Blood amino acid compartmentation in mice bearing Lewis lung carcinoma.

Blood amino acid compartmentation between plasma and red blood cells was studied in mice bearing the Lewis lung carcinoma. The animals showed a change in compartmentation with an increase in the concentration of most amino acids in the plasmatic fraction with the exception of glycine, glutamine, aspartate, asparagine, and taurine. This work focuses on the importance of studying the distribution of amino acids in both fractions when performing studies concerning amino acid metabolism in tumor-bearing animals.

Acclimation of Respiratory O2 Uptake in Green Tissues of Field-Grown Native Species after Long-Term Exposure to Elevated Atmospheric CO2.

C3 and C4 plants were grown in open-top chambers in the field at two CO2 concentrations, normal ambient (ambient) and normal ambient + 340 [mu]LL-1 (elevated). Dark oxygen uptake was measured in leaves and stems using a liquid-phase Clark-type oxygen electrode. High CO2 treatment decreased dark oxygen uptake in stems of Scirpus olneyi (C3) and leaves of Lindera benzoin (C3) expressed on either a dry weight or area basis. Respiration of Spartina patens (C4) leaves was unaffected by CO2 treatment. Leaf dry weight per unit area was unchanged by CO2, but respiration per unit of carbon or per unit of nitrogen was decreased in the C3 species grown at high CO2. The component of respiration in stems of S. olneyi and leaves of L. benzoin primarily affected by long-term exposure to the elevated CO2 treatment was the activity of the cytochrome pathway. Elevated CO2 had no effect on activity and capacity of the alternative pathway in S. olneyi. The cytochrome c oxidase activity, assayed in a cell-free extract, was strongly decreased by growth at high CO2 in stems of S. olneyi but it was unaffected in S. patens leaves. The activity of cytochrome c oxidase and complex III extracted from mature leaves of L. benzoin was also decreased after one growing season of plant exposure to elevated CO2 concentration. These results show that in some C3 species respiration will be reduced when plants are grown in elevated atmospheric CO2. The possible physiological causes and implications of these effects are discussed.

The impairment of respiration by glycolysis in the Lewis lung carcinoma.

Isolated vegetative tumour cells from mice bearing the Lewis lung carcinoma showed low rates of basal respiration with both low oxygen uptake rates and cytochrome-c oxidase activity. The cells were affected by a marked Crabtree effect and a high rate of lactate production in the presence of 10 mM glucose. The glycolytic capacity of the tumour was also assessed through the measurement of the maximum activities for hexokinase, phosphofructokinase, pyruvate kinase and lactate dehydrogenase. These activities were similar to the ones found in other fast-growing, undifferentiated tumours. The concentration of fructose-2,6-bisphosphate in the tumour was 2,3 nmoles/g fresh tissue wt., a value which is of the same order of magnitude as that found in other types of highly glycolytic cells. It is concluded that the Lewis lung carcinoma follows the same pattern as other undifferentiated tumours with a high capacity for both glucose and amino acid utilization.

[Effect of the oxidation of glycine and Krebs cycle substrates on cytochrome activity and alternative pathways of mitochondria from leaves of Pisum sativum L]. / Efecto de la oxidación de glicina y de substratos del ciclo de Krebs en la actividad de las vías citocrómica y alternativa de mitocondrias de hojas de Pisum sativum L.

Isolated mitochondria from adult leaves of Pisum sativum had the capacity to oxidize simultaneously glycine and several substrates of the Krebs cycle (e.g. malate, succinate, citrate, 2-oxo-glutarate), either in the presence of ADP (state three) or in the absence of ADP (state four). The sensitivity of the mitochondrial respiration to inhibitors of the cytochrome (e.g. antimycin A) and the alternative (e.g. salicylhydroxamic acid, SHAM, and tetraethylthiuram disulfide, disulfiram) pathways varied depending on the substrate(s) being used. For instance, the rate of oxygen uptake resistant to antimycin A, which is an estimate of the capacity of the alternative pathway, varied depending on whether glycine was added or not to a medium with malate and succinate. The state four rate of oxygen consumption in the presence of malate or succinate was greatly stimulated by the addition of glycine, and vice versa. This stimulation was apparently mediated by the alternative pathway. The results suggest that part of the electron transport capacity (including the alternative pathway) of these mitochondria is specifically associated with glycine oxidation, and therefore with photo-respiration.

