Quantitative cytochemical changes induced by dexamethasone and adrenalectomy in rat liver chromatin

Physiochemical changes in the state of chromatin shortly following glucocorticoid stimulation of target cells are predicted by the proposed mechanism of steroid action. These changes had not been previously demonstrated in situ. The present experiments demonstrate that in the intact rat, or in one which has been adrenalectomized but given a moderate dose of dexamethasone, the thermal stability of liver cell chromatin is significantly reduced over the level observed in the adrenalectomized untreated animal. This alteration was rated by measuring nuclear acridine orange metachromasia following chromatin denaturation. These data also show an enhanced binding of the dye by the liver cell nuclei under the same conditions. Feulgen dye binding was also found to be enhanced by dexamethasone stimulation but to a level indicative of configurational changes in the chromatin rather than an increase in the amount of DNA in the cells.     

Interphase chromatin of human cells with diploid and haploid genomes

A study was made of the chromatin structure of mature sperm cells from healthy males aged 25 to 40 using fluorescent microscopy and acridine orange staining according to the DNP cell thermal denaturation technique modified by the authors. It was shown that normal human sperm cell chromatin melting profiles represent uniform curves with maxima in the following temperature ranges: 43 (+/- 2) degrees, 55 (+/- 1) degrees, 67 (+/- 2) degrees, 77 (+/- 1) degrees, 82 (+/- 0.5) degrees, 89 (+/- 1) degrees, 92 (+/- 2) degrees (P less than 0.01), that are identical to those obtained with lymphocytes of healthy males with certain deviations from the standard normal variant. No heteromorphism was revealed in the sperm cell chromatin. Marked polymorphism in the chromatin structure occurs but at the diploid cell level. A 10-time decrease in the fluorescence of AO bound with sperm cell chromatin as compared to F530 AO bound with lymphocyte chromatin structure of the same individual supports the data on the over condensation of sperm cell nuclear chromatin as compared to that in lymphocytes.     

Lymphocyte chromatin changes in Down's disease detected by heat denaturation]

By using fluorescent microscopy and acridine orange staining it was shown in the studies on short-term culture of human cells that the melting patterns of chromatin DNA of intact lymphocytes of healthy individuals represented the curves with 6 maxima (F530) at the temperature ranges of 45 degrees C, 65 degrees C, 85 degrees C, and 92 degrees C (P less than 0.01). The melting patterns of lymphocytes from patients with Down's disease represented curves with 4 maxima at the temperature ranges of 65 degrees C, 85 degrees C, 88 degrees C, 92 degrees C (P less than 0.01). No decline in the fluorescence intensity at the temperature intervals of 78 degrees C-85 degrees C was apparently due to a greater degree of condensation of definite regions of the trisomal cell chromatin complex. Possible mechanisms accounting for structural readjustments of the interphasic human lymphocyte chromatin occurring under thermal effects are discussed.     

Different sensitivity of chromatin to acid denaturation in quiescent and cycling cells as revealed by flow cytometry.

The properties of DNA in situ as reflected by its staining with acridine orange are different in quiescent nonstimulated lymphocytes as compared with interphase lymphocytes that have entered the cell cycle after stimulation by mitogens. The difference is seen after cell treatment with buffers at pH 1.5 (1.3-1.9 range) followed by staining with acridine orange at pH 2.6 (2.3-2.9). Under these conditions the red metachromatic fluorescence of the acridine orange-DNA complex is higher in quiescent cells than in the cycling lymphocytes while the orthochromatic green fluorescence is higher in the cycling, interphase cells. The results suggest that DNA in condensed chromatin of quiescent lymphocytes (as in metaphase chromosomes) is more sensitive to acid-denaturation than DNA in dispersed chromatin of the cycling interphase cells. The phenomenon is used for flow cytometric differentiation between G0 and G1 cells and between G2 and M cells. In contrast to normal lymphocytes the method applied to neoplastic cells indicates the presence of cell subpopulations with condensed chromatin but with DNA content characteristic not only of G1 but also of S and G2 cells. The possibility that these cells represent quiescent (resting) subpopulations, arrested in G1, S and/or G2, is discussed.     

