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

1979 ◽  
Vol 27 (1) ◽  
pp. 478-485 ◽  
Author(s):  
Z Darzynkiewicz ◽  
F Traganos ◽  
M Andreeff ◽  
T Sharpless ◽  
M R Melamed
Keyword(s):  
Acridine Orange ◽  
Condensed Chromatin ◽  
Metaphase Chromosomes ◽  
Quiescent Cells ◽  
Acid Denaturation ◽  
Interphase Cells ◽  
The Difference ◽  
Cell Subpopulations ◽  
G1 Cells ◽  
Dna Complex

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.

scholarly journals Different sensitivity of DNA in situ in interphase and metaphase chromatin to heat denaturation.

1977 ◽  
Vol 73 (1) ◽  
pp. 128-138 ◽  
Author(s):  
Z Darzynkiewicz ◽  
F Traganos ◽  
T Sharpless ◽  
M R Melamed
Keyword(s):  
Heat Denaturation ◽  
Published Data ◽  
Dna Denaturation ◽  
Metaphase Chromosomes ◽  
Flow Cytofluorometry ◽  
Dna Reassociation ◽  
Interphase Cells ◽  
The Difference ◽  
The Stability

Heat denaturation of DNA in situ, in unbroken cells, was studied in relation to the cell cycle. DNA in metaphase cells denatured at lower temperatures (8 degrees-10 degrees C lower) than DNA in interphase cells. Among interphase cells, small differences between G1, S, and G2 cells were observed at temperatures above 90 degrees C. The difference between metaphase and interphase cells increased after short pretreatment with formaldehyde, decreased when cells were heated in the presence of 1 mM MgCl2, and was abolished by cell pretreatment with 0.5 N HCl. The results suggest that acid-soluble constituents of chromatin confer local stability to DNA and that the degree of stabilization is lower in metaphase chromosomes than in interphase nuclei. These in situ results remain in contrast to the published data showing no difference in DNA denaturation in chromatin isolated from interphase and metaphase cells. It is likely that factors exist which influence the stability of DNA in situ are associated with the super-structural organization of chromatin in intact nuclei and which are lost during chromatin isolation and solubilization. Since DNA denaturation is assayed after cell cooling, there is also a possibility that the extent of denatured DNA may be influenced by some factors that control strand separation and DNA reassociation. The different stainability of interphase vs. metaphase cells, based on the difference in stability of DNA, offers a method for determining mitotic indices by flow cytofluorometry, and a possible new parameter for sorting cells in metaphase.

scholarly journals Recognition of cells in mitosis by flow cytofluormetry.

1977 ◽  
Vol 25 (7) ◽  
pp. 875-880 ◽  
Author(s):  
Z Darzynkiewicz ◽  
F Traganos ◽  
T Sharpless ◽  
M R Melamed
Keyword(s):  
Cell Cycle ◽  
Acridine Orange ◽  
Chromatin Structure ◽  
Deoxyribonucleic Acid ◽  
Mitotic Cell ◽  
Low Ph ◽  
Interphase Cell ◽  
Neutral Ph ◽  
Interphase Cells ◽  
The Difference

Cells in mitosis may be distinguished from interphase cells based on difference in chromatin structure as revealed by two different methods of staining with acridine orange. In the first method, cells are heated and then stained at neutral pH; the difference in stainability between mitotic and interphase cells reflects the difference in the extent of deoxyribonucleic acid denatured by heat in these cells. At a given temperature the deoxyribonucleic acid of the mitotic cell appears to be more extensively denatured than that of the interphase cell. In the second method, cells are treated with buffer at pH 1.5 (1.3 to 1.9) and then stained at pH 2.6 (2.3 to 2.9). The mechanisms involved in the differential stainability of interphase versus mitotic cells at that low pH are currently under investigation. In both methods, in addition to enumerating cells in mitosis, it is possible to quantitate cells in G1, S and G2 phases of the cell cycle.

