Hyperthermia in a differentiating murine erythroleukemia cell line: cell killing by heat and radiation

The survival response of Friend erythroleukemia cells (a differentiating cell system) to heat and radiation has been examined. The Friend erythroleukemia cells (FELC) were more heat and radiation sensitive than V79 cells, and the heat and radiation survival curves possessed shoulders, showing the ability of the cells to accumulate sublethal damage. Thermal tolerance was expressed after prolonged heating at 41.0-42.0 degrees C. Thermal radiosensitization by heating at 42.0 or 45.0 degrees C was greatest for simultaneous heat and radiation treatments, and recovery occurred when the cells were incubated at 37 degrees C between the heat and radiation or radiation and heat treatments. Arrhenius analysis of the FELC heat survival data showed that the curve for thermal inactivation possessed a break at about 43.0 degrees C and that the thermal inactivation energies above and below the break point were comparable to those for V79 cells and other cell lines reported in the literature.     

The rapid loss of viability ofAzotobacter in aqueous solutions

When a low number ofAzotobacter vinelandii 12837 log phase vegetative cells (2 × 10³ cells/ml) were removed from the culture liquid to water of the same temperature, a rapid loss of viability occurred depending on the procedure of washing and suspending. Death was not accompanied by visible lysis and the rate of loss of viability was less at lower temperatures, and in the presence of salts or cell-free filtrates from heavy cell suspensions in water. The die-off was erratic at increased cell concentrations and was accelerated by utilizable energy sources. Cells standing in a favorable ionic solution (0.1% NaCl) do not lose their viability while cells washed by a series of centrifugations with the same ionic solution show a progressive loss of viability with each washing. Phospholipids were found to leach from the cells into the aqueous solutions. Such cell death suggests instability of the cell membrane and the loss of osmotic or ionic control in the cells.This work was supported by grants AI-02830 and GM 600 from the U.S. Public Health Service.     

Time-temperature relationships for L1A2 cells step-down heated from 38 to 45 degrees C in vitro

The in vitro response of L1A2 cells to a single exposure to one temperature and to step-down heating was investigated. Single heating consisted of heating for a specified time at a constant temperature in the range 38.0-45.0 degrees C, whereas step-down heating involved a pretreatment of either 45.0 degrees C for 10 min or 42.0 degrees C for 90 min. The pretreatments were adjusted to give the same survival level. The survival curves for single heating had an initial shoulder followed by an exponential region, whereas for step-down heating they were strictly exponential and had no shoulder. The time-temperature relationship for cells exposed to single heating showed a biphasic Arrhenius curve with a downward inflection at 40.5 degrees C. Biphasic Arrhenius curves were also observed for step-down heating, but both the 45 degrees C/10 min and the 42 degrees C/90 min pretreatment showed an upward inflection that broke at 42.5 degrees C and 40.5 degrees C, respectively. The downward inflection on the Arrhenius curve for single heating has been attributed to thermotolerance development and the effect of step-down heating to a temporary inhibition of thermotolerance development. However, the present shape of the Arrhenius curves for step-down heating cannot be explained by inhibition of thermotolerance. It is therefore reasonable to assume that step-down heating is more than just the inhibition of thermotolerance, and that step-down heating and thermotolerance are distinct phenomena which act independently.     

Heterogeneity in radiosensitization by heat of cloned cell lines derived from a single human melanoma xenograft

Heterogeneity in radiosensitization by heat was studied using one uncloned and five cloned cell lines isolated from a single tumour of a human melanoma xenograft. Cells from passages 7-12 in vitro were given heat treatments of 42.5 degrees C (45 min), 43.5 degrees C (45 min) or 44.5 degrees C (45 min) immediately after exposure to graded doses of radiation. The survival curves after irradiation alone had similar D0 values but differed in the size of the shoulder. The heterogeneity in heat radiosensitization was reflected in differences in decrease of the D0 values. The thermal enhancement ratios, calculated from the D0 values, were in the ranges 1.2 +/- 0.2-1.7 +/- 0.2 (42.5 degrees C), 1.4 +/- 0.3-2.4 +/- 0.4 (43.5 degrees C) and 2.3 +/- 0.4-3.4 +/- 0.4 (44.5 degrees C). Moreover, at 43.5 degrees C the heterogeneity was also reflected in different modifications of the shape of the survival curves. Two lines showed survival curves with a significant shoulder and a relatively low D0 value whereas two other lines had lost the shoulder almost completely but showed relatively high D0 values. All lines showed survival curves with a broad shoulder after heating at 42.5 degrees C, whereas none of the lines showed survival curves with a significant shoulder after heating at 44.5 degrees C.     