Principle of the supramolecular stacking of the cellular deoxyribonucleoprotein complex

Using cytofluorimetry with acridine orange staining and a modified thermal denaturation technique of cellular DNP, it has been shown that chromatin melting profiles of normal human nuclei (from lymphocytes and granulocytes) have distinct regularities. It is believed that these regularities reflect specific supramolecular chromatin organization. Parallel comparative analysis performed using electrophoretic fractionation and isoelectric focussing of nuclear proteins has revealed that: 1) peculiarities of chromatin melting profiles are independent of the quantity and molecular weights of chromatin proteins; 2) the lack of principal differences in chromatin melting profiles and the data on isoelectric points of nuclear proteins of granulocytes and lymphocytes from the same patient indicate that specific supramolecular organization of DNP-complex depends on the chromatin protein charge.     

In situ factors affecting stability of the DNA helix in interphase nuclei and metaphase chromosomes

The data from earlier cytochemical studies, in which the metachromatic fluorochrome acridine orange (AO) was used to differentially stain single vs double-stranded DNA, suggested that DNA in situ in intact metaphase chromosomes or in condensed chromatin of G0 cells is more sensitive to denaturation, induced by heat or acid, than DNA in decondensed chromatin of interphase nuclei. Present studies show that, indeed, DNA in permeabilized metaphase cells, in contrast to cells in interphase, when exposed to buffers of low pH (1.5-2.8) becomes digestible with the single-strand-specific S1 or mung bean nucleases. A variety of extraction procedures and enzymatic treatments provided evidence that the presence of histones, HMG proteins, and S-S bonds in chromatin, as well as phosphorylation or poly(ADP)ribosylation of chromatin proteins, can be excluded as a factor responsible for the differential sensitivity of metaphase vs interphase DNA to denaturation. Cell treatment with NaCl at a concentration of 1.2 N and above abolished the difference between interphase and mitotic cells, rendering DNA in mitotic cells less sensitive to denaturation; such treatment also resulted in decondensation of chromatin visible by microscopy. The present data indicate that structural proteins extractable with greater than or equal to 1.2 N NaCl may be involved in anchoring DNA to the nuclear matrix or chromosome scaffold and may be responsible for maintaining a high degree of chromatin compaction in situ, such as that observed in metaphase chromosomes or in G0 cells. Following dissociation of histones, the high spatial density of the charged DNA polymer may induce topological strain on the double helix, thus decreasing its local stability; this can be detected by metachromatic staining of DNA with AO or digestion with single-strand-specific nucleases.     

Change in the structure of fibroblast chromatin in a patient with Down's disease and his mother

A clear-cut difference between chromatin melting curves of cells from the patient with Down's disease and his mother and ones from healthy individuals and the proband's father was shown by fluorescence microscopy and acridine orange staining on human fibroblasts incubated in autologous serum. These data suggest the presence of an obvious genetic correlation between phenotypically healthy mother and her sick child. The identity of the chromatin structure of the patients detected both on lymphocytes and other type of cells, human fibroblasts, allows a suggestion that the phenomenon of the altered chromatin structure is typical generally of the given individual's tissues. Certain changes in the cell chromatin structure are mediated by the effect of autologous serum.     

Denaturation and condensation of DNA in situ induced by acridine orange in relation to chromatin changes during growth and differentiation of Friend erythroleukemia cells

