Effects of oxygen deficiency on survival and glycogen content of Chironomus anthracinus (Diptera,Chironomidae) under laboratory and field conditions

Growth and glycogen content of Chironomus anthracinus in Lake Esrom, Denmark was examined during summer stratification in 1992 and 1993. Simultaneously, effects of oxygen deficiency on glycogen utilization and survival were experimentally studied. The population consisted of almost fullgrown 4th instar larvae in 1992 and 2nd and 3rd instar larvae in 1993. Growth rate and glycogen content changed as hypolimnetic oxygen deficiency increased. During a 1st phase of stratification dry weight and glycogen content increased (2nd and 3rd instars) or was almost constant (4th instar) but decreased significantly during the following 2nd phase. This change from growth to degrowth and utilization of endogenous glycogen reserves correlated with a change in the thickness of the microxic layer (<0.2 mg O₂ 1^–1) above the sediment surface. The layer increased from 2–3 m in phase 1 to 4–5 m in phase 2, and we suggest that this deteriorated the oxygen conditions and resulted in a change in larval energy metabolism from fully aerobic during the 1st phase to partly anaerobic in the 2nd phase. During the 2nd phase larval metabolism was estimated at less than 20% of normoxic rate. Experimental exposure of the larvae to anoxia indicated highly different survival of young larvae (2nd and 3rd instars) and older larvae (large 4th instars). The morality of young larvae was 50% after three days in anoxia at 10 °C, whereas only 25% of the older larvae had died after 3–4 weeks under similar conditions. Extending the treatment, however, resulted in increased death rate of the 4th instar larvae with only 10% surviving after seven weeks. The anaerobic metabolism of 4th instar larvae as estimated from glycogen degradation at 10 °C was 5% of normoxia in the interval from 0–5 days but 1.5% in the interval from 20–25 days. It is concluded that survival of C. anthracinus in anoxia is very limited, but traces of oxygen in the environment allowing for faint aerobic metabolism prolong the survival time of the larvae from a few days (2nd and 3rd instars) or a few weeks (4th instar) to probably 3–4 months.     

Glycolysis and the regulation of cryoprotectant synthesis in liver of the freeze tolerant wood frog

Summary Wood frogs,Rana sylvatica, were sampled after freezing at –4°C (a short time course from 2 to 70 min after the appearance of the freezing exotherm) and thawing (20 h at 3°C after 70 min of freezing) and the regulation of liver glycolysis with respect to cryoprotectant glucose synthesis was examined. Within 5 min of the initiation of freezing, cryoprotectant concentrations in blood and liver had begun to increase. This was correlated with a rapid rise in the levels of hexose monophosphates in liver, including a 2.5 fold increase in glucose-6-P and 10 fold rise in fructose-6-P contents within the first 5 min post-exotherm. Contents of fructose-1,6-P₂, fructose-2,6-P₂, triose phosphates, P-enolpyruvate, and pyruvate did not significantly change over the course of freezing. Thawing sharply reduced the levels of hexose monophosphates in liver but raised P-enolpyruvate content by 2.3 fold. Changes in the contents of glycolytic intermediates over the freeze/thaw course are consistent with an inhibitory block of glycolysis at phosphofructokinase during freezing in order to facilitate a rapid glycogenolysis and production of cryoprotectant; during thawing, however, glycolysis appears to be inhibited at the level of pyruvate kinase.Possible regulatory control of cryoprotectant synthesis by covalent modification of liver glycolytic enzymes was examined. Glycogenolysis during freezing was facilitated by an increase in the percentage of glycogen phosphorylase in the activea (phosphorylated) form and also by an increase in the total amount (a+b) of enzyme expressed. For phosphofructokinase, kinetic changes as a result of freezing included a 40% reduction inK _m for fructose-6-P, a 60% decrease inK _a for fructose-2,6-P₂, and a 2 fold increase in I₅₀ for ATP. These changes imply a freezing-induced covalent modification of the enzyme but are not, apparently, the factors responsible for inhibition of glycolytic flux at the phosphofructokinase locus during glucose synthesis. Kinetic parameters of pyruvate kinase were not altered over the freeze/thaw course.     

