Search PubMed⌕ Search

Biomedical subjects

C Nicolini

Publications and source records attributed to C Nicolini.

At least 73 records · Page 4Linked to original sources
PubMed1983

Differential scattering of circularly polarized light as a unique probe of polynucleosome superstructures. A simulation by multiple scattering of dipoles.

A theoretical approach to modeling Circular Intensity Differential Scattering (CIDS) of native chromatin as multiple scattering of dipoles is discussed without the Born approximation. The model can explain the experimental data in the literature. It is shown that CIDS contains more structural information than does total light scattering and to a good approximation is independent of the length of the scattering molecules. Finally, CIDS in conjunction with traditional light scattering measurements should aid in discriminating between various alternative models of higher order chromatin structure now being proposed. Generalization of this theoretical study to other complex biomolecular structures, is also briefly discussed.

Chromatin↗
PubMed1983

The G1 period. Two cycles of chromatin conformational changes monitored by single cell dye intercalation.

In previous papers the existence of two cycles of chromosome condensation-decondensation per cell cycle was suggested based on experiments involving nuclear morphometry measurements of Feulgen-stained nuclei. This conclusion can be criticized since its assumption of a relationship between nuclear morphology and chromatin structure is derived from indirect evidence. In this paper, we report simultaneous measurements of nuclear area and nuclear fluorescence intensity on individual cells stained with the intercalating dye, acridine orange (AO). Using cells in various stages of G1 and synchronized by two different methods, our results demonstrate a linear correlation between nuclear area and fluorescence intensity. They also indicate two cycles of chromatin condensation-decondensation during the G1 period, as assayed by the number of chromatin primary, intercalating AO binding sites. Finally, they show that the first of these cycles involves a transition in early G1 from a very small condensed nucleus (immediately after telephase) to a relatively large, dispersed nucleus that occurs abruptly.

Butyrates↗
PubMed1983

Higher-order structure of chromatin from resting cells. II. High-resolution computer analysis of native chromatin fibres and freeze-etching of nuclei from rat liver cells.

Non-destructive electron microscopy of native chromatin from rat liver nuclei reveals that the 30 nm fibre is formed of four 11 nm nucleofilaments, arranged in a coiled-coil (or rope-like) conformation. At low ionic strength, native fibres show an alternating pattern of compact and unwound regions. Freeze-etching experiments carried out on the same nuclei are compatible with the existence of periodic attachments of the fibres to the nuclear envelope near the pores in a regular, drapery-like fashion. For the first time, computer image analysis has been applied to electron micrographs of giant chromatin fibres and a few essential geometrical parameters characterizing the conformation of the higher-order structures have been determined. No significant difference has been found between calf thymus and rat liver chromatin.

Animals↗
PubMed1982

Biophysical identification and sorting of high metastatic variants from B16 melanoma tumor.

Chromatin structure, in terms of higher order nuclear-DNA condensation (scanning cytometry) and in terms of acridine orange primary binding sites (flow cytometry), is analyzed and shown to be significantly different between high (B16-F10) and low (B16-F1) metastatic variants of B16 melanoma. Furthermore, double staining of B16-F10 and B16-F1 with ethidium bromide (chromatin) and fluorescamine (membranes) provides the identification of a homogeneous subpopulation of cells with enhanced metastatic potential based on differential fluorescamine uptake. Fluorescamine uptake and poststaining viability is shown to be dependent upon the dye/cell ratio at which staining occurs. Utilizing a sterile cell sorting technique, a subpopulation of B16-F10 with increased fluorescamine uptake representing 30% of the total "intact cell" population was isolated by means of a fluorescence activated cell sorter and replated in vitro. This subpopulation when assayed in vivo produced significantly more pulmonary metastases than its parent cell line. Scanning cytometry of the Feulgen stained sorted subpopulation reveals that the cells possess a unique nuclear morphometry characterized by a 2C-3C DNA content and a large nuclear area (disperse chromatin). Finally, when we assay simultaneously for nuclear-DNA organization and cell membrane organization a progressive uncoupling between nuclear and cell morphometry is apparent if B16-F10 (versus B16-F1).

Animals↗
PubMed1982

A comparison of mathematical methods for the analysis of DNA histograms obtained by flow cytometry.

