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G Mazzotti

Publications and source records attributed to G Mazzotti.

At least 37 records · Page 2Linked to original sources

Ultrastructural aspects of the DNA polymerase alpha distribution during the cell cycle.

We studied the nuclear topography of the replicating enzyme DNA polymerase alpha in HeLa cells by transmission electron microscopy and field emission in lens scanning electron microscopy. Cells were synchronized at the G1/S-phase boundary and samples of the different phases of the cell cycle were labeled with an anti-DNA polymerase alpha antibody detected by an immunogold reaction. DNA synthesis was detected by immunogold labeling after bromodeoxyuridine administration. The typical labeling pattern of DNA polymerase alpha observed in G1- and S-phase cells was represented by circular structures 80-100 nm in diameter surrounding an electron-dense area. In double labeled samples these circular structures were associated with bromodeoxyuridine-containing DNA replication sites, forming rosette-like structures. Field emission scanning electron microscopy performed on ultrathin cryosections revealed the chromatin fibers underlying DNA polymerase alpha complexes and showed that the size of the rosette-like structures corresponded to the diameter of chromatin foldings. G2- and M-phase cells showed a spread distribution of DNA polymerase alpha. The evidence of DNA polymerase alpha circular arrangement exclusively in G1- and S-phase cells, obtained by such different approaches, allowed us to consider the three-dimensional structures as DNA replication areas.

Bromodeoxyuridine↗

Immunocytochemical discrimination between early and late S phase: a new approach to the study of gingival epithelium proliferation in rats.

The renewal of the free gingival margin epithelium in rats was studied evaluating 5-bromodeoxyuridine (BrdU) incorporation in proliferating cells by means of an immunocytochemical method. We found a close correspondence between light and electron microscopy patterns of BrdU incorporation at a nuclear level. BrdU was localized in the inner interchromatin regions in cells starting DNA synthesis, while it was localized in the peripheral heterochromatin domains in cells terminating the S phase. This possibility of discriminating cells in early S phase from cells in late S is able to provide far more information as to the time at which a labeled cell starts proliferation than that obtainable with 3H-thymidine autoradiography. This, in turn, permits detection of cells that start proliferation in a wide period of time by means of a single BrdU administration. Rats treated at 7 a.m. demonstrated higher proliferation than rats treated at 7 p.m., supporting the existence of circadian variations in the epithelial renewal. Proliferative events take place by consecutive activation of at least three replication waves, producing clusters of labeled cells which could be observed in rats sacrificed at 10 a.m. In rats treated once with BrdU at 7 a.m., the clusters were localized in both the basal and suprabasal layer of the epithelium; in rats further injected with BrdU at the same time, the clusters increased in size, progressively extending throughout the epithelium. In this way, the renewal of the free gingival margin epithelium does not proceed randomly, but by consecutive activation of discrete units or clusters of basal cells, which then extend to the upper layers. This can be followed at a morphological level as a progression of labeled cells, which move from the basal layer to the epithelium surface in approximately 82-85 hours.

Animals↗

Cytochemistry of the functional domains of the nucleus in normal and in pathologic conditions.

