The metabolic integrity of Fasciola hepatica during in vitro maintenance

Levels of metabolic intermediates and end products in F. hepatica after 24 and 48 h in Hédon-Fleig salt solution with added glucose were compared with levels obtained immediately on removal from the host. Glycogen levels dropped initially, probably due to the expulsion of eggs; thereafter they remained constant. Internal glucose concentrations increased as the parasites equilibrated with the medium. Other changes in internal pool sizes were consistent with regulation to the in vitro conditions. ATP levels increased; ATP/ADP ratios were maintained. Comparisons of mass action ratios and equilibrium constants suggest that hexokinase, pyruvate kinase and phosphofructokinase are regulatory. Output of excretory products approached linearity; from the calculated regressions the proportions of lactate, acetate and propionate were 1: 2: 4. The implications for metabolic regulation in F. hepatica are briefly discussed, and it is concluded that, for at least 48 h in vitro, energy metabolism is not adversely affected.     

The in vivo effects of rafoxanide on the energy metabolism of Fasciola hepatica

Adult F. hepatica were obtained from sheep which had received a single dose of rafoxanide at the therapeutic dose rate (7·5 mg/kg body weight). Flukes were recovered 12 and 24 h after the sheep were treated. No flukes were present after 4 days. Plasma levels of the drug were high after 24 h and remained so at 4 days. Flukes were being expelled from the liver 24 h after treatment. Glycogen levels within the flukes were diminished in the 24 h treated group, as were concentrations of ATP. These effects were not apparent in the 12 h treated group. Fluctuations in glucose, G6P, F6P and pyruvate pools were observed in both groups. The effects of rafoxanide were irreversible after 24 h exposure to the drug. Flukes from the treated sheep were incubated for 6 and 24 h in a simple maintenance medium with added glucose. They showed progressive deterioration in energy status. The results are considered in the context of the mode of action of rafoxanide.     

Flux regulation in glycogen-induced oscillatory glycolysis in cell-free extracts of Saccharomyces carlsbergensis

To localise the controlling point of the glycolytic system, the temporal changes of concentrations of glycolytic intermediates have been analysed after addition of glycogen to a substrate-depleted yeast extract. Three sequential metabolic states are clearly observable: a transition state at which there is continuous accumulation of the intermediates before the glyceraldehydephosphate dehydrogenase (GAPDH, EC 1.2.1.12) step; a stationary state with all glycolytic intermediates having concentrations oscillating at nearly stationary mean values; and a depletion state at which the intermediates before the GAPDH step are being depleted due to the exhaustion of glycogen. In all these states, the mean ethanol production rate and the concentration of ATP and the intermediates beyond the GAPDH-step are maintained fairly constant, while the glycogen consumption rate and intermediate concentrations of the upper part of the glycolytic system change considerably: the glycogen consumption rate varies 4-fold and fructose-bis-phosphate concentration more than 10-fold. Doubling of the initial glycogen concentration and the addition of a great excess of fructose-bis-phosphate do not affect the ethanol production rate and the mean glycerate-3-phosphate (3-PGA) and pyruvate levels. By contrast, ethanol production was accelerated by an increase of the net ATP consumption rate resulting from either the addition of apyrase or by substitution of trehalose for glycogen. Neither the mean absolute ATP level nor the adenylate energy charge were measurably affected, however all this data can be interpreted in terms of a very strong stoichiometric regulation and stabilization of the lower part of the glycolytic system.     

