Metabolomic Comparison of Saccharomyces cerevisiae and the Cryotolerant Species S. bayanus var. uvarum and S. kudriavzevii during Wine Fermentation at Low Temperature

Temperature is one of the most important parameters affecting the length and rate of alcoholic fermentation and final wine quality. Wine produced at low temperature is often considered to have improved sensory qualities. However, there are certain drawbacks to low temperature fermentations such as reduced growth rate, long lag phase, and sluggish or stuck fermentations. To investigate the effects of temperature on commercial wine yeast, we compared its metabolome growing at 12°C and 28°C in a synthetic must. Some species of the Saccharomyces genus have shown better adaptation at low temperature than Saccharomyces cerevisiae. This is the case of the cryotolerant yeasts Saccharomyces bayanus var. uvarum and Saccharomyces kudriavzevii. In an attempt to detect inter-specific metabolic differences, we characterized the metabolome of these species growing at 12°C, which we compared with the metabolome of S. cerevisiae (not well adapted at low temperature) at the same temperature. Our results show that the main differences between the metabolic profiling of S. cerevisiae growing at 12°C and 28°C were observed in lipid metabolism and redox homeostasis. Moreover, the global metabolic comparison among the three species revealed that the main differences between the two cryotolerant species and S. cerevisiae were in carbohydrate metabolism, mainly fructose metabolism. However, these two species have developed different strategies for cold resistance. S. bayanus var. uvarum presented elevated shikimate pathway activity, while S. kudriavzevii displayed increased NAD⁺ synthesis.     

Copper toxicity in the microalga Chlamydomonas reinhardtii: an integrated approach

The effects of copper exposure at five different concentrations on the freshwater alga Chlamydomonas reinhardtii were studied at the biochemical (metabolite), physiological (uptake kinetics and flow cytometry) and growth level. Changes at the physiological level were evident at the lowest exposure concentration while effects on the metabolome and on growth only occurred at the highest copper concentration tested. Flow cytometry revealed the presence of higher reactive oxygen species concentrations in algae exposed to higher copper concentrations and this was confirmed by a significant reduction in glutathione levels as part of the metabolomics assessment. Cu²⁺ uptake kinetic data contributed information on possible mechanisms of copper toxicity, revealing that, a decrease in efflux pumping might be at the basis of an increased metal accumulation at higher exposure levels. This study demonstrates the value of using a comparative approach to investigating the mechanisms of toxicity rather than focusing on a single level of organization or effect.     

Ultra-Fast Analysis of Plasma and Intracellular Levels of HIV Protease Inhibitors in Children: A Clinical Application of MALDI Mass Spectrometry

HIV protease inhibitors must penetrate into cells to exert their action. Differences in the intracellular pharmacokinetics of these drugs may explain why some patients fail on therapy or suffer from drug toxicity. Yet, there is no information available on the intracellular levels of HIV protease inhibitors in HIV infected children, which is in part due to the large amount of sample that is normally required to measure the intracellular concentrations of these drugs. Therefore, we developed an ultra-fast and sensitive assay to measure the intracellular concentrations of HIV protease inhibitors in small amounts of peripheral blood mononuclear cells (PBMCs), and determined the intracellular concentrations of lopinavir and ritonavir in HIV infected children. An assay based on matrix-assisted laser desorption/ionization (MALDI) - triple quadrupole mass spectrometry was developed to determine the concentrations of HIV protease inhibitors in 10 µL plasma and 1×10⁶ PBMCs. Precisions and accuracies were within the values set by the FDA for bioanalytical method validation. Lopinavir and ritonavir did not accumulate in PBMCs of HIV infected children. In addition, the intracellular concentrations of lopinavir and ritonavir correlated poorly to the plasma concentrations of these drugs. MALDI-triple quadrupole mass spectrometry is a new tool for ultra-fast and sensitive determination of drug concentrations which can be used, for example, to assess the intracellular pharmacokinetics of HIV protease inhibitors in HIV infected children.     

Predictability of the covalent binding of acidic drugs in man

Although metabolism via glucuronide conjugation has generally been considered a detoxification route for carboxylic acids, the newly discovered chemical reactivity of these conjugates, leading to covalent binding with proteins, is consistent with the toxicity observed for drugs containing the carboxylic acid moiety. Here we report that degradation rates (intramolecular rearrangement and hydrolysis) for 9 drug glucuronide metabolites show an excellent correlation (r²=0.995) with the extents of drug covalent binding to albumin in vitro. Furthermore, this binding capacity is predictable based on chemical structure of the acid and depends on the degree of substitution at the carbon alpha to the carboxylic acid. The in vivo covalent binding in humans for these drugs is also predictable (r²=0.873) when the extent of adduct formation is corrected for the measured plasma glucuronide concentrations. These results suggest that the structure of a carboxylic acid drug may predict the degree to which the corresponding acyl glucuronides will form covalent adducts that probably/possibly lead to toxicity. This information could be a useful adjunct in drug design.     

