乎全是以酵母和高等生物为材料进行的,为了从起源与进化的角度考察着丝粒蛋白。我们以人喉癌培养细胞ＨｅｐＩＩ作为对照材料,以两种ＡＣＡ血清和ＣＥＮＰ－Ｂ单抗,多抗以及ＣＨＯ动粒蛋白单抗为探针,用间接免疫荧光和免疫印迹技术对嗜热四膜虫作检查,免疫荧光结果表明,ＨｅｐＩＩ细胞的着丝粒抗原间期核中呈点状分布;与ＨｅｐＩＩ细胞的不同,嗜热四膜虫的着丝粒抗原在间期核中的分布不规则     

一组新的核仁组织者区结合蛋白

With the scleroderma autoantinucleolar antiserum which was discribed in our previous paper, we identified a novel group of nucleolar organizer associating proteins, ANOP. By means of immunoblotting, it was found that ANOP mainly consists of three polypeptides with molecular weights of 65, 76, and 78 KD respectively. Immunoelectron microscopy demonstrated that ANOP is localized in the shells of dense fibril components surrounding fibrillar centers of nucleolus. Moreover, indirect immunofluorescence studies on various cells of vertebrate animals indicated that ANOP is a group of highly conserved nucleolar antigens. The fibrillar centers of human, mouse, chinese hamster, chicken, frog and crucian carp cells can all be brightly stained by ANOP specific antiserum. However, it was found that in amphibian, the erythrocytes and the early embryo cells prior to gastrulation are devoid of ANOP. During gastrulation, ANOP begins to appear in the nuclei, and then, the content of ANOP in nucleoli apparently increases gradually. These phenomena suggest that ANOP may take a role in rRNA gene activity.     

Bismuth staining of a nucleolar protein

A major nucleolar protein in Chinese hamster ovary cells with a molecular weight (MW) of 100 kD has been found to stain selectively with the bismuth tartrate technique of Locke & Huie [19]. After glutaraldehyde fixation and bismuth staining of electrophoretic transfers of total nucleolar proteins separated by SDS-PAGE, a single band corresponding to the 100 kD protein is revealed. When the technique is applied to whole cells, small punctate regions of the nucleoli are strongly stained. At the ultrastructural level, bismuth selectively contrasts the fibrillar centers and the adjoining cords of the dense fibrillar component. The remainder of the dense fibrillar component is not stained. It is proposed that the high phosphorylation level of the 100 kD protein is responsible for its glutaraldehyde-insensitive bismuth staining. The concentration of this protein in certain localized regions of the nucleolus suggests that it plays a metabolic rather than a structural role.     

Increased functional load on mouse kidney proximal tubule epithelial cells causes changes in nucleolar 3-D architecture

Ultrastructural 3-D analysis of nucleolar architecture and Ag-NOR protein distribution in mouse kidney-cortex proximal-tubule epithelium has been performed. A principal scheme of structural changes of the nucleolus and organization of its components during the intensification of pre-rRNA synthesis (dynamic model of a nucleolus) based on computer spatial modelling has been advanced. According to the nucleolar composition, three groups of cells, which differ from each other by rRNA synthesis, are defined in normal kidney. Most nephron proximal-section cells (about 52%) are characterized by lower activity of RNA synthesis. Such kind of cells are defined as group I (nucleolar diameter 0.7–1.5 µm) and always contain resting, ring-shaped or close to ring-shaped dense nucleoli, which have 2 or 3 fibrillar centers. Nucleoli of group II cells (about 37%, nucleolar diameter 1.5–2.5 µm) have a higher level of activity, contain 4–7 fibrillar centers, and their structural organization is close to reticulated forms due to the first indications of vacuolar network (identified as prereticulated nucleoli). The most active cells of group III (about 11%, nucleolar diameter 2.5–3.5 µm) include cells with typical reticulated nucleoli with a well expressed vacuolar network and numerous fibrillar centers (18–22). Increased functional load of the epithelium caused by unilateral nephrectomy and diuretic (4-chlor-H [2-furylmethyl] 5-sulphamyl-antranic acid) injection changed the proportion of the different cell groups: group I decreased (about 25%), whereas groups II and III increased (about 8% and 17%, respectively). The increase of nucleolar activity first causes a deformation of the individual fibrillar centers as well as complication and growth of their surface. Further, a progressive fragmentation of the fibrillar centers and the growth of their total volume is observed. The complication and growth of the total volume of Ag-positive zones is another indication of the nucleolar activation. The vacuolar system develops by a gradual fusion of small isolated cavities into a united vacuolar network. Nucleoli with 2–7 fibrillar centers are considered to be intermediate forms reflecting successive stages of its activation or inactivation: from the resting ring-shaped nucleolus via transient stages of increasing functional activity to the active reticulated nucleoli and vice versa. The observed differences in the nucleolar ultrastructure are regarded as evidence of the functional heterogeneity of cell populations within one functional segment of nephron.     

