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Biomedical subjects

C Nicolini

Publications and source records attributed to C Nicolini.

At least 109 records · Page 6Linked to original sources
PubMed1976

Physicochemical alterations in the conformation of rat liver chromatin induced by carcinogens in vivo.

Administration of methylating carcinogens such as methyl methanesulfonate (120 mg/kg), dimethylnitrosamine (5 mg/kg), or methylnitrosourea (80 mg/kg) to rats resulted in an increased ellipticity in circular dichroism spectra and in an enhanced ability to bind ethidium bromide in the liver chromatin. Although shearing of the chromatin preparations increased both the ellipticity and number of binding sites for ethidium bromide, the carcinogen-induced effects were noticeable whether or not chromatin was sheared. Although the doses of the 3 carcinogens used in these studies are equivalent in their ability to induce strand breaks in liver DNA at 4 hr, their effects on the induction of conformational changes in liver chromatin are different. For example, methyl methanesulfonate induced the minimum conformational changes in liver chromatin at 4 hr, whereas methylnitrosourea induced the maximum changes at 4 hr. Methyl methanesulfonate and dimethylnitrosamine, on the other hand, induced maximum changes at 3 days. The conformational changes induced by methyl methanesulfonate and methylnitrosourea, and not by dimethylnitrosamine, tend to be repaired by 14 days.

Animals↗
PubMed1976

Physical-chemical characterization of living cells by laser-flow microfluorometry.

A rapid method for the laser-flow microfluorometry determination of nucleic-acid content per cell is presented. A frequency distribution of fluorescence is obtained from suspensions of living cells treated with ethidium bromide directly in their own medium (or calcium-magnesium-free Hanks' balanced solution). For a fixed number of cells, a frequency distribution of fluorescence is obtained as a function of the amount of ethidium bromide progressively added to the suspension until staturation. At any ratio of added dye per unit of DNA, histograms generated from cells stained with this method give results similar to those generated after fixation and staining by the Feulgen technique, both in terms of cell-cycle phases and ploidy-level determination. The present technique requires a minimal amount of material, is instantaneous, and is conducted directly on living cells. Furthermore, dye concentration-dependence studies of mean fluorescence per cell allow determination of association constant and binding process (primary and secondary) between the intact cell and ethidium bromide. Cells which have the same amount of DNA but vary in the amount of RNA and/or chromatin conformation (like G0 and G1) can then be distinguished.

Animals↗
PubMed1975

Alkaline lysis of mammalian cells for sedimentation analysis of nuclear DNA. Conformation of released DNA as monitored by physical, electron microscopic and enzymological techniques.

The degree of single strandedness of the DNA released from rat liver nuclei by various alkaline lysing solutions (including some with sodium dodecyl sulfate) was determined both before and after sedimentation in alkaline sucrose gradients employing electron microscopy, melting profiles, circular dichroism measurements, and digestibility by S1 nuclease. Regardless of the technique employed, the results obtained following alkaline sucrose gradient centrifugation of the DNA are consistent. The DNA was completely single stranded as judged by electron microscopy, circular dichroism spectra, and digestibility by S1 nuclease, an enzyme that specifically hydrolyzes single-stranded DNA. This was not true if the DNA was analyzed following alkaline lysis of the nuclei but before centrifugation. Under conditions which gave a complete transition to the single-stranded state, as judged by melting profiles and circular dichroism spectra, only 10-15% of the DNA was hydrolyzed by S1 nuclease. An increase in the susceptibility of the released DNA to S1 nuclease was observed with increases in the pH of the lysing solution. In order to release DNA which was single stranded as judged by both physical and enzymological techniques, the rat liver nuclei were lysed for 30 min with a 0.3 M NaOH lysing solution containing 0.5% dodecyl sulfate, 0.3 M NaCl and 0.03 M EDTA.

Animals↗
PubMed1975

Chromatin changes during the cell cycle of HeHa cells.

