adenylate energy charge

Atlantic salmon (Sulmo s&r), mean weight 5.1 kg, were transported live for 1.5 h by a well-boat (fish density 125 kg II-~) from the seacage to a fish processing plant and then kept in the well-boat for 4 h prior to slaughter. Anaerobic white muscle activity due to handling stress during fish loading at the cage, after shipment immediately before slaughter, and after the fish had passed the slaughter line, was evaluated using high-energy phosphates and IMP, the [ATPLIMP] ratio, adenylate energy charge together with pH and redox potential measured directly in the muscle. Water quality parameters, pH, salinity, temperature, dissolved oxygen, carbon dioxide, total carbonate carbon, total alkalinity, ammonia and ammonium were monitored at the cage, during shipment, and in the carbon dioxide anaesthesia tank during commercial fish slaughter. No dramatic effects of handling stress were found, indicating that transport and slaughtering did not have an adverse effect on flesh quality. The results were explained by the ability of the well-boat to maintain good seawater quality during transport, to a quick bulk netting of the fish from well-boat to the slaughter line and to an efficiently run carbon dioxide anaesthesia-tank that minimised struggling prior to killing.

High concentrations of Se in soil might have negative effects on microorganisms. For this reason, the effect of organic substrate addition (glucose+maize straw) on Se volatilisation in relation to changes in microbial biomass and activity indices was investigated using an artificially Se-contaminated soil. Microbial biomass N was reduced on average by more than 50% after substrate addition, but adenylate energy charge (AEC) and metabolic quotient qCO 2 were both increased. The Se content decreased by nearly 30% only with the addition of the organic substrate at 25°C. No significant Se loss occurred without substrate at 25°C or with substrate at 5°C. In the two treatments with substrate addition, the substratederived CO 2 evolution was about 30% lower with Se addition than without. In contrast, Se had no effect on any of the other soil microbial indices analysed, i.e. microbial biomass C, microbial biomass N, adenosine triphosphate (ATP), AEC, ATP-to-microbial biomass C, and qCO 2 .

The objective of the present study was to establish the optimal combination of inhibitors of adenosine metabolism and nucleotide precursors resulting in long-term increase in endogenous adenosine concentration without adverse metabolic consequences in non-ischemic cardiomyocytes and endothelial cells. Cardiomyocytes and endothelial cells were isolated after collagenase digestion of the rat heart. Freshly isolated cardiac myocytes or cultured endothelial cells were incubated for up to 8 h with no inhibitors or substrates or with various combinations of adenosine deaminase inhibitor: 5  erythro-9(2-hydroxy-3-nonyl)adenine (EHNA), adenosine kinase inhibitors: 10  5′-iodotubercidin (ITu) or 10  5′-aminoadenosine (AA) and nucleotide precursors: 100  adenine, 2.5 m ribose and 5 m inorganic phosphate. Nucleotide, nucleoside and base concentrations were evaluated at the end of the incubation by HPLC in cardiomyocyte or endothelial cells extracts and in incubation media. Adenosine content in cardiomyocyte suspension was enhanced after 3 h incubation in the presence of ITu+EHNA as compared to EHNA alone (2.8±0.2 v 0.9±0.2 nmol/mg protein, respectively). ATP decreased from an initial value of 22.7±0.7 nmol/mg protein to 18.9±0.7 in the presence of ITu+EHNA, while ATP was maintained at 21.8±0.7 nmol/mg protein with EHNA. With adenine+ITu+EHNA, the changes were similar to those observed with ITu+EHNA. However, with ribose+adenine+ITu+EHNA, ATP increased to 25.8±1.2 nmol/ mg protein and adenosine concentration was elevated to 3.9±0.3 nmol/mg protein. Similar results were observed if AA was used instead of ITu to inhibit adenosine kinase. All the changes were maintained after 8 h of incubation. Adenosine content was increased in endothelial cells incubated with ITu+EHNA to 3.1±0.4 nmol/mg protein as compared to 1.1±0.2 nmol/mg protein with EHNA alone after 3 h, while ATP decreased (18.1±1.1 v 22.0±1.4 nmol/mg protein with EHNA+ITu or EHNA, respectively). In the presence of adenine+ITu+EHNA, adenosine content increased after 3 h to 6.5±0.9 nmol/mg protein while ATP was elevated to 26.1±0.8 nmol/ mg protein. Additional presence of ribose was without effect. No changes in adenylate energy charge were observed in cardiomyocytes or endothelium under any conditions studied.

Initial rates of sugar uptake (zero-trans rates) are often measured by incubating yeast cells with radiolabeled sugars for 5 to 30 s and determining the radioactivity entering the cells. The yeast cells used are usually harvested from growth medium, washed, suspended in nutrient-free buffer, and stored on ice before they are assayed. With this method, the specific rates of zero-trans lactose uptake by Kluyveromyces lactis or recombinant Saccharomyces cerevisiae strains harvested from lactose fermentations were three-to eightfold lower than the specific rates of lactose consumption during fermentation. No significant extracellular ␤-galactosidase activity was detected. The ATP content and adenylate energy charge (EC) of the yeasts were relatively low before the [ 14 C]lactose uptake reactions were started. A short (1-to 7-min) preincubation of the yeasts with 10 to 30 mM glucose caused 1.5-to 5-fold increases in the specific rates of lactose uptake. These increases correlated with increases in EC (from 0.6 to 0.9) and ATP (from 4 to 8 mol ⅐ g dry yeast ؊1 ). Stimulation by glucose affected the transport V max values, with smaller increases in K m values. Similar observations were made for maltose transport, using a brewer's yeast. These findings suggest that the electrochemical proton potential that drives transport through sugar/H ؉ symports is significantly lower in the starved yeast suspensions used for zero-trans assays than in actively metabolizing cells. Zero-trans assays with such starved yeast preparations can produce results that seriously underestimate the capacity of sugar/H ؉ symports. A short exposure to glucose allows a closer approach to the sugar/H ؉ symport capacity of actively metabolizing cells.

Initial rates of sugar uptake (zero-trans rates) are often measured by incubating yeast cells with radiolabeled sugars for 5 to 30 s and determining the radioactivity entering the cells. The yeast cells used are usually harvested from growth medium, washed, suspended in nutrient-free buffer, and stored on ice before they are assayed. With this method, the specific rates of zero-trans lactose uptake by Kluyveromyces lactis or recombinant Saccharomyces cerevisiae strains harvested from lactose fermentations were three-to eightfold lower than the specific rates of lactose consumption during fermentation. No significant extracellular ␤-galactosidase activity was detected. The ATP content and adenylate energy charge (EC) of the yeasts were relatively low before the [ 14 C]lactose uptake reactions were started. A short (1-to 7-min) preincubation of the yeasts with 10 to 30 mM glucose caused 1.5-to 5-fold increases in the specific rates of lactose uptake. These increases correlated with increases in EC (from 0.6 to 0.9) and ATP (from 4 to 8 mol ⅐ g dry yeast ؊1). Stimulation by glucose affected the transport V max values, with smaller increases in K m values. Similar observations were made for maltose transport, using a brewer's yeast. These findings suggest that the electrochemical proton potential that drives transport through sugar/H ؉ symports is significantly lower in the starved yeast suspensions used for zero-trans assays than in actively metabolizing cells. Zero-trans assays with such starved yeast preparations can produce results that seriously underestimate the capacity of sugar/H ؉ symports. A short exposure to glucose allows a closer approach to the sugar/H ؉ symport capacity of actively metabolizing cells.

