Biomedical subjects

G Mazzotti

Publications and source records attributed to G Mazzotti.

At least 19 recordsLinked to original sources

PubMed1996

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↗

PubMed1996

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↗

PubMed1996

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↗

PubMed1995

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↗

PubMed1995

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↗

PubMed1995

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↗

PubMed1995

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↗

PubMed1995

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

Lymphocyte binding to K562 cells: effect of target cell irradiation and correlation with ICAM-1 and LFA-3 expression.

Tumor irradiation induces modifications in the interaction of surviving target cells with immune cells. This interaction is mediated by adhesion molecules, whose expression can be strongly altered by radiation treatment. Here the probably of K562 tumor cells for lymphocyte binding was studied after exposure of target cells to different doses of gamma-radiation. Results were correlated to the expression of ICAM-1 and LFA-3 adhesion molecules on target cells. Radiation treatment enhanced the expression of both ICAM-1 and LFA-3 on the surface of target cells in a dose and time of culture-dependent fashion, reaching a maximum 24 hrs postirradiation, when also lymphocyte binding was increased. 10-30 Gy irradiation of K562 cells in vitro induces after 24 hrs, an up-regulation of ICAM-1 and LFA-3 expression that, in turn, increase lymphocyte binding, making tumor cells more exposed to cytotoxic attack. The progressive morphological damage induced by radiation, documented by the scattering singlas in flow cytometry and by electron microscopy analysis of irradiated K562 cells, induced, particularly at delayed times of culture in high doses irradiated cells, alterations of the target cell surface that might prevent the correct interaction with immune cells.

Adult↗

PubMed1993

Differential kinetics of propidium iodide uptake in apoptotic and necrotic thymocytes.

Apoptosis and necrosis represent two different mechanisms by which cells die. The dynamics of cellular lesions in these two processes differ. In particular we demonstrate that plasma membrane damage, occurring as a primary event during necrosis represents, on the contrary, a delayed but massive phenomenon during apoptosis. In consequence there are different kinetics of propidium iodide incorporation by necrotic and apoptotic thymocytes. This represents the basis for the flow cytometric identification of different cellular subsets. Analysis of these subsets after sorting showed that clearly apoptotic cells, which are not able to exclude propidium iodide for long incubation periods, do not show any morphologically detectable membrane damage. The kinetics of propidium iodide incorporation in vivo in isolated rat thymocytes can therefore be used in flow cytometric analysis. This technique can be used instead of DNA staining of ethanol-treated cells or nick translation to recognize apoptotic cells, and distinguish apoptosis from necrosis, without killing the cell.

Animals↗

PubMed1993

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↗

PubMed1993

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↗

PubMed1993

Proliferative response of human marginal gingiva to phlogistic process and titanium implant.

In order to detect proliferative processes in human marginal gingiva in pathological conditions and after externalization of titanium implants, we have attempted BrdU incorporation after "in vitro" incubation of tissue fragments. In comparison with healthy controls, immunocytochemical detection of samples from patients affected by hypertrophic gengivitis shows a good number of proliferating cells in the basal layer of the epithelium, while only in one case can positiveness be detected after externalization of titanium implants. Since after reduction of inflammation by hygienic treatment a low number of proliferating cells can be observed only in the regions where pathological alterations are also present, we suggest that the increase in tissue proliferation may be closely dependent on the intensity of the inflammatory process. All these data demonstrate that in vitro BrdU incubation of tissue fragments represents a suitable method to evaluate cell proliferation in human tissue.

Adult↗