Biomedical subjects

V Hatzimanikatis

Publications and source records attributed to V Hatzimanikatis.

10 recordsLinked to original sources

PubMed1999

A mathematical description of regulation of the G1-S transition of the mammalian cell cycle.

A mathematical model of regulation of the G1-S transition of the mammalian cell cycle has been formulated to organize available experimental molecular-level information in a systematic quantitative framework and to evaluate the ability of this manifestation of current knowledge to calculate correctly experimentally observed phenotypes. This model includes nine components and includes cyclin-cdk complexes, a pocket protein (pRb), a transcription factor (E2F-1), and a cyclin-cdk complex inhibitor. Simulation of the model equations yields stable oscillatory solutions corresponding to cell proliferation and asymptotically stable solutions corresponding to cell cycle arrest (quiescence). Bifurcation analysis of the system suggests changes in the intracellular concentrations of either E2F or cyclin E can activate cell proliferation and that co-overexpression of these molecules can prevent cell proliferation. Further analysis suggests that the amount of inhibitor necessary to prevent cell proliferation is independent of the concentrations of cyclin E and E2F and depends only on the equilibrium ratio between the bound and unbound forms of the inhibitor to the complex.

Animals↗

PubMed1999

Nonlinear metabolic control analysis.

Mathematical description of metabolic systems allows the calculation of the expected responses of metabolism to genetic modifications and the identification of the most promising targets for metabolic engineering. Metabolic control analysis (MCA) provides such a description in the form of quantitative indices (elasticities and control coefficients). These indices are determined by perturbation experiments around a reference steady state and, therefore, the predictive power of MCA is limited to small changes in the metabolic parameters. The modeling framework introduced here allows accurate description of the metabolic responses over wide range of changes in the metabolic parameters. The framework requires information about the MCA indices at the reference state and the corresponding values of the metabolic reaction rates, and employs simplifying assumptions about the reaction mechanisms. It is shown that knowledge of the intracellular metabolite concentrations is not necessary for the application of the framework. The performance of the methodology is illustrated using three elementary metabolic systems that display highly nonlinear responses to the modification in their parameters: an unbranched pathway, an interconvertible enzyme system, and a branched pathway subject to feedback inhibition.

Metabolism↗

PubMed1999

Dynamical analysis of gene networks requires both mRNA and protein expression information.

One of the important goals of biology is to understand the relationship between DNA sequence information and nonlinear cellular responses. This relationship is central to the ability to effectively engineer cellular phenotypes, pathways, and characteristics. Expression arrays for monitoring total gene expression based on mRNA can provide quantitative insight into which gene or genes are on or off; but this information is insufficient to fully predict dynamic biological phenomena. Using nonlinear stability analysis we show that a combination of gene expression information at the message level and at the protein level is required to describe even simple models of gene networks. To help illustrate the need for such information we consider a mechanistic model for circadian rhythmicity which shows agreement with experimental observations when protein and mRNA information are included and we propose a framework for acquiring and analyzing experimental and mathematically derived information about gene networks.

Animals↗

PubMed1997

The AlkB monooxygenase of Pseudomonas oleovorans--synthesis, stability and level in recombinant Escherichia coli and the native host.

We have studied the synthesis and stability of the monooxygenase AlkB of Pseudomonas oleovorans in its natural host and in recombinant Escherichia coli. Three strains were investigated: the prototype strain P. oleovorans and the E. coli alk+ recombinants HB101 (pGEc47) and W3110 (pGEc47). Plasmid pGEc47 allows regulated expression of alkB and synthesis of active AlkB in E. coli. The E. coli strains were selected because E. coli HB101 (pGEc47) produces similar amounts of AlkB as P. oleovorans (1.5-2% of total cell protein), whereas E. coli W3110 (pGEc47) is able to make substantially (about fivefold) more AlkB. The AlkB synthesis and degradation rates in batch cultures of the three strains were determined by means of isotopic-labeling and immunological techniques. The mean specific AlkB synthesis rates in P. oleovorans, E. coli HB101 (pGEc47) and E. coli W3110 (pGEc47) were approximately 7, 12.5 and 45 microg x mg protein(-1) x h(-1), respectively. The half-lives of AlkB were estimated to be 80, 3 and 15 for P. oleovorans, E. coli HB101 (pGEc47) and E. coli W3110 (pGEc47), respectively. Thus, the intracellular AlkB level in each of the three strains was the result of their AlkB synthesis and degradation rates. The AlkB level during batch growth was modelled by means of experimentally derived parameters for AlkB synthesis and degradation, and showed good agreement with AlkB levels determined by means of immunoblotting in all strains investigated.

Cytochrome P-450 CYP4A↗

PubMed1997

Metabolic fluxes in riboflavin-producing Bacillus subtilis.

The pentose phosphate pathway and the pyruvate shunt were identified as major pathways of glucose catabolism in a recombinant, riboflavin-producing Bacillus subtilis strain. Reactions connecting the tricarboxylic acid cycle and glycolysis, catalyzed by the malic enzyme and phosphoenolpyruvate carboxykinase, consume up to 23% of the metabolized glucose. These are examples of important fluxes that can be accessed explicitly using a novel analysis based on synergistic application of flux balancing and recently introduced techniques of fractional 13C-labeling and two-dimensional nuclear magnetic resonance spectroscopy. The overall flux distribution also suggests that B. subtilis metabolism has an unusually high capacity for the reoxidation of NADPH. Under the conditions investigated, riboflavin formation in B. subtilis is limited by the fluxes through the biosynthetic rather than the central carbon pathways, which suggests a focus for future metabolic engineering of this system.

Bacillus subtilis↗

PubMed1996

MCA has more to say.

