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C Pellicciari

Publications and source records attributed to C Pellicciari.

At least 55 records · Page 3Linked to original sources
PubMed1995

Study of genome organization by DNA cytochemistry and fluorescence imaging: a review.

DNA analysis by quantitative cytochemistry has been a widely exploited approach to investigate chromatin superstructural changes in relation to cell function and cell cycle progress. To this aim, a number of dyes (especially fluorochromes) for nucleic acids have been used, exhibiting different peculiarities, in terms of both binding mechanism and base specificity. Less attention has been paid to the application of different DNA staining techniques for studying possible differences in genome organization among different taxa. The purpose of this paper is to review and discuss the present reports in the literature, concerning the cytochemical analysis of genome organization, in a comparative perspective. Special attention is given to the integration of quantitative studies based on cytofluorometric and imaging techniques.

Animals↗
PubMed1994

Propidium iodide and the thiol-specific reagent DACM as a dye pair for fluorescence resonance energy transfer analysis: an application to mouse sperm chromatin.

The dyes N-(-7-dimethyl-amino-4-methyl-coumarinyl) maleimide and propidium iodide, specific for the thiol group and DNA, respectively, were considered as a donor-acceptor couple suitable for investigating "in situ" the relative spatial distribution of DNA and protamines in mouse spermatozoa chromatin. The two dyes are characterized by favourable spectral properties, so that a simplified analytical procedure, based on the measurement of both donor and acceptor emission in double-stained samples, can be applied to evaluate the relative efficiency of the energy transfer process and its topological distribution. The results obtained indicate that during the maturation process: 1) the basic arrangement of protamine-DNA complex does not undergo structure changes, and 2) the oxidation of sulfhydryl to disulfide groups, resulting in chromatin stabilization, first involves the protamine thiols spatially closer to DNA. Fluorescence energy transfer imaging suggests that chromatin stabilization starts in the midportion of the sperm head, then spreads towards the periphery.

Animals↗
PubMed1993

Cell cycle kinetic effects of tamoxifen on human breast cancer cells. Flow cytometric analyses of DNA content, BrdU labeling, Ki-67, PCNA, and statin expression.

Tamoxifen is known to inhibit the growth of some human mammary carcinoma cells; this effect is accompanied by a decrease in the proportion of cells synthesizing DNA. In this work, flow cytometry of DNA and of bromodeoxyuridine labeling and the evaluation of the cell cycle-related antigens Ki-67, PCNA, and statin were used to investigate the changes in the proliferation kinetics of MCF-7 cells before and after treatment with 10(-7) M TAM. The treatment with TAM induced a significant decrease in the fraction of S-phase cells and an increase in those with a DNA content typical of G0/1 phase. The TAM-induced block in G0/1 is paralleled by a decrease in the frequency of cells expressing Ki-67 and PCNA, and by an increase in statin-positive (G0) cells. These results confirmed that the TAM-induced inhibition of cell growth is associated with major changes in the cell cycle parameters of MCF-7 cells, and provide the first experimental evidence that two main mechanisms are operating: the accumulation of cells in G1, before the onset of S-phase, and the exit of some cells from the cycling compartment.

Biomarkers, Tumor↗
PubMed1993

Genome size and qualitative and quantitative characteristics of C-heterochromatic DNA in Eulemur species and in a viable hybrid.

The amounts of nuclear DNA and the AT and GC content of four Eulemur (Prosimii, Lemuridae) species and of an E. coronatus x E. macaco hybrid were measured by flow cytometry in peripheral blood leukocytes, following propidium iodide, Hoechst 33258, and mithramycin staining. Hoechst 33258 and mithramycin were also used to evaluate the base composition of genomic DNA in the chromosomes. The amount of DNA resisting C-banding pretreatment (C-heterochromatic DNA) was measured in metaphase chromosomes by static fluorometry. The genome of E. coronatus was significantly larger than the genomes of all other species examined, due to a higher content of pericentromeric, mainly GC-rich, heterochromatic DNA. The restriction banding patterns produced by BamHI digestion and ethidium bromide staining on extracted DNA were studied in the hybrid and its parental species (E. coronatus and E. macaco). The restriction banding pattern of the sole E. coronatus individual showed two bands which were repeated in the restriction banding pattern of the hybrid. The qualitative and quantitative differences of C-heterochromatic DNA in E. coronatus confirm the "splitting" processes and the phylogenetic relationships in the genus Eulemur suggested by Jung et al. (1992) on the basis of the restriction banding patterns produced by endonuclease digestion.

