摘要:

Summary—In mammalian cells, the nucleoli disintegrate during mitosis and some nucleolar proteins disperse at the periphery of allchromosomes forming a novel class of chromosomal passenger proteins. The nucleolar components which participate in the formation of this perichromosomal layer have been investigated to elucidate the role of these perichromosomal proteins in the assembly and disassembly of the nucleoli. i) Electron microscopy immunolabelling reveals that these proteins are predominantly located in the granular component of the nucleoli during interphase,. ii) Immunoprecipitation data suggest that they are distributed at the chromosome periphery in association with U3 small nucleolar RNA (snoRNA). In addition, the distribution of U3 snoRNA visualized byin situhybridization, is similar to that observed for the perichromosomal proteins. iii) In cells which possess a nucleolar remnant during mitosis, U3 snoRNA and perichromosomal proteins were found both in the perichromosomal layer and in the nucleolar remnant. iv) Some of these proteins are conserved from yeast to man such as fibrillarin and a protein of 52 kDa. v) The location of these proteins observed in yeast by confocal microscopy shows that they are not dispersed during mitosis. Their partition between the two daughter cells is performed by scission of nucleolar structures forming a rod during the budding process. Therefore RNP complexes related to the processing steps of ribosome biogenesis in mammalian cells quit the nucleolus in late G2and associate with the chromosome periphery until late telophase. They associate in the perichromosomal layer in human and PtK1cells and both in the perichromosomal layer and the nucleolar remnant in CHO cell

ISSN:0248-4900    

DOI:10.1016/S0248-4900(94)80010-3

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Summary—From several recent studies on monopolar spindles, it is now clear that a phase analogous to metaphase in bipolar spindles exists in the monopolar spindle, denying the validity of the favored model for metaphase which is based on the balance between two oppositely directed poleward forces acting on the unsplit kinetochores. Faced with this new fact, several investigators have proposed new models for metaphase plate formation which work for both the monopolar and the bipolar spindles. Since astral microtubules are thought to play important roles in certain models, we have investigated the role of astral microtubules in maintaining chromosomes at the metaphase position by using monopolar spindles induced in sea urchin embryo cells. When monopolar spindles were exposed to 1 μM nocodazole, a microtubule depolymerizing agent, most of the astral microtubules were rapidly depolymerized while the kinetochore fibers appeared to be little affected. Chromosomes were locked into the metaphase position for as long as 2 min, indicating that the metaphase chromosome position in sea urchin monopolar spindles can be maintained with little or no astral microtubules. By the effect of the antimitotic drug, kinetochore fibers slowly shortened and chromosomes which were attached to the far end of kinetochore fibers moved toward the pole. On the other hand, the chromosome position rapidly shifted farther away from the pole when monopolar spindles were treated with taxol or D2O. These results indicate that the metaphase chromosome position can be altered by affecting microtubule dynamics, particularly that of the kinetochore fiber microtubules as suggested by the results of the nocodazole experimen

ISSN:0248-4900    

DOI:10.1016/S0248-4900(94)80011-1

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Summary—Fallopian tubes were obtained from 25 women undergoing abdominal hysterectomy. Pieces of fallopian tube mucosa were placed in culture flasks containing minimum essential medium in Earle's salts supplemented with fetal bovine serum. First passage was carried out after 7–10 days and subcultures in 4–5 days. For polarised cell culture, epithelial cells were seeded onto an extracellular matrix system. New epithelial cells were seen on day 2–3 of the primary culture and epithelial patches on day 7–10. Cells reached confluence in 4–5 days in subcultures. The cells could be subcultured for 7–11 passages with a life span of 42–60 days. Epithelial origins of the cells were confirmed by immunofluorescence staining with anti‐cytokeratin antibody. Polarised cells showed a columnar pattern, microvilli on their apical surface and basally located nucleus whereas non‐polarised cells were flat. It was concluded that the human fallopian tube epithelial cells can be culturedin vitroto create non‐polarised and polarised cell layers by using a simple and reproducible technique and this system can be a potential model to study function