By means of ultrastructural cytochemistry significant advances have been made in understanding the functional roles of many nuclear domains. This review gives schematic information about the main nuclear domains involved in replication, transcription, processing and transport of the transcripts in normal and in pathologic conditions. Particular attention is paid to a functional domain that appears to be involved in signal transduction. Data are reported on the intranuclear specific localization of key elements of the polyphosphoinositide signal transduction system in different cell types including human osteosarcoma cell lines. Compared with the compartmentalization of the cytoplasm, the nucleus has long been considered as relatively unstructured. On the other hand, fundamental nuclear functions, such as DNA replication and RNA transcription, can be molecularly characterized also in cell-free systems, suggesting that supramolecular organization is not so strictly required as for other cell functions occurring within intact cytoplasmic organelles. Nevertheless, a stringent organization is required for packing about 200 cm of DNA in the about 30 micron 3 of the nucleus. In the absence of membrane-delimited organelles, the nuclear organization is based on functional compartments, or domains, whose spatial localization involves the nuclear matrix, which shares many properties with the cytoskeleton. The nuclear domains are defined as structural compartments, not necessarily stable but dynamically variable, which perform specific metabolic functions through the partitioning of molecular complexes. Their identification has been made possible in the last few years by the development of specific nuclear probes for confocal and electron microscope immunocytochemistry. Therefore, the complex network of structures and enzymatic functions that make up the nucleus is in several cases yielding to molecular analysis, but a large part remains unknown (Strouboulis and Wolffe, 1996; Laemmli and Tjian, 1996). Rapid advances in understanding the functional role of the nuclear domains have been made recently: in particular, of the nuclear envelope, of the nucleolus, and of RNA splicing. In other cases, e.g. the precise localization of the nuclear domains involved in signal transduction, much remains to be clarified (Forbes and Johnson, 1997). It is conceivable that in the near future unexpected new nuclear domains will come to light and new nuclear functions may emerge, especially in field of post-transcriptional processing and transport of RNAs, and in the relationships between the nucleo-skeleton and enzymic fixed sites involved in replication, transcription and signal transduction. The aim of this review is to provide information about the morphological characteristics, the associated functions and the molecular composition of the main nuclear domains found to date. To simplify the exposition, the main data on each nuclear domain are reported in Tables, together with the principal references on the subject. Figures refer to original findings on some aspects of nuclear domain organization.

Cell Nucleus↗

Age- and training-related events in active T subpopulation. Changes in polyphosphoinositide metabolism during mitogenic stimulation.

To examine the effects of age and training on the active T subpopulation we considered elderly amateur cyclists over 65 in comparison with young amateur cyclists and young and aged sedentary healthy controls. Significant differences were observed between trained and sedentary elderly subjects consisting of an increase in the percentage of active E rosettes after 4 and 24 h of in vitro PHA stimulation, and of a decrease in the in vitro phosphorylation of phosphatydylinositol 4,5-bisphosphate (PtdInsP2) and a corresponding increase in phosphatydylinositol 4-phosphate (PtdInsP) in the early steps of the mitotic response. Our findings support the hypothesis of the involvement of inositol lipids in controlling the expression of lymphocyte surface receptors.

Adult↗

Early events of liver regeneration in rats: a multiparametric analysis.

5-Bromodeoxyuridine (BrdU), a synthetic analogue of thymidine, has been utilized in vivo to detect the proliferation which occurs in the liver after two-thirds surgical hepatectomy. Immunocytochemical detection of BrdU incorporation has been carried out at both the morphological and flow cytometrical level, while structural changes of regenerating liver have been investigated, using Mallory-Azan-stained paraffin sections, by means of an image analyser. The results obtained show that in vivo DNA synthesis progression throughout S phase follows a pattern similar to that previously described in vitro in both 3T3 fibroblasts and Friend erythroleukemia cells and also demonstrate a precise correlation between morphological patterns of BrdU incorporating cells and their lobular distribution. Moreover, the activation of at least two proliferation waves can be detected from 18 to 34 h after hepatectomy: the former, starting from adjacent regions of contiguous lobules, apparently induces an irregular increase of lobular dimension; the latter, involving both inner and peripheral lobular domains, seems to be correlated with the appearance of nodule-like structures at the lobule periphery. In view of these results the role of the hepatic acinus and the hypothesis of a streaming of parenchymal cells during liver regeneration have been discussed.

Animals↗

Inositol lipid cycle and autonomous nuclear signalling.

