Localization of single copy gene by PRINS technique.
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Base Sequence↗
Biomedical subjects
N M Maraldi
Publications and source records attributed to N M Maraldi.
Phosphoinositidase C isoforms are specifically localized in the nuclear matrix and cytoskeleton of Swiss 3T3 cells.
The fine subcellular localization of different phosphoinositidase C (PIC) isoforms has been determined by both electron microscope immunocytochemistry and immunoblotting in whole Swiss 3T3 cells as well as in subcellular fractions. PIC-beta, whose signaling activity has been recently demonstrated at the nuclear level (M. A. Martelli, R. S. Gilmour, V. Bertagnolo, L. M. Neri, L. Manzoli, and L. Cocco, Nature, 358, 242-244, 1992), is mainly localized in the interchromatin domains, while it is almost absent from the cytoplasm. PIC-gamma is almost absent from the nucleus and resides in the cytosol, while PIC-delta is undetectable in these cells. PIC-beta is retained in the inner nuclear matrix and lacks the nuclear pore-lamina complex, whereas PIC-gamma is preferentially associated with cytoskeletal filaments. Moreover, PIC-beta is present at the same sites of the nuclear matrix where phospholipids and protein kinase C can be identified. This indicates that some elements of the phosphoinositide signal transduction system are located inside the nucleus. Moreover, PIC-gamma association with the cytoskeleton filaments suggests a possible involvement of this enzyme in cytoskeleton-mediated changes of cell shape.
3T3 Cells↗
Nuclear phosphoinositidase C during growth factor stimulation.
The existence of phosphoinositidase C beta is demonstrated in the nucleus of Swiss 3T3 cells. Moreover, we show that this isoform is specific for the nucleus while the gamma isoform is confined to the cytoplasm of these cells. When Swiss 3T3 fibroblasts are treated with Insulin-like Growth Factor I a rapid and transient activation of the beta isoform occurs at the nucleus whilst the cytoplasmic phosphoinositidase C is unaffected. These results seem to explain the mechanism by which the decrease in the mass of polyphosphoinositol lipids occurs in the nucleus after stimulation with Insulin-like Growth Factor I and actually point out the existence of a nuclear polyphosphoinositide signalling system distinct from the plasma membrane localized system and constituted not only by the previously demonstrated lipid kinases but also by a specific phosphoinositidase C.
3T3 Cells↗
Phosphoinositidase C isozymes in SaOS-2 cells: immunocytochemical detection in nuclear and cytoplasmic compartments.
SaOS-2 cell line presents osteoblastic characteristics which can be modulated by specific agonists involving also phosphoinositide breakdown. In order to determine whether SaOS-2 cells display a phosphoinositide signalling system not only at the cytosol-cell membrane level but also, as recently reported for other cell lines, at the nuclear level, a study has been performed to evaluate the phosphoinositidase C (PIC) activity and to localize different isoforms of PIC in nuclear and cytoplasmic compartments. By immunochemicals methods, and by confocal and electron microscope immunocytochemistry, both PIC beta 1 and gamma 1 have been detected in the nucleus, while only PIC gamma 1 was found in the cytoplasm. A specific association with the inner nuclear matrix has been demonstrated for PIC beta 1 and gamma 1; this latter resulted, on the other hand, in relationship with cytoskeletal filaments after high salt extraction. These findings suggest that these enzymes are not completely soluble but functionally related with cytoskeletal and nucleoskeletal structures.
Cell Line↗
Decrease in nuclear phospholipids associated with DNA replication.
Lipid metabolism in nuclei is very active and appears involved in the transduction of signals to the genome in response to agonists acting at the plasma membrane level. However, the precise topology of nuclear lipid metabolism and the relationship between nuclear lipids and crucial events of the cell function, such as DNA replication, have not been fully elucidated. By using a recently developed cytochemical method for detecting phospholipids inside the nucleus of intact cells at the electron microscope level, we have analyzed the changes in intranuclear phospholipids in DNA-replicating versus resting cells, which are both present in the same sample of regenerating liver after partial hepatectomy. The pattern of DNA synthesis in replicating cells has been monitored by electron microscope immunocytochemistry after bromodeoxyuridine (BrdU) labeling. The data obtained, which allow a fine localization and a quantitative analysis of both DNA synthesis and phospholipid distribution, indicate a significant reduction in the phospholipids detectable inside the nucleus in all steps of the S phase. This could depend on an increased nuclear phospholipid hydrolysis, whose products should in turn activate some of the enzymes involved in the control of DNA replication.
Animals↗
The riboskeleton: the "brain" of the cell?
The complexity of the RNA world has surprised biologists many times in recent years and has led, among other things, to a new definition of a gene. Its implications, however, may go even further than that. It is possible that the ribonucleoproteins form an information processing system which functions as the "brain" of the cell.
Animals↗
DNA synthesis progression in 3T3 synchronized fibroblasts: a high resolution approach.