Survival of Vibrio parahaemolyticus in Cooked Seafood at Refrigeration Temperatures

The growth and survival of two strains of Vibrio parahaemolyticus isolated during food-borne gastroenteritis outbreaks in Japan and surface inoculated on cooked shrimp, shrimp with sauce, or cooked crab were tested at various refrigeration temperatures during a 48-h holding period. On cooked shrimp and crab, the vibrios grew well at 18.3 C, but their numbers declined gradually at 10 C and below. At 12.8 C, vibrios remained static for the most part. Thus, it appeared that 12.8 C was the borderline temperature for growth of the organism on cooked seafood. When cocktail sauce was added to surface-inoculated shrimp at a ratio of 2:1, the vibrio die-off rate was accelerated. In the shrimp and sauce few cells remained after 48 h, but in the sauce alone die-off was complete at 6 h.     

Effect of sub-optimal temperatures on survival of mammalian cells

When survival at low temperatures, in terms of colony-forming ability, is measured in Chinese hamster lung cells (V79), it varies inversely with temperature in the 10–25 °C range; i.e. survival at 10 °C is greater than that at 25 °C. These survival-time curves on semi-log plots have a “shoulder” region followed by a linear region. Survival at these temperatures varies inversely with the macromolecular synthesis rate. Results with cells at 5 °C break the above patterns.     

Factors Influencing the Photodynamic Action of Benzo[a]pyrene on Escherichia coli

Death of Escherichia coli resulted when a buffer suspension was exposed simultaneously to colloidal benzo[a]pyrene (BP) and 355-mmu illumination. Neither hydrocarbon nor illumination alone caused death; oxygen had to be present. The survival curve had a shoulder, and then death proceeded exponentially with time. Death rate was independent of temperature between 6 and 32 C. The duration of the shoulder, however, decreased slightly with increase in temperature. The shoulder was not due to delay in BP entering the cell. Death was influenced by the composition of the medium in which the cells were grown prior to illumination. The amount of BP bound to the cells was determined after three ethyl alcoholether extractions. Appreciable binding occurred in the presence of 355-mmu illumination with air, and relatively little binding occurred under nitrogen; very little binding occurred in the dark with nitrogen or air. At the outset, rate of binding under illumination with air was not temperature-dependent, but with time it became strongly temperature-dependent. Binding under illumination with nitrogen was temperature-independent. Bound BP was associated primarily with cell protein. Cells in growth medium resisted death and BP binding. At 21 and 32 C, deoxyribonucleic acid damage occurred during exponential death. No damage was detected at 21 and 32 C in the dark with BP, under illumination in absence of BP, or under illumination with BP in a nitrogen atmosphere.     

Inhibitors of poly(ADP-ribose) synthetase enhance the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster cells

Two inhibitors of poly(ADP-ribose) synthetase, 5-methylnicotinamide and m-methoxybenzamide, enhanced the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster V79 cells. The inhibitors showed minimal toxicity for cells treated at 37 degrees C, and did not appreciably alter cellular ATP levels under any of the experimental conditions used. Enhanced cell killing occurred when the inhibitors were added after an acute (5-10 min) 45 degrees C heat shock, and after 50 and 100 min exposures to 42 degrees C. When present during heating at 42 degrees C, the inhibitors reduced the shoulder of the 42 degrees C survival curves but did not appreciably affect the slopes. The results suggest a possible role for poly(ADP-ribose) synthetase in the survival response of V79 cells to hyperthermia.     

Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures

Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water-glycerol media. A phase transition occurred at 12 degrees C in water, at 16.5 degrees C at Aw=0.75, and at 19.5 degrees C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 degrees C in pure water and at 27 degrees C in binary water-glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 degrees C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 degrees C in water, to 64 degrees C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.     

