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In the presence of cyanide, populations of yeast cells can exhibit sustained oscillations in the concentration of glycolytic metabolites, NADH and ATP. This study attempts to answer the long-standing question of whether and how oscillations of individual cells are synchronized. It shows that mixing two cell populations that oscillate 180 degrees out of phase only transiently abolishes the macroscopic oscillation. After a few minutes, NADH fluorescence of the mixed population resumes oscillations up to the original amplitude. At low cell densities, addition of acetaldehyde causes transient oscillations. At higher cell densities, where the oscillations are autonomous, 70 microM acetaldehyde causes phase shifts. Extracellular acetaldehyde is shown to oscillate around the 70 microM level. We conclude that acetaldehyde synchronizes the oscillations of the individual cells.

Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified and kinetically characterized. The enzymes were reconstituted in vitro to quantify the contribution of temperature instability of the pathway intermediates to carbon loss from the system. The reconstituted system, consisting of phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase and the fructose 1,6-bisphosphate aldolase/phosphatase, maintained a constant consumption rate of 3-phosphoglycerate and production of fructose 6-phosphate over a 1-h period. Cofactors ATP and NADPH were regenerated via pyruvate kinase and glucose dehydrogenase. A mathematical model was constructed on the basis of the kinetics of the purified enzymes and the measured half-life times of the pathway intermediates. The model quantitatively predicted the system fluxes and metabolite concentrations. Relative enzyme concentrations were chosen such that half the carbon in the system was lost due to degradation of the thermolabile intermediates dihydroxyacetone phosphate, glyceraldehyde 3-phosphate and 1,3-bisphosphoglycerate, indicating that intermediate instability at high temperature can significantly affect pathway efficiency. Database The mathematical models described here have been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.mib.ac.uk/ database/kouril/index.html. The investigation and complete experimental data set is available on the SEEK at https://seek.sysmo-db.org/investigations/51. Four enzymes of the gluconeogenic pathway in Sulfolobus solfataricus were purified, kinetically characterized and reconstituted in vitro at 70 °C to quantify the contribution of heat-unstable pathway intermediates to carbon loss from the system. A mathematical model could quantitatively predict the systems fluxes and metabolite concentrations. Approximately half the carbon in the system was lost due to degradation of thermolabile intermediates. © 2013 FEBS.

It is an accepted paradigm that extended stress predisposes an individual to pathophysiology. However, the biological adaptations to minimize this risk are poorly understood. Using a computational model based upon realistic kinetic parameters we are able to reproduce the interaction of the stress hormone cortisol with its two nuclear receptors, the high-affinity glucocorticoid receptor and the low-affinity pregnane X-receptor. We demonstrate that regulatory signals between these two nuclear receptors are necessary to optimize the body’s response to stress episodes, attenuating both the magnitude and duration of the biological response. In addition, we predict that the activation of pregnane X-receptor by multiple, low-affinity endobiotic ligands is necessary for the significant pregnane X-receptor-mediated transcriptional response observed following stress episodes. This integration allows responses mediated through both the high and low-affinity nuclear receptors, which we predict is an important strategy to minimize the risk of disease from chronic stress., Stress causes cortisol release and this is associated with the development of pathophysiology. Plant and colleagues use a computational model to reproduce the interaction of cortisol with its two nuclear receptors, and reveal how feed-forward and feed-back loops coordinate adaptation to repeated stress.

SUMMARYWe present a comprehensive overview of the hierarchical network of intracellular processes revolving around central nitrogen metabolism inEscherichia coli. The hierarchy intertwines transport, metabolism, signaling leading to posttranslational modification, and transcription. The protein components of the network include an ammonium transporter (AmtB), a glutamine transporter (GlnHPQ), two ammonium assimilation pathways (glutamine synthetase [GS]-glutamate synthase [glutamine 2-oxoglutarate amidotransferase {GOGAT}] and glutamate dehydrogenase [GDH]), the two bifunctional enzymes adenylyl transferase/adenylyl-removing enzyme (ATase) and uridylyl transferase/uridylyl-removing enzyme (UTase), the two trimeric signal transduction proteins (GlnB and GlnK), the two-component regulatory system composed of the histidine protein kinase nitrogen regulator II (NRII) and the response nitrogen regulator I (NRI), three global transcriptional regulators called nitrogen assimilation control (Nac) protein, leucine-responsive regulatory protein (Lrp), and cyclic AMP (cAMP) receptor protein (Crp), the glutaminases, and the nitrogen-phosphotransferase system. First, the structural and molecular knowledge on these proteins is reviewed. Thereafter, the activities of the components as they engage together in transport, metabolism, signal transduction, and transcription and their regulation are discussed. Next, old and new molecular data and physiological data are put into a common perspective on integral cellular functioning, especially with the aim of resolving counterintuitive or paradoxical processes featured in nitrogen assimilation. Finally, we articulate what still remains to be discovered and what general lessons can be learned from the vast amounts of data that are available now.