Twelve methods for analysing FCM-histograms were compared using the same set of data. Some of the histograms that were analysed were simulated by computer and some were taken from experiments. Simulated data were generated assuming synchronously growing cell populations and (i) measurement coefficients of variation (CV) from 2 to 16%; (ii) constant measurement CV or VC's increasing from G1 to G2 phase, and (iii) varying fractions of cells in each phase. Simulated data were also generated assuming synchronous cell populations in which a block in early S phase was applied and released. DNA histograms were measured for L-929 cells at various times after mitotic selection. Labelling indices were also measured for these cells at the same time. The fractions of cells in the G1, S, and (G2 + M) phases were calculated by each analytical method and compared with the actual fractions used for simulation, or in case of experimental data, with autoradiographic results. Generally, all methods yielded reasonably accurate fractions of cells in each phase with relative errors in the range of 10-20%. However, most methods tended to overestimate G1 fractions and underestimate S fractions. In addition, variations in the shape of the S phase distribution often caused considerable errors. Phase fractions were also calculated for histograms of kinetically perturbed populations, simulated as well as experimental. The errors were only slightly larger than for histograms from asynchronously growing cell populations.

Analysis of Variance↗
PubMed1982

Early effects of chemical carcinogens as compared to induced cell proliferation. II. Automated image analysis.

Static automated image analysis was applied to study early variations of chromatin structure in Feulgen-stained liver nuclei from rats injected i.p. with a single dose of dimethylnitrosamine (DMNA), a well known hepatocarcinogen. An increase of nuclear area and a correspondent decrease of average optical density (integrated optical density/area) was observed, as compared with controls, in nuclei from rats treated with 5.4 mg/kg of DMNA. These findings, which were comparable with those induced by partial hepatectomy, indicate the existence in DMNA-treated cells of a chromatin DNA relaxation similar to the G0-G1 transition previously described for human diploid fibroblasts stimulated to proliferate. Because similar results were independently obtained by flow microfluorimetry, it seems reasonable to hypothesize that chromatin decondensation could be a prerequisite for cancer induction.

Animals↗
PubMed1982

Early effects of chemical carcinogens as compared to induced cell proliferation. I. Flow microfluorimetry.

Acridine orange-stained rat liver cells have been analyzed by flow microfluorimetry either after i.p. injection of 0.6 and 5.4 mg/kg of dimethylnitrosamine or 18 hr after partial hepatectomy. Treatment with dimethylnitrosamine induced a marked increase in the fraction of cells with higher green fluorescence intensity which can be related to a greater number of chromatin primary binding sites, and a decrease in the fraction of cells with higher red fluorescence intensity corresponding to a lower amount of cellular RNA. Contrary to the carcinogen-induced early changes towards a more relaxed uncoiled structure, as well as an apparent uncoupling between chromatin-DNA structural alterations and total RNA synthesis, could be first events in the carcinogenetic process.

Acridine Orange↗
PubMed1981

DNA replication, chromatin structure, and histone phosphorylation altered by theophylline in synchronized HeLa S3 cells.

The onset of DNA replication normally is coincident with an increase in histone 1 phosphorylation and a relaxation in chromatin structure. In this paper we show that 5 mM theophylline, added 2 h after selective detachment to synchronized HeLa-S-3 cells, delays the onset and reduces the rate of DNA synthesis while theophylline treatment beginning at 8 h has no effect on subsequent DNA synthesis. These actions of theophylline are accompanied by an inhibition of histone 1 phosphorylation and a prevention of the normal relaxation in chromatin structure between G1 and S phases as revealed by image analysis of Feulgen-stained nuclei. The time courses of intracellular cyclic AMP levels, nonhistone protein phosphorylation, and [3H]lysine incorporation are also compared in the same treated and untreated synchronized HeLa cells. Comparison with experiments using 1-beta-D-arabinofuranosylcytosine (Ara-C) shows that the above phenomena are not a direct result of inhibition of DNA synthesis. We interpret our results as evidence that the associations between histone 1 phosphorylation, chromatin relaxation, and the onset of DNA synthesis are temporally and causally related.

Cell Cycle↗
PubMed1981

Correlation between Barr body and overall chromatin decondensation in vitro.

