Search PubMed⌕ Search

Biomedical subjects

F A Manzoli

Publications and source records attributed to F A Manzoli.

At least 73 records · Page 4Linked to original sources
PubMed1989

Nuclear polyphosphoinositides during cell growth and differentiation.

When highly purified nuclei of Swiss mouse 3T3 cells are incubated with gamma-32P-ATP, radioactivity is incorporated into phosphatidic acid and the two polyphosphoinositol lipids, phosphatidylinositol(4)P and (4,5)P2. If the cells are pre-treated with IGF-I, the incorporation into the polyphosphoinositides is decreased. This effect is maximal by 2 min, is transient in that it disappeared by 1 hr, and is increased markedly by the co-addition of bombesin, even though bombesin alone has no effect. Friend cells exhibit a related phenomenon in that the labelling of PIP2 in isolated nuclei is increased by conditions which cause erythroid differentiation (DMSO addition). We suggest that some aspect of nuclear polyphosphoinositide metabolism is modified when the nucleus is induced to divide or to differentiate, and that this change in inositide metabolism is a very early event in the sequence leading to cell division or differentiation.

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↗
PubMed1988

Rapid changes in phospholipid metabolism in the nuclei of Swiss 3T3 cells induced by treatment of the cells with insulin-like growth factor I.

When highly-purified nuclei of Swiss-mouse 3T3 cells are incubated with [32P]-gamma ATP, radioactivity is incorporated into phosphatidic acid and the two polyphosphoinositol lipids, phosphatidylinositol(4)P and phosphatidylinositol(4,5)P2. If the cells are pre-treated with insulin-like growth factor I, this incorporation into the phospholipids is decreased. The effect is maximal by 2 minutes, is transient in that it has disappeared by one hour, and is increased markedly by the co-addition of bombesin, even though bombesin alone has no effect. We suggest that some aspect of polyphosphoinositide metabolism is altered in the nucleus (leading to a decreased radiolabelling) when the cells are treated with mitogenic growth factors, and that this change in inositide metabolism is a very early event in the sequence leading to cell division.

Adenosine Triphosphate↗
PubMed1988

Natural killer function in flow cytometry. I. Evaluation of NK lytic activity on K562 cell line.

Flow cytometry has been employed to establish an NK assay using the K562 target cell line. These cells show a perpendicular light scatter (PLS) characteristically different from lymphocytes. Other physical parameters, such as forward light scatter (FLS), do not discriminate between the two populations, since dead K562 cells display similar FLS characteristics as effector cells. Killed cells are stained with propidium iodide and followed by flow analysis. It was advisable to add the dye in the medium so that, as long as the target cells are killed they will also be stained. Moreover the flow cytometric and trypan blue evaluation of target cell death rate showed a stronger correlation than did either test with the conventional 51Cr release assay, the first two methods both being based on the same biological mechanism.

Cell Membrane Permeability↗
PubMed1988

Chromatin organization in isolated nuclei: flow cytometric characterization employing forward and perpendicular light scatter.

Flow cytometric perpendicular and forward light scatters have been employed to evaluate whether the changes in chromatin organization due to ionic strength, Mg++ concentration and pH, visible in electron microscopy, can be monitored by flow cytometry. The average intensity of the perpendicular light scatter signal increased as nuclear chromatin became decondensed by lowering the ionic strength or releasing H1 histone at low pH values. These results indicate that flow cytometry signals and in particular the perpendicular light scatter allow the detection of the conformational transitions in chromatin and may therefore be useful for studying cell cycle associated morphological changes in isolated nuclei.

Animals↗
PubMed1988

Gradient fractionation of cycling and resting cells monitored by BrdUrd incorporation.

A number of techniques are currently employed for the fractionation of heterogeneous cell populations or for the separation of cells in different phases of their cycle. With the development of osmotically inert colloidal silica particles media, density gradient centrifugation became an established method for the separation and purification of cells and subcellular particles. We have applied this technique to the separation of cycling from resting Friend erythroleukemia cells, to obtain purified populations for further biological assays. The flow cytometric analysis of DNA content of the different fractions obtained by the gradient and stained with Propidium Iodide (PI), showed the S compartment highly concentrated in the 1.073/77 g/ml interface, while the upper levels of the gradient were highly enriched of cells in G1 phase. Moreover, the dual parameter analysis of DNA content by means of Bromodeoxyuridine (BrdUrd) incorporation and PI staining, showed that part of the cells in the 1.067/73 fraction represented the early S phase even if their DNA level, measured on the basis of PI fluorescence was within the diploid cell cluster. This method seems to be suitable to obtain pure cell fractions even when dealing with numerically large populations.

Animals↗
PubMed1988

Lipid mediated signal transduction in the cell nucleus.

