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Sample records for michaelis-menten mechanism continuous

  1. Leonor Michaelis and Maud Leonora Menten

    Indian Academy of Sciences (India)

    IAS Admin

    Michaelis and Menten's classic paper titled 'Die Kinetik der Invertin wirkung', published ... world is complete without a discussion of the Michaelis–Menten Equation. .... This famous equation must be explored and appreciated in broader contexts.

  2. The Michaelis-Menten-Stueckelberg Theorem

    Directory of Open Access Journals (Sweden)

    Alexander N. Gorban

    2011-05-01

    Full Text Available We study chemical reactions with complex mechanisms under two assumptions: (i intermediates are present in small amounts (this is the quasi-steady-state hypothesis or QSS and (ii they are in equilibrium relations with substrates (this is the quasiequilibrium hypothesis or QE. Under these assumptions, we prove the generalized mass action law together with the basic relations between kinetic factors, which are sufficient for the positivity of the entropy production but hold even without microreversibility, when the detailed balance is not applicable. Even though QE and QSS produce useful approximations by themselves, only the combination of these assumptions can render the possibility beyond the “rarefied gas” limit or the “molecular chaos” hypotheses. We do not use any a priori form of the kinetic law for the chemical reactions and describe their equilibria by thermodynamic relations. The transformations of the intermediate compounds can be described by the Markov kinetics because of their low density (low density of elementary events. This combination of assumptions was introduced by Michaelis and Menten in 1913. In 1952, Stueckelberg used the same assumptions for the gas kinetics and produced the remarkable semi-detailed balance relations between collision rates in the Boltzmann equation that are weaker than the detailed balance conditions but are still sufficient for the Boltzmann H-theorem to be valid. Our results are obtained within the Michaelis-Menten-Stueckelbeg conceptual framework.

  3. Reexamining Michaelis-Menten Enzyme Kinetics for Xanthine Oxidase

    Science.gov (United States)

    Bassingthwaighte, James B.; Chinn, Tamara M.

    2013-01-01

    Abbreviated expressions for enzyme kinetic expressions, such as the Michaelis-Menten (M-M) equations, are based on the premise that enzyme concentrations are low compared with those of the substrate and product. When one does progress experiments, where the solute is consumed during conversion to form a series of products, the idealized conditions…

  4. Exact and approximate solutions for the decades-old Michaelis-Menten equation: Progress-curve analysis through integrated rate equations.

    Science.gov (United States)

    Goličnik, Marko

    2011-01-01

    The Michaelis-Menten rate equation can be found in most general biochemistry textbooks, where the time derivative of the substrate is a hyperbolic function of two kinetic parameters (the limiting rate V, and the Michaelis constant K(M) ) and the amount of substrate. However, fundamental concepts of enzyme kinetics can be difficult to understand fully, or can even be misunderstood, by students when based only on the differential form of the Michaelis-Menten equation, and the variety of methods available to calculate the kinetic constants from rate versus substrate concentration "textbook data." Consequently, enzyme kinetics can be confusing if an analytical solution of the Michaelis-Menten equation is not available. Therefore, the still rarely known exact solution to the Michaelis-Menten equation is presented here through the explicit closed-form equation in terms of the Lambert W(x) function. Unfortunately, as the W(x) is not available in standard curve-fitting computer programs, the practical use of this direct solution is limited for most life-science students. Thus, the purpose of this article is to provide analytical approximations to the equation for modeling Michaelis-Menten kinetics. The elementary and explicit nature of these approximations can provide students with direct and simple estimations of kinetic parameters from raw experimental time-course data. The Michaelis-Menten kinetics studied in the latter context can provide an ideal alternative to the 100-year-old problems of data transformation, graphical visualization, and data analysis of enzyme-catalyzed reactions. Hence, the content of the course presented here could gradually become an important component of the modern biochemistry curriculum in the 21st century. Copyright © 2011 Wiley Periodicals, Inc.

  5. Dynamic disorder in single-molecule Michaelis-Menten kinetics: The reaction-diffusion formalism in the Wilemski-Fixman approximation

    Science.gov (United States)

    Chaudhury, Srabanti; Cherayil, Binny J.

    2007-09-01

    Single-molecule equations for the Michaelis-Menten [Biochem. Z. 49, 333 (1913)] mechanism of enzyme action are analyzed within the Wilemski-Fixman [J. Chem. Phys. 58, 4009 (1973); 60, 866 (1974)] approximation after the effects of dynamic disorder—modeled by the anomalous diffusion of a particle in a harmonic well—are incorporated into the catalytic step of the reaction. The solution of the Michaelis-Menten equations is used to calculate the distribution of waiting times between successive catalytic turnovers in the enzyme β-galactosidase. The calculated distribution is found to agree qualitatively with experimental results on this enzyme obtained at four different substrate concentrations. The calculations are also consistent with measurements of correlations in the fluctuations of the fluorescent light emitted during the course of catalysis, and with measurements of the concentration dependence of the randomness parameter.