Studies of regulatory metabolism in Moniezia expansa: Effects of cambendazole and mebendazole

Effects of cambendazole and mebendazole on the respiratory metabolism of the anterior portion of Moniezia expansa were investigated in vitro. Anaerobically and in the presence of glucose, both drugs inhibited glucose uptake and increased glycogen utilisation. They reduced succinate production, by inhibiting fumarate reductase and (in the case of cambendazole) phosphoenolypyruvate carboxykinase activities, and increased lactate production. The additional lactate formed was accumulated in the worms. The drugs diminished ATP synthesis and/or turnover of adenine nucleotides. Aerobically, the drugs exerted similar effects on glucose uptake, glycogen utilisation and adenine nucleotides but the formation of end products was unaffected. Hexokinase and phosphofructokinase activities were inhibited by the drugs in vitro, but were not inhibited in extracts of parasites preincubated with the drugs.     

A path analysis of the causal relationships between red cell glycolytic intermediates, ADP and ATP in sickle cell anemia

The statistical relationships among the glycolytic intermediates (GI)) of the Embden-Meyerhof pathway, adenine nucleotides (ANs) and various hematological measures were estimated for 34 sickle cell anemia patients. Heterogeneity in linear and quadratic regressions of hemoglobin and hematocrit, both singly and jointly, on the GI and AN variables implied 1) that any single formula to standardize optical density measures of the GIs and ANs on a per gram hemoglobin or per liter cell water basis would not uniformly remove hemoglobin and hematocrit effects: 2) that ignoring significant hematological effects could bias the estimates of correlation among GIs and ANs; and 3) that hemoglobin and hematocrit measures do not reflect the same source of variability. The correlations among the GIs and ANs, after adjustment for hematological variability, were analyzed by path analysis to determine which of five proposed path models for cause and effect relationships were compatible with the data. AMP had a greater influence on ADP (coefficient of determination (CD) = 23%) than all the GIs together, while G6P and ADP influenced ATP variability the most (CD = 33% and 12%). The contributions of unknown factors to ADP and ATP variability were large for all models (CD = 56--77%) possibly due to stress of sickle cell disease. The path model with AMP and the four GIs (G6P, F6P, FDP, DHAP) influencing ADP variation, and the same GIs and ADP influencing ATP was the model most compatible with the data.     

Regulation of glycolysis by adenosine diphosphate in Pisum sativum

The role of ADP in controlling glycolysis has been examined in a soluble extract of germinating pea seeds. A shortage of ADP appears to retard glycolysis principally by restricting the conversion of phosphopyruvate to pyruvate rather than by restricting formation of phosphoglycerate. Upon addition of ADP to the extract there is an immediate decrease in the concentration of phosphopyruvate accompanied by an increase in pyruvate. Apparently the pyruvate-kinase step shows the most marked response to fluctuations in ADP availability. The glycolytic response to ADP depends on the concentration of ATP magnesium ions. The relation of magnesium-ion availability to adenine-nucleotide control of glycolysis is discussed.     

Brain energy metabolism in glutamate-receptor activation and excitotoxicity: Role for APC/C-Cdh1 in the balance glycolysis/pentose phosphate pathway

Recent advances in the field of brain energy metabolism strongly suggest that glutamate receptor-mediated neurotransmission is coupled with molecular signals that switch-on glucose utilization pathways to meet the high energetic requirements of neurons. Failure to adequately coordinate energy supply for neurotransmission ultimately results in a positive amplifying loop of receptor over-activation leading to neuronal death, a process known as excitotoxicity. In this review, we revisited current concepts in excitotoxic mechanisms, their involvement in energy substrate utilization, and the signaling pathways that coordinate both processes. In particular, we have focused on the novel role played by the E3 ubiquitin ligase, anaphase-promoting complex/cyclosome (APC/C)-Cdh1, in cell metabolism. Our laboratory identified 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3) –a key glycolytic-promoting enzyme– as an APC/C-Cdh1 substrate. Interestingly, APC/C-Cdh1 activity is inhibited by over-activation of glutamate receptors through a Ca²⁺-mediated mechanism. Furthermore, by inhibiting APC/C-Cdh1 activity, glutamate-receptors activation promotes PFKFB3 stabilization, leading to increased glycolysis and decreased pentose-phosphate pathway activity. This causes a loss in neuronal ability to regenerate glutathione, triggering oxidative stress and delayed excitotoxicity. Further investigation is critical to identify novel molecules responsible for the coupling of energy metabolism with glutamatergic neurotransmission and excitotoxicity, as well as to help developing new therapeutic strategies against neurodegeneration.     

