Coherent and robust modulation of a metabolic network by cytoskeletal organization and dynamics

In order to investigate the influence of cytoskeletal organization and dynamics on cellular biochemistry, a mathematical model was formulated based on our own experimental evidence. The model couples microtubular protein (MTP) dynamics to the glycolytic pathway and its branches: the Krebs cycle, ethanolic fermentation, and the pentose phosphate (PP) pathway. Results show that the flux through glycolysis coherently and coordinately increases or decreases with increased or decreased levels of polymerized MTP, respectively. The rates of individual enzymatic steps and metabolite concentrations change with the polymeric status of MTP throughout the metabolic network. Negative control is exerted by the PP pathway on the glycolytic flux, and the extent of inhibition depends inversely on the polymerization state of MTP, i.e. a high degree of polymerization relieves the negative control. The stability of the model's steady state dynamics for a wide range of variation of metabolic parameters increased with the degree of polymerized MTP. The findings indicate that the organization of the cytoskeleton bestows coherence and robustness to the coordination of cellular metabolism.     

CONTROL: software for the analysis of the control of metabolic networks

The program CONTROL is based on metabolic control theory anduses the method developed by Reder (1988). In this theory, twosets of parameters are defined in the vicinity of a steady-state:the elasticity coefficients which describe the local behaviourof the isolated enzymes, and the control coefficients whichexpress the response of the whole metabolic network to perturbationsat a given step. The theory shows that relationships exist betweenthe control coefficients (summation relationships or structuralrelationships) and also between the two types of coefficients(control and elasticity coefficients: connectivity relationships).The program CONTROL is divided into two parts (sub-menus). Thefirst one calculates all the control coefficients (flux andconcentrations) of a metabolic network from the elasticity coefficients.Using the second menu, the symbolic relationships are obtainedbetween the control coefficients (summation relationships) andbetween the control coefficients and the elasticity coefficients(connectivity relationships). These two sub-menus can be appliedindependently to any metabolic network (to date limited to 19steps and 19 metabolites).     

Effects of isoflurane, halothane, and enflurane on myocardial flow and energy stores in the perfused rat heart

The effect of three volatile anesthetics (halothane, enflurane, and isoflurane) on coronary flow and metabolic state of isolated rat hearts was studied. These anesthetics are coronary dilators and their effects are dose dependent. At 2 MAC (minimum alveolar concentration), isoflurane, enflurane, and halothane increase coronary flow by 114 +/- 5.9, 93 +/- 6.1, and 77 +/- 6.4%, respectively (p less than 0.001). At these concentrations, they also have a modest but significant metabolic effect causing a 30% reduction in myocardial ATP and phosphocreatine levels, with no significant modification in ADP and AMP concentrations. Energy charge and lactate/pyruvate ratio were also unaffected by these anesthetics. The vascular and metabolic effects were reversible within 2 and 30 min, respectively. Perfusion of the hearts with a Krebs-Henseleit solution without Pi did not interfere with the vascular and the metabolic effect of the anesthetics; however, in this case, ATP and phosphocreatine concentration did not return to control levels after their discontinuation despite full recovery of the vascular effect. These data suggest that the volatile anesthetics have direct coronary vascular and myocardial metabolic effects and that these effects occur independently.     

A probabilistic framework for the exploration of enzymatic capabilities based on feasible kinetics and control analysis

BackgroundAnalysis of limiting steps within enzyme-catalyzed reactions is fundamental to understand their behavior and regulation. Methods capable of unravelling control properties and exploring kinetic capabilities of enzymatic reactions would be particularly useful for protein and metabolic engineering. While single-enzyme control analysis formalism has previously been applied to well-studied enzymatic mechanisms, broader application of this formalism is limited in practice by the limited amount of kinetic data and the difficulty of describing complex allosteric mechanisms.MethodsTo overcome these limitations, we present here a probabilistic framework enabling control analysis of previously unexplored mechanisms under uncertainty. By combining a thermodynamically consistent parameterization with an efficient Sequential Monte Carlo sampler embedded in a Bayesian setting, this framework yields insights into the capabilities of enzyme-catalyzed reactions with modest kinetic information, provided that the catalytic mechanism and a thermodynamic reference point are defined.ResultsThe framework was used to unravel the impact of thermodynamic affinity, substrate saturation levels and effector concentrations on the flux control and response coefficients of a diverse set of enzymatic reactions.ConclusionsOur results highlight the importance of the metabolic context in the control analysis of isolated enzymes as well as the use of statistically sound methods for their interpretation.General SignificanceThis framework significantly expands our current capabilities for unravelling the control properties of general reaction kinetics with limited amount of information. This framework will be useful for both theoreticians and experimentalists in the field.     

Warming and Resource Availability Shift Food Web Structure and Metabolism

Climate change disrupts ecological systems in many ways. Many documented responses depend on species'' life histories, contributing to the view that climate change effects are important but difficult to characterize generally. However, systematic variation in metabolic effects of temperature across trophic levels suggests that warming may lead to predictable shifts in food web structure and productivity. We experimentally tested the effects of warming on food web structure and productivity under two resource supply scenarios. Consistent with predictions based on universal metabolic responses to temperature, we found that warming strengthened consumer control of primary production when resources were augmented. Warming shifted food web structure and reduced total biomass despite increases in primary productivity in a marine food web. In contrast, at lower resource levels, food web production was constrained at all temperatures. These results demonstrate that small temperature changes could dramatically shift food web dynamics and provide a general, species-independent mechanism for ecological response to environmental temperature change.     

Warming reduces the effects of enrichment on stability and functioning across levels of organisation in an aquatic microbial ecosystem

Warming and nutrient enrichment are major environmental factors shaping ecological dynamics. However, cross‐scale investigation of their combined effects by linking theory and experiments is lacking. We collected data from aquatic microbial ecosystems investigating the interactive effects of warming (constant and rising temperatures) and enrichment across levels of organisation and contrasted them with community models based on metabolic theory. We found high agreement between our observations and theoretical predictions: we observed in many cases the predicted antagonistic effects of high temperature and high enrichment across levels of organisation. Temporal stability of total biomass decreased with warming but did not differ across enrichment levels. Constant and rising temperature treatments with identical mean temperature did not show qualitative differences. Overall, we conclude that model and empirical results are in broad agreement due to robustness of the effects of temperature and enrichment, that the mitigating effects of temperature on effects of enrichment may be common, and that models based on metabolic theory provide qualitatively robust predictions of the combined ecological effects of enrichment and temperature.     

Aurintricarboxylic acid, a preferential inhibitor of initiation of protein synthesis

The effect of aurintricarboxylic acid (ATA) was tested on various aspects of protein synthesis directed by the natural messenger ribonucleic acid (RNA) isolated from R17 RNA bacteriophage. The effects of various levels of ATA (up to 1,000 mum) were tested on overall protein synthesis as well as on binding of messenger RNA and fmet-transfer RNA to ribosomes and on the addition of the 50S ribosome to the 30S ribosome initiation complex. All of the reactions tested could be inhibited by ATA, and none of the tested steps was found to be uniquely sensitive to it. However, the total initiation steps were more sensitive to this chemical than the elongation steps; thus, under appropriate conditions this chemical can preferentially inhibit initiation while elongation of the polypeptide chain is not appreciably affected.     

The effect of pressure and temperature on sulphate-reducing bacteria and the action of biocides in oilfield water injection systems

The effects of elevated pressures and temperatures on the growth, morphology and metabolic activity of sulphate-reducing bacteria, isolated from the North Sea, are described. Pressure/temperature profiles, growth curves and sulphate reduction rates are presented for several isolates. The maximum pressure and temperature that supported growth were 65 000 KPa and 45°C respectively. The results are discussed in connection with water injection into oil-bearing reservoirs where there is a concern that generation of hydrogen sulphide by sulphate-reducing bacteria may lead to increased hydrogen sulphide levels (souring) in oil and gas, and to corrosion problems in production facilities. The bacteriostatic effects of a number of commercial biocides were enhanced at elevated hydrostatic pressures and temperatures.     

Mechanisms of vanadium action: insulin-mimetic or insulin-enhancing agent?

The demonstration that the trace element vanadium has insulin-like properties in isolated cells and tissues and in vivo has generated considerable enthusiasm for its potential therapeutic value in human diabetes. However, the mechanisms by which vanadium induces its metabolic effects in vivo remain poorly understood, and whether vanadium directly mimics or rather enhances insulin effects is considered in this review. It is clear that vanadium treatment results in the correction of several diabetes-related abnormalities in carbohydrate and lipid metabolism, and in gene expression. However, many of these in vivo insulin-like effects can be ascribed to the reversal of defects that are secondary to hyperglycemia. The observations that the glucose-lowering effect of vanadium depends on the presence of endogenous insulin whereas metabolic homeostasis in control animals appears not to be affected, suggest that vanadium does not act completely independently in vivo, but augments tissue sensitivity to low levels of plasma insulin. Another crucial consideration is one of dose-dependency in that insulin-like effects of vanadium in isolated cells are often demonstrated at high concentrations that are not normally achieved by chronic treatment in vivo and may induce toxic side effects. In addition, vanadium appears to be selective for specific actions of insulin in some tissues while failing to influence others. As the intracellular active forms of vanadium are not precisely defined, the site(s) of action of vanadium in metabolic and signal transduction pathways is still unknown. In this review, we therefore examine the evidence for and against the concept that vanadium is truly an insulin-mimetic agent at low concentrations in vivo. In considering the effects of vanadium on carbohydrate and lipid metabolism, we conclude that vanadium acts not globally, but selectively and by enhancing, rather than by mimicking the effects of insulin in vivo.     