Nutrient uptake and utilization can be used to select viable day 7 bovine blastocysts after cryopreservation

The ability of individual bovine blastocysts to survive freezing and thawing procedures was assessed by measuring glucose and pyruvate uptake and lactate production immediately before and after cryopreservation. Using glucose and pyruvate uptake and lactate production it was not possible to determine, prior to freezing, which blastocysts would be viable after thawing. However, in the 5 hr immediately after thawing, those blastocysts which expanded their blastocoel had significantly greater glucose and pyruvate uptake and lactate production (P < 0.01) than those embryos which failed to develop after a 14 hr overnight incubation. Interestingly, after thawing, two distinct populations of blastocysts existed with respect to glucose uptake and lactate production, indicating that it is possible to identify those blastocysts immediately after thawing which will reexpand. In contrast, there was a considerable degree of overlap in pyruvate uptakes between the viable and nonviable groups of embryos, indicating that this parameter could not be used to select viable embryos after thawing. There was an increase in the calculated oxidation of carbohydrates after thawing, consistent with a partial uncoupling of the inner mitochondrial membrane. In conclusion, glucose uptake and lactate production can be used to select prospectively viable blastocysts immediately after thawing, indicating that glycolysis is a major energy-generating pathway for the embryo at this time. © 1996 Wiley-Liss, Inc.     

Transmembrane ion distribution during recovery from freezing in the woolly bear caterpillar Pyrrharctia isabella (Lepidoptera: Arctiidae)

During extracellular freezing, solutes in the haemolymph are concentrated, resulting in osmotic dehydration of the cells, which must be reversed upon thawing. Here, we used freeze tolerant Pyrrharctia isabella (Lepidoptera: Arctiidae) larvae to examine the processes of ion redistribution after thawing. To investigate the effect of the intensity of cold exposure on ion redistribution after thawing, we exposed caterpillars to −14 °C, −20 °C or −30 °C for 35 h. To investigate the effect of duration of cold exposure on ion redistribution after thawing, we exposed the caterpillars to −14 °C for up to 6 weeks while sampling several time points. The concentrations of Na⁺, K⁺, Mg²⁺ and Ca²⁺ were measured after thawing in the haemolymph, fat body, muscle, midgut tissue and hindgut tissue. Being frozen for long durations (>3 weeks) or at low temperatures (−30 °C) both result in 100% mortality, although different ions and tissues appear to be affected by each treatment. Both water distribution and ion content changes were detected after thawing, with the largest effects seen in the fat body and midgut tissue. Magnesium homeostasis appears to be vital for post-freeze survival in these larvae. The movement of ions during thawing lagged behind the movement of water, and ion homeostasis was not restored within the same time frame as water homeostasis. Failure to regain ion homeostasis after thawing is therefore implicated in mortality of freeze tolerant insects.     

Energy metabolism of Chironomus anthracinus (Diptera: Chironomidae) from the profundal zone of Lake Esrom,Denmark, as a function of body size,temperature and oxygen concentration

The profundal zone of Lake Esrom, Denmark has a dense population of Chironomus anthracinus, which survives 2–4 months of oxygen depletion each summer during stratification. The metabolism of 3rd and 4th instar larvae was examined in regard to variation in biomass and temperature. Respiration at air saturation was described by a curvilinear multiple regression relating oxygen consumption to individual AFDW and temperature. At 10 °C and varying oxygen regimes the O₂ consumption and CO₂ production of 4th instar larvae were almost unaltered from saturation to about 3 mg O₂ l^–1, but decreased steeply below this level. The respiratory quotient increased from 0.82 at saturation to about 3.4 at oxygen concentrations near 0.5 mg O₂ l^–1. This implied a shift from aerobic to partially anaerobic metabolism. At 0.5 mg O₂ l^–1 the total energy production equalled 20% of the rate at saturation of which more than one third was accounted for by anaerobic degradation of glycogen. This corresponded to a daily loss of 12 µg mg AFDW^–1 or approximately 5% of the body reserves. At unchanged metabolic rate the glycogen store would last three weeks, but long term oxygen deficiency causes a further suppression of the energy metabolism in C. anthracinus.     