Diabetes mellitus is a degenerative disease that has deleterious effects on male reproductive function, possibly through an increase in oxidative stress. This study was conducted in order to clarify the mechanisms by which oxidative stress influences animal models for both type 1 (streptozotocin-treated rats, STZ) and type 2 (Goto-Kakizaki (GK) rats) diabetes. We determined the extent of lipid peroxidation, protein oxidation, lactate levels, adenine nucleotides, adenylate energy charge and the activity of glutathione peroxidase, glutathione reductase and lactate dehydrogenase, in isolated testicular cells of control and diabetic rats. We have also correlated these parameters with sperm count and motility. Sperm concentration and motility were decreased in STZtreated rats. ATP levels were lower in rats treated with STZ for 3 months, in contrast to GK and rats treated with STZ for 1 month, suggesting an adaptative response. STZ-treated rats showed increased lipid peroxidation after 1 week and 3 months of treatment. Glutathione reductase (G-red) activity was found to be higher in GK rats. Glutathione peroxidase activity was lower in GK and rats treated with STZ for 1 month, which is in accordance with the proposal of functional recovery in these animals. We conclude that hyperglycemia has an adverse effect in sperm concentration and motility via changes in energy production and free radical management. Furthermore, both animal models, particularly GK rats and rats treated with STZ for 1 month, present some metabolic adaptations, increasing the efficiency of mitochondrial ATP production, in order to circumvent the deleterious effects promoted by the disease.

It has been suggested that nitric oxide (NO) may contribute to ischemia-induced cell injury. However, the mechanisms underlying NO toxicity have not yet been fully elucidated. In the present study, we investigated the effect of NO on the level of endoplasmic reticulum (ER) calcium stores, on ER Ca 2+ pump activity, on protein synthesis, on concentrations of high-energy phosphates, and on gadd153 mRNA levels. Primary neuronal cells were exposed to the NO-donor (±)-S-Nitroso-N-acetylpenicillamine (SNAP) for 1 h, 2 h, 6 h or 24 h. The level of ER calcium stores was evaluated by measuring the increase in cytoplasmic calcium activity induced by exposing cells to thapsigargin, an irreversible inhibitor of ER Ca 2+ -ATPase; the activity of ER Ca 2+ -ATPase was determined by measuring a phosphorylated intermediate; SNAP-induced changes in gadd153 expression were evaluated by quantitative PCR; SNAP-induced changes in protein synthesis were investigated by measuring the incorporation of L-[4,5-3 H]leucine into proteins, and changes in the levels of ATP, ADP, AMP were measured by HPLC. Exposing cells to SNAP for 1 h to 2 h induced a marked depletion of ER calcium stores through an inhibition of ER Ca 2+ -ATPase (to 58% of control), and a concentration-dependent suppression of protein synthesis which was reversed in the presence of hemoglobin, suggesting NOrelated effects. ATP levels and adenylate energy charge were significantly decreased only when cells were exposed to the highest SNAP concentration for 6 h or 24 h, excluding significant effects of NO on the energy state of cells in the acute state, i.e. when ER calcium stores were already completely depleted and protein synthesis severely suppressed. In light of the regulatory role of ER calcium homeostasis in the control of protein synthesis, the results imply that the suppression of protein synthesis resulted from NO-induced inhibition of ER Ca 2+ -ATPase and depletion of ER calcium stores, and that NO-induced disturbances of energy metabolism are secondary to the effect of NO on ER calcium homeostasis. It is, therefore, concluded that ER calcium stores are a primary target of NO-toxicity.

It has been suggested that nitric oxide (NO) may contribute to ischemia-induced cell injury. However, the mechanisms underlying NO toxicity have not yet been fully elucidated. In the present study, we investigated the effect of NO on the level of endoplasmic reticulum (ER) calcium stores, on ER Ca 2+ pump activity, on protein synthesis, on concentrations of high-energy phosphates, and on gadd153 mRNA levels. Primary neuronal cells were exposed to the NO-donor (±)-S-Nitroso-N-acetylpenicillamine (SNAP) for 1 h, 2 h, 6 h or 24 h. The level of ER calcium stores was evaluated by measuring the increase in cytoplasmic calcium activity induced by exposing cells to thapsigargin, an irreversible inhibitor of ER Ca 2+ -ATPase; the activity of ER Ca 2+ -ATPase was determined by measuring a phosphorylated intermediate; SNAP-induced changes in gadd153 expression were evaluated by quantitative PCR; SNAP-induced changes in protein synthesis were investigated by measuring the incorporation of L-[4,5-3 H]leucine into proteins, and changes in the levels of ATP, ADP, AMP were measured by HPLC. Exposing cells to SNAP for 1 h to 2 h induced a marked depletion of ER calcium stores through an inhibition of ER Ca 2+ -ATPase (to 58% of control), and a concentration-dependent suppression of protein synthesis which was reversed in the presence of hemoglobin, suggesting NOrelated effects. ATP levels and adenylate energy charge were significantly decreased only when cells were exposed to the highest SNAP concentration for 6 h or 24 h, excluding significant effects of NO on the energy state of cells in the acute state, i.e. when ER calcium stores were already completely depleted and protein synthesis severely suppressed. In light of the regulatory role of ER calcium homeostasis in the control of protein synthesis, the results imply that the suppression of protein synthesis resulted from NO-induced inhibition of ER Ca 2+ -ATPase and depletion of ER calcium stores, and that NO-induced disturbances of energy metabolism are secondary to the effect of NO on ER calcium homeostasis. It is, therefore, concluded that ER calcium stores are a primary target of NO-toxicity.

Central infected zone tissue of soybean (Glycine max L. Merr.) nodules was fractionated into separate subcellular compartments using density gradient centrifugation in nonaqueous solvents to better understand how exposure to Ar:O 2 (80:20%, v/v) atmosphere affects C and N metabolism, and to explore a potential role for adenylates in regulating O 2 diffusion. When nodules were switched from air to Ar:O 2 , adenylate energy charge (AEC) in the plant cytosol rose from 0.63 6 0.02 to 0.73 6 0.02 within 7 min and to 0.80 6 0.01 by 60 min. In contrast, AEC of the mitochondrial compartment of this central zone tissue remained high (0.80 6 0.02 to 0.81 6 0.02) following Ar treatment while that of the bacteroid compartment was unchanged, at 0.73 6 0.02, after 7 min, but declined to 0.57 6 0.03 after 60 min. These results were consistent with a simulation model that predicted Ar:O 2 exposure would first reduce ATP demand for ammonia assimilation and rapidly increase cytosolic AEC, before the Ar:O 2-induced decline mediated by a decrease in nodule O 2 permeability reduces bacteroid AEC. The possibility that adenylates play a key, integrating role in regulating nodule permeability to oxygen diffusion is discussed.

Mathematical models were developed to test the likelihood that large cytosolic adenylate concentration gradients exist across the bacteria-infected cells of legume nodules. Previous studies hypothesized that this may be the case to account for the unusually low adenylate energy charge (AEC; 0.65) measured in the plant fraction of metabolically active nodules (M.M.

