Effects of uncoupling of mitochondrial energy conservation on the ultradian clock-driven oscillations in Saccharomyces cerevisiae continuous culture

Protonophores have several different perturbative effects on dissolved O2 concentrations in continuous cultures of Saccharomyces cerevisiae. As well as uncoupling energy conservation from mitochondrial electron transport in vivo, they reset ultradian clock-driven respiratory oscillations and produce cell cycle effects. Thus, additions at low concentration (1.25 microM) of either m-chlorocarbonyl-cyanide phenylhydrazone (CCCP) or 5-chloro-3-t-butyl-2-chloro-4(1)-nitrosalicylanilide (S13) led to phase resetting of the 48 min ultradian clock-driven respiratory oscillations. At 2.5 microM CCCP or 4 microM S13, transient inhibition of oscillatory respiration (for 5 h) preceded synchronisation of the cell division cycle seen as a slow (9 h period) wave that enveloped the 48 min oscillation. At still higher concentrations of CCCP (5 microM), the cell division cycle was prolonged by about 7 h, and during this phase, the respiratory oscillation became undetectable. The significance of these observations with respect to the time-keeping functions of the ultradian clock is discussed.     

Involvement of oxidative stress in the regulation of H(2)S production during ultradian metabolic oscillation of Saccharomyces cerevisiae

Periodic evolution of H(2)S during aerobic chemostat culture of Saccharomyces cerevisiae resulted in ultradian metabolic oscillation via periodic inhibition of respiratory activity. To understand the nature of periodic H(2)S evolution, we investigated whether oxidative stress is associated with H(2)S production. The cellular oxidative states represented by intracellular level of lipid peroxides oscillated out of phase with the oscillation of dissolved O(2). Pulse addition of antioxidant, oxidative agent or inhibitor of antioxidation enzymes perturbed metabolic oscillation producing changes in H(2)S evolution. Analysis of H(2)S production profiles during perturbation of oscillation revealed that the amount of H(2)S production is closely linked with cellular oxidative states. Based on these results and our previous reports, we suggest that oxidative stresses result in periodic depletion of glutathione and cysteine, which in turn causes stimulation of the sulfate assimilation pathway and H(2)S production.     

Pulmonary gas exchange in humans during normobaric hypoxic exercise

Previous studies (J. Appl. Physiol. 58: 978-988 and 989-995, 1985) have shown both worsening ventilation-perfusion (VA/Q) relationships and the development of diffusion limitation during heavy exercise at sea level and during hypobaric hypoxia in a chamber [fractional inspired O2 concentration (FIO2) = 0.21, minimum barometric pressure (PB) = 429 Torr, inspired O2 partial pressure (PIO2) = 80 Torr]. We used the multiple inert gas elimination technique to compare gas exchange during exercise under normobaric hypoxia (FIO2 = 0.11, PB = 760 Torr, PIO2 = 80 Torr) with earlier hypobaric measurements. Mixed expired and arterial respiratory and inert gas tensions, cardiac output, heart rate (HR), minute ventilation, respiratory rate (RR), and blood temperature were recorded at rest and during steady-state exercise in 10 normal subjects in the following order: rest, air; rest, 11% O2; light exercise (75 W), 11% O2; intermediate exercise (150 W), 11% O2; heavy exercise (greater than 200 W), 11% O2; heavy exercise, 100% O2 and then air; and rest 20 minutes postexercise, air. VA/Q inequality increased significantly during hypoxic exercise [mean log standard deviation of perfusion (logSDQ) = 0.42 +/- 0.03 (rest) and 0.67 +/- 0.09 (at 2.3 l/min O2 consumption), P less than 0.01]. VA/Q inequality was improved by relief of hypoxia (logSDQ = 0.51 +/- 0.04 and 0.48 +/- 0.02 for 100% O2 and air breathing, respectively). Diffusion limitation for O2 was evident at all exercise levels while breathing 11% O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of body temperature on the locomotory energetics of lizards