Activation of mitogen-activated protein kinases during natural freezing and thawing in the wood frog

The responses of mitogen-activated protein kinase (MAPK) family members, including ERK (extracellular signal-regulated kinase), JNK (c-Jun NH₂-terminal kinase), and p38, in the metabolic responses to whole animal freezing (up to 24 h frozen at –2.5°C) and thawing (up to 4 h at 5°C after a 12 h freeze) were examined in four organs (liver, kidney, heart, brain) of the freeze-tolerant wood frog Rana sylvatica. Levels of the active phosphorylated form of p38 increased within 20 min as an early response to freezing in liver and kidney but rose later (after 12 h) in heart. Both JNK and p38 were activated during thawing in liver, kidney and heart with temporally-distinct patterns in each organ. The only MAPK response to freeze/thaw in frog brain was a transient elevation of p38 after 90 min thawing. ERK activity did not respond to freeze/thaw in any organ. The levels of c-Fos increased during freezing in kidney and brain whereas c-Jun was unaffected by freeze/thaw. Organ-specific responses by MAPKs, particularly p38, suggest that these may have roles in regulating metabolic or gene expression responses that may be adaptive in dealing with freezing stress or metabolic recovery during thawing.     

Metabolic responses in two species of crayfish (Parastacus defossus and Parastacus brasiliensis) to post-hypoxia recovery

The period of post-hypoxia recovery is essential for the rapid replenishment of energy reserves and for the removal of metabolic end products formed during hypoxia. Periods of post-hypoxia recovery were analyzed in two crayfish species, where Parastacus defossus is a fossorial species, and Parastacus brasiliensis lives in lotic environments with higher oxygen levels. After 4 h of hypoxia (2 mg O(2)/L), groups of animals were placed in tanks with oxygenated water and were then removed at intervals of 1, 3, 6, and 9 h. Hemolymph and tissues (hepatopancreas, muscle, and anterior and posterior gills) were extracted for the determination of glucose, lactate, free glucose, glycogen, total proteins, total lipids, arginine phosphate, and arginine. As expected, lactate levels were restored more rapidly in P. defossus than in P. brasiliensis. P. defossus restored its glycogen reserves of the hepatopancreas and muscle tissue. Free glucose was quickly restored in all tissues of both species. In relation to arginine phosphate reserves, P. defossus showed a greater ability to restore this metabolite in the hepatopancreas. Both species recovered their arginine phosphate reserves, but they also used this metabolite in longer periods of recovery. Mainly in P. brasiliensis the reserves of total lipids seem to be an important source of energy during the recovery period. The animals developed various metabolic strategies to post-hypoxia recovery, mainly P. defossus which restored its reserves more completely and more rapidly than did P. brasiliensis.     

Freeze-preservation of buds from Scots pine trees

A procedure has been developed for freeze-preservation of buds of the Scots pine (Pinus sylvestris L.). Instead of liquid nitrogen, cold storage in –80°C was used. The partly dormant material used in the experiments was obtained directly from a natural stand in Northern Finland and no prefreezing or cryoprotectants for preconditioning were used. Cooling velocity was 1°C/min up to a terminal freezing temperature of –39°C, after which the buds were immersed in liquid nitrogen at –196°C for 10 minutes. The material was then transferred to a deepfreezer at –80°C and stored up to 6 months. After rapid thawing, the buds were sterilized and their viability was tested by FDA staining and by culturing meristems on 1/2 MS medium for at least two weeks. All the freezing experiments were performed during March and April. The best survival of buds (90–100%) was achieved at the beginning of April, after which a pronounced decline in survival occurred obviously due to a rise in the water content of the buds.     

