Temperature and oxygen regulation of oleate desaturation in developing sunflower (Helianthus annuus) seeds

The effect of low (10°C) and high (30°C) temperature on in vivo oleate desaturation has been studied in developing sunflower ( Helianthus annuus L.) seeds under conditions of different oxygen availability (capitulum, detached achenes or peeled seeds). In seeds remaining in the capitulum, only a part of the oleate newly synthesized at high temperature was desaturated to linoleate, whereas more oleate than that synthesized de novo was desaturated at low temperature. Achenes were only able to significantly desaturate oleate at low temperatures. In contrast, oleate desaturation was detected in peeled seeds incubated at low and high temperatures, showing the highest rate at 20°C. Hull removing dramatically increased the activity of the microsomal oleate desaturase (FAD2, EC 1.3.1.35) at all studied temperatures, although a long-term inactivation of the enzyme was observed at high temperatures. Low oxygen concentration (1–2%) obtained by respiration of peeled seeds incubated in sealed vials, brought about the inactivation of the enzyme. All these data suggest that temperature regulates oleate desaturation controlling the amount of oleate and the FAD2 activity. In addition, this enzyme seems to be also regulated by the availability of oxygen, which is affected inside the achene by its diffusion through the hull, and the competition with respiration, both factors being temperature-dependent.     

Oxygen-independent temperature regulation of the microsomal oleate desaturase (FAD2) activity in developing sunflower (Helianthus annuus) seeds

The temperature and oxygen regulation of the microsomal oleate desaturase (FAD2, EC 1.3.1.35) activity has been studied in developing sunflower ( Helianthus annuus L.) seeds. In plants cultivated in growth chambers, the linoleic acid content in the seed lipids increased along the 25/15°C (day/night) cycle, except during the first hours of the warm period, where it decreased significantly. In contrast, FAD2 activity decreased notably at the beginning of the warm period, showing a small and continuous increase during the rest of the cycle. The temperature effect on the linoleic acid content and the FAD2 activity was also investigated using peeled seeds and detached achenes subjected to temperature changes. In peeled seeds, a change of temperature from 10 to 30°C brought about a significant decrease of FAD2 activity. On the contrary, when the temperature shifted from 30 to 10°C, FAD2 activity only increased slightly. Unlike peeled seeds, detached achenes showed a fast and dramatic increase or decrease in the level of FAD2 activity in response to a temperature change from 30 to 10°C, or from 10 to 30°C, respectively. The in vivo and in vitro thermal properties of the FAD2 enzyme were also studied. Optimal temperature and heat-resistance profile showed similar patterns in both conditions. All these data support the hypothesis that temperature regulates FAD2 activity by two different and independent mechanisms: a direct effect, and an indirect effect affecting oxygen availability. Furthermore, these results suggest that the low thermal stability of the enzyme is the main factor responsible for the direct temperature effect on FAD2 activity.     

Effect of water temperature on the immune response and infectivity pattern of white spot syndrome virus (WSSV) in freshwater crayfish