We present an experimental and computational pipeline for the generation of kinetic models of metabolism, and demonstrate its application to glycolysis in Saccharomyces cerevisiae. Starting from an approximate mathematical model, we employ a “cycle of knowledge” strategy, identifying the steps with most control over flux. Kinetic parameters of the individual isoenzymes within these steps are measured experimentally under a standardised set of conditions. Experimental strategies are applied to establish a set of in vivo concentrations for isoenzymes and metabolites. The data are integrated into a mathematical model that is used to predict a new set of metabolite concentrations and reevaluate the control properties of the system. This bottom-up modelling study reveals that control over the metabolic network most directly involved in yeast glycolysis is more widely distributed than previously thought.

The molecular basis of many forms of signal transfer in living organisms is provided via the transient phosphorylation of regulatory proteins by transfer of phosphoryl groups between these proteins. The dominant form of signal transduction in prokaryotic microorganisms proceeds via so-called two-component regulatory systems. These systems constitute phosphoryl transfer pathways, consisting of two or more components. Most of these pathways are linear, but some converge and some are divergent. The molecular properties of some of the well-characterised representatives of two-component systems comply with the requirements to be put upon the elements of a neural network: they function as logical operators and show the phenomenon of autoamplification. Because there are many phosphoryl transfer pathways in parallel and because there also appears to be cross-talk between these pathways, the total of all two-component regulatory systems in a single prokaryotic cell may show the typical characteristics of a 'phospho-neural network'. This may well lead to signal amplification, associative responses and memory effects, characteristics which are typical for neural networks. One of the main challenges in molecular microbial physiology is to determine the extent of the connectivity of the constituting elements of this presumed 'phospho-neural network', and to outline the extent of intelligence-like behaviour this network can generate. Escherichia coli is the organism of choice for this characterization.

The postulated strictly linear descriptions of the rate dependence of oxidative phosphorylation in skeletal muscle on the free energy of ATP hydrolysis (delta GP) over the range of physiological steady states fail to harmonize with reported findings of identical basal respiration rates in mammalian muscles at different delta GP values. The relevance of an extension of the strictly linear description to a description deriving from enzyme kinetics that predicts a sigmoidal dependence was investigated in human finger flexor muscle using 31P-nuclear magnetic resonance spectroscopy. At constant pH 7.0, the experimental variation of adenine nucleotide concentrations with power output, which reflects the rate of oxidative phosphorylation, was compared with predictions by various formulations of adenine nucleotide control of respiration. The quasi-linear sigmoidal description was found to be statistically equivalent but physiologically superior to the strictly linear description. The predicted maximal oxidatively sustained steady-state power output and rate-dependent sensitivity of respiration to changes in delta GP were in agreement both with theoretical considerations and with experimental observations in the present study and other studies of intact mammalian skeletal muscle.

Molecular biology for flux control P. R. ]ensen*, J. L. Snoept, D. Molenaart, W. C. van HeeswijktS, B. N. Kholodenkotts, A. A. van der Gugtenll and H. V. WesterhofftSlI *Department of Microbiology, Technical University of Denmark, Lyngby, Denmark, tDepartment of Microbial Physiology, Vrije Universiteit Amsterdam, de Boelelaan 1087, I08 I HV Amsterdam, The Netherlands, $E.C. Slater Institute, BioCentrum, University of Amsterdam, Amsterdam, The Netherlands, $A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia, and JIDepartment of Tumor Biology, The Netherlands Cancer InstitutdAvL, Amsterdam, The Netherlands 367

Allowing for creatine kinase buffering of changes in adenine nucleotide concentrations, and the known relationship between muscle performance and rate of ATP hydrolysis by myosin, the variation of exerted force with intracellular Gibbs enery of ATP hydrolysis is calculated for voluntary muscle contraction. The resulting relationship is sigmoidal, most of the operating range coinciding with the quasi-linear range around the inflection point. Finger-flexor muscle magnetic resonance spectroscopy data are shown to be in line with this prediction.

Relay or group-transfer pathways are important for metabolism and signal transduction. Yet, they are not addressed by standard metabolic control analysis. In this paper the control theory for this type of pathways is developed. Control coefficients are defined both with respect to modulation of enzyme concentration (enzyme control coefficient) and with respect to modulation of ‘elemental’ process activity (process control coefficient). Whereas the latter obeys the theorems of standard metabolic control theory, the more operational, former type of control coefficient obeys new control theorems: (i) the sum of enzyme control coefficients on the flux of group transfer equals 2 minus the control by pathway boundary substrates and products, divided by the extent to which the pathway enzymes are complexed, (ii) the sum of the controls on the concentration of any of the non-complexed pathway components is 1 (iii) the sum of the controls on the concentration of any of enzyme-enzyme complexes is 2, with the same corrections as above, (iv) the control exerted by enzyme concentrations can be calculated from the kinetic properties (elasticity coefficients).The implications for metabolism and signal transduction of the special control properties of relay pathways are discussed.