Anaerobic metabolism during activity in lizards

Summary A new technique developed for the determination of total lactate production in small animals was used to evaluate the role of anaerobiosis during activity at different temperatures in lizards. Measurements on six species of small lizards indicate little interspecific variation or thermal effect in resting lactate levels (0.35 mg lactate/g body weight) or maximal lactate levels achieved at exhaustion (1.4 mg lactate/g). Normally activeAnolis in captivity had a lactate content of 0.5 mg lactate/g. Rates of lactate formation were most rapid during the first 30 sec of activity and had a low thermal dependence (Q₁₀=1.1–1.3 above 20 °C). The lactate formed during activity persists for long periods; e.g., for 30 to 60 min between 20 and 37 °C inAnolis carolinensis (Fig. 1). Recovery rate generally increases with temperature. Muscle lactate concentrations peak at the end of activity, but liver and blood lactate are not maximal until 10 and 30 min, respectively, after activity (Fig. 2). The decrease in the blood lactate is shown to be a poor estimator of total recovery. An estimated 80–90% of the total energy utilized during initial vigorous activity comes from anaerobic sources. Because of its low thermal dependence, anaerobiosis permits high levels of activity in lizards at all body temperatures without requiring high levels of aerobic resting metabolism.Support for this research was provided by a Miller Post-doctoral Research Felowship to AFB and NSF Grant GB 22642 to PL.     

Tumor Cells Switch to Mitochondrial Oxidative Phosphorylation under Radiation via mTOR-Mediated Hexokinase II Inhibition - A Warburg-Reversing Effect

A unique feature of cancer cells is to convert glucose into lactate to produce cellular energy, even under the presence of oxygen. Called aerobic glycolysis [The Warburg Effect] it has been extensively studied and the concept of aerobic glycolysis in tumor cells is generally accepted. However, it is not clear if aerobic glycolysis in tumor cells is fixed, or can be reversed, especially under therapeutic stress conditions. Here, we report that mTOR, a critical regulator in cell proliferation, can be relocated to mitochondria, and as a result, enhances oxidative phosphorylation and reduces glycolysis. Three tumor cell lines (breast cancer MCF-7, colon cancer HCT116 and glioblastoma U87) showed a quick relocation of mTOR to mitochondria after irradiation with a single dose 5 Gy, which was companied with decreased lactate production, increased mitochondrial ATP generation and oxygen consumption. Inhibition of mTOR by rapamycin blocked radiation-induced mTOR mitochondrial relocation and the shift of glycolysis to mitochondrial respiration, and reduced the clonogenic survival. In irradiated cells, mTOR formed a complex with Hexokinase II [HK II], a key mitochondrial protein in regulation of glycolysis, causing reduced HK II enzymatic activity. These results support a novel mechanism by which tumor cells can quickly adapt to genotoxic conditions via mTOR-mediated reprogramming of bioenergetics from predominantly aerobic glycolysis to mitochondrial oxidative phosphorylation. Such a “waking-up” pathway for mitochondrial bioenergetics demonstrates a flexible feature in the energy metabolism of cancer cells, and may be required for additional cellular energy consumption for damage repair and survival. Thus, the reversible cellular energy metabolisms should be considered in blocking tumor metabolism and may be targeted to sensitize them in anti-cancer therapy.     

