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The ultrastructural modifications produced by anisotonic NaCl treatment of Chinese hamster mitotic cells were observed at three NaCl concentrations which have been frequently used in radiosensitization studies: 0.05, 0.5 and 1.5 M. After exposure to 0.05 M NaCl, many well-spread chromosomes are visible. The chromatin fibres are well dispersed and membraneous material is associated with the chromosomes. After hypertonic treatment with 0.5 M NaCl, the chromosomes have a uniform, structureless appearance with some coalescing into larger anaphase-like masses. At 1.5 M NaCl, large scale cellular dehydration is apparent, and filamentous structures such as microfilaments are tightly constricted. The degree of chromosome staining is also reduced below the level of the cytoplasm. After both hypo- and hypertonic NaCl treatment the chromosomes appear swollen relative to untreated cells, but hypertonic treatment causes chromosome clumping and dissociates chromatin. Conformational changes in the chromatin may restrict the capacity for DNA repair and be related to cellular radiosensitivity.

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Isodose radiation survival of V79 Chinese hamster cells, pretreated with strongly hypertonic concentrations of NaC1 at 22 degrees C, or at 37 degrees C, has been determined and correlated with ultrastructural changes within the nucleus. After an exposure of less than 10 min to 1.5 M NaC1, at both temperatures, the cells are radioprotected, but after longer exposures, the cells treated at 37 degrees C are radiosensitive, whereas those treated at 22 degrees C still show protection. The cells are radiosensitized at both temperatures by pretreatment with 0.5 M and 0.05 M NaC1. The ultrastructure of the nucleus observed after the anisotonic treatments suggests that contraction or swelling of chromatin may be associated with the observed variation in radiation sensitivity.

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Ultrastructural changes produced in cultured cells after irradiation by X-rays reflect the temporal development of the original lesions. Alterations in membrane structures, especially the plasma and nuclear membranes, are documented, supporting the suggestion that membrane and membrane-DNA attachment sites are relevant to radiation-induced cell killing. Alterations produced by the radiosensitizer diamide and by anisotonic salt treatments were also observed. Diamide, at 0.4 to 0.6 mmol/L, rapidly decreased the active uptake of K+ ions at the plasma membrane and produced clear, organelle-free regions of cytoplasm and distorted nuclei. Anisotonic NaCl treatment of mitotic cells swells chromosomes under both hypo- and hypertonic conditions, leaving the chromatin open to free-radical attack. In interphase, the cell is drastically shrunken during the first few minutes of hypertonic (1.5 mol/L NaCl) salt treatment, after which breaks and localized blebs appear at the plasma membrane, while the nucleus swells, leaving the chromatin in an open state. This process occurs more quickly at 37 degrees C than at room temperature (22 degrees C) and correlates with the relative radiosensitivity of cells treated at these temperatures.

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Evidence is presented supporting the hypothesis that reovirus intermediate subviral particles (ISVP), which show increased infectivity relative to intact virions, can gain entry into host L cells by two alternative pathways. One pathway is by the process of viropexis, involving phagocytic vacuoles. A second entry pathway is via direct penetration of the plasma membrane of the cell, without involvement of a phagocytic vacuole. Using electron microscopy, a kinetic analysis of the uptake process was carried out. Results indicate that at 37 degrees C ISVP gain entry into host cells primarily by direct entry, although viropexis also occurs, while intact virions gain entry by viropexis almost exclusively. A second line of experimental evidence consistent with the idea that ISVP can 'melt' their way through the plasma membrane is provided by studies on the release of pre-loaded radioactive 51Cr from host cells following infection. 51Cr release data demonstrate that infection with ISVP leads to an immediate increased leakiness of the cell plasma membrane, whereas no such increase takes place following infection with an equivalent number of intact virions. This demonstrates that ISVP can interact with the plasma membrane of the cell in a manner which is qualitatively different from the interaction between intact virions and the plasma membrane. The ability of ISVP to directly penetrate the plasma membrane of the host cell, which intact virions apparently cannot do, could explain the decreased duration of the eclipse phase, as well as the increased infectivity of ISVP, relative to that observed for infection with intact virions.

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We have shown that radiation affects the nuclear envelope, a membrane structure-closely associated with DNA. The density of nuclear pores visible on freeze-etch surfaces decreased at a rate of 0.042 (pores/mum2)/100 rad with respect to unirradiated cells. This is interpreted as a radiation-induced delay in development of the nuclear envelope.

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Specific monovalent cations control the modification of reovirus infectivity by chymotrypsin. Digestion in K(+), Rb(+), or Cs(+) reduces infectivity several logs, whereas in Na(+) or Li(+) digestion markedly enhances infectivity.

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Reovirus virions, grown in suspension cultures of L cells and extensively purified by density gradient and velocity gradient centrifugation after their release from cell debris by fluorocarbon extraction, are characterized by a mean particle diameter of 73 nm and a density in CsCl of 1.36 to 1.37 g/cm(3). Treatment of intact virions by chymotrypsin (CHT) digestion in vitro converts them to subviral particles (SVP) having characteristics which are determined by the species of monovalent cation present during the digestion. In the presence of Cs(+) ions, CHT converts the virions to SVP of mean diameter 51 nm and density 1.43 to 1.44 g/cm(3). In the presence of K(+) ions, the conversion is to SVP of diameter 51 nm and density 1.39 to 1.40 g/cm(3). The SVP made in the presence of either Cs(+) or K(+) possess an extremely active RNA polymerase and nucleoside triphosphate phosphohydrolase (NTPase) activity in vitro and are resistant to further digestion by CHT. Treatment of intact virions with CHT in the presence of Na(+) or Li(+) ions results in their conversion to SVP of mean diameter 64 nm and density 1.37 to 1.38 g/cm(3). Such SVP are not active in in vitro RNA synthesis or NTP hydrolysis and are resistant to further digestion by CHT even during prolonged exposure to high concentrations of enzyme. Addition of Cs(+) or K(+) ions to the digestion mixture allows conversion of the 64-nm diameter SVP to 51-nm diameter SVP in which the RNA polymerase and NTPase are active in vitro. Analysis of the proteins present in intact virions and in the different SVP reveals clear differences which indicate that the conversions are accomplished by removal or cleavage of particular species of polypeptides.

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