The Effect of Light, Temperature, and Anaesthetics on ATP Levels in the Leaves of Chenopodium rubrum and Phaseolus vulgaris

The effects of light, temperature, and anaesthetics on ATP levelsin the leaves of Chenopodium rubrum and of Phaseolus vulgarishave been determined by an UV-enzymic method. ATP levels were found to vary inversely with temperature. Moreover,the anaesthetics, ether and ohloroform, were found to elevateleaf ATP. The effect of light was to reduce ATP levels althougha tendency for increased ATP at high light intensities was detected. A correlation is deduced between elevated ATP level and decreasedcytoplasmic viscosity, for cold, light, and anaesthetics. On the basis that cold, and some anaesthetic substances, havebeen shown to release plants from dormancy, it is suggestedthat ATP levels may play a fundamental role in vernalizationand in the breaking of dormancy, and that the interaction oflight may also be on this basis. An explanation is suggested for these effects, and their widerimplications are discussed.     

A bimodal germination response to temperature in cocklebur seeds. II. ATP and adenylate pool

Abstract. The mechanism involved in a bimodal germination-temperature response in pre-soaked cocklebur (Xanthium pennsylvanicum Wallr.) seeds was studied with special reference to adenylate metabolism. Exposure to either low (optimal at 8°C) or high (optimal at 34°C) temperature which was effective in inducing the germination of the seeds brought about the accumulation of ATP in them. The ATP level remained unchanged at temperatures around 23°C. Pretreatment with KCN, stimulating germination even at 23°C, subsequently increased the ATP content, total adenylate pool and energy charge (EC) in the axial tissue prior to germination above those of the untreated controls. The lower the treatment temperature, the greater the inhibitory effect of KCN on ATP formation. An increase in germination following an increasing duration of pre-soaking at 8°C was comparable to increasing both the ATP content and total adenylate pool of axes, but not the EC value. Similarly, changes in germination following an increased exposure duration at 8°C correlated with changes in ATP content rather than EC value in the axes. Unlike the case of chilling, an increase in ATP level in response to 34°C was greater in the early period of water imbibition, during which times its germination-stimulating effect appeared more striking than in the later period, and it occurred without a concomitant rise in EC value because of the increased supply of AMP. Such a supply of AMP was reduced in the presence of benzohydroxamic acid or propyl gallale, inhibitors of an alternative respiratory pathway. It was thus concluded that both low temperature, coupled with warm temperature, and high temperature, by itself, can induce seed germination by increasing the ATP level as well as the total adenylate pool, but not the EC value, in the axial tissue. Further, that increases in both the ATP level and the adenylate pool especially are required for seed germination to proceed, probably depending on the activities of the cytochrome and alternative respiration pathways, respectively.     

Effects of ambient temperatures on blood viscosity and plasma protein concentration of broiler chickens (Gallus domesticus)

This paper describes the effects of ambient temperatures on whole blood viscosity and plasma protein concentration in broiler chickens. Whole blood viscosity and haematocrit, compared at 7 and 20°C, decreased significantly at or above 25°C of ambient temperature. However, no marked difference were found between 7 and 20°C or between the temperatures of 25, 30 and 35°C. The heat-induced decrease of whole blood viscosity was found after exposure for 1 h. These results suggest that the heat-induced decrease in blood viscosity is only a level change, which occurs between thermoneutral and high ambient temperature.     