Oxygen consumption (VO2), carbon dioxide production (VCO2), and stamina were measured in the lizard Tupinambis nigropunctatus running at sustainable and non-sustainable velocities (v) on a motor-driven treadmill. Three experimental groups were measured: field-fresh animals at body temperature (Tb) = 35 degrees C and laboratory-maintained animals at Tb = 35 and 25 degrees C. Mean preferred Tb was determined to be 35.2 degrees C. At 35 degrees C, field-fresh animals had a greater maximal oxygen consumption (VO2max corr) (4.22 vs 3.60 ml O2 g-0.76h-1) and a greater endurance. The net cost of transport (slope of VO2 on v) did not differ between the groups (= 2.60 ml O2 g-0.76)km-1). Velocity at which VO2max is attained (MAS) is 0.84 km h-1. The respiratory exchange ratio (R) exceeded 1.0 at v above MAS, indicating supplementary anaerobic metabolism. At 25 degrees C, VO2max corr was lower (2.34 ml O2 g-0.76h-1) as was endurance, MAS occurring at 0.5 km h-1. Net cost of transport was not significantly different than at 35 degrees C. The effect of Tb on locomotory costs was analyzed for this lizard and other species. It was concluded that the net cost of transport is temperature independent in all species examined and the total cost of locomotion (VO2 v-1) is temperature dependent in Tupinambis (Q10 = 1.4-2.0) and all other species examined except one. The energetic cost of locomotion [(VO2active-VO2rest)v-1], previously reported to be temperature independent in lizards, is temperature dependent in Tupinambis (Q10 = 1.3-1.6) and in two other species.2r     

Composition and structure of whey protein/gum arabic coacervates

Complex coacervation in whey protein/gum arabic (WP/GA) mixtures was studied as a function of three main key parameters: pH, initial protein to polysaccharide mixing ratio (Pr:Ps)(ini), and ionic strength. Previous studies had already revealed under which conditions a coacervate phase was obtained. This study is aimed at understanding how these parameters influence the phase separation kinetics, the coacervate composition, and the internal coacervate structure. At a defined (Pr:Ps)(ini), an optimum pH of complex coacervation was found (pH(opt)), at which the strength of electrostatic interaction was maximum. For (Pr:Ps)(ini) = 2:1, the phase separation occurred the fastest and the final coacervate volume was the largest at pH(opt) = 4.0. The composition of the coacervate phase was determined after 48 h of phase separation and revealed that, at pH(opt), the coacervate phase was the most concentrated. Varying the (Pr:Ps)(ini) shifted the pH(opt) to higher values when (Pr:Ps)(ini) was increased and to lower values when (Pr:Ps)(ini) was decreased. This phenomenon was due to the level of charge compensation of the WP/GA complexes. Finally, the structure of the coacervate phase was studied with small-angle X-ray scattering (SAXS). SAXS data confirmed that at pH(opt) the coacervate phase was dense and structured. Model calculations revealed that the structure factor of WP induced a peak at Q = 0.7 nm(-1), illustrating that the coacervate phase was more structured, inducing the stronger correlation length of WP molecules. When the pH was changed to more acidic values, the correlation peak faded away, due to a more open structure of the coacervate. A shoulder in the scattering pattern of the coacervates was visible at small Q. This peak was attributed to the presence of residual charges on the GA. The peak intensity was reduced when the strength of interaction was increased, highlighting a greater charge compensation of the polyelectrolyte. Finally, increasing the ionic strength led to a less concentrated, a more heterogeneous, and a less structured coacervate phase, induced by the screening of the electrostatic interactions.     

Diffusion limitation in normal humans during exercise at sea level and simulated altitude

The relative roles of ventilation-perfusion (VA/Q) inequality, alveolar-capillary diffusion resistance, postpulmonary shunt, and gas phase diffusion limitation in determining arterial PO2 (PaO2) were assessed in nine normal unacclimatized men at rest and during bicycle exercise at sea level and three simulated altitudes (5,000, 10,000, and 15,000 ft; barometric pressures = 632, 523, and 429 Torr). We measured mixed expired and arterial inert and respiratory gases, minute ventilation, and cardiac output. Using the multiple inert gas elimination technique, PaO2 and the arterial O2 concentration expected from VA/Q inequality alone were compared with actual values, lower measured PaO2 indicating alveolar-capillary diffusion disequilibrium for O2. At sea level, alveolar-arterial PO2 differences were approximately 10 Torr at rest, increasing to approximately 20 Torr at a metabolic consumption of O2 (VO2) of 3 l/min. There was no evidence for diffusion disequilibrium, similar results being obtained at 5,000 ft. At 10 and 15,000 ft, resting alveolar-arterial PO2 difference was less than at sea level with no diffusion disequilibrium. During exercise, alveolar-arterial PO2 difference increased considerably more than expected from VA/Q mismatch alone. For example, at VO2 of 2.5 l/min at 10,000 ft, total alveolar-arterial PO2 difference was 30 Torr and that due to VA/Q mismatch alone was 15 Torr. At 15,000 ft and VO2 of 1.5 l/min, these values were 25 and 10 Torr, respectively. Expected and actual PaO2 agreed during 100% O2 breathing at 15,000 ft, excluding postpulmonary shunt as a cause of the larger alveolar-arterial O2 difference than accountable by inert gas exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Temperature on Rates of Alternative and Cytochrome Pathway Respiration and Their Relationship with the Redox Poise of the Quinone Pool