In legume nodules the [O 2 ] in the infected cells limits respiration and nitrogenase activity, becoming more severe if nodules are exposed to subambient O 2 levels. To identify the site of O 2 limitation, adenylate pools were measured in soybean (Glycine max) nodules that were frozen in liquid N 2 before being ground, lyophilized, sonicated, and separated on density gradients of nonaqueous solvents (heptane/tetrachloroethylene) to yield fractions enriched in bacteroid or plant components. In nodules maintained in air, the adenylate energy charge (AEC ‫؍‬ [ATP ؉ 0.5 ADP]/[ATP ؉ ADP ؉ AMP]) was lower in the plant compartment (0.65 ؎ 0.04) than in the bacteroids (0.76 ؎ 0.095), but did not change when the nodulated root system was exposed to 10% O 2. In contrast, 10% O 2 decreased the bacteroid AEC to 0.56 ؎ 0.06, leading to the conclusion that they are the primary site of O 2 limitation in nodules. To account for the low but unchanged AEC in the plant compartment and for the evidence that mitochondria are localized in O 2-enriched microenvironments adjacent to intercellular spaces, we propose that steep adenylate gradients may exist between the site of ATP synthesis (and ADP use) in the mitochondria and the extramitochondrial sites of ATP use (and ADP production) throughout the large, infected cells.

Two soils from temperate sites (UK; arable and grassland) were incubated aerobically at 0, 5, 15 or 258C for up to 23 days. During this period both soils were analysed for soil microbial biomass carbon (biomass C) and adenosine 5 ' triphosphate contents (ATP). Biomass C did not change signi®cantly in either soil at any temperature throughout, except during days 0 to 1 in the grassland soil. Soil ATP contents increased slowly throughout the 23 days of incubation, from 2.2 to a maximum of 3.1 nmol ATP g À1 soil in the arable soil (a 40% increase) and from 6.2 to a maximum of 11.2 nmol ATP g À1 soil in the grassland soil (an increase of 81%), both at 258C. Since biomass C did not change either with increasing temperature or increasing time of incubation, it was concluded that an increase in ATP was either due to an increase in adenylate energy charge or de novo synthesis of ATP, or both. During the incubation, biomass ATP concentrations ranged from about 5 to 12 mmol ATP g À1 biomass C but trends between biomass ATP and incubation temperatures were not very obvious until about day 13. On day 23, biomass ATP concentrations were positively and linearly related to temperature: (mmol ATP g À1 biomass C = 6.98 2 0.35 + 0.134 2 0.023 T 0 (r 2 = 0.77) with no signi®cant dierence in the slope between the grassland and arable soils. At 258C the biomass ATP concentration was 10.3 mmol g À1 biomass C, remarkably close to many other published values. It was concluded that, although the biomass increased its ATP concentration in response to increasing temperature, the increase was comparatively small. Also, at all temperatures tested, the biomass maintained its ATP concentration within the range commonly reported for microorganisms growing expontentially in vitro. This is despite the fact that the biomass normally exhibits other features more typical of a``resting'' or dormant population Ð a paradox which still is not resolved.

Samples from the bio-dynamic, bio-organic, and conventional trial, Therwil, Switzerland, were analyzed with the aim of determining the effects of organic land use management on the energy metabolism of the soil microbial biomass and on the fraction of microbial residues. The contents of adenylates, adenosine triphosphate (ATP), glucosamine, muramic acid, and galactosamine were significantly largest in the biodynamic organic farming (BYODIN) treatment and significantly lowest in the conventional farming treatment with inorganic fertilization (CONMIN). In contrast, the ergosterol-to-ATP ratio and fungal C-to-bacterial C ratios were significantly lowest in the BYODIN treatment and significantly largest in the CONMIN treatment. No clear treatment effects were observed for the ergosterol content and the adenylate energy charge (AEC), the ATP-to-microbial biomass C ratio and the ergosterol-to-fungal C ratio. Ergosterol, an indicator for saprotrophic fungal biomass, and fungal residues were significantly correlated. The microbial biomass carbon-to-nitrogen ratio showed a negative relationship with the AEC and strong positive relationships with the ratios ergosterol-to-microbial biomass C, ergosterol-to-ATP and fungal C-to-bacterial C. In conclusion, the long-term application of farmyard manure in combination with organic farming practices led to an increased accumulation of bacterial residues.

The first step in the separation of adenine nucleotides from different types of tissues or cells is deproteinization. Several sample preparation methods successfully used for a number of tissues or cells failed to work with erythrocytes. Use of strong acids or bases for deproteinization resulted in a low yield due to the hydrolysis of adenine nucleotides. Moreover, the neutralization of these acids or bases increased the ionic strength, resulting in broad and overlapping peaks. In neutral salt precipitation methods, saturated salts caused clogging of the capillaries. A new deproteinization procedure method was developed. The samples were deproteinized by heating of erythrocytes in boiling distilled water at 958C for 5 min. The denatured proteins were removed by centrifugation and membrane filtration. The adenine nucleotides were then separated using a polyacrylamide coated capillary. Depending on the type, diameter, length of the capillary and the voltage applied, an average of 16.50 min was sufficient for the separation of adenine nucleotides. All adenine nucleotides were clearly resolved and gave very sharp peaks. The amount of each adenine nucleotide was calculated from the areas under the peaks and AEC values were calculated using the integrator software. The AEC value of 0.9160.04 (n 5 10) obtained for healthy persons was in good agreement with the literature value of 0.85-0.95. These reported method for sample preparation and capillary electrophoresis is simple, fast and inexpensive compared to the previously reported sample preparation, HPLC and enzymatic methods for the determination of AEC.

The phosphate potential (A G'p) and the proton motive force (Ap) were recorded simultaneously in Streptococcus cremoris under different conditions. If thermodynamic equilibrium exists the ratio A G'p/A p gives the number of protons translocated by the ATPase per ATP hydrolyzed or synthesized. In resting cells ATP-synthesis could be energized by a valinomycin-induced potassiumdiffusion potential. In the subsequent phase ATP-hydrolysis occurred and the A G'p/A p approached a value close to 2. In growing and fermenting cells the A G'p/Ap ratio was considerably higher (almost 5). This indicates that under growing conditions the A G'p is not near thermodynamic equilibrium with A p and that the membrane bound ATPase of S. cremoris acts exclusively as an ATP hydrolase. During logarithmic growth the phosphate potential, the A p and the adenylate energy charge remain essentially constant at-4 6 0 m V ,-1 0 0 m V and 0.81, respectively. When the growth sustaining substrate is consumed a rapid dissipation of the phosphate potential to about-3 6 0 mV and of the A p to zero mV occurs.