Nucleolar Changes and Fibrillarin Redistribution Following Apatone Treatment of Human Bladder Carcinoma Cells

Ascorbate and menadione (Apatone) in a ratio of 100:1 kills tumor cells by autoschizis. In this study, vitamin-induced changes in nucleolar structure were evaluated as markers of autoschizis. Human bladder carcinoma (T24) cells were overlain with vitamins or with culture medium. Supernatants were removed at 1-hr intervals from 1 to 4 hr, and the cells were washed with PBS and prepared for assay. Apatone produced marked alterations in nucleolar structure including redistribution of nucleolar components, formation of ring-shaped nucleoli, condensation and increase of the proportion of perinucleolar chromatin, and the enlargement of nucleolar fibrillar centers. Immunogold labeling of the nucleolar rRNA revealed a granular localization in treated and sham-treated cells, and immunogold labeling of the rDNA revealed a shift from the fibrillar centers to the condensed perinucleolar chromatin. Fibrillarin staining shifted from the fibrillar centers and adjacent regions to a more homogeneous staining of the entire nucleolus and was consistent with the percentage of autoschizic cells detected by flow cytometry. Because autoschizis entails sequential reactivation of DNase I and DNase II, and because the fibrillarin redistribution following DNase I and Apatone treatment is identical, it appears that the nucleolar and fibrillarin changes are markers of autoschizis. (J Histochem Cytochem 58:635–651, 2010)     

Scattering of the silver-stained proteins of nucleolar organizer regions in Ishikawa cells by actinomycin D.

Scattering of the silver-stained proteins of nucleolar organizer regions (Ag-NOR proteins) was produced by actinomycin D in Ishikawa cells. Scattering of Ag-NOR proteins was found only in cells treated with actinomycin D and various other agents had no effect. Scattering was dose-dependent up to 10(-2) micrograms/ml of actinomycin D, but it was not found at higher concentrations that caused marked inhibition of total DNA and RNA synthesis. Actinomycin D (10(-2) micrograms/ml) caused the following changes: (i) nucleolar segregation and (ii) emergence of dense fibrillar bodies in the nucleoplasm. Ag-NOR proteins were observed on the fibrillar centers and surrounding fibrillar components in control nucleoli, on the fibrillar and amorphous zones in segregated nucleoli, and on the dense fibrillar bodies emerging in the nucleoplasm. The scattering of Ag-NOR proteins was due to the argyrophilic nature of the dense fibrillar bodies. Actinomycin D (10(-1) micrograms/ml) also caused similar morphological alterations in the nucleolus and nucleoplasm, but Ag-NOR proteins were observed only on nucleolar remnants.     

Fibrillarin: a new protein of the nucleolus identified by autoimmune sera

Autoimmune serum from a patient with scleroderma was shown by indirect immunofluorescence to label nucleoli in a variety of cells tested including: rat kangaroo PtK2, Xenopus A6, 3T3, HeLa, and human peripheral blood lymphocytes. Immunoblot analysis of nucleolar proteins with the scleroderma antibody resulted in the labeling of a single protein band of 34 kD molecular weight with a pI of 8.5. Electron microscopic immunocytochemistry demonstrated that the protein recognized by the scleroderma antiserum was localized exclusively in the fibrillar region of the nucleolus which included both dense fibrillar and fibrillar center regions. Therefore, we have named this protein "fibrillarin". Fibrillarin was found on putative chromosomal nucleolar organizer regions (NORs) in metaphase and anaphase, and during telophase fibrillarin was found to be an early marker for the site of formation of the newly forming nucleolus. Double label indirect immunofluorescence and immunoelectron microscopy on normal, actinomycin D-segregated, and DRB-treated nucleoli showed that fibrillarin and nucleolar protein B23 were predominantly localized to the fibrillar and granular regions of the nucleolus, respectively. RNase A and DNase I digestion of cells in situ demonstrated that fibrillarin was partially removed by RNase and completely removed by DNase. These results suggest that fibrillarin is a widely occurring basic nonhistone nucleolar protein whose location and nuclease sensitivity may indicate some structural and/or functional role in the rDNA-containing dense fibrillar and fibrillar center regions of the nucleolus.     

