Search PubMed⌕ Search

Biomedical subjects

F A Manzoli

Publications and source records attributed to F A Manzoli.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1990

Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei. Role of phosphatidylinositol transfer protein.

The incorporation of phosphatidyl[2-3H]inositol ([3H]PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP). The nuclei are able to phosphorylate [3H]PI, with the production of phosphatidylinositol 4-phosphate (PIP). Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle. Incubation with [gamma-32P]ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity. The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.

Animals↗
PubMed1990

Temperature-dependent association of DNA polymerase alpha activity with the nuclear matrix.

We have investigated the effect of preincubating isolated nuclei at the physiological temperature of 37 degrees C on the recovery of DNA polymerase alpha and beta activities bound to the nuclear matrix. In HeLa cells, when purified nuclei are incubated for at least 30 min at 37 degrees C prior to extraction with 2 M NaCl and digestion with DNase I, about 30% of nuclear DNA polymerase alpha activity is associated with the final matrix along with about 20% of nuclear protein. If the preincubation is carried out at 0 degrees C, less than 5% of the enzyme activity is resistant to high salt extraction and the protein recovery drops to about 12%. On the contrary, the recovery of nuclear DNA polymerase beta activity bound to the matrix fraction is independent of the temperature at which the preincubation is performed. The same levels of DNA polymerase alpha activity are found to be matrix associated even if reducing and chelating agents are present during the exposure of isolated nuclei to 37 degrees C, suggesting that this phenomenon does not depend on the in vitro formation of disulfide bonds or on some metal ion-protein interaction. Our data could explain why, in the past, different results have been obtained when the association of DNA polymerase alpha with the nuclear matrix has been analyzed.

Cell Nucleus↗
PubMed1990

Nuclear inositol lipids. Relationship between growth factor induced metabolic changes and protein kinase C activity.

We have sought to establish the effect of mitogen treatment on nuclear inositol lipids and the relationship between inositol cycle products and hyperphosphorylation of nuclear proteins via PKC during the lag phase leading to the onset of DNA synthesis. Swiss 3T3 cells were labelled for 36 hr with high levels of [3H]-myo-inositol and the radioactivity in nuclear inositol phospholipids was measured. Treatment of cells for 2 min, but not for 4 hr, with mitogenic concentrations of insulin-like growth factor I and bombesin caused a marked decrease in PtdInsP and PtdInsP2. Moreover, in vivo phosphorylation of some nuclear proteins occurs later on. Among these proteins, histone H1 and 0.75 M PCA soluble polypeptide, with an apparent Mr of 21,000 as revealed by electrophoretic analysis, are phosphorylated in vitro by protein kinase C in isolated nuclei purified from 3T3 cells treated for 90 min with IGF-I and bombesin. Since these phosphorylative events follow the earlier changes in nuclear polyphoinositide metabolism induced by the same mitogen combination, it seems possible that these two phenomena are related to each other and trigger the synthetic machinery responsible for replicating DNA.

Adenosine Triphosphate↗
PubMed1990

Nuclear inositol lipids in Friend erythroleukemia cells. Changes related to differentiation induced by hexamethylenebisacetamide.

Subcellular distribution of inositol lipids has been studied in Friend Erythroleukemia Cells following induction to erythroid differentiation with hexamethylenebisacetamide, after labelling with [3H]myo-inositol. In situ autoradiography indicated that inositol-derived molecules were present also in the nuclear compartment of uninduced and induced cells. Fractionation studies showed that the nuclear polyphosphoinositides were deeply changed after short induction times, while the whole cell inositol lipids resulted only slightly modified by the inducer. The nuclear recovery of phosphatidylinositol 4,5-bisphosphate was largely increased after 2 hrs of induction, suggesting that inositol lipid metabolism is involved in the early differentiation events occurring at the nuclear level.

Acetamides↗
PubMed1990

A morphological study of membrane lesions during natural killer-mediated lysis.

Early and late events occurring during cell-mediated cytolysis have been examined ultrastructurally using transmission, scanning and freeze-fracture electron microscopy. K562 erythroleukemic cells were used as target for human natural killer lymphocytes, a subpopulation involved in host defense against tumor, virus infected and allogeneic cells. Cell binding between target and effector is already evident after 15 min, but membrane lesions and target hydration appear after 30 min incubation. At 60 min K562 cells appear swollen and microvilli-deprived. Progressive membrane disruption is also evident and the underlying cytoplasmic material is clearly exposed after 120 min. Submicroscopic membrane lesions appear to be early events in comparison with 51Cr (isotope) release from damage cells.

Cell Membrane↗
PubMed1989

Lipid phosphorylation in isolated rat liver nuclei. Synthesis of polyphosphoinositides at subnuclear level.

Isolated rat liver nuclei and subnuclear fractions synthesize polyphosphoinositides in vitro in a mode dependent on the presence of nuclear membrane, detergent and exogenous substrates. The nuclear membrane is not essential as a source of lipid kinases, since the addition of exogenous phosphatidylinositol or phosphatidylinositol monophosphate to reaction mixtures lacking membranes restores the synthesis of phosphatidylinositol mono- and bisphosphate, respectively. Inositide phosphorylation is best accomplished by high-salt extracted nuclei and pre-detergent lamina. These data suggest that the nucleus, and especially the nuclear periphery, is a cell compartment in which polyphosphoinositide synthesis occurs; this might be related to the progression of phosphatidylinositol metabolism-dependent signals to the genetic apparatus.

Adenosine Triphosphate↗
PubMed1989

Changes in nuclear inositol phospholipids induced in intact cells by insulin-like growth factor I.

Swiss 3T3 cells were labelled for 36 hours with high levels of 3H-myo-inositol and the radioactivity in nuclear inositol phospholipids was measured. Treatment of cells for 2 minutes, but not for 4 hours, with mitogenic concentrations of insulin-like growth factor I and bombesin caused a slight decrease in PtdIns and more marked decreases in PtdInsP and PtdInsP2. These effects were not seen if isolated nuclei were incubated with IGF-I and bombesin. We interpret these results to mean that rapid mass changes occur in nuclear inositol phospholipids in the early stages of the mitotic response.

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

Mitogen-stimulated phosphorylation of nuclear proteins in Swiss 3T3 cells: evidence for a protein kinase C requirement.

When Swiss 3T3 fibroblasts are treated with a combination of IGF-I2 and bombesin at mitogenic concentrations, in vivo phosphorylation of some nuclear proteins occurs within 45-90 min. Among these proteins, histone H1 and a 0.75 M PCA soluble polypeptide with an apparent Mr of 21,000, as revealed by electrophoretic analysis, are phosphorylated in vitro by protein kinase C in isolated nuclei purified from 3T3 cells treated for 90 min with IGF-I and bombesin. Since these phosphorylative events follow the earlier changes, recently demonstrated, in nuclear polyphosphoinositide metabolism induced by the same mitogen combination, it seems possible that these two phenomena are related to each other and trigger the synthetic machinery responsible for replicating DNA.

Animals↗