摘要:

Summary—To separate and concentrate various cytoplasmic organelles in wild type and albinoXenopusoocytes, defolliculated cells were loaded on a Ficoll‐400 gradient and centrifuged. Optimum results were obtained with centrifugations at 10 000gfor 5 min at 20°C. The cells became pear‐shaped and appeared stratified with the white lipid yolk on top, an intermediate transparent zone of about 100–300μm, and the greenish protein yolk at the bottom. To determine the cellular constituents, particularly of the transparent zone, electron microscopy was performed. The transparent zone was found to contain (from animal to vegetal) the various endoplasmic reticula, a layer of mitochondria, cytoplasm enriched in ribosomes and the depressed nucleus. In centrifuged stratified wild type oocytes, most of the pigment was layered on top of the protein yolk. The typical cortical aspects of the oocyte persisted. Centrifuged albino oocytes had a very pronounced transparent zone with sharp transitions to the lipid phase and to the protein yolk. The resting membrane potentials of centrifuged oocytes were between −35 and −65 mV, and the membrane resistances were in the 500 kΩ to 1 MΩ range. Under voltage clamp conditions, the oocytes exhibited Ca2+‐activated Cl−currents with biphasic kinetics and spontaneous oscillations of these currents. It is concluded that centrifuged stratified oocytes have normal electrophysiological properties, and that they are a suitable preparation to study the contribution of various cellular organelles to the propagation of second messen

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)89422-6

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—In order to determine the localization of actin, growing and fully grown rat oocytes were immunocytochemically examined using a post‐embedding ultrastructural protein‐A gold technique. In quiescent oocytes, the nucleoplasm showed slightly lower levels of actin signal when compared to the surrounding cytoplasm. The highest levels of labeling were found on nucleoli showing a reticular type morphology. In oocytes at the diakinesis stage in which nucleolar compaction had occurred, the levels of labeling increased by 5–6 times those found in quiescent oocytes. Except for conspicuous accumulation of actin under the plasma membrane, compact nucleoli had significantly higher levels of labeling when compared with those found on the general cytoplasm, while the nucleoplasm with homogeneously dispersed chromatin showed significantly lower levels of associated actin signal than the general cytoplasm. In oocytes at metaphase I, the cytoplasmic region had comparable or lower levels of labeling than the cytoplasm of oocytes at diakinesis. The meiotic spindle embedded in material with medium electron density showed a similar level of labeling as the surrounding cytoplasm. On the other hand, significantly higher levels of associated actin were observed on the chromosomes of metaphase I. The actin signals were dispersed over the chromosomes and not concentrated on a specific region. These results suggest that nuclear actin may be involved in the process of chromosome construction and also the formation of the compacted structure of the nu

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)89423-8

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—Recent evidence suggests that multiple pathways exist in PMN activation and that specific leukocyte response may be due to the activation of a particular signaling pathway. Using flow cytometry, PMN activation pathways were studied through the parallel comparison of n‐formyl‐Met‐Leu‐Phe (fMLP)‐ and phorbol‐12‐myristate‐13‐acetate (PMA)‐induced stimulation and by simultaneous assays for CD11b expression and morphology. The maximal CD11b expression was higher with PMA than with fMLP, suggesting different activation pathways. Under these experimental conditions, a morphological response to fMLP was not observed. However, significant shape change was detected in PMA treated samples and was suppressed by either the removal of extracellular calcium or staurosporine at the concentrations above 14.5μM. Calcium ionophore induced a similar light scattering pattern to that by PMA and enhanced CD11b expression, both of which were not inhibitable by staurosporine. These observations, for the first time, indicated that Ca2+was a mediator in activation processes and that the treatment of PMN with PMA

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)89424-X

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—Several studies have shown that kinases and phosphatases can interact with the centrosome during interphase and mitosis suggesting that centrosomal components might be the targets of these enzymes. The association of the cAMP‐dependent protein kinase type II and the mitotic kinase p34cdc2with centrosomes from human lymphoblast cells has previously been shown (Keryeret al, 1993,Exp Cell Res204, 230–240; Baillyet al, 1989,EMBO J8, 3985–3995). In this paper we demonstrate that isolated centrosomes are able to phosphorylate a few number of centrosomal proteins (Mr230–220000; 135000 and 50000) and also H1 histone. The phosphorylation of H1‐histone is cell cycle dependent and modulated by phosphatases. The use of kinase and phosphatase inhibitors and the addition of the catalytic subunit of cAMP‐dependent kinase or of cyclinB‐p34cdc2kinase showed that both kinases phosphorylate the same centrosomal substrates. In addition two centrosomal proteins (Mr100000 and 37000) were phosphorylated only by p34cdc2kinase. Although the low amount of centrosomal proteins precluded a full characterization of these substrates we discuss the identity of the major centrosomal phosphoproteins by comparison with proteins known to associate with microtubule‐organizing centres or mitotic spindles. Our results raise also the intriguing possibility that the cAMP‐dependent protein kinase could be regulated by the mitotic kinase at t