The response of oscillating glycolysis to perturbations in the NADH/NAD system: a comparison between experiments and a computer model.

The glycolytic pathway is described by a set of coupled non linear differential equations of first order with respect to time. The individual terms of these equations consist of enzyme velocities assuming a steady state hypothesis for the enzymatic forms. These are specified and the system is solved numerically. Oscillations are explained by interaction of PFK with the adenylate system. The conditions for the occurrence of oscillations are tested in a series of computer runs. The phase relations between intermediates of the model agree with those found in yeast cells. As an application of the model the disturbation of oscillations by the addition of acetaldehyde is simulated. The predictions of the model agree with experimental results.     

Fermentative glycolysis with purified Escherichia coli enzymes for in vitro ATP production and evaluating an engineered enzyme

Each of the twelve enzymes for glycolytic fermentation, eleven from Escherichia coli and one from Saccharomyces cerevisiae, have been over-expressed in E. coli and purified with His-tags. Simple assays have been developed for each enzyme and they have been assembled for fermentation of glucose to ethanol. Phosphorus-31 NMR revealed that this in vitro reaction accumulates fructose 1,6-bisphosphate while recycling the cofactors NAD⁺ and ATP. This reaction represents a defined ATP-regeneration system that can be tailored to suit in vitro biochemical reactions such as cell-free protein synthesis. The enzyme from S. cerevisiae, pyruvate decarboxylase 1 (Pdc1; EC 4.1.1.1), was identified as one of the major ‘flux controlling’ enzymes for the reaction and was replaced with an evolved version of Pdc1 that has over 20-fold greater activity under glycolysis reaction conditions. This substitution was only beneficial when the ratio of glycolytic enzymes was adjusted to suit greater Pdc1 activity.     

Molecular aspects of sucrose transport and its metabolism to starch during seed development in wheat: A comprehensive review

Wheat is one of the most important crops globally, and its grain is mainly used for human food, accounting for 20% of the total dietary calories. It is also used as animal feed and as a raw material for a variety of non-food and non-feed industrial products such as a feedstock for the production of bioethanol. Starch is the major constituent of a wheat grain, as a result, it is considered as a critical determinant of wheat yield and quality. The amount and composition of starch deposited in wheat grains is controlled primarily by sucrose transport from source tissues to the grain and its conversion to starch. Therefore, elucidation of the molecular mechanisms regulating these physiological processes provides important opportunities to improve wheat starch yield and quality through biotechnological approaches. This review comprehensively discusses the current understanding of the molecular aspects of sucrose transport and sucrose-to-starch metabolism in wheat grains. It also highlights the advances and prospects of starch biotechnology in wheat.     

A new regulatory principle for in vivo biochemistry: Pleiotropic low affinity regulation by the adenine nucleotides – Illustrated for the glycolytic enzymes of Saccharomyces cerevisiae

Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme’s catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology.     

Systems biology of the metabolic network regulated by the Akt pathway

Cancer has been proposed as an example of systems biology disease or network disease. Accordingly, tumor cells differ from their normal counterparts more in terms of intracellular network dynamics than single markers. Here we shall focus on a recently recognized hallmark of cancer, the deregulation of cellular energetics. The constitutive activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway has been confirmed as an essential step toward cell transformation. We will consider how the effects of Akt activation are connected with cell metabolism; more precisely, we will review existing metabolic models and discuss the current knowledge available to construct a kinetic model of the most relevant metabolic processes regulated by the PI3K/Akt pathway. The model will enable a systems biology approach to predict the metabolic targets that may inhibit cell growth under hyper activation of Akt.     

Mapping photoautotrophic metabolism with isotopically nonstationary (13)C flux analysis

Understanding in vivo regulation of photoautotrophic metabolism is important for identifying strategies to improve photosynthetic efficiency or re-route carbon fluxes to desirable end products. We have developed an approach to reconstruct comprehensive flux maps of photoautotrophic metabolism by computational analysis of dynamic isotope labeling measurements and have applied it to determine metabolic pathway fluxes in the cyanobacterium Synechocystis sp. PCC6803. Comparison to a theoretically predicted flux map revealed inefficiencies in photosynthesis due to oxidative pentose phosphate pathway and malic enzyme activity, despite negligible photorespiration. This approach has potential to fill important gaps in our understanding of how carbon and energy flows are systemically regulated in cyanobacteria, plants, and algae.     

Molecular and functional characterization of the plastid-localized Phosphoenolpyruvate enolase (ENO1) from Arabidopsis thaliana

The Arabidopsis thaliana gene At1g74030 codes for a putative plastid phosphoenolpyruvate (PEP) enolase (ENO1). The recombinant ENO1 protein exhibited enolase activity and its kinetic properties were determined. ENO1 is localized to plastids and expressed in most heterotrophic tissues including trichomes and non-root-hair cells, but not in the mesophyll of leaves. Two T-DNA insertion eno1 mutants exhibited distorted trichomes and reduced numbers of root hairs as the only visible phenotype. The essential role of ENO1 in PEP provision for anabolic processes within plastids, such as the shikimate pathway, is discussed with respect to plastid transporters, such as the PEP/phosphate translocator.     

Lysine as a heme iron ligand: A property common to three truncated hemoglobins from Chlamydomonas reinhardtii

Background

The nuclear genome of Chlamydomonas reinhardtii encodes a dozen hemoglobins of the truncated lineage. Four of these, named THB1–4, contain a single ~130-residue globin unit. THB1, which is cytoplasmic and capable of nitric oxide dioxygenation activity, uses a histidine and a lysine as axial ligands to the heme iron. In the present report, we compared THB2, THB3, and THB4 to THB1 to gain structural and functional insights into algal globins.

Quantitative evaluation of respiration induced metabolic oscillations in erythrocytes

The changes in the partial pressures of oxygen and carbon dioxide (P_O2 and P_CO2) during blood circulation alter erythrocyte metabolism, hereby causing flux changes between oxygenated and deoxygenated blood. In the study we have modeled this effect by extending the comprehensive kinetic model by Mulquiney and Kuchel [P.J. Mulquiney, and P.W. Kuchel. Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement, Biochem. J. 1999, 342, 581–596.] with a kinetic model of hemoglobin oxy-/deoxygenation transition based on an oxygen dissociation model developed by Dash and Bassingthwaighte [R. Dash, and J. Bassingthwaighte. Blood HbO₂ and HbCO₂ dissociation curves at varied O₂, CO₂, pH, 2,3-DPG and temperature levels, Ann. Biomed. Eng., 2004, 32(12), 1676–1693.]. The system has been studied during transitions from the arterial to the venous phases by simply forcing P_O2 and P_CO2 to follow the physiological values of venous and arterial blood. The investigations show that the system passively follows a limit cycle driven by the forced oscillations of P_O2 and is thus inadequately described solely by steady state consideration. The metabolic system exhibits a broad distribution of time scales. Relaxations of modes with hemoglobin and Mg²⁺ binding reactions are very fast, while modes involving glycolytic, membrane transport and 2,3-BPG shunt reactions are much slower. Incomplete slow mode relaxations during the 60 s period of the forced transitions cause significant overshoots of important fluxes and metabolite concentrations – notably ATP, 2,3-BPG, and Mg²⁺. The overshoot phenomenon arises in consequence of a periodical forcing and is likely to be widespread in nature – warranting a special consideration for relevant systems.