Anaerobiosis and acid-base status in marine invertebrates: a theoretical analysis of proton generation by anaerobic metabolism

Summary In animals, various organic acids are accumulated during hypoxia or anoxia as products of anaerobic energy metabolism. The diversity of such acids is largest in marine invertebrates where succinate, propionate, acetate, lactate, alanine, octopine, strombine, and alanopine, are produced mainly from glycogen and aspartate. The effect of these substances on the acid-base status was assessed by a theoretical analysis of the respective metabolic pathways. This resulted in a general rule which was applied to evaluate the proton balance of the reactions in energy metabolism: net changes in the number of carboxyl groups or changes in the degree of dissociation of other groups (e.g. phosphate or ammonia) determine the net amount of H⁺ ions released or bound by the substrates and the metabolic end products.For marine invertebrates the results of the analysis can be summarized as follows: In glycogenolysis one mol of protons per mol of end products is released during cytosolic glycolysis, independent of the type of metabolic end product (lactate, octopine, alanopine, strombine, or alanine). The same applies for mitochondrial production of propionate and acetate, whereas formation of succinate results in dissociation of two mol H⁺ per mol. Fermentation of aspartate, however, diminishes the amount of protons which is produced in the succinate-propionate pathway. Net metabolisation of Mg ATP^2– yields extra protons, whereas the cleavage of phosphagens (e.g. creatine phosphate, arginine phosphate) consumes protons.Additionally the break-down of energy-rich phosphates to inorganic phosphate has to be taken into account because of the shift of the intracellular buffer curve caused by changes of the respective effective pK values.     

Thermal biology of horseshoe crab embryos and larvae: A role for heat shock proteins

Eggs of the American horseshoe crab, Limulus polyphemus L., develop on sandy estuarine beaches during the spring and summer, and are potentially vulnerable to thermal stress during the 3-4 weeks of development to the first instar (trilobite) larval stage. In many marine taxa, heat shock (stress) proteins (Hsp's) help individuals acclimate to stresses by restoring the proper folding of cellular proteins whose shape has been altered by temperature shock or other forms of environmental stress. We examined the survival of embryos and first instar (trilobite) larvae following heat shock, and compared the levels of Hsp70 in heat shocked and control animals. Animals acclimated to 13 or 22 °C had close to 100% survival when heat shocked for 3 h at 35 or 40 °C, but exposure to 45 °C for 3 h was lethal. To study the effect of heat shock on Hsp70 production under environmentally realistic conditions, animals were acclimated to either 13 or 22 °C, heat-shocked at 35 °C for 3 h, and soluble proteins were extracted following 0, 2, 4, or 6 h recovery at 22 °C. The relative amounts of Hsp70 in horseshoe crab embryos and larvae were examined using SDS-PAGE and Western blotting. Relative to controls animals held at a constant temperature, there was a slight elevation of Hsp70 only among heat shocked trilobite larvae in the 6 h recovery treatment. Hsp70 levels did not differ significantly between control and heat shocked embryos. Horseshoe crabs have adapted to living in a thermally stressful environment by maintaining a high baseline (constitutive) level of cellular stress proteins such as Hsp70, rather than by synthesizing inducible Hsp's when stressful temperatures are encountered. This may be an effective strategy given that the heat shocks encountered by intertidal embryos and larvae occur regularly as a function of diurnal and tidal temperature changes.     

Improved cardiac function following hormonal therapy in brain dead pigs: relevance to organ donation

Deterioration of function in brain dead baboons is associated with depletion of both myocardial energy stores and certain circulating hormones, notably thyroxine, cortisol, and insulin. We have therefore investigated the effect of the administration of these three hormones to the brain dead pig; their value has been assessed on both the freshly excised and stored donor heart. Brain death was induced by ligation of the two arteries to the upper part of the body which arise from the aortic arch. Storage of selected hearts was by continuous hypothermic perfusion for 20 to 24 hr. Hearts were biopsied for estimation of adenosine triphosphate, creatine phosphate, lactate, and glycogen, and were subsequently functionally tested. Six groups of pigs were studied. Hearts were tested from control pigs which had not undergone brain death (A1), from brain dead pigs which had received intravenous fluid and inotropic support for 4 hr (B1), and from brain dead pigs which had in addition received 2 hr of hormonal therapy (thyroxine 2 micrograms cortisol 100 mg, and insulin 5-10 IU hourly) (C1). A further 3 groups (A2-C2) underwent management identical to A1-C1, but in addition the hearts were stored for 24 hr. Brain death in pigs was followed by a consumption of myocardial energy stores, despite anaerobic glycolysis; this was associated with reduced myocardial function. The administration of hormones to the brain dead pig led to some replenishment of myocardial energy and glycogen reserves and reduction in lactate, with associated improvement in hemodynamic function. A period of hypothermic perfusion storage appeared to reverse the anaerobic metabolism occurring in the heart in the nonhormonally treated brain dead animal, though not in the hormonally treated animal, and led to replenishment of glycogen reserves in nontreated animals. The observation that both better function and an increase in myocardial energy stores occurred in hormonally treated, stored hearts, even though perfusate lactate dehydrogenase rose to significantly higher levels during hypothermic perfusion storage, and tissue lactate levels remained high, suggests that thyroxine promotes both aerobic and anaerobic metabolism in brain dead animals.     