DNA in situ is progressively denatured when the cells or nuclei are treated with increasing concentration of acridine orange (AO). This transition can be monitored by flow cytometry as a decrease in green fluorescence. The complexes of denatured DNA and AO undergo immediate condensation and aggregation; this step is manifested by appearance of red luminescence and formation of precipitates that can be detected by electron microscopy. The precipitates form preferentially in heterochromatin as well as in ribosomes and polysomes. Their formation and further aggregation affects cellular light scatter properties in both the forward and right-angle direction. The AO-induced DNA denaturation and condensation was studied in nuclei of Friend erythroleukemia cells from exponentially growing, differentiated or quiescent cells. The DNA in nuclei of quiescent cells, from plateau-phase cultures, was the most sensitive to denaturation; it denatured (measured by changes in luminescence) at an AO concentration between 50 and 80 microM with the midpoint of the transition (Cd) at 70 microM. DNA in nuclei of differentiated cells (dimethyl-sulfoxide-induced erythroid differentiation) was more resistant (Cd = 77-83 microM), whereas DNA in exponentially growing cells was the most resistant (Cd = 86 microM). Extraction of proteins with 0.1 M HCl at 0 degree C abolished the differences between the cells and shifted the transition to a lower AO concentration (Cd = 46 microM). For comparison, the midpoint transitions representing condensation of free, nucleic acids measured as light scatter changes occurred at 13, 22, 31 and 53 microM of AO, for rRNA, tRNA, and denatured and native-calf thymus DNA, respectively. Denaturation and condensation of DNA, which can be induced by AO either in isolated nuclei or viable permeabilized or fixed cells provides a new approach to discriminate cell subpopulations with different chromatin structure by flow cytometry. The molecular mechanisms of this phenomenon are discussed.     

Thymus involution induced by mouse hepatitis virus A59 in BALB/c mice.

Mouse hepatitis virus A59 (MHV-A59) infection of adult BALB/c mice induced a severe, transient atrophy of the thymus. The effect was maximal at 1 week after infection, and thymuses returned to normal size by 2 weeks after infection. There was no effect of glucocorticoids, since thymus atrophy was also found in adrenalectomized, infected mice. In infected thymus, immature CD4+ CD8+ lymphocytes were selectively depleted, and apoptosis of lymphocytes was increased. The MHV receptor glycoprotein MHVR was detected on thymus epithelial cells but not on T lymphocytes. In a small number of stromal epithelial cells, but in very few lymphocytes, the viral genome was detectable by in situ hybridization. These observations suggested that MHV-A59-induced thymic atrophy results not from a generalized lytic infection of T lymphocytes but rather from apoptosis of immature double-positive T cells that might be caused by infection of a small proportion of thymus epithelial cells or from inappropriate secretion of some factor, such as a cytokine.     

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.     

Acridine orange binding to chromatin of individual cells and nuclei under different staining conditions. Thermadenaturation of Chromatin.

The thermodenaturation of chromatin in situ was studied by staining heat-treated nuclei with acridine orange. It was found that formaldehyde, which under the present conditions had to be used to prevent extensive renaturation of DNA, seriously affects the results of standard acridine orange staining in an unspecific manner. In particular the acetylation step involved in this staining method is strongly inhibited. Thus the standard method of staining can only give qualitative information about the effects of thermodenaturation. On the other hand, acridine orange staining at defined equilibrium, without prior acetylation is insensitive to formaldehyde and multiphasic thermodenaturation profiles are obtained with this method. At low temperatures these profiles mainly reflect changes in the protein-DNA interaction whereas at higher temperatures DNA denaturation also contributes to the curves. Although these two processes cannot be separately quantitated by simple measurements of dye binding, the thermodenaturation profiles still contain biologically significant information about the properties of chromatin in situ.     

DNA Segments Sensitive to Single-Strand-Specific Nucleases Are Present in Chromatin of Mitotic Cells