For an ‘ideal’ metabolic pathway, genetic modulation of the enzyme concentration and titration with different types of specific inhibitor all leads to the determination of the same quantitative indicator of the extent to which an enzyme controls the flux and metabolite concentrations: the control coefficient with respect to the enzyme concentration. By contrast, for a group-transfer pathway these methods reveal various modes of the control exerted by an enzyme on the flux and on the concentrations of pathway components. Modulation of gene expression allows one to determine the (classical) control coefficient with respect to the enzyme concentration. Titration with inhibitors (competitive or uncompetitive) that do not bind to enzyme-enzyme complexes leads to information on the classical control coefficient of the inhibited enzyme and on the relative concentrations of its different forms. Should such inhibitors be irreversible, the classical control coefficients can be measured directly. Titration with a purely non-competitive inhibitor (binding to all the complexes of the target enzyme) reveals the impact control coefficient, a measure of the total kinetic effect of that enzyme on the system. Combined approaches applied to intact systems will detect an expected variety of control properties that cannot be measured after the system has been disassembled.

Metabolic Control Analyses have led to significant advances in understanding the control of cell metabolism. However, the classical theory does not address all complex cases of the organization of cellular metabolism. Here the control theory is extended to include (i) pathways with high enzyme concentrations and moiety conservation and (ii) metabolic channelling in general. The new theory descends the microlevel of elemental steps into the reaction cycle of the enzymes and turns back to the macrolevel of complete enzyme reactions. We showed how the elemental control coefficients are related to the traditional flux control coefficients of the enzymes. We derived what the sums of the enzyme control coefficients are equal to in various non-ideal cell pathways.

The living cell functions by virtue of an enormous number of different processes. It is one of the most difficult challenges of modern biology to elucidate how all those processes are coordinated quantitatively so as to lead to a viable system with optimal responses to various changes in the environment. The biochemical and biophysical processes of the living cell do not constitute a network with random connections. In this paper we shall discuss that cell function is organized in hierarchical substructures. We will briefly touch on the topics of (i) metabolic control and regulated gene expression, (ii) time dependent metabolism in intact yeast cells, and (iii) metabolite channelling.

Xenopus laevis skin secretion contains a mixture of magainins, which are small positively charged oligopeptides with antimicrobial activity. In this study, we show that two of these peptides, i.e. magainin-2 and PGLa, are much more active in biological functions when added together than when added alone. This synergy applies for the antimicrobial activity of these peptides, and for the toxic effects on tumor cells. We show that this peptide combination is also synergistic when permeabilizing protein-free liposomes for glucose, when dissipating the membrane potential in cytochrome oxidase liposomes and Escherichia coli, and, reversibly, when stimulating respiration in the liposomes. The occurrence of synergy in these diverse systems (complex and simple) suggests that the biological synergy results from synergy in the primary activity of the magainin peptides, namely the permeabilization of free-energy transducing membranes, possibly by forming a multimeric transmembrane pore of mixed peptide composition. The antimicrobial activity of X. laevis skin secretions may be greatly enhanced by the application of this binary weapon.

The effect of calcium on the control exerted by the adenine nucleotide translocator over respiration in isolated heart mitochondria was investigated in order to determine whether calcium directly stimulates the translocator. At respiration rates intermediate between states 3 and 4, Ca2+is shown to increase the control over 2-oxoglutarate oxidation exerted by the adenine nucleotide translocator in rat heart mitochondria. This did not occur when succinate was the respiratory substrate, even though the control exerted by the translocator was substantial, indicating that Ca2+does not have a direct effect on the adenine nucleotide translocator. Ca2+increased the uncoupled oxidation rate of 2-oxoglutarate, but not succinate. Using the summation theorem for flux control, the effect of Ca2+is explained by a shift of the control over respiration rate toward the adenine nucleotide translocator, from the respiratory chain, presumably as the result of the activation of the 2-oxoglutarate dehydrogenase complex.

The usual equations expressing the enzyme control coefficients (quantitative indicators of ‘global’ control properties of a pathway) via the elasticity coefficients (reflecting local kinetic properties of an enzyme reaction), cannot be applied to a variety of ‘non-ideal’ pathways, in particular to pathways with metabolic channelling. Here we show that the relationship between the control and elasticity coefficients can be obtained by considering such a metabolic pathway as a network of elemental chemical conversions (steps). To calculate the control coefficients of enzymes one should first determine the elasticity coefficients of such elemental steps and then take their appropriate combinations. Although the method is illustrated for a channelled pathway it can be used for any non-ideal pathway including those with high enzyme concentrations where the sequestration of metabolites by enzymes cannot be neglected.

To study how changes in the [ATP]/[ADP] ratio affect the level of DNA supercoiling in Escherichia coli, the cellular content of H(+)-ATPase was modulated around the wild-type level. A relatively large drop in the [ATP]/[ADP] ratio from the normal ratio resulted in a small increase in the linking number of our reporter plasmid (corresponding to a small decrease in negative supercoiling). However, when cells depleted their carbon and energy source, the ensuing drop in energy state was accompanied by a strong increase in linking number. This increase was not due to reduced transcription of the DNA in the absence of growth substrate, since rifampin had virtually no effect on the plasmid linking number. To examine whether DNA supercoiling depends more strongly on the cellular energy state at low [ATP]/[ADP] ratios than at high ratios, we used cells that were already at a low energy state after substrate depletion; after the addition of an uncoupler to these cells, the [ATP]/[ADP] ratio decreased further, which resulted in a strong increase in plasmid linking number. Our results suggest that the strong thermodynamic control of DNA supercoiling takes over at low [ATP]/[ADP] ratios, whereas at high ratios homeostatic control mechanisms attenuate thermodynamic control.