We investigated the effect of short-term changes in temperature on alternative (Alt) and cytochrome (Cyt) pathway respiration, both in intact tissues and isolated mitochondria of 14-d-old cotyledons of soybean (Glycine max L. cv Stevens). We also established the extent to which temperature alters the interaction between the oxidizing pathways and the level of ubiquinone (UQ) reduction (UQ(r)/UQ(t)). No difference was found between the temperature coefficient of respiration (Q(10); proportional change per 10 degrees C) of Alt and Cyt pathway respiration in cotyledon slices (Q(10) = 1.92 and 1.86, respectively). In isolated mitochondria, the Q(10) of the fully activated Alt pathway (Q(10) = 2.24-2.61) was always equal to, or higher than, that of Cyt c oxidase (COX) alone (Q(10) = 2.08) and the complete Cyt pathway (Q(10) = 2.40-2.55). This was true regardless of substrate or whether ADP was present. There was little difference in the Q(10) of the Cyt pathway with or without ADP; however, the Q(10) of COX was substantially lower in the presence of an uncoupler (Q(10) = 1.61) than its absence (Q(10) = 2.08). The kinetics of Alt and Cyt pathway activity in relation to UQ(r)/UQ(t) were not affected by temperature. For a given UQ(r)/UQ(t) value, the proportion of maximum flux taking place was similar at all temperatures for both pathways (+/-ADP). However, the Q(10) of the Alt and the Cyt pathways (+ADP) increased with increasing UQ(r)/UQ(t). We conclude that the Alt pathway is not less temperature sensitive than the Cyt pathway or COX per se and that changes in the degree of control exerted by individual steps in the respiratory apparatus could result in changes in the Q(10) of mitochondrial O(2) uptake.     

Effects of temperature and temperature-steps on circadian locomotor rhythmicity in the blow fly Calliphora vicina

The free-running period (in darkness) of the locomotor activity rhythm in adult blow flies (Calliphora vicina) was temperature-compensated between 15 and 25 degrees C, showing Q(10) values between 0.98 and 1.04. Single steps-up (20 to 25 degrees C) or steps-down (20 to 15 degrees C) in temperature caused stable phase shifts of the activity rhythm, giving rise to temperature-step phase response curves (PRCs) with both advances and delays. Phase advances, however, were dominant for steps-up, and phase delays for steps-down; the two PRCs were almost "mirror images" of each other. Following protocols introduced by Zimmerman et al. [(1968) Temperature compensation of the circadian oscillation in Drosophila pseudoobscura and its entrainment by temperature cycles, Journal of Insect Physiology, 14, 669-684] for the rhythm of pupal eclosion in Drosophila pseudoobscura, the steps-up and steps-down PRCs for C. vicina were used to compute a theoretical PRC for a 6 h low temperature pulse, and from this a theoretical steady-state phase relationship of the locomotor activity rhythm to a train of such pulses making up a temperature cycle (18 h at 20 degrees and 6 h at 15 degrees C).     