The Synthesis and Migration of Nuclear Proteins during Mitosis and differentiation of Cells in Rats

1965 ◽  
Vol s3-106 (75) ◽  
pp. 229-240
Author(s):  
R. T. SIMS
Keyword(s):  
Granular Cell ◽  
Nuclear Proteins ◽  
Photographic Emulsion ◽  
Metaphase Chromosomes ◽  
Interphase Nuclei ◽  
Cell Layers ◽  
The Difference ◽  
Mitotic Figures ◽  
And Migration ◽  
Silver Grains

Hooded rats were given an intraperitoneal injection of 3H-tyrosine, and killed in pairs 10 min, 30 min, 12 h, 36 h, 7 days, and 30 days later. A piece of skin with white growing hair, and the tongue, were taken from each animal and radioautographs were prepared. Silver grains were counted over whole nuclei and whole mitotic figures of the germinal cells and whole nuclei of differentiating cells of both tissues. It was found that the interphase nuclei have significantly more silver grains over them than the chromosomes at all stages of mitosis and there are virtually no grains over metaphase, anaphase, and early telophase chromosomes in both tissues of all the animals killed up to 36 h after the injection. The difference between the grain counts over the interphase nuclei and the chromosomes of dividing cells is at least 20-fold at 30 min in the hair matrix, at least 5-fold at 30 min in the tongue and at 36 h in both tissues. It was established that the differences observed between the radioactivities of the nuclei and chromosomes of mitotic figures are real from estimates of: the radioactivity of the cell cytoplasm, volumes of the metaphase chromosomes and interphase nuclei within 1µ of the photographic emulsion, and the volumes of cytoplasm separating the photographic emulsion and these structures. No protein synthesis was demonstrable in the chromosomes during metaphase, anaphase, and early telophase. Nuclear proteins leave the chromosomes during prophase and prometaphase and return to the nucleus during late telophase. The cells in the matrix and upper bulb of the growing hair follicle and those in the germinal, prickle, and granular cell layers of the tongue are in different functional states; 30 min after injection of 3H-tyrosine they have different amounts of it in their nuclear proteins. It is suggested that the amount incorporated into each nucleus is related to the rate at which proteins are being synthesized by the cell.

scholarly journals PHA Responsiveness and Subpopulations of Circulating Lymphocytes in Pernicious Anemia

Blood ◽  
1974 ◽  
Vol 44 (6) ◽  
pp. 849-855 ◽  
Author(s):  
A. C. MacCuish ◽  
S. J. Urbaniak ◽  
A. H. Goldstone ◽  
W. J. Irvine
Keyword(s):  
Pernicious Anemia ◽  
Lymphocyte Transformation ◽  
Cell Subpopulation ◽  
Significant Difference ◽  
Beta Counting ◽  
Significant Depression ◽  
Circulating Lymphocytes ◽  
Immunologic Abnormality ◽  
The Difference ◽  
Cell Subpopulations

Abstract Lymphocyte transformation responses to the mitogen phytohemagglutinin (PHA) were measured in 20 patients with proven pernicious anemia (PA) and 20 matched controls using 3H-thymidine label. The patients with PA showed significant depression of lymphocyte transformation to the three doses of PHA employed, as judged by beta counting; however, radioautographic examination of PHA-stimulated cells indicated that the results were due to a failure of intranuclear incorporation of 3H-thymidine by PA lymphocytes, rather than a failure of PHA to induce blastogenesis. The percentages and numbers of T and B lymphocytes in peripheral blood were measured in 30 patients and controls by rosette and immunofluorescence techniques, respectively. There was no significant difference in the B cell subpopulations between patients and controls; the T cell subpopulation was slightly lower in the PA patients (mean 62.4%) than in the controls (mean 65.5%), but the difference was not statistically significant. The depressed uptake of 3H-thymidine by stimulated lymphocytes in PA would seem to reflect a chemical defect rather than inherent immunologic abnormality.

The effects of diazepam on mitosis and the microtubule cytoskeleton. I. Observations on the diatoms Hantzschia amphioxys and Surirella robusta

1994 ◽  
Vol 107 (9) ◽  
pp. 2643-2651
Author(s):  
T.P. Spurck ◽  
J.D. Pickett-Heaps
Keyword(s):  
Time Lapse ◽  
Detectable Effect ◽  
Metaphase Chromosomes ◽  
Central Spindle ◽  
Cell Cleavage ◽  
Spindle Poles ◽  
Transmission Electron ◽  
Live Diatoms ◽  
Interphase Cells ◽  
Chromosome Motion

The effects of diazepam (DZP) on mitosis and the microtubule (MT) cytoskeleton in the live diatoms Hantzschia amphioxys and Surirella robusta were followed using time-lapse video microscopy. Similarly treated cells were fixed and later examined for immunoflouresence staining of MTs or for transmission electron microscopy. DZP treatment (250 microM) had no effect on interphase cells but affected mitosis, resulting in the majority of prometaphase and metaphase chromosomes releasing from one or both spindle poles and collecting irregularly along the central spindle. Chromosomes remaining attached to one pole continued to display slight prometaphase oscillations; however, this activity was never observed in metaphase spindles. Following removal of DZP, some chromosomes still bipolarly attached, immediately released elastically from one pole. Within the first 2 minutes of recovery, all chromosomes recommenced spindle attachment, exhibiting normal prometaphase oscillations and proceeded through mitosis. DZP treatment during anaphase had no detectable effect on chromosome motion or cell cleavage. These results suggest that DZP acts as an anti-MT agent, selectively affecting polar MTs at prophase, prometaphase and metaphase, and thereby weakening kinetochore connection to the poles. From these and other results (unpublished), its mode of action is different to that of most anti-MT agents.