Animals↗
PubMed1993

A single-step staining procedure for the detection and sorting of unfixed apoptotic thymocytes.

In this study we describe a cytometric method to sort apoptotic cell fractions, suitable for biochemical and morphological analyses. Rat thymocytes were used as a model system, as apoptosis naturally occurs in the thymus, where the negative selection of the T cell repertoire takes place. Massive apoptosis was induced in vitro by the topoisomerase-II inhibitor, etoposide. After etoposide treatment, a large fraction of thymocytes showed the morphological and electrophoretic features of apoptotic cells. Unfixed thymocytes were stained for 30 min with propidium iodide (PI: 50 micrograms/ml containing RNase type A and detergent), and analyzed by flow cytometry. Apoptotic thymocytes typically showed sub-G1 DNA contents. Compared to non-apoptotic thymocytes, sub-G1 cells had lower values of low-angle (FSC) scatter and higher values of right-angle (SSC) scatter, so that, in dual parameter cytograms of FSC versus SSC, two distinct cell populations were apparent, and were separated by flow sorting. The purified cell fractions obtained by this procedure had a very well preserved ultrastructural morphology; both early and late apoptotic stages (until the onset of cytoplasm segmentation) were present in the sorted apoptotic fraction.

Animals↗
PubMed1991

A 33 kDa protein band is enhanced during long-term adaptation of EUE cells to a hypertonic medium.

A cell line derived from human embryonic epithelium (EUE cells) shows an enhanced expression of a 33 kDa protein when adapted to grow in a hypertonic medium containing 0.246 M NaCl (1.8 x the isotonic concentration). The maximum amount of this protein, followed by SDS-PAGE electrophoresis, was found after 4 days of adaptation; thereafter, the protein band remained fairly constant up to 30 days. When the cells were transferred back to a medium containing 0.137 M NaCl (isotonic medium), the protein pattern reverted to that of control cells. This protein is mainly localized in the cytosol, although a small part is associated with the 150,000 g pellet and needs detergents to be extracted. The molecular weight and the cellular location suggest a possible analogy with the so-called amphitropic proteins, that are known to interact with both the epidermal growth factor receptor and hydrophobic structures, such as the membrane phospholipids and the cytoskeletal components.

Adaptation, Physiological↗
PubMed1991

Proliferating cell nuclear antigen (PCNA)/cyclin expression during the cell cycle in normal and leukemic cells.

Bivariate flow cytometric analysis of the cell proliferation-associated nuclear protein, identified as the "proliferating cell nuclear antigen" (PCNA)/cyclin and of nuclear DNA content, was performed in quiescent and mitogen-stimulated human peripheral blood lymphocytes, in EUE (human embryonic epithelium) cells, before and after a long-term exposure to a hypertonic (HT) medium, in 4 human leukemic cell lines and in fresh bone marrow (BM) cells from 10 patients with untreated acute non-lymphoblastic leukemia (ANLL). The PCNA/cyclin was detected using both an autoantibody extracted from sera of systemic lupus erythematosus patients and the recently produced mouse monoclonal antibody (MoAb) IgG, named 19F4. The distribution of cells in the different phases of the cycle and the percentage of S-phase cells were obtained in duplicate samples, by DNA flow cytometry (FCM) and by dual parameter FCM of DNA content and bromodeoxyuridine (BUDR) incorporation. In all cell types, the non-specific cytoplasmic background fluorescence was significantly lower with the MoAb compared to that obtained with the polyclonal Ab. The percentage of PCNA-positive cells (both with the autoantibody and the 19F4 MoAb) was always higher than that of S-phase cells by DNA FCM and of BUDR-labeled cells. The pattern of PCNA-expression in both normal proliferating cells and acute leukemia cells, showed that most G0/G1 cells did not express significant amounts of PCNA; an increase in PCNA immunofluorescence was found in late G1 cells, and further increases were observed in S- and G2-M phase cells. PCNA/cyclin, as revealed both with autoantibodies and with the 19F4 MoAb, is associated with all actively or potentially dividing (i.e. G1, S and G2-M) cells thus identifying the proliferative cellular compartment. Combined with the use of multiparameter FCM techniques, the PCNA immunolocalization offers a useful tool to study cell kinetics in normal and leukemic human cell populations.