Influence of cell crowding on toxicity of nitroheterocycles

Cell density (no. of cells per unit area or volume) during drug treatment may play a role of considerable importance in the interpretation of drug toxicity experiments performed in vitro. Chinese hamster V-79 and mouse L-929 cells exposed to nitroheterocycles under aerobic conditions are considerably more sensitive to the cytotoxic effects of these drugs when incubated at low cell density (10² cells/cm² or 10⁴ cells/ml) than at higher cell density (10⁴ cells/cm² or 10⁶ cells/ml). This may be related to diffusion limitations when cells are in contact and to the ability of dense cell suspensions to inactivate drugs. In contrast, under anaerobic conditions, more toxicity is observed at high cell density than at low cell density, perhaps due to local effects of toxic metabolites. Toxicity appears to correlate with intracellular drug levels under both aerobic and hypoxic conditions.The chemical nature of the fluorescent species has not yet been determined. However, it is likely that loss of the nitro group reduces but does not abolish fluorescence. When L-cells were used to metabolize AF-2 under hypoxia so that only 20% of the parent compound remained (with nitro group intact), 50% of the fluorescence was still present (unpublished results). Cells incubated with AF-2 under air or nitrogen show little decrease in fluorescence intensity for several hours after drug removal suggesting that the fluorescent compound being observed was bound intracellularly. As yet, we have no reason to suspect that the fluorescent products bound under hypoxia differ from those bound under air. Therefore, cell density dependent toxicity of nitroheterocycles under aerobic conditions may be related to diffusion limitations when cells are in close contact, as well as drug inactivation by cells at high cell densities.     

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode’s ATP levels.^1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.^4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.     

The Affinity of DNA-Microtubule Protein Complexes and their Disruption by Tubulin Binding Drugs

Abstract

Using the gel shift assay system, we have measured the apparent affinity constant for the interaction of two different DNAs with MAP proteins found in both total calf brain microtubules and heat stable brain preparations. Both DNAs studied contained centromere/kinetochore sequences- one was enriched in the calf satellite DNA; the other was a large restriction fragment containing the yeast CEN11 DNA sequence. Complexes formed using both DNAs had similar K_app values in the range of 2.1×10⁷ M^?1 to 2.0×10⁸ M^?1. CEN11 DNA-MTP complexes had by far the highest K_app value of 2.0×10⁸ M^?1. The CEN11 DNA sequence is where the yeast kinetochore of chromosome 11 is formed and where the single yeast microtubule is bound in vivo. The CEN11 conserved region II known binding sites -(dA/dT)n runs- for mammalian MAP2 protein, are in good agreement with this higher K_app value. The effects of the classical tubulin binding drugs colchicine, podophyllotoxin and vinblastine on the DNA-MAP protein complex stability were investigated by determining the drug concentrations where the complexes were destabilized. Only the complexes formed from total microtubule protein (tubulin containing) were destabilized over a wide drug concentration range. Heat stable brain protein complexes (no tubulin) were largely unaffected. Furthermore, it took 10–100 fold higher drug concentrations to disrupt the CEN 11 DNA complexes compared to the calf thymus satellite DNA enriched complexes. These data support our previous results suggesting that there is a DNA sequence dependent interaction with MAP proteins that appears to be conserved in evolution (Marx et. al., Biochim. Biophys. Acta. 783, 383–392,1984; Marx and Denial, Molecular Basis of Cancer 172B,65-15 1985). In addition, these results imply that the classical tubulin binding drugs may exert their biological effects in cells at least in part by disrupting DNA-Protein complexes of the type we have studied here.     

