A dynamically coherent pattern of rhythms that matches between distant species across the evolutionary scale.

We address the temporal organization of circadian and ultradian rhythms, crucial for understanding biological timekeeping in behavior, physiology, metabolism, and alignment with geophysical time. Using a newly developed five-steps wavelet-based approach to analyze high-resolution time series of metabolism in yeast cultures and spontaneous movement, metabolism, and feeding behavior in mice, rats, and quails, we describe a dynamically coherent pattern of rhythms spanning over a broad range of temporal scales (hours to minutes). The dynamic pattern found shares key features among the four, evolutionary distant, species analyzed. Specifically, a branching appearance given by splitting periods from 24 h into 12 h, 8 h and below in mammalian and avian species, or from 14 h down to 0.07 h in yeast. Scale-free fluctuations with long-range correlations prevail below ~ 4 h. Synthetic time series modeling support a scenario of coexisting behavioral rhythms, with circadian and ultradian rhythms at the center of the emergent pattern observed.

Restoration of energy homeostasis by SIRT6 extends healthy lifespan.

Aging leads to a gradual decline in physical activity and disrupted energy homeostasis. The NAD+-dependent SIRT6 deacylase regulates aging and metabolism through mechanisms that largely remain unknown. Here, we show that SIRT6 overexpression leads to a reduction in frailty and lifespan extension in both male and female B6 mice. A combination of physiological assays, in vivo multi-omics analyses and 13C lactate tracing identified an age-dependent decline in glucose homeostasis and hepatic glucose output in wild type mice. In contrast, aged SIRT6-transgenic mice preserve hepatic glucose output and glucose homeostasis through an improvement in the utilization of two major gluconeogenic precursors, lactate and glycerol. To mediate these changes, mechanistically, SIRT6 increases hepatic gluconeogenic gene expression, de novo NAD+ synthesis, and systemically enhances glycerol release from adipose tissue. These findings show that SIRT6 optimizes energy homeostasis in old age to delay frailty and preserve healthy aging.

Antioxidant defences of Spironucleus vortens: Glutathione is the major non-protein thiol.

The aerotolerant hydrogenosome-containing piscine diplomonad, Spironucleus vortens, is able to withstand high fluctuations in O2 tensions during its life cycle. In the current study, we further investigated the O2 scavenging and antioxidant defence mechanisms which facilitate the survival of S. vortens under such oxidizing conditions. Closed O2 electrode measurements revealed that the S. vortens ATCC 50386 strain was more O2 tolerant than a freshly isolated S. vortens intestinal strain (Sv1). In contrast to the related human diplomonad, Giardia intestinalis, RP-HPLC revealed the major non-protein thiols of S. vortens to be glutathione (GSH, 776 nmol/107 cells) with cysteine and H2S as minor peaks. Furthermore, antioxidant proteins of S. vortens were assayed enzymatically and revealed that S. vortens possesses superoxide dismutase and NADH oxidase (883 and 37.5nmol/min/mg protein, respectively), but like G. intestinalis, lacks catalase and peroxidase activities. Autofluorescence of NAD(P)H and FAD alongside the fluorescence of the GSH-adduct in monochlorobimane-treated live organisms allowed the monitoring of redox balances before and after treatment with inhibitors, metronidazole and auranofin. H2O2 was emitted into the exterior of S. vortens at a rate of 2.85 pmol/min/106 cells. Metronidazole and auranofin led to depletion of S. vortens intracellular NAD(P)H pools and an increase in H2O2 release with concomitant oxidation of GSH, respectively. Garlic-derived compounds completely inhibited O2 consumption by S. vortens (ajoene oil), or significantly depleted the intracellular GSH pool of the organism (allyl alcohol and DADS). Hence, antioxidant defence mechanisms of S. vortens may provide novel targets for parasite chemotherapy.

Redox-optimized ROS balance: a unifying hypothesis.