The cryopreservation of Chlorella

The temperature at which Chlorella 211/8h was grown determined the response to a subsequent stress of freezing to and thawing from-196°C. Cells cultured at 20°C were the most sensitive to freezing injury; at both higher and lower growth temperatures resistance to damage induced by freezing developed. At all culture temperatures examined the freezing tolerance varied with the age of culture.     

Accumulation of lactate by frozen painted turtles (Chrysemys picta) and its relationship to freeze tolerance

Hatchling painted turtles (Chrysemys picta) survived freezing at -2 degrees C for 4 d, few recovered from freezing lasting 6 d, and none survived being frozen for 8 d. Whole-body glucose and lactate were low in animals that had not been subjected to cold and ice but increased precipitously in animals that were frozen for 2 d. Both metabolites continued to increase, but at a somewhat lower rate, in animals frozen for 4, 6, or 8 d. The increase in whole-body lactate reflects a reliance by frozen hatchlings on anaerobiosis, whereas the increase in glucose presumably results from mobilization of glycogen reserves to support anaerobic metabolism. Mortality of frozen hatchlings is correlated with the increase in whole-body lactate. Factors that may contribute to the observed correlation include a compromised capacity for individual organs to cope with the lactic acidosis that accompanies anaerobic metabolism and organ-specific depletion of energy reserves. Individual organs must rely on buffering and glucose reserves available in situ because blood of frozen hatchlings does not circulate. Thus, buffer from the shell cannot be transported to other organs, lactate cannot be sequestered in the shell, and glucose mobilized from liver glycogen is not available to supplement glucose reserves of other tissues. This integrated suite of physiological disruptions may limit tolerance of freezing to conditions with little or no ecological relevance.     

Ontogenic changes in tolerance to hypoxia and energy metabolism of larval and juvenile Japanese flounder Paralichthys olivaceus

Changes in tolerances to hypoxia and sodium azide, an indicator of cellular respiration, and activities of various energy metabolism-related chemical components were studied in Japanese flounder Paralichthys olivaceus during its early life stages from 3.5 to 20.5 mm in total length (TL). They showed flexion stage around 10.4 mm TL. Lethal levels of hypoxia increased with growth from 3.5 to 8 mm total TL, and the levels remained high in larvae, until 10.4 mm TL, decreased significantly thereafter. The 50% lethal concentration of sodium azide temporarily increased at 4.5 mm TL, diminished drastically between 4.5 and 10.4 mm TL, and then increased again in post-flexion larvae. Cytochrome c oxidase activity was highest in larvae around flexion, at 10.4 mm TL, and subsequently decreased. In post-flexion larvae at 13.0 mm TL, lactate dehydrogenase (LDH) and creatine kinase activities increased; LDH activity decreased at the juvenile stage. The adenosine triphosphate content and energy charge in fish were consistently higher in the larval stage than in the juvenile stage. These results indicated that, from just before flexion to the post-flexion stage, the energy metabolism of larvae is higher due to activated aerobic and subsequent anaerobic metabolism for metamorphosis; as a consequence, hypoxia tolerance in fish is the lowest during the increase of aerobic metabolism just before and around flexion.     