ISSN:0248-4900    

DOI:10.1016/S0248-4900(94)80012-X

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Summary—We studied the response to differentin vitroculture conditions and the ability for polarization in three‐dimensional (3‐D) and two‐dimensional (2‐D) cell culture systems of the commonly used human bladder carcinoma cell line RT112. In the case of 2‐D culture the cells were grown on glass or plastic coverslips, filter membranes, or bovine lens capsules. The alginate culture technique (ACT) was used to test the effects of 3‐D cell culture on the polarization of the RT112 epithelial cells. Our studies show clear differences in the arrangement of the cells depending on the cultivation procedure. The RT112 cells cultured on glass and plastic supports were irregular and flattened in shape whereas the cells grown on filters or on lens capsules developed into 2–3 layers consisting of markedly polarized cells. However, ACT was superior to cell culture on either artificial supports or even on lens capsule. During the 3‐D cultivation the transformed epithelial cells regenerated multicellular spheroids which maintained a tissue‐like, geometrically well‐ordered and highly prismatic organization. This ACT‐induced morphogenesis is novel and distinct from that reported with conventional culture conditions. Microscopic investigations showed that RT112 cells grown in alginate were both much more tightly packed and more regularly organized compared to 2‐D cultures. In addition, the spheroidal organized cells exhibited well developed cell‐cell contacts, a distinct endoplasmatic reticulum, and a marked Golgi apparatus. In summary, ACT can be used for 3‐Din vitrogrowth of the transformed human epithelial cell line RT112 that offers substantial advantages over conv

ISSN:0248-4900    

DOI:10.1016/S0248-4900(94)80013-8

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Summary—The ciliature in most entodiniomorphid ciliates is restricted to the cell apex, forming ciliary crowns that can be rapidly retracted under stress conditions. In a previous study, calmyonemin, a 23 kDa analog of centrin, has been characterized from ciliated cortical zones inEudiplodinium maggii; its localization in subkinetal myonemes, ie bundles of contractile filaments powering retraction of the ciliature, was demonstrated by immunoelectron microscopy (David and Vigues (1994)Cell Motil Cytoskeleton, 27, 169–179). Here we used immunofluorescence microscopy to determine the distribution of calmyonemin and its variations throughout the cell cycle inEntodinium bursa. Labelling of morphostatic cells with anti‐calmyonemin antibody reveals immunoreactivity at the base of the ciliature, in circumciliary lips, around the cytoproct and in an endoplasmic array originating from the post‐oral region and extending down to the posterior part of the cell. All arrays of calmyonemin from the parental cell remain unaffected during cell division except the endoplasmic array which undergoes a transient resorption followed by synchronous reassembly in the two future products of division. The assembly of subkinetal myonemes in the sub‐equatorial zone is described in relation with the main features of oral morphogenesis previously revealed by others using silver impregnation techniques and light m

ISSN:0248-4900    

DOI:10.1016/S0248-4900(94)80014-6

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Summary—Immunological cross‐reactivity studies between the ApicomplexaBabesia divergensandPlasmodium falciparumallowed usto identify aP falciparum60 kDa protein (Pf60) using an antiserum directed against aB divergens37 kDa culture‐derived exoantigen. In immunofluorescence assays (IFA), Pf60 appears as a doublet of fluorescent spots associated to the apical pole of merozoites. The doublet co‐locates with two rhoptry components: the protein RAP‐1 and the 140/130/110 (105) kDa rhoptry protein complex suggesting the rhoptry location of Pf60. The biosynthesis of Pf60, established by labeling experiments with [35S]methionine on synchronized cultures, and by immunofluorescence detection, occurred during late schizogony. The physico‐chemical properties of Pf60, the absence of identified precursor forms and the absence of co‐precipitation with other proteins indicated a new class of rhoptry protein. Pf60 was detected in all the different geographicP falciparumstrains so far tested, with a slight variability in molecular mass ranging from 58 to 60 kDa. During the invasion process of erythrocytes by merozoites, the IFA showed the presence of the Pf60 in the apex of free merozoites, but not in invading merozoite, as well as in new ring‐infected erythrocytes. Furthermore, immunoprecipitation assays indicated the presence of Pf60 in the culture medium, and its absence in new ring‐infected erythrocytes. All together these results suggest a possible involvement of the Pf60 protein in the

ISSN:0248-4900    

DOI:10.1016/S0248-4900(94)80015-4

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Summary—Paramecium, like other ciliates, has a system of regulated secretion. The secretory storage granules, known as trichocysts, are of large size and elaborate architecture and occupy in wild type cells a significant fraction of the cellular volume. They thus provide an excellent model system for studies of secretory granule biogenesis. Previous analysis of secretory mutants unable to produce functional trichocysts (Gautier et al (1994).J Cell Biol124, 893–902) suggested that one of the mutants studied,trichless, might have defective organelle acidification. We have therefore applied a technique (Anderson et al (1984)Proc Natl Acad Sci USA81, 4838–4842) for the postembedding characterization of acidic compartments using the weak base 3‐(2,4‐dinitroanilino)‐3′‐amino‐N‐methyldipropylamine (DAMP) to Paramecium. Quantitative immunolocalization experiments using double label (antibodies against DAMP and against regulated secretory proteins) show that secretory granules are not significantly acidic at any stage of their development inParamecium. Analysis of trichless mutant cells shows that organelle acidification is not defective and allows us to follow the regulated secretory proteins from apparently normal immature secretory granules to a probable degradative compartment. Consideration of the ensemble of the results leads us to discuss the possibility that immature secretory granules are a sorting compartment inParamecium, as originally proposed by Kuliawat and Arvan ((1994)J Cell Biol126, 77–86) for the insulin producing β‐ce