The effects of hypoxia on contractile tension and on tissue adenylate pool content, nicotinamide adenine nucleotide, NAD, nicotinamide adenine dinucleotide phosphate, NADP, and creatine phosphate, CrP, were investigated in isolated, spontaneously beating, guinea pig atria. When two different degrees of hypoxia were induced by lowering oxygen tension from 95 % 02 (control) to 40 % (moderate hypoxia) and 20 % (severe hypoxia) for 30 min, contractile tension slowly decreased to 60 % and 40 % of control, respectively. In 40 % 02 hypoxie atria, ATP was not significantly decreased, AMP slightly increased, TAN (total adenylate nucleotides) and adenylate energy charge [(ATP + 0.5 ADP)/(ATP + ADP + AMP)] did not change and creatine phosphate was decreased down to 53 %. Hypoxic atria in 20 % Oz showed a significant decrease of 26 % in ATP, while ADP and AMP increased four and seven times, respectively. The adenylate energy-charge value was reduced from 0.93 to 0.70. Creatine phosphate decreased to below the analytical detection limit. Moderate hypoxia (40 % O2), which induced a significant decrease of contractile tension but only minor changes of energetic tissue metabolism, was further investigated 2, 5, and 10 min after low oxygen tension was applied. Two stages of variations were evident during 30 min of experimental hypoxia. Within the first 10 min, concomitantly with atrial tension decrease, ATP, NAD, NADP, ATP/AMP, ATP/ADP, and TAN decreased, CrP began to decrease, inosine and xanthine showed no significant change. During the following 20 min of hypoxia, all parameters returned to the control levels with the exception of creatine phosphate. Adenylate energy charge did not change. The electrophysiological analysis of atrial cells did not show any major change in action potential configuration and resting potential, during 40 % Oz hypoxia. The differences at metabolic level between moderate and more severe hypoxia suggest that the energetic state may be extremely unbalanced, in atrial tissue, as long as hypoxia is aggravated. Moreover, the time-course study, during 30 min of 40% 02, suggests that the early decrease of contractile tension does not depend on lowered energy availability, instead it might be, at least in part, a preventive measure to maintain energy balance in myocardial tissue to counteract hypoxic damage and, in this mechanism of defense, creatine phosphate shuttle seems to play a relevant role.

Legume seeds are heterotrophic and dependent on mitochondrial respiration. Due to the limited diffusional gas exchange, embryos grow in an environment of low oxygen. O2 levels within embryo tissues were measured using microsensors and are lowest in early stages and during night, up to 0.4% of atmospheric O2 concentration (1.1 μm). Embryo respiration was more strongly inhibited by low O2 during earlier than later stages. ATP content and adenylate energy charge were lowest in young embryos, whereas ethanol emission and alcohol dehydrogenase activity were high, indicating restricted ATP synthesis and fermentative metabolism. In vitro and in vivo experiments further revealed that embryo metabolism is O2 limited. During maturation, ATP levels increased and fermentative metabolism disappeared. This indicates that embryos become adapted to the low O2 and can adjust its energy state on a higher level. Embryos become green and photosynthetically active during differentiation. Photosynthetic ...

The role of oxygen and energy state in development and storage activity of cereal grains is an important issue, but has remained largely uninvestigated due to the lack of appropriate analytical methods. Metabolic pro®ling, bioluminescence-based in situ imaging of ATP, and oxygen-sensitive microsensors were combined here to investigate barley seed development. For the ®rst time temporal and spatial maps of O 2 and ATP distribution in cereal grains were determined and related to the differentiation pattern. Steep O 2 gradients were demonstrated and strongly hypoxic regions were detected within the caryopsis (<0.1% of atmospheric saturation). Growing lateral and peripheral regions of endosperm remained wellsupplied with O 2 due to pericarp photosynthesis. ATP distribution in the developing grain was coupled to endosperm differentiation. High ATP concentrations were associated with the local onset of starch storage within endosperm, while low ATP overlapped with the hypoxic regions. Temporally, the building of steep gradients in ATP coincided with overall elevating metabolite levels, speci®c changes in the metabolite pro®les (glycolysis and citrate cycle), and channelling of metabolic¯uxes towards storage (increase of starch accumulation rate). These ®ndings implicate an inhomogenous spatial arrangement of metabolic activity within the caryopsis. It is suggested that the local onset of starch storage is coupled with the accumulation of ATP and elevated metabolic activity. Thus, the ATP level re¯ects the metabolic state of storage tissue. On the basis of these ®ndings, a hypothetical model for the regulation of starch storage in barley seeds is proposed.

Patients undergoing hepatectomy have an increased susceptibility to infection. We therefore studied the energy metabolism of the polvmorphonuclear leucocvte (PMN), focusing on energy charge and function, especially superoxide anion (O2−) generation, in relation to the hepatic mitochondrial redox state. By labelling the PMN adenine nucleotide pool with radioactive adenine and by superoxide dismutase-inhibitahle reduction of ferricytochrome c, the energy charge and O2− production was measured in 18 patients with hepatoma (non-cirrhotic, seven; cirrhotic, 11) undergoing hepatectomy. Their arterial ketone body ratios (KBRs), reflecting the hepatic mitochondrial redox potential, were above 0.7 before operation. After surgery, the 18 patients were divided into two groups: group A. KBR &gt; 0.7, n = 10; and group B, KBR &lt; 0.7, n = 8. The energy charge and O2− release in group B decreased significantly from preoperative values (P &lt; 0.001 and P &lt; 0.01 respectively) and when compared...

Soil waterlogging responses were examined in three Spartina patens populations along a steep flooding gradient in coastal Louisiana. Root anatomy and physiological indicators of anaerobic metabolism were examined to identify and compare flooding responses in dune, swale and marsh populations, while soil physicochemical factors were measured to characterize the three habitats. Soil waterlogging increased along the gradient from dune to marsh habitats and was accompanied by increases in root porosity (aerenchyma). Aerenchyma in marsh roots was apparently insufficient to provide enough oxygen for aerobic respiratory demand, as indicated by high root alcohol dehydrogenase activities and low energy charge ratios. Patterns of root metabolic indicators suggest that dune and swale roots generally respired aerobically, while anaerobic metabolism was important in marsh roots. However, in each population, relatively greater soil waterlogging was accompanied by differences in enzyme activities leading to malate accumulation. In dune and swale roots under these circumstances, depressed adenylate energy charge ratios may have been the result of an absence of increased ethanol fermentation. These trends suggest that: 1) Aerenchyma formation was an important, albeit incomplete, long-term adaptation to the prevalent degree of soil waterlogging. 2) All populations adjusted root metabolism in response to a relative (short-term) increase in soil waterlogging,

The effects of varying the steady-state rate of non-cyclic photosynthetic electron transport on the leaf adenylate energy charge and the epoxidation state of the xanthophyll-cycle pigments were determined in leaves of cotton (Gossypium hirsutum L.) and the mangrove (Aegialitis annulata R.Br.). Different photosynthetic rates were obtained by varying the intercellular CO 2 concentration and/or the leaf temperature, and in some cases, by changing the leaf conductance to CO2 diffusion. Also determined were the effects of these treatments on the changes in the adenylate energy charge and the epoxidation state of the xanthophyll-cycle pigments that occur after darkening of the leaves. The leaf adenylate pool remained close to equilibrium with the adenylate kinase both in the light at steady state and during dark relaxation. The adenylate energy charge increased as the photosynthetic rate decreased and maximal levels were obtained when CO 2 assimilation and, therefore, non-cyclic electron flow were maximally inhibited. This implies that, in nature, photophosphorylation may provide energy needed for ion-pumping and biosynthetic and repair processes, even under stress conditions that severely restrict or prevent photosynthetic gas exchange. High levels of de-epoxidized violaxanthin in the light did not necessarily indicate or depend on a high adenylate energy charge. Dithiothreitol, an inhibitor of the violaxanthin de-epoxidase and ascorbate peroxidase, did not inhibit the adenylate energy charge in the light. Thus we conclude that coupled electron transport during inhibited CO2 fixation was not driven by a dithiothreitol-sensitive Mehler ascorbate-peroxidase reaction. The changes in the adenylate energy charge and xanthophyll re-epoxidation that fol

The present study is a first attempt at evaluating the effects of repeated disturbance, such as that caused by mechanical fishing systems, on the target species, Tapes philippinarum, intensively exploited in the Lagoon of Venice (North-East Italy). In particular, in free-access fishing grounds, detrimental effects of dredging may be suffered by under-sized clams, which are disturbed by fishing tools many times throughout their lives before they are collected. Similar mechanical stress conditions are experienced by T. philippinarum in licensed areas, where they are farmed in aquaculture conditions and undergo the harmful impact of hydraulic dredging. To evaluate the effects of short-term mechanical stress, under-sized clams were subjected to experimental shaking in the laboratory, and detrimental effects on their well-being were investigated by applying the biomarker approach. Changes in physiological, biochemical and behavioural responses were evaluated by determining scope for growth, adenylate energy charge, survival in air time, reburrowing time, and shell damage level. Responses highlighted general worsening in clam condition as mechanical stress increased. Among the various measurements, survival in air and reburrowing time appeared particularly suitable as indices, their responsiveness and applicability suggesting their use in assessing mechanical stress due to dredging in field conditions.