261 LABELING SEX-SPECIFIC DNA SEQUENCES IN MAMMALIAN SPERM

2008 ◽  
Vol 20 (1) ◽  
pp. 210
Author(s):  
B. A. Didion ◽  
R. Bleher
Keyword(s):  
Dna Sequences ◽  
Somatic Cells ◽  
Complete Separation ◽  
Cell Nuclei ◽  
Metaphase Chromosomes ◽  
Telomeric Sequences ◽  
Synthetic Dna ◽  
Y Chromosomes ◽  
Boar Sperm ◽  
The Difference

While flow cytometric separation of X- andY-chromosome- bearing sperm has advanced to the point of acceptance in the commercial production of sex-preselected cattle, it is important to continue researching this area to improve efficiencies. For example, the difference in DNA sequence between the X- andY-chromosomes has merit as a foundation for an alternative sperm sexing approach that could enable the complete separation and use of an entire ejaculate. We used synthetic DNA mimics conjugated to a fluorescent dye for in situ detection of Y-chromosomes in metaphase preparations of porcine somatic cells and spermatozoa. Peptide nucleic acids (PNA) are synthetic compounds with higher affinity and stability than conventional DNA probes and are used as specific hybridization probes to complementary DNA. The application of PNA probes was demonstrated previously in telomere analysis studies, and we confirmed their efficacy using a CY3-(CCCTAA)3 PNA to probe bull and boar sperm telomeric sequences. Using male porcine somatic cells and theY-chromosome as a template, we arranged for the synthesis of a CY3-conjugated PNA to bind 13-15 base pairs of unique, Y-chromosome sequence. By testing different labeling conditions, we found that brief incubation of metaphase chromosomes with the PNA produced a localized signal on theY-chromosome. No signals were present when chromosomes of porcine female somatic cells were incubated with the PNA probes. Because chromosomes occupy non-random territories in all cell nuclei including those in sperm, we expected to find centrally located signals in 50% of fixed boar sperm when these were treated with the same PNA as used for the somatic cells. We found the signals present in 161 of 302 (53.3%) sperm to consist of a single, centrally located, round fluorescent dot in the sperm head. Further research is required to establish the uptake of PNA in live sperm toward evaluation of this approach for sperm sexing.

scholarly journals Thiazole Orange as an Alternative to Antibody Binding for Detecting Triple-helical DNA in Heterochromatin of Drosophila and Rhynchosciara

2017 ◽  
Vol 66 (3) ◽  
pp. 143-154 ◽  
Author(s):  
Eduardo Gorab ◽  
Peter Lees Pearson
Keyword(s):  
Fluorescent Dyes ◽  
Triplex Dna ◽  
Thiazole Orange ◽  
Metaphase Chromosomes ◽  
Specificity And Sensitivity ◽  
Rhynchosciara Americana ◽  
The Difference ◽  
Nucleotide Specificity ◽  
Nucleic Acid Structures ◽  
Immune Detection

The standard method for detecting triple-stranded DNA over the last 1.5 decades has been immune detection using antibodies raised against non-canonical nucleic acid structures. Many fluorescent dyes bind differentially to nucleic acids and often exhibit distinctive staining patterns along metaphase chromosomes dependent upon features, including binding to the major and minor DNA grooves, level of chromatin compaction, nucleotide specificity, and level of dye stacking. Relatively recently, the fluorochrome Thiazole Orange (TO) was shown to preferentially bind to triplex DNA in gels. Here, we demonstrate that TO also detects triplex DNA in salivary gland chromosomes of Drosophila melanogaster and Rhynchosciara americana identical in location and specificity to observations using antibodies. This finding may enable triple-stranded DNA investigations to be carried out on a much broader and reproducible scale than hitherto possible using antibodies, where a frequently encountered problem is the difference in detection specificity and sensitivity between one antibody and another.

scholarly journals Histoplasma capsulatum modulates the acidification of phagolysosomes.

