Engineering a living cell to desired metabolite concentrations and fluxes: pathways with multifunctional enzymes

With molecular genetics enabling modulation of the concentrations of cellular enzymes, metabolic engineering becomes limited by the question of which modulations of the enzyme concentrations are required to bring about a desired pattern of cellular metabolism. In an earlier paper (Kholodenko et al. (1998). Biotechnol. Bioeng. 59, 239-247) we derived a method to determine the required modulations. This method, however, cannot be immediately applied to cellular pathways with enzymes catalyzing more than one step in metabolism (multifunctional enzymes). In the present paper we show to which extent the presence of multifunctional enzymes limits biotechological ambitions, which one might otherwise pursue in vain. In particular, it is impossible to change the concentration of a single intermediate and leave the rest of metabolism unperturbed if that intermediate interacts directly with a multifunctional enzyme. The analytical machinery of Metabolic Control Analysis is used to relate the desired and ensuing changes in the metabolic pattern. An explicit solution to this problem of engineering metabolism is then given in the form of a single matrix equation.     

Trophic interception: how a boundary-foraging organism influences cross-ecosystem fluxes

The rate at which subsidies move between habitats is a function not only of those factors that facilitate such transfers, but also of factors that limit or restrict the movement of subsidies. The interruption or redirection of subsidies by organisms foraging at the boundary between habitats, or trophic interception, has the potential to substantially restructure the food webs of recipient habitats. In this study we describe how a waterstrider, Aquarius remigis , limits the transfer of a subsidy across the land-water boundary. Prey interception varied with the type of stream habitat; on average, waterstriders in isolated pools intercepted 71.8% of experimental prey inputs of all sizes compared with 21.5% in connected pools and 0.8% in riffles. Across all stream habitat types, waterstriders consumed 43.2% of the experimental inputs of the smallest prey used, the prey size class most similar to natural inputs in our study area. We estimate that foraging waterstriders may intercept 0.3–1.2 kg of terrestrial prey subsidy for every 100 m of stream channel during three summer months, resources that could otherwise support 13–58 young-of-the-year salmonids. In controlled trials, waterstriders significantly altered the amount of terrestrial prey directly consumed by fish, while fish also altered waterstrider interception of those prey. Interestingly, when waterstriders and fish were present together, more terrestrial prey were lost to the streambed than when either or both of these consumers were absent, making this resource available to benthic detritivores, and facilitating the direct incorporation of terrestrial nutrients into stream detrital webs. Overall, we demonstrate that organisms that forage at habitat boundaries can control the quantity and quality of subsidies arriving in recipient habitats, potentially altering food web structures within those habitats.     

Metabolic networks: enzyme function and metabolite structure

Metabolism is one of the most complex cellular processes. Connections between biochemical reactions via substrate and product metabolites create complex metabolic networks that may be analyzed using network theory, stoichiometric analysis, and information on protein structure/function and metabolite properties. These frameworks take into consideration different aspects of enzyme chemistry, enzyme structure and metabolite structure, and demonstrate the impact of metabolic biochemistry on the systemic properties of metabolism. The integration of these approaches and the systematic classification of enzyme function and the chemical structure of metabolites will enhance our understanding of metabolism, and could improve our ability to predict enzyme function and novel metabolic pathways.     

Teaching cell biology to nonscience majors through forensics, or how to design a killer course

Nonscience majors often do not respond to traditional lecture-only biology courses. However, these students still need exposure to basic biological concepts. To accomplish this goal, forensic science was paired with compatible cell biology subjects. Several topics such as human development and molecular biology were found to fulfill this purpose. Another goal was to maximize the hands-on experience of the nonscience major students. This objective was fulfilled by specific activities such as fingerprinting and DNA typing. One particularly effective teaching tool was a mock murder mystery complete with a Grand Jury trial. Another objective was to improve students' attitudes toward science. This was successful in that students felt more confident in their own scientific abilities after taking the course. In pre/post tests, students answered four questions about their ability to conduct science. All four statements showed a positive shift after the course (p values ranging from.001 to.036, df = 23; n = 24). The emphasis on experiential pedagogy was also shown to increase critical thinking skills. In pre/post testing, students in this course significantly increased their performance on critical thinking assessment tests from 33.3% correct to 45.3% (p =.008, df = 4; n = 24).     

Myocardial density and composition: a basis for calculating intracellular metabolite concentrations

Systems for describing myocardial cellular metabolism with appropriate thermodynamic constraints on reactions have to be on the basis of estimates of intracellular and mitochondrial concentrations of metabolites as driving forces for reactions. This requires that tissue composition itself must be modeled, but there is marked inconsistency in the literature and no full data set on hearts of any species. To formulate a self-consistent set of information on the densities, contents, or concentrations of chemical components and volumes of tissue spaces, we drew on information mostly on rats. From the data on densities, volumes, volume fractions, and mass fractions observed mainly on left ventricular myocardium, cytoplasm, and mitochondria and from morphometric data on cellular components and the vasculature, we constructed a matrix based on conservation laws for density, volume, and constituent composition. The four constituents were water, protein, fat, and solutes (or ash). To take into account the variances in the observed data sets, we used a constrained nonlinear least squares optimization to minimize the differences between the final results and the data sets. The results provide a detailed estimate of cardiac tissue composition, previously unavailable, for the translation of whole tissue concentrations or concentrations per gram protein into estimated local concentrations that are relevant to reaction processes. An example is that the concentrations of phosphocreatine and ATP in cytosolic water space are twice as high as their mean tissue concentrations. This conservation optimization method is applicable to any tissue or organ.     

