Microcalorimetric study of iodized and noniodized cells and C-phycocyanin of Spirulina platensis

It was shown that eight stages of transition are observed in the heating process of Spirulina platensis cells in temperature range 5-140 degrees C. The first stage covers the temperature range 5-53 degrees C with maximum approximately 45 degrees C. The heat evolved in this temperature range is equal to 380 +/- 20 J/g of dry biomass, it does not change at scanning rate lower than 0.083 degrees C/min and belongs, mainly, to cell respiration in a stationary regime, in the dark. It was shown that endotherm approximately 66 degrees C belongs to denaturation of C-phycocyanin which denaturates in solutions with Td = 64.2 degrees C, deltaHd = 34.7 +/- 2.1 J/g and for it deltaHd(cal)/deltaH(V.H) is equal to 10.8 +/- 1.2. The endotherms with Td equal to 58 and 88 degrees C are connected with denaturation of phycobilisome proteins and endotherm with Td = 48 degrees C and deltaHd = 4.2J/g of dry biomass-with denaturation of protein which, apparently, is connected with cell respiration.     

Influence of heat transmission mode on heating rates and on the selection of patches for heating in a mediterranean lizard

Heliothermy (heat gain by radiation) has been given a prominent role in basking lizards. However, thigmothermy (heat gain by conduction) could be relevant for heating in small lizards. To ascertain the importance of the different heat transmission modes to the thermoregulatory processes, we conducted an experimental study where we analyzed the role of heat transmission modes on heating rates and on the selection of sites for heating in the Mediterranean lizard Acanthodactylus erythrurus (Lacertidae). The study was conducted under laboratory conditions, where two situations of different operative temperatures (38 degrees and 50 degrees C) were simulated in a terrarium. In a first experiment, individuals were allowed to heat up during 2 min at both temperatures and under both heat transmission modes. In a second experiment, individuals were allowed to select between patches differing in the main transmission mode, at both temperatures, to heat up. Experiences were conducted with live, nontethered lizards with a starting body temperature of 27 degrees C. Temperature had a significant effect on the heating rate, with heat gain per unit of time being faster at the higher operative temperature (50 degrees C). The effect of the mode of heat transmission on the heating rate was also significant: at 50 degrees C, heating rate was greater when the main heat transmission mode was conduction from the substrate (thigmothermy) than when heating was mainly due to heat gain by radiation (heliothermy); at 38 degrees C, heating rates did not significantly differ between transmission modes. At 38 degrees C, selection of the site for heating was not significantly different from that expected by chance. However, at 50 degrees C, the heating site offering the slowest heating rate (heliothermic patch) was selected. These results show that heating rates vary not only with environmental temperature but also with different predominant heat transmission modes. Lizards are able to identify and exploit this heterogeneity, selecting the source of heat gain (radiation) that minimizes the risk of overheating when temperature is high.     

The effect of media tonicity on Escherichia coli resistance to heating with different gradient

The influence of NaCl water solutions and glycerine hypertonic concentration on the survival of bacteria Escherichia coli B/r heated with different values of heat drop was investigated. It was shown that the transfer of cell suspensions from isotonic conditions to media with raised osmotic pressure, preliminarily heated up to 60 degrees C, and the following heating at this temperature inhibited differences in cell sensitivity to heating at different heat drop. Unlike, it was found that the transfer of cell suspensions from isotonic conditions to hypertonic media before and after heating at 60 degrees C increased differences in resistance of these microorganisms to heating at different heat drop. It is proposed that different resistance of bacteria to damaging action of hyperthermia at different heat drop, and a modified influence of hypertonic solutions on these differences may be due to heat induced destabilization of cell osmotic homeostasis. The extent of expression of this destabilization may be determined by a quantitative ratio of osmotic pressure values in the cell-suspension medium system in particular temperature and tonic environmental conditions.     