  6. Classical Michaelis-Menten and system theory approach to modeling metabolite formation kinetics.

    Science.gov (United States)

    Popović, Jovan

    2004-01-01

    When single doses of drug are administered and kinetics are linear, techniques, which are based on the compartment approach and the linear system theory approach, in modeling the formation of the metabolite from the parent drug are proposed. Unlike the purpose-specific compartment approach, the methodical, conceptual and computational uniformity in modeling various linear biomedical systems is the dominant characteristic of the linear system approach technology. Saturation of the metabolic reaction results in nonlinear kinetics according to the Michaelis-Menten equation. The two compartment open model with Michaelis-Menten elimination kinetics is theorethicaly basic when single doses of drug are administered. To simulate data or to fit real data using this model, one must resort to numerical integration. A biomathematical model for multiple dosage regimen calculations of nonlinear metabolic systems in steady-state and a working example with phenytoin are presented. High correlation between phenytoin steady-state serum levels calculated from individual Km and Vmax values in the 15 adult epileptic outpatients and the observed levels at the third adjustment of phenytoin daily dose (r=0.961, p<0.01) were found.

  7. Stability in a Simple Food Chain System with Michaelis-Menten Functional Response and Nonlocal Delays

    Directory of Open Access Journals (Sweden)

    Wenzhen Gan

    2013-01-01

    Full Text Available This paper is concerned with the asymptotical behavior of solutions to the reaction-diffusion system under homogeneous Neumann boundary condition. By taking food ingestion and species' moving into account, the model is further coupled with Michaelis-Menten type functional response and nonlocal delay. Sufficient conditions are derived for the global stability of the positive steady state and the semitrivial steady state of the proposed problem by using the Lyapunov functional. Our results show that intraspecific competition benefits the coexistence of prey and predator. Furthermore, the introduction of Michaelis-Menten type functional response positively affects the coexistence of prey and predator, and the nonlocal delay is harmless for stabilities of all nonnegative steady states of the system. Numerical simulations are carried out to illustrate the main results.

  8. At the centennial of Michaelis and Menten, competing Michaelis-Menten steps explain effect of GLP-1 on blood-brain transfer and metabolism of glucose.

    Science.gov (United States)

    Gejl, Michael; Rungby, Jørgen; Brock, Birgitte; Gjedde, Albert

    2014-08-01

    Glucagon-like peptide-1 (GLP-1) is a potent insulinotropic incretin hormone with both pancreatic and extrapancreatic effects. Studies of GLP-1 reveal significant effects in regions of brain tissue that regulate appetite and satiety. GLP-1 mimetics are used for the treatment of type 2 diabetes mellitus. GLP-1 interacts with peripheral functions in which the autonomic nervous system plays an important role, and emerging pre-clinical findings indicate a potential neuroprotective role of the peptide, for example in models of stroke and in neurodegenerative disorders. A century ago, Leonor Michaelis and Maud Menten described the steady-state enzyme kinetics that still apply to the multiple receptors, transporters and enzymes that define the biochemical reactions of the brain, including the glucose-dependent impact of GLP-1 on blood-brain glucose transfer and metabolism. This MiniReview examines the potential of GLP-1 as a molecule of interest for the understanding of brain energy metabolism and with reference to the impact on brain metabolism related to appetite and satiety regulation, stroke and neurodegenerative disorders. These effects can be understood only by reference to the original formulation of the Michaelis-Menten equation as applied to a chain of kinetically controlled steps. Indeed, the effects of GLP-1 receptor activation on blood-brain glucose transfer and brain metabolism of glucose depend on the glucose concentration and relative affinities of the steps both in vitro and in vivo, as in the pancreas. © 2014 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  9. Occurrence of dead core in catalytic particles containing immobilized enzymes: analysis for the Michaelis-Menten kinetics and assessment of numerical methods.