A Novel Mathematical Model Describing Adaptive Cellular Drug Metabolism and Toxicity in the Chemoimmune System

Cells cope with the threat of xenobiotic stress by activating a complex molecular network that recognizes and eliminates chemically diverse toxic compounds. This “chemoimmune system” consists of cellular Phase I and Phase II metabolic enzymes, Phase 0 and Phase III ATP Binding Cassette (ABC) membrane transporters, and nuclear receptors regulating these components. In order to provide a systems biology characterization of the chemoimmune network, we designed a reaction kinetic model based on differential equations describing Phase 0–III participants and regulatory elements, and characterized cellular fitness to evaluate toxicity. In spite of the simplifications, the model recapitulates changes associated with acquired drug resistance and allows toxicity predictions under variable protein expression and xenobiotic exposure conditions. Our simulations suggest that multidrug ABC transporters at Phase 0 significantly facilitate the defense function of successive network members by lowering intracellular drug concentrations. The model was extended with a novel toxicity framework which opened the possibility of performing in silico cytotoxicity assays. The alterations of the in silico cytotoxicity curves show good agreement with in vitro cell killing experiments. The behavior of the simplified kinetic model suggests that it can serve as a basis for more complex models to efficiently predict xenobiotic and drug metabolism for human medical applications.     

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world''s most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly¹. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited ²;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance ³. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.In vitro promastigotes ⁴ and axenic amastigotes assays⁵ are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).     

Staphylococcus aureus Alpha-Toxin Mediates General and Cell Type-Specific Changes in Metabolite Concentrations of Immortalized Human Airway Epithelial Cells

Staphylococcus aureus alpha-toxin (Hla) is a potent pore-forming cytotoxin that plays an important role in the pathogenesis of S. aureus infections, including pneumonia. The impact of Hla on the dynamics of the metabolome in eukaryotic host cells has not been investigated comprehensively. Using ¹H-NMR, GC-MS and HPLC-MS, we quantified the concentrations of 51 intracellular metabolites and assessed alterations in the amount of 25 extracellular metabolites in the two human bronchial epithelial cell lines S9 and 16HBE14o⁻ under standard culture conditions and after treatment with sub-lethal amounts (2 µg/ml) of recombinant Hla (rHla) in a time-dependent manner. Treatment of cells with rHla caused substantial decreases in the concentrations of intracellular metabolites from different metabolic pathways in both cell lines, including ATP and amino acids. Concomitant increases in the extracellular concentrations were detected for various intracellular compounds, including nucleotides, glutathione disulfide and NAD⁺. Our results indicate that rHla has a major impact on the metabolome of eukaryotic cells as a consequence of direct rHla-mediated alterations in plasma membrane permeability or indirect effects mediated by cellular signalling. However, cell-specific changes also were observed. Glucose consumption and lactate production rates suggest that the glycolytic activity of S9 cells, but not of 16HBE14o⁻ cells, is increased in response to rHla. This could contribute to the observed higher level of resistance of S9 cells against rHla-induced membrane damage.     

A Mapping of Drug Space from the Viewpoint of Small Molecule Metabolism

Small molecule drugs target many core metabolic enzymes in humans and pathogens, often mimicking endogenous ligands. The effects may be therapeutic or toxic, but are frequently unexpected. A large-scale mapping of the intersection between drugs and metabolism is needed to better guide drug discovery. To map the intersection between drugs and metabolism, we have grouped drugs and metabolites by their associated targets and enzymes using ligand-based set signatures created to quantify their degree of similarity in chemical space. The results reveal the chemical space that has been explored for metabolic targets, where successful drugs have been found, and what novel territory remains. To aid other researchers in their drug discovery efforts, we have created an online resource of interactive maps linking drugs to metabolism. These maps predict the “effect space” comprising likely target enzymes for each of the 246 MDDR drug classes in humans. The online resource also provides species-specific interactive drug-metabolism maps for each of the 385 model organisms and pathogens in the BioCyc database collection. Chemical similarity links between drugs and metabolites predict potential toxicity, suggest routes of metabolism, and reveal drug polypharmacology. The metabolic maps enable interactive navigation of the vast biological data on potential metabolic drug targets and the drug chemistry currently available to prosecute those targets. Thus, this work provides a large-scale approach to ligand-based prediction of drug action in small molecule metabolism.     

Are Pharmaceuticals with Evolutionary Conserved Molecular Drug Targets More Potent to Cause Toxic Effects in Non-Target Organisms?