Survival of Escherichia coli and Salmonella spp. in estuarine environments

Survival of Escherichia coli and Salmonella spp. in estuarine waters was compared over a variety of seasonal temperatures during in situ exposure in diffusion chambers. Sublethal stress was measured by both selective-versus-resuscitative enumeration procedures and an electrochemical detection method. E. coli and Salmonella spp. test suspensions, prepared to minimize sublethal injury, were exposed in a shallow tidal creek and at a site 7.1 km further downriver. Bacterial die-off and sublethal stress in filtered estuarine water were inversely related to water temperature. Salmonella spp. populations exhibited significantly less die-off and stress than did E. coli at water temperatures of less than 10 degrees C. Although the most pronounced reductions (ca. 3 log units) in test bacteria occurred during seasonally warm temperatures in the presence of the autochthonous microbiota, 10(2) to 10(4) test cells per ml remained after 2 weeks of exposure to temperatures of greater than 15 degrees C. Reductions in test bacteria were associated with increases in the densities of microflagellates and plaque-forming microorganisms. These studies demonstrated the survival potential of enteric bacteria in estuarine waters and showed that survival was a function of interacting biological and physical factors.     

Survival of Escherichia coli and Salmonella spp. in estuarine environments.

Survival of Escherichia coli and Salmonella spp. in estuarine waters was compared over a variety of seasonal temperatures during in situ exposure in diffusion chambers. Sublethal stress was measured by both selective-versus-resuscitative enumeration procedures and an electrochemical detection method. E. coli and Salmonella spp. test suspensions, prepared to minimize sublethal injury, were exposed in a shallow tidal creek and at a site 7.1 km further downriver. Bacterial die-off and sublethal stress in filtered estuarine water were inversely related to water temperature. Salmonella spp. populations exhibited significantly less die-off and stress than did E. coli at water temperatures of less than 10 degrees C. Although the most pronounced reductions (ca. 3 log units) in test bacteria occurred during seasonally warm temperatures in the presence of the autochthonous microbiota, 10(2) to 10(4) test cells per ml remained after 2 weeks of exposure to temperatures of greater than 15 degrees C. Reductions in test bacteria were associated with increases in the densities of microflagellates and plaque-forming microorganisms. These studies demonstrated the survival potential of enteric bacteria in estuarine waters and showed that survival was a function of interacting biological and physical factors.     

Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures

Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water-glycerol media. A phase transition occurred at 12 °C in water, at 16.5 °C at Aw=0.75, and at 19.5 °C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 °C in pure water and at 27 °C in binary water-glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 °C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 °C in water, to 64 °C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.     

Effect of oxygen on liquid holding recovery of Bacteroides fragilis.

Liquid holding recovery (LHR) in ultraviolet-irradiated Bacteroides fragilis cells occurred under aerobic conditions but was inhibited by anaerobic conditions. The increase in survival after aerobic LHR resulted in an increase in the shoulder regions of the ultraviolet survival curves. Maximum LHR was obtained after holding the cells for 2 to 3 h. LHR was temperature dependent, and in stationary-phase cells LHR was independent of nutrients. Higher levels of LHR occurred in exponential-phase cells than in stationary-phase cells, and LHR was affected by nutrients in exponential-phase cells. Sublethal concentrations of caffeine and acriflavine inhibited LHR. In addition to LHR, minimal medium recovery also occurred in the concentration of [3H]thymine-containing dimers in the acid-insoluble fraction of the cells. A corresponding increase in [3H]thymine-containing dimers was observed in the acid-soluble fraction after LHR. Although a small proportion of irradiated cells produced filaments, this phenomenon was not directly related to LHR in B. fragilis.     

Effects of growth at different temperatures on the physical state of lipids in native microsomal membranes from Tetrahymena