The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.

Adenylate (i.e. adenosine tri-(ATP), di-(ADP) and monophosphates (AMP)) and microbial biomass C data were collected over a wide range of sites including forest floor layers and forest, grassland and arable soils. Microbial biomass C was measured by fumigation extraction and adenylates after alkaline Na 3 PO 4 /DMSO/EDTA extraction and HPLC detection. Our aims were (1) to test whether the sum of adenylates is a better estimate for microbial biomass than the determination of ATP, (2) to compare our conversion values with those proposed by others, and (3) to analyse whether soil properties or land use form affect the relationships between ATP, adenylates and microbial biomass C. A close relationship was found between microbial biomass C and ATP ðr ¼ 0:96Þ; but also with the sum of adenylates ðr ¼ 0:96Þ within all appropriately conditioned soil samples ðn ¼ 112Þ: In the mineral soil ðn ¼ 98Þ; the geometric means of the ATP-to-microbial biomass C ratio and the adenylates-to-microbial biomass C ratio were 7.4 and 11.4 mmol g 21 , respectively. The mean ratios did not differ significantly between the different texture classes and land use forms. In the forest floor, the ATP-to-microbial biomass C ratio and the adenylates-tomicrobial biomass C ratio were both roughly two-thirds of those of the mineral soil. The average adenylate energy charge (AEC) of all soil samples was 0.79 and showed a strong negative relationship with the soil pH ðr ¼ 20:69Þ: However, the AEC is presumably only indirectly affected by the soil pH.

Storage and transport of litchi fruit at low temperature is widely practiced commercially. In this study, no pericarp browning was evident at out-turn on litchi fruit stored for 10 and 20 days at cold temperature of 3-5 • C, but fruit stored for 10 days at 3-5 • C gradually browned after 12 h of shelf time at ambient temperature of 25 • C, with the browning index (BI) being elevated to 2.5 at 24 h of shelf life. Furthermore, fruit stored for 30 days began to rot and had a decay incidence of about 30% after 24 h on the shelf. The temperature increment from 3-5 to 25 • C induced marked increases in activities of lipase, phospholipase D (PLD) and lipoxygenase (LOX). Litchi fruit stored for 10 days at 3-5 • C followed by the shelf time at 25 • C had lower activities of lipase, PLD and LOX, and also lower levels of membrane permeability, than did fruit stored for 20 and 30 days. Energy level of the pericarp tissue of cold-stored litchi fruit was similarly dependent on storage time at 3-5 • C plus shelf time at 25 • C. Adenosine triphosphate (ATP) content and adenylate energy charge (AEC) level in pericarp tissues decreased as cold storage progressed. However, ATP and adenosine diphosphate (ADP) contents, and the AEC levels, in cold-stored litchi fruit increased during shelf time at 25 • C, reaching a peak after 6 h, and then decreasing. Fruit stored for 30 days at 3-5 • C had much lower ATP content and AEC level than fruit stored for 10 and 20 days. Increased activities of lipase, PLD and LOX, and energy shortage in cold-stored pericarp tissues during subsequent shelf time at 25 • C suggest that the deterioration of membrane integrity and loss of compartmentation gives rise to accelerated browning and fruit quality deterioration.

The aim of this study was to examine the relationships between the concentrations of adenylate nucleotides (ATP, ADP, AMP), total nucleotide pool (TAN), adenylate energy charge (AEC) and 2,3-biphosphoglycerate (2,3-BPG) in the erythrocytes of young horses in the period of their rapid growth and development. The studies were conducted on 10 young Wielkopolska breed stallions for two years; Group A: 1-month-old, Group B: 3-month-old, Group C: 6-month-old, Group D: 1year-old, and Group E: 2-year-old. ATP, ADP and AMP concentrations in the erythrocytes were determined by HPLC. 2,3-BPG was determined using the Calbiochem USA kit. We observed that erythrocyte ATP, ADP, AMP and TAN concentrations significantly decreased with age (p < 0.001), while 2,3-BPG concentration and AEC values significantly increased with age. A significant correlation between the ATP and TAN concentrations was found within all the examined groups (p < 0.05). A significant correlation was also found between ATP concentration and AEC values (except 12 month-old stallions). Moreover, an age-dependent negative correlation between ATP and 2,3-BPG (r =-0.81) was found. The conclusion is that the adenylate energy charge and erythrocyte 2,3-BPG variables in the Wielkopolska stallions mainly depend on age, thus warranting the use of specific age-related reference values for this breed's foals.

Light, besides initiating primary photochemical processes, alters the redox state of soluble components in chloroplast. The present review attempts to cover the mechanism of reductive photoactivation of enzymes of photosynthetic carbon reduction cycle using key enzymes as examples. The reduced soluble components-ferredoxin, thioredoxin and NADPH, in turn, cause the reduction of disulphides to dithiols of chloroplastic enzymes. NADP-malate dehydrogenase is subject to activation by light through changes in NADPH/NADP. The key enzyme of C 4 photosynthesis-PEP carboxylase, though cytosolic, has been shown to be activated by disulphide/sulphhydryl interconversion by reductants generated in light through chloroplast electron transport flow. Pyruvate P i dikinase activity is controlled by the adenylate energy charge. It remains unclear how light controls the activation of cytosolic enzymes.

Ecosystems are under the pressure of 10 complex mixtures of contaminants whose effects 11 are not always simple to assess. Biomarkers, 12 acting as early warning signals of the presence 13 of potentially toxic xenobiotics, are useful tools 14 for assessing either exposure to, or the effects of 15 these compounds providing information about 16 the toxicant bioavailability. In fact, it has been 17 argued that a full understanding of ecotoxicolog-18 ical processes must consider an integrated multi-19 level approach, in which molecular impact is 20 related with higher-order biological consequences at the individual, population and community levels. Monitoring programs should make use of this tool to link contaminants and ecological responses fulfilling strategies like those launched by OSPAR (Commissions of Oslo and Paris) Convention on the protection of the marine environment of the NorthEast Atlantic and the International Council for the Exploration of the Sea (ICES). An overview of the work done in the past few years using biomarkers as in situ tools for pollution assessment in Portuguese coastal waters is presented as a contribution to the set up of a biomonitoring program for the Portuguese coastal zone. Considering the data set available the biomonitoring proposal should include the analysis of biomarkers and effects at individual levels. The aim of the program will include a spatial and temporal characterization of the biomarkers acetyl-cholinesterase, metallothioneins, DNA damage, adenylate energy charge and scope-for-growth levels. The investigation of the spatial variation of biomarkers is crucial to define sites for long term monitoring, which will be integrated with a chemical monitoring program. This framework will be a major contribution to the implementation of a national database for the use of biomarkers along the Portuguese coast.