While it is generally accepted that mitochondrial reactive oxygen species (ROS) balance depends on the both rate of single electron reduction of O2 to superoxide (O2.-) by the electron transport chain and the rate of scavenging by intracellular antioxidant pathways, considerable controversy exists regarding the conditions leading to oxidative stress in intact cells versus isolated mitochondria. Here, we postulate that mitochondria have been evolutionarily optimized to maximize energy output while keeping ROS overflow to a minimum by operating in an intermediate redox state. We show that at the extremes of reduction or oxidation of the redox couples involved in electron transport (NADH/NAD+) or ROS scavenging (NADPH/NADP+, GSH/GSSG), respectively, ROS balance is lost. This results in a net overflow of ROS that increases as one moves farther away from the optimal redox potential. At more reduced mitochondrial redox potentials, ROS production exceeds scavenging, while under more oxidizing conditions (e.g., at higher workloads) antioxidant defenses can be compromised and eventually overwhelmed. Experimental support for this hypothesis is provided in both cardiomyocytes and in isolated mitochondria from guinea pig hearts. The model reconciles, within a single framework, observations that isolated mitochondria tend to display increased oxidative stress at high reduction potentials (and high mitochondrial membrane potential, Psim), whereas intact cardiac cells can display oxidative stress either when mitochondria become more uncoupled (i.e., low Psim) or when mitochondria are maximally reduced (as in ischemia or hypoxia). The continuum described by the model has the potential to account for many disparate experimental observations and also provides a rationale for graded physiological ROS signaling at redox potentials near the minimum.

From mitochondrial dynamics to arrhythmias.

The reactive oxygen species (ROS)-dependent mitochondrial oscillator described in cardiac cells exhibits at least two modes of function under physiological conditions or in response to metabolic and oxidative stress. Both modes depend upon network behavior of mitochondria. Under physiological conditions cardiac mitochondria behave as a network of coupled oscillators with a broad range of frequencies. ROS weakly couples mitochondria under normal conditions but becomes a strong coupling messenger when, under oxidative stress, the mitochondrial network attains criticality. Mitochondrial criticality is achieved when a threshold of ROS is overcome and a certain density of mitochondria forms a cluster that spans the whole cell. Under these conditions, the slightest perturbation triggers a cell-wide collapse of the mitochondrial membrane potential, Delta psi(m), visualized as a depolarization wave throughout the cell which is followed by whole cell synchronized oscillations in Delta psi(m), NADH, ROS, and GSH. This dynamic behavior scales from the mitochondrion to the cell by driving cellular excitability and the whole heart into catastrophic arrhythmias. A network collapse of Delta psi(m) under criticality leads to: (i) energetic failure, (ii) temporal and regional alterations in action potential (AP), (iii) development of zones of impaired conduction in the myocardium, and, ultimately, (iv) a fatal ventricular arrhythmia.

Fluorescein diacetate hydrolysis as a measure of fungal biomass in soil.

The fatty acid methyl esters of lipids extracted from an agricultural soil in the preharvest period of soybean or middle growth cycle from wheat were characterized and quantified by gas-liquid chromatography. The fatty acids 18:2omega6 and 16:1omega5 were used as markers of saprotrophic and arbuscular mycorrhizal fungi. In parallel, biomass estimation through plate counts in selective media for cellulolytic and saprotrophic fungi was also performed all throughout a soybean crop or middle growth cycle of wheat. As an enzymatic method, the fluorescein diacetate (FDA) hydrolytic activity of the samples was determined. Owing to the high relationship exhibited by FDA hydrolysis with organic carbon and total nitrogen content of soil, the enzymatic activity was correlated with the microbial biomass estimated through marker lipids or plate counts. The results obtained point out that FDA hydrolysis may be used as a rapid, cheap, and reliable estimator of fungal biomass.

Measurement of the glycogen synthetic pathway in permeabilized cells of cyanobacteria.