Antibodies, Monoclonal↗
PubMed1991

Expression of cell cycle related proteins--proliferating cell nuclear antigen (PCNA) and statin--during adaptation and de-adaptation of EUE cells to a hypertonic medium.

EUE cells adapted to grow for long times in a hypertonic medium have a longer cell cycle than those growing in isotonic medium. To elucidate whether this lengthening involves specific cycle phases to differing extents, the expression of two cycle-related protein, PCNA and statin, was studied by dual parameter flow cytometry of indirect immunofluorescence protein labelling and DNA content. In isotonic medium, most cells, in all the cycle phases, were PCNA positive; in contrast, PCNA negative cells and statin positive cells were very few in number and only fell in the G0/1 range of DNA contents. In hypertonic medium, the frequency of PCNA positive cells was lower, and that of statin positive cells higher, than in isotonic medium, particularly in the G0/1 range of DNA contents: this suggests that a G0 block occurs under long-term hypertonic stress. Consistently, dual parameter flow cytometric measurement of BrdUrd immunofluorescence labelling and DNA content showed that fewer cells entered S phase in hypertonic medium and their progression through the S phase was slower; evidence was also found for the occurrence of a G2 block. These kinetics changes were fully reversible in isotonic medium, thus indicating the adaptive nature of the EUE response to hypertonicity.

Adaptation, Physiological↗
PubMed1991

Changes in cell cycle and chromatin distribution in C6 glioma cells treated by dibutyryl cyclic AMP.

C6 glioma cells in culture were treated with 1 mM dibutyryl cyclic AMP (Db-cAMP) for 5, 8, 24 and 72 h. The cells were labelled with [3H]-thymidine before either the end, or the beginning, of the Db-cAMP treatment. The cell cycle passage was monitored by the simultaneous determination of DNA content and DNA synthesis in propidium iodide stained autoradiograms. The data revealed an early (t less than or equal to 3-8 h) and moderate inhibitory effect of Db-cAMP on all phases of the cell cycle except mitosis; some cells (2%) were completely blocked in the S phase. Later (8 less than t less than 24-72 h), the cycling of a substantial part of the population became inhibited in G1 phase. Microdensitometric texture analysis of Feulgen-stained nuclei, performed 24 h after administration of Db-cAMP, showed a higher inhomogeneity of the DNA distribution in cell nuclei, caused by the condensation of a part of the chromatin. This may reflect either changes in genome expression taking part in the process of cAMP induced differentiation or transit of some cells into quiescent G0 or S0 phases.

Animals↗
PubMed1990

Cytochemical evaluation of C-heterochromatic-DNA in metaphase chromosomes.

A method is proposed to evaluate the amount of DNA resistant to the C-banding pretreatments (C-heterochromatic-DNA) in metaphase chromosomes. Measurements were performed by microfluorometry on propidium iodide stained metaphases of man, gorilla and mouse; in these species, C-heterochromatin exhibits significant differences of both base composition and distribution along the chromosomes. The amount of C-heterochromatic-DNA was found to be about 16%, 28% and 58% of the total DNA content (genome size) in man, gorilla and mouse, respectively. The areas of C-bands after Giemsa staining were also assessed by microdensitometry, and corresponded to about 8%, 15% and 14% of the total karyotype area of man, gorilla and mouse respectively. No direct relation thus exists between C-band areas and the amount of DNA resistant to the C-banding pretreatments. In man and gorilla, the amount of C-heterochromatic-DNA accounts for the differences observed in genome size.

Animals↗
PubMed1990

Changes of gene expression during long term adaptation of human EUE cells to a hypertonic medium: electrophoretic protein patterns and DNase I digestion in situ.

