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F A Manzoli

Publications and source records attributed to F A Manzoli.

At least 19 recordsLinked to original sources
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↗
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↗
PubMed1993

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↗
PubMed1993

Immunocytochemical analysis of phosphatidylinositol-specific phospholipase C in PC12 cells: predominance of the delta isoform during neural differentiation.

The rat pheochromocytoma PC12 cell line, which differentiates into sympathetic neurons under nerve growth factor (NGF) treatment, contains at least three phosphoinositidase C (PIC) isozymes, PIC beta, PIC gamma, PIC delta. These isozymes have been previously shown to display a different subcellular localization. To determine whether or not NGF induces changes in the presence and/or distribution of PIC isozymes during PC12 neural differentiation, studies were carried out by means of in situ immunocytochemistry. After NGF administration the proliferative activity was progressively reduced to very low levels, as measured by bromodeoxy Uridine incorporation, and a neuron-like morphology was displayed by almost all cells. In unstimulated PC12 cells, PIC beta was detected in the nucleus whereas PIC delta was only cytoplasmic; PIC gamma was found in both cell compartments. In cells treated with NGF for 3 days, neural processes extended to twice the diameter of the cell body; the gamma isoform was concentrated near the nucleus, while the immunoreactivity of the beta form remained constant and the delta form was increased. After 10 days of treatment with NGF, PIC beta was hardly detectable and PIC gamma immunostaining was considerably decreased. On the contrary, PIC delta progressively increased and, after 14 days of NGF exposure, fully differentiated cells displayed an intense labelling of cell body and neurites. In the same cells, PIC beta and PIC gamma were almost negative. These results suggest that NGF dependent neural differentiation is related to the selective down regulation of PIC beta and gamma and the increase of PIC delta isozyme associated with the decrease of cell proliferation.

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↗
PubMed1992

Discrete subcellular localization of phosphoinositidase C beta, gamma and delta in PC12 rat pheochromocytoma cells.

Phosphoinositidase C activity was revealed in nuclei isolated from PC12 rat pheochromocytoma cells incubated with tritiated phosphatidylinositol, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Phosphoinositide breakdown was found to be optimal at neutral pH and Ca++ concentrations ranging from endogenous levels to millimolar values. To characterize the enzymes involved, three monoclonal antibodies directed against the beta, gamma and delta phosphoinositidase C isoforms were employed. A combination of Western blot immunochemical analysis on cytoplasmic and nuclear fractions and of in situ immunocytochemistry on intact cells and isolated nuclei indicated that phosphoinositidase C gamma, though predominantly cytoplasmic, was present in both cell compartments. On the contrary, phosphoinositidase C beta was exclusively localized in the nucleus, whereas phosphoinositidase C delta was restricted to the cytoplasm. These data suggest that inositol lipid breakdown is controlled by different phosphoinositidase C isozymes in the various cell compartments, and support the notion that a separate phosphoinositide signalling system is located in the nucleus.

Animals↗
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

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↗
PubMed1992

Changes in inositol lipid metabolism and protein kinase C translocation in nuclei of mitogen stimulated Swiss 3T3 cells.

The correlation between changes in nuclear polyphosphoinositide levels preceding PKC translocation to the nucleus and the onset of DNA synthesis has been discussed. Using two different clones of Swiss 3T3 fibroblasts belonging to the same original cell line, one of which is unresponsive to mitogenic stimulation with IGF-I on its own or in combination with bombesin, it has been observed that a rapid and transient breakdown of nuclear PIP and PIP2 occurs only in responsive cells and this precedes the translocation of PKC to the nucleus, as evidenced by immunochemical analysis as well as by enzymatic activity. Therefore, it seems that a direct link exists between nuclear polyphosphoinositide metabolism, PKC translocation to the nucleus and cell division. Since IGF-I acts at the plasma membrane through a tyrosine kinase receptor it seems that the mitogenic stimulation induced by this factor utilizes different signalling pathways at the plasma membrane and at the nucleus. Because of the evidence that type I IGF receptor is expressed in both responsive and unresponsive cells and that the receptor machinery at the plasma membrane is active the lack of the transient changes in nuclear inositol lipids and of PKC translocation in unresponsive cells further suggests that the cell nucleus is capable of an autonomous signalling system based on polyphosphoinositide metabolism.

3T3 Cells↗
PubMed1992

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↗
PubMed1991

Temporal changes in intracellular distribution of protein kinase C in Swiss 3T3 cells during mitogenic stimulation with insulin-like growth factor I and bombesin: translocation to the nucleus follows rapid changes in nuclear polyphosphoinositides.