    Science.gov (United States)

    Pereira, Félix Monteiro; Oliveira, Samuel Conceição

    2016-11-01

    In this article, the occurrence of dead core in catalytic particles containing immobilized enzymes is analyzed for the Michaelis-Menten kinetics. An assessment of numerical methods is performed to solve the boundary value problem generated by the mathematical modeling of diffusion and reaction processes under steady state and isothermal conditions. Two classes of numerical methods were employed: shooting and collocation. The shooting method used the ode function from Scilab software. The collocation methods included: that implemented by the bvode function of Scilab, the orthogonal collocation, and the orthogonal collocation on finite elements. The methods were validated for simplified forms of the Michaelis-Menten equation (zero-order and first-order kinetics), for which analytical solutions are available. Among the methods covered in this article, the orthogonal collocation on finite elements proved to be the most robust and efficient method to solve the boundary value problem concerning Michaelis-Menten kinetics. For this enzyme kinetics, it was found that the dead core can occur when verified certain conditions of diffusion-reaction within the catalytic particle. The application of the concepts and methods presented in this study will allow for a more generalized analysis and more accurate designs of heterogeneous enzymatic reactors.

  10. Stability estimation of autoregulated genes under Michaelis-Menten-type kinetics

    Science.gov (United States)

    Arani, Babak M. S.; Mahmoudi, Mahdi; Lahti, Leo; González, Javier; Wit, Ernst C.

    2018-06-01

    Feedback loops are typical motifs appearing in gene regulatory networks. In some well-studied model organisms, including Escherichia coli, autoregulated genes, i.e., genes that activate or repress themselves through their protein products, are the only feedback interactions. For these types of interactions, the Michaelis-Menten (MM) formulation is a suitable and widely used approach, which always leads to stable steady-state solutions representative of homeostatic regulation. However, in many other biological phenomena, such as cell differentiation, cancer progression, and catastrophes in ecosystems, one might expect to observe bistable switchlike dynamics in the case of strong positive autoregulation. To capture this complex behavior we use the generalized family of MM kinetic models. We give a full analysis regarding the stability of autoregulated genes. We show that the autoregulation mechanism has the capability to exhibit diverse cellular dynamics including hysteresis, a typical characteristic of bistable systems, as well as irreversible transitions between bistable states. We also introduce a statistical framework to estimate the kinetics parameters and probability of different stability regimes given observational data. Empirical data for the autoregulated gene SCO3217 in the SOS system in Streptomyces coelicolor are analyzed. The coupling of a statistical framework and the mathematical model can give further insight into understanding the evolutionary mechanisms toward different cell fates in various systems.

  11. Patterns induced by super cross-diffusion in a predator-prey system with Michaelis-Menten type harvesting.

    Science.gov (United States)

    Liu, Biao; Wu, Ranchao; Chen, Liping

    2018-04-01

    Turing instability and pattern formation in a super cross-diffusion predator-prey system with Michaelis-Menten type predator harvesting are investigated. Stability of equilibrium points is first explored with or without super cross-diffusion. It is found that cross-diffusion could induce instability of equilibria. To further derive the conditions of Turing instability, the linear stability analysis is carried out. From theoretical analysis, note that cross-diffusion is the key mechanism for the formation of spatial patterns. By taking cross-diffusion rate as bifurcation parameter, we derive amplitude equations near the Turing bifurcation point for the excited modes by means of weakly nonlinear theory. Dynamical analysis of the amplitude equations interprets the structural transitions and stability of various forms of Turing patterns. Furthermore, the theoretical results are illustrated via numerical simulations. Copyright © 2018. Published by Elsevier Inc.

  12. Diffusion influence on Michaelis Menten kinetics: II. The low substrate concentration limit

    Science.gov (United States)

    Kim, Hyojoon; Shin, Kook Joe

    2007-02-01

    The diffusion-influenced Michaelis-Menten kinetics in the low substrate concentration limit is studied in one and three dimensions. For the initial pair distribution of enzyme and substrate, we obtain the exact analytical results. We find that at short times the diffusion effect can make the reaction rate faster. The concentration deviations of the substrate and enzyme show t-1/2 and t-3/2 power-law behaviours in one and three dimensions, respectively, at long times. On the other hand, the average lifetime of the intermediate is independent of the initial state in one dimension, while it depends on the initial state in three dimensions. The ultimate production yield approaches unity in one dimension but it reaches a different value depending on other parameters in three dimensions. We also obtain the analytical results for the initial random distribution.

