Biomedical subjects

E Falcieri

Publications and source records attributed to E Falcieri.

At least 73 records · Page 4Linked to original sources

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

Morphological aspects of steroid hormone action on rat hepatocyte.

A morphological study was performed on the effect of HC treatment on normal and adrenalectomized rat liver. The observation of liver fragments shows that the hormone treatment can balance only partially the hormonal deficiency caused by ADX, resulting in particular changes of both hepatic architecture and fine ultrastructural organization.

Adrenalectomy↗

PubMed1992

A combined ultrastructural approach to the study of nuclear matrix thermal stabilization.

Using mouse erythroleukaemia cells and different ultrastructural techniques, the morphology was investigated of the nuclear matrix obtained after incubation at 37 degrees C of isolated nuclei. If purified nuclei were heated for 45 min at 37 degrees C, the final matrix exhibited well-recognizable nucleolar remnants, an inner network and a peripheral lamina. Without such incubation only the peripheral lamina was seen surrounding homogeneous, finely granular material. Similar results were obtained with both araldite-embedded and freeze-fractured nuclear matrices, although in the latter case the loose granular material was not evident. Observations of araldite-embedded, heat-treated nuclei revealed clumping of heterochromatin in small, very electron-dense masses with large interchromatin spaces. These ultrastructural aspects were even more striking in freeze-fractured nuclei. Cytochemical matrix analysis by osmium-amine staining for nucleic acids and DNase-gold labelling for DNA localization demonstrated that also matrix residual nucleic acids, mostly RNA, are stabilized by heat exposure of isolated nuclei. The results demonstrate that the morphology of heat-stabilized nuclear matrix is not artefactually affected during the preparation for conventional electron microscopy and suggest a possible involvement of nucleic acids in the heat-induced stabilization of the nuclear matrix.

Animals↗

PubMed1992

Changes in polyphosphoinositide levels in rat liver nuclei in response to prolactin, a known hepatic mitogen.

The effect of prolactin action on nuclear polyphosphoinositide synthesis was investigated in isolated rat liver nuclei. An increased uptake of phosphate from [gamma 32P] adenosinetriphosphate was observed in both phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate with a maximum response at 10(-12) M concentration of hormone. Pulse-chase experiments in isolated nuclei following prolactin treatment indicate that the observed increase in accumulation of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate is mainly due to a decrease in their rate of turnover possibly induced by a change in activity of polyphosphoinositide-specific monoesterases. In vitro prolactin also reduces the activity of nuclear phospholipase C specific for phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. Moreover, this feature is strongly supported by the concomitant decrease in nuclear diacylglycerol mass. Thus these data suggest that once prolactin reaches the nucleus an intranuclear signalling is evoked through inositol lipid metabolism.

Animals↗

PubMed1992

Further considerations on the thermal stabilization of the nuclear matrix in mouse erythroleukemia cells.

The morphology and the polypeptide composition of the nuclear matrix obtained from 37 degrees C incubated nuclei has been studied in mouse erythroleukemia cells. From a structural point of view, in the absence of heat treatment, the matrix lacked identifiable nucleolar remnants and the internal fibrogranular meshwork whereas a peripheral lamina was seen. On the contrary, the matrix obtained from heat exposed nuclei displayed very electrondense nucleolar remnants and an abundant inner network. These results were obtained irrespective of the type of extracting agent (2M NaCl or 0.2 M (NH4)2SO4) used to remove histones and other soluble proteins. The heat stabilization of the matrix could not be prevented by sulfhydryl blocking chemicals such as iodoacetamide and n-ethylmaleimide, thus suggesting that heat does not stabilize the matrix by inducing the formation of disulfide bonds. Only limited differences in the polypeptide pattern of matrix isolated under different conditions were seen using one-dimensional pore gradient polyacrylamide gels stained with both Coomassie Brilliant Blue and silver despite the fact that the matrix fraction from heat treated nuclei retained about three fold more protein in comparison with controls. The same results were obtained also by means of two-dimensional non-equilibrium gel electrophoresis.

Ammonium Sulfate↗

PubMed1991

Heat-induced stabilization of the nuclear matrix: a morphological and biochemical analysis in murine erythroleukemia cells.