HeLa S3 cells were synchronized by selective mitotic detachment and chromatin was isolated from synchronized populations at various times during the cell cycle. The isolated chromatins were studied by circular dichroism and for their ability to bind the intercalating dye, ethidium bromide. Chromatin from cells in the middle of the S phase showed an increase in maximum ellipticity in the 250- to 300-nm region of circular dichroism spectra and an increased ability to bind ethidium bromide, when compared to chromatin isolated from mitotic cells. Chromatin from G1 cells had values intermediate between mitotic and mid-S chromatins.

Binding Sites↗
PubMed1975

Effect of cell trypsinization on nuclear proteins of WI-38 fibroblasts in culture.

When resting confluent monolayers of WI-38 fibrolasts are trypsinized and replated at a lower density they are stimulated to proliferate again with an interval of 18 hours between replating and the onset of DNA synthesis. Trypsinization of resting cells causes a 40% loss of nuclear proteins as well as of cytoplasmic proteins. The amount of nuclear proteins remains low for the first six hours after the cells have been replated and then it increases rapidly, reaching the same level of non-trypsinized resting cells by ten hours after plating. The proteins that are lost from the nucleus immediately after trypsinization are chromatin-associated proteins and most of them are non-histone chromosomal proteins, although a modest loss of histones cannot be ruled out. The loss of non-histone chromosomal proteins from cells that have been trypsinized causes changes in the structure of chromatin that can be detected by circular dichroism and by viscosity measurements. These results show that cell trypsinization causes an extensive loss of proteins from chromatin and that the loss is restored only several hours after the cells have been replated at a lower density.

Cell Line↗
PubMed1975

Circular dichroism and ethidium bromide binding studies of chromatin from WI-38 fibroblasts stimulated to proliferate.

Confluent monolayers of WI-38 diploid fibroblasts can be stimulated to proliferate by fresh serum. In the first 3 h after stimulation (that is, several hours before DNA replication) the chromatin of stimulated cells show structrual changes which include: (1) an increase in maximum positive ellipticity and a blue shift in the 250-300 nm region of circular dichroism spectra; and (2) an increase,in isolated chromatin, of the number of binding sites for the intercalating dye, ethidium bromide.The differences between chromtin of stimulated and chromatin of unstimulated cells are abolised when bother chromatins are treated with 0.25 M NaCL.

Binding Sites↗
PubMed1975

Effect of chromosomal proteins extractable with low concentrations of NaCl on chromatin structure of resting and proliferating cells.

When quiescent WI-38 cells are stimulated to proliferate by nutritional changes, the chromatin of stimulated cells shows an increased ellipticity in circular dichroism spectra and an increased ability to bind ethidium bromide. The differences between the chromatins of quiescent and stimulated cells are abolished if both chromatins are treated with 0.25 M NaCl. However, when salt-treated chromatins are reconstituted by dialysis agains 10 mM Tris-HCl, the difference in circular dichroism spectra and ethidium bromide binding re-appear. Furthermore, addition of the 0.25 M NaCl extract from chromatin of stimulated cells to chromatin of quiescent cells causes an increased ellipticity in circular dichroism spectra similar to that of stimulated chromatin. Since only proteins (mostly nonhistones) are detectable in the 0.25 M NaCl extract, these results strongly suggest that the chromosomal proteins extractable with low concentrations of salt are responsible for the functional and structural changes occurring in chromatin of WI-38 cells stimulated to proliferate.

Binding Sites↗
PubMed1975

The discrete phases of the cell cycle: autoradiographic, physical, and chemical evidences.