Thermotolerance induced by heat, sodium arsenite, or puromycin: its inhibition and differences between 43 degrees C and 45 degrees C

When CHO cells were treated either for 10 min at 45-45.5 degrees C or for 1 hr with 100 microM sodium arsenite (ARS) or for 2 hr with 20 micrograms/ml puromycin (PUR-20), they became thermotolerant to a heat treatment at 45-45.5 degrees C administered 4-14 hr later, with thermotolerance ratios at 10(-3) isosurvival of 4-6, 2-3.2, and 1.7, respectively. These treatments caused an increase in synthesis of HSP families (70, 87, and 110 kDa) relative to total protein synthesis. However, for a given amount of thermotolerance, the ARS and PUR-20 treatments induced 4 times more synthesis than the heat treatment. This decreased effectiveness of the ARS treatment may occur because ARS has been reported to stimulate minimal redistribution of HSP-70 to the nucleus and nucleolus. Inhibiting protein synthesis with cycloheximide (CHM, 10 micrograms/ml) or PUR (100 micrograms/ml) after the initial treatments greatly inhibited thermotolerance to 45-45.5 degrees C in all cases. However, for a challenge at 43 degrees C, thermotolerance was inhibited only for the ARS and PUR-20 treatments. CHM did not suppress heat-induced thermotolerance to 43 degrees C, which was the same as heat protection observed when CHM was added before and during heating at 43 degrees C without the initial heat treatment. These differences between the initial treatments and between 43 and 45 degrees C may possibly be explained by reports that show that heat causes more redistribution of HSP-70 to the nucleus and nucleolus than ARS and that redistribution of HSP-70 can occur during heating at 42 degrees C with or without the presence of CHM. Heating cells at 43 degrees C for 5 hr after thermotolerance had developed induced additional thermotolerance, as measured with a challenge at 45 degrees C immediately after heating at 43 degrees C. Compared to the nonthermotolerant cells, thermotolerance ratios were 10 for the ARS treatment and 8.5 for the initial heat treatment. Adding CHM (10 micrograms/ml) or PUR (100 micrograms/ml) to inhibit protein synthesis during heating at 43 degrees C did not greatly reduce this additional thermotolerance. If, however, protein synthesis was inhibited between the initial heat treatment and heating at 43 degrees C, protein synthesis was required during 43 degrees C for the development of additional thermotolerance to 45 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of cycloheximide or puromycin on induction of thermotolerance by sodium arsenite in Chinese hamster ovary cells: involvement of heat shock proteins

After sodium arsenite (100 microM) treatment, the synthesis of three major heat shock protein families (HSPs; Mr = 110,000, 87,000, and 70,000), as studied with one-dimensional gels, was enhanced twofold relative to that of unheated cells. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed as a 100,000-fold increase in survival from 10(-6) to 10(-1) after 4 hr at 43 degrees C, and as a thermotolerance ratio (TTR) of 2-3 at 10(-3) isosurvival for heating at 45.5 degrees C. Cycloheximide (CHM: 10 micrograms/ml) or puromycin (PUR: 100 micrograms/ml), which inhibited total protein synthesis and HSP synthesis by 95%, completely suppressed the development of thermotolerance when either drug was added after sodium arsenite treatment and removed prior to the subsequent heat treatment. Therefore, thermotolerance induced by arsenite treatment correlated with an increase in newly synthesized HSPs. However, with or without arsenite treatment, CHM or PUR added 2-6 hr before heating and left on during heating caused a 10,000-100,000-fold enhancement of survival when cells were heated at 43 degrees C for 4 hr, even though very little synthesis of heat shock proteins occurred. Moreover, these cells manifesting resistance to heating at 43 degrees C after CHM treatment were much different than those manifesting resistance to 43 degrees C after arsenite treatment. Arsenite-treated cells showed a great deal of thermotolerance (TTR of about 10) when they were heated at 45 degrees C after 5 hr of heating at 43 degrees C, compared with less thermotolerance (TTR of about 2) for the CHM-treated cells heated at 45 degrees C after 5 hr of heating at 43 degrees C. Therefore, there are two different phenomena. The first is thermotolerance after arsenite treatment (observed at 43 degrees C or 45.5 degrees C) that apparently requires synthesis of HSPs. The second is resistance to heat after CHM or PUR treatment before and during heating (observed at 43 degrees C with little resistance at 45.5 degrees C) that apparently does not require synthesis of HSPs. This phenomenon not requiring the synthesis of HSPs also was observed by the large increase in thermotolerance to 45 degrees C caused by heating at 43 degrees C, with or without CHM, after cells were incubated for 6 hr following arsenite pretreatment. For both phenomena, a model based on synthesis and redistribution of HSPs is presented.     

Thermal stress and Ca-independent contractile activation in mammalian skeletal muscle fibers at high temperatures.

Temperature dependence of the isometric tension was examined in chemically skinned, glycerinated, rabbit Psoas, muscle fibers immersed in relaxing solution (pH approximately 7.1 at 20 degrees C, pCa approximately 8, ionic strength 200 mM); the average rate of heating/cooling was 0.5-1 degree C/s. The resting tension increased reversibly with temperature (5-42 degrees C); the tension increase was slight in warming to approximately 25 degrees C (a linear thermal contraction, -alpha, of approximately 0.1%/degree C) but became more pronounced above approximately 30 degrees C (similar behavior was seen in intact rat muscle fibers). The extra tension rise at the high temperatures was depressed in acidic pH and in the presence of 10 mM inorganic phosphate; it was absent in rigor fibers in which the tension decreased with heating (a linear thermal expansion, alpha, of approximately 4 x 10(-5)/degree C). Below approximately 20 degrees C, the tension response after a approximately 1% length increase (complete < 0.5 ms) consisted of a fast decay (approximately 150.s-1 at 20 degrees C) and a slow decay (approximately 10.s-1) of tension. The rate of fast decay increased with temperature (Q10 approximately 2.4); at 35-40 degrees C, it was approximately 800.s-1, and it was followed by a delayed tension rise (stretch-activation) at 30-40.s-1. The linear rise of passive tension in warming to approximately 25 degrees C may be due to increase of thermal stress in titin (connectin)-myosin composite filament, whereas the extra tension above approximately 30 degrees C may arise from cycling cross-bridges; based on previous findings from regulated actomyosin in solution (Fuchs, 1975), it is suggested that heating reversibly inactivates the troponin-tropomyosin control mechanism and leads to Ca-independent thin filament activation at high temperatures. Additionally, we propose that the heating-induced increase of endo-sarcomeric stress within titin-myosin composite filament makes the cross-bridge mechanism stretch-sensitive at high temperatures.     

Control of total peripheral resistance during hyperthermia in rats

To elucidate the effect of blood volume on the circulatory adjustment to heat stress, we studied alpha-chloralose-anesthetized rats at three levels of blood volume: normovolemia (NBV), hypervolemia (HBV; +32% plasma volume by isotonic albumin solution infusion), and hypovolemia (LBV; -16% plasma volume by furosemide administration). Body surface heating was performed with an infrared lamp to raise arterial blood temperature (Tb) at the rate of approximately 0.1 degree C/min. Before heating, central venous pressure (CVP) was significantly higher in HBV (0.41 +/- 0.25 mmHg) and lower in LBV (-1.44 +/- 0.22 mmHg) than in NBV (-0.41 +/- 0.10 mmHg). The Tb at which CVP started to decrease was approximately 40 degrees C in HBV, approximately 41 degrees C in NBV, and approximately 42 degrees C in LBV, and it decreased by 1.53 +/- 0.14, 1.92 +/- 0.24, and 0.62 +/- 0.14 mmHg from 37 to 43 degrees C of Tb in HBV, NBV, and LBV, respectively. Stroke volume was closely correlated with CVP, and this relationship was not affected by Tb. Heart rate responses to the raised Tb were similar among the three groups. Mean arterial pressure (MAP) was not affected by blood volume modification or CVP and was maintained at preheating (Tb 37 degrees C) level until Tb rose to 40 degrees C. Above this Tb, MAP increased until Tb reached 43 degrees C (+30-40 mmHg) for all three groups. Total peripheral resistance (TPR) was inversely correlated with CVP, and the slope of the linear relationship between TPR and CVP in LBV was three- to fourfold steeper than in NBV or HBV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding activity of glucocorticoid receptors after heat shock

The response of glucocorticoid receptors (GR) to heat was measured by the change in ligand binding activity both in control cells and in cells made tolerant to heat by a prior mild heat exposure. The study was prompted by earlier data showing that one of the heat shock proteins (HSP90) is an essential component of the GR complex and that treatment of mammalian cells with hydrocortisone induces resistance to heat damage. The GR rapidly loses binding activity after commencement of heating. There is a 50% loss of activity after 4 min at 45 degrees C, 8 min at 44 degrees C, or 17 min at 43 degrees C. The reduction in binding is due mainly to a reduction in affinity of binding to the ligand. The ability to bind glucocorticoid recovers quickly after heat treatment. Activity returns to levels 60-80% of normal by 2 h after a heat treatment that initially reduces binding to less than 20% of normal. However, complete restoration of binding activity takes approximately 3 days. The recovery of binding activity does not require protein synthesis. Pretreatment of cells with hydrocortisone, using conditions that induce heat resistance, reduces the activity to 10-20% of control, but residual receptors display a heat sensitivity similar to that of control cells. There was evidence for a limited degree of protection of GR from heat damage in thermotolerant cells.     

Evidence for localized cell heating induced by infrared optical tweezers.

The confinement of liposomes and Chinese hamster ovary (CHO) cells by infrared (IR) optical tweezers is shown to result in sample heating and temperature increases by several degrees centigrade, as measured by a noninvasive, spatially resolved fluorescence detection technique. For micron-sized spherical liposome vesicles having bilayer membranes composed of the phospholipid 1,2-diacyl-pentadecanoyl-glycero-phosphocholine (15-OPC), a temperature rise of approximately 1.45 +/- 0.15 degrees C/100 mW is observed when the vesicles are held stationary with a 1.064 microns optical tweezers having a power density of approximately 10(7) W/cm2 and a focused spot size of approximately 0.8 micron. The increase in sample temperature is found to scale linearly with applied optical power in the 40 to 250 mW range. Under the same trapping conditions, CHO cells exhibit an average temperature rise of nearly 1.15 +/- 0.25 degrees C/100 mW. The extent of cell heating induced by infrared tweezers confinement can be described by a heat conduction model that accounts for the absorption of infrared (IR) laser radiation in the aqueous cell core and membrane regions, respectively. The observed results are relevant to the assessment of the noninvasive nature of infrared trapping beams in micromanipulation applications and cell physiological studies.     

Heat storage in horses during submaximal exercise before and after humid heat acclimation.

The effect of humid heat acclimation on thermoregulatory responses to humid and dry exercise-heat stress was studied in six exercise-trained Thoroughbred horses. Horses were heat acclimated by performing moderate-intensity exercise for 21 days in heat and humidity (HH) [34.2-35.7 degrees C; 84-86% relative humidity (RH); wet bulb globe temperature (WBGT) index approximately 32 degrees C]. Horses completed exercise tests at 50% of peak O(2) uptake until a pulmonary arterial temperature (T(pa)) of 41.5 degrees C was attained in cool dry (CD) (20-21.5 degrees C; 45-50% RH; WBGT approximately 16 degrees C), hot dry (HD 0) [32-34 degrees C room temperature (RT); 45-55% RH; WBGT approximately 25 degrees C], and HH conditions (HH 0), and during the second hour of HH on days 3, 7, 14, and 21, and in HD on the 18th day (HD 18) of heat acclimation. The ratios of required evaporative capacity to maximal evaporative capacity of the environment (E(req)/E(max)) for CD, HD, and HH were approximately 1.2, 1.6, and 2.5, respectively. Preexercise T(pa) and rectal temperature were approximately 0.5 degrees C lower (P < 0. 05) on days 7, 14, and 21 compared with day 0. With exercise in HH, there was no effect of heat acclimation on the rate of rise in T(pa) (and therefore exercise duration) nor the rate of heat storage. In contrast, exercise duration was longer, rate of rise in T(pa) was significantly slower, and rate of heat storage was decreased on HD 18 compared with HD 0. It was concluded that, during uncompensable heat stress in horses, heat acclimation provided modest heat strain advantages when E(req)/E(max) was approximately 1.6, but at higher E(req)/E(max) no advantages were observed.     

Characterization of thermal stability of the Escherichia coli Fapy-DNA glycosylase

Thermal stability of Escherichia coli Fpg protein was studied using far-UV circular dichroism and intrinsic fluorescence. Experimental data indicate that Fpg irreversibly aggregates under heating above 35 degrees C. Heat aggregation is preceded by tertiary conformational changes of Fpg. However, the secondary structure of the fraction that does not aggregate remains unchanged up to approximately 60 degrees C. The kinetics of heat aggregation occurs with an activation enthalpy of approximately 21 kcal/mol. The fraction of monomers forming aggregates decreases with increasing urea concentration, with essentially no aggregation observed above approximately 3 M urea, suggesting that heat aggregation results from hydrophobic association of partially unfolded proteins. With increasing urea concentration, Fpg unfolds in a two-state reversible transition, with a stability of approximately 3.6 kcal/mol at 25 degrees C. An excellent correlation is observed between the unfolded fraction and loss of activity of Fpg. A simple kinetic scheme that describes both the rates and the extent of aggregation at each temperature is presented.     

Rapid and uniform electromagnetic heating of aqueous cryoprotectant solutions from cryogenic temperatures

Devitrification (ice formation during warming) is one of the primary obstacles to successful organ vitrification (solidification without ice formation). The only feasible approach to overcoming either devitrification or its damaging effects in a large organ appears at present to be the use of some form of electromagnetic heating (EH) to achieve the required high heating rates. One complication of EH in this application is the need for warming within a steel pressure vessel. We have previously reported that resonant radiofrequency (RF) helical coils provide very uniform heating at ambient temperatures and low heating rates and can be modified for coaxial power transmission, which is necessary if only one cable is to penetrate through the wall of the pressure vessel. We now report our initial studies using a modified helical coil, high RF input power, and cryogenic aqueous cryoprotectant solutions [60% (w/v) solution of 4.37 M dimethylsulfoxide and 4.37 M acetamide in water and 50% (w/w) 1,2-propanediol]. We also describe the electronic equipment required for this type of research. Temperatures were monitored during high-power conditions with Luxtron fiberoptic probes. Thermometry was complicated by the use of catheters needed for probe insertion and guidance. The highest heating rates we observed using catheters occurred at temperatures ranging from about -70 to -40 degrees C, the temperature zone where devitrification usually appears in unstable solutions during slow warming. We find that in this range we can achieve measured heating rates of approximately 300 degrees C/min in 30- to 130-ml samples using 200 to 700 W of RF power without overheating the sample at any point. However, energy conservation calculations imply that our measured peak heating rates may be considerably higher than the true heating rates occurring in the bulk of our solutions. We were able to estimate the overall true heating rates, obtaining an average value of about 20 degrees C/min/100 W/100 ml, which implies a heating efficiency close to 100%. It appears that it should be possible to warm vitrified rabbit kidneys rapidly enough under high-pressure conditions to protect them from devitrification.     

The influence of pre-treatment temperature on the thermal sensitivity of a mouse tumour

The response of tumours to hyperthermia was tested by giving graded heat treatments and assessing local control at 90 days. Mice were divided into three groups which were pre-treated for 3 days in ambient temperatures of 4, 21 or 35 degrees C. This enabled the mean tumour resting temperature to be varied by up to 11 degrees C, before subsequent heat treatment. For the heat treatments, the tumours were clamped in order to eliminate blood flow, resulting in uniform temperature distributions and hence more uniform thermal sensitivity. TCD50 values were used to construct Arrhenius plots. For all three pre-treatment temperatures, these plots demonstrated a factor of 1.6 increase in heating time per degree Celsius reduction in heating temperature. However, tumours kept in a 4 degrees C environment before treatment were more thermally sensitive than those kept in 21 degrees C conditions, while those in a 35 degrees C environment were more resistant. Pretreatment at 4 degrees C was equivalent to an increase of either 0.5 degree C in heating temperature or 28 per cent in heating time, compared with pre-treatment at 21 degrees C. Pre-treatment at 35 degrees C was equivalent to a reduction of either 0.6 degree C in heating temperature or 25 per cent in heating time. These data indicate that the pre-treatment tumour temperature is an important parameter, but the effect of heat treatment is more closely related to absolute heating temperature rather than to the increase in temperature above the normal resting level.     

A small temperature rise may contribute towards the apparent induction by microwaves of heat-shock gene expression in the nematode Caenorhabditis Elegans

We have previously reported that low intensity microwave exposure (0.75-1.0 GHz CW at 0.5 W; SAR 4-40 mW/kg) can induce an apparently non-thermal heat-shock response in Caenorhabditis elegans worms carrying hsp16-1::reporter genes. Using matched copper TEM cells for both sham and exposed groups, we can detect only modest reporter induction in the latter exposed group (15-20% after 2.5 h at 26 degrees C, rising to approximately 50% after 20 h). Traceable calibration of our copper TEM cell by the National Physical Laboratory (NPL) reveals significant power loss within the cell (8.5% at 1.0 GHz), accompanied by slight heating of exposed samples (approximately 0.3 degrees C at 1.0 W). Thus, exposed samples are in fact slightly warmer (by < or =0.2 degrees C at 0.5 W) than sham controls. Following NPL recommendations, our TEM cell design was modified with the aim of reducing both power loss and consequent heating. In the modified silver-plated cell, power loss is only 1.5% at 1.0 GHz, and sample warming is reduced to approximately 0.15 degrees C at 1.0 W (i.e., < or =0.1 degrees C at 0.5 W). Under sham:sham conditions, there is no difference in reporter expression between the modified silver-plated TEM cell and an unmodified copper cell. However, worms exposed to microwaves (1.0 GHz and 0.5 W) in the silver-plated cell also show no detectable induction of reporter expression relative to sham controls in the copper cell. Thus, the 20% "microwave induction" observed using two copper cells may be caused by a small temperature difference between sham and exposed conditions. In worms incubated for 2.5 h at 26.0, 26.2, and 27.0 degrees C with no microwave field, there is a consistent and significant increase in reporter expression between 26.0 and 26.2 degrees C (by approximately 20% in each of the six independent runs), but paradoxically expression levels at 27.0 degrees C are similar to those seen at 26.0 degrees C. This surprising result is in line with other evidence pointing towards complex regulation of hsp16-1 gene expression across the sub-heat-shock range of 25-27.5 degrees C in C. elegans. We conclude that our original interpretation of a non-thermal effect of microwaves cannot be sustained; at least part of the explanation appears to be thermal.     

Influence of the sporulation temperature upon the heat resistance of Bacillus subtilis.

The influence of different sporulation temperatures (30, 37, 44 and 52 degrees C) upon heat resistance of Bacillus subtilis was investigated. Heat resistance was greater after higher sporulation temperatures. Relation of heat resistance and temperature of sporulation was not linear over all the range of temperatures tested. Heat resistance increased about tenfold in the range of 30-44 degrees C. Sporulation at 52 degrees C did not show any further increase in heat resistance. This effect was constant over all the range of heating temperatures tested (100-120 degrees C). z value remained constant (z = 9 degrees C). Greater heat resistances at higher temperatures of sporulation were not due to selection of more heat resistant cells by a higher sporulation temperature. Spores obtained from cells incubated at 32 or 52 degrees C always possessed heat resistances that corresponded to the sporulation temperature regardless of the incubation temperature of their vegetative cells.     

Thermal susceptibility of Streptococcus faecium strains isolated from frankfurters

The heat resistance of nine strains of Streptococcus faecium isolated from frankfurters was determined at 63 and 68 degrees C in brain heart infusion broth. Exponential-phase cultures (approximately 10(7) colonies/mL) were used as inoculants. The heat resistance of S. faecium DP2181, a moderately resistant isolate, was further examined in broth (55, 63, and 68 degrees C) and frankfurter emulsion (63 and 68 degrees C). The decimal reduction times (D values) were determined by regression. In broth, both time-temperature combinations resulted in a 3-4 log decline in bacterial numbers for the nine S. faecium strains tested. For S. faecium DP2181, the survivor curves deviated from the logarithmic order of death at all three heating temperatures. An initial slow period of death was evident at 55 degrees C and a resistant tail of organisms was observed at 55, 63, and 68 degrees C. The D55D63, and, D68 values for the logarithmic portion of the corresponding survivor curves were 105.6, 9.36, and 3.34 min, respectively. The survival of DP2181 was enhanced by the frankfurter emulsion. The results indicate that populations of S. faecium existed that were very heat resistant and could survive normal frankfurter processing if initially present in high numbers.     

Induction of heat shock proteins in Chinese hamster ovary cells and development of thermotolerance by intermediate concentrations of puromycin

During 4 hr after puromycin (PUR: 20 micrograms/ml) treatment, the synthesis of three major heat shock protein families (HSPs: Mr = 110,000, 87,000, and 70,000) was enhanced 1.5-fold relative to that of untreated cells, as studied by one-dimensional gel electrophoresis. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. In parallel, thermotolerance was observed at 10(-3) isosurvival as a thermotolerance ratio (TTR) of either 2 or greater than 5 after heating at either 45.5 degrees C or 43 degrees C, respectively. However, thermotolerance was induced by only intermediate concentrations (3-30 micrograms/ml) of puromycin that inhibited protein synthesis by 15-80%; a high concentration of PUR (100 micrograms/ml) that inhibited protein synthesis by 95% did not induce either HSPs or thermotolerance. Also, thermotolerance was never induced by any concentration (0.01-10 micrograms/ml) of cycloheximide that inhibited protein synthesis by 5-94%. Furthermore, after PUR (20 micrograms/ml) treatment, the addition of cycloheximide (CHM: 10 micrograms/ml), at a concentration that reduces protein synthesis by 94%, inhibited both thermotolerance and synthesis of HSP families. Thus, thermotolerance induced by intermediate concentrations of PUR correlated with an increase in newly synthesized HSP families. This thermotolerance phenomenon was compared with another phenomenon termed heat resistance and observed when cells were heated at 43 degrees C in the presence of CHM or PUR immediately after a 2-hr pretreatment with CHM or PUR. Heat protection increased with inhibition of synthesis of both total protein and HSP families. Moreover, this heat protection decayed rapidly as the interval between pretreatment and heating increased to 1-2 hr, and did not have any obvious relationship to the synthesis of HSP families. Therefore, there are two distinctly different pathways for developing thermal resistance. The first is thermotolerance after intermediate concentrations of PUR treatment, and it requires incubation after treatment and apparently the synthesis of HSP families. The second is resistance to heat after CHM or PUR treatment immediately before and during heating at 43 degrees C, and it apparently does not require synthesis of HSP families. This second pathway not requiring the synthesis of HSP families also was observed by the increase in thermotolerance at 45.5 degrees C caused by heating at 43 degrees C after cells were incubated for 2-4 hr following pretreatment with an intermediate concentration of PUR.     

Superoxide dismutase protects against aerobic heat shock in Escherichia coli.

Exposure of a superoxide dismutase-null (sodA sodB) strain of Escherichia coli to aerobic heat stress (45 to 48 degrees C) caused a profound loss of viability, whereas the same heat stress applied anaerobically had a negligible effect. A superoxide dismutase-competent parental strain was resistant to the lethal effect of the aerobic heating. It follows that aerobic heating imposes an oxidative burden of which O2- must be a major component. This effect is not seen at 53 degrees C, presumably because, at this higher temperature, direct thermolability of vital cell components overrides the effect of superoxide radicals.     

Hyperthermic killing and hyperthermic radiosensitization in Chinese hamster ovary cells: effects of pH and thermal tolerance

To quantitatively relate heat killing and heat radiosensitization, asynchronous or G1 Chinese hamster ovary (CHO) cells at pH 7.1 or 6.75 were heated and/or X-irradiated 10 min later. Since no progression of G1 cells into S phase occurred during the heat and radiation treatments, cell cycle artifacts were minimized. However, results obtained for asynchronous and G1 cells were similar. Hyperthermic radiosensitization was expressed as the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to that of the D0 of the radiation survival curve for heat plus radiation. The TEF increased continuously with increased heat killing at 45.5 degrees C, and for a given amount of heat killing, the amount of heat radiosensitization was the same for both pH's. When cells were heated chronically at 42.4 degrees C at pH 7.4, the TEF increased initially to 2.0-2.5 and then returned to near 1.0 during continued heating as thermal tolerance developed for both heat killing and heat radiosensitization. However, the shoulder (Dq) of the radiation survival curve for heat plus radiation did not manifest thermal tolerance; i.e., it decreased continuously with increased heat killing, independent of temperature, pH, or the development of thermotolerance. These results suggest that heat killing and heat radiosensitization have a target(s) in common (TEF results), along with either a different target(s) or a difference in the manifestation of heat damage (Dq results). For clinical considerations, the interaction between heat and radiation was expressed as (1) the thermal enhancement ratio (TER), which is the dose of X rays alone divided by the dose of X rays combined with heat to obtain an isosurvival, e.g., 10(-4), and (2) the thermal gain factor (TGF), the ratio of the TER at pH 6.75 to the TER at pH 7.4. Since low pH reduced the rate of development of thermal tolerance during heating at low temperatures, low pH enhanced heat killing more at 42-42.5 degrees C than at 45.5 degrees C where thermal tolerance did not develop. Therefore, the increase in the TGF after chronic heating at 42-42.5 degrees C was greater than after acute heating at 45.5 degrees C, due primarily to the increase in heat killing causing an even greater increase in heat radiosensitization. These findings agree with animal experiments suggesting that in the clinic, a therapeutic gain for tumor cells at low pH may be greater for temperatures of 42-42.5 degrees C than of 45.5 degrees C.     

Response of murine bone marrow granulocyte-macrophage colony-forming units to hyperthermia in situ

A detailed understanding of how bone marrow stem cell progenitors are affected by heat is prerequisite to predicting how whole-body or regional hyperthermia protocols may affect bone marrow function. This investigation reports the reproductive integrity of murine tibial bone marrow granulocyte-macrophage colony-forming units (CFU-GM) after in situ hyperthermia. Heat was applied by water bath immersion of the leg of male BALB/c mice anesthetized with 90 mg/kg pentobarbital given subcutaneously. Tibial and rectal temperatures were monitored in representative animals by microthermocouples (tip diameter approximately 100 microns). By approximately 3 min after immersion of the limb, marrow temperature was within 0.3 degree C of water bath temperature (O'Hara et al., Int. J. Hyperthermia 5, 589-601, 1989) and was within 0.1 degree C by 5 min after immersion. The CFU-GM were cultured in "lung-conditioned" McCoy's 5A medium supplemented with 15% fetal calf serum and 0.3% Bacto agar. In situ heating of tibial marrow to exposure temperatures of 42, 42.5, 43, 44, and 45 degrees C gave D0's (+/- 95% CI) of 91 +/- 44, 44 +/- 27, 27 +/- 2.2, 16 +/- 6, and 7 +/- 4 min, respectively. Heating to 41.5 degrees C for up to 180 min did not result in cytotoxicity. Development of thermotolerance after approximately 100 min of heating was apparent by the presence of a "resistant tail" of the 42 degrees C survival curve. A plot of D0 vs water bath temperature was bimodal with an inflection point at approximately 42.5 degrees C. The inactivation enthalpy for temperatures above 42.5 degrees C was 586 kJ/mol (140 kcal/mol) and for temperatures below 42.5 degrees C was estimated to be 1205 kJ/mol (288 kcal/mol). These results show that CFU-GM can be heated predictably in situ, can be inactivated with thermal exposures as low as 42 degrees C, and are capable of developing thermotolerance. These findings underscore the necessity to understand stem cell inactivation by hyperthermia in situ prior to widespread implementation of clinical hyperthermia protocols where bone marrow may be included in the treatment field.