Using mouse erythroleukemia cells we performed a comprehensive morphological and biochemical study of the nuclear matrix obtained after exposure of isolated nuclei to 37 degrees C or from cells heat shocked in vivo at 43 or 45 degrees C. At the ultrastructural level it was possible to see that in the absence of a 37 degrees C incubation of purified nuclei, the final matrix lacked well-defined nucleolar remnants but a peripheral lamina was clearly visible, as well as a sparse fibrogranular network which was located at the periphery of the structures. On the contrary, after a 37 degrees C nuclear incubation, very electron-dense nucleolar remnants were observed along with an abundant meshwork dispersed throughout the interior of the structures. When intact cells were heat shocked in vivo, electron-dense residual nucleoli were present only when isolated nuclei had been exposed to 37 degrees C in vitro, whereas without such an incubation, they were not as easily distinguishable and appeared less electron-dense. In the latter case the inner network was more evenly distributed. After purified nuclei were incubated at 37 degrees C for 45 min, the high salt and DNase I resistant fraction retained about 18% of the nuclear protein whereas if the heating was omitted protein recovery dropped to 6%. An increase in the recovery of intact structures in the matrix fraction was the main reason for the higher protein recovery. Heating nuclei in vitro further increased the amount of nuclear protein present in the matrix fraction even if intact cells had been heat shocked in vivo. No major qualitative differences were seen when the polypeptide pattern of the various types of nuclear matrices was analyzed on one-dimensional polyacrylamide gels and this finding was further supported by Western blot analysis with a monoclonal antibody to lamins A and C. These results show that heating mainly stabilizes the nucleolar remnants of the matrix and to a lesser extent the inner network, but the morphology of the final structures is different depending on whether the stabilization is performed in vivo or in vitro.

Animals↗

PubMed1990

Membrane modifications in the course of hepatocyte isolation.

A transmission E/M, scanning E/M and freeze fracture ultrastructural study has been performed on the rat hepatocyte in the course of isolation from the liver parenchyma. The cell submicroscopic aspect indicates a good morpho-functional preservation from the liver perfusion to the final stages of cell isolation. The freeze fracture membrane analysis evidentiates the constant presence of gap junctions and tight junctions, characterized by particular structural alterations, probably due to progressive functional uncoupling. The persistence of these cell differentiations until complete cell isolation may be considered a further morphological expression of the maintenance of the differentiated stage of the hepatocyte. Fragments of membranes from adjacent cells, still adherent to isolated hepatocyte surfaces, can also be occasionally detected by freeze-fracture techniques.

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

Image analysis of the chromatin organization in the nuclear domains of freeze fractured hepatocytes and lymphocytes.

The complex organization of the interphase nucleus can be analyzed, by way of thin sectioning and also freeze-fracture. This approach has previously been utilized in association with image analysis to quantitatively describe the organization of isolated rat liver nuclei and nuclear matrices. The main nuclear domains which, in section, present marked differences due to their electron-density, can be identified in replicas with more complex procedures, based on the quantitative evaluation of the number of particles per unit area and mainly by using image analysis. A quantitative analysis of the nuclear substructures has been performed by way of image analysis on in situ nuclei of freeze-fractured cells presenting marked differences in the heterochromatin quantity, such as hepatocytes and lymphocytes. The replicated nuclear particles have been classified according to their diameter and the obtained histograms have been quantitatively evaluated. The nuclear domains, heterochromatin, interchromatin, nucleolus, present characteristic ratios among the three main classes of particles; that is, ribonucleoproteins, solenoid filaments and solenoid fibre aggregates. The typical patterns of the nuclear domains can be further stressed by selecting a single class of particles and by examining its topographic localization. While interchromatin and nucleolar domains present a similar quantitative pattern in hepatocytes and lymphocytes, the heterochromatin of lymphocytes contains a significative higher percentage of solenoid aggregates than that of hepatocytes.

Animals↗

PubMed1990

A preliminary study of human pineal gland concretions: structural and chemical analysis.

Acervuli and fragments of pineal gland obtained from 33 subjects of both sexes and age ranging from 1 to 87 years, (30 autopsy and 3 biopsy specimens) were analyzed by light microscopy, transmission and scanning electron microscopy, X-ray diffraction and X-ray energy dispersive microanalysis. It was found that primary mineralization occurs in an organic matrix formed by pinealocytes and that hydroxyapatite also takes place in mineral deposition. From our analysis, the formation of acervuli appears to be age and sex independent and can be possibly related to the secretory activity of the gland.

Adolescent↗

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

Natural killer function in flow cytometry. II. Evaluation of NK lytic activity by means of target cell morphological changes detected by right angle light scatter.

Morphological changes that occur in K562 cells after natural killing produce profound changes in cellular light scattering properties. The possibility of gating out all the effector cells by thresholding on perpendicular light scatter and the subsequent identification of two distinct clusters of cells, which correspond to dead and viable targets, have permitted the measurement of natural killer activity in vitro. The changes in scattering properties after cell death are mainly determined by the variation of internal refractive index of the dying cell. A comparison of the scattering and propidium iodide staining procedures showed good correlation. The morphological detection and measurement of cellular death is therefore used to estimate NK lytic activity. This methodology permits the measurement of NK activity without staining the target and the measurement of perpendicular light scatter provides an alternative approach to the study of lytic processes in vitro.

Cytotoxicity, Immunologic↗

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↗

PubMed1989

Histochemical and ultrastructural study of the human pineal gland in the course of aging.