In order to investigate at ultrastructural level the mechanism of DNA synthesis progression during the different moments of S-phase a bromodeoxyuridine-anti bromodeoxyuridine (BrdU-anti BrdU) method has been applied to synchronized 3T3 fibroblasts. After 30 min BrdU incorporation, five different labelling patterns can be identified and should be related to early, middle and late S-phase. These patterns are represented mainly by diffuse labelling localized in different nuclear domains and by quite rare cases in which the labelling is limited to isolated clusters of gold particles. After a 5-min pulse with BrdU it is possible to observe isolated clusters of gold particles at each moment of S-phase, which, however, exhibit the same distribution of the five principal labelling patterns observed after 30 min incorporation. In both cases labelling can be detected in the interchromatin regions during early S-phase, at the boundary between interchromatin and heterochromatin during middle S-phase and in the heterochromatin domains during late S-phase. Considering their size, the isolated spots of labelling could be interpreted as single replication units which are subsequently activated throughout the different moments of the S-phase.
3T3 Cells↗
Nuclear matrix-bound replicational sites detected in situ by 5-bromodeoxyuridine.
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 2 M NaCl and DNase I digestion. Such an observation was also confirmed by spatial confocal microscopy studies. The intensity of fluorescence, 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.
3T3 Cells↗
A combined ultrastructural approach to the study of nuclear matrix thermal stabilization.
Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37 degrees C of isolated nuclei. If purified nuclei were heated for 45 min at 37 degrees 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-amine 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.
Animals↗
Human cytomegalovirus nuclear and cytoplasmic dense bodies.
One of the characteristic features of cytomegalovirus (CMV) replication is the formation of cytoplasmic dense bodies. Recent findings revealed similar structures also in the nuclei of CMV-infected cells. By transmission electron microscopy, immuno electronmicroscopy, and cytochemistry, we have studied the morphogenetic steps and macromolecular composition of both structures. Our results show that both structures contain DNA, RNA and viral antigenic proteins. Nuclear dense bodies are probably an expression of a stimulated cellular metabolism, while cytoplasmic dense bodies may represent the site where surplus cellular and viral molecules are stored before being eliminated.
Cell Nucleus↗
Monoclonal antibodies to human DNA topoisomerase I and the two isoforms of DNA topoisomerase II: 170- and 180-kDa isozymes.
Several monoclonal antibodies of different isotypes specific to human DNA topoisomerase I, to 170- and 180-kDa DNA topoisomerase II isozymes, were produced and characterized. The specificity of monoclonal antibodies was confirmed by comparison with polyclonal antibodies by Western blot, by immunoprecipitation of enzyme activity, and by immunoprecipitation of DNA topoisomerases with characterized polyclonal antisera. Morphological studies performed by immunofluorescence indicate that the three groups of monoclonal antibodies (MoAbs) stain the nucleus with characteristic patterns, which can be compared with those obtained with polyclonal antibodies. In particular the MoAbs to the 100-kDa DNA topoisomerase I stain the nucleolus and the nucleoplasm; the MoAbs to 170- and 180-kDa DNA topoisomerase II give completely distinct intranuclear patterns: those to the 170-kDa protein stain mainly the nucleoplasm, whereas those to the 180-kDa protein stain only the nucleolus. The two DNA topoisomerase II isozymes clearly exhibit fluctuations in their expression during cell growth: the 170-kDa isozyme is more abundant during the logarithmic phase of growth, while the 180-kDa isozyme is mainly present during the plateau phase of growth.
Antibodies, Monoclonal↗
The 180-kDa isoform of topoisomerase II is localized in the nucleolus and belongs to the structural elements of the nucleolar remnant.
Monoclonal antibodies raised against two isoforms (170 and 150/180 kDa) of DNA topoisomerase II showed distinct fluorescence patterns in HeLa cells in different moments of the cell cycle (C. Negri et al., 1992, Exp. Cell Res. 200, 452-459). The ultrastructural distribution of the 150/180-kDa isoform, which in immunofluorescence showed a localization into the nucleolar region, has been analyzed by electron microscopy with a gold-conjugated secondary antibody in HeLa and K562 cells. The results indicate that this isoform of the enzyme is exclusively localized in the nucleolus, mainly in the dense fibrillar component, while the nucleoplasm of interphase cells and the chromosomes of mitotic cells are completely negative. The antibody also reacts with the nucleolus of isolated nuclei and with the nucleolar remnant of purified nuclear matrices. A quantitative evaluation of the label distribution indicates that the percentage of label in the nucleolar remnant of isolated matrix is almost identical to that of the nucleolus in whole cells. The interaction with the insoluble proteins of the isolated nuclear matrix is also demonstrated by quantitative immunoblotting in which the MoAb specifically stains a unique band corresponding to the 150/180-kDa isoform of topoisomerase II. The localization of the 150/180-kDa isoform of topoisomerase II in the nucleolar remnant strongly suggests that it represents a structural element for the spatial organization and for the regulation of transcription of the ribosomal genes.
Blotting, Western↗
Evaluation of osteonectin as a diagnostic marker of osteogenic bone tumors.
