J. Borsa

Reovirus: evidence for a second step in the intracellular uncoating and transcriptase activation process

Abstract Intracellular uncoating of reovirus has been reexamined. Biochemical and electron microscopy techniques were used. Present findings demonstrate that intracellular uncoating to the level of activated transcriptase proceeds via at least two distinct steps. In the first step, intact virions are converted to subviral particles which, by several criteria, appear to be identical to intermediate subviral particles (ISVP) generated in vitro . The endogenous transcriptase in such particles is in a switched-off state. Cells were infected with ISVP in an attempt to demonstrate further uncoating. Incubation of ISVP-infected cells at 37° for an appropriate time interval converts the input ISVP, which are totally refractory to proteolytic digestion, to a form in which a single major polypeptide is either lost or becomes protease sensitive. In electron micrographs of thin sections of cells which have been infected with ISVP, and subsequently incubated at 37°, virus particles of reduced diameter can be seen within the cytoplasm. Particles with activated transcriptase can be extracted from infected cells which have been incubated at 37° for an appropriate time. Extraction of these particles requires treatment of the cell homogenate with proteinase K. No active particles can be extracted with identical treatment of infected cells which have not been incubated at 37° prior to cell homogenization. These findings strongly suggest that the intracellular uncoating of reovirus to the level of active transcriptase proceeds via a pathway which is mechanistically identical to that elucidated for uncoating and transcriptase activation in vitro .

RADIOSENSITIZATION OF MAMMALIAN CELLS BY p-NITROACETOPHENONE. I. CHARACTERIZATION IN ASYNCHRONOUS AND SYNCHRONOUS POPULATIONS.

SummaryThe effectiveness of p-nitroacetophenone (PNAP) as a sensitizing agent to x-rays has been measured in asynchronous and synchronous populations of Chinese hamster cells. PNAP was found to sensitize hypoxic cells selectively (those equilibrated with an environment of 96 per cent N2 + 4 per cent CO2 containing less than 2 p.p.m. O2) with a maximum dose-modifying factor (DMF) of ∼ 1·50 at concentrations of 400 µM and greater in complete medium. In a parallel radiation scheme, the DMF for cells saturated with air was consistently between 2·75 and 3·05. In synchronously-growing Chinese hamster cells, the cyclic variation in surviving fraction measured for cells in air experiencing a radiation dose of 1055 rads could be reproduced for cells irradiated in hypoxic conditions with 2840 rads. We conclude that O2 sensitizes Chinese hamster cells in a dose-modifying fashion, irrespective of position in the cell-cycle. Hypoxic synchronized cells irradiated in the presence of PNAP with 1980 rads are also sensitiz...

Radiation-induced Strand Breakage in Mammalian Cell DNA: I. Enhancement of Single-strand Breaks by Chemical Radiosensitizers

SummaryChinese hamster cells irradiated with x-rays in complete medium while attached to glass surfaces and equilibrated with either air or nitrogen have been subsequently assayed for both colony-forming ability and size distribution of single-strand DNA fragments. Compounds evaluated for their ability to radiosensitize hypoxic cells and the concentrations used were: 1 mM para-nitroacetophenone (PNAP), 500µM N-(5-nitro-2-furfurylidine)-1-aminohydantoin (nitrofurantoin or NF2), 500µM 5-nitrofuraldehyde-2-semicarbazone (nitrofurazone or NF1), and 500µM 5-nitrofuraldehyde-2-oxime (nifuroxime or NF3). D0 values of 323, 227, 179, 155, 160, and 105 rads were obtained for N2, N2 + 1 mM PNAP, N2 + 500µM NF2, N2 + 500µM NF1, N2 + 500µM NF3, and air, respectively, which give enhancement ratios [ER(Survival)] of 1·00, 1·45, 1·80, 2·09, 2·01 and 3·08, respectively, for these conditions. The values of electron volt / single-strand break determined from alkaline sucrose sedimentation analyses for N2, N2 + 1 mM PNAP, N2...

Radiosensitization of mammalian cells by p-nitroacetophenone. 3. Effectiveness of nitrobenzene analogues

SummaryNitrobenzene analogues of p-nitroacetophenone (PNAP) have been tested on Chinese-hamster cells for their ability to sensitize hypoxic cells to x-rays. Seven compounds which are more effective than PNAP at a given non-toxic concentration are reported. One of these, 1-(2,4-dinitrophenyl)pyridinium chloride (DNPC), approaches the nitrofuran class of radiosensitizers in effectiveness. A correlation between cellular radiosensitization and drug electron affinity is examined for its usefulness in predicting radiosensitizing potential of compounds in the nitrobenzene series.

RADIOSENSITIZATION OF MAMMALIAN CELLS BY p-NITROACETOPHENONE. II. EFFECTIVENESS OF ANALOGUES.

SummaryAnalogues of p-nitroacetophenone (PNAP) have been tested on Chinese-hamster cells for their ability to inhibit cell proliferation (measure of toxicity) and to sensitize hypoxic cells to x-rays. Compounds with different substituents at the para position on the benzene ring showed capabilities of interfering with cell proliferation which could be correlated with their electronegativities. Using the 50 per cent ‘inhibition of proliferation’ value as an estimate of toxicity, the analogues showed decreasing toxicity in the order p-NO2 > p-Cl, p-Br > p-OCH2 > p-CH3 > p-NH2 > acetophenone. Of these compounds only PNAP sensitized hypoxic mammalian cells to x-rays in the concentration ranges tested.Studies with isomers of PNAP showed that m-nitroacetophenone was less toxic, as well as less effective as a sensitizer, than either o-nitroacetophenone or or PNAP. Mechanisms of toxicity and radiosensitization are discussed in relation to the electron affinities of the compounds.