Geometric and densitometric properties of the Barr body of early and late phase II confluent human fibroblasts are analysed by the automated image analyser Quantimet 720 D. In cells with the same 2C DNA content, the state of condensation of the Barr body varies proportionally wih the state of condensation of the entire nucleus yielding a correlation between nuclear and Barr body area. In light of these results, indicating the participation of the Barr body in the overall process of chromatin condensation and decondensation, a definitive "'static" separation between dense ("heterochromatic") and dispersed ("euchromatic") regions of chromatin seems to be arbitrary. The implications of these results in terms of a possible attachment of interphase chromatin to the nuclear envelope are briefly discussed.

Cell Nucleus↗
PubMed1980

Normal versus abnormal cell proliferation. A unitary and analytical overview.

The most recent findings on the molecular and cellular characterization of normal and abnormal cell proliferation are summarized. They include molecular spectroscopy, nucleic acid conformation, protein modifications, premature chromosome condensation, nucleoli changes, nuclear and cell morphometry, image analysis, flow microfluorimetry, and time-lapse cinematography. Biophysical and biochemical evidence in favor or against two cycles of chromatin condensation, followed by two abrupt random decondensations, per cell cycle are presented. Other biphasic changes at the molecular and cellular levels that favor the existence of two random transitions, or restriction points, per cell cycle are discussed. A comprehensive unitary model of the cell cycle is then outlined; this model is able to explain most findings on continuously dividing cells and on quiescent cells induced to proliferate. Within this analytical framework the physical-chemical and biological properties are given, in either normal or tumor cells, for the various types of "noncycling" cells that are here viewed as necessary steps in mammalian cell growth rather than separates states. The implications of the coupling of higher-order chromatin structure with cell geometry and growth, high in fibroblast-like cells but low in transformed cells, are also discussed. Molecular mechanisms likely responsible for the chromatin conformational changes occurring at the G0 leads to G1, G1 leads to S, G2 leads to M transitions are finally discussed in terms of polyelectrolyte theory.

Animals↗
PubMed1980

Reversible (G0) and nonreadily reversible (Q) noncycling cells in human peripheral blood. Immunological, structural, and biological characterization.

PHA-stimulated human lymphocytes (normal-resting-proliferating) at 0, 24, 48, 72, and 144 h were studied with Acridine Orange (AO) staining. By viable cell sorting, by subsequent subculturing, and by the use of biochemical, biophysical, and immunological assays, not only have the G0 resting and G1 (cycling) cell cycle phases been objectively characterized, but a separate subpopulation of quiescent cells that are functionally viable and deeply committed to nonproliferation, the Q cells, has been identified. Multiparameter cytofluorimetric analysis, methyl14C-thymidine incorporation, automated image analysis, and mitogen stimulation studies have shown that the "Q" cell, compared to the "G0" resting but easily recruitable cell, exhibits quite lower red and green AO emission, possesses 2c to 4c DNA content (rather than only 2c), has a higher average optical density, and is either nonrecruitable or recruitable-with-difficulty in PHA-stimulated lymphocyte cultures.

Acridine Orange↗
PubMed1980

The quinternary chromatin-DNA structure. Three-dimensional reconstruction and functional significance.

Nuclear DNA-space images from Feulgen-stained HeLa cells synchronized at 1, 3, 5, 8, 12, 15, and 18 h following mitosis are digitized and their densitometric-geometric patterns are analyzed by means of a Quantimet 720-D image analyzer on line with a PDP11/40 computer. Frequency distributions of picture point optical densities for the phases and subphases as seen in nuclear images show that DNA packing changes are evident by means of ordinary optical microscopy. Radii of gyration of the images, and optical density profiles and distributions for several squashes of similar cells reveal that in particular instances chromatin DNA is distributed mostly towards the periphery, and usually with high circular isotropy. Cross power spectra of individual scan lines suggest that existence of higher order "quinternary" periodic structure for chromatin that modulates during the cell cycle. Three-dimensional reconstruction 2- micrometer sections of intact, Feulgen-stained mammalian tumor tissue show stainable material only toward the nuclear perimeter and not in the center (compatible with the evidence that initial thymidine incorporation in HeLa cells is generally at the nuclear border). Densitometric properties of reconstructed interphase chromatin-DNA bodies are highly coupled with similar properties of the whole nucleus, showing that a more condensed nucleus is always accompanied by a more condensed interphase chromatin DNA. The effect of micrococcal nuclease digestion on the digitized nuclear images is also presented. All the above data are then discussed in terms of a quinternary chromatin-DNA structure and its modulation during the cell cycle.

Cell Cycle↗