The ubiquitous use of pharmaceuticals has resulted in a continuous discharge into wastewater and pharmaceuticals and their metabolites are found in the environment. Due to their design towards specific drug targets, pharmaceuticals may be therapeutically active already at low environmental concentrations. Several human drug targets are evolutionary conserved in aquatic organisms, raising concerns about effects of these pharmaceuticals in non-target organisms. In this study, we hypothesized that the toxicity of a pharmaceutical towards a non-target invertebrate depends on the presence of the human drug target orthologs in this species. This was tested by assessing toxicity of pharmaceuticals with (miconazole and promethazine) and without (levonorgestrel) identified drug target orthologs in the cladoceran Daphnia magna. The toxicity was evaluated using general toxicity endpoints at individual (immobility, reproduction and development), biochemical (RNA and DNA content) and molecular (gene expression) levels. The results provide evidence for higher toxicity of miconazole and promethazine, i.e. the drugs with identified drug target orthologs. At the individual level, miconazole had the lowest effect concentrations for immobility and reproduction (0.3 and 0.022 mg L⁻¹, respectively) followed by promethazine (1.6 and 0.18 mg L⁻¹, respectively). At the biochemical level, individual RNA content was affected by miconazole and promethazine already at 0.0023 and 0.059 mg L⁻¹, respectively. At the molecular level, gene expression for cuticle protein was significantly suppressed by exposure to both miconazole and promethazine; moreover, daphnids exposed to miconazole had significantly lower vitellogenin expression. Levonorgestrel did not have any effects on any endpoints in the concentrations tested. These results highlight the importance of considering drug target conservation in environmental risk assessments of pharmaceuticals.     

Hydrogel Based 3-Dimensional (3D) System for Toxicity and High-Throughput (HTP) Analysis for Cultured Murine Ovarian Follicles

Various toxicants, drugs and their metabolites carry potential ovarian toxicity. Ovarian follicles, the functional unit of the ovary, are susceptible to this type of damage at all stages of their development. However, despite of the large scale of potential negative impacts, assays that study ovarian toxicity are limited. Exposure of cultured ovarian follicles to toxicants of interest served as an important tool for evaluation of toxic effects for decades. Mouse follicles cultured on the bottom of a culture dish continue to serve an important approach for mechanistic studies. In this paper, we demonstrated the usefulness of a hydrogel based 3-dimensional (3D) mouse ovarian follicle culture as a tool to study ovarian toxicity in a different setup. The 3D in vitro culture, based on fibrin alginate interpenetrating network (FA-IPN), preserves the architecture of the ovarian follicle and physiological structure-function relationship. We applied the novel 3D high-throughput (HTP) in vitro ovarian follicle culture system to study the ovotoxic effects of an anti-cancer drug, Doxorobucin (DXR). The fibrin component in the system is degraded by plasmin and appears as a clear circle around the encapsulated follicle. The degradation area of the follicle is strongly correlated with follicle survival and growth. To analyze fibrin degradation in a high throughput manner, we created a custom MATLAB® code that converts brightfield micrographs of follicles encapsulated in FA-IPN to binary images, followed by image analysis. We did not observe any significant difference between manually processed images to the automated MATLAB® method, thereby confirming that the automated program is suitable to measure fibrin degradation to evaluate follicle health. The cultured follicles were treated with DXR at concentrations ranging from 0.005 nM to 200 nM, corresponding to the therapeutic plasma levels of DXR in patients. Follicles treated with DXR demonstrated decreased survival rate in greater DXR concentrations. We observed partial follicle survival of 35% ± 3% (n = 80) in 0.01nM treatment and 48% ± 2% (n = 92) in 0.005nM, which we identified as the IC50 for secondary follicles. In summary, we established a 3D in vitro ovarian follicle culture system that could be used in an HTP approach to measure toxic effects on ovarian follicles.     

Bacterially-Derived Nanocells for Tumor-Targeted Delivery of Chemotherapeutics and Cell Cycle Inhibitors

Chemotherapeutic drug therapy in cancer is seriously hampered by severe toxicity primarily due to indiscriminate drug distribution and consequent collateral damage to normal cells. Molecularly targeted drugs such as cell cycle inhibitors are being developed to achieve a higher degree of tumor cell specificity and reduce toxic side effects. Unfortunately, relative to the cytotoxics, many of the molecularly targeted drugs are less potent and the target protein is expressed only at certain stages of the cell cycle thus necessitating regimens like continuous infusion therapy to arrest a significant number of tumor cells in a heterogeneous tumor mass. Here we discuss targeted drug delivery nanovectors and a recently reported bacterially-derived 400nm sized minicell that can be packaged with therapeutically significant concentrations of chemotherapeutic drugs, targeted to tumor cell surface receptors and effect intracellular drug delivery with highly significant anti-tumor effects in-vivo. We also report that molecularly targeted drugs can also be packaged in minicells and targeted to tumor cells with highly significant tumor growth-inhibition and regression in mouse xenografts despite administration of minute amounts of drug. This targeted intracellular drug delivery may overcome many of the hurdles associated with the delivery of cytotoxic and molecularly targeted drugs.     