Recently the discrete model of the cell cycle, described as a series of metabolic progressions through four distinct phases, has been challenged by a few curves of percent labeled mitoses (PLM) and related autoradiographic data, which have been questionably interpreted in terms of a "continuous" cell cycle model. This conclusion is based in part on a few questionable observations which we summarize, along with our objections and explanations. We present a critical analysis of the autoradiographic data obtained with the PLM, in terms of the limitations and inconveniences of the technique itself. Some attention is paid to the usefulness of the "radioautographic transfer function," which seems an arbitrary mathematical expression of old autoradiographic observations. Finally, a review is presented of the old (by autoradiography) and new (by physical, chemical, and computer image analysis) evidences of the existence of dicrete phases in the cell cycle, which in turn support the idea of an all-or-none phenomenon for DNA synthesis, and which rule out the hypothesis of a continuous cell cycle model with a varying DNA synthesis rate function.

Autoradiography↗
PubMed1975

A clarification of the complex spectrum observed with the ultraviolet circular dichroism of ethidium bromide bound to DNA.

Ethidium bromide intercalation strongly effects the circular dichroism spectrum of DNA in the region of 230-300 mu, in a complex manner. In this report we present a study that quantitizes the relationships of the circular dichroism spectrum in the region of 230-300 mu and the ethidium bromide induced optical activity centered around 308 mu. We present evidence of two hidden cooperative bands that are probably the negative counterparts of the 308 mu band and 330 mu shoulder positive cooperative bands. The hidden band is quantitatively characterized. We confirm that the direct effect of ethidium bromide on the DNA spectrum is simply linearly proportional to the amount of intercalated dye. We also observe that the ethidium bromide enters freely when there is a molecule intercalated for every 3 sites, but that the intercalation is more difficult when the molecule intercalates at every second site.

Animals↗
PubMed1975

Early, reversible plasma membrane injury in galactosamine-induced liver cell death.

Administration to rats of D-galactosamine (400 mg/kg) produces liver cell death that develops during the first 24 hours. Plasma membranes isolated within the first few hours from these animals show a 40% reduction in 5'-nucleotidase activity and a two-fold increase in maximum negative ellipticity determined by circular dichroism. Simultaneous administration of uridine prevents liver cell death and these early alterations in the plasma membranes. Uridine also prevents cell death if administered for up to 3 hours after galactosamine. The 5'nucleotidase activity reduced when uridine is administered for up to 2-1/2 hours after galactosamine. Changes in the liver calcium ion concentration accompany these plasma membrane alterations. Uridine will prevent and reverse the changes in calcium content in parallel to its ability to reverse the membrane alterations. The significance of these findings with respect to the mechanism of galactosamine-induced liver cell death is discussed.

Animals↗
PubMedDate not supplied

Conformational changes in chromatin from density inhibited WI-38 fibroblasts stimulated to proliferate.

Quiescent confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to proliferate by replacing the old medium with fresh medium plus 10% serum. Circular dichroism spectra of chromatin from stimulated cells between 2 and 10 hrs after stimulation show an increase in positive ellipticity maxima and a blue shift in the 250-300 nm region. These changes are reversed when the stimulated cells enter DNA synthesis (which, in the present conditions, begins to increase at 12-15 hrs and reaches a peak at 20 hrs). The circular dichroism changes occurring 3 hrs after stimulation have been studied in greater detail. They consist in a 35% (average) increase in positive ellipticity and a blue shift in the 250-300 nm region. Changes in the gamma less than 244 nm region are less consistent. The differences between chromatins of stimulated and unstimulated cells are abolished when both chromatins are washed with 0.25 M NaC1. This procedure removes 10-12% of chromosomal proteins, which chromatograph with non-histone proteins. DNA, RNA and histones could not be detected in the 0.25 M NaC1 extract. In gel electrophoretic profiles of radioactively labelled chromosomal proteins from stimulated and unstimulated WI-38 cells there were no detectable differences between histones. The non-histone proteins of stimulated cells showed one radioactive peak which was increased above the level of non-histone proteins from control cells. These results show that structural changes occur in the chromatin of WI-38 cells stimulated to proliferate several hrs before the onset of DNA synthesis. The fact that differences in the chromatins can be abolished by washing with 0.25 M NaC1 could give a clue as to the mechanisms responsible for these structural changes.

Animals↗