ISSN:0248-4900    

DOI:10.1016/S0248-4900(94)80016-2

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Summary—In order to determine whether the human lactotransferrin receptor recently described on platelets was also present onhematopoietic precursors, we investigated its presence and characteristics on the megakaryocytic Dami cell line. The reversible binding of human 5‐([2‐(carbo(hydrazino)methyl]thio)acetyl)aminofluorescein‐labeled lactotransferrin showed that such a receptor was only present on the subpopulation of the largest cells. The increase in numbers of large cells during culture was paralleled by a concurrent increase in lactotransferrin receptor positive cells. Scatchard analysis of the binding of [125I]‐labeled lactotransferrin showed that a single affinity class of binding site was present (Kd= 446 ± 40 nM) and that there were 52 ± 3 × 105sites per cell. The mouse monoclonal antibody DP5B3G10, specific for the human lactotransferrin receptor, allowed its characterization as a 105 kDa protein on Western blots. The same monoclonal antibody was used to separate the small and large cell subpopulations of Dami cells by panning. Separate culture of the small cells showed that the receptor appeared prior to and independent from endomitosis. In contrast, GPIb was expressed only by large megakaryocytes. The use of conditioned medium from cultures of whole Darni cell populations indicated that a soluble factor is involved in differentiation, but not in the appearance of the lactotransfer

ISSN:0248-4900    

DOI:10.1016/S0248-4900(94)80017-0

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Summary—This paper reports a chemico‐enzymatic synthesis of β‐CD derivatives. The recognition properties of these derivativeswere tested using flocculating yeast and isolated lectins. It was observed that the substitution of β‐cyclodextrins with galactose end arms induces the better recognition by a cell‐linked galactose‐specific lectin. The physicochemical effects of the β‐CD derivatives on membranes were estimated using red blood cells and the effects on the viability of yeast and human rectal tumor cells were appreciated by measuring the mitochondrial deshydrogenase activity. The substitutions of the β‐CD ring by sugar antennae decrease the negative physicochemical effects of the β‐CD, ie their, hemolytic properties. However, these substitutions induce significant modifications of the biological properties of the molecules, particularly the cytotoxicity and the grow

ISSN:0248-4900    

DOI:10.1016/S0248-4900(94)80018-9

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY

摘要:

Summary—Prolactin (PRL) is a trophic hormone which acts mainly at the plasma membrane level of hepatocyte. The mechanismsinvolved in the transduction of the signal after binding of PRL to its receptors are not yet well documented. In the present study we have examined the subcellular patterns of PRL receptor expression in rat liver by ultrastructuralin situhybridization and immunocytology.In situhybridization was performed using digoxigenin‐labeled oligonucleotide probes revealed by indirect immunogold reaction. The expression of both the long and short forms of PRL‐receptor mRNA was readily identified in the cytoplasmic matrix, and in association with the endoplasmic reticulum, but a low expression of these forms was detected in the nucleus of hepatocyte. Moreover, this expression appeared clearly higher in female rather than in male hepatocytes. On the other hand, immunogold detection of PRL‐receptor protein was performed using two monoclonal antibodies (U5 and T6), specific to the extracellular domain of the PRL‐receptor. Indirect immunocytological detection confirmed the presence of PRL receptor‐like immunoreactivity at the level of the plasma membrane, and in the cytoplasmic matrix associated or not with endocytotic vesicles, the endoplasmic reticulum, the peroxisomes, the Golgi complex, and the nuclei of both male and female hepatocytes. No clear difference was found between U5 and T6 mAbs, with regard to the subcellular localization. These results show the distribution of both PRL‐receptor mRNA and PRL receptor protein in numerous subcellular compartments of hepatocyte, and evidence that these compartments are involved in the early stage of t

ISSN:0248-4900    

DOI:10.1016/S0248-4900(94)80019-7

出版商:Blackwell Publishing Ltd    

年代:1994

数据来源: WILEY