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Biomedical subjects

F A Manzoli

Publications and source records attributed to F A Manzoli.

At least 91 records · Page 5Linked to original sources
PubMed1987

Increased number of circulating Leu 11+ (CD 16) large granular lymphocytes and decreased NK activity during human ageing.

The phenotype and functional activity of circulating T and natural killer (NK) cells was investigated in a group of selected normal elderly donors in comparison with a group of normal young individuals. Old subjects exhibit a decline in circulating T cells and an impaired response to mitogens (Con A, PHA) associated with a relative increase in cells reacting with Leu 7 and Leu 11a monoclonal antibodies, directed against large granular lymphocytes with NK activity. The rise in circulating NK cells observed in the elderly was not associated with a concomitant increase in NK cytolytic activity against K 562 tumour cells, and time course kinetics of the cytotoxic reaction was similar to that observed in young subjects. When a greater than 95% pure population of Leu 11a+ cells from old individuals was sorted by flow cytometry and their functional cytotoxic activity examined, a significant decrease in NK cytotoxicity was detected. The target cell binding capacity of effector cells, morphologically evaluated at single cell level, did not differ in old and young subjects, even though bound cells from young donors have a higher lytic capacity. These data show that an increase in circulating NK granular lymphocytes occurs during human ageing, but suggest that in old subjects only a subset of NK cells is active in order to maintain a functional response similar to that observed in the young.

Adult↗
PubMed1987

The application of flow cytometry to the study of nuclear matrix. A multiparametric analysis.

The nuclear matrix, which in situ corresponds essentially to the interchromatin ribonucleoprotein particles, once isolated appears constituted by a peripheral lamina connected to the nucleolar remnant by a fibrous network. The features of the matrix components depend on the procedure employed during its purification, a multistep method commonly involving nuclease digestion, low and high salt extractions and treatment with a non ionic detergent. Here is reported the application of flow cytometry to the analysis of matrix purification. The experimental data indicate that, despite deep changes of the nuclear content due to the selective extraction of almost all nucleohistone and membrane components, the scatter pattern of the matrix, which is drastically lowered as the nucleic acid content decreases, closely resembles that of the starting nuclei. This behaviour permits one to follow the different matrix populations deriving from diploid and polyploid parenchymal liver nuclei throughout the isolation procedure and confirms the feasibility of flow cytometry for both analysis and sorting of nuclei and nuclear matrices. In addition, an ultrastructural analysis on thin sections and a morphometric study by means of semiautomated image analysis of phase contrast micrographs have been performed. The image analysis showed a decrease in particle dimension to about 50% of the nuclear value, which could partially explain the reduction of forward light scatter.

Animals↗
PubMed1986

Flow cytometric analysis of liposome-nuclei interaction: transfer and intranuclear release of carboxyfluorescein.

Flow cytometric analysis of the transfer of liposome-encapsulated carboxyfluorescein to isolated rat liver nuclei indicated that the fluorophore is actively taken up in this form, while negligible amounts are transferred when the dye is free in the reaction medium. The kinetic analysis of the uptake indicated a time- and dose-dependent reduction of the slope in the absence of transport saturable sites on the nuclear surface and of quenching phenomena. The comparison between entire and membrane-deprived nuclei demonstrated that the initial rate of uptake was higher in the absence of the complete nuclear envelope. The intranuclear binding sites were considered on the basis of the fluorescence distribution and of quantitative estimates of the amount of linked dye. The possibility of employing flow cytometry to monitor the interactions between liposomes and isolated nuclei by means of a fluorescent probe is discussed.

Animals↗
PubMed1986

Morphometric analysis and topological organization of nuclear matrix in freeze-fractured electron microscopy.

The ultrastructural organization of nuclear matrix, purified from intact or membrane-denuded rat liver nuclei, has been analysed by means of freeze-fracturing technique. This method avoids dehydration and embedding which, in conventional thin sectioning, partly distort or mask the matrix ultrastructure. The various matrix components, and mainly the peripheral lamina and the inner network revealed complex arrangements undetectable with conventional techniques. Morphometric analyses performed with a Texture Analysis System (TAS) Leitz, allowed to obtain precise information on the matrix constituents, based on the histograms of their size distribution. These textural characteristics have been utilized in order to identify, by means of a particular computer programme, the putative matrix localization within intact freeze-fractured nuclei.

Animals↗
PubMed1986

Changes in nucleosome structure and histone H3 accessibility. Iodoacetamidofluorescein labelling after treatment with phosphatidylserine vesicles.