Fluorescence measurements of the probe 1,6-diphenyl-1,3,5-hexatriene in native Tetrahymena pyriformis microsomal membranes revealed characteristic "break points" in curves of polarization vs. temperature. In the 5--35 degree C range, membranes from cells grown at 39 degrees C exhibited two break points, one at 11.6 +/- 0.6 degrees C and another at 23.1 +/- 1.6 degrees C. Membranes from 15 degrees C grown cells also showed two break points, one at 8.0 +/- 1.7 degrees C and another at 17.7 +/- 1.7 degrees C. Complementary measurements of turbidity (absorbance at 360 nm) vs. temperature revealed break points at approximately the same temperatures as observed with the fluorescent probe, thus strengthening the likelihood that the break points signify the onset or termination of lipid phase separations or some other significant structural alteration of lipids. In general, break points measured in the native membrane samples occurred at slightly lower temperatures than did break points in lipids extracted from comparable membranes. This suggests two possible types of protein--lipid interaction. First, there may be a selective withdrawal of relatively highly saturated phospholipid molecular species from the bulk lipid phase and into protein annulus regions. Alternatively, the configuration of the hydrophobic core of certain key membrane proteins may be such that nonspecific interactions with the lipids stabilize the liquid-crystalline phase.     

pH- and thermal-dependent conformational transition of PGAIPG, a repeated hexapeptide sequence from tropoelastin

The secondary structure of PGAIPG (Pro-Gly-Ala-IIe-Pro-Gly), a repeated hexapeptide of tropoelastin, in buffer solution of different pH was determined by using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The thermal-dependent structural change of PGAIPG in aqueous solution or in solid state was also examined by thermal FTIR microspectroscopy. The conformation of PGAIPG in aqueous solution exhibited a pH-dependent structural characterization. A predominant peak at 1614 cm(-1) (aggregated beta-sheet) with a shoulder near 1560 cm(-1) (beta-sheet) appeared in pH 5.5-8.5 buffer solutions. A new broad shoulder at 1651 cm(-1) (random coil and/or alpha-helix) with 1614 cm(-1) was observed in the pH 4.5 buffer solution. However, the broad shoulder at 1651 cm(-1) was converted to a maximum peak at 1679 cm(-1) (beta-turn/antiparallel beta-sheet) when the pH shifted from 4.5 to 3.5, but the original pronounced peak at 1614 cm(-1) became a shoulder. Once the pH was lowered to 2.5, the IR spectrum of PGAIPG was dominated by major absorption at 1679 cm(-1) with a minor peak at 1552 cm(-1) (alpha-helix/random coil). The result indicates that the pH was a predominant factor to transform PGAIPG structure from aggregated beta-sheet (pH 8.5) to beta-turn/intermolecular antiparallel beta-sheet (pH 2.5). Moreover, a partial conformation of PGAIPG with minor alpha-helix/random coil structures was also explored in the lower pH buffer solution. There was no thermal-dependent structural change for solid-state PGAIPG. The thermal-induced formation of aggregated beta-sheet for PGAIPG in aqueous solution was found from 28 to 30 degrees C, however, which might be correlated with the formation of an opaque gel that turned from clear solution. The formation of aggregated beta-sheet structure for PGAIPG beyond 30 degrees C might be due to the intermolecular hydrogen bonded interaction between the hydrophobic PGAIPG fragments induced by coacervation.     

Cell survival and recovery processes in Chinese hamster AA8 cells and in two radiosensitive clones

Cell survival and recovery after gamma irradiation were investigated in a Chinese hamster ovary cell line (AA8) and in two radiosensitive clones (EM9 and NM2) derived from it. When analyzed by the multitarget and linear-quadratic equations, the dose-response curves for survival of both EM9 and NM2 cells, compared with AA8 cells, were characterized by a decreased magnitude of the shoulder or single-hit region (as reflected by Dq or alpha, respectively) but no difference in the terminal slope or double-hit region (as reflected by DO or beta, respectively). Recovery from sublethal damage (SLD) and potentially lethal damage (PLD) was measured in the three cell lines to examine the relationship between the shoulder width of the survival curve and the magnitude of cellular recovery. NM2 cells exhibited a reduced shoulder on their survival curve and a reduced capacity for SLD recovery, compared with AA8 cells, after equitoxic doses of radiation. EM9 cells, which also had a reduced shoulder on their survival curve, displayed the same rate and extent of recovery as AA8 cells for both SLD and PLD. PLD recovery, as assayed in fed plateau-phase NM2 cells by delayed plating, occurred with slower initial kinetics but to the same final extent as that in AA8 cells, resulting in modification of both the shoulder and the slope of the survival curve. However, PLD recovery, as assayed in log-phase NM2 cells by postirradiation treatment with hypertonic salt, was normal and affected predominantly the slope of the survival curve. These data demonstrate that although both SLD and PLD recovery play a role in determining cell survival, cell-survival curve parameters may not always be useful in predicting cellular recovery capacity.     