A simple, rapid and reliable procedure for permeabilizing cyanobacterial cells and measuring the glycogen synthetic pathway in situ, is presented. Cells from Anabaena sp. strain PCC 7120 were permeabilized with a mixture of toluene:ethanol (1:4 v/v). Fluorescence microscopy of cells incubated with fluorescein diacetate showed Anabaena non-permeabilized cells as green fluorescents, whereas permeabilized (viable) cells exhibited the intrinsic red fluorescence. Labelled alpha-1,4-glucan was recovered when permeabilized cells were incubated with the substrates of ADP-glucose pyrophosphorylase or glycogen synthase. The kinetic and regulatory properties of both enzymes could be reproduced in situ. The simplicity of the procedure and the ability to measure in situ glucan fluxes show the methodology as useful for studying the intracellular regulation of storage polysaccharides in a photosynthetic prokaryote.

Ultrasensitivity in (supra)molecularly organized and crowded environments.

The ultrasensitive response of biological systems is a more sensitive one than that expected from the classical hyperbola of Michaelis-Menten kinetics, and whose physiological relevance depends upon the range of variation of substrate or effector for which ultrasensitivity is observed. Triggering and modulation of the ultrasensitive response in enzymatic and cellular systems are reviewed. Several demonstrations of ultrasensitive behavior in cellular systems and its impact on the amplification properties in signalling cascades and metabolic pathways are also highlighted. It is shown that ubiquitous cytoskeletal proteins may up- or downmodulate ultrasensitivity under physico-chemical conditions resembling those predominant in cells.

Why homeodynamics, not homeostasis?

Ideas of homeostasis derive from the concept of the organism as an open system. These ideas can be traced back to Heraclitus. Hopkins, Bernard, Hill, Cannon, Weiner and von Bertalanffy developed further the mechanistic basis of turnover of biological components, and Schoenheimer and Rittenberg were pioneers of experimental approaches to the problems of measuring pool sizes and dynamic fluxes. From the second half of the twentieth century, a biophysical theory mainly founded on self-organisation and Dynamic Systems Theory allowed us to approach the quantitative and qualitative analysis of the organised complexity that characterises living systems. This combination of theoretical framework and more refined experimental techniques revealed that feedback control of steady states is a mode of operation that, although providing stability, is only one of many modes and may be the exception rather than the rule. The concept of homeodynamics that we introduce here offers a radically new and all-embracing concept that departs from the classical homeostatic idea that emphasises the stability of the internal milieu toward perturbation. Indeed, biological systems are homeodynamic because of their ability to dynamically self-organise at bifurcation points of their behaviour where they lose stability. Consequently, they exhibit diverse behaviour; in addition to monotonic stationary states, living systems display complex behaviour with all its emergent characteristics, i.e., bistable switches, thresholds, waves, gradients, mutual entrainment, and periodic as well as chaotic behaviour, as evidenced in cellular phenomena such as dynamic (supra)molecular organisation and flux coordination. These processes may proceed on different spatial scales, as well as across time scales, from the very rapid processes within and between molecules in membranes to the slow time scales of evolutionary change. It is dynamic organisation under homeodynamic conditions that make possible the organised complexity of life.

Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase.

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor P(i). Molecular 'crowding' renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

Chaotic dynamics and fractal space in biochemistry: simplicity underlies complexity.

In this work we attempt to analyze the coupling between the dynamics of biochemical reactions (especially chaotic dynamics), and the geometry of cytoarchitecture (especially fractal ultrastructure), because of its importance and consequences for the ultradian dynamic behaviour of cells. Fractal geometry in intracellular macromolecular assemblies suggests that chaotic dynamics occur during their organization. Non-linear interactions in and between spatial and temporal domains and over wide ranges of scales underlie the emergent properties of complex biological systems.

Dynamics of metabolism and its interactions with gene expression during sporulation in Saccharomyces cerevisiae.