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43°C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20°C, but in a liquid crystalline state when cells were grown at 37 and 43°C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

Low temperature alters plasma membrane lipid composition and ATPase activity of pineapple fruit during blackheart development

Plasma membrane (PM) plays central role in triggering primary responses to chilling injury and sustaining cellular homeostasis. Characterising response of membrane lipids to low temperature can provide important information for identifying early causal factors contributing to chilling injury. To this end, PM lipid composition and ATPase activity were assessed in pineapple fruit (Ananas comosus) in relation to the effect of low temperature on the development of blackheart, a form of chilling injury. Chilling temperature at 10 °C induced blackheart development in concurrence with increase in electrolyte leakage. PM ATPase activity was decreased after 1 week at low temperature, followed by a further decrease after 2 weeks. The enzyme activity was not changed during 25 °C storage. Loss of total PM phospholipids was found during postharvest senescence, but more reduction was shown from storage at 10 °C. Phosphatidylcholine and phosphatidylethanolamine were the predominant PM phospholipid species. Low temperature increased the level of phosphatidic acid but decreased the level of phosphatidylinositol. Both phospholipid species were not changed during storage at 25 °C. Postharvest storage at both temperatures decreased the levels of C18:3 and C16:1, and increased level of C18:1. Low temperature decreased the level of C18:2 and increased the level of C14:0. Exogenous application of phosphatidic acid was found to inhibit the PM ATPase activity of pineapple fruit in vitro. Modification of membrane lipid composition and its effect on the functional property of plasma membrane at low temperature were discussed in correlation with their roles in blackheart development of pineapple fruit.     

Simultaneous monitoring of intracellular ATP and oxygen levels in chondrogenic differentiation using a dual‐color bioluminescence reporter

A number of assay methods which measure cellular metabolic activity have only measured intracellular ATP levels because it has been speculated that ATP production and oxygen consumption are obligatorily coupled to each other under normal conditions. However, there exist many cases in which ATP production and oxygen consumption are uncoupled. Therefore, measurement of only intracellular ATP levels has a limit for understanding the overall metabolic states during various cellular functions. Here, we report a novel system for simultaneously monitoring intracellular ATP and oxygen levels using a red‐emitting Phrixothrix hirtus luciferase (PxRe) and a blue‐emitting Renilla luciferase (Rluc). Using this system, we monitored the dynamic changes in both intracellular ATP and oxygen levels during chondrogenesis. We found that the oxygen level oscillated at twice the frequency of ATP in chondrogenesis and the oxygen oscillations have an antiphase mode to the ATP oscillations; we also found an independent mode for the ATP oscillations. This result indicates that both mitochondrial and non‐mitochondrial respiration oscillate and thus play a role in chondrogenesis. This dual‐color monitoring system is useful for studying metabolic regulations that underlie diverse cellular processes. Copyright © 2013 John Wiley & Sons, Ltd.     

Stability of ATP in Antarctic mineral soils

The stability of exogenous ATP in Antarctic Ross desert soils has been assessed using bioluminescence monitoring of ATP-supplemented samples. Under typical east Antarctic dry valley summer conditions (−3 to +15°C), exogenous ATP was degraded with a half-life of between 0.5 and 30 h. The rate of degradation was affected, in order of significance, by soil biomass levels, temperature and water content. Such rapid removal of exogenous ATP strongly suggests that extracellular ATP from lysed cells in cold desiccated soils does not make a significant contribution to the standing ATP titre     

Approaches to the measurement of intracellular adenine and the discrimination between adenine transport and metabolism in L1210 leukemia cells

Incubation of L1210 leukemia cells with 10 μM [³H]adenine in the absence of energy substrate results in a very rapid accumulation of ³H within the cells. By 20 s intracellular adenine is near steady-state; beyond this the rate of accumulation of intracellular ³H reflects nucleotide synthesis, predominantly the rate of ATP accumulation within the cell as determined by liquid chromatography. Adenine incorporation into the nucleotides proceeds via adenine-phosphoribosyl transferase, which is rate-limiting to AMP formation and subsequently the formation of ADP and ATP. Acceleration of this pathway by the addition of glucose and phosphate decreases the intracellular adenine level far below equilibrium as metabolism is increased relative to transport. Assessment of methodology to evaluate intracellular adenine and its metabolites indicates that (i) a 4°C wash removes the major portion of intracellular adenine and (ii) at 4°C, transport of adenine remains rapid and while nucleotide synthesis is decreased, ATP still accumulates within the cell. Hence, measurement of cellular uptake of radioactive label at 4°C after cells are washed free of adenine cannot be used as a measurement of adenine surface binding since this radioactive label represents, at least in part, phosphorylated derivatives of adenine within the cell. Unlabeled adenine and structurally related compounds were found to inhibit [³H]adenine net uptake under conditions where metabolism of adenine was reduced, suggesting that base transport is mediated by a facilitated diffusion mechanism. This is consistent with other studies from this laboratory that demonstrate exchange diffusion between adenine and other bases.     