Survival of Escherichia coli and Vibrio harveyi in Dead Sea water

Abstract When suspensions of Escherichia coli or the marine luminescent bacterium Vibrio harveyi were mixed with Dead Sea water, the number of viable bacteria decreased by 90% in a time varying from less than 1 h to several hours, depending on the bacterial strain tested. Survival was better at low temperatures, and diluting the Dead Sea water permitted prolonged survival of both coliform bacteria and V. harveyi . The death rate of E. coli in Dead Sea water was comparable to that in water from the Great Salt Lake (Utah). The high concentrations of calcium and magnesium in Dead Sea water, rather than the high total salinity, was identified as the main factor responsible for the rapid die-off. Exposure to direct solar irradiation significantly increased the die-off rate of E. coli in Dead Sea water.
Large numbers of coliform bacteria were recovered from the lake at distances of at least 20 m from a sewage discharge site on the western shore of the Dead Sea.     

Factors influencing survival and growth of mammalian cells exposed to hypothermia. I. Effects of temperature and membrane lipid perturbers

The Arrhenius plot of the rate of V79 Chinese hamster cell inactivation due to hypothermia has a "break" around 7-10 degrees C with optimum storage temperature for unprotected cells being about 10 degrees C. Addition of the membrane lipid perturber, butylated hydroxytoluene, improves survival of cells when compared to controls at temperatures below this break but not above. Arrhenius plots of growth rates of the cells show breaks at 30 and 40 degrees C. Measurements of membrane fluidity by electron spin resonance or membrane polarization anisotropy by fluorescence spectrophotometry techniques as a function of temperature in these cells also reveal "breaks" centered around 8 and 30 degrees C. Hence, the changes in the rate of cell inactivation and growth as a function of temperature may be related to membrane lipid phase changes.     

Protective effect of casein toward Salmonella typhimurium in acid-milk

Lactic acid is the inhibitory agent in yoghurt responsible for the inhibition of Salmonella typhimurium. Casein, however, may exert a protective effect toward the survival of the salmonella in acid-milk products. Salmonella typhimurium was found to die-off 21.2% more rapidly in 18-h yoghurt-whey than in 18-h yoghurt at 37 degrees C with a pH of 3.85 and 1.42% lactic acid. When casein was added to yoghurt-whey, the die-off rate of the salmonellas was reduced to that found in yoghurt. The rate remained unchanged when 4.8% sodium caseinate was added to the whey. When 0 to 14% casein was added to the acid-whey the die-off rate changed from 9.7 to 24.0 min/log reduction of cells, respectively. There was a direct correlation between the increase in casein concentration and length of survival of the salmonellas. At a pH of 3.85, 4.2 or 4.5, the die-off rate was 6.5, 13.0 or 40 min/log reduction of cells in milk containing 1.42% lactic acid, and was 4.0, 10.0 or 33.3 min/log reduction, respectively, in whey with 1.42% lactic acid. Thus, the protective effect of casein toward Salm. typhimurium increased as the pH increased. This indicated that casein exerts a protective effect on Salm. typhimurium in acid dairy products and the degree of protection depends on the casein concentration, the form of the casein molecule and the pH.     

X-Ray and Ultraviolet Sensitivity of Synchronized Chinese Hamster Cells at Various Stages of the Cell Cycle

Populations of Chinese hamster cells, synchronized by selecting for cells at or close to division, were exposed to 250 kvp x-rays and to ultraviolet light at different stages of the cell cycle and colony-forming ability examined thereafter. These cells were found to be most resistant to x-rays during the latter part of the DNA synthetic period (S) and to be about equally sensitive before (G₁) and after (G₂) this period. Multitarget type curves of the same slope (D_o ~ 200 rad) only approximately fitted the survival data at different stages in the cycle. The changes in response were primarily due to variations in the shoulders (or extrapolation numbers) of the curves however. The response to ultraviolet light differed from that to x-rays. Resistance was greatest in G₂ and changes in both shoulder and slope of the survival curves occurred throughout the cell cycle. The x-ray and ultraviolet responses for component stages of the cell cycle were respectively compounded into expected survival data for a log phase asynchronous population of hamster cells and found to agree well with direct experiment.