Effect of Oxygen on the Contribution of Respiration to the CO(2) Compensation Point in Wheat and Bean Leaves.

The CO(2) compensation point at 21% O(2) (Gamma(21)) and at 2% O(2) (Gamma(2)), and the rate of dark CO(2) efflux at 21% O(2) (R(n)) were measured in adult wheat (Triticum aestivum L, cv Gabo) leaves at the end of the night and after a period of photosynthesis of 5 h at 800 mubar CO(2). The values of Gamma(21) and R(n) significantly increased after the light period, due to the stimulation of respiration by carbohydrates. In contrast, Gamma(2) did not increase after the same period of photosynthesis, suggesting that the respiratory component of Gamma(2) was not stimulated by carbohydrates. In a different experiment, Gamma(21), Gamma(2), and R(n) were studied during the growth period of bean (Phaseolus vulgaris L, cv Hawkesbury Wonder) leaves. The values of Gamma(21) and R(n) were high in young leaves, and decreased rapidly in parallel during maturation. However, Gamma(2) presented relatively low values in growing bean leaves, and a model predicted that the observed values of Gamma(2) should have been considerably higher if their respiratory component was considered to be as large as that of Gamma(21). The results suggest that the rate of respiration in the light contributing to the CO(2) compensation point in wheat and bean leaves is smaller at low O(2) levels than at ambient levels.

Inhibition of photosynthesis by carbohydrates in wheat leaves.

The rate of net CO(2) assimilation of mature wheat (Triticum aestivum L.) leaves in ambient air (21% O(2), 340 microbars CO(2)) declined with time of illumination at temperatures lower than 25 degrees C, but not at higher temperatures, and the rate of decline increased when maintained in air with higher CO(2) concentration (700-825 microbars). In this latter case, the decline in the rate of net CO(2) assimilation also occurred at high temperatures. Stomatal conductance also declined with time in some cases and stomata became more sensitive to CO(2), but this was not the primary cause of the decrease in CO(2) assimilation because internal partial pressure of CO(2) remained constant. Treatments which reduced the rate of translocation (e.g. lower temperatures, chilling the base of the leaf) produced a marked decline in CO(2) assimilation of leaves in atmospheric and high CO(2) concentrations. The decreased net CO(2) assimilation was correlated with carbohydrate accumulation in each case, suggesting end product inhibition of photosynthesis. Analysis of CO(2) assimilation in high carbohydrate leaves as a function of intercellular CO(2) partial pressure showed reduction in the upper part of the curve. The initial slope of this curve, however, was not affected. Photosynthetic rates in the upper part of this curve generally recovered after a short period in darkness in which carbohydrates were removed from the leaf. The stimulation of net CO(2) assimilation by 2% O(2) (Warburg effect), and the apparent quantum yield, decreased after several hours of light.

The metabolic environment of cancer.

The tumor cell has a very distinctive metabolism. It acts as a metabolic trap for host nutrients thus taking vital compounds for the metabolism of the host. Depending on the particular tumor growing pattern, cancer cells use preferentially glucose or amino acids for their energetic or biosynthetic needs. Lipids, fatty acids in particular, can also be taken up by the tumor cell. In addition, it can also release some compounds into the host circulation which are not normally produced by the original cell before neoplastic transformation. Some of these compounds affect the metabolism of the host in an unfavorable way since they can oppose the host's metabolic responses, which sustain homeostasis. The final product is that the metabolic machinery of these cells allows them to grow continuously in an uncontrolled manner. The consequences of tumor invasion on the host's metabolism are varied. They have, however, one thing in common: the reduction of the metabolic efficiency of the host. Muscular protein depletion, increased gluconeogenesis, uncoupling of oxidative phosphorylation constitute the main metabolic responses of the host as a result of tumor invasion. The net result of all these metabolic changes is profound energy imbalance which normally ends with cachexia and, eventually, death.