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)89425-1

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—Localization of p53 in human cultured lymphocytes and in cultured skin fibroblasts was studied by immuno‐fluorescent microscopy and post‐embedded immunoelectron microscopy using Lowicryl K4M. In quiescent lymphocytes, p53 was found in small amounts in both the cytoplasm and the nucleus. p53 in the nucleus was found associated with the non‐chromatin structure. At 24 h or 72 h of PHA stimulation, p53 increased markedly just beneath the plasma membrane and in the nucleus, which stained diffusely with anti‐p53. In resting fibroblasts, small amounts of p53 were present in both the cytoplasm and the nucleus. After 16 h of stimulation of confluent‐resting fibroblasts by trypsinization and replating, a phase just prior to the initiation of DNA synthesis, p53 slightly increased in both the cytoplasm and the nucleus. Afterwards, p53 was present predominantly in the cytoplasm, closely associated with the cytoskeletal actin filaments. In mitotic cells, p53 was distributed throughout the cytoplasm. When fibroblasts were extracted with saponin, p53 was still associated with the actin filaments, as well as mitochondrial membranes and granular structures of the nuclear matrix. Our data suggest that the initial increase of p53 in cells that enter the cell cycle through G1 first bind to the actin cytoskeleton, and that some of the p53 then move into the nucleus to initiate gene activation and DNA synthesis for cell proliferation. This implies that there is some functionally significant interaction between p53 and actin i

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)89426-3

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—The wild‐type human p53 tumor suppressor gene was tested for its ability to modulate cytotoxic activity ofin vitroactivated peripheral blood lymphocytes. Peripheral blood mononuclear cells (PBMCs) were stimulated by phytohemagglutinin (PHA), interferonα2b (IFNα2b), interleukin 2 (IL‐2) or their combinations to induce cytotoxicity. This stimulation significantly increased the percentage of cells expressing p53, which was at its maximum when induced by IL‐2 combined with IFNα2b. The role of p53 in the modulation of different aspects of cytotoxic activity of these cells was analyzed by studying the effects of p53 abrogation by antisense oligonucleotide (p53 AS) treatment in comparison with p53 sense or scrambled (missense) oligonucleotide (p53 S or p53 MS) treatment. We show that p53 plays a key role through induction of apoptosis in target cells (tumor necrosis factor pathway) rather than through osmolytic degeneration (perforin pathway) which is only slightly increased by p53 abrogation. Meanwhile,in vitroabrogation of p53 expression in PBL was found to be accompanied by an increase of CD8+ lymphocytes and an important increase of the CD56 ‘bright’ NK cell

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)89427-5

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—Thirty endometrial biopsies were cultured in order to separate stromal and epithelial cells. Using epidermal growth factor (EGF), cortisol, cholera toxin, insulin with 5% horse serum for epithelial cells or a medium with 20% fetal calf serum for stromal cells, we could specifically enrich endometrial culture in epithelial or stromal cells and culture them for 1 or 2 months. These cultures retained the phenotypic characteristics of epithelial (cytokeratins, mucin HMFG 1) and stromal (vimentin, smooth muscle actin, desmin) lineage by immunostaining analysis. Epithelial and stromal cultures from one individual were respectively immortalized by the SV 40 large T antigen. The immortalized cell lines kept the phenotype of the normal cells from which they derive

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)89428-7

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—Multidrug‐resistant (MDR) variants of a human osteosarcoma cell line (U‐2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P‐glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell mor

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)89429-9

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—All the ganglia belonging to the central nervous system of adults of the milkweed bugSpilostethus pandurus(Hemiptera) were screened immunohistochemically for vertebrate gastrin‐cholecystokinin (CCK‐8(s))‐like peptides. Several large reactive perikarya are present in the median part of the protocerebrum, their processes extending to the dorsal ‘aorta’. These cell bodies are also paraldehyde fuchsin‐positive,iethey are A‐type cells. In the lateral part of the protocerebrum, in the deutocerebrum and tritocerebrum, and in the suboesophageal, prothoracic and abdominal ganglia, a few immunoreactive cell bodies send axonal processes into their respective neuropiles. The A‐type cells reactive to CCK antiserum were identified, at the ultrastructural level, by combining paraldehyde fuschin staining of semithin sections with a post‐embedding immunogold technique carried out on adjacent ultrathin sections. The neurosecretory cells contain numerous vesicles of elevated electron density. These data suggest that members of the CCK peptide family are present in the central nervous system ofSp

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)89430-5

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

Summary—TGFβis supposed to play an important role in the process of epithelial maturation in the developing fetal lung. Using an immunofluorescence approach, we showed that fetal rat lung fibroblasts elaborate the three TGFβisoforms known in mammals (TGFβ1,β2 andβ3) whereas epithelial cells appear to synthesize only TGFβ1 andβ3. Isolated fibroblasts secrete the three isoforms. Biological assay of TGFβactivity in fibroblast‐conditioned media did not reveal significant changes according to the stage when fibroblasts we

ISSN:0248-4900    

DOI:10.1016/0248-4900(96)89431-7

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY