Changes In Energy Charge Research Articles

Phosphorescent Differential Sensing of Physiological Phosphates with Lanthanide Ions-Modified Mn-Doped ZnCdS Quantum Dots.

Phosphates, both inorganic and organic, play fundamental roles in numerous biological and chemical processes. The biological functions of phosphates connect with each other, analysis of single phosphate-containing biomolecule therefore cannot reveal the exact biological significance of phosphates. Sensor array is therefore the best choice for differentiation analysis of physiological phosphates. Lanthanide ions possess high affinity toward physiological phosphates, while lanthanide ions can also efficiently quench the luminescence of quantum dots (QDs). Taking lanthanide ions as cartridges, here we proposed a sensor array for sensing of physiological phosphates based on lanthanide ions-modified Mn-doped ZnCdS phosphorescent QDs in the manner of indicator-displacement assay. A series of lanthanide ions were selected as quencher for phosphorescent QDs. Physiological phosphates could subsequently displace the quencher and recover the phosphorescence. Depending on their varied phosphorescence restoration, a sensor array was thus developed. The photophysics of phosphorescence quenching and restoration were studied in detail for better understanding the mechanism of the sensor array. The exact contribution of each sensor element to the sensor array was evaluated. Those sensor elements with little contribution to the differentiation analysis were removed for narrowing the size of the array. The proposed sensor array was successfully explored for probing nucleotide phosphates-involved enzymatic processes and their metabolites, simulated energy charge changes, and analysis of physiological phosphates in biological samples.

Survival During Long-Term Starvation: Global Proteomics Analysis ofGeobacter sulfurreducensunder Prolonged Electron-Acceptor Limitation

The bioavailability of terminal electron acceptors (TEAs) and other substrates affects the efficiency of subsurface bioremediation. While it is often argued that microorganisms exist under "feast or famine", in the laboratory most organisms are studied under "feast" conditions, whereas they typically encounter "famine" in nature. The work described here aims to understand the survival strategies of the anaerobe Geobacter sulfurreduces under TEA-starvation conditions. Cultures were starved for TEA and at various times sampled to perform global comparative proteomic analysis using iTRAQ to obtain insight into the dynamics of change in proteins/enzymes expression associated with change in nutrient availability/environmental stress. Proteins varying in abundance with a high level of statistical significance (p < 0.05) were identified to understand how cells change from midlog to (i) stationary phase and (ii) conditions of prolonged starvation (survival phase). The most highly represented and significantly up-regulated proteins in the survival phase cells are involved in energy metabolism, cell envelope, and transport and binding functional categories. The majority of the proteins were predicted to be localized in the cell membranes. These results document that changes in the outer and cytoplasmic membranes are needed for survival of Geobacter under starvation conditions. The cell shuts down anabolic processes and becomes poised, through changes in its membrane proteins, to sense nutrients in the environment, to transport nutrients into the cell, and to detect or utilize TEAs that are encountered. Under TEA-limiting conditions, the cells turned from translucent white to red in color, indicating higher heme content. The increase in heme content supported proteomics results showing an increase in the number of cytochromes involved in membrane electron transport during the survival phase. The cell is also highly reduced with minimal change in energy charge (ATP to total adenine nucleotide ratio). Nonetheless, these proteomic and biochemical results indicate that even under TEA starvation cells remain poised for bioremediation.

Development of an ion‐pair HPLC method for investigation of energy charge changes in cerebral ischemia of mice and hypoxia of Neuro‐2a cell line

The determination of adenine nucleotides and energy charge (EC) has great importance in the characterization of cerebral ischemic injury and post-ischemic recovery. An IP-HPLC method was developed for the quantification of AMP, ADP, ATP and EC in cerebral ischemia and hypoxia of the Neuro-2a cell line. The chromatographic conditions were: a Zorbax SB-C18 reversed-phase column; mobile phase 100 mM KH(2)PO(4), 1 mM tetrabutylammonium hydroxide, and 2.5% acetonitrile, brought to pH 7.0 with potassium hydroxide (4 M), filtered through a 0.45 microm Millipore filter and degassed prior to use. The flow-rate was 1.0 mL/min. The injection volume was 20 microL. Detection was performed at a wavelength of 254 nm under a constant temperature (27 +/- 1 degrees C). The method was validated by means of linearity, using calibration curves constructed with five concentration levels of each compound. The limit of detection was also determined. The system precision was calculated as the coefficient of variation for five injections for each compound tested. Cerebral tissue was homogenized (4 degrees C) in 1 mL of an ice-cold 6% trichloroacetic acid that contained ATPase inhibitor and obtained good recovery (>90%). The results show that the described method for the determination of adenine nucleotides by HPLC has good linearity, limit of detection, precision and specificity, and is simple and rapid to perform.

Intermediary metabolism in mudskippers,Periophthalmodon schlosseriandBoleophthalmus boddarti, during immersion or emersion

This study aimed to examine effects of 48 h emersion on intermediary metabolism in the mudskippers Periophthalmodon schlosseri (Pallas, 1770) and Boleophthalmus boddarti (Pallas, 1770). The glycogen content increased significantly, while the lactate content remained unchanged, in the muscle of P. schlosseri during 48 h of emersion. Thus, emersion led to either a decrease in glycogen utilization or an increase in glycogen synthesis. Because there was no change in energy charge, P. schlosseri probably used energy stores other than glycogen during emersion. The phosphofructose kinase-1 (PFK) from the muscle of P. schlosseri became more sensitive to ATP inhibition after 6 h of emersion, indicating that a transient decrease in the glycolytic flux indeed occurred at this locus that led to an increase in glycogen content. In contrast, such phenomena were not observed in the muscle of B. boddarti during 48 h of emersion. Emersion also led to a significant increase in the muscle fructose-2,6-bisphosphate (F26P2) content in P. schlosseri, but not in B. boddarti. Based on results obtained from P. schlosseri undergoing forced exercise, we propose that a correlation may exist between the F26P2content and the utilization of amino acids as an energy source in this mudskipper during emersion.

AMP-deaminase from normal and cirrhotic human liver: A comparative study

AMP-deaminase (EC 3.5.4.6) is an enzyme of nucleotide breakdown involved in regulation of adenine nucleotide pool in mammalian cells. Reaction catalysed by AMP-deaminase constitutes a rate-limiting step in adenine nucleotide catabolism in liver. In this study kinetic and regulatory properties of AMP-deaminase purified from normal and cirrhotic human liver were investigated. In comparison to AMP-deaminase extracted from the normal human liver, AMP-deaminase extracted from the cirrhotic liver was less sensitive towards substrate analogues, and only a very limited response towards pH and adenylate energy charge changes tested for enzyme isolated from this tissue source had been observed. At physiological pH 7.0, in the absence and in the presence of important allosteric effectors (ATP, ADP, GTP and orthophosphate), AMP-deaminases from the two sources studied manifested different regulatory profiles, with half-saturation constant (S0.5) values being distinctly higher for the enzyme extracted from the pathological organ. In contrast to AMP-deaminase isolated from the normal, healthy liver, where presence of relatively large (68 kDa) protein fragment was also detected, only smaller protein fragments were identified, while SDS-PAG electrophoresis of AMP-deaminase isolated from the cirrhotic liver was performed. The obtained results indicate clearly that advanced proteolytic processes occurring in the cirrhotic liver may affect structural integrity of AMP-deaminase studied, making enzyme less active and less sensitive to regulatory action of important allosteric effectors.

Organ-specific changes in high-energy phosphates after hemorrhagic shock and resuscitation in the rat.

Adenosine nucleotides provide energy for many essential cellular functions. Liver and intestinal ATP and energy charge are known to decrease during hemorrhagic shock, and the ability to regenerate high-energy phosphates may have important implications for recovery. We measured organ-specific changes in energy charge after hemorrhagic shock and after shock followed by resuscitation. Anesthetized Sprague-Dawley rats were bled and maintained at a mean arterial pressure (MAP) of 40 mmHg for 1, 2, 3, or 4 hours. Some animals were resuscitated with normal saline and shed blood (1:1) to a mean arterial pressure of 80 to 90 mmHg for 1 hour. Control animals were anesthetized, but not hemorrhaged. At the conclusion, blood gases and adenine nucleotides were measured. Arterial pO2 and pCO2 were normal in all groups. Unresuscitated hemorrhage caused metabolic acidosis, but bicarbonate was normal in controls and after hemorrhage followed by resuscitation. Energy charge (EC) in the gastrocnemius was unaffected by hemorrhage or resuscitation. Liver EC decreased after hemorrhage (p = 0.0001), but recovered partially after resuscitation. Kidney EC was decreased after only 3 hours of hemorrhage and 1 hour of resuscitation (p = 0.005), but not with shorter periods of hemorrhage. Lung EC decreased with shock, but was substantially worse after resuscitation (p < 0.05). After hemorrhage and resuscitation, EC decreased in lung, liver, kidney, and intestine, but the time course, extent of decline, and ability to recover after resuscitation varied from organ to organ. Inability to regenerate high-energy phosphates after hemorrhagic shock may be a marker for more severe cellular damage.

Influence of the antioxidants vitamin E and idebenone on retinal cell injury mediated by chemical ischemia, hypoglycemia, or oxidative stress

A role for the antioxidants vitamin E and idebenone in decreasing retinal cell injury, after metabolic inhibition induced by chemical ischemia and hypoglycemia, was investigated and compared with oxidative stress conditions. Preincubation of the antioxidants, vitamin E (20 μM) and idebenone (10 μM), effectively protected from retinal cell injury after oxidative stress or hypoglycemia, whereas the protection afforded after postincubation of both antioxidants was decreased. Delayed retinal cell damage, mediated by chemical ischemia, was attenuated at 10 or 12 h postischemia, only after exposure to the antioxidants during all the experimental procedure. An antagonist of the N-methyl- d-aspartate (NMDA) receptors, an inhibitor of nitric oxide synthase (NOS) or a blocker of l-type Ca 2+ channels were ineffective in reducing cell injury induced by chemical ischemia, hypoglycemia or oxidative stress. Oxidative stress and hypoglycemia increased (about 1.2-fold) significantly the fluorescence of the probe DCFH 2-DA, that is indicative of intracellular ROS formation. Free radical generation detected with the probe dihydrorhodamine 123 (DHR 123) was enhanced after oxidative stress, chemical ischemia or hypoglycemia (about 2-fold). Nevertheless, the antioxidants vitamin E or idebenone were ineffective against intracellular ROS generation. Cellular energy charge decreased greatly after chemical ischemia, was moderately affected after hypoglycemia, but no significant changes were observed after oxidative stress. Preincubation with vitamin E prevented the changes in energy charge upon 6 h posthypoglycemia. We can conclude that irreversible changes occurring during chemical ischemia mainly reflect the alterations taking place at the ischemic core, whereas hypoglycemia situations may reflect changes occurring at the penumbra area, whereby vitamin E or idebenone may help to increase cell survival, exerting a beneficial neuroprotective effect.

Energy Charge Is Not Decreased in Lymphocytes of Patients With Leber??s Hereditary Optic Neuropathy With the 11778 Mutation

A defect in mitochondrial energy conservation is strongly suggested to be involved in the pathogenesis of Leber's hereditary optic neuropathy (LHON). The authors therefore compared the energy charge in lymphocytes among patients with LHON, their asymptomatic maternal lineages, and normal control subjects. Blood samples were obtained from 7 patients, 10 asymptomatic maternal relatives, and 16 normal subjects. Molecular analysis confirmed that all had the homoplasmic 11,778 point mutation in the mtDNA of their blood cells. The concentrations of adenosine triphosphate (ATP), diphosphate (ADP), and monophosphate (AMP) were determined by high-performance liquid chromatography. The energy charge was calculated as (ATP + 1/2 ADP)/(ATP + ADP + AMP). The mean energy charges of lymphocytes were 0.871 +/- 0.049 in patients with LHON, 0.884 +/- 0.061 in their asymptomatic maternal relatives, and 0.885 +/- 0.061 in normal controls, respectively. No statistically significant difference was found among the three groups. Although the study did not find the anticipated change in energy charge in peripheral blood cells, this neither confirms nor rejects the notion that a defect in the mitochondrial oxidative phosphorylation system is involved in the pathogenesis of LHON.