The involvement of phospholipids and in particular polyphosphoinositides in cellular signalling has been documented in detail in the last 20 years. In addition to the plasma membrane localization also the nucleus is shown to be a site for both synthesis and hydrolysis of the phosphorylated forms of phosphatidylinositol. Previous observation have established that the nucleus possesses a specific PLC for inositol lipids, i.e., the PLC beta 1 isoform, which undergoes rapid and transient activation after IGF-I stimulation of quiescent Swiss 3T3 cells and is down-regulated after treatment of Friend erythroleukemia cells with DMSO. Here we have reviewed: (i) the potential of nuclear PLC beta 1 to be a target for anti-cancer drug, (ii) the capability of this PLC isoform, when activated by IGF-I, to be a key signalling molecule in the onset of DNA synthesis, via DAG generation and PKC alpha translocation to the nucleus, (iii) the chromosome mapping of PLC beta 1 gene. The differentiation program of Friend cells can be activated by other agents besides DMSO including tiazofurin, an anti-tumor drug, also capable of affecting the nuclear inositol lipid cycle. Tiazofurin induces a lowering of the activity of PLC beta 1 due to down regulation of this isoform as revealed by both Western blotting and Northern blotting analyses. Using Swiss 3T3 cells stably transformed with an antisense PLC beta 1 construct, the knock-out of the PLC beta 1 gene induces both a loss of PLC beta 1 expression, as determined by Western blots, and a loss of the mitogenic responsiveness to IGF-I. These events show a direct relationship between nuclear PLC beta 1 evoked signals and IGF-I induced cell proliferation. Finally, the assignment of the PLC beta 1 gene to the band q35-36 of rat chromosome 3 paves the way for further genetic studies given the fact that the region where PLC beta 1 gene maps is a hot spot for genetic alterations in a number of experimentally induced rat tumors. Taken as a whole, these results assign a key role to the regulation of nuclear PLC activity and expression both in growth-factor activated mitogenesis and in in vitro erythroid differentiation.

Antineoplastic Agents↗

Nuclear inositol lipid cycle and differentiation.

Previous investigations from our laboratory and others have shown the existence of an autonomous intranuclear inositide cycle endowed with conventional lipid kinases and PLC which in PC12 pheochromocytoma cells, human osteosarcoma SaOS-2 cells, rat liver and Swiss 3T3 cells is the isoform beta 1, which in the latter cells is activated upon IGF-I stimulation. The behavior of the nuclear inositol lipid cycle has been investigated in nuclei of Friend erythroleukemia cells. These nuclei possess both lipid kinases and PLC. The cycle upon treatment with differentiating agents (i.e., DMSO and tiazofurin) is characterized by an accumulation of polyphosphoinositides and a decrease of DAG due to down-regulation of a specific PLC. Indeed, even if both beta 1 and gamma 1 isoforms are present in these nuclei, when Friend cells undergo terminal erythroid differentiation only the PLC beta 1 isoform is down-regulated as shown by immunochemical and immunocytochemical analysis, by direct determination of enzymatic activity and in the presence of neutralizing monoclonal antibodies as well as by Northern blot for PLC beta 1 message, whilst the amount of PLC gamma 1 and its activity are unaffected by erythroid differentiation. In conclusion, the presence of a specific nuclear PLC whose activity and expression are down-regulated during differentiation of erythroleukemia cells points out a role for nuclear phosphoinositide signalling in the processes of cell differentiation and hints at the nuclear PLC beta 1 as an important step of the cycle in relation to the erythroid differentiative commitment of murine erythroleukemia cells.

Animals↗

Immunocytochemical detection of the intranuclear variations of phosphatidylinositol 4,5-bisphosphate amount associated with changes of activity and amount of phospholipase C beta 1 in cells exposed to mitogenic or differentiating agonists.

The intracellular localizations of phosphatidylinositol 4,5-bisphosphate (PIP2) and of its hydrolyzing enzyme phospholipase C (PLC; in this case the beta 1 isoform) have been evaluated by electron microscope immunocytochemistry in cells exposed to mitogenic or differentiating agents. These cells have been previously demonstrated to present a signal transduction system based on the polyphosphoinositide hydrolysis localized at the nuclear level, which can be specifically modulated by agonists. The results demonstrate that in Swiss 3T3 mouse fibroblasts mitogenically stimulated by insulin-like growth factor I (IGF-I), a rapid and transient decrease of the PIP2 detectable by immunogold labeling occurs at the nuclear interior. This effect appears due to the activation of the PLC beta 1 isozyme already present in the nucleus, since no significant variations of the enzyme amount and distribution can be detected by immunolabeling. However, after 30 min of exposure to IGF-I, when the PLC beta 1 activity is returned to basal level, a slight but significant increase of the enzyme amount is detected both in the nucleus and in the cytoplasm. On the other hand, an increased accumulation of PIP2 in the nucleus, accompanied by a decrease of the intranuclear amount of PLC beta 1 isozyme, have been observed in mouse erythroleukemia Friend cells, induced to erythroid differentiation by dimethylsulfoxide (DMSO). These results indicate that quantitative immunocytochemistry represents an increment in the available methodologies to investigate the complex regulation of nuclear PI-signalling.