The recent development of two mathematical frameworks for the analysis and design of metabolic systems is reviewed here. Both frameworks employ a (log)linear kinetic metabolic model that is constructed making direct, explicit use of the individual enzyme kinetic parameters which are a foundation of Metabolic Control Analysis (MCA) information. The first framework allows the description of the dynamic responses of metabolic systems subject to fluctuations in their parameters. The second framework considers the problem of optimizing the regulatory structure of metabolic networks. Both frameworks are powerful in enabling greater insights into metabolism based on MCA quantities.

Animals↗

PubMed1996

Physiology and metabolic fluxes of wild-type and riboflavin-producing Bacillus subtilis.

Continuous cultivation in a glucose-limited chemostat was used to determine the growth parameters of wild-type Bacillus subtilis and of a recombinant, riboflavin-producing strain. Maintenance coefficients of 0.45 and 0.66 mmol of glucose g-1 h-1 were determined for the wild-type and recombinant strains, respectively. However, the maximum molar growth yield of 82 to 85 g (cell dry weight)/mol of glucose was found to be almost identical in both strains. A nonlinear relationship between the specific riboflavin production rate and the dilution rate was observed, revealing a coupling of product formation and growth under strict substrate-limited conditions. Most prominently, riboflavin formation completely ceased at specific growth rates below 0.15 h-1. For molecular characterization of B. subtilis, the total amino acid composition of the wild type was experimentally determined and the complete building block requirements for biomass formation were derived. In particular, the murein sacculus was found to constitute approximately 9% of B. subtilis biomass, three- to fivefold more than in Escherichia coli. Estimation of intracellular metabolic fluxes by a refined mass balance approach revealed a substantial, growth rate-dependent flux through the oxidative branch of the pentose phosphate pathway. Furthermore, this flux is indicated to be increased in the strain engineered for riboflavin formation. Glucose catabolism at low growth rates with reduced biomass yields was supported mainly by the tricarboxylic acid cycle.

Amino Acids↗

PubMedDate not supplied

Application of mathematical tools for metabolic design of microbial ethanol production.

Many attempts to engineer cellular metabolism have failed due to the complexity of cellular functions. Mathematical and computational methods are needed that can organize the available experimental information, and provide insight and guidance for successful metabolic engineering. Two such methods are reviewed here. Both methods employ a (log)linear kinetic model of metabolism that is constructed based on enzyme kinetics characteristics. The first method allows the description of the dynamic responses of metabolic systems subject to spatiotemporal variations in their parameters. The second method considers the product-oriented, constrained optimization of metabolic reaction networks using mixed-integer linear programming methods. The optimization framework is used in order to identify the combinations of the metabolic characteristics of the glycolytic enzymes from yeast and bacteria that will maximize ethanol production. The methods are also applied to the design of microbial ethanol production metabolism. The results of the calculations are in qualitative agreement with experimental data presented here. Experiments and calculations suggest that, in resting Escherichia coli cells, ethanol production and glucose uptake rates can be increased by 30% and 20%, respectively, by overexpression of a deregulated pyruvate kinase, while increase in phosphofructokinase expression levels has no effect on ethanol production and glucose uptake rates.

Bacteriological Techniques↗

PubMedDate not supplied

Metabolic consequences of phosphotransferase (PTS) mutation in a phenylalanine-producing recombinant Escherichia coli.

E. coli strain PPA305, which has a wild-type PTS system, and PPA316, which utilizes a proton-galactose symport system for glucose uptake, were used as host strains to harbor a phenylalanine overproduction plasmid pSY130-14 and to study the effects of using different glucose uptake systems on phenylalanine production. The non-PTS strain (PPA316/pSY130-14) produced much less phenylalanine, ranging from 0 to 67% of that produced by the PTS strain (PPA305/pSY130-14) depending on cultivation conditions used. The non-PTS strain PPA316/pSY130-14 had an intracellular PEP concentration only one-sixth that of the PTS strain, PPA305/pSY130-14. Additionally, PPA316/pSY130-14 had a substantially lower energy state in terms of the size of the pool of high-energy phosphate compounds and the magnitude of the pH difference across the cytoplasmic membrane. The non-PTS strain consumed oxygen at a higher rate, attained lower biomass concentration, and produced no acetate and phenylalanine during fermentation, suggesting more carbon was oxidized to CO2, most likely through the TCA cycle. Analysis of intracellular fluxes through the central carbon pathways was performed for each strain utilizing exponential phase data on extracellular components and assuming quasi-steady state for intermediate metabolites. The non-PTS strain had a higher flux through pyruvate kinase (PYK) and TCA cycle which, in agreement with the observed higher oxygen uptake rate, suggests that more carbon was oxidized to CO2 through the TCA cycle. Further analysis using rate expression data for PYK and NMR data for the intracellular metabolites identified the regulatory properties of PYK as the probable cause for lower intracellular PEP levels in PPA316/pSY130-14.

Adenosine Triphosphate↗

PubMedDate not supplied

Proteomics: theoretical and experimental considerations.

Cellular engineering relies on the ability to decipher the genetic basis of various phenotypes. Emerging technologies for analyzing the biological function of the information encoded in the genome of particular organisms and/or tissues focus on the monitoring of transcription (mRNA) and translation (protein) processes. Elementary theoretical considerations presented in this article strongly suggest that a combination of mRNA and protein expression patterns should be simultaneously considered to fully develop a conceptual understanding of the functional architecture of genomes and gene networks. We propose a framework of experimental and mathematical methods for acquiring and analyzing quantitative proteomic information and discuss recent developments in proteome analytical technology.

Bacterial Proteins↗