The susceptibility of two species of freshwater crayfish, Pacifastacus leniusculus and Astacus astacus, to white spot syndrome virus (WSSV) by intramuscular injection was compared and the results show that both species are susceptible to WSSV. The effect of water temperature on the development of white spot disease in crayfish was also studied. Crayfish were exposed to different temperatures after WSSV injection or oral exposure and the mortalities were recorded over a period of 45 days. No mortality was observed when crayfish were held at 4+/-2 degrees C or 12+/-2 degrees C and reached 100% when these crayfish transferred to 22+/-2 degrees C. The mortalities of nearly moribund crayfish at 22+/-2 degrees C with WSSV could be delayed after transfer to temperature below 16 degrees C. These results clearly show that low temperature affects the WSSV pathogenicity in crayfish. Moreover, haemocyte counts, phenoloxidase activity, mRNA levels of prophenoloxidase (proPO) and the lipopolysaccharide and beta-1,3-glucan binding protein (LGBP) in crayfish exposed to various water temperatures were studied. Total haemocyte and granular cell counts of crayfish held at different temperatures were not significantly (P>0.05) different, except for the total haemocyte number at 18 degrees C was significantly (P<0.05) higher than in crayfish at 4 degrees C. The percentage of granular cells in crayfish held at 4 degrees C was the highest compared to crayfish maintained at other temperatures. The phenoloxidase activities in haemocyte lysate supernatant (HLS) of crayfish at all temperature groups remained similar. The amount of proPO-mRNAs in haemocytes was much higher than the amount of LGBP-m RNAs in all the experimental groups. However, there was no change in the level of pro PO-mRNA at the tested temperatures. Interestingly, the level of LGBP-mRNA of crayfish kept at 22 degrees C was much lower than in those held at lower temperatures. Proliferation of the haematopoietic tissues was higher at high temperatures which may support replication of WSSV, and explain the high mortality of crayfish with WSSV infection at high temperature. Based on these studies it is concluded that crayfish might act as a carrier of WSSV at low water temperature and could develop white spot disease if the water temperature is increased.     

Preliminary characterization of the delta-9 desaturase of Tetrahymena pyriformis W.

1. In vitro assay conditions have been defined for measurement of delta 9 desaturase activity in Tetrahymena pyriformis W. 2. The reaction depends on the presence of oxygen and a reduced pyridine nucleotide cofactor. FAD supports a low level of enzymatic activity. 3. Both stearyl-CoA and palmityl-CoA are acceptable substrates. Oleate formation is maximal at 30 degrees C. 4. Delta-9 desaturase activity appears to be localized in the microsomal fraction. Delta-6 and/or delta 12 desaturase activities have also been observed. 5. When the specificity of the delta 9 desaturase towards stearyl-CoA and palmityl-CoA was observed at 30 and 16 degrees C it was found that lowering the assay temperature did not affect specificity. Stearyl-CoA was more readily desaturated at both temperatures. 6. Exogenous oleyl-CoA and diisopropylfluorophosphate had little effect on delta 9 desaturase activity. However, cyanide strongly inhibited desaturation and a sensitivity to sulfhydryl-binding reagents has also been demonstrated.     

Influence of hypoxia and of hypoxemia on the development of cardiac activity in zebrafish larvae

Cardiac activity and anaerobic metabolism were analyzed in zebrafish larvae raised under normoxia (PO(2) = 20 kPa) and under chronic hypoxia (PO(2) = 10 kPa) at three different temperatures (25, 28, and 31 degrees C). Heart rate increased with development and with temperature. Under normoxia, cardiac output increased significantly at high temperature (31 degrees C), but not at 28 or at 25 degrees C. Under chronic hypoxia, however, heart rate as well as cardiac output increased at all temperatures in larvae at about hatching time or shortly thereafter. Cardiac activity of larvae raised for 2 wk after fertilization with a reduced hemoglobin oxygen-carrying capacity in their blood (hypoxemia; due to the presence of CO or of phenylhydrazine in the incubation water) was not different from control animals. Whole body lactate content of these animals did not increase. Thus there was no indication of a stimulated anaerobic energy metabolism. The increase in cardiac activity observed during hypoxia suggests that at about hatching time receptors are present that sense hypoxic conditions, and this information can be used to induce a stimulation of convective oxygen transport to compensate for a reduction in bulk oxygen diffusion in the face of a reduced oxygen gradient between environmental water and tissues. Under normoxia, however, the PO(2) gradient between environmental water and tissues and diffusional oxygen transport assure sufficient oxygen supply even if hemoglobin oxygen transport in the blood is severely impaired. Thus, under normoxic conditions and with a normal metabolic rate of the tissues, convective oxygen transport is not required until approximately 2 wk after fertilization.     