A histochemical and ultrastructural study was carried out on the pineal gland of 2 day- to 86-year-old subjects. Specimens were obtained by stereotaxis in the course of neurosurgery; other specimens were autoptical pineal fragments. Histochemical and ultrastructural analyses showed lipofuscin pigments, frequently localized in the proximity of blood capillaries and of cytoplasmic microacervuli. The brain sand amount does not appear to be age-related because microacervuli are barely present or frequently absent in the oldest subjects. Its involvement in the secretory activity rather than in gland atrophy is also suggested. The close relationship between cytoplasmic microacervuli and intermediate filaments suggests a possible role of the cytoskeleton in the formation of the brain sand.

Aged↗

PubMed1988

A high yield technique for freeze-fracturing of small fractions of isolated cells.

A simple, high-yield technique for the freeze-fracturing of small amounts of isolated cells is described. A drop of cells fixed in suspension is deposited on a polylysine-treated coverslip, forming a monolayer through electrostatic forces. After cryoprotection, the coverslip is inverted on a gold carrier covered with Vinol and then frozen in liquid nitrogen. The monolayer will be fractured by advancing the knife under the coverslip. Large areas of cell surface can be exposed despite their low number, such as that obtainable after cell sorting by flow cytometry.

Erythrocytes↗

PubMed1987

Role of bacterial exopolymers and host factors on adherence and phagocytosis of Staphylococcus aureus in foreign body infection.

Using a previously developed guinea pig model of foreign body infection, we examined ultrastructural and functional surface alterations of Staphylococcus aureus strain Wood 46 during the early phase of infection. Exopolymer-free bacteria were prepared and inoculated into subcutaneously implanted tissue cages. After three hours, the bacteria showed abundant capsular and intercellular exopolymers, which were visualized by transmission electron microscopy. Exopolymers were also produced by S. aureus exposed in electron microscopy. Exopolymers were also produced by S. aureus exposed in vitro to fluid from the tissue cage. In contrast, human serum albumin prevented exopolymer production by S. aureus. The influence of exopolymers on the susceptibility of S. aureus to ingestion and phagocytic killing by neutrophils was tested in vitro and found to be negligible. Furthermore, adherence of S. aureus to fibronectin-coated surfaces was unaffected by the presence or absence of exopolymers. Thus, in our experimental model, exopolymers are produced early during the onset of infection, but they have little impact on adherence and phagocytosis.

Animals↗

PubMed1987

Modifications of the chromatin arrangement induced by ethidium bromide in isolated nuclei, analyzed by electron microscopy and flow cytometry.

Ethidium bromide (EB) is widely used for investigating the DNA conformation in chromatin both with conventional and cytofluorimetric techniques. Since the interaction of the dye with DNA should result in structural deformations which can be different in isolated or in situ chromatin, a study has been performed on the effects caused by different amounts of EB and the analogous propidium iodide on isolated nuclei, in which chromatin maintains its native relationships with the other nuclear structures (envelope, nucleolus, interchromatin RNP, nuclear matrix). The results obtained by comparing ultrastructural observations in thin sections and in freeze-fracturing with conformational analysis in multiparameter flow cytometry indicate that the phenanthridinic fluorochromes, especially at the high concentrations used for cytofluorimetric analyses, cause deep rearrangements of the chromatin in situ. These effects consist both in aggregation and condensation of the fibers into the dense chromatin domains, and in an increase of the supernucleosomal configuration associated with an enlargement of interchromatin spaces in which the RNP particles appear particularly evident. These results, discussed with those available on isolated chromatin, suggest that any unwinding effect of the intercalating dyes on the DNA cause a general condensation of chromatin as a consequence of the constraints which characterize the organization of the chromatin inside the nucleus.

Animals↗

PubMed1984

Polyalbumin receptors on hepatitis B virus and on 22 nm hepatitis B surface antigen (HBsAg)2 particles.

Receptors for polymerized human serum albumin ( pHSA ) were studied by solid-phase radioimmunoassay on different hepatitis B surface antigen (HBsAg) particles subpopulations prepared both from hepatitis B e antigen (HBeAg) and from anti-HBe-positive sera. HBsAg particles in HBeAg-positive serum showed higher expression of the receptor compared with HBsAg particles from anti-HBe-positive serum. Analysis of different morphological forms of virus particles was performed after separation by density-gradient ultracentrifugation. Maximum receptor expression was detected in HBV particles containing fractions while the 22-nm HBsAg particles had significantly lower receptor activity. These observations support the hypothesis of a pathogenetic role of the pHSA receptor in mediating virus access to hepatocytes. Indeed, the higher pHSA binding activity on HBV particles could allow selective attachment of the infectious virion to liver cells that bear similar albumin receptors on their surface.

Hepatitis B Antigens↗