Relationship between Photosynthesis and Respiration: The Effect of Carbohydrate Status on the Rate of CO(2) Production by Respiration in Darkened and Illuminated Wheat Leaves.

The rate of dark CO(2) efflux from mature wheat (Triticum aestivum cv Gabo) leaves at the end of the night is less than that found after a period of photosynthesis. After photosynthesis, the dark CO(2) efflux shows complex dependence on time and temperature. For about 30 minutes after darkening, CO(2) efflux includes a large component which can be abolished by transferring illuminated leaves to 3% O(2) and 330 microbar CO(2) before darkening. After 30 minutes of darkness, a relatively steady rate of CO(2) efflux was obtained. The temperature dependence of steady-state dark CO(2) efflux at the end of the night differs from that after a period of photosynthesis. The higher rate of dark CO(2) efflux following photosynthesis is correlated with accumulated net CO(2) assimilation and with an increase in several carbohydrate fractions in the leaf. It is also correlated with an increase in the CO(2) compensation point in 21% O(2), and an increase in the light compensation point. The interactions between CO(2) efflux from carbohydrate oxidation and photorespiration are discussed. It is concluded that the rate of CO(2) efflux by respiration is comparable in darkened and illuminated wheat leaves.

Cyanide-resistant respiration in photosynthetic organs of freshwater aquatic plants.

THE RATE AND SENSITIVITY TO INHIBITORS (KCN AND SALICYLHYDROXAMIC ACID[SHAM]) OF RESPIRATORY OXYGEN UPTAKE HAS BEEN INVESTIGATED IN PHOTOSYNTHETIC ORGANS OF SEVERAL FRESHWATER AQUATIC PLANT SPECIES: six angiosperms, two bryophytes, and an alga. The oxygen uptake rates on a dry weight basis of angiosperm leaves were generally higher than those of the corresponding stems. Leaves also had a higher chlorophyll content than stems. Respiration of leaves and stems of aquatic angiosperms was generally cyanide-resistant, the percentage of resistance being higher than 50% with very few exceptions. The cyanide resistance of respiration of whole shoots of two aquatic bryophytes and an alga was lower and ranged between 25 and 50%. These results suggested that the photosynthetic tissues of aquatic plants have a considerable alternative pathway capacity. The angiosperm leaves generally showed the largest alternative path capacity. In all cases, the respiration rate of the aquatic plants studied was inhibited by SHAM alone by about 13 to 31%. These results were used for calculating the actual activities of the cytochrome and alternative pathways. These activities were generally higher in the leaves of angiosperms. The basal oxygen uptake rate of Myriophyllum spicatum leaves was not stimulated by sucrose, malate or glycine in the absence of the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP), but was greatly increased by CCCP, either in the presence or in the absence of substrates. These results suggest that respiration was limited by the adenylate system, and not by substrate availability. The increase in the respiratory rate by CCCP was due to a large increase in the activities of both the cytochrome and alternative pathways. The respiration rate of M. spicatum leaves in the presence of substrates was little inhibited by SHAM alone, but the SHAM-resistant rate (that is, the cytochrome path) was greatly stimulated by the further addition of CCCP. Similarly, the cyanide-resistant rate of O(2) uptake was also increased by the uncoupler.

Effect of photosynthesis and carbohydrate status on respiratory rates and the involvement of the alternative pathway in leaf respiration.