The dynamics of metabolism has been shown to be involved in the triggering of events that are concurrent with sporulation of the budding yeast Saccharomyces cerevisiae. Indeed, quantitative correlations have been demonstrated between sporulation and the rate of carbon substrate or oxygen consumption, and the fluxes through gluconeogenic and glyoxylate cycle pathways. The results suggest that an imbalance between catabolic and anabolic fluxes influences the occurrence of the differentiation process. The hypothesis that the initiation of sporulation is triggered by the accumulation of an intracellular metabolite is confronted with the notion that intermediary metabolism and the expression of genes involved in sporulation interact to trigger the differentiation process. Several pieces of evidence indicate that derepression of the gluconeogenic pathway is crucial for the initiation of sporulation. One of the possible pathways through which glucose repression hampers sporulation might be the repression of gluconeogenesis as well as that of respiratory activity, in turn modulating the expression of IMEL++. The stages defined in the dynamics of sporulating cultures, namely readiness and commitment, are related to metabolic events associated with sporulation. An interpretation in terms of metabolic flux dynamics is given to the reversal of commitment occurring when the normal progression to sporulation is somehow blocked. The quantitative data are here integrated in a model attempting to simulate the dynamics of metabolic as well as cellular events during sporulation. The model is envisaged as a test of the hypothesis that an imbalance between anabolism and catabolism is involved in initiation of the sporulation process. It is proposed that such an imbalance may be a signal for differential gene expression associated with the differentiation pathway.

Modulation of urokinase-type plasminogen activator and metalloproteinase activities in cultured mouse mammary-carcinoma cells: enhancement by paclitaxel and inhibition by nocodazole.

Paclitaxel is a potent anti-tumor drug used in the treatment of breast cancer. It induces de-centralization of the microtubular system in tumor cells, blocking cell division. In the search for dissemination to a secondary site, cancer cells are capable of degrading most components of the extracellular matrix via an extracellular proteolytic cascade, including urokinase-type plasminogen activator (uPA) and the matrix metalloproteinases (MMPs). In the present study, the effects of paclitaxel and nocodazole, 2 drugs known to affect microtubules with opposite mechanisms of action, have been tested for their effect on the secretion of uPA and MMPs in cultures of F3II mouse mammary-tumor cells. Tumor-derived uPA activity significantly increased after pre-treatment of tumor cells for 24 hr with micromolar concentrations of paclitaxel (4 microM), while decreasing after pre-treatment with nocodazole (1 microM). A similar modulation was found for MMP-9 by zymographic analysis. Immunofluorescence and Western-blot analysis confirmed the formation of parallel microtubule fragments in paclitaxel-treated cells and almost complete de-polymerization of microtubules in nocodazole-treated ones. Our data suggest that, through opposite actions on microtubule organization and dynamics, paclitaxel and nocodazole exert inverse modulation of tumor-derived proteolytic activity in mammary tumor cells.

Ultrasensitive glycogen synthesis in Cyanobacteria.

Cyanobacter ADPglucose pyrophosphorylase exhibits a ultrasensitive response in activity towards its allosteric effector 3-phosphoglycerate, elicited by orthophosphate and polyethyleneglycol-induced molecular crowding. The ultrasensitive response was observed either when the enzyme operates in the zero or first order region for its physiological substrates. The ultrasensitivity exhibited maximal amplification factors of 15-19-fold with respect to 1% of the maximal system velocity. Only a 2.4-3.8-fold increase in 3PGA concentration was necessary to augment the flux from 10% to 90% through AGPase as compared with 200-fold required for the control. The results are discussed in terms of finely tuned regulatory mechanisms of polysaccharide synthesis in oxygenic photosynthetic organisms.

Quantitation of the effects of disruption of catabolite (de)repression genes on the cell cycle behavior of Saccharomyces cerevisiae.

We have studied the effect of disrupting catabolite (de)repression genes SNF1, SNF4, and MIG1 on the cell cycle behavior of the CEN. PK122 wild type (WT) strain of Saccharomyces cerevisiae by flow cytometry in glucose-limited chemostat cultures or batch growth in the presence of different carbon sources. Through a combination of flow cytometry of propidium iodide-stained cells and mathematical modeling we showed that the deletion of the SNF4 gene provoked a decrease in the length of G1 with respect to the WT strain along with a smaller difference in the cell cycle length of parent and daughter cells. snf1 and mig1 mutants exhibited slightly shorter G1 respect to the WT. Additionally, in the mig1 mutant the cell cycle length of parent and daughter cells was slightly altered. The results obtained are in agreement with the view that the SNF4 gene is involved in the regulation of cell cycle in yeast.