The effect of long-term waste water irrigation (up to 80 years) on soil organic matter, soil microbial biomass and its activities was studied in two agricultural soils (Vertisols and Leptosols) irrigated for 25, 65 and 80 years respectively at Irrigation District 03 in the Valley of Mezquital near Mexico City. In the Vertisols, where larger amounts of water have been applied than in the Leptosols, total organic C (TOC) contents increased 2.5-fold after 80 years of irrigation. In the Leptosols, however, the degradability of the organic matter tended to increase with irrigation time. It appears that soil organic matter accumulation was not due to pollutants nor did microbial biomass:TOC ratios and qCO 2 values indicate a pollutant effect. Increases in soil microbial biomass C and activities were presumably due to the larger application of organic matter. However, changes in soil microbial communities occurred, as denitrification capacities increased greatly and adenylate energy charge (AEC) ratios were reduced after long-term irrigation. These changes were supposed to be due to the addition of surfactants, especially alkylbenzene sulfonates (effect on denitrification capacity) and the addition of sodium and salts (effect on AEC) through waste water irrigation. Heavy metals contained in the sewage do not appear to be affecting soil processes yet, due to their low availability. Detrimental effects on soil microbial communities can be expected, however, from further increases in pollutant concentrations due to prolonged application of untreated waste water or an increase in mobility due to higher mineralization rates.

Very little is known on European hake&#39;s reproductive biology and especially on biological characteristics of its sperm despite a growing interest in its aquaculture potential. This study reports, for the first time some hake sperm characteristics. After activation, the swimming phase lasts 3 min (8 min when activated with 50% sea water (SW) but lower initial velocity). The initial flagellar beat frequency (BF), velocity and percentage of motile cells decreased after 100 s. After 2 days at 4°C, the mean Adenylate Energy Charge (AEC) level was 0.71. Sperm stored at 4°C still showed motility after 9 days for 2 individuals. When cryopreserved, the motility recovery index of thawed spermatozoa ranged from 11.8% to 29.6%.

The first step in the separation of adenine nucleotides from different types of tissues or cells is deproteinization. Several sample preparation methods successfully used for a number of tissues or cells failed to work with erythrocytes. Use of strong acids or bases for deproteinization resulted in a low yield due to the hydrolysis of adenine nucleotides. Moreover, the neutralization of these acids or bases increased the ionic strength, resulting in broad and overlapping peaks. In neutral salt precipitation methods, saturated salts caused clogging of the capillaries. A new deproteinization procedure method was developed. The samples were deproteinized by heating of erythrocytes in boiling distilled water at 958C for 5 min. The denatured proteins were removed by centrifugation and membrane filtration. The adenine nucleotides were then separated using a polyacrylamide coated capillary. Depending on the type, diameter, length of the capillary and the voltage applied, an average of 16.50 min was sufficient for the separation of adenine nucleotides. All adenine nucleotides were clearly resolved and gave very sharp peaks. The amount of each adenine nucleotide was calculated from the areas under the peaks and AEC values were calculated using the integrator software. The AEC value of 0.9160.04 (n 5 10) obtained for healthy persons was in good agreement with the literature value of 0.85-0.95. These reported method for sample preparation and capillary electrophoresis is simple, fast and inexpensive compared to the previously reported sample preparation, HPLC and enzymatic methods for the determination of AEC.

In legume nodules the [O2] in the infected cells limits respiration and nitrogenase activity, becoming more severe if nodules are exposed to subambient O2 levels. To identify the site of O2 limitation, adenylate pools were measured in soybean (Glycine max) nodules that were frozen in liquid N2 before being ground, lyophilized, sonicated, and separated on density gradients of nonaqueous solvents (heptane/tetrachloroethylene) to yield fractions enriched in bacteroid or plant components. In nodules maintained in air, the adenylate energy charge (AEC = [ATP + 0.5 ADP]/[ATP + ADP + AMP]) was lower in the plant compartment (0.65 ± 0.04) than in the bacteroids (0.76 ± 0.095), but did not change when the nodulated root system was exposed to 10% O2. In contrast, 10% O2decreased the bacteroid AEC to 0.56 ± 0.06, leading to the conclusion that they are the primary site of O2 limitation in nodules. To account for the low but unchanged AEC in the plant compartment and for the evidence that mitoc...

Research was carried out to test the effectiveness of delayed controlled atmosphere (CA) storage (21 days at 0 8C in air before CA storage) on the occurrence of browning disorders and related physiological changes such as energy status and fatty acid metabolism in 'Conference' pears during long-term CA storage. After delayed CA, fruit were stored for up to 6 months in 3% CO 2 '/1% O 2 at 0 8C. Fruit stored without delay had a high incidence of flesh and core browning and cavities, whereas delayed CA-fruit had only a very low incidence of flesh browning and no incidence of core browning and cavities. No significant losses of flesh firmness, acidity, skin colour and soluble solids contents between treatments were detected. Fruit subjected to delayed CA had higher ethylene production and respiration activity during the first 2 months of storage and higher ATP concentrations in the fruit tissue. A higher adenylate energy charge was maintained during storage, indicating a higher energy status in delayed CA-fruit. Delayed CA-fruit showed higher contents of total and free fatty acids and polar lipids than fruit stored in CA without delay. The results suggest that by delaying CA storage conditions, 'Conference' pears can become adapted to CA during the early stages of the storage period, making them more resistant to browning disorders. Reasons for this may be found in the energy status of fruit and integrity of membranes which may play an important role in this adaptation process.

Initial rates of sugar uptake (zero-trans rates) are often measured by incubating yeast cells with radiolabeled sugars for 5 to 30 s and determining the radioactivity entering the cells. The yeast cells used are usually harvested from growth medium, washed, suspended in nutrient-free buffer, and stored on ice before they are assayed. With this method, the specific rates of zero-trans lactose uptake by Kluyveromyces lactis or recombinant Saccharomyces cerevisiae strains harvested from lactose fermentations were three- to eightfold lower than the specific rates of lactose consumption during fermentation. No significant extracellular β-galactosidase activity was detected. The ATP content and adenylate energy charge (EC) of the yeasts were relatively low before the [14C]lactose uptake reactions were started. A short (1- to 7-min) preincubation of the yeasts with 10 to 30 mM glucose caused 1.5- to 5-fold increases in the specific rates of lactose uptake. These increases correlated with i...

Light, besides initiating primary photochemical processes, alters the redox state of soluble components in chloroplast. The present review attempts to cover the mechanism of reductive photoactivation of enzymes of photosynthetic carbon reduction cycle using key enzymes as examples. The reduced soluble components-ferredoxin, thioredoxin and NADPH, in turn, cause the reduction of disulphides to dithiols of chloroplastic enzymes. NADP-malate dehydrogenase is subject to activation by light through changes in NADPH/NADP. The key enzyme of C 4 photosynthesis-PEP carboxylase, though cytosolic, has been shown to be activated by disulphide/sulphhydryl interconversion by reductants generated in light through chloroplast electron transport flow. Pyruvate P i dikinase activity is controlled by the adenylate energy charge. It remains unclear how light controls the activation of cytosolic enzymes.