The effects of long term (1 to 10 days) growth in a hypertonic medium have been studied in human EUE cells. Following polyacrylamide gel electrophoresis, a change in the protein pattern has been found, with the progressive enhancement, during adaptation, of a 33 kDa band. Experiments of DNA digestion in situ by DNase I showed that chromatin DNA of cells grown in a hypertonic medium is more available to the enzyme cleavage. These findings show that long term hypertonic stress is able to induce a change in gene expression in EUE cells.

Cells, Cultured↗
PubMed1990

Cell kinetics of PHA-activated lymphocytes are slowed by prolonged hypertonic stress.

The effect of prolonged exposure to a hypertonic medium on human lymphocytes during mitogenic stimulation with phytohemagglutinin was investigated. The process of chromatin decondensation during the first 24 hrs stimulation (G0 to G1 transition) and the changes in kinetic parameters and the occurrence of chromosome aberrations from 48 hrs to 72 hrs of stimulation were studied. In HT medium, lymphocyte transition from G0 to G1 was slowed; there were fewer S-phase cells, after 48 hrs PHA stimulation, whereas after 72 hrs the resistant cells showed the same frequency of S-phase cells as the controls. The mitotic index was always smaller, and the frequency of G0/G1 cells larger. No significant increase in the frequencies of chromosome aberrations were found. These findings suggest that human peripheral lymphocytes can survive and grow in a hypertonic medium; chromosome damages, if not repaired, may be lethal, and only lymphocytes with normal karyotypes can survive for long times in the HT medium, although with modified kinetic characteristics.

Cell Cycle↗
PubMed1989

Effect of hypertonic medium on human cell growth: III. Changes in cell kinetics of EUE cells.

The effects of hypertonicity on cell kinetics of EUE cells in culture have been investigated. After 4 days of growth in a hypertonic medium, the plating efficiency of EUE cells was reduced and cell growth was significantly slowed. Flow cytometric measurements of DNA content in synchronized cells, as well as flow cytometric determinations of DNA content and bromodeoxyuridine incorporation in asynchronous cells, also showed that the cell cycle is slowed in a hypertonic medium. In addition, the fraction of cycling cells is smaller and their progression through the S phase slower than in an isotonic medium.

Cell Cycle↗
PubMed1989

The harderian gland of the frog, Rana esculenta, during the annual cycle: histology, histochemistry and ultrastructure.

The Harderian gland in Rana esculenta has been studied during the annual cycle at the histological, histochemical and ultrastructural levels. The Harderian gland has an acinar structure and is the only orbital gland in anuran amphibia. It develops at the medial corner of the orbit from the conjunctival epithelium at the premetamorphic stage. In the adult the glandular secretion reaches a maximum during the months of July and August, drops in September and resumes slowly from October onwards. The secretion is seromucoid and the secretory granules are released into the acinar lumen, mainly by exocytosis. Porphyrins were not detected. No sexual dimorphism was observed in the glandular cells. The resumption of secretory activity in October and the enhancement of secretion in May are marked by the appearance of "blue nuclei" (Mallory stain) in a relatively high percentage of glandular cells. This unusual blue colour, using the Mallory stain (by which nuclei stain red), disappears after digestion of paraffin sections with RNAase, but not with DNAase and trypsin. The blue staining may, therefore, indicate an increased amount of nuclear RNA. The Harderian gland in the frog most probably serves to lubricate and moisten the eye in the absence of the lacrimal gland. However, the gland may also represent an immunoactive organ owing to the presence of numerous mast cells and plasma cells in the interacinar spaces.

Animals↗
PubMed1988

Fluorescein mercuric acetate as a dye for fluorescence and electron microscope detection of -SH groups in mouse sperm protamines: a critical evaluation.

Fluorescein Mercuric Acetate (FMA), a specific fluorescent dye for thiols, was tested as a possible reagent for the ultrastructural detection of -SH groups in sperm chromatin protamines. It was shown that FMA may be profitably used for staining at electron microscopy the chromatin -SH groups of caput epididymidis spermatozoa. Like other mercury compounds, FMA proved to be unstable under the electron beam; therefore the use of low beam intensity, and cooling of the specimens are recommended. The main advantages of this staining method are the relatively lower toxicity of FMA, compared to other organic mercurials, and the opportunity to compare the fluorescence patterns on semithin sections with the corresponding ultrastructural findings on thin sections.

Animals↗