Addition of superoxide dismutase mimics during cooling process prevents oxidative stress and improves semen quality parameters in frozen/thawed ram spermatozoa

High levels of reactive oxygen species (ROS), which may be related to reduced semen quality, are detected during semen cryopreservation in some species. The objectives of this study were to measure the oxidative stress during ram semen cryopreservation and to evaluate the effect of adding 2 antioxidant mimics of superoxide dismutase (Tempo and Tempol) during the cooling process on sperm motility, viability, acrosomal integrity, capacitation status, ROS levels, and lipid peroxidation in frozen and/or thawed ram spermatozoa. Measuring of ROS levels during the cooling process at 35, 25, 15, and 5 °C and after freezing and/or thawing showed a directly proportional increase (P < 0.05) when temperatures were lowering. Adding antioxidants at 10 °C confered a higher motility and sperm viability after cryopreservation in comparison with adding at 35 °C or at 35 °C/5 °C. After freezing and/or thawing, sperm motility was significantly higher (P < 0.05) in Tempo and Tempol 1 mM than that in control group. Percentage of capacitated spermatozoa was lower (P < 0.05) in Tempo and Tempol 1 mM in comparison with that in control group. In addition, ROS levels and lipid peroxidation in group Tempo 1 mM were lower (P < 0.05) than those in control group. These results demonstrate that ram spermatozoa are exposed to oxidative stress during the cooling process, specifically when maintained at 5 °C and that lipid peroxidation induced by high levels of ROS decreases sperm motility and induces premature sperm capacitation. In contrast, the addition of Tempo or Tempol at 0.5 to 1 mM during the cooling process (10 °C) protects ram spermatozoa from oxidative stress.     

Uniconazole-induced alleviation of freezing injury in relation to changes in hormonal balance, enzyme activities and lipid peroxidation in winter rape

Winter rape (Brassica napus L. cv. 601) seedlings were treated with 50 mg.l-1 of foliar-applied uniconazole and then exposed to freezing stress with a light/dark temperature regime of 2 °C/–3 °C for 5 days at the seedling stage. Stressed plants contained lower endogenous GA3 and IAA contents than the controls, while zeatin and ABA contents and ethylene levels were significantly increased. Uniconazole-treated plants had lower endogenous GA3 and IAA contents, and higher zeatin and ABA contents and ethylene levels. Leaf chlorophyll content and respiratory capacity of roots were reduced significantly after plants were subjected to freezing stress, and foliar sprays of uniconazole retarded the degradation of chlorophyll and increased respiratory capacity of roots. Uniconazole-induced freezing tolerance was accompanied by increased activities of various antioxidant enzymes, including superoxide dismutase, catalase and peroxidase. Foliar applications of uniconazole reduced electrolyte leakage and malondialdehyde accumulation caused by freezing stress, suggesting that uniconazole may have decreased freezing-induced lipid peroxidation and membrane damage.     

Changes in the carbohydrate metabolism of mitogenically stimulated human peripheral lymphocytes : II. Relative importance of glycolysis and oxidative phosphorylation on phytohaemagglutinin stimulation

The effect of inhibition of the oxidative phosphorylation of human blood lymphocytes in the presence and absence of phytohaemagglutinin has been investigated. It was found that the incorporation of inorganic phosphate into acid-soluble nucleotides is dependent on, though not a direct measure of oxidative phosphorylation. Optimal concentration for inhibition of oxidative phosphorylation with oligomycin and the uncoupler 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB) were determined. Under conditions of maximally inhibited P_i incorporation into acid-soluble nucleotides (80%) and maximally increased oxygen consumption and lactate production (4–5 times), the stimulatory effect of phytohaemagglutinin on several glycolytic parameters could still be observed. Therefore, stimulation of cellular processes by PHA is still possible when energy is provided by glycolysis only.