It was observed before that DNAin situin chromatin of mitotic cells is more sensitive to denaturation than DNA in chromatin of interphase cells. DNA sensitivity to denaturation, in these studies, was analyzed by exposing cells to heat or acid and using acridine orange (AO), the metachromatic fluorochrome which can differentially stain double-stranded (ds) vs single-stranded (ss) nucleic acids, as a marker of the degree of DNA denaturation. However, without prior cell treatment with heat or acid no presence of single-stranded DNA in either mitotic or interphase cells was detected by this assay. In the present experiments we demonstrate that DNAin situin mitotic cells, without any prior treatment that can induce DNA denaturation, is sensitive to ss-specific S1 and mung bean nucleases. Incubation of permeabilized human T cell leukemic MOLT-4, promyelocytic HL-60, histiomonocytic lymphoma U937 cells, or normal PHA-stimulated lymphocytes with S1 or mung bean nucleases generated extensive DNA breakage in mitotic cells. DNA strand breaks were detected using fluorochrome-labeled triphosphonucleotides in the reaction catalyzed by exogenous terminal deoxynucleotidyl transferase. Under identical conditions of the cells’ exposure to ss-specific nucleases, DNA breakage in interphase cells was of an order of magnitude less extensive compared to mitotic cells. The data indicate that segments of DNA in mitotic chromosomes, in contrast to interphase cells, may be in a conformation which is sensitive to ss nucleases. This may be a reflection of the differences in the torsional stress of DNA loops between interphase and mitotic chromatin. Namely, greater stress in mitotic loops may lead to formation of the hairpin-loop structures by inverted repeats; such structures are sensitive to ss nucleases. The present method of detection of such segments appears to be more sensitive than the use of AO. The identification of mitotic cells based on sensitivity of their DNA to ss nucleases provides an additional method for their quantification by flow cytometry.     

Use of acridine orange in the estimation of lymphoid cell migration in an organism

Lymphoid cell migration was studied on the model of their transplantation from animal-donors to recipients. The studied organs were crushed small in the saline solution. The number of nuclei-containing elements has been determined in the obtained suspensions. Smears have been prepared and studied with the help of luminescence microscope combined with phase contrast device. We have determined and counted the ratio of "acridine orange cells" glittering in ultra-violet rays to all visible in phase-contrast nuclei containing cells. Then we have found the percentage of the marked cells in the organ from the total number of the injected lymphocytes. The usage of the given technique shown the enhancement of thymus cell migration to the liver after partial hepatectomy.     

Characteristics of the reactions to N-alloantigens of lymphoid cells from hydrocortisone-stimulated and adrenalectomized mice]

The capacity of the spleen, bone marrow and thymus cells from CBA mice (intact, adrenalectomized, and those treated with single or repeated hydrocortisone injections) to induce the lymph node type of "graft-versus-host" reaction (GVHR) in (CBA X C57BL) F1 hybrid recipients was evaluated. Two days after 2.5 mg hydrocortisone injection the capacity of the spleen and bone marrow cells to induce GVHR increased while that of the thymus cells remained unchanged. Seven and particularly 15 days after hydrocortisone injection the spleen cells became less active. Two days following repeated daily hormone injections in a dose of 0.25 mg within 18 days the thymocyte activity in GVHR increased, while that of the spleen and bone marrow cells did not change.     

Conformation of control and acetylated HeLa stripped chromatin after reassociation with H1

The effect of histone H1 on the conformation and stability of control and acetylated HeLa high-molecular-weight chromatin that had been stripped of H1 and nonhistone proteins was compared by circular dichroism (CD) and thermal denaturation measurements. Two different preparations of H1, originating from rat thymus and chicken erythrocyte, were used in the reconstitution studies. The control and acetylated stripped chromatin had identical CD and thermal denaturation properties, as did their reconstitutes with rat thymus H1. Reconstitutes of the two chromatins with chicken erythrocyte H1 had similar CD properties, but thermal denaturation studies showed that the acetylated reconstitute was destabilized compared to the control reconstitute. Reconstitutes of both chromatins with chicken erythrocyte H1 had a more condensed and stabilized structure than the reconstitutes with rat thymus H1. Thus, acetylation caused a decrease in the stability of chromatin in the presence of erythrocyte H1, but more marked differences were detected in the structure of stripped chromatin after reassociation with different H1 preparations.     