Cell growth and differentiation can be affected by the transduction of extracellular signals involving cyclic nucleotides, inositol phospholipids and phospholipid dependent protein kinase C systems. Since we previously reported existence of lipids inside the nucleus and nuclear fractions, it seems of interest to examine the possible presence of the cascade of inositol lipids in isolated nuclei as well as the presence of the protein kinase C, whose activity is tightly related to the phosphoinositide cycle, and requires the presence of phosphatidylserine, which has been previously demonstrated to deeply affect nuclear structure and function. Here we show that highly purified nuclei from both rat liver and Friend cells, free of nuclear membrane, can incorporate radiolabel from ATP-[32P] into phosphatidic acid, phosphatidyl-inositol phosphate and phosphatidylinositol (4', 5')bisphosphate. The degree of radiolabelling of phosphatidylinositol bisphosphate is highly dependent on the state of differentiation of the cells. Moreover, a doublet of immunoreactive bands has been identified in rat liver nuclei by means of a polyclonal antibody against protein kinase C. The two polypeptides appear to be tightly bound to the nuclear matrix. These two forms of the enzyme might be translational products specifically located in the nucleus, involved in the transduction to the genomic apparatus of regulatory signals generated by growth factors and tumor promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Membrane-bound immunoglobulins increase during red blood cell aging.

A flow cytofluorimetric method was used to detect the immunoglobulin molecules present on human red blood cells. Normal human erythrocytes were separated into seven fractions of increasing mean age by density centrifugation on discontinuous gradients. Some biochemical and morphological properties of these cells were determined as well as their IgG content. The results obtained suggest that IgG binding during red cell aging is a cumulative process.

Erythrocyte Aging↗
PubMed1988

Chromatin structural transitions following histone H1 displacement by phosphatidylserine vesicles and low pH treatment. A multiparametric analysis involving flow cytometry, electron microscopy, and nuclease digestion.

We describe several morphological and functional modifications in isolated rat liver nuclei incubated in the presence of phosphatidylserine (PS) multilamellar vesicles (MLV). These effects, which occur through the release of histone H1, induce chromatin decondensation, as shown by electron microscopy and nuclease digestion. Flow cytometry was employed to monitor these changes in chromatin structure in isolated nuclei by means of perpendicular light scatter (PLS) and fluorescence signals. Chromatin decondensation induced by PS or by low pH treatment was accompanied by an increase in perpendicular light scatter and by less efficient binding of ethidium bromide. These flow cytometric findings are peculiar to chromatin decondensation induced by displacement of histone H1. Conversely, chromatin decondensation caused by lowering of the divalent ion concentration, without displacement of histone H1, is characterized only by an increase in perpendicular light scatter.

Animals↗
PubMed1988

1H-NMR relaxation study of the liposome--nuclei interaction.

Phospholipidic liposomes can influence DNA template availability for RNA synthesis when interacting with isolated nuclei by enhancing (when the phospholipids are negatively charged) or by inhibiting (when they are neutral) trascriptional activity. The analysis of the 1H-NMR relaxation behaviour of isolated nuclei, of Small Unilamellar Vesicles (SUV) and of the SUV-Nuclei system has been undertaken to elucidate the mechanism of the interaction between the nuclear core and the phospholipids.

Animals↗
PubMed1987

Synthesis of polyphosphoinositides in nuclei of Friend cells. Evidence for polyphosphoinositide metabolism inside the nucleus which changes with cell differentiation.

Previous work demonstrated the existence of phosphatidylinositol kinase and phosphatidylinositol phosphate kinase in rat liver nuclei, with the suggestion that these activities are in the nuclear membrane [Smith & Wells (1983) J. Biol. Chem. 258, 9368-9373]. Here we show that highly purified nuclei from Friend cells, washed free of nuclear membrane by Triton, can incorporate radiolabel from [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate. The degree of radiolabelling of phosphatidylinositol bisphosphate is highly dependent on the state of differentiation of the cells, being barely detectable in growing cells and much greater after dimethyl sulphoxide-induced differentiation; this difference is mostly due to different amounts of phosphatidylinositol phosphate in the isolated nuclei. We suggest that polyphosphoinositides are made inside the nucleus and that they have a role in chromatin function; either the phospholipids themselves play a role, or there is a possibility of intranuclear signalling by inositide-derived molecules.

Animals↗
PubMed1987

Immunochemical characterization of protein kinase C in rat liver nuclei and subnuclear fractions.

A doublet of immunoreactive bands has been identified in rat liver nuclei, nuclear matrix and lamina by means of a polyclonal antibody against protein kinase C. The two polypeptides show an apparent molecular weight of 77 and 74 kDa on SDS-polyacrylamide gels, and appear to be tightly bound nuclear components, resistant to detergent and high salt extraction. Given the complexity of the genes encoding for protein kinase C, these two forms of the enzyme might be translational products specifically located in the nucleus, involved in the transduction to the genomic apparatus of regulatory signals generated by growth factors and tumor promoters.