Oxidative Activity of Mitochondria Isolated from Plant Tissues Sensitive and Resistant to Chilling Injury

Arrhenius plots of the respiration rates of mitochondria isolated from chilling sensitive plant tissues (tomato and cucumber fruit, and sweet potato roots) showed a linear decrease from 25 C to about 9 to 12 C (with Q(10) values of 1.3 to 1.6), at which point there was a marked deviation with an increased slope as temperatures were reduced to 1.5 C (Q(10) of 2.2 to 6.3). The log of the respiration rate of mitochondria from chilling resistant tissues (cauliflower buds, potato tubers, and beet roots) showed a linear decrease over the entire temperature range from 25 to 1.5 C with Q(10) values of 1.7 to 1.8. Phosphorylative efficiency of mitochondria from all the tissues, as measured by ADP:O and respiratory control ratios, was not influenced by temperatures from 25 to 1.5 C. These results indicate that an immediate response of sensitive plant tissues to temperatures in the chilling range (0 to 10 C) is to depress mitochondrial respiration to an extent greater than that predicted from Q(10) values measured above 10 C. The results are also consistent with the hypothesis that a phase change occurs in the mitochondrial membrane as the result of a physical effect of temperature on some membrane component such as membrane lipids.     

Regulation of branched-chain, and sulfur-containing amino acid metabolism by glutathione during ultradian metabolic oscillation of Saccharomyces cerevisiae

Autonomous ultradian metabolic oscillation (T approximately or =50 min) was detected in an aerobic chemostat culture of Saccharomyces cerevisiae. A pulse injection of GSH (a reduced form of glutathione) into the culture induced a perturbation in metabolic oscillation, with respiratory inhibition caused by H2S burst production. As the production of H2S in the culture was controlled by different amino acids, we attempted to characterize the effects of GSH on amino acid metabolism, particularly with regard to branched chain and sulfur-containing amino acids. During stable metabolic oscillation, concentrations of intracellular glutamate, aspartate, threonine, valine, leucine, isoleucine, and cysteine were observed to oscillate with the same periods of dissolved O2 oscillation, although the oscillation amplitudes and maximal phases were shown to differ. The methionine concentration was stably maintained at 0.05 mM. When GSH (100 microM) was injected into the culture, cellular levels of branched chain amino acids increased dramatically with continuous H2S production, whereas the cysteine and methionine concentrations were noticeably reduced. These results indicate that GSH-dependent perturbation occurs as the result of the promotion of branched chain amino acid synthesis and an attenuation of cysteine and methionine synthesis, both of which activate the generation of H2S. In a low sulfate medium containing 2.5 mM sulfate, the GSH injections did not result in perturbations of dissolved O2, NAD(P)H redox oscillations without burst H2 production. This suggests that GSH-dependent perturbation is intimately linked with the metabolism of branched-chain amino acids and H2 generation, rather than with direct GSH-GSSG redox control.     

Temperature dependence of cardiac sarcoplasmic reticulum Ca²⁺-ATPase from rainbow trout Oncorhynchus mykiss

In this work, the temperature dependence of the sarco-endoplasmic reticulum Ca(2+) -ATPase (SERCA2) activity from rainbow trout Oncorhynchus mykiss cardiac ventricles was measured and compared with the mammalian SERCA2 isoform. The rate of ATP-dependent Ca(2+) transport catalysed by O. mykiss vesicles was totally abolished by thapsigargin and the Ca(2+) ionophore A(23187) . At warm temperatures (25 and 30° C), the SERCA2 from O. mykiss ventricles displayed the same rate of Ca(2+) uptake. At 35° C, the activity of the O. mykiss enzyme decreased after 20 min of reaction time. The rate of Ca(2+) uptake catalysed by the mammalian SERCA2 was temperature dependent exhibiting its maximal activity at 35° C. In contrast to the rate of Ca(2+) uptake, the rate of ATP hydrolysis catalysed by O. mykiss SERCA2 was not significantly different at 25 and 35° C, but the rate of ATP hydrolysis catalysed by the rat Rattus norvegicus SERCA2 isoform at 35° C was two-fold higher than at 25° C. At low temperatures (5 to 20° C), the rate of Ca(2+) uptake from O. mykiss SR was less temperature dependent than the R. norvegicus isoform, being able to sustain a high activity even at 5° C. The mean ±s.e. Q(10) values calculated from 25 to 35° C for ATP hydrolysis were 1·112 ± 0·026 (n = 3) and 2·759 ± 0·240 (n = 5) for O. mykiss and R. norvegicus, respectively. Taken together, the results show that the O. mykiss SERCA2 was not temperature dependent over the 10 to 25° C temperature interval commonly experienced by the animal in vivo. The Q(10) value of SERCA2 was significantly lower in O. mykiss than R. norvegicus which may be key for cardiac function over the wide environmental temperatures experienced in this eurythermal fish.     