1993 ◽  
Vol 177 (6) ◽  
pp. 1605-1611 ◽  
Author(s):  
L G Eissenberg ◽  
W E Goldman ◽  
P H Schlesinger
Keyword(s):  
Drug Delivery ◽  
Acridine Orange ◽  
Antigen Processing ◽  
Lysosomal Enzymes ◽  
Histoplasma Capsulatum ◽  
Hydrolytic Enzymes ◽  
Fluorescein Isothiocyanate ◽  
Neutral Ph ◽  
Microbial Survival ◽  
The Difference

The phagolysosome is perhaps the most effective antimicrobial site within macrophages due both to its acidity and to its variety of hydrolytic enzymes. Few species of pathogens survive and multiply in these vesicles. However, one strategy for microbial survival would be to induce a higher pH within these organelles, thus interfering with the activity of many lysosomal enzymes. Altering the intravesicular milieu might also profoundly influence antigen processing, antimicrobial drug delivery, and drug activity. Here we report the first example of an organism proliferating within phagolysosomes that maintain a relatively neutral pH for a sustained period of time. We inoculated P388D1 macrophages with fluorescein isothiocyanate (FITC)-labeled Histoplasma capsulatum or zymosan. Using the ratio of fluorescence excitations at 495 and 450 nm, we determined that vesicles containing either virulent or avirulent FITC-labeled H. capsulatum yeasts had a pH one to two units higher than vesicles containing either zymosan or methanol-killed H. capsulatum. The difference in pH remained stable for at least 5.5 h postinoculation. Longer-term studies using cells preincubated with acridine orange indicated that phagolysosomes containing live Histoplasma continued to maintain a relatively neutral pH for at least 30 h. Many agents raise the pH of multiple vesicles within the same cell. In contrast, H. capsulatum affects only the phagolysosome in which it is located; during coinoculation of cells with unlabeled Histoplasma and labeled zymosan, organelles containing zymosan still acidified normally. Similarly, unlabeled zymosan had no influence on the elevated pH of vesicles housing labeled Histoplasma. Thus, zymosan and Histoplasma were segregated into separate phagolysosomes that responded independently to their phagocytized contents. This localized effect might reflect an intrinsic difference between phagosomes housing the two particle types, active buffering by the microbe, or altered ion transport across the phagolysosomal membrane such that acidification is inhibited.

scholarly journals PROPHASING OF INTERPHASE NUCLEI AND INDUCTION OF NUCLEAR ENVELOPES AROUND METAPHASE CHROMOSOMES IN HELA AND CHINESE HAMSTER HOMO- AND HETEROKARYONS

1974 ◽  
Vol 62 (1) ◽  
pp. 104-113 ◽  
Author(s):  
Yoshitaka Obara ◽  
Lee S. Chai ◽  
Herbert Weinfeld ◽  
Avery A. Sandberg
Keyword(s):  
Nuclear Envelope ◽  
Hela Cells ◽  
Interphase Nucleus ◽  
Chinese Hamster ◽  
Binucleate Cell ◽  
Metaphase Chromosomes ◽  
Interphase Nuclei ◽  
Multinucleate Cells ◽  
Interphase Cells ◽  
Nuclear Envelope Formation

Fusing human HeLa metaphase cells with HeLa interphase cells resulted within 30 min in either of two phenomena in the resultant binucleate cell: either prophasing of the interphase nucleus or formation of a normal-appearing nuclear envelope around the metaphase chromosomes. The frequency of either occurrence was strongly dependent on environmental pH. At pH's of 6.6–8.0, prophasing predominated; at pH 8.5 nuclear envelope formation predominated. Additionally, the frequencies of the two events in multinucleate cells depended on the metaphase/interphase ratio. When the ratio was 0.33 nuclear envelope formation predominated; when it was 2.0 prophasing predominated. In their general features, the results with fused HeLa cells resembled those reported earlier with fused Chinese hamster Don cells. However, the results provided an indication that between pH 6.6 and 8.0 the HeLa metaphase cells possessed a much greater capacity than the Don metaphase cells to induce prophasing. Fusion of Don metaphase cells with HeLa interphase cells or of Don interphase cells with HeLa metaphase cells at pH 8.0 resulted in nuclear envelope formation or prophasing in each kind of heterokaryon. As in the homokaryons, the frequencies of the two events in the heterokaryons depended on the metaphase/interphase ratio. The statistics of prophasing and nuclear envelope formation in the homo- and heterokaryon populations were consistent with the notion that disruption or formation of the nuclear envelope depends on the balance attained between disruptive and formative processes.

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