Diabetes mellitus is a degenerative disease that has deleterious effects on male reproductive function, possibly through an increase in oxidative stress. This study was conducted in order to clarify the mechanisms by which oxidative stress influences animal models for both type 1 (streptozotocin-treated rats, STZ) and type 2 (Goto-Kakizaki (GK) rats) diabetes. We determined the extent of lipid peroxidation, protein oxidation, lactate levels, adenine nucleotides, adenylate energy charge and the activity of glutathione peroxidase, glutathione reductase and lactate dehydrogenase, in isolated testicular cells of control and diabetic rats. We have also correlated these parameters with sperm count and motility. Sperm concentration and motility were decreased in STZtreated rats. ATP levels were lower in rats treated with STZ for 3 months, in contrast to GK and rats treated with STZ for 1 month, suggesting an adaptative response. STZ-treated rats showed increased lipid peroxidation after 1 week and 3 months of treatment. Glutathione reductase (G-red) activity was found to be higher in GK rats. Glutathione peroxidase activity was lower in GK and rats treated with STZ for 1 month, which is in accordance with the proposal of functional recovery in these animals. We conclude that hyperglycemia has an adverse effect in sperm concentration and motility via changes in energy production and free radical management. Furthermore, both animal models, particularly GK rats and rats treated with STZ for 1 month, present some metabolic adaptations, increasing the efficiency of mitochondrial ATP production, in order to circumvent the deleterious effects promoted by the disease.

Adenine nucleotides—adenosine monophosphate, diphosphate, and triphosphate—are of utmost importance to all living organisms, where they play a critical role in the energy metabolism and are tied to allosteric regulation in various regulatory enzymes. Adenylate energy charge represents the precise relationship between the concentrations of adenosine monophosphate, diphosphate, and triphosphate and indicates the amount of metabolic energy available to an organism. The experimental conditions of adenylate extraction in freshwater amphipod Gammarus fossarum are reported here for the first time and are crucial for the qualitative and quantitative determination of adenylate nucleotides using efficient and sensitive ion-pair reverse phase LC. It was shown that amphipod calcified exoskeleton impeded the neutralization of homogenate. The highest adenylate yield was obtained by homogenization in perchloric acid and subsequent addition of potassium hydroxide and phosphate buffer to achieve a p...

Using an especially-designed facility, the Mode Stirred Reverberation Chamber, we exposed tomato plants (Lycopersicon esculentum Mill. VFN8) to low level (900 MHz, 5 V m ¡1 ) electromagnetic Welds for a short period (10 min) and measured changes in abundance of three speciWc mRNA soon after exposure. Within minutes of electromagnetic stimulation, stress-related mRNA (calmodulin, calcium-dependent protein kinase and proteinase inhibitor) accumulated in a rapid, large and 3-phase manner typical of an environmental stress response. Accumulation of these transcripts into the polysomal RNA also took place (indicating that the encoded proteins were translated) but was delayed (indicating that newly-synthesized mRNA was not immediately recruited into polysomes). Transcript accumulation was maximal at normal Ca 2+ levels and was depressed at higher Ca 2+ , especially for those encoding calcium-binding proteins. Removal of Ca 2+ (by addition of chelating agents or Ca 2+ channel blocker) led to total suppression of mRNA accumulation. Finally, 30 min after the electromagnetic treatment, ATP concentration and adenylate energy charge were transiently decreased, while transcript accumulation was totally prevented by application of the uncoupling reagent, CCCP. These responses occur very soon after exposure, strongly suggesting that they are the direct consequence of application of radio-frequency Welds and their similarities to wound responses strongly suggests that this radiation is perceived by plants as an injurious stimulus.

Two soils from temperate sites (UK; arable and grassland) were incubated aerobically at 0, 5, 15 or 258C for up to 23 days. During this period both soils were analysed for soil microbial biomass carbon (biomass C) and adenosine 5 ' triphosphate contents (ATP). Biomass C did not change signi®cantly in either soil at any temperature throughout, except during days 0 to 1 in the grassland soil. Soil ATP contents increased slowly throughout the 23 days of incubation, from 2.2 to a maximum of 3.1 nmol ATP g À1 soil in the arable soil (a 40% increase) and from 6.2 to a maximum of 11.2 nmol ATP g À1 soil in the grassland soil (an increase of 81%), both at 258C. Since biomass C did not change either with increasing temperature or increasing time of incubation, it was concluded that an increase in ATP was either due to an increase in adenylate energy charge or de novo synthesis of ATP, or both. During the incubation, biomass ATP concentrations ranged from about 5 to 12 mmol ATP g À1 biomass C but trends between biomass ATP and incubation temperatures were not very obvious until about day 13. On day 23, biomass ATP concentrations were positively and linearly related to temperature: (mmol ATP g À1 biomass C = 6.98 2 0.35 + 0.134 2 0.023 T 0 (r 2 = 0.77) with no signi®cant dierence in the slope between the grassland and arable soils. At 258C the biomass ATP concentration was 10.3 mmol g À1 biomass C, remarkably close to many other published values. It was concluded that, although the biomass increased its ATP concentration in response to increasing temperature, the increase was comparatively small. Also, at all temperatures tested, the biomass maintained its ATP concentration within the range commonly reported for microorganisms growing expontentially in vitro. This is despite the fact that the biomass normally exhibits other features more typical of a``resting'' or dormant population Ð a paradox which still is not resolved.

White muscle high-energy phosphate levels showed that the bleeding procedure applied during slaughter of Atlantic salmon (Salmo salar) did not overshadow the total pre-slaughter handling stress. The energy status of the muscle seemed to increase as a result of bleeding and the bleeding per se did not have adverse effects on flesh freshness resulting from excessive handling stress. The time for onset of rigor mortis and rigor strength were clearly related to the initial stress level and the rate of ATP depletion. The onset of rigor in stressed fish occurred about one day earlier compared with rested fish and the stressed fish also became stiffer.

salmon (Sulmo s&r), mean weight 5.1 kg, were transported live for 1.5 h by a well-boat (fish density 125 kg II-~) from the seacage to a fish processing plant and then kept in the well-boat for 4 h prior to slaughter. Anaerobic white muscle activity due to handling stress during fish loading at the cage, after shipment immediately before slaughter, and after the fish had passed the slaughter line, was evaluated using high-energy phosphates and IMP, the [ATPLIMP] ratio, adenylate energy charge together with pH and redox potential measured directly in the muscle. Water quality parameters, pH, salinity, temperature, dissolved oxygen, carbon dioxide, total carbonate carbon, total alkalinity, ammonia and ammonium were monitored at the cage, during shipment, and in the carbon dioxide anaesthesia tank during commercial fish slaughter. No dramatic effects of handling stress were found, indicating that transport and slaughtering did not have an adverse effect on flesh quality. The results were explained by the ability of the well-boat to maintain good seawater quality during transport, to a quick bulk netting of the fish from well-boat to the slaughter line and to an efficiently run carbon dioxide anaesthesia-tank that minimised struggling prior to killing.