Animals↗
PubMed1987

Use of poligonal windows for physical discrimination among mononuclear subpopulations in flow cytometry.

Peripheral blood mononuclear cells from ten healthy normal donors were analyzed by means of monoclonal antibodies and flow cytometry (FACS IV). In order to apply optimal gating for the identification and exclusion of monocytes from lymphocyte populations, mononuclear cells were analyzed on the scattergram using rectangular or poligonal computer-generated windows. Two main windows on lymphoid population were used which mainly differed for the up-right corner, in the border area between lymphocytes and monocytes. While the number of lymphocytes contained either in the regular or in the poligonal windows was the same, the number of contaminating monocytes decreased by two-fold in the poligonal one. Besides, the use of tight lymphocyte gating, in order to reach lower monocyte contamination, leads to a loss of lymphoid cells which does not appear to be random, but seems to affect mainly the Leu11c+ population with natural killer activity. These cells produce forward and perpendicular scatter signals higher than other lymphocyte subsets, and, therefore, are mainly located in the area of the scattergram which divides lymphocytes from monocytes. These data are in accordance with the large granular morphology of natural killer cells. The use of the poligonal windows seems to be useful to reduce monocyte contamination with no selective loss of natural killer lymphocytes, and may be particularly helpful in the analysis of pathological samples.

Antibodies, Monoclonal↗
PubMed1987

Flow cytometric analysis of isolated rat liver nuclei during growth.

The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non-parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.

Animals↗
PubMed1987

Unfolding of nucleosome core induced by phosphatidylserine.

The main experimental findings on the actual presence of lipids among the minor chromatin components are revised and discussed especially in the light of the reported effects that exogenous lipids induce in DNA and RNA synthesis by using purified templates. Moreover, all the available evidence of the influence of phospholipid liposomes on the activities and structure of isolated nuclei are reported. In order to further clarify the possible mechanism by which phospholipids could affect gene expression, the modifications at the nucleosome core level have been investigated by means of IAF staining and electron microscopy. The results obtained indicate that the increased transcriptional activity induced by PS MLV in isolated nuclei requires both the removal of histone H1, which causes the unfolding of the solenoid into the nucleosome fiber configuration of the chromatin, and the subsequent splitting of the H3 dimer. This latter process, monitored by IAF accessibility to H3 in isolated nucleosomes incubated with PS, causes the transition from the nucleosome to the lexosome structure, which is the configuration favoring the activity of RNA polymerases.

Animals↗
PubMed1987

Influence of phosphatidylserine on endogeneous RNA synthesis in isolated rat liver nuclei.

The possible involvement of anionic phospholipids in the transcriptional process was studied in isolated rat liver nuclei synthesizing RNA in the presence of phosphatidylserine, which was employed in the form of multillamellar liposomes as a means of delivering the lipid to the nuclei in aqueous medium. The divalent ion requirement for RNA synthesis and the properties of the incubation mixture were not significantly modified by the phospholipid, which increased the rate and the extent of the incorporation of 3H-UMP without changing the endogeneous degradation pattern of the product or affecting the activity of a particular RNA polymerase, as indicated by the sensitivity to amanitin. The thin layer chromatography analysis of the alkaline hydrolysates of the RNA showed that the stimulation involved an increase of the total polyribonucleotide elongation rate. The size of the product was essentially unchanged in the presence of phosphatidylserine, as demonstrated by the qualitative overlapping of the sedimentation profiles of control and lipid treated samples in formamide-sucrose gradients. The release of the H1 fraction from intact nuclei occurring with phosphatidylserine indicated that the DNA template availability was increased by a partial removal of the restrictions imposed by histones, as suggested also by the comparison with heparin and Sarkosyl. These evidences, together with the data accumulated on the occurrence of lipids in chromatin and nuclear matrix, and on their changes related to cell growth, differentiation and malignant transformation, allow a better definition of the role that phospholipids might play in regulating the DNA template availability in the cell.

Amanitins↗
PubMed1987

Changes of chromatin organization induced by phospholipids.

Isolated nuclei represent a suitable model for studying the influence of exogenous phospholipids, normally found as minor chromatin components, on the nuclear structure, which, in turn, could be related to the observed modifications of DNA and RNA synthesis. The morphological modifications induced on chromatin RNP granules and nuclear matrix have been analyzed both with conventional thin sectioning and with an original method based on image analysis of freeze-fractured and replicated nuclear samples. The results obtained support the hypothesis that anionic phospholipids, by removing histone H1, induce a transition of the chromatin from solenoid to nucleosome conformation and favour the RNA polymerizing activity which results in an increased release of RNP particles, while neutral phospholipids, probably affecting the matrix structure, partly impare the RNP maturation and transport, with consequent increase of chromatin condensation.

Animals↗