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  • 1
    Electronic Resource
    Electronic Resource
    Nuclear matrix-bound replicational sites detected in situ by 5-bromodeoxyuridine (1992)
    Springer
    Histochemistry and cell biology 98 (1992), S. 19-32 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The nuclear matrix was prepared in situ from Swiss 3T3 cells, which were synchronized by contact inhibition and serum starvation and pulse-labelled for very short periods of time with 5-bromodeoxyuridine (5-BrdU). For the first time 5-BrdU has been employed to demonstrate the association of newly synthesized DNA with a nucleoskeleton. Immunofluorescence analysis using a monoclonal antibody to 5-BrdU revealed five different intranuclear staining patterns at different stages of the S phase. These patterns were observed also in intact cells and did not change during the matrix preparation steps which involve extraction with 2M NaCl and DNase I digestion. Such an observation was also confirmed by spatial confocal microscopy studies. The intensity of lfuorescence, which was evaluated by cytofluorometry, increased to reach a maximum during mid-S phase and then decreased. Because no significant difference was found in the time to label residual DNA of different 5-BrdU staining patterns, this strongly suggests that a different number of replicons is activated at different stages of the S phase. These results strengthen the hypothesis that eukaryotic DNA replication occurs in close association with an insoluble protein nuclear skeleton, which determines the three-dimensional spatial organization of chromosome duplication.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00716934
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  • 2
    Electronic Resource
    Electronic Resource
    A combined ultrastructural approach to the study of nuclear matrix thermal stabilization (1992)
    Springer
    Histochemistry and cell biology 98 (1992), S. 121-129 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37° C of isolated nuclei. If purified nuclei were heated for 45 min at 37° C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-ammine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00717003
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  • 3
    Electronic Resource
    Electronic Resource
    Immunocytochemical evaluation of protein kinase C translocation to the inner nuclear matrix in 3T3 mouse fibroblasts after IGF-I treatment (1995)
    Springer
    Histochemistry and cell biology 103 (1995), S. 447-457 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The complex pathway which links the agonist-cell membrane receptor binding to the response at the genome level involves, among other elements, protein kinase C (PKC). Agonists acting at the cell membrane can affect an autonomous nuclear polyphosphoinositide signaling system inducing an activation of nuclear phosphoinositidase activity and a subsequent translocation of PKC to the nuclear region. The fine localization of PKC has been investigated by means of electron microscopy quantitative immunogold labeling in 3T3 mouse fibroblasts, mitogenically stimulated by IGF-I. The enzyme, which in untreated cells is present in the cytoplasm, except for the organelles, and in the nucleoplasm, after IGF-I treatment is reduced in the cytoplasm and almost doubled in the nucleus. The PKC isoform translocated to the nucleus is the α isozyme, which is found not only associated with the nuclear envelope but mainly with the interchromatin domains. By using in situ matrix preparations, PKC appears to be retained at the nuclear matrix level, both at the nuclear lamina and at the inner nuclear matrix, suggesting a direct involvement in the phosphorylation of nuclear proteins which are responsible for the regulation of DNA replication.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01457544
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  • 4
    Electronic Resource
    Electronic Resource
    The effect of sodium tetrathionate stabilization on the distribution of three nuclear matrix proteins in human K562 erythroleukemia cells (1995)
    Springer
    Histochemistry and cell biology 104 (1995), S. 29-36 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Using both conventional fluorescence and confocal laser scanning microscopy we have investigated wheter or not stabilization of isolated human erythroleukemic nuclei with sodium tetrathionate can maintain in the nuclear matrix the same spatial distribution of three polypeptides (Mr 160 kDa and 125 kDa, previously shown to be components of the internal nuclear matrix plus the 180-kDa nucleolar isoform of DNA topoisomerase II) as seen in permeabilized cells. The incubation of isolated nuclei in the presence of 2 mM sodium tetrathionate was performed at 0° C or 37° C. The matrix fraction retained 20–40% of nuclear protein, depending on the temperature at which the chemical stabilization was executed. Western blot analysis revealed that the proteins studied were completely retained in the high-salt resistant matrix. Indirect immunofluorescence experiments showed that the distribution of the three antigens in the final matrix closely resembled that detected in permeabilized cells, particularly when the stabilization was performed at 37° C. This conclusion was also strengthened by analysis of cells, isolated nuclei and the nuclear matrix by means of confocal laser scanning microscopy. We conclude that sodium tetrathionate stabilization of isolated nuclei does not alter the spatial distribution of some nuclear matrix proteins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01464783