3T3 Cells↗

Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate localization sites within the nucleus.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key element of signal transduction, being the preferential substrate of specific phospholipases that produce second messengers such as inositol trisphosphate (IP3) and diacylglycerol (DG). Because PIP2 has been cytochemically identified by monoclonal antibodies not only in the cytoplasmic membranes but also in the nuclear envelope and within the nucleus, we performed a study by immunoblotting and by confocal and electron microscopic immunocytochemistry to identify the nuclear sites of PIP2 localization and to exclude any cross-reactivity of the antibody with other nuclear molecules. The results confirm the specificity of the immunolabeling and indicate that PIP2 is localized at precise intranuclear sites both in in situ and in isolated nuclei. They also show that a significant amount of the phospholipid is retained by the cytoskeleton and by the inner nuclear matrix in in situ matrix preparations. Moreover the sensitivity of the immunocytochemical reaction is capable of detecting quantitative variations of PIP2 nuclear content induced by agonists that modulate the signal transduction system at the nuclear level.

3T3 Cells↗

High-resolution FEISEM detection of DNA centromeric probes in HeLa metaphase chromosomes.

HeLa metaphase chromosome spreads were hybridized with centromeric biotinylated DNA probes and detected with gold-conjugated anti-biotin antibodies. Chromosomes were observed by an in-lens field emission scanning electron microscope (FEISEM), which permits detection of biological samples without any coating. DNA probes were well localized in the centromeric region of chromosomes and there was clear discrimination between 10 nm fibers that hybridized to DNA probes and those that did not hybridize. This approach shows that in situ hybridization can be directly visualized at the FEISEM level by evaluating only secondary electron emission, which allows physical localization of the hybridized probe with high resolution so that backscatter detection represents only a control. Because chromosomes maintain the 10-nm fiber organization after in situ hybridization procedures, our data suggest that this fiber represents the lowest order of chromatin arrangement that permits transitory DNA denaturation.

Centromere↗

High resolution detection of human metaphase chromosomes.

The new generation of field emission in lens scanning electron microscopy with the sample located within the objective lens allows the observation of uncoated biological samples. Here we describe HeLa metaphase chromosomes isolated by two procedures and the localization within these chromosomes of a DNA centromeric probe. Chromosomes isolated in methanol acetic acid show 10 nm fibers organized in a three dimensional network while the structure of chromosomes obtained by the polyamine method's characterized by very dense short protusions of 30-50 nm in thickness. In situ hybridization with a DNA centromeric probe revealed by colloidal gold particles demonstrate that the hybridization occurs at the level of the 10 nm fiber with a high definition in the localization of specific DNA sequences.

Centromere↗

Immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate in Epon embedded specimens.

A comparison between the time-consuming freeze-substitution Lowicryl HM 20 embedding procedure with the Epon embedding method for the immunocytochemical detection of phosphatidylinositol 4,5-bisphosphate (PIP2) by means of a monoclonal antibody is described. The results indicate that the intracellular localization of the phospholipid and the efficiency of its identification are similar, opening the possibility of performing the immunoreaction also on samples that are routinely processed for pathological investigations.

Animals↗

High resolution detection of uncoated metaphase chromosomes by means of field emission scanning electron microscopy.

HeLa metaphase chromosomes were examined by means of "in lens" field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.

Chromosomes, Human↗

Age-related events in active T lymphocyte subpopulation. A morphological study.

The incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of active T lymphocytes from healthy aged donors was evaluated after in vitro PHA stimulation by means of light microscopy, electron microscopy and flow cytometry. The percentage of BrdU-labelled cells differed markedly between aged and young donors after 48 h of PHA stimulation, due to an enlarged early S phase compartment. In contrast, after 72 h the percentage of positive cells was quite similar in both age groups and no significant differences in the distribution within S phase nor changes in the patterns of ultrastructural localization of DNA replicon sites were observed. Our study provides evidence that an altered synchronization and a substantial delay in the in vitro cell proliferation occur in this peculiar T subpopulation as a consequence of the ageing process.

Adult↗

Lipid-dependent nuclear signalling: morphological and functional features.

Enzymes involved in lipid metabolism exist within the nucleus and are responsive to external stimuli. In particular, the kinases which phosphorylate phosphatidylinositol and phosphatidylinositol-4-monophosphate have been demonstrated in nuclei of both undifferentiated and differentiated Friend cells and of quiescent Swiss 3T3 cells as well as of those exposed to insulin-like growth factor I. Besides the lipid kinases, also the phosphoinositidases C (PIC) are active inside the nucleus. In Swiss 3T3 cells the nuclear PIC beta 1 is activated and its activation by IGF-I temporally precedes the translocation to the nucleus of protein kinase C. In Friend cell nuclei, on the other hand, when erythroid differentiation is induced, the PIC beta 1 activity is reduced. Another aspect of the nuclear signalling transduction system which appears quite interesting is its actual localization at subcellular level. By using electron microscope immunogold labelling, the nuclear PIC isoforms (the beta 1 isoform in Swiss 3T3 cells, the beta 1 and gamma 1 in Friend cells) are localized mainly in the interchromatin domains. This localization has been further confirmed on in situ matrix preparations of 3T3 cells in which PIC beta 1 is associated with the inner nuclear matrix but not with the nuclear pore-lamina complex. Colocalization experiments indicate that nuclear PIC beta 1 is present in sites in which both nuclear phospholipids and PKC can be detected, while the cytoplasmic PIC gamma 1 can be identified in close association with cytoskeletal filaments identified by anti-actin antibodies. The precise localization of the different PIC isoforms strongly indicates that the signal transduction system operating at the nuclear level may be part of a cross-talk between the cytoplasm and the nucleus controlling either cell proliferation or differentiation.

3T3 Cells↗

Evidence for an early and transient involvement of nuclear inositol lipids in subcellular signalling events related to DNA repair processes.

The involvement of nuclear inositol lipids in the processes related to DNA repair upon ionizing radiation has been investigated in Murine Erythroleukaemia cells. Early changes in the in vitro phosphatidylinositol-bisphosphate phosphorylation in isolated nuclei were found to precede transiently the marked increase in DNA synthesis occurring after irradiation. Such an increase detected by anti-BrdU monoclonal antibodies has been found to be related mainly to DNA polymerase beta activity as revealed by the kinetic analysis of in vitro DNA synthesis. The results here presented allow us to speculate on a possible involvement of nuclear inositol lipids in the cascade of the early events leading to the regulation of DNA repair in the nucleus.

Animals↗

Ultrastructural patterns of cell damage and death following gamma radiation exposure of murine erythroleukemia cells.

Radiation causes damage to cell surface membranes, cytoplasmic organelles, and the nuclear process of DNA synthesis and repair, and this eventually results in different modes of cell death. In this study we examined murine erythroleukemia (MEL) cells, exposed to 15 and 60 Gy of 10 MeV photonic energy, and left in culture for up to 96 hours. Electron microscopical analysis was performed on conventionally embedded samples and freeze-fracture replicas, in order to detect ultrastructural patterns of cell damage and death. Of interest was the observation of chromatin condensates, nuclear membrane associations and nuclear pore redistribution during early apoptosis. Pronounced rearrangements of transmembrane particles during late stages of cellular necrosis were also found. The morphological damage induced by both doses of radiation as a function of time after exposure was only quantitatively but not qualitatively different.

Animals↗