Growth Inhibition by Amino Acids in Saccharomyces cerevisiae

Amino acids are essential metabolites but can also be toxic when present at high levels intracellularly. Substrate-induced downregulation of amino acid transporters in Saccharomyces cerevisiae is thought to be a mechanism to avoid this toxicity. It has been shown that unregulated uptake by the general amino acid permease Gap1 causes cells to become sensitive to amino acids. Here, we show that overexpression of eight other amino acid transporters (Agp1, Bap2, Can1, Dip5, Gnp1, Lyp1, Put4, or Tat2) also induces a growth defect when specific single amino acids are present at concentrations of 0.5-5 mM. We can now state that all proteinogenic amino acids, as well as the important metabolite ornithine, are growth inhibitory to S. cerevisiae when transported into the cell at high enough levels. Measurements of initial transport rates and cytosolic pH show that toxicity is due to amino acid accumulation and not to the influx of co-transported protons. The amino acid sensitivity phenotype is a useful tool that reports on the in vivo activity of transporters and has allowed us to identify new transporter-specific substrates

Structural Kinetic Modeling of Metabolic Networks

To develop and investigate detailed mathematical models of cellular metabolic processes is one of the primary challenges in systems biology. However, despite considerable advance in the topological analysis of metabolic networks, explicit kinetic modeling based on differential equations is still often severely hampered by inadequate knowledge of the enzyme-kinetic rate laws and their associated parameter values. Here we propose a method that aims to give a detailed and quantitative account of the dynamical capabilities of metabolic systems, without requiring any explicit information about the particular functional form of the rate equations. Our approach is based on constructing a local linear model at each point in parameter space, such that each element of the model is either directly experimentally accessible, or amenable to a straightforward biochemical interpretation. This ensemble of local linear models, encompassing all possible explicit kinetic models, then allows for a systematic statistical exploration of the comprehensive parameter space. The method is applied to two paradigmatic examples: The glycolytic pathway of yeast and a realistic-scale representation of the photosynthetic Calvin cycle.Comment: 14 pages, 8 figures (color

Asymmetry in inward- and outward-affinity constant of transport explain unidirectional lysine flux in Saccharomyces cerevisiae

Contains fulltext : 171926.pdf (publisher's version ) (Open Access)The import of basic amino acids in Saccharomyces cerevisiae has been reported to be unidirectional, which is not typical of how secondary transporters work. Since studies of energy coupling and transport kinetics are complicated in vivo, we purified the major lysine transporter (Lyp1) of yeast and reconstituted the protein into lipid vesicles. We show that the Michaelis constant (KM) of transport from out-to-in is well in the millimolar range and at least 3 to 4-orders of magnitude higher than that of transport in the opposite direction, disfavoring the efflux of solute via Lyp1. We also find that at low values of the proton motive force, the transport by Lyp1 is comparatively slow. We benchmarked the properties of eukaryotic Lyp1 to that of the prokaryotic homologue LysP and find that LysP has a similar KM for transport from in-to-out and out-to-in, consistent with rapid influx and efflux. We thus explain the previously described unidirectional nature of lysine transport in S. cerevisiae by the extraordinary kinetics of Lyp1 and provide a mechanism and rationale for previous observations. The high asymmetry in transport together with secondary storage in the vacuole allow the cell to accumulate basic amino acids to very high levels

Structural basis for the enhanced activity of cyclic antimicrobial peptides:The case of BPC194

AbstractWe report the molecular basis for the differences in activity of cyclic and linear antimicrobial peptides. We iteratively performed atomistic molecular dynamics simulations and biophysical measurements to probe the interaction of a cyclic antimicrobial peptide and its inactive linear analogue with model membranes. We establish that, relative to the linear peptide, the cyclic one binds stronger to negatively charged membranes. We show that only the cyclic peptide folds at the membrane interface and adopts a β-sheet structure characterised by two turns. Subsequently, the cyclic peptide penetrates deeper into the bilayer while the linear peptide remains essentially at the surface. Finally, based on our comparative study, we propose a model characterising the mode of action of cyclic antimicrobial peptides. The results provide a chemical rationale for enhanced activity in certain cyclic antimicrobial peptides and can be used as a guideline for design of novel antimicrobial peptides

Weak Acid Permeation in Synthetic Lipid Vesicles and Across the Yeast Plasma Membrane

We present a fluorescence-based approach for determination of the permeability of small molecules across the membranes of lipid vesicles and living cells. With properly designed experiments, the method allows us to assess the membrane physical properties both in vitro and in vivo. We find that the permeability of weak acids increases in the order of benzoic > acetic > formic > lactic, both in synthetic lipid vesicles and the plasma membrane of Saccharomyces cerevisiae, but the permeability is much lower in yeast (one to two orders of magnitude). We observe a relation between the molecule permeability and the saturation of the lipid acyl chain (i.e., lipid packing) in the synthetic lipid vesicles. By analyzing wild-type yeast and a manifold knockout strain lacking all putative lactic acid transporters, we conclude that the yeast plasma membrane is impermeable to lactic acid on timescales up to ∼2.5 h.BT/Industrial Microbiolog