Free energy dissipation of the pyruvate kinase reaction has a minimum at cell metabolite concentrations

The ratio of substrates and products (mass action ratio) for the reaction catalyzed by the enzyme pyruvate kinase is measured under the constraint of constant reaction rate for pyruvate kinase (EC 2.7.1.40) from brewers yeast and Eschcrichiacoli. For both organisms, a maximum of the ratio is found at concentrations comparable to those obtained from cell metabolite measurements. This observation suggests an optimum principle for free energy transduction in the glycolytic reaction pathway. as a maximum of the mass action ratio corresponds to a minimum dissipation of free energy.     

Circular permutation: a different way to engineer enzyme structure and function

Protein engineers traditionally rely on amino acid substitutions to alter the functional properties of biomacromolecules, yet have largely overlooked the potential benefits of reorganizing the polypeptide chain of a protein by circular permutation (CP). By connecting the native protein termini via a covalent linker and introducing new ends through the cleavage of an existing peptide bond, CP can perturb local tertiary structure and protein dynamics, as well as introduce possible quaternary structure changes. In several recent studies, these effects have successfully been exploited to manipulate protein scaffolds, resulting in improved catalytic activity and altered substrate or ligand binding affinity, as well as enabling the design of novel biocatalysts and biosensors.     

Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart

About 3,000 individuals in the United States are awaiting a donor heart; worldwide, 22 million individuals are living with heart failure. A bioartificial heart is a theoretical alternative to transplantation or mechanical left ventricular support. Generating a bioartificial heart requires engineering of cardiac architecture, appropriate cellular constituents and pump function. We decellularized hearts by coronary perfusion with detergents, preserved the underlying extracellular matrix, and produced an acellular, perfusable vascular architecture, competent acellular valves and intact chamber geometry. To mimic cardiac cell composition, we reseeded these constructs with cardiac or endothelial cells. To establish function, we maintained eight constructs for up to 28 d by coronary perfusion in a bioreactor that simulated cardiac physiology. By day 4, we observed macroscopic contractions. By day 8, under physiological load and electrical stimulation, constructs could generate pump function (equivalent to about 2% of adult or 25% of 16-week fetal heart function) in a modified working heart preparation.     

Intravital microscopy: a novel tool to study cell biology in living animals

Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criterion. Indeed, first we will focus on those studies in which organs were imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.     

When and how to kill a plant cell: Infection strategies of plant pathogenic fungi

Fungi cause severe diseases on a broad range of crop and ornamental plants, leading to significant economical losses. Plant pathogenic fungi exhibit a huge variability in their mode of infection, differentiation and function of infection structures and nutritional strategy. In this review, advances in understanding mechanisms of biotrophy, necrotrophy and hemibiotrophic lifestyles are described. Special emphasis is given to the biotrophy-necrotrophy switch of hemibiotrophic pathogens, and to biosynthesis, chemical diversity and mode of action of various fungal toxins produced during the infection process.     

Integration of molecular genetics and proteomics with cell metabolism: how to proceed; how not to proceed!

There now exists a resurgence of interest in the role of intermediary metabolism in medicine; especially in relation to medical disorders. Coupled with this is the contemporary focus on molecular biology, genetics and proteomics and their integration into studies of regulation and alterations in cellular metabolism in health and disease. This is a marriage that has vast potential for elucidation of the factors and conditions that are involved in cellular metabolic and functional changes, which heretofore could not be addressed by the earlier generations of biochemists who established the major pathways of intermediary metabolism. The achievement of this present potential requires the appropriate application and interpretation of genetic and proteomic studies relating to cell metabolism and cell function. This requires knowledge and understanding of the principles, relationships, and methodology, such as biochemistry and enzymology, which are involved in the elucidation of cellular regulatory enzymes and metabolic pathways. Unfortunately, many and possibly most contemporary molecular biologists are not adequately trained and knowledgeable in these areas of cell metabolism. This has resulted in much too common inappropriate application and misinformation from genetic/proteomic studies of cell metabolism and function. This presentation describes important relationships of cellular intermediary metabolism, and provides examples of the appropriate and inappropriate application of genetics and proteomics. It calls for the inclusion of biochemistry, enzymology, cell metabolism and cell physiology in the graduate and postgraduate training of molecular biology and other biomedical researchers.     

Chinese hamster ovary cell mitosis and its response to ionizing radiation: a morphological analysis of the living cell

Repeated microscopic observations of exponentially growing Chinese hamster ovary cells were made and the times and mitotic stages were recorded in control and irradiated cultures at 37 degrees C. As determined by autoradiography, the time from the end of S phase to early prophase (the G2 phase) was 46 min, to breakdown of the nuclear envelope was 91 min, and to restoration of the nuclear envelope was 116 min. The time spent in morphologically distinguishable phases of mitosis and the effects of 0.5, 1.0, 1.5, 2.0, and 4.0 Gy of gamma or X radiation on cells at each phase were determined. Affected cells were found to be delayed without or with reversion to an earlier mitotic stage before recovering and advancing through mitosis. Cells were timed in the five steps comprising delay with reversion: inertia, cessation I, regression, cessation II, and reprogression. No cells treated in late prophase, i.e., within 8-10 min of nuclear envelope breakdown, were delayed by the doses used; therefore the critical or transition point must be situated in middle prophase. Cells irradiated in this stage were not delayed by 0.5 or 1.0 Gy, but suffered a dose-dependent delay with or without reversion after 1.5, 2.0, and 4.0 Gy. Cells irradiated in early prophase and very late interphase responded similarly, but a greater percentage of the latter reverted.