The accessibility of the sulfhydryl-specific dye 6-iodoacetamidofluorescein (IAF) to H3 histone has been studied in isolated rat liver nuclei and mononucleosomal core particles after treatment with phosphatidylserine (PS) multilamellar vesicles (MLV). In isolated nuclei, despite the enhancement of total RNA synthesis and the massive chromatin decondensation produced by liposomes, the amount of histone H3 which can be labelled with the dye remains essentially the same in PS-treated as in control nuclei. However, when mononucleosomal core particles, treated with PS vesicles, are reacted with IAF, H3 becomes derivatized by the dye, while controls do not. These data provide additional evidence that the metabolic and structural changes observed in isolated nuclei treated with PS MLV, are due mainly to the reported removal of histone H1. Moreover, the experiments reported confirm the usefulness of IAF in studying the changes of nucleosome organization, since PS is able to affect the nucleosomal core configuration in isolated nucleosome particles, derivatizing the buried cysteine groups of H3 histone.

Animals↗
PubMed1986

Effect of phospholipids on transcription and ribonucleoprotein processing in isolated nuclei.

The response of isolated rat liver and murine erythroleukemia nuclei to phospholipid liposomes has been monitored with different techniques, by studying the endogenous RNA synthesis, the release of transcripts in the medium, the pattern of acid-extractable nuclear proteins and the ultra-structural morphology. Total transcription in rat liver and beta-globin mRNA synthesis in MEL nuclei are increased by PS and reduced by PC. These changes of RNA polymerase activity, and the transport of RNAs from nucleus as well as the nuclear protein changes, correlate with structural transitions which occur in both types of nuclei, consisting of euchromatization with loss of RNP particles in the case of PS and opposite effects with PC. The significance of these modifications in relationship to the possible involvement of phospholipids in the control of gene expression is discussed.

Animals↗
PubMed1986

Morphometric and topologic analysis of freeze-fractured interphase nuclei.

Interphase rat liver nuclei were studied by freeze fracturing followed by electron microscopic observations. This method permits information on the native organization of the nuclear components in the hydrated state to be obtained. Morphometric analyses, performed with a Leitz Texture Analysis System, gave precise information on the different nuclear components, based on the histograms of their size distribution in heterochromatin, interchromatin and nucleolar areas. The textural characteristics were analyzed by computer to determine the topologic distribution of the solenoid chromatin fibers, the nucleosome filaments and the ribonucleoproteins in the different nuclear domains.

Animals↗
PubMed1985

Response of isolated nuclei to phospholipid vesicles: a two dimensional gel electrophoresis analysis of H2SO4 soluble nuclear proteins after treatment with phosphatidylserine.

H2SO4 soluble proteins extracted from nuclei incubated with phosphatidylserine multilamellar vesicles (PS MLV) have been analysed by means of two-dimensional gel electrophoresis with particular attention to the uH2A fraction. A reduction of H1, H1 degrees and proteins A5, B7, B15 and B23 has been observed in lipid treated nuclei, while the core histones, as well as uH2A are unaffected by liposome treatment. Since these proteins show in vitro the same binding affinity for PS, their behaviour appears to be related to difference in localization in the nucleosome, responsible for their variable accessibility in the chromatin. These results might explain how this phospholipid induces a decondensation of chromatin and a stimulation of RNA synthesis.

Animals↗
PubMed1985

Increase of globin RNA synthesis induced by phosphatidylserine liposomes in isolated erythroleukemic cell nuclei. Morphological and functional features.

The effect of phospholipid vesicles on chromatin structure, protein composition and globin RNA synthesis has been analysed in nuclei isolated from murine erythroleukemia cells. In terms of chromatin organisation, PC vesicles with neutral surface charge do not affect the structure of chromatin fibres, whereas negatively charged PS vesicles induce chromatin decondensation to a great extent. Indeed the fibres appear uniformly dispersed lacking also the perinucleolar heterochromatin. These morphological features are accompanied by depletion of lysine-rich histones H1 and H1(0) and of histone-like protein A5, due to PS liposomes. Functionally, PS vesicles induce enhancement both of total RNA and specific mRNA synthesis, as analysed by in vitro transcription of beta globin gene. On the contrary, PC vesicles do not seem to affect significantly total RNA and globin mRNA synthesis. These observations fit well with previous data obtained in other experimental systems, and support both the use of these molecules as probes for chromatin structure and function and their possible involvement in transcriptional events.

Amanitins↗
PubMed1984

Conformational changes of nuclear chromatin related to phospholipid induced modifications of the template availability.

The phospholipid involvement in the regulation of the functional and structural properties of isolated nuclei has been studied by analyzing the composition and the possible function of the nuclear matrix bound phospholipids in rat liver and murine erythroleukemic cells. The digestion of the matrix phospholipids with phospholipases results in the release of essentially all the newly replicated matrix DNA. The exogenous addition of liposomal phosphatidylserine to rat liver nuclei induces chromatin structural changes consisting in a disaggregation of the heterochromatin, probably mediated by the matrix remodeling, and in a transition from the solenoid fiber to the nucleosome filament, due to the removal of the histone H1. These effects occur through a direct interaction of phospholipid molecules with the inner nuclear components, as demonstrated by carboxyfluorescein transfer and electron microscope autoradiography.

