J. Kruuv

The effects of cotyledon senescence on the composition and physical properties of membrane lipid

The phospholipid content of rough and smooth microsomal fractions from cotyledons of germinating bean declines as the tissue becomes senescent. Both types of membrane contain comparable proportions of three major phospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, which collectively comprise about 90% of the total. This proportionality does not change appreciably during senescence. Only small quantities of lysophosphatides were noted at all stages of senescence. The unsaturated:saturated fatty acid ratio for total extracted lipid declined only slightly in both membrane systems, but pronounced differences in this ratio were observed among the major phospholipids of the membranes. The most striking alteration in lipid composition with advancing senescence was an increase in the sterol:phospholipid ratio; this rose by about 50% for rough microsomes and 400% for smooth microsomes. For both types of membrane the patterns of change in this ratio correlated with previously reported changes in bulk lipid transition temperature, suggesting that the increase in sterol level may contribute to changes in phase behaviour of the membranes during senescence. Arrhenius plots of rotational correlation times for the electron spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide (2N14) partitioned into the membrane lipid showed an increase in viscosity with advancing senescence and a corresponding increase in activation energy for both types of membrane. These changes in activation energy and viscosity correlated closely with the increase in sterol:phospholipid ratio. However, no phase transitions were detectable between temperatures of 2 and 55 degrees C despite the fact that transitions from a lipid-crystalline to gel state are detectable within this temperature range by wide angle X-ray diffraction.

Correlation of (Na+K+)-ATPase activity with growth of normal and transformed cells

Abstract The (Na+K+)-stimulated Mg2+-dependent ATPase activities of 3T3 and SV40 transformed 3T3 cells were compared as a function of cell population density. For normal cells the enzyme activity remained relatively constant during exponential growth, but decreased sharply coincident with contact inhibition of growth at confluence. This decrease in activity could be reversed by stimulating contact-inhibited cultures to undertake renewed short-term growth either by adding fetal calf serum or changing the medium completely. Transformed cells did not experience a decrease in (Na+K+)-ATPase activity upon reaching confluence, but this is consistent with the fact that they were still growing exponentially at this stage. However, non-confluent cultures of both normal and transformed cells incurred a marked decrease in levels of the enzyme when growth was inhibited by serum depletion. The results have been interpreted as indicating that levels of (Na+K+)-ATPase in both normal and transformed cells are correlated with growth. Hence the different patterns of ATPase activity displayed by malignant cells and their normal counterparts with increase in cell number appear to be a reflection of their dissimilar growth behaviours rather than of any innate difference between them.

Variation in Radiation Response of Mammalian Cells as a Function of Oxygen Tension

Complete radiation survival curves for mammalian cells as a function of partial pressure of oxygen have been obtained with considerable medium present. The results show an apparent decrease in extrapolation number (n) when irradiation is carried on during conditions of moderate hypoxia (5000 ppm oxygen) with the n value returning to the air level when the hypoxia becomes extreme. This phenomenon can be explained by radiochemical depletion of oxygen. At less than 25 ppm oxygen, the cells are not capable of sublethal repair, while at 200 ppm oxygen repair proceeds at almost the aerobic rate.

Relationship between hyperthermic cell killing and protein denaturation by alcohols

The monohydric alcohols methanol, ethanol, 2-propanol, and tert.-butanol dramatically sensitize V79 Chinese hamster lung cells to hyperthermia. for concentrations less than 3% alcohol by weignt, the rate of cell inactivation (k) appears to vary exponentially as a function of alcohol concentration. The degree of sensitization increases with increasing chain length, and for a constant concentration the ratio of the ks is 1:1.3:1.8:3.1 for methanol, ethanol, 2-propanol, and t-butanol, respectively. Mono-, di-, and trihydric alcohols have similar effects upon the rate of protein denaturation. Membrane lipid fluidity of V79 cells was determined from 23 to 63/sup 0/C by measuring the rotational correlation time of the spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide (2N14). A straight line was found for the Arrhenius plot of tau/sub c/, with an activation energy of 4.82 +- 0.07 kcal/mole. From the Arrhenius plot of the rate of cell killing from 42.0 to 46.8/sup 0/C, the enthalpy (..delta..H) and entropy (..delta..S) of activation were found to be 146 +- 9 kcal/mole and 388 cal/mole K, respectively. These values are consistent with protein denaturation (or possibly the denaturation of some other macromolecule) being the rate-limiting step in hyperthermic cell killing.

