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The metabolic capabilities of cells determine their biotechnological potential, niches in multicellular host-organisms, fitness in ecosystems, and threat levels as a pathogen. Their comprehensive experimental characterisation is generally not feasible, in particular for unculturable and extremophile microorganisms. In principle, the full range of metabolic capabilities can be computed from an organism's annotated genome sequence using a metabolic network reconstruction. However, current computational methods cannot deal with genome-scale metabolic networks. Part of the problem is that these methods generally aim to enumerate all feasible metabolic pathways, while computation of all possible (elementally balanced) conversions between nutrients and products (elementary conversion modes, ECMs) would suffice. All the ECMs of a metabolic network capture its full metabolic capability. We have implemented ECM enumeration in ecmtool and illustrate its usage in several examples. This work contributes to the elucidation of the full metabolic footprint of any cell and provides an exhaustive list of its metabolic capabilities.

In single cells, all molecules fluctuate in concentration because synthesis and degradation events occur asynchronously in a probabilistic manner. Since molecules generally influence multiple reactions, concentration fluctuations can propagate through the entire molecular circuit of a cell. This causes single isogenic cells to vary in their phenotypic properties, which affects the fitness of the associated genotype. Currently, we have a good understanding of how fluctuations arise and propagate in small molecular circuits. Convincing experimental observations exist of fluctuations in the growth and fitness characteristics of single cells. We have however a poor understanding of which molecule concentration fluctuations cause emergent fluctuations in the phenotype of a single cell, how extensive fluctuation propagation is, and what its effects on fitness are. In this opinion paper, we discuss those aspects of current single-cell systems biology.

Most fermented dairy products are manufactured using mixed starter cultures. These cultures are usually a well-balanced mixture of several different lactic acid bacteria. For the development of novel or improved dairy products, flavour characteristics are generally a primary target. In recent years, a number of high-throughput screening tools have emerged allowing the targeted selection of flavour-forming cultures and these will be illustrated with relevant examples. This includes methods directly targeting flavour production such as GC–MS analysis of flavour profiles on the head space of fermentation fluids. High-throughput tools and DNA microarrays are valuable tools for elucidating the regulatory responses to different substrates and processing conditions thus allowing rational intervention in fermentations to improve flavour production. Finally, genome-scale metabolic models are used to predict the production of relevant (flavour) components and to expand our knowledge about flavour-forming pathways. The integration of these approaches is anticipated to strengthen culture development programs enabling the production of well-tasting, fermented dairy products.

Hepatic VLDL and glucose production is enhanced in type 2 diabetes and associated with hepatic steatosis. Whether the derangements in hepatic metabolism are attributable to steatosis or to the increased availability of FA metabolites is not known. We used methyl palmoxirate (MP), an inhibitor of carnitine palmitoyl transferase I, to acutely inhibit hepatic FA oxidation and investigated whether the FAs were rerouted into VLDL secretion and whether this would affect hepatic glucose production. After an overnight fast, male APOE3*Leiden transgenic mice received an oral dose of 10 mg/kg MP. Administration of MP led to an 83% reduction in plasma β-hydroxybutyrate (ketone body) levels compared with vehicle-treated mice (0.47 ± 0.07 vs. 2.81 ± 0.16 mmol/l, respectively; P < 0.01), indicative of impaired ketogenesis. Plasma FFA levels were increased by 32% and cholesterol and insulin levels were decreased by 17% and 50%, respectively, in MP-treated mice compared with controls. MP treatment led to a 30% increase in liver triglyceride (TG) content. Surprisingly, no effect on hepatic VLDL-TG production was observed between the groups at 8 h after MP administration. In addition, the capacity of insulin to suppress endogenous glucose production was unaffected in MP-treated mice compared with controls. In conclusion, acute inhibition of FA oxidation increases hepatic lipid content but does not stimulate hepatic VLDL secretion or reduce insulin sensitivity. Copyright © 2005 by the American Society for Biochemistry and Molecular Biology, Inc. Chemicals / CAS: 3 hydroxybutyrate dehydrogenase, 9028-38-0; cholesterol, 57-88-5; insulin, 9004-10-8; palmoxiric acid, 68170-97-8, 81556-15-2; apolipoprotein E3 (Leidein); Apolipoprotein E3; Apolipoproteins E; Fatty Acids, Nonesterified; Lipoproteins, VLDL; RNA, Messenger

Familial dysbetalipoproteinemia associated with the apolipoprotein E2 (APOE2) genotype is a recessive disorder with low penetrance. We have investigated whether additional expression of full-length APOE3, APOE4, or a truncated variant of APOE4 (APOE4-202) can reduce APOE2-associated hyperlipidemia. This was achieved using adenovirus-mediated gene transfer to mice transgenic for human APOE2 and deficient for endogenous Apoe (APOE2.Apoe-/- mice). The hyperlipidemia of APOE2.Apoe-/- mice was readily aggravated by APOE3 and APOE4 overexpression. Only a very low dose of APOE4 adenovirus was capable of reducing the serum cholesterol and triglyceride (TG) levels. Expression of higher doses of APOE4 was associated with an increased VLDL-TG production rate and the accumulation of TG-rich VLDL in the circulation. In contrast, a high dose of adenovirus carrying APOE4-202 reduced both the cholesterol and TG levels in APOE2.Apoe-/- mice. Despite the absence of the C-terminal lipid-binding domain, APOE4-202 is apparently capable of binding to lipoproteins and mediating hepatic uptake. Moreover, overexpression of APOE4-202 in APOE2.Apoe-/- mice does not aggravate their hypertriglyceridemia. These results extend our previous analyses of APOE4-202 expression in Apoe-/- mice and demonstrate that apoE4-202 functions even in the presence of clearance-defective apoE2. Thus, apoE4-202 is a safe and efficient candidate for future therapeutic applications. Chemicals/CAS: Apolipoprotein E2; Apolipoprotein E4; Apolipoproteins E; Cholesterol, 57-88-5; Lipids; Lipoproteins; Lipoproteins, VLDL; Protein Isoforms; Triglycerides; very low density lipoprotein triglyceride

Apolipoprotein (apo) E stimulates the secretion of very low density lipoproteins (VLDLs) by an as yet unknown mechanism. Recently, a working mechanism for apoE was proposed (Twisk, J., Gillian-Daniel, D. L., Tebon, A., Wang, L., Barrett, P. H., and Attie, A. D. (2000) J. Clin. Invest. 105, 521-532) in which apoE prevents the inhibitory action of the low density lipoprotein receptor (LDLr) by binding to it. We have first tested whether this newly described effect of the LDLr on VLDL secretion, obtained in vitro, is also observed in vivo. In LDLr knockout mice (LDLr-/-), the production of VLDL triglycerides and apoB was 30% higher than that in controls. Also the ratio of apoB1OO:apoB48 secretion was increased in the LDLr-/- mice. The composition of nascent VLDL was similar in both strains. To test whether the action of apoE depends on the presence of the LDLr, VLDL production was measured in LDLr-/- and apoE-/- LDLr-/- mice. Deletion of apoE on a LDLr-/- background still caused a 50% decrease of VLDL triglycerides and apoB production. The composition of nascent VLDL was again similar for both strains. We conclude that the effect of apoE on hepatic VLDL production is independent of the presence of the LDLr. Chemicals/CAS: Apolipoproteins E; Lipoproteins, VLDL; Receptors, LDL

It is becoming accepted that steady-state fluxes are not necessarily controlled by single rate-limiting steps. This leaves open the issue whether cellular dynamics are controlled by single pacemaker enzymes, as has often been proposed. This paper shows that yeast sugar transport has substantial but not complete control of the frequency of glycolytic oscillations. Addition of maltose, a competitive inhibitor of glucose transport, reduced both average glucose consumption flux and frequency of glycolytic oscillations. Assuming a single kinetic component and a symmetrical carrier, a frequency control coefficient of between 0.4 and 0.6 and an average-flux control coefficient of between 0.6 and 0.9 were calculated for hexose transport activity. In a second approach, mannose was used as the carbon and free-energy source, and the dependencies on the extracellular mannose concentration of the transport activity, of the frequency of oscillations, and of the average flux were compared. In this case the frequency control coefficient and the average-flux control coefficient of hexose transport activity amounted to 0.7 and 0.9, respectively. From these results, we conclude that 1) transport is highly important for the dynamics of glycolysis, 2) most but not all control resides in glucose transport, and 3) there should at least be one step other than transport with substantial control.

The three main existing models for glycolytic oscillations in yeast were re-examined to investigate how these oscillations are controlled. We implemented the operational definitions provided by metabolic control analysis to quantify the control properties of enzymes with regard to glycolytic oscillations. In all three models, the control of the frequency and that of the amplitudes of the metabolites were distributed among the enzymes. There was no obvious correlation between the control of the average flax and the control of the frequency. Most importantly, the so-called ‘oscillophore’ of the system, traditionally the enzyme primarily held responsible for the generation of the oscillation, was not the only controlling step. We conclude that just like steady-state flux control is not necessarily limited to a rate-limiting step, oscillations are not dictated by a single ‘oscillophore’.