In spinach (Spinacia oleracea Hybrid 102 [New World seeds]) and wheat (Triticum aestivum L. cv Gabo) leaves, O(2) uptake rates in the dark were faster after the plants had been allowed to photosynthesize for a period of several hours. Alternative path activity also increased following a period of photosynthesis in these leaves. No such effects were observed with isolated mitochondria. In spinach and wheat leaves, the level of fructose plus glucose decreased during a period of darkness. In pea (Pisum sativum cv Alaska) leaves, the level of these sugars did not vary significantly during the day, and respiratory rates were also constant. In slices cut from wheat leaves harvested at the end of the night, addition of sugars increased the rate of respiration and engaged the previously latent alternative oxidase. In pea leaves, O(2) uptake in the first few minutes following illumination was faster than that observed before illumination, but declined during the next 15 to 20 minutes. Adding the alternative oxidase inhibitor salicylhydroxamic acid, or imposing high bicarbonate concentrations during the period of photosynthesis, prevented the rise in O(2) uptake rate during the immediate post illumination period.We conclude that the level of respiratory substrate in leaves determines their rate of O(2) uptake, and the degree to which the alternative path contributes to that O(2) uptake.

Effects of temperature, oxygen concentration, leaf age and seasonal variations on the CO2 compensation point of Lolium perenne L. : Comparison with a mathematical model including non-photorespiratory CO2 production in the light.

Various factors affect the CO2 compensation point of detached leaves of Lolium perenne L. These include oxygen concentration, temperature, leaf age, and season (spring and summer). Analysis of the results using the model of G.D. Farquhar, S. von Caemmerer and J.A. Berry (1980) Planta 149, 78-90, indicates that some of the CO2 evolved by leaves in the light is derived from sources other than photorespiration. It is suggested that the operation of the tricarboxylic acid in the light can account for most of this CO2.

Short-term effects of carbon dioxide on carnation callus cell respiration.

The addition of potassium bicarbonate to the electrode cuvette immediately stimulated the rate of dark O(2) uptake of photomixotrophic and heterotrophic carnation (Dianthus caryophyllus L.) callus, of Elodea canadensis (Michx) leaves, and of other plant tissues. This phenomenon occurred at pH values lower than 7.2 to 7.8, and the stimulation depended on the concentration of gaseous CO(2) in the solution. These stimulatory responses lasted several minutes and then decreased, but additional bicarbonate or gaseous CO(2) again stimulated respiration, suggesting a reversible effect. Carbonic anhydrase in the solution increased the stimulatory effect of potassium bicarbonate. The CO(2)/bicarbonate dependent stimulation of respiration did not occur in animal tissues such as rat diaphragm and isolated hepatocytes, and was inhibited by salicylhydroxamic acid in carnation callus cells and E. canadensis leaves. This suggested that the alternative oxidase was engaged during the stimulation in plant tissues. The cytochrome pathway was severely inhibited by CO(2)/bicarbonate either in the absence or in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone. The activity of cytochrome c oxidase of callus tissue homogenates was also inhibited by CO(2)/bicarbonate. The results suggested that high carbon dioxide levels (mainly free CO(2)) partially inhibited the cytochrome pathway (apparently at the oxidase level), and this block in electron transport elicited a large transient engagement of the alternative oxidase when present uninhibited.

Amino acid metabolism in tumour-bearing mice.

Mice bearing the Lewis lung carcinoma showed a high tumour glutaminase activity and significantly higher concentrations of most amino acids than in both the liver and the skeletal muscle of the host. Tumour tissue slices showed a marked preference for glutamine, especially for oxidation of its skeleton to CO2. It is proposed that the metabolism of this particular carcinoma is focused on amino acid degradation, glutamine being its preferred substrate.

Leaf Photosynthesis and Respiration of High CO(2)-Grown Tobacco Plants Selected for Survival under CO(2) Compensation Point Conditions.