The utilization and desaturation of oleate and linoleate during glycerolipid biosynthesis in olive (Olea europaea L.) callus cultures

Callus cultures from olive (Olea europaea L.) were used to study characteristics of desaturation in this oil-rich tissue. The incorporation of [1-(14)C]oleate and [1-(14)C]linoleate into complex lipids and their further desaturation was followed in incubations of up to 48 h. Both radiolabelled fatty acids were rapidly incorporated into lipids, especially phosphatidylcholine and triacylglycerol. Radiolabelling of these two lipids peaked after 1-4 h, after which it fell. In contrast, other phosphoglycerides and the galactosylglycerides were labelled in a more sustained manner. [1-(14)C]Linoleate was almost exclusively found in the galactolipids. With [1-(14)C]linoleate as a precursor, the only significant desaturation to linolenate was in the galactolipids. Monogalactosyldiacylglycerol was the first lipid in which [1-(14)C]linoleate and [1-(14)C]linolenate appeared after incubation of the calli with [1-(14)C]oleate and [1-(14)C]linoleate, respectively. The presence of radioactivity in the plastidial lipids shows that both [1-(14)C]oleate and [1-(14)C]linoleate can freely enter the chloroplast. Two important environmental effects were also examined. Raised incubation temperatures (30-35 degrees C) reduced oleate desaturation and this was also reflected in the endogenous fatty acid composition. Low light also caused less oleate desaturation. The data indicate that lysophosphatidylcholine acyltransferase is important for the entry of oleate and linoleate into olive callus lipid metabolism and phospholipid:diacylglycerol acyltransferase may be involved in triacylglycerol biosynthesis. In addition, it is shown that plastid desaturases are mainly responsible for the production of polyunsaturated fatty acids. Individual fatty acid desaturases were differently susceptible to environmental stresses with FAD2 being reduced by both high temperature and low light, whereas FAD7 was only affected by high temperature.     

An analysis of the effect of changes in growth temperature on proteolysis in vivo in the psychrophilic bacterium Vibrio sp. strain ANT-300.

In the psychrophilic bacterium Vibrio sp. strain ANT-300, the rate of protein degradation in vivo, measured at fixed temperatures, increased with elevation of the growth temperature. A shift in growth temperature induced a marked increase in this rate. Dialysed cell-free extracts hydrolysed exogenous insulin, globin and casein (in decreasing order of activity) but did not hydrolyse exogenous cytochrome c. Cells contained at least seven protease separated by DEAE-Sephacel chromatography, one of which was an ATP-dependent serine protease. The ATP-dependent proteolytic activity in extracts of cells incubated for 3 h at 16 degrees C after a shift-up from 0 degrees C increased to a level 36% and 17% higher than that of cells grown at 0 degrees C and 13 degrees C, respectively. A shift-down to 0 degrees C from 13 degrees C induced only a slight increase in the proteolytic activity. Extracts of all cells, whether exposed to temperature shifts or not, showed the same temperature dependence with respect to both ATP-dependent and ATP-independent protease activity. In all the extracts these proteases also exhibited the same heat lability. The ATP-dependent protease was inactivated by incubation at temperatures above 25 degrees C. There was an increase in ATP-independent protease activity during incubation at temperatures between 25 and 30 degrees C, but a decrease at 35 degrees C and higher. These results suggest that the marked increases in proteolysis in vivo, caused by a shift in temperature, may result not only from increases in levels of ATP-dependent serine protease(s) but also from increases in the susceptibility of proteins to degradation.     

