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  • 1
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Human and mouse sperm nuclei obtained by sonication or mechanical agitation of freshly isolated sperm in the presence of anionic detergent were purified through a sucrose gradient and stained with acridine orange (AO); their fluorescence intensity was measured by flow cytometry. The green fluorescence, characteristic of AO binding to DNA by intercalation, was twice lower per unit of DNA for human sperm nuclei than for human peripheral blood lymphocytes. After extraction of basic proteins with 0.08 N HCl, AO binding to DNA increased 3.2-fold for lymphocytes and only 1.3-fold for sperm indicating that, in contrast to somatic cells, the proteins restricting AO binding to DNA are essentially non-extractable from sperm at that low pH. Treatment of human and mouse nuclei with dithiothreitol (DTT), a sulfhydryl reducing agent, and trypsin, removed constraints responsible for the restriction of AO binding. Specifically, as a result of DTT treatment alone there was up to a 20–30% increase of AO binding; upon subsequent addition of trypsin there was a further rapid rise in AO binding up to a final level of approximately 5 times the original AO binding to isolated sperm nuclei. Electron microscopy of DTT-treated human sperm nuclei showed that the reducing agent caused chromatin decondensation to a level whereby 20–30 Å diameter fibers interconnecting chromatin bodies about 30–75 nm in diameter were revealed. Trypsin digestion in the presence of DTT converted the chromatin bodies into a network of fibrous structures about 150 Å in diameter. Both electron microscopy and flow cytometry demonstrated an extremely large intercellular variation among human sperm nuclei in response to DTT and trypsin treatment indicating heterogeneity of chromatin structure. In contrast, AO staining of mouse sperm nuclei increased homogeneously in response to DTT and trypsin treatment.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 100 (1979), S. 425-438 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The metachromatic fluorochrome acridine orange was used to differentially stain DNA and RNA in Chinese hamster ovary (CHO) cells and in mitogen-stimulated human lymphocytes during their progression through the cell cycle. Green and red fluorescence of individual cells, representing cellular DNA and RNA, respectively, was measured by flow cytometry.CHO cells were synchronized by selective detachment at mitosis. Their rate of progression through G1 and subsequently through S phase correlated with the content of stainable RNA. The mean duration of the G1 phase was 5.2 hours for cells with high RNA content (highest 25 percentile population) and 8.1 hours for cells with low RNA (lowest 25 percentile). The duration of S phase was 5.9 and 7.5 hours for high- and low-RNA, 25 percentile subpopulations, respectively.Lymphocytes synchronized at the G1/S boundary by hydroxyurea or 5-fluorodeoxyuridine showed extremely high intecellular variation with respect to content of stainable RNA. After release from the block they traversed S phase at rates linearly proportional to the content of stainable RNA. The duration of S phase was five hours for cells with high RNA-, six to nine hours for cells with moderate RNA- and up to 27 hours for cells with minimal RNA-content.The data suggest that the rate of progression the cell cycle of individual cells within a population may be correlated with the number of ribosomes per cell.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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