1,721,024 research outputs found
Redistribution of DNA topoisomerase II β after in vitro stabilization of human erythroleukemic nuclei by heat or Cu++ revealed by confocal microscopy
No full textUsing confocal laser scanning microscope and a monoclonal antibody we have examined by means of indirect immunofluorescence techniques the distribution of DNA topoisomerase IIβ (the 180-kDa nucleolar isoform of topoisomerase II) following stabilization of isolated nuclei by exposure to moderate heat (37°or 42°C) or Cu++. In intact cells the antibody specifically decorated the nucleoli. The same pattern was maintained if nuclei were incubated at 0°C in a buffer containing spermine/spermidine/KC1 or stabilized by means of 0.5 mM Cu++ for 10 minutes at 0°C in the same buffer. On the contrary, if stabilization was performed by incubating the nuclei either at 37°or 42°C, the immunoreactivity dispersed all over the nucleus, forming numerous speckles. This phenomenon was not detected if, in addition to spermine/spermidine/KC1, the incubation buffer also contained 5 mM Mg++ and the temperature was 37°C. If the stabilization was performed at 42°C, Mg++ failed to maintain the original distribution of DNA topoisomerase IIβ, as seen in intact cells. The analysis on 2-D optical section showed the alteration of the nucleolar profile, particularly at 37°C, even when the samples were treated with Mg++. The 3-D reconstruction figured out the irregularity of the surface at 37°C and the variations of the volume occupied by the fluorescent figures. These were in close proximity to each other both in intact cells and in 0°C incubated nuclei; they showed a certain degree of shrinkage in 0°C plus Cu++ exposed samples (-20% of the volume), and, on the contrary, the labeled structures were scattered in a volume increased two- or threefold when exposed to 37°or 42°C, respectively. The addition of Mg++ restored the original spatial relationship and volume at 37°C, but not at 42°C, where the volumetric analysis showed an increase of about 50%. Our results demonstrate that heat stabilization of isolated nuclei in a buffer without Mg++ (i.e., a technique often employed to prepare the nuclear matrix or scaffold) cannot be considered an optimal procedure to maintain the original distribution of protein within the nucleus
The use of in situ nick translation to relate DNA cleavage sites and chromatin rearrangement during apoptosis.
No full textImmunocytochemical detection of the specific association of different PIC isoforms with cytoskeletal and nuclear matrix compartments in PC12 cells.
No full textThe increasing evidence of discrete roles of phosphoinositidase C (PIC) isoforms and the assessment of their localization in the cytoskeleton and in the nucleus support the involvement of particular isotypes of this enzyme in signal transduction at multiple levels. PC12 rat pheochromocytoma is one of the few cell lines expressing three immunologically distinct isoforms of PIC. We have analyzed the subcellular distribution of the PIC beta 1, gamma 1 and delta 1 isoforms using confocal and electron microscope immunocytochemistry. PIC beta 1 is mainly found in the nucleus and is associated with interchromatin domains. On the other hand, the PIC gamma 1 isoform is found in the nucleus and in the cytosol, while PIC delta 1 is exclusively cytoplasmic. Immunoblot and immunocytochemical experiments indicate that the various PIC isoforms are differently bound to structural cell compartments, such as cytoskeletal and nuclear matrix elements. In fact, PIC beta 1 and PIC gamma 1 isoforms are tightly associated with the nuclear matrix, while only about 50\% of PIC gamma 1 is associated with the cytoskeleton after DNase I and high salt extractions. PIC gamma 1 is almost completely soluble under these conditions. These results further confirm the complexity of the inositide signal transduction mechanism, which involves several PIC isoforms, specifically localized in different cell compartments and support the existence of a membrane-unrelated inositol lipid-dependent signalling in the nuclear interior
Increase of nuclear phosphatidylinositol 4,5-bisphosphate and phospholipase C beta 1 is not associated to variations of protein kinase C in multidrug-resistant Saos-2 cells.
No full textThe multidrug resistance (MDR) phenotype that is mediated by an overexpression of P-glycoprotein, has been suggested to be related also to an increased activity of protein kinase C (PKC) and to changes in phospholipid pattern. By electron microscope quantitative immunocytochemistry, we investigated whether PKC and other elements of the polyphosphoinositide signal transduction system are affected in an MDR variant of the human osteosarcoma cell line Saos-2. These cells, which are characterized by an increased expression of P-glycoprotein not only at the plasma membrane but also at the nuclear level, showed increased intranuclear amounts of phosphatidylinositol 4,5-bisphosphate and of phospholipase C beta 1, while both the amount and activity of both nuclear and cellular PKC were not modified with respect to sensitive cells. These results suggest that, in this model, the changes observed in the elements of nuclear signal transduction could be related to previously reported modifications of the MDR phenotype, but that P-glycoprotein phosphorylation is not dependent from increased PKC activity
Interleukin-1alfa stimulates nuclear phosphoinositidase C activity in human osteosarcoma SaOS-2 cells.
No full textInterleukin-1alfa stimulates nuclear phosphoinositidase C activity in human osteosarcoma SaOS-2 cell
Interleukin-1-receptor-associated kinase 2 (IRAK2)-mediated interleukin-1-dependent nuclear factor kappa B transactivation in Saos2 cells requires the Akt/protein kinase B kinase
No full textThe post-receptor pathway that leads to nuclear factor kappaB (NF-kappaB) activation begins with the assembly of a membrane-proximal complex among the interleukin 1 (IL-1) receptors and the adaptor molecules, myeloid differentiation protein 88 (MyD88), IL-1-receptor-associated kinases (IRAKs) and tumour-necrosis-factor-receptor-associated factor 6. Eventually, phosphorylation of the inhibitor of NF-kappaB (IkappaB) by the IkappaB kinases releases NF-kappaB, which translocates to the nucleus and modulates gene expression. In this paper, we report that IRAK2 and MyD88, but not IRAK1, interact physically with Akt, as demonstrated by coimmunoprecipitation and pull-down experiments. Interestingly, the association of Akt with recombinant IRAK2 is decreased by stimulation with IL-1, and is favoured by pre-treatment with phosphatase. Likewise, Akt association with IRAK2 is increased considerably by overexpression of PTEN (phosphatase and tensin homologue deleted on chromosome 10), while it is completely abrogated by overexpression of phosphoinositide-dependent protein kinase 1. These data indicate that Akt takes part in the formation of the signalling complex that conveys the signal from the IL-1 receptors to NF-kappaB, a step that is much more membrane-proximal than was reported previously. We also demonstrate that Akt activity is necessary for IL-1-dependent NF-kappaB transactivation, since a kinase-defective mutant of Akt impairs IRAK2- and MyD88-dependent, but not IRAK1-dependent, NF-kappaB activity, as monitored by a gene reporter assay. Accordingly, IRAK2 failed to trigger inducible nitric oxide synthase and IL-1beta production in cells expressing dominant-negative Akt. However, NF-kappaB binding to DNA was not affected by inhibition of Akt, indicating that Akt regulates NF-kappaB at a level distinct from the dissociation of p65 from IkappaBalpha and its translocation to the nucleus, possibly involving phosphorylation of the p65 transactivation domain
3-D reconstruction of nuclear lamina shape
No full textThe localization of lamins A and C has been revealed by a FITC-conjugated monoclonal antibody in Swiss 3T3 fibroblasts by using confocal microscopy. 3-D reconstruction reveals that in these flattened cells, the nuclear lamina delimits coin-shaped nuclei.
The relationship between the lamina and the chromatin has been studied by double labelling with the anti-lamin antibody and propidium iodide
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