Thermal inactivation of glucose oxidase. Mechanism and stabilization using additives

Thermal inactivation of glucose oxidase (GOD; beta-d-glucose: oxygen oxidoreductase), from Aspergillus niger, followed first order kinetics both in the absence and presence of additives. Additives such as lysozyme, NaCl, and K2SO4 increased the half-life of the enzyme by 3.5-, 33.4-, and 23.7-fold respectively, from its initial value at 60 degrees C. The activation energy increased from 60.3 kcal mol-1 to 72.9, 76.1, and 88.3 kcal mol-1, whereas the entropy of activation increased from 104 to 141, 147, and 184 cal x mol-1 x deg-1 in the presence of 7.1 x 10-5 m lysozyme, 1 m NaCl, and 0.2 m K2SO4, respectively. The thermal unfolding of GOD in the temperature range of 25-90 degrees C was studied using circular dichroism measurements at 222, 274, and 375 nm. Size exclusion chromatography was employed to follow the state of association of enzyme and dissociation of FAD from GOD. The midpoint for thermal inactivation of residual activity and the dissociation of FAD was 59 degrees C, whereas the corresponding midpoint for loss of secondary and tertiary structure was 62 degrees C. Dissociation of FAD from the holoenzyme was responsible for the thermal inactivation of GOD. The irreversible nature of inactivation was caused by a change in the state of association of apoenzyme. The dissociation of FAD resulted in the loss of secondary and tertiary structure, leading to the unfolding and nonspecific aggregation of the enzyme molecule because of hydrophobic interactions of side chains. This confirmed the critical role of FAD in structure and activity. Cysteine oxidation did not contribute to the nonspecific aggregation. The stabilization of enzyme by NaCl and lysozyme was primarily the result of charge neutralization. K2SO4 enhanced the thermal stability by primarily strengthening the hydrophobic interactions and made the holoenzyme a more compact dimeric structure.     

Ultralow oxygen treatment for control of Latrodectus hesperus (Araneae: Theridiidae) on harvested table grapes

The spider Latrodectus hesperus Chamberlin & Ivie (Araneae: Theridiidae) was subjected to low and ultralow oxygen (ULO) treatments at different temperatures. Complete control of the spiders was achieved in 24-h ULO treatments with 0.5% O2 or lower at 1 degrees C and in a 24-h low oxygen (2%) treatment at 15 degrees C. Oxygen level and temperature greatly affected spider mortality. At 1 degrees C, as oxygen level was decreased from 2 to 0.5%, spider mortality increased from 0 to 100%. At 2% O2, as temperature was increased from 1 to 15 degrees C, spider mortality increased from 0 to 100%. Grape clusters from two table grape (Vitis spp.) cultivars, 'Thompson Seedless' and 'Flame Seedless', were subjected to the 24-h ULO treatment with 0.5% O2 at 1 degrees C. The ULO treatment had no negative effects on grape quality. Because of the relatively short treatment time, effectiveness at low storage temperature and the easily attained oxygen level, we conclude that the ULO treatment have good potential to be implemented commercially for control of black widow spiders on harvested table grapes.     

Respiratory response to temperature and hypoxia in the zebra mussel Dreissena polymorpha

The effects of temperature acclimation, acute temperature variation and progressive hypoxia on oxygen consumption rates (VO2) were determined for the zebra mussel Dreissena polymorpha. In the first experiment, after acclimation to 5, 15 or 25 degrees C for at least 2 weeks, VO2 was determined at 5 degrees C increments from 5 to 45 degrees C. VO2 increased in all three acclimation groups from 5 to 30 degrees C, corresponding to the normal ambient temperature range for this species. Mussels displayed imperfect temperature compensation at temperatures above 15 degrees C, but exhibited little acclimatory ability below 15 degrees C. In the hypoxia experiment, VO2 was determined over the course of progressive hypoxia, from full saturation (oxygen tension [PO2]=160 Torr [21.3 kPa]) to a PO2 at which oxygen uptake ceased (<10 Torr [1.3 kPa]). Mussels were acclimated to either 5, 15 or 25 degrees C for at least 2 weeks and their respiratory response to progressive hypoxia was measured at three test temperatures (5, 15 and 25 degrees C). The degree of oxygen regulation increased with increasing test temperature, particularly from 5 to 15 degrees C, but decreased with increasing acclimation temperature. The decreased metabolic rate observed for warm-acclimated animals, particularly in the upper portion of the temperature range of the zebra mussel, may allow for conservation of organic energy stores during warm summer months. Compared to other freshwater bivalves, D. polymorpha is a relatively poor oxygen regulator, corresponding with its preference for well-oxygenated aquatic habitats. In addition, a new quantitative method for determining the degree of oxygen regulation is presented.     