A method for accounting for maintenance costs in flux balance analysis improves the prediction of plant cell metabolic phenotypes under stress conditions

Flux balance models of metabolism generally utilize synthesis of biomass as the main determinant of intracellular fluxes. However, the biomass constraint alone is not sufficient to predict realistic fluxes in central heterotrophic metabolism of plant cells because of the major demand on the energy budget due to transport costs and cell maintenance. This major limitation can be addressed by incorporating transport steps into the metabolic model and by implementing a procedure that uses Pareto optimality analysis to explore the trade‐off between ATP and NADPH production for maintenance. This leads to a method for predicting cell maintenance costs on the basis of the measured flux ratio between the oxidative steps of the oxidative pentose phosphate pathway and glycolysis. We show that accounting for transport and maintenance costs substantially improves the accuracy of fluxes predicted from a flux balance model of heterotrophic Arabidopsis cells in culture, irrespective of the objective function used in the analysis. Moreover, when the new method was applied to cells under control, elevated temperature and hyper‐osmotic conditions, only elevated temperature led to a substantial increase in cell maintenance costs. It is concluded that the hyper‐osmotic conditions tested did not impose a metabolic stress, in as much as the metabolic network is not forced to devote more resources to cell maintenance.     

Modeling of uncertainties in biochemical reactions

Mathematical modeling is an indispensable tool for research and development in biotechnology and bioengineering. The formulation of kinetic models of biochemical networks depends on knowledge of the kinetic properties of the enzymes of the individual reactions. However, kinetic data acquired from experimental observations bring along uncertainties due to various experimental conditions and measurement methods. In this contribution, we propose a novel way to model the uncertainty in the enzyme kinetics and to predict quantitatively the responses of metabolic reactions to the changes in enzyme activities under uncertainty. The proposed methodology accounts explicitly for mechanistic properties of enzymes and physico‐chemical and thermodynamic constraints, and is based on formalism from systems theory and metabolic control analysis. We achieve this by observing that kinetic responses of metabolic reactions depend: (i) on the distribution of the enzymes among their free form and all reactive states; (ii) on the equilibrium displacements of the overall reaction and that of the individual enzymatic steps; and (iii) on the net fluxes through the enzyme. Relying on this observation, we develop a novel, efficient Monte Carlo sampling procedure to generate all states within a metabolic reaction that satisfy imposed constrains. Thus, we derive the statistics of the expected responses of the metabolic reactions to changes in enzyme levels and activities, in the levels of metabolites, and in the values of the kinetic parameters. We present aspects of the proposed framework through an example of the fundamental three‐step reversible enzymatic reaction mechanism. We demonstrate that the equilibrium displacements of the individual enzymatic steps have an important influence on kinetic responses of the enzyme. Furthermore, we derive the conditions that must be satisfied by a reversible three‐step enzymatic reaction operating far away from the equilibrium in order to respond to changes in metabolite levels according to the irreversible Michelis–Menten kinetics. The efficient sampling procedure allows easy, scalable, implementation of this methodology to modeling of large‐scale biochemical networks. Biotechnol. Bioeng. 2011;108: 413–423. © 2010 Wiley Periodicals, Inc.     