Endogenous protective mechanisms in remodeling of rat heart mitochondrial membranes in the acute phase of streptozotocin-induced diabetes

The aim of present study was to investigate functional and physical alterations in membranes of heart mitochondria that are associated with remodeling of these organelles in acute phase of streptozotocin-induced diabetes and to elucidate the role of these changes in adaptation of the heart to acute streptozotocin-induced diabetes (evaluated 8 days after single dose streptozotocin application to male Wistar rats). Action of free radicals on the respiratory chain of diabetic-heart mitochondria was manifested by 17 % increase (p<0.05) in oxidized form of the coenzyme Q(10) and resulted in a decrease of states S3 and S4 respiration, the respiratory control index, rate of phosphorylation (all p<0.01) and the mitochondrial transmembrane potential (p<0.05), but the ADP/O ratio decreased only moderately (p>0.05). On the contrary, membrane fluidity and the total mitochondrial Mg2+-ATPase activity increased (both p<0.05). In diabetic heart mitochondria, linear regression analysis revealed a reciprocal relationship between the increase in membrane fluidity and decrease in trans-membrane potential (p<0.05, r = 0.67). Changes in membrane fluidity, transmembrane potential, Mg2+-ATPase activity and the almost preserved ADP/O ratio appear as the manifestation of endogenous protective mechanisms participating in the functional remodeling of mitochondria which contributes to adaptation of the heart to diabetes.     

Biphasic changes in isometric tension after a temperature jump in skinned Ca-activated skeletal muscles of the frog

Joulean temperature jump from 4--7 degrees to 10--25 degrees C completed in 0.2 ms induced biphasic rise of tension in suspended in the air segments of chemically skinned Ca-activated (pCa = 5.5 divided by 6) muscle fibres of the frog. Amplitudes of the 1st and 2nd phases grew up to 30--40% and 60--70% of the initial tension correspondingly when the amplitude of temperature jump increased to 17--21 degrees C. The time constant of the 1st phase (1.3--0.5 ms) decreased with temperature (Q10 = 1.8). The time constant of the 2nd phase was about 10 ms.     

Oxygen consumption and body temperature of active and resting honeybees

We measured the energy turnover (oxygen consumption) of honeybees (Apis mellifera carnica), which were free to move within Warburg vessels. Oxygen consumption of active bees varied widely depending on ambient temperature and level of activity, but did not differ between foragers (>18 d) and middle-aged hive bees (7-10 d). In highly active bees, which were in an endothermic state ready for flight, it decreased almost linearly, from a maximum of 131.4 microl O(2) min(-1) at 15 degrees C ambient temperature to 81.1 microl min(-1) at 25 degrees C, and reached a minimum of 29.9 microl min(-1) at 40 degrees C. In bees with low activity, it decreased from 89.3 microl O(2) min(-1) at 15 degrees C to 47.9 microl min(-1) at 25 degrees C and 14.7 microl min(-1) at 40 degrees C. Thermographic measurements of body temperature showed that with increasing activity, the bees invested more energy to regulate the thorax temperature at increasingly higher levels (38.8-41.2 degrees C in highly active bees) and were more accurate. Resting metabolism was determined in young bees of 1-7 h age, which are not yet capable of endothermic heat production with their flight muscles. Their energy turnover increased from 0.21 microl O(2) min(-1) at 10 degrees C to 0.38 microl min(-1) at 15 degrees C, 1.12 microl min(-1) at 25 degrees C, and 3.03 microl min(-1) at 40 degrees C. At 15, 25 and 40 degrees C, this was 343, 73 and 10 times below the values of the highly active bees, respectively. The Q(10) value of the resting bees, however, was not constant but varied in a U-shaped manner with ambient temperature. It decreased from 4.24 in the temperature range 11-21 degrees C to 1.35 in the range 21-31 degrees C, and increased again to 2.49 in the range 30-40 degrees C. We conclude that attempts to describe the temperature dependence of the resting metabolism of honeybees by Q(10) values can lead to considerable errors if the measurements are performed at only two temperatures. An acceptable approximation can be derived by calculation of an interpolated Q(10) according to the exponential function (V(O(2))=0.151 x 1.0784(T(a))) (interpolated Q(10)=2.12).     