High-Oleate Oilseeds Fail to Develop at Low Temperature

The fad2 mutants of Arabidopsis thaliana are deficient in activity of the endoplasmic reticulum oleate desaturase that is the main enzyme responsible for polyunsaturated lipid synthesis in developing seeds of oil crops. A comparison of wild-type and fad2 seeds developing on heterozygous (FAD2/-) plants was used as a model for genetically engineered high-oleate oilseeds of species such as soybean and canola. When fad2 seeds developed at normal temperatures (22[deg]C), they showed high viability compared to wild-type seeds. When a portion of seed development took place at 6[deg]C, germination of the wild-type siblings remained high but germination of fad2 segregants declined considerably. This was true even when exposure to low temperature was limited to the final stages of seed filling and maturation. Compared to wild-type seeds, fully viable fad2 seeds produced at 22[deg]C had reduced lipid contents and were slower to germinate at 10 and 6[deg]C. Taken together, these results indicate that for some oilseed species at least, molecular genetic manipulation of oleate levels in the oil may result in plant lines with unacceptable performance in the field.     

Activation of plasma membrane H(+)-ATPase and expression of PMA1 and PMA2 genes in Saccharomyces cerevisiae cells grown at supraoptimal temperatures.

During exponential growth at temperatures of 30 to 39 degrees C, the specific activity of H(+)-ATPase in the plasma membrane of Saccharomyces cerevisiae (assayed at the standard temperature 30 degrees C) increased with increases in growth temperature. In addition, the optimal temperature for in vitro activity of this ATPase was 42 degrees C. Therefore, the maximum values of ATPase activity were expected to occur in cells that grew within the supraoptimal range of temperatures. Activation induced by supraoptimal temperatures was not the result of increased synthesis of this membrane enzyme. When the growth temperature increased from 30 to 40 degrees C, expression of the essential PMA1 gene, monitored either by the level of PMA1 mRNA or the beta-galactosidase activity of the lacZ-PMA1 fusion, was reduced. Consistently, quantitative immunoassays showed that the ATPase content in the plasma membrane decreased. Like ATPase activity, the efficiency of the PMA2 promoter increased with increases in growth temperature in cells that had been grown at 30 to 39 degrees C, but its level of expression was several hundred-fold lower than that of PMA1. These results suggest that the major PMA1 ATPase is activated at supraoptimal temperatures.     

The ontogeny of physiological response to temperature in early stage spiny lobster (Jasus edwardsii) larvae

The physiological response to temperature, in terms of oxygen consumption, nitrogen excretion and feed intake was examined in Jasus edwardsii larvae at mid-stages I-III. From stage I to stage III, the mass-specific oxygen consumption increased in a sigmoid pattern over the temperature range of 10-22 degrees C. The Q(10) value declined significantly from 14-18 to 18-22 degrees C range, indicating a reduced temperature dependence of larval metabolism at higher temperatures. At all stages, feed intake increased with increasing temperature but reached a plateau at the higher temperatures for stages I and II larvae. In contrast, nitrogen excretion increased linearly over this temperature range for all larval stages. Therefore, higher temperatures ( approximately 22 degrees C) may cause an energetic imbalance and reduce growth potential in early stage larvae. While the convection requirement index (quotient of feed intake and oxygen consumption) indicated an equivalent metabolic feeding efficiency from 14 to 22 degrees C, a consistent decline of the O/N ratio above 16-18 degrees C from stage I to stage III suggested that exposure to elevated temperatures may result in an increase in the amount of protein being diverted from growth to catabolic processes. Based on these results, a temperature of 18 degrees C is recommended for the culture of early stage J. edwardsii larvae.     