Catabolite repression mutants of Saccharomyces cerevisiae show altered fermentative metabolism as well as cell cycle behavior in glucose-limited chemostat cultures.

In glucose-limited continuous cultures, a Crabtree positive yeast such as Saccharomyces cerevisiae displays respiratory metabolism at low dilution rates (D) and respiro-fermentative metabolism at high D. We have studied the onset of ethanol production and cell cycle behavior in glucose-limited chemostat cultures of the wild type S. cerevisiae strain CEN.PK122 (WT) and isogenic mutants, snf1 (cat1) and snf4 (cat3) defective in proteins involved in catabolite derepression and the mutant in glucose repression mig1 (cat4). The triggering of fermentative metabolism was dependent upon catabolite repression properties of yeast and was coincident with a significant decrease of G1 length. WT cells of the strain CEN.PK122 displayed respiratory metabolism up to a D of 0.2 h-1 and exhibited longer G1 lengths than the snf1 and snf4 mutants that started fermenting after a D of 0.1 and 0.15 h-1, respectively. The catabolite derepression mutant snf4 showed a significant decrease in the duration of G1 with respect to the WT. An increase of 300% to 400% in the expression of CDC28 (CDC28-lacZ) with a noticeable shortening in G1 to values lower than approximately 150 min, was detected in the transformed wild type CEN.SC13-9B in glucose-limited chemostat cultures. The expression of CDC28-lacZ was analyzed in the wild type and isogenic mutant strains growing at maximal rate on glucose or in the presence of ethanol or glycerol. Two- to three-fold lower expression of the CDC28-lacZ fusion gene was detected in the snf1 or snf4 disruptants with respect to the WT and mig1 strains in the presence of all carbon sources. This effect was further shown to be growth rate-dependent exhibiting apparently, a threshold effect in the expression of the fusion gene with respect to the length of G1, similar to that shown in chemostat cultures. At the onset of fermentation, the control of the glycolytic flux was highly distributed between the uptake, hexokinase, and phosphofructokinase steps. Particularly interesting was the fact that the snf1 mutant exhibited the lowest fluxes of ethanol production, the highest of respiration and correspondingly, the branch to the tricarboxylic acid cycle was significantly rate-controling of glycolysis.

Modulation of sporulation and metabolic fluxes in Saccharomyces cerevisiae by 2 deoxy glucose.

Quantitative studies of metabolic fluxes during Saccharomyces cerevisiae sporulation on acetate in the presence of the glucose analog, 2-deoxy glucose (2dG) are reported. We have studied the inhibition of sporulation and associated catabolic or anabolic fluxes by 2dG. Sporulation frequencies decreased from 50% to 2% asci per cell at 2dG concentrations in the range of 0.03 to 0.30 g l-1, respectively. Under the same conditions, the acetate consumption flux was inhibited up to 60% and the glyoxylate cycle and gluconeogenic fluxes decreased from 0.7 and 0.3 mmol h-1 g-1 dw, respectively, to negligible values. We observed a linear correlation of the acetate consumption rate with the sporulation frequency by varying the 2dG concentration. The linear correlation was also verified between the frequency of sporulation and the fluxes through glyoxylate cycle and gluconeogenic pathways. In addition, the same association of inhibition of sporulation and metabolic fluxes was found in other S. cerevisiae strains displaying different potentials of sporulation. The results presented suggest that inhibition of sporulation in the presence of the glucose analog may be attributed, at least in part, to the inhibition of anabolic fluxes and might be associated with catabolite repression.

Heterogeneous distribution and organization of cytoskeletal proteins drive differential modulation of metabolic fluxes.

On the basis of experimental data obtained in vitro, we propose that differential segregation of actin and tubulin in the cytoplasm may be a regulatory mechanism of metabolic fluxes. The results presented point out that the same enzymes may be differentially modulated at different locations in the cytoplasm, depending on the cytoskeletal protein present at that location, its concentration, polymeric status, or geometric arrangement. Essentially, actin or microtubular protein would exert their effect on enzymatic catalysis through displacement of the equilibrium of enzyme oligomers either to active or less active species. The latter was corroborated by mathematical modeling of the dynamic coupling between microtubular protein assembly-disassembly and pyruvate kinase activity. From these results, a precise biochemical meaning can be given to the putative linkage existing between the mechanisms by which cells rearrange their cytoplasmic architecture and the dynamics of biochemical reactions taking place concomitantly.

