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Biomedical subjects

G Mazzotti

Publications and source records attributed to G Mazzotti.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1992

Natural killer function in flow cytometry. III. Surface marker determination of K562-conjugated lymphocytes by dual laser flow cytometry.

The recognition of effector cell populations that are able to actively from conjugates with target cells is of major importance in studies of lymphocyte cytotoxicity. A number of methodologies have been described to identify the conjugates and count them, but there have been few studies of the binding capability of the different subsets of effector cells involved in the conjugation phenomenon. Here we describe a methodology that permits the study of two surface markers on lymphocytes conjugated to K562 target cells. In particular, the expression of low density CD8 (CD8dim) has been studied on both CD3+ and CD16+ lymphocytes bound to K562 target cells. Previously described methodologies, either optical microscopy or flow cytometry, were not able to identify the effector population by mAb double staining, especially in the case of antigens expressed at low density. The flow cytometric methodology described here permits the measurement of the binding activity of small lymphocyte subsets such as the CD3+ 8dim+ population. However, the method could be used to study the binding activity of any effector population defined by mAb double staining.

Antigens, Differentiation↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1992

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↗
PubMed1991

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↗
PubMed1991

Characterization and cell cycle kinetics of hepatocytes during rat liver regeneration: in vivo BrdUrd incorporation analysed by flow cytometry and electron microscopy.

The incorporation of bromodeoxyuridine (BrdUrd) into newly synthesized DNA has been analysed during hepatocellular regeneration induced by partial hepatectomy in young rats. The kinetic state of the liver has been studied by flow cytometric analysis of the incorporated BrdUrd, while the fine localization of DNA replication sites through the cell cycle has been investigated at the ultrastructural level by the immunogold technique. Eighteen hours after partial hepatectomy flow cytometry revealed an early S phase distribution which corresponded to a specific staining of the interchromatin domains of the hepatocyte nucleus. Thirty-four hours after hepatectomy, on the other hand, when most cells were in late S, a specific staining of heterochromatin domains was observed. The effect of the BrdUrd technique on nuclear aggregation has also been analysed and discussed. The results demonstrate that specific patterns of DNA replication can be recognized during the cell cycle and that flow cytometry and electron microscopy appear to be complementary in the kinetic study of liver regeneration.

Animals↗
PubMed1990

High-resolution detection of newly synthesized DNA by anti-bromodeoxyuridine antibodies identifies specific chromatin domains.

We analyzed the incorporation of bromodeoxyuridine (BrdUrd) into DNA in exponentially growing murine erythroleukemia cells (FLC-745), using fluorescent anti-BrdUrd antibodies with light microscopy and flow cytometry. The fine localization of the DNA replicating sites was investigated at the ultrastructural level by using a second antibody conjugated with colloidal gold. The latter approach, which does not require acidic denaturation of the DNA, enables preservation of good morphology and obtains a better resolution power than that of electron microscopic autoradiography, the percentage of labeled cells obtained with the two techniques being comparable. After short BrdUrd pulses, characteristic distribution of the labeling can be identified in the heterochromatin, in interchromatin domains, or at the boundary between the dispersed and the condensed chromatin. Similar patterns are also observable in the nuclear structures which condense after acid denaturation, suggesting that DNA replication takes place at fixed sites associated with the nuclear matrix.

Animals↗
PubMed1990

Radiosensitivity of human natural killer cells: binding and cytotoxic activities of natural killer cell subsets.

The sensitivity of human natural killer (NK) cell activities (both binding and killing) after exposure of peripheral blood mononuclear cells to different doses of gamma radiation was studied. A panel of monoclonal antibodies was used to identify the NK and T-lymphocyte subsets and to evaluate their radiosensitivity. Peripheral blood mononuclear cells were irradiated with low (2-6 Gy) and high (10-30 Gy) doses and NK cell binding and cytotoxic activity against K562 target cells were studied after 3 h and 48 h in culture. The primary damage to NK cell activity was identified at the postbinding level and affected mainly the lytic machinery. After 48 h culture postirradiation, an overall depression of cytotoxic activity was observed, but ionizing radiation produced either a selection of the more cytotoxic NK cell subsets, which therefore might be considered more resistant to radiation damage than the less cytotoxic NK cells, or a long-term stimulation of cytotoxic activity in surviving cells.

Cesium Radioisotopes↗
PubMed1989

Cytochemical localization of DNA loop attachment sites to the nuclear lamina and to the inner nuclear matrix.

The rat liver nuclear matrix, obtained by endogenous nuclease digestion and extraction with low and high ionic strength media, contains residual DNA fragments that are considered to represent the attachment sites of the chromatin domains to the nucleoskeleton. These sites, protected against nuclease digestion by their binding with the nucleoskeleton proteins, should be either mainly linked to the peripheral lamina or to the inner nuclear matrix. The DNA fragment distribution at the level of the different components of the nuclear matrix has been evaluated in samples embedded in Epon and in hydrophilic resins by means of the DNase-gold technique. The labeling obtained suggests that the chromatin loops are prevailingly associated with the interior of the matrix; in fact about twice of the label is present in the inner matrix with respect to the peripheral lamina area. These results confirm the hypothesis that in interphase the chromatin maintains an organization similar to that of chromosomes, with loops radiating from a central scaffold, instead of being mainly attached to the lamina as otherwise suggested.

Animals↗
PubMed1989

Phospholipase C digestion induces the removal of nuclear RNA: a cytochemical quantitative study.

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 (Cocco et 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.

Animals↗
PubMed1989

Impaired lymphocyte stimulation induced by long-term training.

In recent there has been increasing interest in the definition of hormone influence on the immune system. Physical stress provides a suitable model for studying the interactions between the immune system and the neuroendocrine factors which have been shown to modulate the lymphoid cellular compartment. Our approach has been devoted to defining the stable modifications induced in the immune system in athletes during agonistic training. The results show that the circulating compartment of the immune system tends to modulate its different subsets under the continuous influence of stress hormones, together with a specific functional impairment of the helper subset in the proliferative response after stimulation with PHA, and particularly with PWM.

Antibodies, Monoclonal↗
PubMed1989

Esophageal motility in cirrhotics with and without esophageal varices.

Esophageal manometry was performed in 45 cirrhotics with varices, in 15 cirrhotics without varices, and in 20 normal subjects, to define the effect of varices on esophageal motility. Cirrhotics with varices showed a decreased amplitude of motor waves in the lower half of the esophagus (p less than 0.01), an increased duration of primary peristaltic waves along the entire length of the esophagus (upper esophagus, p less than 0.05; lower esophagus, p less than 0.01), and an increased peak-to-peak speed of primary peristaltic waves (p less than 0.01). Resting lower esophageal sphincter pressure and duration of sphincter relaxation were similar in patients and controls. The above-mentioned abnormalities might be due to the mechanical effect of the presence of varices.

Adolescent↗