Adenine nucleotides—adenosine monophosphate, diphosphate, and triphosphate—are of utmost importance to all living organisms, where they play a critical role in the energy metabolism and are tied to allosteric regulation in various regulatory enzymes. Adenylate energy charge represents the precise relationship between the concentrations of adenosine monophosphate, diphosphate, and triphosphate and indicates the amount of metabolic energy available to an organism. The experimental conditions of adenylate extraction in freshwater amphipod Gammarus fossarum are reported here for the first time and are crucial for the qualitative and quantitative determination of adenylate nucleotides using efficient and sensitive ion-pair reverse phase LC. It was shown that amphipod calcified exoskeleton impeded the neutralization of homogenate. The highest adenylate yield was obtained by homogenization in perchloric acid and subsequent addition of potassium hydroxide and phosphate buffer to achieve a p...

The activity and some kinetic properties of RBC Na+,K+-ATPase (EC 3.6.1.37) were investigated in essential hypertensives (EH; 40 subjects) and normotensives (NT; 20 subjects). A decrease in ouabain-sensitive *'Rb uptake as well as ouabain-sensitive ATPase activity was found in EH. [Na+]i and [K']i of EH did not show any statistical difference from NT. Na+,K+-ATPase showed a reduced Mg2+ activation and the apparent K,,, value for Mg2+ was 2-fold increased in the EH group. The influence of temperature on the Na+,K+-ATPase showed a reduced modulation and a minor activity peak at 37°C in the patients, consequently the calculated activation energy of the enzyme was increased at temperatures lower than 40%. Increased RBC adenylate energy charge (EC) was observed in EH when compared with NT. A negative correlation between EC and total Na+,K+-ATPase activity was found when all subjects were compared and also in both groups, showing a possible pump involvement in the regulation of the RBC metabolic flux in EH. These data provide evidence about some m~i~cations in active Na+,K+ transport and in EC in RBC which allows a further characterization of membrane cation fluxes in EH.

Samples from the bio-dynamic, bio-organic, and conventional trial, Therwil, Switzerland, were analyzed with the aim of determining the effects of organic land use management on the energy metabolism of the soil microbial biomass and on the fraction of microbial residues. The contents of adenylates, adenosine triphosphate (ATP), glucosamine, muramic acid, and galactosamine were significantly largest in the biodynamic organic farming (BYODIN) treatment and significantly lowest in the conventional farming treatment with inorganic fertilization (CONMIN). In contrast, the ergosterol-to-ATP ratio and fungal C-to-bacterial C ratios were significantly lowest in the BYODIN treatment and significantly largest in the CONMIN treatment. No clear treatment effects were observed for the ergosterol content and the adenylate energy charge (AEC), the ATP-to-microbial biomass C ratio and the ergosterol-to-fungal C ratio. Ergosterol, an indicator for saprotrophic fungal biomass, and fungal residues were significantly correlated. The microbial biomass carbon-to-nitrogen ratio showed a negative relationship with the AEC and strong positive relationships with the ratios ergosterol-to-microbial biomass C, ergosterol-to-ATP and fungal C-to-bacterial C. In conclusion, the long-term application of farmyard manure in combination with organic farming practices led to an increased accumulation of bacterial residues.

The purpose of this study was to examine the responses of the oyster C. gigas to oxygen levels at subcellular and whole-organism levels. Two experiments were carried out. The first experiment was designed to measure at 15°C, 20°C and 25°C for 20 hours the clearance (CR) and oxygen consumption (OC) rates of oysters exposed at different concentrations of oxygen. The goal of this first part was to estimate the hypoxic threshold for oysters below which their metabolism shifts toward anaerobiosis, by estimating the oxygen critical point (PcO 2) at 15, 20 and 25°C. The second experiment was carried out to evaluate the metabolic adaptations to hypoxia for 20 days at three temperatures: 12°C, 15°C and 20°C. The metabolic pathways were characterized by the measurement of the enzymes pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) and the alanine and succinate content and the adenylate energy charge (AEC). The respiratory chain functioning was estimated by the measurement of the activity of the electron transport system (ETS). The values of PcO 2 were 3.02±0.15, 3.43±0.20 and 3.28±0.24 mg O 2 L-1 at 15, 20 and 25°C, respectively. In whole oysters, hypoxia involved the inhibition of PK whatever the temperature but PEPCK was not stimulated. Succinate accumulated significantly only at 12°C and alanine at 12 and 15°C. A negative relationship between the PK activity and the alanine content was found only in hypoxic oysters. Lastly, hypoxia increased significantly the activity of ETS. With high PcO 2 values, the metabolic depression occurred quickly showing that oysters had a low capacity to regulate their respiration when oxygen availability is reduced, particularly in summer.

The purpose of this study was to examine the responses of the oyster C. gigas to oxygen levels at subcellular and whole-organism levels. Two experiments were carried out. The first experiment was designed to measure at 15°C, 20°C and 25°C for 20 hours the clearance (CR) and oxygen consumption (OC) rates of oysters exposed at different concentrations of oxygen. The goal of this first part was to estimate the hypoxic threshold for oysters below which their metabolism shifts toward anaerobiosis, by estimating the oxygen critical point (PcO 2) at 15, 20 and 25°C. The second experiment was carried out to evaluate the metabolic adaptations to hypoxia for 20 days at three temperatures: 12°C, 15°C and 20°C. The metabolic pathways were characterized by the measurement of the enzymes pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK) and the alanine and succinate content and the adenylate energy charge (AEC). The respiratory chain functioning was estimated by the measurement of the activity of the electron transport system (ETS). The values of PcO 2 were 3.02±0.15, 3.43±0.20 and 3.28±0.24 mg O 2 L-1 at 15, 20 and 25°C, respectively. In whole oysters, hypoxia involved the inhibition of PK whatever the temperature but PEPCK was not stimulated. Succinate accumulated significantly only at 12°C and alanine at 12 and 15°C. A negative relationship between the PK activity and the alanine content was found only in hypoxic oysters. Lastly, hypoxia increased significantly the activity of ETS. With high PcO 2 values, the metabolic depression occurred quickly showing that oysters had a low capacity to regulate their respiration when oxygen availability is reduced, particularly in summer.

The purpose of this study is to examine the response of the oyster Crassostrea gigas to sulphide at different biological scales. A first experiment was designed to measure for 20 h the clearance (CR) and oxygen consumption (OC) rates of oysters exposed at different concentrations of sulphide (0, 3, 6, 12, 20, and 40 mM). The second experiment was carried out to evaluate the metabolic adaptations to chronic sulphide exposure for 10 days by measuring PK and PEPCK enzyme activities, succinate and alanine content, the adenylate energy charge (AEC), and the activity of the electron transport system (ETS) in whole oysters at above 20 mM. PK and PEPCK mRNAs and enzyme activities have been measured in adductor muscle. When exposed to sulphide above 20 mM, CR stopped, whereas oyster maintained their average OC rate between 1 and 2 mg O 2 h-1 g-1 dw. In the second experiment, sulphide exposure of oysters resulted in higher mortality, reduced the glycolytic flux by inhibiting the PK activity and decreased the ETS activity leading to a lower AEC. At day 10, a kind of recovery took place: the ETS activity and AEC of oysters exposed to sulphide increased to the level of the control oysters. In the adductor muscle, sulphide did not affect the transcriptional level of PK; PK was only regulated at enzyme level. Conversely, PEPCK was regulated only at transcriptional level. Nevertheless, muscle exhibited a wholly anaerobic metabolism by the significant increase of alanine and succinate contents followed by the significantly decrease of the ATP content during the 10 days of the experiment. This experiment showed that sulphide involved partially the whole oyster and wholly the adductor muscle in an anaerobic pathway of energy production; but oxygen was needed for the detoxification of the organism.