The concept of a single rate-limiting step was proven to be too simplistic for understanding control and regulation of metabolism. Consequently, searches have identified relatively few steps with high control. Here we review a number of such searches and indicate what mechanisms may be responsible for this elusiveness of control. It turns out that this elusiveness of control has itself led to increased understanding of the roles played in metabolic control and regulation of such diverse factors as distributiveness of control, condition dependence, enzyme elasticity, homeostasis, control hierarchies, the input into a pathway, coenzyme sequestration, and redundancy and diversity of control function.

Various factors appear to control muscle energetics, often in conjunction. This calls for a quantitative approach of the type provided by Metabolic Control Analysis for intermediary metabolism and mitochondrial oxidative phosphorylation. To the extent that direct transfer of high energy phosphates and spatial organization plays a role in muscle energetics however, the standard Metabolic Control Theory does not apply, neither do its theorems regarding control. This chapter develops the Control Theory that does apply to the muscle system. It shows that direct transfer of high energy phosphates bestows a system with enhanced control: the sum of the control exerted by the participating enzymes on the flux of free energy form the mitochondrial matrix to the actinomyosin may well exceed the 100% mandatory for ideal metabolic pathways. It is also shown how sequestration of high energy phosphates may allow for negative control on pathway flux. The new control theory gives methods functionally to diagnose the extent to which channelling and metabolite sequestration occur.

The success rate of introducing new functions into a living species is still rather unsatisfactory. Much of this is due to the very essence of the living state, i.e. its robustness towards perturbations. Living cells are bound to notice that metabolic engineering is being effected, through changes in metabolite concentrations. In this study, we asked whether one could engage in such engineering without changing metabolite concentrations. We have illustrated that, in silico, one can do so in principle. We have done this for the case of substituting the yeast glucose transporter plus hexokinase for the Lactococcus lactis phosphotransferase system, in an L. lactis network, this engineering is 'silent' in terms of metabolite concentrations and almost all fluxes. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The control and regulation of metabolic systems are determined by their responses to changes in the internal metabolites (the internal state) and parameters of the system. In many cases, the concentrations of the intermediates are constrained by moiety conservations, for example those requiring that all intermediate forms of any enzyme sum to the conserved total concentration of that enzyme. In this study, we show how responses to changes in the internal state are related to responses to changes in the total amounts of conserved moieties. The relationship between these two different measures of control leads to a generalisation of the connectivity theorems. The results have important implications for the study of a variety of phenomena such as metabolite (coenzyme) sequestration, group-transfer and channelling. The relationships we derive make it possible to determine the control features of these pathways. As an illustration, two examples are chosen. The first shows the effect of sequestration of substrate moiety while the second deals with the sequestration of the enzyme moieties and enzyme/enzyme interactions.

The analogue of metabolic control theory is developed for the control of reactions catalyzed by single enzymes. The control exerted by any of the elemental transitions of enzyme catalytic cycles on reaction rate and on concentrations (probabilities) of enzyme states is quantified in line with the principle of detailed balance. For enzyme reactions with arbitrary kinetic schemes, e.g., with several enzyme cycles, reflecting coupling and slipping of reactions, it is derived what the various sums of the control coefficients are equal to (cycle summation theorems). Total control on flux, state probability and ratios of branch fluxes are 1, 0 and 0, respectively. The general connectivity theorems are derived which indicate how control is determined by the kinetics of the elemental steps. In addition, for enzymes catalyzing single (or completely coupled) processes the control coefficients are expressed in terms of actual and standard free energy differences across the steps. The prevalent qualitative contention that the step with the smallest forward rate constant, or with the largest free energy drop is the step limiting the performance of the enzyme is shown to fail. The new theory should allow subtle analysis of the control of an enzyme catalyzed reaction.

The extents to which different rate constants limit the steady-state rate of an isolated enzyme can be quantified as the control coefficients of those constants and elemental steps. We have found that the sum of the control coefficients of rate constants characterising unidirectional rates depleting a particular enzyme intermediate is equal to the concentration of that enzyme intermediate as a fraction of the total enzyme concentration. Together with simple measurements this powerful relation may be used (i) to estimate certain enzyme intermediate levels, in particular the free enzyme concentration, and (ii) to estimate the control coefficients of rate constants and steps.

Glycolytic oscillations can be induced by adding glucose to starved Saccharomyces cerevisiae cells and, after a steady state has been established, cyanide. Transient oscillations or limit-cycle oscillations can be induced depending on the growth phase in which the cells are harvested. To find what causes these differences in the dynamic behaviour, we analyzed glycolytic enzyme activities at different growth phases. The hexokinase activity increased by a factor of three after growth substrate transition from glucose to ethanol; the other measured activities remained constant. Cyanide was found not only to block respiration, but also to trap acetaldehyde. Both cyanide actions appear necessary for the occurrence of sustained glycolytic oscillations.

Metabolic control analysis can relate control properties of an intact system to kinetic properties (elasticity coefficients) of the enzymes within that system. The method formulating the former as matrix inverse of the latter is elaborated here for the general case and founded in standard metabolic control theory. Then a method is developed that accomplishes the reverse: it is shown that a matrix containing all elasticity coefficients and information concerning the pathway structure equals the inverse of a matrix containing flux and concentration control coefficients. As a consequence, by measuring the control properties of an intact system, one is able to deduce its in situ pathway structure and enzyme kinetic properties: This solves the ever-present question of whether the kinetic properties of enzymes in their isolated state differ from those under the conditions prevailing in the cell.

The spatial organization in photosystems containing chloroplast membranes is such that some of the relevant distances are of the order of the wavelength of the absorbed light. Consequently, interference between incident and reflected light may occur. This paper calculates the heterogeneity to be expected in the resultant light intensity. The pattern is determined by the wavelength-dependent phase shift of light during scattering. We show that in stroma-membranes this heterogeneity may well be responsible for paradoxical polarities of the photovoltaic effect in which flash illumination of a chloroplast suspension generates a macroscopic electric potential opposite to that expected from shading. The grana-stack dimensions are calculated to lead to the classical picture of shading, consistent with a “normal” photovoltaic effect. We demonstrate that the simultaneous presence of stroma- and grana-membranes may explain the observed wavelength dependence of the polarity of the photovoltaic effect.

Initial attempts to increase fermentative capacity of baker's yeast focussed on the overproduction of single enzymes, which proved to be insufficient. Nowadays many components of the system are monitored simultaneously in a search for a correlation with fermentative capacity. However, this strategy has not yet proven fruitful either. Here we investigate an element previously neglected, the glucose transporter, and find that a loss of glucose transport capacity correlates with a decrease of fermentative capacity during nutrient starvation. However the correlation is not unique, suggesting that the loss of fermentative capacity cannot be attributed to an inactivation of glucose transport alone. Our results suggest the necessity to use a detailed kinetic model as an underlying working hypothesis and to use Metabolic Control Analysis to examine the pathway's control properties.

In vitro enzymatic assays of cell-free extracts offer an opportunity to assess in vivo enzyme concentrations. If performed under conditions that resemble the conditions in vivo, they may also reveal some of the capacities and properties of the same enzymes in vivo; we shall call this the ex vivo approach. The kinetic characterization of purified enzymes has yet a different utility for systems biology, as does the in vivo determination of enzyme activities. All these approaches are different, and it is becoming important that the appropriate approach be used for the intended purpose. Here, we therefore discuss five approaches to the measurement of enzyme activity in terms of the source of the enzyme activity, the identity of the assay medium, and the purpose of the assay.

Absorption spectroscopy is one of the most widely used techniques employed for determining the concentrations of absorbing species (chromophores) in solutions. It is a nondestructive technique which biologists and biochemists and now systems biologists use to quantify the cellular components and characteristic parameters of functional molecules. This quantification is most relevant in the context of systems biology. For creating a quantitative depiction of a metabolic pathway, a number of parameters and variables are important and these need to be determined experimentally. This chapter describes the UV-visible absorption spectroscopy used to produce experimental data for bottom-up modeling approaches of systems biology which uses concentrations and kinetic parameters (K(m) and V(max)) of enzymes of metabolic/signaling pathways, intracellular concentrations of metabolites and fluxes. It also briefly describes the application of this technique for quantification of biomolecules and investigating biomolecular interactions.

Regulation analysis is a methodology that quantifies to what extent a change in the flux through a metabolic pathway is regulated by either gene expression or metabolism. Two extensions to regulation analysis were developed over the past years: (i) the regulation of V(max) can be dissected into the various levels of the gene-expression cascade, such as transcription, translation, protein degradation, etc. and (ii) a time-dependent version allows following flux regulation when cells adapt to changes in their environment. The methodology of the original form of regulation analysis as well as of the two extensions will be described in detail. In addition, we will show what is needed to apply regulation analysis in practice. Studies in which the different versions of regulation analysis were applied revealed that flux regulation was distributed over various processes and depended on time, enzyme, and condition of interest. In the case of the regulation of glycolysis in baker's yeast, it appeared, however, that cells that remain under respirofermentative conditions during a physiological challenge tend to invoke more gene-expression regulation, while a shift between respirofermentative and respiratory conditions invokes an important contribution of metabolic regulation. The complexity of the regulation observed in these studies raises the question what is the advantage of this highly distributed and condition-dependent flux regulation.

Systems biology has come of age. In most scientifically active countries, significant research programs are funded. Various scientific journals, standards, repositories, and Web sites are devoted to the topic. Systems biology has spun off new subdisciplines such as synthetic biology and systems medicine. There are training courses at the M.Sc. and Ph.D. level at various Universities. And various industries are engaging systems biology in their RD. Systems biology has also developed numerous new methodologies. This chapter attempts to organize these methodologies from the perspectives of the unique aims of systems biology, and by comparing with one of its parents, molecular biology.