Osteonectin (ON), a 32,000-kd glycoprotein involved in the early steps of mineralization of skeletal tissue, is a recognized differentiation marker of normal osteogenic cells. The expression of ON was evaluated in vitro and in tissue sections by the polyclonal antibody bON II. In different cell cultures immunocytochemistry and molecular biology displayed a nonspecific reaction for the antibody, which showed itself to be useless for the in vitro identification of cells of the osteoblastic lineage. The diagnostic use of bON II antibody was investigated by immunohistochemistry on a series of osteogenic and nonosteogenic bone tumors. A strongly positive stain of the entire neoplastic component of osteosarcoma and osteoblastoma and a weaker stain of the mononuclear component of giant cell tumor and chondroblastoma were observed. On the other hand, stains for chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant fibrous histiocytoma, and brown tumor from hyperparathyroidism were entirely negative. Our results indicate that ON may be helpful in the histologic diagnosis of bone tumors, particularly in differentiating small cell osteosarcoma from other small round cell tumors.
Biomarkers, Tumor↗
Morphological evidence of function-related localization of phospholipids in the cell nucleus.
The evidence accumulated in recent years on the presence of phospholipids inside the interphase nucleus needs a precise localization of the nuclear sites of accumulation, transport and degradation of these molecules. A very useful approach for monitoring the fine localization of nuclear phospholipids is represented by a recently developed technique using gold-conjugated phospholipases. In fact, in addition to the phospholipids organized in bilayers in the membrane, this technique identifies amorphous lipoprotein complexes present in different cell areas as well as in the nucleus. In this way and using sample preparation systems which reduce lipid removal and translocation, such as cryofixation, cryosectioning, embedding in hydrophylic resins and cryofracturing, we have analyzed the subnuclear localization of phospholipids in different experimental conditions. The results indicate that: in interphase the nuclear phospholipids are localized mainly in the interchromatin spaces and in the nucleolar domain; the observed co-localization of phospholipids and ribonucleoproteins suggests that phospholipids are involved in the mechanism of transport and release of the transcripts; the demonstrated release of ribonucleoproteins after phospholipase digestion suggests that phospholipids mediate the binding between ribonucleoproteins and the nuclear matrix; significant changes of the phospholipid localization occur in the different phases of the cell cycle or in the course of induced cell differentiation.
Animals↗
Localization of dystrophin COOH-terminal domain by the fracture-label technique.
The precise localization of dystrophin in the skeletal muscle cell should contribute to a better understanding of the yet unclear functional role of this protein, both in normal and in Duchenne muscular dystrophy. Immunocytochemical studies did not give conclusive results on the localization of dystrophin with respect to the sarcolemma and to the cytoskeletal components. To improve the reliability of the electron microscopic immunocytochemical localization of dystrophin, a mAb against the COOH-terminus of the molecule has been used in association with the fracture-label technique, which, causing a partition of the membrane in protoplasmic and exoplasmic halves, allows a more precise dystrophin localization. The results obtained indicate that dystrophin is associated with the protoplasmic half of the plasmalemma, and the observation that it does not randomly follow the partition of the membrane is consistent with a stable association with the cytoskeleton.
Cell Membrane↗
Intranuclear localization of phospholipids by ultrastructural cytochemistry.
The presence of phospholipids within the interphase nucleus and in isolated chromatin, previously demonstrated by analytical biochemical methods, has been only rarely documented by cytochemical procedures, especially at the ultrastructural level. By means of a gold-conjugated phospholipase technique, we investigated the fine localization of endogenous phospholipids in the different nuclear domains in rat pancreas and in cell cultures. To reduce possible removal or displacement of phospholipids, different specimen preparation procedures such as cryofixation, cryosectioning, and freeze-fracturing were utilized. Apart from slight differences in efficiency among these methods, phospholipids have been cytochemically identified in the same nuclear domains: the interchromatin granules and fibers and the dense fibrillar component of the nucleolus. These results suggest that the phospholipids are an actual nuclear component, not randomly distributed in the nucleoplasm but mainly localized in the nuclear domains involved in the synthesis, maturation, and transport of ribonucleoproteins.
Animals↗
Inositol lipid phosphorylation in the cell nucleus.
Inositol lipid metabolism has been analyzed in isolated rat liver nuclei and nuclear fractions, in order to determine the subcellular distribution of the sites of lipid phosphorylation and breakdown. Lipid kinases and phosphoesterases appear to be tightly bound nuclear components, and can utilize exogenous substrates administered to membrane-depleted structures. The possible involvement of specific carrier protein in the nuclear metabolism of inositol lipids has also been analysed by studying the uptake and processing of phosphatidylinositol transferred to the isolated nuclei by phosphatidylinositol transfer protein (PI-TP). PI-TP greatly stimulates the incorporation of phosphatidylinositol from microsomal membranes and synthetic vesicles, and the lipid taken up is available for phosphorylation and breakdown by enzymes associated to the nucleus. The results obtained support previous data on the metabolic and structural role of nuclear lipids, and suggest that the cell nucleus is a site of lipid phosphorylation, not necessarily involving enzymes and substrates located on the nuclear membrane. They also indicate that an integrated signalling pathway can exist at the nuclear level utilizing inositol lipid-derived second messengers and PKC to control replication and transcription.
Animals↗
Image analysis techniques. The problem of the quantitative evaluation of the chromatin ultrastructure.
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Animals↗