Respiration and viability of thermally injured Saccharomyces cerevisiae.

Resting cells of Saccharomyces cerevisiae Y25 were heated at 56 degrees C for 0 to 2 min. Respiratory activity of the cells reflected the severity of the heat stress. The endogenous respiration was approximately 50 microliter of O2/mg per h for cells heated for 2 min at 56 degrees C as compared with 2 microliter of O2/mg per h for nonheated cells. There was a distinct decrease in respiration after 1 to 3 h, and after 20 h the respiration rate of heated cells was less than that of nonheated cells. Along with increased rates of endogenous respiration, respiratory quotients of cells were altered after heat stress. Addition of 2,4-dinitrophenol stimulated O2 (uptake) in nonheated cells but decreased O2 (uptake) of heated cells. Due to the high rate of endogenous respiration, addition of glucose resulted in no substantial change in the rate of respiration of heated cells. However, addition of glucose prolonged the presence of the high rates of respiration observed in heated cells.     

The supply of metabolic substrate from glia to photoreceptors in the retina of the honeybee drone

1. The drone retina is composed essentially of only two types of cells: a population of identical photoreceptor cells occupying 38% of the volume is embedded in a syncytium of glia (called outer pigment cells). Nearly all the mitochondria are in the photoreceptors. 2. A retinal slice consumes 18 microliter O2 (ml tissue)-1 min-1 in the dark for up to 6 h, even without exogenous substrate; in 6 h this would require the equivalent of 127 mM glucose in the photoreceptors or 8.7 mg glycogen (ml tissue)-1. 3. Freshly dissected retinas contain about 45 mg glycogen (ml tissue)-1, but this appears, from electron micrographs and from the PAS reaction, to be exclusively in the glia. After superfusion with substrate-free Ringer solution for 30 min, slices of retina contained less than 20 microM glucose. It therefore appears that to sustain respiration, carbohydrate substrate must be transferred from the glia to the photoreceptors. 4. Even after 6 h superfusion with substrate-free Ringer solution O2 consumption (QO2) was not increased by exogenous glucose, pyruvate, trehalose or lactate, nor decreased by 2-deoxy-D-glucose. QO2 was increased 2-3 fold by either light stimulation or (for at least 20 min) by 50 microM dinitrophenol. 5. QO2 was only slightly reduced when Na-dependent glucose transport was inhibited either by reduction of extracellular [Na+], or the presence of phlorizin. 6. It is suggested that drone retinal function does not require the uptake of glucose by the photoreceptors, but that the glia do take up glucose.     

Acute heat stress protects rats against endotoxin shock.

The purpose of this study was to determine 1) whether prior (24-h) heat stress could render rats cross-resistant to the lethal activity of bacterial lipopolysaccharide (LPS) and 2) whether this acquired state of resistance is associated with endotoxemia during the heat stress event. Four groups (n = 7/group) of rats were examined: 1) saline treated, 2) LPS treated, 3) heat stressed and saline treated, and 4) heat stressed and LPS treated. Saline or LPS (Escherichia coli, serotype 0111:B4, 20 mg/kg body wt) was given intravenously 24 h after exposure to heat (ambient temperature 47-50 degrees C, relative humidity 30%) for heat-stressed rats and at the same time of day for nonheated rats; survival was monitored for 48 h. Thermal responses were similar (P > 0.05); values for maximum core temperature (Tc) and time above Tc of 40 degrees C were 42.7 +/- 0.1 and 42.6 +/- 0.1 degrees C (SE) and 44.0 +/- 2.1 and 47.9 +/- 3.7 (SE) min for the heat-stressed saline-treated and heat-stressed LPS-treated rats, respectively. Administration of LPS to nonheated rats resulted in 71.4% (5 of 7 rats) lethality. In contrast, all (7 of 7) rats subjected to a single nonlethal heat stress event 24 h before LPS treatment survived (P < 0.05). Endotoxin was not detected in arterial plasma immediately after heat stress in rats (n = 6) exposed to a Tc of 42.9 +/- 0.1 degrees C. These findings demonstrate that acute heat stress can protect rats from the lethal activity of LPS.     

Properties of glucose uptake in vegetative and sporulating cells of Saccharomyces cerevisiae

The effect of digitonin, acetic acid, urea and ethanol treatment on the glucose uptake of vegetative cells and of sporulating cells (3 h after transfer to sporulation medium) was examined in Saccharomyces cerevisiae. Both glucose uptake activities decreased at a similar rate, and a slightly different rate, in treatment with various concentrations of digitonin and of acetic acid, respectively, at 25 degrees C for 10 min. The glucose uptake activity of the sporulating cells was much more stable to urea treatment than that of the vegetative cells; the activity decreased about 36% and 76% in the sporulating cells and the vegetative cells, respectively, under conditions of 2.5 M urea at 25 degrees C for 10 min. The glucose uptake activity of the vegetative cells was more stable to ethanol treatment than that of the sporulating cells; the activity decreased about 56% and 88% in the vegetative cells and the sporulating cells, respectively, in 25% ethanol at 25 degrees C for 10 min.     

Monosaccharide transport into lactating-rat mammary acini.

The uptake and release of 3-O-methyl-D-[3H]glucose at 37 degrees C by acini, prepared from lactating-rat mammary gland with collagenase, was inhibited by glucose, phloretin, cytochalasin B, HgCl2 and low temperature. Uptake and phosphorylation of 2-deoxy-D-[3H]glucose, studied in greater detail, could be ascribed to a specific, saturable, inhibitable, process of apparent Km 16 mM and Vmax. approx. 56 nmol/min per mg of protein, plus a non-specific, non-inhibitable process that was monitored with [14C]fructose. The mean rate of uptake of 5 mM-2-deoxyglucose (16 nmol/min per mg of protein) was similar to the rate of consumption of 5 mM-glucose, suggesting that transport was a rate-limiting step in the overall metabolism of glucose. This accords with evidence for a glucose gradient across the plasma membrane.     

Uptake studies in adipocytes isolated from rainbow trout (Salmo gairdnerii). A comparison with adipocytes from rat and cat

Adipocytes were isolated from mesenteric adipose tissue of rainbow trout (Salmo gairdnerii) by incubation of tissue slices at 20 degrees C in a buffer containing 3 mg collagenase per ml. These cells were compared to adipocytes from the cat and the rat, isolated by conventional technique (1 mg collagenase per ml buffer, incubation temperature 37 degrees C). Uptake studies of some metabolites were performed with fish, rat and in some cases cat adipocytes. At a glucose concentration of 0.33 mM, the glucose uptake into rat cells was more than twice as fast as in cells from the cat, and more than five times as fast as in trout cells. 2-Amino butyrate resembled glucose in relative uptake rates between species. Metabolite uptake into rat cells was specific, with different uptake rates for different metabolites. The uptake into trout adipocytes proceeded at similar rates for all metabolites tested, provided the concentrations were the same. The uptake rate of glucose into rat cells was stimulated by insulin. Insulin had no effect on glucose uptake into adipocytes from trout.     

Differential metabolism of cellobiose and glucose by Clostridium thermocellum and Clostridium thermohydrosulfuricum.

Clostridium thermohydrosulfuricum consumed glucose in preference to cellobiose as an energy source for growth. The rates of substrate uptake in glucose- and cellobiose-grown cell suspensions were 45 and 24 nmol/min per mg (dry weight), respectively, at 65 degrees C. The molar growth yields (i.e., grams of cells per mole of glucose equivalents) were similar on cellobiose and glucose (19 and 16, respectively). Both glucose- and cellobiose-grown cells contained a glucose permease activity and high levels of hexokinase (greater 0.34 mumol/min per mg of protein at 40 degrees C). Growth on cellobiose was associated with induction of a cellobiose permease activity. In contrast, Clostridium thermocellum metabolized cellobiose in preference to glucose as an energy source and displayed lower growth rates on both substrates. The substrate uptake rates in cellobiose- and glucose-grown cell suspensions were 18 and 17 nmol/min per mg (dry weight), respectively. The molar yields were 38 on cellobiose and 20 on glucose. Extracts of glucose- and cellobiose-grown cells both contained cellobiose phosphorylase and phosphoglucomutase activities, whereas only glucose-grown cells contained detectable levels of glucose permease and hexokinase activities. The general catalytic and kinetic properties of the glucose- and cellobiose-catabolizing enzymes in the two species are described, and a model is proposed to distinguish differential saccharide metabolism by these thermophilic ethanologens.     

The role of oxidative stress in the induction of Drosophila heat-shock proteins

The role of oxidative stress in the induction of heat-shock proteins (HSPs) was studied in Drosophila Kc cells by comparing the effects of two different inducers, temperature stress and reoxygenation following a period of anoxia, on cellular respiration, thiol status, and the accumulation of HSPs. A heat shock from 25 to 37 degrees C caused a 60% increase in the rate of O2 uptake but caused little oxidative stress as indicated by a constant level of reduced glutathione, a slight increase in oxidized glutathione, and no change in protein sulfhydryls. Heat shock resulted in a pronounced accumulation of HSPs which was not inhibited by anoxic conditions. A different HSP inducer, reoxygenation following anoxia, resulted in an overall inhibition of respiration, the appearance of CN -insensitive O2 uptake, a 50% decrease in the level of reduced glutathione and a fourfold increase in the ratio of oxidized to reduced glutathione. Despite these indicators of oxidative stress, HSP synthesis was less pronounced than observed during heat shock and was not affected by antioxidants. Oxidative stress may induce HSP synthesis in some cases but is not responsible for HSP synthesis during a heat shock.     

Microcalorimetric investigations of the host-parasite relationship between Biomphalaria glabrata and Schistosoma mansoni (author's transl)

1. The ratio of dry weight to wet weight of infected snails (5.5 +/- 2.2%) is significantly smaller than that of uninfected animals (8.3 +/- 1.0%). 2. The specific values of oxygen consumption and heat-production per dry weight are significantly higher for infected snails (2.41 +/- 0.44 microliter O2/h/mg; 12.63 +/- 1.24 muW/mg) than for uninfected ones (1.88 +/- 0.24 microliter O2/h/mg, 8.94 +/- 0.91 muW/mg). 3. The rate of oxygen consumption always suffices to explain the measured heat flows for aerobic catabolism of carbohydrates or fats and proteins, respectively. Neither for infected nor for uninfected snails is it necessary to assume an anaerobic energy metabolism. 4. Structures in the calorimetrically obtained heat production curves are discussed.     

Uptake of free hemoglobin by rat liver parenchymal cells

Parenchymal cells isolated from rat liver are capable of taking up free hemoglobin. Uptake was saturable with a concentration for half-maximal velocity of 1.35 mg/ml (1.99 X 10(-5) M) hemoglobin. At a concentration of 0.088 mg/ml, the endocytic index for hemoglobin uptake was 4.5 microliters/h per mg of cell protein. This may be compared with the rate of fluid pinocytosis by these cells of 0.025 microliter/h per mg of cell protein (determined with yeast invertase as the marker). Free beta globin chains were also taken up with an endocytic index of 26.7 microliters/h per mg of cell protein at a beta chain concentration of 0.075 mg/ml. Hemoglobin inhibited the uptake of labeled beta globin. Hemoglobin-haptoglobin complex at a concentration of 0.12 mg/ml (as hemoglobin) was cleared at a rate of 0.89 microliter/h per mg cell protein and its uptake was also inhibited by free hemoglobin. We conclude that haptoglobin serves to conserve the iron of hemoglobin by preventing its renal clearance and not by promoting its hepatic uptake.     

Biochemical changes during sucrose deprivation in higher plant cells

The mobilization of stored carbohydrates (sucrose and starch) during sucrose starvation was studied with sycamore (Acer pseudoplatanus) cells. When sucrose was omitted from the nutrient medium, vacuolar sucrose was first consumed. When a threshold of intracellular sucrose concentration was attained the cytoplasmic phosphorylated compounds decreased whereas cytoplasmic Pi increased symmetrically. Such a situation triggered starch breakdown. When almost all the intracellular sucrose pool had disappeared, the cell respiration rates (normal and uncoupled) declined progressively. The decrease in the rate of respiration triggered by sucrose starvation was attributable neither to the availability of substrate for mitochondrial respiration nor to a decrease in the maximal rate of O2 consumption by mitochondria expressed in terms of nanomole of O2 consumed per min/mg of mitochondrial protein. In fact, the uncoupled respiration rates decreased in parallel with the decrease in total intracellular cardiolipin or cytochrome aa3. These results demonstrate therefore that after a long period of sucrose starvation the progressive decrease in the uncoupled rate of O2 consumption by sycamore cells was attributable to a progressive diminution of the number of mitochondria/cell.     

Effect of prior heat shock on heat resistance of Listeria monocytogenes in meat

The effect of prior heat shock on the thermal resistance of Listeria monocytogenes in meat was investigated. A sausage mix inoculated with approximately 10(7) L. monocytogenes per g was initially subjected to a heat shock temperature of 48 degrees C before being heated at a final test temperature of 62 or 64 degrees C. Although cells heat shocked at 48 degrees C for 30 or 60 min did not show a significant increase in thermotolerance as compared with control cells (non-heat shocked), bacteria heat shocked for 120 min did, showing an average 2.4-fold increase in the D64 degrees C value. Heat-shocked cells shifted to 4 degrees C appeared to maintain their thermotolerance for at least 24 h after heat shock.     

Effect of prior heat shock on heat resistance of Listeria monocytogenes in meat.

The effect of prior heat shock on the thermal resistance of Listeria monocytogenes in meat was investigated. A sausage mix inoculated with approximately 10(7) L. monocytogenes per g was initially subjected to a heat shock temperature of 48 degrees C before being heated at a final test temperature of 62 or 64 degrees C. Although cells heat shocked at 48 degrees C for 30 or 60 min did not show a significant increase in thermotolerance as compared with control cells (non-heat shocked), bacteria heat shocked for 120 min did, showing an average 2.4-fold increase in the D64 degrees C value. Heat-shocked cells shifted to 4 degrees C appeared to maintain their thermotolerance for at least 24 h after heat shock.     

Kinetics of substrate utilization in adenine-limited chemostat cultures of Bacillus subtilis KYA741.

The specific rates of limiting substrate utilization were investigated in adenine- or glucose-limited chemostat cultures of Bacillus subtilis KYA741, an adenine-requiring strain, at 37 degrees C. With the glucose-limited cultures, the specific rate of glucose consumption versus dilution rate gave a linear relationship from which the true growth yield and maintenance coefficient were determined to be 0.09 mg of bacteria per mg of glucose and 0.2 mg of glucose per mg of bacteria per h, respectively. With the adenine-limited cultures, adenine as the limiting substrate was not completely consumed at lower dilution rates (e.g., D less than 0.1), unlike in the glucose-limited cultures. When a linear relationship of specific rate of adenine consumption versus dilution rate was extrapolated to zero dilution rate, a negative value for the specific rate of adenine consumption, -0.01 mg of adenine per mg of bacteria per h, was obtained, giving a true growth yield for adenine of 5.2 mg of bacteria per mg of adenine. On the other hand, the maintenance coefficient of oxygen uptake gave a positive value of 8.1 x 10(-3) mmol/mg of bacteria per h. Based on previous results showing that adenine is resupplied by lysing cells, we developed kinetic models of adenine utilization and cell growth that gave a good estimation of the peculiar behavior of cell growth and adenine utilization in adenine-limited chemostat cultures.     

Effects of cell density and glucose and glutamine levels on the respiration rates of hybridoma cells

The effects of cell density as well as the concentration levels of glucose and glutamine on the specific respiration rate of a hybridoma cell line were investigated. The experimental oxygen consumption rate was found to be constant over a wide range of dissolved oxygen levels if the suspension medium contained glutamine. In glutamine-free medium, however, the rate of oxygen consumption decreased slowly with time.In a stationary flask batch culture, the specific respiration rate decreased from about 7 to 2.9 mumol/min per 10(9) cells as the cell density increased exponentially from 1 x 10(5) to 1.2 x 10(6)/mL. To isolate the effect of cell density, cells were re suspended in fresh culture medium so that nutrient concentrations were the same for all experiments. The specific respiration rate decreased with increasing cell density in the same manner as in the stationary flask culture, falling from 8 to 4 mumol/min per 10(9) cells as the cell density increased from 10(5) to 10(6) cells/mL, then declining to 2 mumol/min per 10(9) cells when the cell density reached 10(7) cells/mL.Cells suspended in Hanks balanced sale solution (HBSS) were used to elucidate the effect of glucose and glutamine levels on respiration. The addition of glucose in concentrations of 0.25, 0.50, and 0.75 g/L had no observable effect on the specific oxygen uptake rate; however, a glucose concentration of 1 g/L reduced the uptake rate by 22%. Glutamine in a concentration of 0.30 g/L increased the specific respiration rate in HBSS containing 0 and 1 g/L glucose by approximately 13%.     

Respiratory activity of yeast Yarrowia lipolytica under oxidative stress and heat shock

Heat shock (45 degrees C) and the effect of oxidants (H2O2) resulted in a decrease of the respiratory activity of yeast cells and their survival rate. Increased resistance to stress effects after mild heat treatment (37 degrees C) or treatment with a nonlethal dose of oxidants (0.5 mM H2O2 for 60 min) was accompanied by appearance of an alternative (cyanide-resistant) oxidative pathway in the mitochondria, which promotes survival due to retention of the capacity for ATP synthesis in the first coupling point at the level of endogenous NADH dehydrogenase. The alternative oxidative pathway is more resistant to the effect of stressors that disrupt electron transfer in the cytochrome site of the respiratory chain.     

The relation of sodium and potassium ion transport to the respiration and adenine nucleotide content of liver slices treated with inhibitors of respiration

1. The dependence of the net transport of Na(+) and K(+) by rat liver on the respiration has been determined by incubating slices in the presence of varying concentrations of respiratory inhibitors. 2. Neither the rate of net transport nor the total amount of each ion transported was inhibited unless the rate of endogenous respiration was decreased below a critical value of about 330mmol of O(2)/h per kg of protein (i.e. 50% of the total endogenous respiration). 3. The uninhibited rate of respiration could be varied over a twofold range (380-770mmol of O(2)/h per kg of protein) by the use of different substrates, but the critical value for the onset of transport inhibition was quite constant (290-360mmol/h per kg of protein) under these different conditions. 4. Slices incubated at 38 degrees C without inhibitors showed an increase of their ATP content and the concentration ratio ATP/ADP. The final ATP content and concentration ratio, ATP/ADP, of slices treated with different concentrations of inhibitors were closely related to the rate of respiration. 5. The increased ATP content of the control slices during incubation was equal to the increase of total adenine nucleotides. At increasing degrees of respiratory inhibition the relative contributions of ADP and AMP to the total adenine nucleotide content increased. 6. The critical rate of respiration for the onset of inhibition of ion transport and the corresponding contents of adenine nucleotides provide estimates of the maximal values of certain parameters of energy metabolism required for the support of alkali-cation transport in the liver slices.     

Influence of body temperature on the development of fatigue during prolonged exercise in the heat.

We investigated whether fatigue during prolonged exercise in uncompensable hot environments occurred at the same critical level of hyperthermia when the initial value and the rate of increase in body temperature are altered. To examine the effect of initial body temperature [esophageal temperature (Tes) = 35.9 +/- 0.2, 37.4 +/- 0. 1, or 38.2 +/- 0.1 (SE) degrees C induced by 30 min of water immersion], seven cyclists (maximal O2 uptake = 5.1 +/- 0.1 l/min) performed three randomly assigned bouts of cycle ergometer exercise (60% maximal O2 uptake) in the heat (40 degrees C) until volitional exhaustion. To determine the influence of rate of heat storage (0.10 vs. 0.05 degrees C/min induced by a water-perfused jacket), four cyclists performed two additional exercise bouts, starting with Tes of 37.0 degrees C. Despite different initial temperatures, all subjects fatigued at an identical level of hyperthermia (Tes = 40. 1-40.2 degrees C, muscle temperature = 40.7-40.9 degrees C, skin temperature = 37.0-37.2 degrees C) and cardiovascular strain (heart rate = 196-198 beats/min, cardiac output = 19.9-20.8 l/min). Time to exhaustion was inversely related to the initial body temperature: 63 +/- 3, 46 +/- 3, and 28 +/- 2 min with initial Tes of approximately 36, 37, and 38 degrees C, respectively (all P < 0.05). Similarly, with different rates of heat storage, all subjects reached exhaustion at similar Tes and muscle temperature (40.1-40.3 and 40. 7-40.9 degrees C, respectively), but with significantly different skin temperature (38.4 +/- 0.4 vs. 35.6 +/- 0.2 degrees C during high vs. low rate of heat storage, respectively, P < 0.05). Time to exhaustion was significantly shorter at the high than at the lower rate of heat storage (31 +/- 4 vs. 56 +/- 11 min, respectively, P < 0.05). Increases in heart rate and reductions in stroke volume paralleled the rise in core temperature (36-40 degrees C), with skin blood flow plateauing at Tes of approximately 38 degrees C. These results demonstrate that high internal body temperature per se causes fatigue in trained subjects during prolonged exercise in uncompensable hot environments. Furthermore, time to exhaustion in hot environments is inversely related to the initial temperature and directly related to the rate of heat storage.     

Effects in vitro of alloxan on the glucose metabolism of mouse pancreatic B-cells

To facilitate detailed studies of the B-cytotoxic action of alloxan we developed a model using isolated pancreatic islets of normal mice. An essential feature of this model is the low temperature employed during exposure to alloxan, which minimizes the degradation of the drug. The islets were incubated with alloxan for 30min at 4 degrees C and subsequently various aspects of their metabolism were studied. The O(2) consumption was measured by the Cartesian-diver technique. Islets exposed to 2mm-alloxan and control islets had the same endogenous respiration, whereas the O(2) uptake of the alloxan-treated islets was inhibited and that of the control islets stimulated when they were incubated with 28mm-glucose as an exogenous substrate. The islet glucose oxidation was estimated by measurement of the formation of (14)CO(2) from [U-(14)C]glucose at 37 degrees C. Compared with the controls, alloxan-treated islets showed a decrease in the glucose-oxidation rate in a dose-dependent manner. Pretreatment of the islets with 28mm-glucose for 30min at 37 degrees C completely protected against this effect, whereas preincubations at glucose concentrations below 16.7mm failed to exert any protective effect. The glucose utilization was estimated as the formation of (3)H(2)O from [5-(3)H]glucose. Alloxan (2mm) failed to affect islet glucoseutilization rate in the presence of either 2.8 or 28mm-glucose. In contrast, islets exposed to 5 or 10mm-alloxan exhibited lowered glucose utilization. It is concluded that in vitro alloxan has an acute inhibitory effect on the islet glucose metabolism, and that this action can be prevented by previous exposure to a high glucose concentration. The results are consistent with the idea that the B-cytotoxicity of alloxan reflects an interaction with intracellular sites involved in the oxidative metabolism of the B-cell.     

Thermoregulation in the meerkat (Suricata suricatta Schreber, 1776)

Tre of the suricates exhibits a marked diurnal rhythm (mean Tre at night 36.3 +/- 0.6 degrees C and 38.3 +/- 0.5 degrees C during the day). Oxygen consumption is lowest at Ta 30-32.5 degrees C (mean 0.365 +/- 0.022 ml O2 g-1 hr-1); this is 42% below the value expected from body mass. At Ta below the TNZ, oxygen uptake rises rapidly, minimal thermal conductance (0.040 ml O2 g-1 h-1 degrees C-1) being 18% above the mass-specific level. Lowest heart rates occur at Ta 30 degrees C (mean 109.6 +/- 9.8 beats min-1) and oxygen pulse is minimal at Ta 30-35 degrees C with 40-45 microliter O2 beat-1. At Ta 15-32.5 degrees C total evaporative water loss is between 0.46-0.63 ml H2O kg-1 hr-1 and increases markedly during heat stress (to a mean of 5.35 ml H2O kg-1 hr-1 at Ta 40 degrees C). This rise of TEWL is mainly attributable to the onset of panting at Ta above 35 degrees C.