Temperature-sensitive intracellular Mg2+ block of L-type Ca2+ channels in cardiac myocytes

We examined the concentration-dependent blocking effects of intracellular Mg2+ on L-type Ca2+ channels in cardiac myocytes using the whole cell patch-clamp technique. The increase of L-type Ca2+ channel current (I(Ca)) (due to relief of Mg2+ block) occurred in two temporal phases. The rapid phase (runup) transiently appeared early (<5 min) in dialysis of the low-Mg2+ solution; the slow phase began later in dialysis (>10 min). Runup was not blocked by intracellular GTP (GTP(i)). The late phase of the I(Ca) increase (late I(Ca)) was suppressed by GTP(i) (0.4 mM) and was observed in myocytes of the guinea pig or frog at higher (32 or 24 degrees C, respectively) rather than lower temperatures (24 or 17.5 degrees C, respectively). At pMg = 6.0, raising the temperature from 24 to 32 degrees C evoked late I(Ca) with a Q10 of 14.5. Restoring the temperature to 24 degrees C decreased I(Ca) with a Q10 of only 2.4. The marked difference in the Q10 values indicated that late I(Ca) (pMg = 5-6) is an irreversible phenomenon. Phosphorylation suppressed the intracellular [Mg2+] dependency of late I(Ca). This effect of phosphorylation together with the inhibitory action of GTP(i) on Mg2+-dependent blocking of I(Ca) are common properties of mammalian and amphibian cardiomyocytes.     

Oscillatory metabolism of Saccharomyces cerevisiae in continuous culture

Short-period (40-50 min) synchronized metabolic oscillation was found in a continuous culture of yeast Saccharomyces cerevisiae under aerobic conditions at low-dilution rates. During oscillation, many parameters changed cyclically, such as dissolved oxygen concentration, respiration rate, ethanol and acetate concentrations in the culture, glycogen, ATP, NADH, pyruvate and acetate concentrations in the cells. These changes were considered to be associated with glycogen metabolism. When glycogen was degraded, the respiro-fermentative phase was observed, in which ethanol was produced and the respiration rate decreased. In this phase, the levels of intracellular pyruvate and acetate became minimum, ATP became high and intracellular pH at its lowest level. When glycogen metabolism changed from degradation to accumulation, the respiratory phase started, during which ethanol was re-assimilated from the culture and the respiration rate increased. Intracellular pyruvate and acetate became maximum, ATP decreased and the intracellular pH appeared high. These findings may indicate new aspects of the control mechanism of glycogen metabolism and how respiration and ethanol fermentation are regulated together under aerobic conditions.     

Estimation of protease activity in soils at low temperatures by casein amendment and with substitution of buffer by demineralized water

The aim of this work was to modify the method of Ladd and Buttler (1972), by substituting Tris-HCl buffer (pH 8.52) with demineralized water (DEMI H(2)O), in order to assess its suitability for measurement of casein-protease activity at pH levels close to those of real soil in H(2)O. Measurements were undertaken over a range of incubation temperatures from 3 to 49 degrees C. Testing was performed on one organic soil and two different mineral soils. The substitution of Tris-HCl buffer by DEMI H(2)O at 49 degrees C decreased casein-protease activity to 67.25% in mineral soil and to 53.76% in organic soil. With decreasing temperature casein-protease activity decreased the most in organic soil, i.e., 0.07% of original its value at 3 degrees C. The incubation period was extended to maximally 336 h at 3 degrees C to totally obtain >10.0% of L-tyrosine equivalents released at optimum or close to optimum temperature and pH conditions. The Q(10) values of casein-protease activity measured after substituting Tris-HCl buffer with DEMI H(2)O were unexpectedly high. Between the temperatures of 3 and 49 degrees C Q(10) ranged from 3.46 to 4.25, whereas between 3 and 25 degrees C Q(10) ranged from 6.78 to 11.08. Therefore, the modified method of Ladd and Buttler (1972) presented can be used for measurement of soil casein-protease activity under pH conditions close to that of real soil pH and at an averaged soil temperatures measured in the field. This modification makes possible an expression of soil casein-protease activity potential - when being combined with measurements of casein-protease activity under optimum or close to optimum temperature and pH conditions, if high concentration of casein is present.     