Does the synthesis of ribosomal RNA take place-within nucleolar fibrillar centers or dense fibrillar components?

By means of immunocytochemistry performed on cryosections of cultured cells, RNA polymerase I was localized mainly to nucleolar fibrillar centers. The labelling of nucleolar dense fibrillar components was low and depended on the cell type. In contrast, DNA topoisomerase I and RNP complexes containing U3 snRNA were enriched in dense fibrillar components, their occurrence in fibrillar centers being usually much less.     

A comparative study of the main components of the nucleolus in different populations of rat trophoblast cells during differentiation. II. The nucleoli of the trophospongium cells, the glycogen cells and the secondary giant cells

A comparative study was performed of the arrangement of different nucleolar components during differentiation of trophoblast cell populations in the junctional zone of placenta (glycogen cells and trophospongium) and in the secondary giant cells. Each cell type is characterized by specific interrelation of nucleolar components. Some glycogen cells show signs of segregation of nucleolar components: strands of nucleolar components with fibrillar centers (FCs) are displaced to the periphery of the nucleolus and contact with the perinucleolar chromatin. Large reticular nucleoli in trophospongium cells contain many FCs which are gathered into several "chains" by strands of dense fibrillar component. Such a "chain" has also been found in nucleoli of secondary giant cells, with greater number of FCs in each "chain". Relationship between the arrangement of nucleolar components and the level of cell differentiation is discussed.     

The relationships between ribosomal genes and fibrillar centers in thyroid cells cultivated in vitro

Despite the fact that the fibrillar centers of the nucleolus and the chromosomal nucleolar organisers (NORs) are similarly stained with the NOR-silver technique, there remain some questions about the identification of fibrillar centers as NORs. The distinct delineation of the fibrillar centers in porcine thyroid cells allowed us to determine whether there was a numerical equivalence or correlation between fibrillar centers and NORs. Hybridization in situ and silver staining performed on pig chromosomes showed that pairs 8 and 10 contained rDNA sites. Silver staining of thyroid cells in electron microscopy showed that the fibrillar centers and their surrounding layer of dense fibrils were the sites of silver deposit. Chromatin fibers were demonstrated within the fibrillar centers through the aid of the osmiumammine reaction and with the oxidized diaminobenzidine technique. It was observed that in cultured thyroid cells the fibrillar centers could be identified in the light microscope as argyrophilic spherules, and easily counted. The number of fibrillar centers was variable according to culture conditions. In cells cultured for 5 hr, the mean number of fibrillar centers was 1.7. After 5 days of culture, the number of fibrillar centers increased, reaching a mean value of 5.93. When thyroid cells were stimulated with thyrotropin, the number of fibrillar centers again increased to a mean value of 7.54. These results demonstrate that the relationship between fibrillar centers and NORs is not a simple proportionality: the number of fibrillar centers increases with increased cellular activity. These data imply that in active cells each NOR may pass through several fibrillar centers.     

3-dimensional organization of the nucleolus and the nucleolus-organizing regions in differentiated cells. I. Ring-shaped nucleoli in the endotheliocytes, smooth-muscle cells and fibroblasts in the mouse

The method of ultra-thin serial sections was used to study the three-dimensional structure and to perform the quantitative analysis of ring-shaped nucleoli of kidney and liver endotheliocytes, smooth muscle cells of kidney arterioles and fibroblasts of mice. Spatial models of ring-shaped nucleoli with one fibrillar centre are given. For the quantitative analysis the following parameters were measured: the number and volumes of nucleoli, fibrillar centers, RNP-containing structures, the vacuolar system and the RNP-index (the latter is a ratio of RNP-part and fibrillar center volumes). Nucleoli of the same type of cells, occasionally in the same nucleus, were found to differ sharply in their fibrillar center shape. Differences in the mean volume values of nucleoli, fibrillar centers and the RNP-part between some cell populations are sufficiently well pronounced. Within the same population ring-shaped nucleoli have, as a rule, specific volume values of nucleoli, RNP and fibrillar centers. The comparison of quantitative data obtained on different cell types showed that the mean RNP-index values were the most stable parameter. The structural relation between fibrillar centers, intra- and perinucleolar chromatin and lacunar region is shown. The structural organization of intranucleolar chromatin and rRNA in the nucleolar body and in fibrillar centers is discussed.