Animals↗
PubMed1984

Distribution of acid alpha-naphthyl acetate esterase among human T lymphocyte subsets.

Human T lymphocyte subsets, identified by means of OKT3, 4 and 8 monoclonal antibodies, were isolated by a fluorescence activated cell sorter (FACS IV) and analyzed for distribution of alpha-naphthyl acetate esterase (ANAE) activity. As compared to OKT8+ lymphocytes a higher proportion of OKT4+ lymphocytes was ANAE-positive exhibiting a spot or dot-like pattern in the cytoplasm. OKT8 and 4 positive subsets showed a similar ANAE distribution in diffuse granular form. Although OKT4 and OKT8 populations presented a different ANAE dot-like reactivity, this marker did not allow as clear a distinction between them as that reported for TG and TM lymphocytes.

Cell Separation↗
PubMed1984

Response of isolated nuclei to phospholipid vesicles: analysis of the nuclear proteins after treatment with phosphatidylserine and phosphatidylcholine and comparison with heparin.

Phospholipid liposomes affect the histone pattern of isolated rat liver nuclei. Multilamellar vesicles (MLV) obtained with phosphatidylserine (PS) release a large amount of the lysine rich histones, while those obtained with phosphatidylcholine (PC) do not induce significant changes with respect to controls. This different response has been compared to the effects obtained with Heparin, which slightly modifies the relative ratio of the histone fractions. These data might account for the mode by which phospholipids induce transitions of the chromatin structure and changes of the endogenous RNA polymerase activity.

Animals↗
PubMed1984

Response of isolated nuclei to phospholipid vesicles: analysis of chromatin sensitivity to DNase I and micrococcal nuclease.

Phosphatidylserine (PS) and phosphatidylcholine (PC) multilamellar vesicles (MLV) affect chromatin structure as analysed by DNase I sensitivity. The kinetics of DNA solubilisation during the digestion of nuclei indicates that phosphatidylserine causes an increase in DNase accessibility while phosphatidylcholine slightly reduces this accessibility. The effect of phosphatidylserine has also been analysed by means of isokinetic sucrose gradients and agarose gel electrophoresis of nuclear DNA solubilised by micrococcal nuclease. This analysis indicates that phosphatidylserine induces a very rapid production of mononucleosome subunits as compared with untreated nuclei.

Animals↗
PubMed1982

Role of chromatin phospholipids on template availability and ultrastructure of isolated nuclei.

The influence of phospholipid vesicles has been tested on isolated nuclei by evaluating the endogenous DNA-dependent RNA polymerase and the chromatin ultrastructure. Negatively charged phosphatidylserine liposomes have a stimulating effect on RNA synthesis, while the vesicles obtained with the neutral sphingomyelin, phosphatidylethanolamine and phosphatidylcholine, and the acidic phosphatidylinositol, are inhibitory. The enhancement of transcription by phosphatidylserine seems to be due to both elongation and initiation of RNA transcripts and very likely depends on interactions of the lipid with the template rather than with the enzymes. The morphological analysis indicates deep changes of the chromatin organization, mainly concerning the size and distribution of the fibers, without any variation of the nuclear volume. The rearrangement of the chromatin could account for the variations of the template availability for RNA synthesis induced by the vesicles and might be associated with changes of the nuclear matrix.

Animals↗
PubMed1981

[Karyotype analysis during development of chronic myelocytic leukemia].

We report cytogenetic data concerning 9 patients with chronica granulocytic leukemia and eosinophilia at the onset and during the chronic phase of the disease before the blastic crisis. The cariotype analises in the intermediate phases allowed to determine the first changes which could be involved in the evolutionary events of the disease up to the moment in which the typical markers of the blastic crisis can be found.

Adult↗
PubMed1981

Effect of phospholipid vesicles on endogenous RNA polymerase activity of isolated rat liver nuclei.

Negatively charged phosphatidylserine vesicles increase RNA polymerase activity of isolated rat liver nuclei, whereas inhibition is obtained with sphingomyelin, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol vesicles. The effect of phosphatidylserine on template activity is dose-dependent and similar to that reported for acidic polymers like heparin. These data indicate that the nature of the polar head is mainly responsible for the different effects and suggest that the phospholipids may be involved in mediating gene expression.

Animals↗
PubMed1981

Non-histone chromatin proteins in human thymocytes and T lymphocytes from blood.

The composition of nuclear proteins from human thymocytes and T lymphocytes from peripheral blood was analyzed. Total thymocytes and total peripheral blood T lymphocytes differed markedly in non-histone chromatin proteins (both phosphorylated and non-phosphorylated), but did not differ in histones. When the cells were separated according to density, T-lymphocyte fractions with a close specific gravity showed restricted differences in non-histone chromatin patterns.

Cell Count↗