In vitro effects of 50 Hz magnetic fields on oxidatively damaged rabbit red blood cells

The aim of this study was to investigate the effects of 50 Hz magnetic fields (0.2-0.5 mT) on rabbit red blood cells (RBCs) that were exposed simultaneously to the action of an oxygen radical-generating system, Fe(II)/ascorbate. Previous data obtained in our laboratory showed at the exposure of rabbit erythrocytes or reticulocytes to Fe(II)/ascorbate hexokinase inactivation, whereas the other glycolytic enzymes do not show any decay. We also observed depletion of reduced glutathione (GSH) content with a concomitant intracellular and extracellular increase in oxidized glutathione (GSSG) and a decrease in energy charge. In this work we investigated whether 50 Hz magnetic fields could influence the intracellular impairments that occur when erythrocytes or reticulocytes are exposed to this oxidant system, namely, inactivation of hexokinase activity, GSH depletion, a change in energy charge, and hemoglobin oxidation. The results obtained indicate the a 0.5 mT magnetic field had no effect on intact RBCs, whereas it increased the damage with Fe(II)/ascorbate to a 0.5 mT magnetic field induced a significant further decay in hexokinase activity (about 20%) as well as a twofold increase in methemoglobin production compared with RBCs that were exposed to the oxidant system alone. Although further studies will be needed to determine the physiological implications of these data, the results reported in this study demonstrate that the effects of the magnetic fields investigated are able to potentiate the cellular damage induced in vitro by oxidizing agents.

Sequential Changes of Energy Charge, Lipoperoxide Level, and DNA Synthesis Rate of the Liver Following Biliary Obstruction in Rats

To clarify the effects of obstructive jaundice on the liver, sequential changes of hepatic energy charge, the concentrations of adenine nucleotides and malondialdehyde, DNA synthesis rate, and histology of the liver were examined on the day before and Days 1, 2, 4, 7, and 14 after biliary obstruction in rats and compared with those of sham-operated controls. Foci of necrotic hepatocytes were present on Days 1 and 2 and mitoses of the hepatocytes were frequently observed with a peak on Day 2 in the jaundiced liver. Marked proliferation of bile ductules were subsequently observed on Days 7 and 14, resembling biliary cirrhosis. The DNA synthesis rate was significantly activated after bile duct obstruction with its peak on Day 2, more than nine times higher than the control value and returned to the control level on Day 14. Hepatic ATP concentration and energy charge gradually declined with prolonged jaundice and significantly lower levels persisted after Day 7 compared with the controls. The malondialdehyde level in the jaundiced liver gradually increased and became significantly higher on Day 14. We conclude that obstructive jaundice decreases hepatic energy charge and increases the lipoperoxide level. In the initial stage of obstructive jaundice, the hepatocytes proliferate associated with activated DNA synthesis probably to compensate hepatic damage; however, prolonged obstructive jaundice induces functional hepatic injury possibly necessitating biliary drainage.

The dephosphorylation of AMP and IMP by a soluble low Km 5′nucleotidase from human seminal plasma: Some regulatory aspects

In this study, a soluble low K m 5′nucleotidase, dephosphorylating IMP with a V max/ K m ratio 10-times higher than that of AMP, has been purified from human seminal plasma. The effect of inorganic phosphate (P i) and adenylate energy charge variations on the activity of this enzyme has also been investigated. In the physiological range, with IMP as substrate, the activity of the enzyme does not change whereas the hydrolysis of AMP increases with decreasing energy charge values. In the presence of both the substrates, phosphate exerts an inhibitory effect on the enzyme activity with a similar concentration dependence pattern. The results show that AMP-hydrolysing activity responds to variations of energy charge by increasing the AMP degradation thus protecting the value of energy charge at the expense of a decrease in the total adenylate pool. In contrast, the dephosphorylation of IMP is not regulated by changes in energy charge. This data suggests that the degradation of IMP and AMP, although carried out by the same enzyme, is controlled by different regulatory mechanisms.