Seasonal acclimation of prairie deer mice

Prairie deer mice responded to long nights by reducing their metabolic rates, core temperatures, thermal conductances and incremental metabolic responses to cold stimulus, while increasing their capacities for nonshivering thermogenesis. Some winter animals spontaneously entered daily torpor in the mornings and thereby further reduced their metabolic rates and core temperatures. Provision of exogenous melatonin (by subdermal implants) mimiced short photoperiod effects on metabolic rates and core temperatures of wild-caught, laboratory maintained animals. Provision of supplemental dietary tryptophan to laboratory animals conditioned to natural light cycles mimiced metabolic effects of long nights in summer animals, and further reduced metabolic rates of winter mice, but did not affect their core temperature levels. Newly caught, laboratory maintained deer mice responded to natural seasonal clues of shortphotoperiod and increased dietary tryptophan by reducing their resting energy requirements through both lower metabolic and lower core temperature levels. Short photoperiod and seasonal change also promoted gonadal involution, and resulted in more socially tolerant huddling by mice with reduced core temperature. Reduced 24-hour LH excretion rates were also observed in winter animals which were exposed to seasonal light cycles at warm (25°C) room temperatures. We propose that seasonal acclimatization involves pineal effects on sex hormone-influenced social behaviors and on resting metabolism. These effects serve to conserve resting energy expenditure and promote hypothermic insulation by wild prairie deer mice.     

The Ecological Component of Ecological Risk Assessment: Lessons from a Field Experiment

To improve ecological relevance, regulatory agencies are promoting assessments of effects at higher levels of organization, an objective that requires an understanding of current ecological theories. One such theory, hierarchy theory, contends that the effects of a disturbance acting at one level of organization (e.g., population) are not, as a rule, transmitted to higher levels of organization (e.g., community). Conversely, effects at higher levels of organization only occur if lower level variables have been affected. Further, responses to disturbance depend on disturbance history. In this study, I determined the effects of a disturbance treatment at the population, guild, and community levels of organization for vegetation in five wetlands with a disturbance history ranging from highly to rarely disturbed. The 2-year field experiment revealed that the effects of the disturbance treatment were most strongly felt at the population level of organization in wetlands without a history of disturbance. These observed impacts took place against a backdrop of constant change. Thus, the eventual disappearance of treatment effects was not due to a return to the pre-treatment state, but rather a return to a trajectory similar to that exhibited by the control plots. The implications of these results for ecological risk assessment are: (1) the observed effects of a stressor in a system cannot be extrapolated to other systems unless they have similar disturbance histories, (2) detecting effects before they become serious requires monitoring at lower levels of organization, (3) recovery to a naturally innate state is not a viable concept, and (4) the traditional approach of using one post-treatment measurement to determine if reference and impact sites differ is of very questionable value.     

The Cystic Fibrosis-causing Mutation ΔF508 Affects Multiple Steps in Cystic Fibrosis Transmembrane Conductance Regulator Biogenesis

The deletion of phenylalanine 508 in the first nucleotide binding domain of the cystic fibrosis transmembrane conductance regulator is directly associated with >90% of cystic fibrosis cases. This mutant protein fails to traffic out of the endoplasmic reticulum and is subsequently degraded by the proteasome. The effects of this mutation may be partially reversed by the application of exogenous osmolytes, expression at low temperature, and the introduction of second site suppressor mutations. However, the specific steps of folding and assembly of full-length cystic fibrosis transmembrane conductance regulator (CFTR) directly altered by the disease-causing mutation are unclear. To elucidate the effects of the ΔF508 mutation, on various steps in CFTR folding, a series of misfolding and suppressor mutations in the nucleotide binding and transmembrane domains were evaluated for effects on the folding and maturation of the protein. The results indicate that the isolated NBD1 responds to both the ΔF508 mutation and intradomain suppressors of this mutation. In addition, identification of a novel second site suppressor of the defect within the second transmembrane domain suggests that ΔF508 also effects interdomain interactions critical for later steps in the biosynthesis of CFTR.     

Wall structure of a bitunicate ascus

Summary Ultraviolet light-induced, bleached Euglena clones exhibit synchronous steps of cell division in response to daily cycles of light and dark. The cyclic division activity, in the bleached cells, will persist in constant lighting conditions with a period, independent of temperature, of about 24 h. This persisting rhythm of cell division supports the hypothesis that this phase of the cell cycle may be coupled to the fluctuations of the endogenous circadian clock of the cell.Newly isolated bleached clones are sensitive to light in their growth rates and metabolic characteristics, showing light induced difference in substrate-stimulated respiration, and production of the polyglucan, paramylon. After repeated subculturing of a bleached clone the photosensitivity of the metabolic characteristics and of the growth rate are diminished along with the ability to photo-entrain division synchrony. Division control and the induction of cell synchrony in this organism apparently involve both the temporal influence of the endogenous cell clock and one or more other photosensitive reactions in the metabolism of the cell.A unique culture mixing technique utilizing the bleached Euglena, failed to support the hypothesis of the involvement of intercellular communication in the maintenance of cell synchrony in constant lighting conditions.