Reovirus Transcriptase Activation in vitro: Further Studies on the Facilitation Phenomenon

Activation of the transcriptase in purified reovirus by chymotrypsin requires facilitating conditions in the digestion mix. In the presence of facilitation the virions are uncoated to cores. Without facilitation, uncoating is blocked at an intermediate stage, with transcriptase latent. Two independent types of facilitation have been discovered. The first type is mediated by K+, Rb+ or Cs+ ions. A second type, described here, is mediated by suitably high concentrations of virus in the digestion mix and requires collisions between intermediate subviral particles for its occurrence. The virus-concentration-mediated process is reversibly inhibited by high levels of active chymotrypsin.

Switch-on of transcriptase function in reovirus: Analysis of polypeptide changes using 2-D gels

Two-dimensional gel electrophoresis (IEF and SDS-PAGE) was used to examine virion polypeptide changes associated with switch-on of transcriptase function in reovirus. Results reveal that switch-on is correlated with altered electrophoretic behavior of a specific minor polypeptide (delta 1) which is present in intermediate subviral particles. A second finding is that each of the molecular weight classes of viral polypeptides exists as a series of subspecies with different isoelectric points. This suggests that extensive posttranslational modification of progeny viral polypeptides occurs during particle morphogenesis. These findings have important theoretical and practical implications.

Circular dichroism of intermediate subviral particles of reovirus Elucidation of the mechanism underlying the specific monovalent cation effects on uncoating

1. Circular dichroic (CD) spectra of purified intermediate subviral particles of reovirus were determined in the presence of different monovalent cations. 2. The CD spectra reveal that reo intermediate subviral particles can exist in two conformationally different forms. The two forms are readily distinguished by comparison of their ellipticities in the wavelength regions 210 nm and 220 nm, with a Na+-induced form exhibiting a reduced negative ellipticity relative to a Cs+-induced form. 3. The transition between the Na+- and Cs+-induced forms is reversible by manipulation of the species of monovalent cation present and appears to be temperature independent. 4. Temperature variation studies on dilute suspensions of particles indicate that the Na+-induced form is stable, whereas the Cs+-induced from undergoes a second transition, temperature dependent and irreversible, to become a viral core. 5. A model is presented relating these observations to the known properties of reovirus uncoating and transcriptase activation.

DISCUSSION PAPER: CYTOTOXIC MECHANISMS OF FOLATE ANTAGONISTS

In this discussion I should like to comment on three separate points. The first will deal with some of the differences observed by different investigators with regard to the characteristics of cell kill by amethopterin (Methotrexate) (MTX). The second point is brief and is related to the selection of new folic acid analogues which, in some mammalian cell systems, may be superior to MTX as cell killing agents. The third point will deal, again briefly, with one aspect of the problem of predicting the clinical outcome of treatment. The area relating to the first point is best defined by comparing the findings of Dr. Hryniuk and associates on the L5178Y system as presented earlier in this volume and in the literature 1 with the published results of Borsa and Whitmore,a who worked with an L-cell system. In the L5178Y system cell killing begins immediately upon addition of MTX, and there is more cell kill during a four-hour exposure in the absence of exogenous purine than in its presence. In the L-cell system, cell killing begins only after a delay of approximately 10 hours, and the rate of cell killing is much greater in the presence of exogenous purine than in its absence. The delayed onset type of MTX-induced cell killing is also operative in the Hela cell system of Rueckert and Mueller.:’ Thus in the L5178Y system, cell kill is very rapid and is diminished in the presence of exogenous purine, whereas in the L-cell system and in the Hela cell system the cell kill is of a delayed type and in L-cells at least, is greatly augmented in the presence of exogenous purine. Two possibilities exist to explain these two types of cell kill characteristics. One is that there is a basic difference in killing mechanism operative in the different cell systems employed. The other possibility is that the differences are due to procedural artifacts. One possible source of such variation stems from the different conditions under which cells can be assayed for viability following drug treatment. In our work with L-cells, we assayed for cells which, following treatment with MTX, could grow into colonies in medium that contained both thymidine (TdR) and purines. Thus the MTX-induced thymineless and/or purineless state was abruptly brought to an end at the time of plating of the cells. Our reasoning in this was that the assay condition for viability should simulate the worst possible condition in vivo where TdR and purines might be made available to treated cells from degradation of other cells, perhaps previously killed by the MTX treatment. Rueckert and Mueller also included TdR in their plating medium. Under this assay condition, the cell kill was of the delayed type as previously mentioned. One might get different results if the assay for viable cells were carried out in medium lacking TdR and purines, as it was in Dr. Hryniuk’s L5178Y system. In such a case the MTX-induced thymineless and/or

Buckwheat lipoxygenase: Inactivation by gamma-irradiation

Abstract Lipoxygenase (LOX) activity was detected in buckwheat extracts, and was shown to have a pH optimum of 6·5. LOX activity was not affected during 6 days of germination. The specific activity increased when ground buckwheat seeds were extracted with 0·01% or 0·1% solutions of calcium chloride or magnesium chloride, compared to extractions with distilled water or 1·0% solutions of either of these salts. Increase in specific activity was greater in the presence of calcium ions compared to magnesium ions. Gamma-irradiation of buckwheat seeds up to 4 kGy resulted in a decrease in LOX activity to a level of approximately 22% of the untreated control, but increased to approximately 43% of the control at a dose of 6 kGy. These results suggest that gamma-irradiation reduces LOX activity, but is not likely to completely prevent lipid oxidation in buckwheat.