Polarized Caco-2 cells

Reactive oxygen metabolites are implicated in gastrointestinal disease and enterocyte injury associated with ischemia-reperfusion, bacterial translocation, inflammatory bowel disease, and necrotizing enterocolitis. The ileal-like, human colon carcinoma cell line, Caco-2, was used to investigate oxidative damage. After challenging Caco-2 cells with reactive oxygen metabolites, the permeability, viability, and energy charge of Caco-2 cells were assessed. Permeability was determined by transepithelial electrical potential and flux of small molecules. Viability was determined by release of 51Cr. Cell energy was evaluated by determining adenylate energy charge. The source of reactive oxygen metabolites, with the exception of menadione, did not affect viability of Caco-2 cells; cell permeability was increased. The increased varied with the source and location of the reactive oxygen metabolite. There was no change in energy charge. This study suggests that reactive oxygen metabolites could cause enterocyte damage and that the source of the reactive oxygen metabolite is an important variable in determining the extent of damage. Antioxidants might prevent injury.

Energy charge and mitotic activity in regenerating rat liver during parenteral nutrition.

The relationship among parenteral nutrition, hepatic energy charge, and mitotic activity was investigated in partially hepatectomized rats fed diets based on glucose, ketone bodies, and saline (starvation). Male Sprague-Dawley rats underwent 70% hepatectomy and jugular vein catheterization for parenteral feeding. All rats were infused with saline for 6 hours, then randomly assigned to one of three parenteral infusions. Rats received either 0.9% saline only (group A rats), 25% glucose + 4% amino acid (group B rats), or 18% monoacetoacetin + 7% glucose + 4% amino acid (group C rats). Three rats receiving saline infusion were killed at 2, 4, and 6 hours after surgery, and three rats from each diet group were killed at 2- to 4-hour intervals from 10 through 32 hours. Hepatic energy charge and mitotic index were measured at each time point. Energy charge was similar for each treatment until 18 hours but was depressed from 6 through 16 hours and began to increase between 16 and 18 hours. Energy charge at 22 hours for group B rats was significantly higher than energy charge for group A and C rats. This difference was maintained through 32 hours. Mitosis started between 24 and 26 hours for all treatments, and group A and C rats exhibited a much higher mitotic index than did group B rats. Adenosine triphosphate was the main driving force for changes in energy charge. The data showed that energy charge and mitotic index were inversely related. It is speculated that high energy charge may negatively influence activity of enzymes inasmuch as activity of these enzymes is altered by phosphorylation-dephosphorylation.

Changes in energy charge and phosphorylation potential during electrical conditioning of skeletal muscle: I.Y. Christlieb, R.L. Kao, G.J. Magovern Jr., S.B. Park, and G.J. Magovern. Allegheny-Singer Research Inst., Pittsburgh, PA

Changes in energy charge and phosphorylation potential during electrical conditioning of skeletal muscle: I.Y. Christlieb, R.L. Kao, G.J. Magovern Jr., S.B. Park, and G.J. Magovern. Allegheny-Singer Research Inst., Pittsburgh, PA

Adenosine triphosphate and adenylate energy charge in relation to dormancy of wild oat seeds

Adenosine phosphates, expressed as ATP and energy charge levels, were compared in genetically identical dormant and nondormant caryopses of wild oat (Avena fatua L.). Air-dry nondormant caryopses had a larger pool of ATP and a higher energy charge than dormant caryopses. During the early hours of imbibition of water by dormant caryopses, ATP levels and energy charge increased indicating they are not limiting factors for germination. Moreover, during the induction of germination in dormant caryopses by gibberellic acid (GA3), changes in energy charge were similar to those observed on water, indicating that GA3 does not overcome dormancy simply by elevating energy charge levels. In a second study, ATP and energy charge were measured in initially dormant caryopses incubated under conditions of temperature that either removed or retained dormancy. Both embryo and endosperm extracts demonstrated correlations between levels of ATP or energy charge with temperature of incubation but not with the state of dormancy. The conclusions drawn from both lines of investigation do not support the hypothesis that release of dormancy is related to the metabolism of adenosine phosphates in caryopses of wild oat.