Temperature dependence of nitrate reductase in the psychrophilic unicellular alga Koliella antarctica and the mesophilic alga Chlorella sorokiniana

Temperature responses of nitrate reductase (NR) were studied in the psychrophilic unicellular alga, Koliella antarctica, and in the mesophilic species, Chlorella sorokiniana. Enzymes from both species were purified to near homogeneity by Blue Sepharose (Pharmacia, Uppsala, Sweden) affinity chromatography and high-resolution anion-exchange chromatography (MonoQ; Pharmacia; Uppsala, Sweden). Both enzymes have a subunit molecular mass of 100 kDa, and K. antarctica NR has a native molecular mass of 367 kDa. NR from K. antarctica used both NADPH and NADH, whereas NR from C. sorokiniana used NADH only. Both NRs used reduced methyl viologen (MVH) or benzyl viologen (BVH). In crude extracts, maximal NADH and MVH-dependent activities of cryophilic NR were found at 15 and 35 degrees C, respectively, and retained 77 and 62% of maximal activity, respectively, at 10 degrees C. Maximal NADH and MVH-dependent activities of mesophilic NR, however, were found at 25 and 45 degrees C, respectively, with only 33 and 23% of maximal activities being retained at 10 degrees C. In presence of 2 microM flavin adenine dinucleotide (FAD), activities of cryophilic NADH:NR and mesophilic NADH:NR were stable up to 25 and 35 degrees C, respectively. Arrhenius plots constructed with cryophilic and mesophilic MVH:NR rate constants, in both presence or absence of FAD, showed break points at 15 and 25 degrees C, respectively. Essentially, similar results were obtained for purified enzymes and for activities measured in crude extracts. Factors by which the rate increases by raising temperature 10 degrees C (Q10) and apparent activation energy (E(a)) values for NADH and MVH activities measured in enzyme preparations without added FAD differed slightly from those measured with FAD. Overall thermal features of the NADH and MVH activities of the cryophilic NR, including optimal temperatures, heat inactivation (with/without added FAD) and break-point temperature in Arrhenius plots, are all shifted by about 10 degrees C towards lower temperatures than those of the mesophilic enzyme. Transfer of electrons from NADH to nitrate occurs via all three redox centres within NR molecule, whereas transfer from MVH requires Mo-pterin prosthetic group only; therefore, our results strongly suggest that structural modification(s) for cold adaptation affect thermodynamic properties of each of the functional domains within NR holoenzyme in equal measure.     

Regulation of Crassula argentea phosphoenolpyruvate carboxylase in relation to temperature.

The effect of temperature on the kinetic parameters of phosphoenolpyruvate carboxylase purified from Crassula argentea was such that both the Vmax and Km(MgPEP) values tended upward over the range from 11 to 35 degrees C. The increased rate at low temperatures due to the low Km is at least partially offset by the increased Vmax at higher temperatures, potentially leading to a broad plateau of enzyme activity and a relatively small effect of temperature on the enzyme. The cooperativity was negative at 11 degrees C, but above 15 degrees C it became positive. The presence of 5 mM glucose-6-phosphate has relatively little effect on Vmax but it clearly reduces Km and overcomes any effect of temperature on this parameter in the range studied. Positive cooperativity is observed only at temperatures above 25 degrees C. The size of the native enzyme, as determined by dynamic light scattering, was strongly toward the tetrameric form. At a temperature of 40 degrees C and above, a considerable oligomerization takes place. No loss of activity can be observed in this range of temperature. In the presence of either glucose-6-phosphate or magnesium phosphoenolpyruvate, at temperatures under 25 degrees C, the equilibrium is displaced toward higher levels of aggregation. Maximal accumulation of lead malate occurred at 10 to 12 degrees C in vivo with reduction to about 25% at 35 degrees C. Glucose-6-phosphate followed a similar curve in response to temperature, but the overall difference was about 50%. The sum of phosphoenolpyruvate plus pyruvate is level at night temperatures below 25 degrees C, doubling at 35 degrees C. Calculated concentrations of malate, glucose-6-phosphate, and phosphoenolpyruvate plus pyruvate indicate that the concentrations present are equal to or greater than Ki, Ka, and Km values for these metabolites, respectively.     