Decreased mitochondrial biogenesis in temperature-sensitive cell division cycle mutants of Saccharomyces cerevisiae.

The temperature-sensitive cell division cycle (cdc) G1 mutants cdc28 and cdc35 show decreased mitochondrial volumes with respect to the wild type strain A364A (WT) at the restrictive temperature. Of the three criteria of mitochondrial biogenesis studied, that is, number of mitochondria per cell, relative area of the cell occupied by mitochondria, or relative area of mitochondria occupied by inner membranes, only the second indicator was significantly lower in cdc mutants than in the WT. The mitochondrial inner membranes development did not compensate for the decrease in the organelles volume. Apparently, the reduced mitochondrial biogenesis was not due to the temperature shift because the relative area of the cell occupied by mitochondria was already significantly lower at 25 degrees C in cdc mutants. The specific fluxes of oxygen consumption confirmed that the respiratory capacity of cdc mutants is largely impaired in respect to the WT. Cdc28 and cdc35 mutants of Saccharomyces cerevisiae had been previously shown to exhibit high respiratory quotients (from 3 to 7) in respect to the WT (RQ approximately 1.0), which correlated with carbon and energy uncoupling probably the result of glucose-induced catabolite repression [Aon MA, Mónaco ME, Cortassa S (1995) Exp Cell Res 217, 42-51; Mónaco ME, Valdecantos PA, Aon MA (1995) Exp Cell Res 217, 52-56].

Fluxes of carbon, phosphorylation, and redox intermediates during growth of saccharomyces cerevisiae on different carbon sources.

In the present work we develop a method for estimating anabolic fluxes when yeast are growing on various carbon substrates (glucose, glycerol, lactate, pyruvate, acetate, or ethanol) in minimal medium. Fluxes through the central amphibolic pathways were calculated from the product of the total required amount of a specified carbon intermediate times the growth rate. The required amount of each carbon intermediate was estimated from the experimentally determined macromolecular composition of cells grown in each carbon source and the monomer composition of macromolecules.Substrates sharing most metabolic pathways such as ethanol and acetate, despite changes in the macromolecular composition, namely carbohydrate content (34% +/- 1 and 21% +/- 3, respectively), did not show large variations in the overall fluxes through the main amphibolic pathways. For instance, in order to supply anabolic precursors to sustain growth rates in the range of 0.16/h to 0.205/h, similar large fluxes through Acetyl CoA synthase were required by acetate (4.2 mmol/hr g dw) or ethanol (5.2 mmol/h g dw).The V(max) activities of key enzymes of the main amphibolic pathways measured in permeabilized yeast cells allowed to confirm, qualitatively, the operation of those pathways for all substrates and were consistent on most substrates with the estimated fluxes required to sustain growth.When ATP produced from oxidation of the NADH synthesized along with the key intermediary metabolites was taken into account, higher Y(ATP) (max) values (36 with respect to 24 g dw/mol ATP) were obtained for glucose. The same result was obtained for glycerol, ethanol, and acetate. A yield index (YI) was defined as the ratio of the theoretically estimated substrate flux required to sustain a given growth rate over the experimentally measured flux of substrate consumption. Comparison of Yl between growth on various carbon sources led us to conclude that ethanol (Yl = 0.84), acetate (Yl = 0.77), and lactate (Yl = 0.77) displayed the most efficient use of substrate for biomass production. For the other substrates, the Yl decayed in the following order: pyruvate > glycerol > glucose.An improvement of the quantitative understanding of yeast metabolism, energetics, and physiology is provided by the present analysis. The methodology proposed can be applied to other eukaryotic organisms of known chemical composition. (c) 1995 John Wiley & Sons, Inc.