Temperature-related divergence in experimental populations of Drosophila melanogaster. II. Correlation between fitness and body dimensions

From a laboratory stock of Drosophila melanogaster (Oregon), reared for more than 20 years at 18° C, a new population was derived and maintained at 28° C for 8 years. The chromosomal and cytoplasmic contribution to genetic divergence between the two populations was estimated. Six body traits and reproductive fitness were taken into account. The third chromosome is responsible for the adaptive difference for temperature between the two lines. Temperature-selected genes which control body size are located on the second and third chromosomes, although the contribution of each chromosome depends on the environment in which the flies develop. The correlation between the chromosomal and cytoplasmic contributions to different traits and fitness, changes with temperature. At 28° C the correlation between fitness and each body trait is proportional to the response to selection exhibited by each of them, but this is not true at 18° C. Body size has, therefore, an adaptive significance in relation to temperature, which is expressed only in the environment where selection occurs. Cytoplasmic genes affect almost all characters to an extent similar to that of chromosomal genes. Inter-chromosomal and nucleo-cytoplasmic interactions are present and also change with temperature. In general, genes selected in a given environment produce greater phenotypic changes in that environment than in another. The population that experienced both temperatures is fitter in both environments, suggesting that the capacity to adapt to warm temperatures depends on genes other than those which are involved in the adaptation to cold.     

Intracellular pH and energy metabolism in the highly stenothermal Antarctic bivalve Limopsis marionensis as a function of ambient temperature

Changes in oxygen consumption, ammonia excretion and in the acid-base and energy status of various tissues were investigated in the cold stenothermal Antarctic bivalve, Limopsis marionensis, and compared to similar data in the limpet, Nacella concinna, for an assessment of thermal sensitivity. Oxygen consumption of L. marionensis varied between −1.5 and 2°C with a Q ₁₀ of 2.2. Ammonia excretion could only be detected in animals exposed to elevated temperature for periods in excess of 45 days and close to death and it is interpreted as the onset of protein and amino acid catabolism with starvation under temperature stress. In L. marionensis any change in temperature as well as starvation stress at constant temperature induced a decrease in phospho-l-arginine and ATP levels. However, only temperature stress resulted in a drop in the Gibb's free energy change of ATP hydrolysis. Intracellular pH rose in all tissues during upward or downward temperature changes of only 1.5 or 2°C for 24 h with a concomitant trend to accumulate succinate and acetate in the tissues. These changes are seen to reflect disturbances of the tissue acid-base and energy status with any under- or overshoot in aerobic metabolic rate during a temperature decrease or increase. Elevated temperature at 2°C during 2 weeks of incubation resulted in continued net ATP depletion, at low levels of ATP free energy. This indicates long-term stress, which was also mirrored in the inability to establish a new steady-state mean rate of oxygen consumption. Incubation at even higher temperatures of 4 and 7°C led to an aggravation of energetic stress and transition to an intracellular acidosis, as well as a fall in oxygen consumption. In N. concinna a drop in energy levels was also visible at 2°C but was compensated for during long-term incubation. In conclusion, L. marionensis will be able to compensate for a temperature change only in a very narrow range whereas the thermal tolerance window is much wider in N. concinna. The inability of the metabolic rate to rise continually and the concomitant transition to anaerobic metabolism and long-term energetic stress characterize the upper critical temperature. Stenothermality is discussed, not only as reflecting the permanent and very stable low temperature in the natural environment, but also regarding dif- ferences in the level of activity and aerobic scope. Accepted: 21 December 1998     