Four self-pollinated, doubled-haploid tobacco, (Nicotiana tabacum L.) lines (SP422, SP432, SP435, and SP451), selected as haploids by survival in a low CO(2) atmosphere, and the parental cv Wisconsin-38 were grown from seed in a growth room kept at high CO(2) levels (600-700 parts per million). The selected plants were much larger (especially SP422, SP432, and SP451) than Wisconsin-38 nine weeks after planting. The specific leaf dry weight and the carbon (but not nitrogen and sulfur) content per unit area were also higher in the selected plants. However, the chlorophyll, carotenoid, and alkaloid contents and the chlorophyll a/b ratio varied little. The net CO(2) assimilation rate per unit area measured in the growth room at high CO(2) was not higher in the selected plants. The CO(2) assimilation rate versus intercellular CO(2) curve and the CO(2) compensation point showed no substantial differences among the different lines, even though these plants were selected for survival under CO(2) compensation point conditions. Adult leaf respiration rates were similar when expressed per unit area but were lower in the selected lines when expressed per unit dry weight. Leaf respiration rates were negatively correlated with specific leaf dry weight and with the carbon content per unit area and were positively correlated with nitrogen and sulfur content of the dry matter. The alternative pathway was not involved in respiration in the dark in these leaves. The better carbon economy of tobacco lines selected for low CO(2) survival was not apparently related to an improvement of photosynthesis rate but could be related, at least partially, to a significantly reduced respiration (mainly cytochrome pathway) rate per unit carbon.

The respiratory behavior of Lewis carcinoma cells--existence of a cyanide-resistant respiration.

The respiratory O2 consumption of vegetative, non-necrotic, Lewis lung carcinoma cells was found to be very low compared with that of non-tumor tissues and was highly resistant to cyanide. However, the resistant rate was inhibited by salicylhydroxamic acid (SHAM) in either isolated cells or tissue fragments. In addition, this compound did not affect their cytochrome-c oxidase activity. The results support the existence of an alternative oxidase system that significantly contributes to oxygen uptake in Lewis carcinoma cells. To our knowledge, this is the first report showing significant SHAM-sensitivity of tumor respiration and perhaps of higher animal cell respiration.

Sulfide-Resistant Respiration in Leaves of Elodea canadensis Michx: Comparison with Cyanide-Resistant Respiration.

The rate of dark O(2) uptake of Elodea canadensis leaves was titrated with either cyanide or sulfide in the presence and in the absence of 5 millimolar salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase. The inhibition of O(2) uptake by SHAM alone was very small (3-6%), suggesting that actual respiration mainly occurred through the cytochrome pathway. O(2) uptake was slightly stimulated by cyanide at concentrations of 50 micromolar or higher, but in the presence of SHAM respiration was strongly suppressed. The effects of sulfide on O(2) uptake were similar to those of cyanide, except that the percent stimulation of O(2) uptake by sulfide alone was somewhat higher than that of cyanide. However, the estimates of the capacity of the alternative pathway were similar with both inhibitors. Another difference is that maximal inhibition of respiration in the presence of SHAM was observed with lower concentrations of sulfide (50 micromolar) than cyanide (250 micromolar). The results suggest that sulfide can be used as a suitable inhibitor of cytochrome c oxidase in studies with intact plant tissues, and that sulfide does not apparently inhibit the alternative oxidase.

Regulation of nonphosphorylating electron transport pathways in soybean cotyledon mitochondria and its implications for fat metabolism.

The respiration of mitochondria isolated from germinating soybean cotyledons was strongly resistant to antimycin and KCN. This oxygen uptake was not related to lipoxygenase which was not detectable in purified mitochondria. The antimycin-resistant rate of O(2) uptake was greatest with succinate as substrate and least with exogenous NADH. Succinate was the only single substrate whose oxidation was inhibited by salicyl hydroxamic acid alone, indicating engagement of the alternative oxidase. Concurrent oxidation of two or three substrates led to greater involvement of the alternative oxidase. Despite substantial rotenone-resistant O(2) uptake with NAD-linked substrates, respiratory control was observed in the presence of antimycin, indicating restriction of electron flow through complex I. Addition of succinate to mitochondria oxidizing NAD-linked substrates in state four stimulated O(2) uptake substantially, largely by engaging the alternative oxidase. We suggest that these properties of soybean cotyledon mitochondria would enable succinate received from the glyoxysome during lipid metabolism to be rapidly oxidized, even under a high cytosolic energy charge.