Selective robustness is a key feature of biochemical networks. It confers a fitness benefit to organisms living in dynamic environments. The (in-)sensitivity of a network to external perturbations results from the interplay between network dynamics, structure and enzyme kinetics. In this work, we focus on the subtle interplay between robustness and control (fragility). We describe a quantitative method for defining the fragility and robustness of system fluxes to perturbations. We find that for many mathematical models of metabolic pathways, the robustness of fluxes vis-à-vis perturbations of all the enzyme activities is captured by a broad distribution of the robustness coefficients. We find that in cases where a metabolic pathway flux is made less robust with respect to the perturbation of a particular network step, the average robustness may still be increased. We then show that fragility is conserved upon a perturbation of network processes and equate fragility with control as defined in metabolic control analysis. This highlights the non-intuitive nature of the interplay between fragility and robustness and the need for a dynamic network understanding. © 2012 The Authors Journal compilation © 2012 FEBS.

5-Oxoproline (5-OP; pyroglutamate) is an intermediate in the biosynthesis of the endogenous tripeptide glutathione and has been seen to be elevated in the biofluids and tissues of rats following the administration of glutathione-depleting hepatotoxic xenobiotics such as acetaminophen (paracetamol), bromobenzene and ethionine. As 5-OP is a potential biomarker for hepatotoxicity HPLC-MS/MS methods have been developed for its quantification in in vitro cell culture media and rat plasma. For the cell culture media the lower limit of quantification (LLOQ), defined as the lowest concentration on the calibration curve, was 10 ng/ml. Minimal carry over was observed for cell culture media between injections (less than 5% at all concentrations examined), precision and accuracy were generally better than 20% for within and between day analyses. For rat plasma a LLOQ of 50 ng/ml was obtained. Carry over for plasma was less than 5% for all concentrations, within and between batch accuracy and precision were generally better than 20%. The methods were linear for both sample types from the LLOQ up to 1. μg/ml. For samples obtained from rats subjected to chronic administration of the hepatotoxin methapyrilene, concentrations of 5-OP were not observed to increase significantly at any time point compared to controls. 5-OP was also determined in the culture media of human liver epithelial (THLE) cells transfected with cytochrome P450 2E1 (THLE-2E1). Following exposure of THLE-2E1 cells to acetaminophen, large increases in the concentrations of 5-OP were observed, which correlated with reduced cellular glutathione content and with cell toxicity. These results show that LC-MS/MS can be used to perform rapid, sensitive, and quantitative determination of 5-OP in vivo and in vitro and will enable additional investigations into the utility of 5-OP as a biomarker of liver drug-induced liver injury. © 2011 Elsevier B.V.

Chemical equations are normally written in terms of specific ionic and elemental species and balance atoms of elements and electric charge. However, in a biochemical context it is usually better to write them with ionic reactants expressed as totals of species in equilibrium with each other. This implies that atoms of elements assumed to be at fixed concentrations, such as hydrogen at a specified pH, should not be balanced in a biochemical equation used for thermodynamic analysis. However, both kinds of equations are needed in biochemistry. The apparent equilibrium constant K' for a biochemical reaction is written in terms of such sums of species and can be used to calculate standard transformed Gibbs energies of reaction Δ(r)G'°. This property for a biochemical reaction can be calculated from the standard transformed Gibbs energies of formation Δ(f)G(i)'° of reactants, which can be calculated from the standard Gibbs energies of formation of species Δ(f)G(j)° and measured apparent equilibrium constants of enzyme-catalyzed reactions. Tables of Δ(r)G'° of reactions and Δ(f)G(i)'° of reactants as functions of pH and temperature are available on the web, as are functions for calculating these properties. Biochemical thermodynamics is also important in enzyme kinetics because apparent equilibrium constant K' can be calculated from experimentally determined kinetic parameters when initial velocities have been determined for both forward and reverse reactions. Specific recommendations are made for reporting experimental results in the literature.

The utility of model repositories is discussed in the context of systems biology (SB). It is shown how such repositories, and in particular their live versions, can be used for computational SB: we calculate the robustness of the yeast glycolytic network with respect to perturbations of one of its enzyme activities and one transport system. The robustness with respect to perturbations in the key enzyme phosphofructokinase is surprisingly large. We then note the upcoming convergence of pharmacokinetics–pharmacodynamics (PK–PD) and bottom-up SB. In PK alone, quite a few one-, two- or more compartment models provide valuable initial guesses and insights into the absorption, distribution, metabolism and excretion of particular drugs. These models are phenomenological however, forbidding implementation of molecule-based tools and network information. In order to facilitate a fruitful synergy between SB and PK–PD, and between PK and PD, we present a new platform that combines standard phenomenological models used in the PK/PD field with mechanism-based SB models and approaches.

Dupuytren's disease (DD) is an ill-defined fibroproliferative disorder of the palm of the hands leading to digital contracture. DD commonly occurs in individuals of northern European extraction. Cellular components and processes associated with DD pathogenesis include altered gene and protein expression of cytokines, growth factors, adhesion molecules, and extracellular matrix components. Histology has shown increased but varying levels of particular types of collagen, myofibroblasts and myoglobin proteins in DD tissue. Free radicals and localised ischaemia have been suggested to trigger the proliferation of DD tissue. Although the existing available biological information on DD may contain potentially valuable (though largely uninterpreted) information, the precise aetiology of DD remains unknown. Systems biology combines mechanistic modelling with quantitative experimentation in studies of networks and better understanding of the interaction of multiple components in disease processes. Adopting systems biology may be the ideal approach for future research in order to improve understanding of complex diseases of multifactorial origin. In this review, we propose that DD is a disease of several networks rather than of a single gene, and show that this accounts for the experimental observations obtained to date from a variety of sources. We outline how DD may be investigated more effectively by employing a systems biology approach that considers the disease network as a whole rather than focusing on any specific single molecule. © 2011 BioMed Central Ltd.

This chapter discusses the silicon cell paradigm , i.e. the existing systems biology activity of making experiment-based computer replica of parts of biological systems. Now that such mathematical models are accessible to in silico experimentation through the World-Wide Web , a new future has come to biology. Some experimentation can now be done in silico, leading to significant discoveries of new mechanisms of robustness , of new drug targets , as well as to harder validations or falsifications of biological hypotheses. One aspect of this future is the association of such live models into models that simulate larger parts of the human body, up to organs and the whole individual. Reasons to embark on this type of systems biology, as well as some of the challenges that lie ahead, are discussed. It is shown that true silicon cell models are hard to obtain. Shortcut solutions are indicated. One of the major attempts at silicon cell systems biology, in the Manchester Centre for Integrative Systems Biology, is discussed in some detail. Early attempts at higher order, human, silicon cell models are described briefly, one addressing interactions between intracellular compartments and a second trying to deal with interactions between organs .

Absorption spectroscopy is one of the most widely used techniques employed for determining the concentrations of absorbing species (chromophores) in solutions. It is a nondestructive technique which biologists and biochemists and now systems biologists use to quantify the cellular components and characteristic parameters of functional molecules. This quantification is most relevant in the context of systems biology. For creating a quantitative depiction of a metabolic pathway, a number of parameters and variables are important and these need to be determined experimentally. This chapter describes the UV–visible absorption spectroscopy used to produce experimental data for bottom-up modeling approaches of systems biology which uses concentrations and kinetic parameters (Km and Vmax) of enzymes of metabolic/signaling pathways, intracellular concentrations of metabolites and fluxes. It also briefly describes the application of this technique for quantification of biomolecules and investigating biomolecular interactions.

In vitro enzymatic assays of cell-free extracts offer an opportunity to assess in vivo enzyme concentrations. If performed under conditions that resemble the conditions in vivo, they may also reveal some of the capacities and properties of the same enzymes in vivo; we shall call this the ex vivo approach. The kinetic characterization of purified enzymes has yet a different utility for systems biology, as does the in vivo determination of enzyme activities. All these approaches are different, and it is becoming important that the appropriate approach be used for the intended purpose. Here, we therefore discuss five approaches to the measurement of enzyme activity in terms of the source of the enzyme activity, the identity of the assay medium, and the purpose of the assay.

Systems biology has come of age. In most scientifically active countries, significant research programs are funded. Various scientific journals, standards, repositories, and Web sites are devoted to the topic. Systems biology has spun off new subdisciplines such as synthetic biology and systems medicine. There are training courses at the M.Sc. and Ph.D. level at various Universities. And various industries are engaging systems biology in their R&D. Systems biology has also developed numerous new methodologies. This chapter attempts to organize these methodologies from the perspectives of the unique aims of systems biology, and by comparing with one of its parents, molecular biology.

A multidrug resistant (MDR) human non-small cell lung carcinoma cell line, SW-1573/2R120 (2R120), not containing the drug-efflux pump P-glycoprotein (PgP), has been studied for the transport of daunorubicin (DN) across the cellular plasma membrane. Earlier, reduced initial DN-uptake rates and lower cellular DN steady-state concentrations were found for this cell line, when it was compared to the SW-1573 wild-type cell line. This finding was an indication for the presence of another cellular drug-efflux pump. However, we found similar DN-efflux rates in drug-free medium for the two cell lines, while for Pgp-containing MDR SW-1573/2R160 (2R160) cells the efflux rate was increased compared to wild-type cells. In order to elucidate differences in DN transport across the cellular plasma membrane, the association of DN with plasma membranes of intact cells was investigated, using fluorescence-resonance-energy transfer. For this purpose, the plasma-membrane probe 1-(4-trimethyl-ammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) was chosen since, because of the overlap between the emission spectrum of TMA-DPH and the excitation spectrum of DN, transfer of energy can be achieved from TMA-DPH to DN. Cells were loaded with TMA-DPH and, after addition of 10 microM DN, the TMA-DPH fluorescence was quenched. Rapid initial quenching proved to be similar in the MDR 2R160 (Pgp-containing) cells and in the SW-1573 wild-type cells (21 +/- 1% and 20 +/- 2%, respectively), but was less in the MDR 2R120 cells not containing Pgp (14 +/- 1%). This finding correlated with a lowered amount of DN dissolved in the plasma membrane of 2R120 cells. We interpret these data to be the result of a 'vacuum-cleaner' pumping system other than Pgp which removes DN from a plasma membrane compartment and equilibrates relatively slowly with the interior of the cell.