Temperature-dependent development of cardiac activity in unrestrained larvae of the minnow Phoxinus phoxinus

The minnow (Phoxinus phoxinus) was raised up to the stage of swim bladder inflation at temperatures between 10 degrees C and 25 degrees C, and the time of development significantly decreased at higher temperatures. Accordingly, initiation of cardiac activity was observed at day 2 in 25 degrees C animals and at day 4 in 12.5 degrees C animals. Only a minor increase in body mass was observed during the incubation period, and, at the end of the incubation period, animals raised at 25 degrees C did not have a significantly lower body mass compared with animals raised at 15 degrees C. Metabolic activity, determined as the rate of oxygen consumption of a larva, increased from 3.3 to 19.5 nmol/h during development at 15 degrees C and from 5.6 to 47.6 nmol/h during development at 25 degrees C. Heart rate showed a clear correlation to developmental stage as well as to developmental temperature, but at the onset of cardiac activity, diastolic ventricular volume and also stroke volume were higher at the lower temperatures. Furthermore, stroke volume increased with development, except for the group incubated at 12.5 degrees C, in which stroke volume decreased with development. Initial cardiac output showed no correlation to incubation temperature. Although metabolic activity increased severalfold during development from egg to the stage of swim bladder inflation at 15 degrees C and at 25 degrees C, weight-specific cardiac output increased only by approximately 40% with proceeding development. At 12.5 degrees C, cardiac output remained almost constant until opening of the swim bladder. The data support the notion that oxygen transport is not the major function of the circulatory system at this stage of development. The changes in heart rate with temperature appear to be due to the intrinsic properties of the pacemaker; there was no indication for a regulated response.     

The effect of progressive hypoxia on respiration in the dogfish (scyliorhinus canicula) at different seasonal temperatures.

1. Dogfish were acclimated to 7, 12 or 17 degrees C and exposed to progressive hypoxia at the temperature to which they had been acclimated. During normoxia, the Q10 values for oxygen uptake, heart rate, cardiac output and respiratory frequency over the full 10 degrees C range were: 2.1, 2.1, 2.1 and 2.5 respectively. Increased acclimation temperature had no effect on cardiac stroke volume or systemic vascular resistance, although there was a decrease in branchial vascular resistance, pHa and pHv. 2. Progressive hypoxia had no effect on heart rate or oxygen uptake at 7 degrees C, whereas at 12 degrees C and 17 degrees C there was bradycardia, and a reduction in O2 uptake, with the critical oxygen tension for both variables being higher at the higher temperature. Cardiac stroke volume increased during hypoxia at each temperature, such that cardiac output did not change significantly at 12 and 17 degrees C. Neither pHa nor pHv changed significantly during hypoxia at any of the three temperatures. 3. The influence of acclimation temperatures on experimental results from poikilotherms is pointed out. Previously-published results show quantitative differences. 4. The significance of the present results with respect to the functioning and location of oxygen receptors is discussed. It is argued that as the metabolic demand and critical oxygen tension of the whole animal are increased at high acclimation temperatures the same must be the case with the oxygen receptor. This would raise the stimulation threshold and could account for the bradycardia seen during hypoxia becoming manifest at higher values of PI,O2, Pa,O2 and Pv,O2 as the acclimation temperature is raised.     

