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F Traganos

Publications and source records attributed to F Traganos.

33 records · Page 2Linked to original sources
PubMed1977

Cell cycle-related changes in nuclear chromatin of stimulated lymphocytes as measured by flow cytometry.

Flow cytometric techniques have been developed to assay lymphocyte stimulation as reflected by the increase in the cell transcriptional activity and cell progression through the cell cycle. The metachromatic fluorescent dye, acridine orange, is used to (a) stain DNA and RNA differentially in individual cells, and (b) stain nuclear chromatin after removal of cellular RNA BY RNase and cell pretreatment at acidic pH. Stimulated cells with diploid DNA content (G1) have an increased content of stainable RNA that makes it possible to distinguish them from nonstimulated (G0) cells. G0 cells can also be distinguished from G1 cells based on differences in stainability of their nuclear chromatin after treatment with acid. Mitotic indices can be scored automatically, inasmuch as the metaphase chromatin stains differently than does chromatin in the interphase cells. Altogether, the numbers of cells in the G0, G1, S, G2, and M phases may be obtained rapidly and with great accuracy. The cell transciptional activity can be correlated with changes in nuclear chromatin (e.g., during the transition from G0 to G1). The two independent techniques may also prove to be useful in recognizing and quantitating noncycling cells in other cell systems. The possible mechanisms responsible for differential stainability of nuclear chromatin in cells at different phases of the cell cycle are discussed.

Cell Cycle↗
PubMed1977

Nucleic acid content and nuclear chromatin structure of human bladder cell culture lines as studied by flow cytofluorometry.

Two human bladder cell lines, T-24 and HCV-29, are studied with flow cytofluorometry and acridine orange staining to determine relative DNA and RNA content per cell and to measure resistance to thermal denaturation of DNA in situ. The RNA/DNA ratio for HCV-29 is over twofold higher than that for T-24, a difference that is consistent with the differences in cytological morphology and staining characteristics of these two cell lines and is sufficient to distinguish them completely, although measurements of DNA or RNA alone may not. In addition, the two cell lines show differences in DNA "melting" curves that indicate structural or conformational differences in nuclear chromatin. It is evident that the features are related to nuclear and cellular morphology, and they may be of value as additional parameters for characterizing tissue culture cell lines.

Cell Line↗
PubMed1976

Lymphocyte stimulation: a rapid multiparameter analysis.

Several parameters of stimulation of individual lymphocytes are measured simultaneously by flow-cytofluorometry after differential staining of cellular DNA and RNA with the metachromatic fluorescent dye acridine orange. The method provides a means of analyzing the progression of stimulated cells through the cell cycle (G1, S and G2 + M), in addition to measuring the accumulation of RNA per cell; the RNA parameter is useful in distinguishing G1 from G0 cells. The multiparameter analysis may be of unique value in discriminating cases where the transcriptional and proliferative responses of lymphocytes to stimulants are not correlated.

Acridines↗
PubMed1976

DNA denaturation in situ. Effect of divalent cations and alcohols.

Heat denaturation profiles of rat thymus DNA, in intact cells, reveal the presence of two main DNA fractions differing in sensitivities to heat. The thermosensitive DNA fraction shows certain properties similar to those of free DNA: its stability to heat is decreased by alcohols and is increased in the presence of the divalent cations Ca2+, Mn2+, or Mg2+ at concentrations of 0.1-1.0 mM. Unlike free DNA, however, this fraction denatures over a wide range of temperature, and is heterogeneous, consisting of at least two subfractions with different melting points. The thermoresistant DNA fraction shows lowered stability to heat in the presence of Ca2+, Mn2+, or Mg2+ and increased stability in the presence of alcohols. It denatures within a relatively narrow range of temperature, consists of at least three subfractions, and, most likely, represents DNA masked by histones. The effect of Ca2+, Mn2+, or Mg2+ in lowering the melting point of the thermoresistant DNA fraction is seen at cation concentrations comparable to those required to maintain gross chromatin structure in cell nuclei or to support superhelical DNA conformation in isolated chromatin (0.5-1.0 mM). It is probable that factors involved in the maintenance of gross chromatin organization in situ and/or related to DNA superhelicity also have a role in modulating DNA-histone interactions, and that DNA-protein interactions as revealed by conventional methods using isolated chromatin may be different from those revealed when gross chromatin morphology remains intact.