Antipsychotic drugs scavenge radiation-induced hydroxyl radicals and intracellular ROS formation,and protect apoptosis in human lymphoma U937 cells

Antioxidant activity has been reported for some atypical antipsychotic drugs; however, the detailed mechanism is not well known. Here, we investigated the effects of atypical antipsychotic drugs on ^?OH radical formation, intracellular reactive oxygen species (ROS), and apoptosis induced by ionising radiation. The reaction rate constants with ^?OH radicals were determined for five antipsychotic drugs as follows, in descending order: olanzapine, aripiprazole, clozapine, haloperidol, and risperidone. Experiments with aminophenyl fluorescein, a fluorescent dye, showed that olanzapine and clozapine could scavenge intracellular ROS. However, experiments with hydroxyphenyl fluorescein showed that only olanzapine inhibited ROS generation. X-irradiation-induced apoptosis in human lymphoma U937 cells was inhibited by clozapine at relatively low concentrations and by olanzapine at higher concentrations. Clozapine inhibited caspase-8 and caspase-3 activation and prevented loss of mitochondrial membrane potential. In contrast, olanzapine inhibited X-irradiation-induced p-JNK activation. Although the atypical antipsychotic drugs used here have relatively high reaction rate constants with ^?OH radicals in aqueous solutions, inhibition of intracellular ROS was not due to ^?OH radical scavenging. In addition, suppression of X-irradiation-induced apoptosis was not directly linked with intracellular ROS scavenging. When apoptosis signalling pathways were studied, clozapine-mediated inhibition of apoptosis was dependent on caspase-3 and caspase-8. In contrast, olanzapine inhibited apoptosis via down regulation of X-irradiation-induced p-JNK. These results suggested that both olanzapine and clozapine have antioxidative and antiapoptotic activities via distinct pathways, and provide useful information for better understanding of drug characteristics.     

Off-target effects of psychoactive drugs revealed by genome-wide assays in yeast

To better understand off-target effects of widely prescribed psychoactive drugs, we performed a comprehensive series of chemogenomic screens using the budding yeast Saccharomyces cerevisiae as a model system. Because the known human targets of these drugs do not exist in yeast, we could employ the yeast gene deletion collections and parallel fitness profiling to explore potential off-target effects in a genome-wide manner. Among 214 tested, documented psychoactive drugs, we identified 81 compounds that inhibited wild-type yeast growth and were thus selected for genome-wide fitness profiling. Many of these drugs had a propensity to affect multiple cellular functions. The sensitivity profiles of half of the analyzed drugs were enriched for core cellular processes such as secretion, protein folding, RNA processing, and chromatin structure. Interestingly, fluoxetine (Prozac) interfered with establishment of cell polarity, cyproheptadine (Periactin) targeted essential genes with chromatin-remodeling roles, while paroxetine (Paxil) interfered with essential RNA metabolism genes, suggesting potential secondary drug targets. We also found that the more recently developed atypical antipsychotic clozapine (Clozaril) had no fewer off-target effects in yeast than the typical antipsychotics haloperidol (Haldol) and pimozide (Orap). Our results suggest that model organism pharmacogenetic studies provide a rational foundation for understanding the off-target effects of clinically important psychoactive agents and suggest a rational means both for devising compound derivatives with fewer side effects and for tailoring drug treatment to individual patient genotypes.     

Changes in substrate availability in Escherichia coli lead to rapid metabolite,flux and growth rate responses

The interactions between the intracellular metabolome, fluxome and growth rate of Escherichia coli after sudden glycolytic/gluconeogenic substrate shifts are studied based on pulses of different substrates to an aerobic glucose-limited steady-state (dilution rate=0.1 h⁻¹). After each added glycolytic (glucose) and gluconeogenic (pyruvate and succinate) substrate pulse, no by-products were secreted and a pseudo steady state in flux and metabolites was achieved in about 30–40 s. In the pulse experiments a large oxygen uptake capacity of the cells was observed. The in vivo dynamic responses showed massive reorganization and flexibility (1/100–14-fold change) of extra/intracellular metabolic fluxes, matching with large changes in the concentrations of intracellular metabolites, including reversal of reaction rate for pseudo/near equilibrium reactions. The coupling of metabolome and fluxome could be described by Q-linear kinetics. Remarkably, the three different substrate pulses resulted in a very similar increase in growth rate (0.13–0.3 h⁻¹). Data analysis showed that there must exist as yet unknown mechanisms which couple the protein synthesis rate to changes in central metabolites.