Early changes in liver chromatin in response to partial hepatectomy]

Guinea-pig and mouse liver chromatin responds to the partial hepatectomy by an increase in binding of a basic dye acridine orange (AO) and by a decrease of its stability to heat in thermal denaturation test in situ. Degree of the changes in AO chromatin binding is identical in the cells of different ploidy and proportional to their DNA content. Treatment of the preparations by 0.6 M NaCl solutions under conditions bringing about the selective removal of histone H1 from the cells produces in vitro changes in DNA properties taking place in cells in vivo in the course of their activation. The treatment of cells with 0.35 M NaCl solution results in the disappearance of changes occurring in the chromatin of activated cells whereas the properties of control cells remain unchanged. The data obtained are interpreted as a result of the removal of some non-histone regulatory proteins from the chromatin of activated cells that is accompanied by changes in the character of DNA-histone interaction. At the time of maximum increase of AO binding a significant intensification of endogenous RNA polymerase activity was found, the incorporation of [3H] UTP in the nucleolus being higher than that in the extranucleolar part of the nucleus. High ionic strength in the incubation medium (0.4 M (NH4)2SO4) results in drastic increase of radioactive label in the nucleus and in the disappearance of differences between activated and non-activated chromatin. It is concluded that the intensification of RNA synthesis under the influence of proliferative stimulus is more likely dependent on the additional opening of DNA-matrix than on the direct activation of the enzyme.     

The influence of 0.35 M sodium chloride treatment on physico-chemical properties of liver chromatin in partially hepatectomized guinea pigs

The treatment of fixed preparations of guinea pig liver cells by 0.35 M NaCl solution results in the disappearance of changes (increase in acridine orange binding and decrease in thermal stability) arising in chromatin activated by partial hepatectomy. The data obtained are interpreted to be the result of the removal from activated chromatin of some newly synthesized non-histone proteins.     

Studies on the role of polyamines associated with the ribosomes from Bacillus stearothermophilus

1. Spermine and spermidine were the main polyamines detectable in Bacillus stearothermophilus. 2. When grown at 65 degrees B. stearothermophilus contained lower concentrations of polyamines per mg. of RNA than when grown at 45 degrees or at 55 degrees . 3. Ribosomes isolated from B. stearothermophilus in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride contained sufficient polyamines to neutralize between 4% and 9% of their RNA phosphorus. 4. Removal of polyamines from the ribosomes by dialysis against m-potassium chloride did not appreciably alter the hypochromicity or thermal denaturation profiles of the ribosomes when measured in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride, though it did cause a loss of ribosome particles sedimenting at greater than 78s. 5. When ribosomes were dialysed against acridine orange solutions acridine orange bound to the ribosomes and did not displace spermine, but when a mixture of ribosomal RNA and spermine was dialysed against acridine orange the acridine orange displaced the spermine. It is concluded that polyamines in the ribosomes are less accessible for displacement by acridine orange than when polyamines are bound to ribosomal RNA.     

The differential fluorescence of bacteria stained with acridine orange and the effects of heat

Some factors affecting the fluorescence of bacteria stained with acridine orange and the direct epifluorescent filter technique (DEFT) were studied. When bacterial cells from a chemostat operated at dilution rates between 0.1 and 0.7/h were used the differential fluorescence observed in the DEFT related to cell 'activity' and the orange fluorescence, which was predominant at high growth rates, may be related to an increase in the RNA content of the cells. Heat affected the colour of cell fluorescence and this was dependent on the cell type and, in particular, age. Uptake of acridine orange into the cells was also found to be an important factor determining the colour of fluorescence. However, with heat-treated cells there was no correlation between the amount of uptake and colour of fluorescence. The relative amounts and degree of denaturation of the different types of nucleic acids remaining in the cells after heat treatment appeared primarily to determine the colour of fluorescence.     

Unknown G0/G1 Cell Subpopulation Revealed by Hydrochloric Acid/Acridine Orange Staining

An unknown cell subpopulation was observed in mouse and rat thymus, spleen and bone marrow cells, as well as in human peripheral blood mononuclear cells (resting and stimulated by PHA) using equilibrium HCl/acridine orange staining. This subpopulation includes cells with decreased green and unchanged red fluorescence. The staining does not affect cells in S- and G₂/M-phases. The mechanism and biological meaning of the effect await further investigation.