Adaptation of Ribonucleic Acid Metabolism to Anoxia in Rice Embryos

Rice (Oryza sativa var. Cigalon) is a plant which can adapt to very stringent anoxic conditions. It has previously been shown that during the adaptation period the energy charge can be used as a marker for metabolic activity. We have studied RNA metabolism during this period and correlated it with changes in the energy charge. Uptake of labeled precursor, UTP-specific activity, and incorporation were measured. Immediately after transfer to anaerobic conditions, the UTP pool size is reduced and the overall rate of incorporation drops. During adaptation, the rate of incorporation increases and stabilizes at about half of its value in aerobic conditions. Analysis of RNA shows that rRNA and mRNA are synthesized and that the processing of ribosomal RNA precursor is altered. Polyribosomes are present throughout the adaptation period although their amount is reduced during the first hour of anoxia. Changes in poly(A) content were noticed, indicating that some mRNA are rapidly degraded. Taken together, the results show that the RNA metabolism can be modulated during adaptation to anoxia in a parallel manner with energy charge changes.

Experimental and clinical study on ATP-MgCl2 administration for postischemic acute renal failure.

The present study was undertaken to investigate the effect of ATP-MgCl2 on the recovery of renal function following renal ischemia. Bilateral renal ischemia was produced for 90 minutes in dogs. Immediately after the release of ischemia, ATP-MgCl2 (50 mumoles/kg) was given intravenously. Serum creatinine and FeNa were measured following the release of ischemia. Renal cellular energy charge, glomerular endothelial thickness and per cent circularity of interstitial cells were measured. Creatinine and FeNa were significantly lower in ATP-MgCl2 treated dogs compared to those in saline treated controls. Changes in energy charge, glomerular endothelial thickness and per cent circularity indicated ischemically induced renal cellular edema was reversed with ATP-MgCl2 through the improvement of energy metabolism. Taking those experimental data into consideration, ATP-MgCl2 was given to 16 acute renal failure patients and 13 patients survived. ATP-MgCl2 administration is effective for the treatment of acute renal failure.

Changes in hepatic energy charge, blood ketone body ratio, and indocyanine green clearance in relation to DNA synthesis after hepatectomy

The changes in the energy charge (ATP+0.5ADP)/ (ATP+ADP+AMP) levels of the remnant liver were studied in relation to changes in the incorporation rate of methyl- 3H-thymidine into DNA, the blood ratio of acetoacetate/β-hydroxybutyrate and indocyanine green (ICG) clearance in 70% hepatectomized rabbits. The energy charge levels of the remnant liver decreased rapidly to 0.767 from 0.856 of normal, despite a marked enhancement of mitochondrial phosphorylative activity, concomitant with the fall in blood ketone body ratio, before a maximal increase of DNA synthesis after hepatectomy. Blood ketone body ratio was correlated with hepatic energy charge (r=0.696, p<0.01). After a maximal increase of DNA synthesis, hepatic energy charge levels and the blood keton body ratios increased gradually to normal levels, concomitant with a rise in ICG clearance. Energy charge levels of the remnant liver can be evaluated by the blood ketone body ratio.

Regulation of nitrogenase activity in aerobes by MG 2+ availability: An hypothesis

Nitrogenase functions at or near its maximum capacity in vivo , despite a reported energy charge in the cell that should severely inhibit the enzyme. Deenergizing cellular membranes, which is postulated to release magnesium in mitochondria, has been reported to produce rapid inhibition of nitrogenase activity while giving only small changes in energy charge and NAD + NADH ratio. It is proposed that the level of magnesium available for complexation by the potent inhibitor ADP is the rate controlling variable for nitrogenase activity.

Changes in energy charge and block of protein synthesis in rabbit reticulocytes under the action of valinomycin.

Valinomycin blocks protein synthesis at the elongation stage in intact reticulocyte while being without action on a cell free system. We demonstrate here that this membrane mediated inhibition is accompanied by a change of the energy charge (ATP/ADP/AMP ratio) of the cells. There seems to be an accumulation of AMP without a proportional gain in ADP. The change in energy charge in itself can be the cause of the inhibition of protein synthesis. We show also that valinomycin can cause a decrease of the energy charge at concentrations where its action as an ionophore is not seen and independent of a possible activation of the Na-K dependent ATPase.