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  • 5
    Electronic Resource
    Electronic Resource
    The nuclear matrix and apoptosis (1997)
    Springer
    Histochemistry and cell biology 108 (1997), S. 1-10 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Apoptosis is a form of active cell death, genetically encoded, that plays a key role during several physiological and pathological conditions. During the apoptotic process, striking morphological and biochemical changes take place in the cell nucleus. However, the molecular mechanisms underlying these changes have escaped clarification for many years. Recently, attention has been devoted to identifying the modifications that occur during apoptosis in the nuclear matrix, a mainly proteinaceous framework structure which is thought to play a fundamental role in organizing nuclear structure and function. In this review, we focus our attention on the biochemical and morphological changes detected in the nuclear matrix during the apoptotic process. Particular emphasis will be placed on the proteolysis that some nuclear matrix proteins undergo early during the apoptotic process, as well as on the detachment of DNA loops from the matrix by the action of endonuclease(s). Future research in this field may provide important information about the principal mechanisms that cause nuclear destruction in apoptotic cells.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004180050140
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  • 6
    Electronic Resource
    Electronic Resource
    Changes in the subnuclear distribution of two RNA metabolism-related proteins can be detected in nuclear scaffold or matrix prepared by different techniques (1997)
    Springer
    Histochemistry and cell biology 108 (1997), S. 525-536 
    Details
    ISSN: 1432-119X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The nuclear scaffold or matrix is a mainly proteinaceous structure thought to act as a nucleoskeleton determining the higher order organization of eukaryotic chromatin. These structures are prepared from isolated nuclei by a series of extraction steps involving the use of ionic detergents or high salt, and restriction enzymes or non-specific nucleases to remove chromatin and other loosely bound components. Since these treatments are harsh and unphysiological, the question remains open as to whether or not these structures, isolated in vitro, correspond to a nucleoskeleton existing in vivo. Recently, it has been demonstrated that the majority of nuclear matrix proteins are involved in RNA metabolism. In this study we have employed a morphological approach involving the use of confocal laser scanning microscopy and indirect immunofluorescence techniques to analyze whether two widely employed methods to prepare the nuclear scaffold or matrix can maintain the spatial distribution of two polypeptides involved in RNA metabolism, i.e., a 105-kDa component of spliceosomes and a ribonucleoprotein antigen. We demonstrate that the localization of these polypeptides changes, in some cases dramatically, in the final nucleoskeletal structures when compared with intact cells. Only when isolated nuclei were stabilized in vitro with the cross-linking agent sodium tetrathionate (NaTT) prior to extraction with 2 M NaCl and DNase I digestion, were the immunofluorescent patterns displayed by the nuclear matrix indistinguishable from those detected in intact cells. These results emphasize the usefulness of NaTT in studying putative nucleoskeletal structures, but also show that the methods currently employed to prepare the nuclear scaffold or matrix may create in vitro artifacts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004180050193
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  • 7
    Electronic Resource
    Electronic Resource
    Phospholipase C digestion induces the removal of nuclear RNA: A cytochemical quantitative study (1989)
    Springer
    Journal of molecular histology 21 (1989), S. 491-500 
    Details
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary It has been reported that the incubation of isolated rat liver nuclear matrices with phospholipase C causes the digestion of the matrix-bound phospholipids and the release of most matrix-linked RNAs (Coccoet al., 1980). In this paper, the presence of phospholipids in nuclear substructures and the effects of their removal by phospholipase C digestion have been investigated by means of enzyme—colloidal gold cytochemistry. The nuclear phospholipids appear to be localized in the interchromatin areas and in the nucleolus and are virtually absent in the heterochromatin, when labelled with phospholipase C—colloidal gold. The double labelling test with ribonuclease A and phospholipase C conjugated with gold particles of different diameters shows that the nuclear phospholipids are co-localized with RNA-containing structures. The enzymatic digestion of phospholipids on thin sections of either isolated nuclei or pancreas embedded in LR White resin results in the decrease of the RNase-A colloidal gold labelling of nuclear RNA-containing structures, but not of the rough endoplasmic reticulum. The data confirm the presence of phospholipids in the nucleus in the absence of possible translocation due to isolation procedures and strengthen the hypothesis that they are involved in interactions between nucleic acids and proteins of the nuclear matrix.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01845799
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