Plateau Phase in Growth Induced by Hypoxia

SummaryMammalian fibroblasts were incubated for four days at 37°C under hypoxic conditions. After one day the cells reached a plateau-phase in growth. After replating under aerobic conditions, various properties of the cells were investigated. It was found that they had accumulated in a pre-DNA-synthetic state. However, growth curves, DNA-synthesis data, and radiation-survival curves indicated that these cells behaved like an asynchronous population.

Repair of Potentially Lethal Damage in X-Irradiated V79 Cells

Repair of potentially lethal damage (PLD) was investigated in V79 Chinese hamster cells exposed to X radiation under various conditions of growth and cell-to-cell contact. It was found that these c...

The effect of hypoxia on the generation time of mammalian cells.

Growth curves for Chinese hamster fibroblasts are determined for various partial pressures of oxygen. It is found that for oxygen tensions greater than 500 parts per million, growth is only minimal...

Hyperthermia-induced inhibition of respiration and mitochondrial protein denaturation in CHL cells

Respiration of Chinese hamster lung V79 cells, as assayed by O2 consumption, increases linearly from 8 to 40 degrees C when plotted in the Arrhenius fashion but is strongly inhibited above 40 degrees C. The protein of mitochondria isolated from V79 cells undergoes structural transitions at 28 and 40 degrees C. This is supported by changes in the fluorescence excitation spectrum of conjugated pyrene maleimide and, to a lesser extent, intrinsic protein fluorophores. Electron spin resonance labelling studies with a derivative of tempo maleimide imply that extensive protein unfolding coincides with the 40 degrees C transition. The structural transition at 40 degrees C correlates well with inhibition of O2 consumption, is irreversible and is probably due to protein denaturation, while the change at 28 degrees C is reversible and has no effect on O2 consumption. Previous studies indicate the presence of a broad lipid transition extending from approximately 8 to 30 degrees C in mitochondrial membranes with all lipids being in the liquid-crystalline state above 30 degrees C. Thus, the onset of the lipid transition may induce the observed protein conformational change at 28 degrees C, but inhibition of respiration above 40 degrees C can be explained by protein denaturation alone. The region from 28 to 40 degrees C of stable protein conformation corresponds to the temperature range of V79 cell growth.

Lack of a correlation between hyperthermic cell killing, thermotolerance, and membrane lipid fluidity.

Butylated hydroxytoluene (BHT) is an effective membrane lipid perturber. Uptake studies using (/sup 3/H)BHT showed that it is effectively taken up by V79 Chinese hamster lung cells. The correlation time of rotation (tau/sub c/) of the spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide (2N14) was decreased 4.0 and 12.9% by addition of 0.03 and 0.07 mM BHT, respectively. This corresponds to increases in membrane fluidity produced by temperature increases of 1.8 and 6.0/sup 0/C, respectively. Neither BHT treatment sensitized the cells to hyperthermia. Also no decrease in membrane lipid fluidity, again as measured with the spin label 2N14, was found in thermotolerant V79 cells compared to control cells. Thermotolerance was induced by a 20-min exposure to 44.5/sup 0/C and the membrane fluidity measurements were made 16 hour later, when the maximum level of thermotolerance was observed. Thus no evidence was found for a correlation between membrane lipid fluidity and hyperthermic killing of V79 cells.

The effect of extreme hypoxia on recovery after radiation by synchronized mammalian cells.

An evacuation system for the production of extreme hypoxia is described. With this system survival curves using asynchronous Chinese hamster cells for extremely hypoxic and aerobic conditions have a common extrapolation number and a dose-modifying factor of 3.0-3.1. When synchronized hypoxic cells are irradiated during the most resistant phase of the age-response cycle and kept hypoxic between doses, no recovery from sublethal damage is observed.