Using a polyclonal antibody raised against a synthetic peptide of the catalytic region of protein kinase C, we have carried out a combined immunocytochemical and immunochemical analysis to follow the subcellular localisation of this enzyme in response to mitogenic stimulation with insulin-like growth factor I and bombesin. These investigations show a time dependent translocation of protein kinase C from the cytoplasm to the nucleus since 5 min stimulation reaching a maximal effect after 45 min. These results show clearly that mitogen induced translocation of protein kinase C to the nucleus follows temporally the earlier changes in nuclear polyphosphoinositide metabolism previously demonstrated.

Amino Acid Sequence↗
PubMed1991

Inositol lipids in Friend erythroleukemia cells: evidence for changes in nuclear metabolism after differentiation.

The incorporation of 32Pi into phospholipids was studied in Friend erythroleukemia cells either induced or not to erythroid differentiation with 4 mM hexamethylenebisacetamide (HMBA). The effect of the differentiating agent on the recovery of radiolabelled phospholipids was compared in whole cells, isolated nuclei and nuclear matrix after in vivo labelling for 1 hr. The procedure employed for the isolation of nuclei was demonstrated to allow only negligible lipid redistribution caused by cell manipulations. Among the lipids extractable from nuclei, acidic phospholipids, and particularly polyphosphoinositides, were more represented than in whole cells, while small differences were found in the other phospholipid classes examined. The comparison between the uninduced and induced condition showed that the relative amounts of nuclear inositol lipids were modified by HMBA treatment of the cells, with a decreased recovery of phosphatidylinositol 4,5 bisphosphate. These results indicate that phosphatidylinositol and its phosphorylation products synthesized in vivo show a different metabolism in nuclei and whole cells. They appear to be tightly bound nuclear components, also present in membrane-deprived nuclei and nuclear matrix, and are probably related to the nuclear events involved in erythroid differentiation.

Acetamides↗
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

On the association of DNA polymerase alpha activity with the nuclear matrix in HeLa cells.

The association of DNA polymerase alpha activity with the nuclear matrix has been reinvestigated in HeLa cells. Isolated nuclei were extracted with 2M NaCl and then digested with Dnase I and the final structures were recovered by centrifugation through a sucrose cushion. Typically over 98% of the total DNA synthesized in the matrix fraction on either endogenous matrix-associated DNA or activated calf thymus DNA was due to DNA polymerase alpha as defined by inhibition to n-ethylmaleimide or aphidicolin. DNA polymerase beta activity was absent or recovered in only trace amounts. Matrix-bound DNA polymerase alpha activity demonstrated a remarkable degree of stability: DNA synthesis was essentially linear up to 3 hours at 37 degrees C. Overall, these results substantiate previous findings from regenerating rat liver, unlike other data obtained from tissue culture cells.

Aphidicolin↗
PubMed1991

In vitro phosphorylation of lamin B by protein kinase C in friend erythroleukemia. Effect of chemically induced differentiation.

Nuclear matrix isolated from murine erythroleukemia cells (Friend cells) has been phosphorylated with gamma 32P-ATP and purified protein kinase C in order to identify specific nuclear substrates for the enzyme. HMBA has been employed to induce the cell to differentiate and to compare the changes of phosphorylation profile after erythroid differentiation. Lamin B has been found to be hyperphosphorylated by rat brain PK-C in nuclear matrix purified from uninduced cells. This difference characterizes the cells from 14 to 72 hrs of HMBA treatment and indicates that the ability of lamin B to be phosphorylated by PK-C is linked to the differentiated state. The involvement of PK-C in lamin phosphorylation might represent an early step of the signalling pathway utilized by erythroid differentiating agents to target the cell nucleus.

Acetamides↗
PubMed1990

Calcium free inositol (1,4,5)-trisphosphate stimulates protein kinase C dependent protein phosphorylation in nuclei isolated from mitogen-treated Swiss 3T3 cells.

As a step towards the elucidation of the role played by nuclear polyphosphoinositides, we have investigated the effect of exogenous calcium free inositol (1,4,5)-trisphosphate on the in vitro phosphorylation of proteins in nuclei prepared from Swiss 3T3 cells treated with bombesin and insulin-like growth factor I. When present in combination with phosphatidylserine, inositol (1,4,5)-trisphosphate enhanced the phosphorylation of two nuclear proteins, Mr 21,000 and 31,000, as well as of exogenous histone H1, to the same extent as a combination of phosphatidylserine and diacylglycerol. Inositol (1,4,5)-trisphosphate alone had no effect. This stimulation could be abolished by the protein kinase C inhibitor sphingosine and by EGTA, while could be restored by a combination of phosphatidylserine and exogenous Ca+(+) ions. These results raise the possibility that inositol (1,4,5)-trisphosphate is capable of liberating Ca+(+) ions from a nuclear store thus stimulating protein kinase C activity.

Animals↗