Haemophilus influenzae type b reemergence after combination immunization

An increase in Haemophilus influenzae type b (Hib) in British children has been linked to the widespread use of a diphtheria/tetanus/acellular pertussis combination vaccine (DTaP-Hib). We measured anti-polyribosyl-ribitol phos- phate antibody concentration and avidity before and after a Hib booster in 176 children 2–4 years of age who had received 3 doses of DTP-Hib (either DT whole cell pertus- sis-Hib or DTaP-Hib) combination vaccine in infancy. We also measured pharyngeal carriage of Hib. Antibody con- centrations before and avidity indices after vaccination were low (geometric mean concentration 0.46μg/mL, 95% confidence interval [CI] 0.36–0.58; geometric mean avidity index 0.16, 95% CI 0.14–0.18) and inversely related to the number of previous doses of DTaP-Hib (p = 0.02 and p<0.001, respectively). Hib was found in 2.1% (95% CI 0.7%–6.0%) of study participants. Our data support an association between DTaP-Hib vaccine combinations and clinical Hib disease through an effect on antibody concen- tration and avidit

Tele work: dispersed organizational activity and new forms of spatial - temporal coordination and control

This article focuses on a set of new flexible work arrangements commonly referred to as cctelework) or cctelecommuting,). Although it is not possible to give an unambiguous and precise definition of telework, there is some working consensus on the subject: the belief is held that information technology has made it possible to relocate many types of work involving the electronic processing of information geographically, and telework is the term used to describe workers who have been relocated in this way. An attempt is made to develop theoretical framework in which telework cart be studied. For this purpose the changes that take place: in the environment of organizations have been taken into consideration. It is suggested that as a result of the redesign process new organizational forms emerge that are characterized by a ((dispersed activity pattern)). Dispersed Organizations, are designed to overcome time-andplace constraints associated with rigid bureaucratic structuring and can be distinguished from existing bureaucratic organizations because they are based on it different design philosophy with regard to how, where and when work is done. Dispersed organizations are being characterized by different time and place constraints with respect to interaction between operational units. Although organizations with dispersed activities can be found nowadays in almost every institutional area, little systematic analysis has yet been undertaken. It is assumed that different forms of telework can be studied within this context. The concept of (dispersed organizations) will be discussed and illustrated with results from a case-study conducted in an energycompany in the Netherlands

Tensor Analyzing Powers for Quasi-Elastic Electron Scattering from Deuterium

We report on a first measurement of tensor analyzing powers in quasi-elastic electron-deuteron scattering at an average three-momentum transfer of 1.7 fm$^{-1}$. Data sensitive to the spin-dependent nucleon density in the deuteron were obtained for missing momenta up to 150 MeV/$c$ with a tensor polarized $^2$H target internal to an electron storage ring. The data are well described by a calculation that includes the effects of final-state interaction, meson-exchange and isobar currents, and leading-order relativistic contributions.Comment: 4 pages, 3 figure

Spin-Momentum Correlations in Quasi-Elastic Electron Scattering from Deuterium

We report on a measurement of spin-momentum correlations in quasi-elastic scattering of longitudinally polarized electrons with an energy of 720 MeV from vector-polarized deuterium. The spin correlation parameter $A^V_{ed}$ was measured for the $^2 \vec{\rm H}(\vec e,e^\prime p)n$ reaction for missing momenta up to 350 MeV/$c$ at a four-momentum transfer squared of 0.21 (GeV/c)$^2$. The data give detailed information about the spin structure of the deuteron, and are in good agreement with the predictions of microscopic calculations based on realistic nucleon-nucleon potentials and including various spin-dependent reaction mechanism effects. The experiment demonstrates in a most direct manner the effects of the D-state in the deuteron ground-state wave function and shows the importance of isobar configurations for this reaction.Comment: 4 pages, 3 figures, submitted to Phys. Rev. Lett. for publicatio

Functional recovery is considered the most important target: a survey of dedicated professionals

Background: The aim of this study was to survey the relative importance of postoperative recovery targets and perioperative care items, as perceived by a large group of international dedicated professionals. Methods: A questionnaire with eight postoperative recovery targets and 13 perioperative care items was mailed to participants of the first international Enhanced Recovery After Surgery (ERAS) congress and to authors of papers with a clear relevance to ERAS in abdominal surgery. The responders were divided into categories according to profession and region. Results: The recovery targets ‘To be completely free of nausea’, ‘To be independently mobile’ and ‘To be able to eat and drink as soon as possible’ received the highest score irrespective of the responder's profession or region of origin. Equally, the care items ‘Optimizing fluid balance’, ‘Preoperative counselling’ and ‘Promoting early and scheduled mobilisation’ received the highest score across all groups. Conclusions: Functional recovery, as in tolerance of food without nausea and regained mobility, was considered the most important target of recovery. There was a consistent uniformity in the way international dedicated professionals scored the relative importance of recovery targets and care items. The relative rating of the perioperative care items was not dependent on the strength of evidence supporting the items