Influence of oxygen on the growth of Saccharomyces cerevisiae in continuous culture

Baker's yeast, Saccharomyces cerevisiae, was investigated for the combined influence of dissolved oxygen and glucose concentration in continuous culture. A reactor was operated at a range of dilution rates (0.1, 0.2, 0.25, 0.27, and 3.0 h(-1)), above and below the critical value that separates the oxidative and fermentation regions. For each dilution rate (D), steady states were established at each of five to ten different dissolved oxygen concentrations (DO) in the range of 0.01-5 mg/L. The use of on-line mass spectrometry facilitated the measurement of gaseous and dissolved O(2), CO(2), and ethanol. Intracellular carbohydrate, protein, RNA, DNA, lipid, and cytochrome concentrations were measured. Cell size measurements were reduced to specific surface areas. Cytochrome content showed up to 100% variation during a 20-day period of adaptation at D = 0.2 h(-1) to low DO. Eventually, the culture behaved the same at DO = 0.05 mg/L as it did initially at 3 mg/L. At D = 0.2, 0.25, and 0.27 h(-1), the transition between oxidation and fermentation was characterized by a critical DO which decreased with decreasing D. The X-D curves were shifted such that the critical D value was reduced with decreasing DO. Specific oxygen update rates varied with DO according to the saturation kinetics. Specific cell surface areas increased with decreasing DO. Cytochrome content generally decreased with decreasing DO, and Q(O(2) ) could be linearly related to the total cytochrome content, which exhibited a maximum at D = 0.27 h(-1).     

Simulation of oxygen carrier mediated oxygen transport to C3A hepatoma cells housed within a hollow fiber bioreactor

A priori knowledge of the dissolved oxygen (O2) concentration profile within a hepatic hollow fiber (HF) bioreactor is important in developing an effective bioartificial liver assist device (BLAD). O2 provision is limiting within HF bioreactors and we hypothesize that supplementing a hepatic HF bioreactor's circulating media with bovine red blood cells (bRBCs), which function as an O2 carrier, will improve oxygenation. The dissolved O2 concentration profile within a single HF (lumen, membrane, and representative extra capillary space (ECS)) was modeled with the finite element method, and compared to experimentally measured data obtained on an actual HF bioreactor with the same dimensions housing C3A hepatoma cells. Our results (experimental and modeling) indicate bRBC supplementation of the circulating media leads to an increase in O2 consumed by C3A cells. Under certain experimental conditions (pO2,IN) = 95 mmHg, Q = 8.30 mL/min), the addition of bRBCs at 5% of the average in vivo human red blood cell concentration (% hRBC) results in approximately 50% increase in the O2 consumption rate (OCR). By simply adjusting the operating conditions (pO2,IN) = 25 mmHg, Q = 1.77 mL/min) and increasing bRBC concentration to 25% hRBC the OCR increase is approximately 10-fold. However, the improved O2 concentration profile experienced by the C3A cells could not duplicate the full range of in vivo O2 tensions (25-70 mmHg) typically experienced within the liver sinusoid with this particular HF bioreactor. Nonetheless, we demonstrate that the O2 transport model accurately predicts O2 consumption within a HF bioreactor, thus setting up the modeling framework for improving the design of future hepatic HF bioreactors.     

Effect of stimulus cycle time on acute respiratory responses to intermittent hypercapnic hypoxia in unsedated piglets.

To determine whether stimulus frequency affects physiological compensation to an intermittent respiratory stimulus, we studied piglets (n = 43) aged 14.8 +/- 2.4 days. A 24-min total hypercapnic hypoxia (HH) (10% O(2)-6% CO(2)-balance N(2) = HH) was delivered in 24-, 8-, 4-, or 2-min cycles alternating with air. Controls (n = 10) breathed air continuously. Minute ventilation and temperature were not different between the 2-min and 24-min groups, with neither different from controls during recovery. Piglets exposed to 8-min cycles had ventilatory stimulation, whereas those exposed to 4-min cycles had significant depression of ventilation. Despite this, piglets in these intermediate intermittent HH (IHH) groups (8- and 4-min cycles) showed more severe acidosis and attenuated temperature changes (P < 0.001 and P < 0.01 for pH and temperature vs. 24 min, respectively). Cycle time affected the ability of young piglets to tolerate IHH. More severe respiratory acidosis developed when IHH was delivered in intermediate (4 min or 8 min) cycles compared with the same total dose as a single episode or in short (2 min) cycles.