Activity of supplemental enzymes and their effect on nutrient utilization and growth performance of growing chickens as affected by pelleting temperature

Activity of supplemental enzymes in a barley‐soybean‐maize based diet at 60, 75 and 90°C pelleting temperatures was studied using feed viscosity, in‐vitro enzyme activity and broiler performance data.

High pelleting temperatures increased feed viscosity but supplemented enzymes reduced the viscosity at all three temperatures levels by 11, 14 and 17%, respectively. Water intake and losses in excreta of birds were found to be affected by feed viscosity. Activity of cellulase enzyme, measured using the radial diffusion method, was unaffected at 60 and 75°C, but reduced by 73% in feed processed at 90°C. Enzymes increased the weight gain of broilers by 11.1% at 90°C, but no effect could be seen at low pelleting temperatures possibly due to high dietary protein and energy contents. Feed intake was unaffected by enzymes. Birds consumed 6% more feed and grew 9% faster when the pelleting temperature was increased from 60 to 75°C. Reduced feed intake and daily weight gain observed at 90° C could be fully compensated by the enzyme supplementation. High pelleting temperature reduced energy metabolizability (3.2%) and nitrogen utilization (4%) but enzyme almost compensated them (by 3.3% and 2.6%, respectively). No interaction could be detected between the pelleting temperatures and enzymes.

It is concluded that pelleting temperatures as high as 90°C drastically reduce cellulase activity, energy and nitrogen utilization thus lowering broiler performance. Either the remaining activity of cellulase or other thermostable enzymes can prevent the losses.     

Effects of hypothermia on canine kidney mitochondria

The effect of hypothermia on the function of isolated dog kidney cortex mitochondria was determined with an FAD- and NAD⁺-linked substrate. In dog kidney mitochondria, temperatures of 10 °C or less suppress ADP stimulation of respiration but have little or no effect upon uncoupler, Ca²⁺ or valinomycin-K⁺ stimulation of respiration. This suggests that the adenine nucleotide translocase which catalyses the transport of ADP into the mitochondria limits the rate of respiration and generation of ATP at 10 °C in kidneys undergoing preservation. The coupling of oxidation to phosphylation, as determined by measuring the amount of ATP formed at low temperatures, indicates, however, that mitochondria are fully coupled at both 10 and 5 °C. The respiratory control index at 15 °C is greater (with pyruvate plus malate) than at 30 or 10 °C and suggests that 15 °C may be the optimum perfusion temperature for maintaining adenine nucleotide levels in the perfused kidney.     

Effect of temperature and viscosity on the motility of the spirochete Treponema denticola

Treponema denticola is an oral spirochete associated with periodontal diseases. Because bacterial motility is likely to be a potential virulence factor, we investigated the effect of viscosity and temperature on cell speed. In agreement with the work of others, translational motility was a function of the macroscopic viscosity of the medium. In addition, we found that although the speed of spirochetes was slow at 25°C (4 μm s⁻¹), it increased quite markedly at 35°C (19 μm s⁻¹). The results indicate that both viscosity and temperature are critical factors in T. denticola translational motility.     

Heat shock effect on glutathione and superoxide dismutase in Tetrahymena pyriformis

Intracellular levels of total glutathione and cytosolic superoxide dismutase activity were assayed in cells from Tetrahymena pyriformis either exposed to sub-lethal (34°C) or to lethal heat shock (39°C). The results showed that glutathione levels decrease to 60% of normal values after a sub-lethal heat shock for 1 hour. This change is part of the heat shock response, since the effect is reversed as soon as cells are brought to their normal growing temperature. Using actinomycin D, which blocks the synthesis of high molecular weight hsp (Galego and Rodrigues-Pousada, 1985), prior to thermal stress, the fall in total glutathione is not observed, suggesting a partial correlation with the synthesis of these stress proteins. Using cells pre-exposed to a sub-lethal heat shock, a subsequent short severe heat shock does not lead to a significant decrease of the glutathione content. Superoxide dismutase (SOD) activity is not significantly induced after either a short period at 34°C or a prolonged treatment at the same temperature.     