The control of enzymes and substrates on the flux through microbial metabolic pathways can be quantified in terms of flux control coefficients. In pathways involving group transfer, the summation theorem for flux control by the enzymes has to be modified: the sum of control by all enzymes is between 1 and 2. The phosphoenolpyruvate:glucose phosphotransferase system is such a pathway. Experimental determination of the control by the enzymes in this pathway is under way. The control of the enzymes on the glycolytic flux in yeast is low, with the possible exception of the uptake step. In Klebsiella pneumoniae potassium and ammonium ions can simultaneously be 'limiting', (i.e. have significant control on growth) at pH 6, but not at pH 8. This may be due to the fact that at pH 8 the high-affinity potassium uptake system is absent.

The H(+)-ATPase plays a central role in Escherichia coli free-energy transduction and hence in E. coli physiology. We here investigate the extent to which this enzyme also controls the growth rate, growth yield, and respiratory rate of E. coli. We modulate the expression of the atp operon and determine the effect on said properties. When quantified in terms of control coefficients, we find that, in the wild-type cell growing on glucose in minimal medium, this key enzyme (H(+)-ATPase) exerts virtually no control on growth rate (magnitude of C < 0.01), a minor positive control on growth yield (C = 0.15), and a small but negative control on respiration rate (C = -0.25). The control the enzyme exerts on the consumption rate of the carbon and free-energy substrate is negative (C = -0.15). We also studied how the control coefficients themselves vary with the expression of the atp operon. As the level of expression of the atp operon was reduced, the control exerted by the H(+)-ATPase on growth rate and growth yield increased slightly; the control on growth rate passed through a maximum (C = 0.1) and disappeared when the atp operon was not expressed at all, reflecting that with this substrate there are alternative routes for ATP synthesis. At elevated levels of the H(+)-ATPase compared to the wild type, the control exerted by the enzyme on growth rate became negative. The evolutionary context of the absence of control by the atp operon on growth rate is discussed.

In ‘simple’ metabolic pathways the response to an external signal is readily described in terms of the effect of the signal on its receptor enzyme and the control exerted by that enzyme. We show here that in the response of ‘channelled’ pathways to such a signal, additional terms appear that reflect the direct enzyme-enzyme interactions. They tend to enhance the responsiveness of the pathway. The normalized value of the response is called the signal transduction coefficient. We show that in channelled pathways these coefficients are usually larger than in corresponding non-channelled (simple) pathways.

In simple metabolic pathways the control exerted by enzyme concentrations on the pathway flux adds up to one when the control is quantified in terms of control coefficients. In this paper we demonstrate that this classical summation theorem has to be modified in pathways where the enzymes participate by transferring a group between each other. We derive the corresponding new control theorem and show how it is consistent with standard metabolic control analysis. In group-transfer pathways lacking enzyme complexes, the sum of the flux control by enzyme concentrations and by the donor and acceptor couples of the pathway, equals two. In group-transfer pathways with enzyme-enzyme interactions the flux control by the dissociation rate constants of the enzyme-enzyme complexes must be added to obtain this sum of two. In all cases, the sum of the controls by all reaction activities remains one. Both by using the new theorem and by numerical simulations, we then demonstrate that, in group-transfer pathways with or without enzyme interactions, the sum of the control of enzymes on the pathway flux is higher than one and can reach a value of two. The total control of all enzymes on the concentration of any intermediate either with or without the transferred group can be equal to one, rather than to the zero found in the classical case. Examples of group-transfer pathways are the bacterial phosphoenolpyruvate:sugar phosphotransferase system, the main pathway for uptake of sugars in Enterobacteriaceae, and the electron-transfer chain in free-energy transducing membranes.

The, submaximal, thermodynamic efficiency of aerobic microbial growth depends on whether no consumption / production of 02, or no carbon product other than biomass, is admitted into the assimilation equation. When the growth substrate has a low degree of reduction, the former efficiency approaches 22 % whilst the latter approaches 54 %. Expectations for maximal growth rate at optimum efficiency and for maximum economic growth rate at optimum efficiency are 24 and 54 %, respectively. A new control analysis method is derived that may be used to deduce properties of intracellular processes from the control properties of the cell as a whole.

With the aid of recA-lacZ fusion strains, the in vivo regulation of the Pseudomonas stutzeri recA gene has been studied. It is shown that expression of this gene can be induced with a variety of DNA damaging agents, as well as with agents that interfere with DNA replication. For this induction, the presence of an active RecA protein is essential. Sequence analysis of the promoter region of the P. stutzeri recA gene showed that its open reading frame is preceded by an SOS-box, suggesting a regulation of its expression, similar to the regulation of recA expression in Escherichia coli.

The fact that information flows from DNA to RNA to protein to function suggests that regulation is ‘hierarchical’, i.e. dominated by regulation of gene expression. In the case of dominant regulation at the metabolic level, however, there is no quantitative relationship between mRNA levels and function. We here develop a method to quantitate the relative contributions of metabolic and hierarchical regulation. Applying this method to the glycolytic flux in three species of parasitic protists, we conclude that it is rarely regulated by gene expression alone. This casts strong doubts on whether transcriptome and proteome analysis suffices to assess biological function.