信阳桃花水母对几种生态因子胁迫的耐受反应

在不同温度、盐度、pH等因子胁迫条件下,研究观察了信阳桃花水母(Craspedacusta sowerbyi xinyangensis)的生活状态、形态变化及存活等耐受反应,并测定了信阳桃花水母在不同温度下的耗氧率和最低耐氧能力.结果表明,信阳桃花水母的最适生活温度范围为15 ℃～25 ℃,pH为6.0～8.2.信阳桃花水母对盐度耐受力差,盐度为2时,仅能存活96 h;但具有较强的耐低氧能力,20 ℃时窒息点为026 mg·L^-1,平均耗氧量和平均耗氧率随温度的升高而升高.     

Oxidative Stress at High Temperatures in Lactococcus lactis Due to an Insufficient Supply of Riboflavin

Lactococcus lactis MG1363 was found to be unable to grow at temperatures above 37°C in a defined medium without riboflavin, and the cause was identified to be dissolved oxygen introduced during preparation of the medium. At 30°C, growth was unaffected by dissolved oxygen and oxygen was consumed quickly. Raising the temperature to 37°C resulted in severe growth inhibition and only slow removal of dissolved oxygen. Under these conditions, an abnormally low intracellular ratio of [ATP] to [ADP] (1.4) was found (normally around 5), which indicates that the cells are energy limited. By adding riboflavin to the medium, it was possible to improve growth and oxygen consumption at 37°C, and this also normalized the [ATP]-to-[ADP] ratio. A codon-optimized redox-sensitive green fluorescent protein (GFP) was introduced into L. lactis and revealed a more oxidized cytoplasm at 37°C than at 30°C. These results indicate that L. lactis suffers from heat-induced oxidative stress at increased temperatures. A decrease in intracellular flavin adenine dinucleotide (FAD), which is derived from riboflavin, was observed with increasing growth temperature, but the presence of riboflavin made the decrease smaller. The drop was accompanied by a decrease in NADH oxidase and pyruvate dehydrogenase activities, both of which depend on FAD as a cofactor. By overexpressing the riboflavin transporter, it was possible to improve FAD biosynthesis, which resulted in increased NADH oxidase and pyruvate dehydrogenase activities and improved fitness at high temperatures in the presence of oxygen.     

Effects of temperature and physical activity on blood flow shunts and intracardiac mixing in the toad Bufo marinus.

Blood flow in systemic (.Qsys) and pulmocutaneous (.Qpul) arteries was measured as a function of body temperature (10 degrees, 20 degrees, and 30 degrees C) at rest and following enforced physical activity in conscious, adult cane toads (Bufo marinus). Arterial and mixed venous hemoglobin concentration (CHb) and total oxygen content (Co2, tot) were measured in a separate group under identical conditions. Heart rate (fH) and total flow (.Qtot) increased significantly (P<0.001) with elevated temperature and with activity, whereas stroke volume (VS) increased (P<0.001) only with activity. .Qtot ranged about 10-fold, from 10 degrees C (rest) to 30 degrees C (activity); increases in both fH and VS contributed to the increase in .Qtot. The overall distribution of blood to the pulmocutaneous circuit (net L-R shunt) increased with both temperature and activity and was significantly correlated with .Qtot. These data indicate that blood flow distribution in toads is a direct function of cardiac output, and this is linked to relative changes in resistance in the major outflow vessels. Arterial O2 saturation (Sa) was high (mean=93%) in all conditions except activity at 30 degrees C, when it decreased to 74% and contributed to a decrease in the arteriovenous O2 difference. Venous O2 saturation (Sv) was high at rest (76%) and dropped significantly during activity to about 30% at all temperatures. Intracardiac arterial-venous mixing (systemic mixing index) showed the strongest correlation with variation in fH with minimal mixing (17%) occurring at about 50 beats min-1. The most mixing occurred at the lowest fH (13 beats min-1) and at the highest fH (103 beats min-1). The results indicate that the heart of a 0.25-kg toad becomes more efficient from an oxygen transport perspective from low fH to 50 beats min-1 and then less efficient at higher fH, contributing to an uncoupling of blood flow and metabolic rates at these high rates.