Heat-induced stimulation of 3-O-methylglucose transport in rat thymocytes.

Cells incubated at 41–46 °C show a gradual increase in the initial rate of 3-O-methylglucose uptake when subsequently assayed at 37 °C. Cellular ATP levels remain constant throughout this temperature range, but at temperatures higher than 46 °C, ATP levels decline as does the extent of transport stimulation. Cells incubated at 45 °C for 5 min continue to show a gradual increase in transport activity throughout a subsequent 25-min incubation period at 37 °C. The increase in transport activity is characterized by an increase in the proportion of the rapid phase of 3-O-methylglucose uptake, with little or no change in the half-time of either the rapid phase or the slow phase. Transport stimulation at high temperatures is blocked by inhibitors of oxidative phosphorylation. Cells depleted of intracellular exchangeable Ca²⁺ by treatment with the ionophore A23187 in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid show nearly the same degree of stimulation at high temperatures as untreated cells, suggesting that exchangeable Ca²⁺ ions do not play an obligatory role in the mechanism of transport stimulation. It is suggested that structural changes occur at 41–46 °C in the membrane proteins controlling glucose transport activity.     

Growth at 37°C of the EGs strain of the amoeboflagellate Naegleria gruberi containing viruslike particles. II. Cytoplasmic changes

The EG_s strain of the amoeboflagellate Naegleria gruberi contains viruslike particles (VLP) apparently responsible for the development of cytoplasmic structures in infected cells. Growth of amoebae at 37°C produced changes in the normal pattern of development of the cytoplasmic units. Structures referred to as bacterium-like bodies, which developed in infected amoebae grown at 21°C, did not form at the elevated temperature. Amoeba cytoplasm at the elevated temperature exhibited regions of varying densities and bundles of microtubule-like fibrils. Presumed transmissive stages which were seen in cells grown at 21°C were not formed at 37°C. These changes are of significance in that they parallel cytoplasmic changes in cytopathic chick embryo fibroblasts exposed to lysates made from VLP-infected cultures of amoebae.     

The effect of high temperatures on leaf cells of Valerianella: relative heat stability of the tonoplast membrane of mesophyll vacuoles

The effect of short-term heat stress on the tonoplast membrane of lamb's lettuce (Valerianella locusta (L.) Betcke) mesophyll vacuoles has been investigated. The maintainance of a proton concentration difference (δpH) across the tonoplast membrane served as a criterion for the integrity of the vacuoles. After heat treatment, δpH was measured at room temperature using the fluorescent amine, 9-aminoacridine. It was found with this method that thermal damage to isolated vacuoles mainly occurred in the temperature range above 50°C. Compared with this results, the photosynthetic functions of isolated lettuce protoplasts proved to be markedly more thermolabile, e.g. photosynthetic CO₂ fixation and light-induced chlorophyll fluorescence were drastically reduced at temperatures between 40° and 50°C. Heating of whole leaves and protoplasts and subsequent isolation of vacuoles showed that tonoplast-membrane integrity is not affected by heat stress in situ up to 45°C. Measurement of 9-aminoacridine fluorescence in protoplasts, which allowed conclusions to be drawn regarding the integrity of the tonoplast membrane in its natural cytoplasmic environment, revealed that heat treatment up to 55°C did not significantly affect vacuolar compartmentation. The data provide evidence that the tonoplast membrane is relatively heat stable compared with photosynthetic membranes.