Animals↗
PubMed1976

Cytofluorometric studies on conformation of nucleic acids in situ. I. Restriction of acridine orange binding by chromatin proteins.

Binding of the fluorochrome acridine orange (AO) to nucleic acids in situ is studied by automated cytofluorometry in two differentiating cell systems: Friend virus-transformed murine erythroleukemia induced to differentiate by dimethyl sulfoxide, and phytohemagglutinin-stimulated human lymphocytes. The specificity of the stain for deoxyribonucleic acid is discussed on the basis of data obtained by cell treatment with nucleases. Evidence is presented that in the case of Friend leukemia cells, but not phytohemagglutinin-stimulated lymphocytes, a significant change in the number of AO-intercalating sites in DNA occurrs during differentiation. These results suggest that changes in nuclear chromatin occurring during cell differentiation may be correlated, in some but not all systems, with changes in accessibility of DNA in situ to intercalating dyes. The role of divalent cations, especially Mg2+, in the conformation of nuclear chromatin and in modulation of the accessibility of nucleic acids to AO is discussed. The method provides a tool for the study of nucleic acid-protein interaction in situ, and in some cell systems it may be applicable as a marker for recognition of cell transformation, differentiation or neoplasia.

Acridines↗
PubMed1976

Cytofluorometric studies on conformation of nucleic acids in situ. II. Denaturation of deoxyribonucleic acid.

Thermal denaturation of deoxyribonucleic acid (DNA) in situ in individual unbroken cells is studied by a cytofluorometric method. This method allows us to investigate DNA denaturation in the presence of divalent cations at concentrations reported to be necessary to maintain native structure of nuclear chromatin. Under these conditions the pattern of DNA denaturation is very different than when studied in the presence of ethylenediaminetetraacetate or citrate. The results suggest that with divalent cations present, the histone basic charges are more uniformly distributed along whole nuclear DNA. Various cell types exhibit great differences in sensitivity to DNA denaturation when assayed in the presence of 1 mM MgCl2. Human lymphocytes, monocytes and certain kinds of human leukemic cells show differences large enough to be used as a parameter for their recognition in mixed samples. Possible applications of the method in basic research on chromatin conformation and as a tool for cell recognition in diagnostic cytology or in the classification of human leukemia are proposed.

Animals↗
PubMed1976

Urinary cytology automation. Preliminary studies with acridine orange stain and flow-through cytofluorometry.

Preliminary results are reported in an ongoing program to develop automated cytologic examinations for the detection of bladder cancer from exfoliated urinary epithelium. A metachromatic fluorescent dye, acridine orange, was used to stain the cells in suspension in such a way that their nuclei (DNA) fluoresced green in blue light, and their cytoplasm (chiefly RNA) fluoresced red. The intensities of green and red fluorescence per cell were measured for up to 5000 cells per sample by a flow-through cytofluorometer, and differences were found between cell types that served to identify bladder epithelial cells, leukocytes, squamous cells, and other particulates. Benign and malignant bladder epithelial cells differed principally in their cytoplasmic RNA staining intensity, and methods are discussed for amplifying this and other differences that may be of diagnostic value in an automated instrumental system.

Automation↗
PubMed1975

Denaturation of deoxyribonucleic acid in situ effect of formaldehyde.

In situ denaturation of nuclear deoxyribonucleic acid (DNA) is studied by use of acridine orange to differentially stain native versus denatured DNA, and a flow-through cytofluorometer for measurements of cell fluorescence. Thermal- or acid-induced DNA denaturation is markedly influenced by formaldehyde. Two mechanisms of the formaldehyde action are distinguished. If cells are exposed to the agent during heating, DNA denaturation is facilitated, most likely by the direct action of formaldehyde as a "passive" denaturing agent on DNA. If cells are pretreated with formaldehyde which is then removed, DNA resistance to denaturation increases, presumably due to chromatin cross-linking. It is believed that both effects occur simultaneously in conventional techniques employing formaldehyde to study DNA in situ, and that the extent of each varies with the temperature and cell type (chromatin condensation). Thus, profiles of DNA denaturation of cells heated with formaldehyde do not represent characteristics of DNA denaturation in situ; DNA denaturation under these conditions is modulated by the reactivity of chromatin components with formaldehyde rather than by DNA interactions with the macromolecules of nuclear mileu.

Acridines↗