  13. Diffusion influence on Michaelis-Menten kinetics: II. The low substrate concentration limit

    International Nuclear Information System (INIS)

    Kim, Hyojoon; Shin, Kook Joe

    2007-01-01

    The diffusion-influenced Michaelis-Menten kinetics in the low substrate concentration limit is studied in one and three dimensions. For the initial pair distribution of enzyme and substrate, we obtain the exact analytical results. We find that at short times the diffusion effect can make the reaction rate faster. The concentration deviations of the substrate and enzyme show t -1/2 and t -3/2 power-law behaviours in one and three dimensions, respectively, at long times. On the other hand, the average lifetime of the intermediate is independent of the initial state in one dimension, while it depends on the initial state in three dimensions. The ultimate production yield approaches unity in one dimension but it reaches a different value depending on other parameters in three dimensions. We also obtain the analytical results for the initial random distribution

  14. Complex dynamics of a stochastic discrete modified Leslie-Gower predator-prey model with Michaelis-Menten type prey harvesting

    Directory of Open Access Journals (Sweden)

    A. Elhassanein

    2014-06-01

    Full Text Available This paper introduced a stochastic discretized version of the modified Leslie-Gower predator-prey model with Michaelis-Menten type prey harvesting. The dynamical behavior of the proposed model was investigated. The existence and stability of the equilibria of the skeleton were studied. Numerical simulations were employed to show the model's complex dynamics by means of the largest Lyapunov exponents, bifurcations, time series diagrams and phase portraits. The effects of noise intensity on its dynamics and the intermittency phenomenon were also discussed via simulation.

  15. Why plankton modellers should reconsider using rectangular hyperbolic (Michaelis-Menten, Monod descriptions of predator-prey interactions

    Directory of Open Access Journals (Sweden)

    Kevin John Flynn

    2016-09-01

    Full Text Available Rectangular hyperbolic type 2 (RHt2; Michaelis-Menten or Monod -like functions are commonly used to describe predation kinetics in plankton models, either alone or together with a prey selectivity algorithm deploying the same half-saturation constant for all prey types referenced to external prey biomass abundance. We present an analysis that indicates that such descriptions are liable to give outputs that are not plausible according to encounter theory. This is especially so for multi-prey type applications or where changes are made to the maximum feeding rate during a simulation. The RHt2 approach also gives no or limited potential for descriptions of events such as true de-selection of prey, effects of turbulence on encounters, or changes in grazer motility with satiation. We present an alternative, which carries minimal parameterisation effort and computational cost, linking allometric algorithms relating prey abundance and encounter rates to a prey-selection function controlled by satiation. The resultant Satiation-Controlled-Encounter-Based (SCEB function provides a flexible construct describing numeric predator-prey interactions with biomass-feedback control of grazing. The SCEB function includes an attack component similar to that in the Holling disk equation but SCEB differs in having only a single (satiation-based handling constant and an explicit maximum grazing rate. We argue that there is no justification for continuing to deploy RHt2 functions to describe plankton predator-prey interactions.

  16. Biphasic character of ribosomal translocation and non-Michaelis-Menten kinetics of translation

    Science.gov (United States)

    Xie, Ping

    2014-12-01

    We study theoretically the kinetics of mRNA translocation in the wild-type (WT) Escherichia coli ribosome, which is composed of a small 30 S and large 50 S subunit, and the ribosomes with mutations to some intersubunit bridges such as B1a, B4, B7a, and B8. The theoretical results reproduce well the available in vitro experimental data on the biphasic kinetics of the forward mRNA translocation catalyzed by elongation factor G (EF-G) hydrolyzing GTP, which can be best fit by the sum of two exponentials, and the monophasic kinetics of the spontaneous reverse mRNA translocation in the absence of the elongation factor, which can be best fit by a single-exponential function, in both the WT and mutant ribosomes. We show that both the mutation-induced increase in the maximal rate of the slow phase for the forward mRNA translocation and that in the rate of the spontaneous reverse mRNA translocation result from a reduction in the intrinsic energy barrier to resist the rotational movements between the two subunits, giving the same degree of increase in the two rates. The mutation-induced increase in the maximal rate of the fast phase for the forward mRNA translocation results mainly from the increase in the rate of the ribosomal unlocking, a conformational change in the ribosome that widens the mRNA channel for the mRNA translocation to take place, which could be partly due to the effect of the mutation on the intrasubunit 30S head rotation. Moreover, we study the translation rate of the WT and mutant ribosomes. It is shown that the translation rate versus the concentration of EF-G-GTP does not follow the Michaelis-Menten (MM) kinetics, which is in sharp contrast to the general property of other enzymes that the rate of the enzymatic reaction versus the concentration of a substrate follows the MM kinetics. The physical origin of this non-MM kinetics for the ribosome is revealed.

  17. Utilization of integrated Michaelis-Menten equations for enzyme inhibition diagnosis and determination of kinetic constants using Solver supplement of Microsoft Office Excel.

    Science.gov (United States)

    Bezerra, Rui M F; Fraga, Irene; Dias, Albino A

    2013-01-01

    Enzyme kinetic parameters are usually determined from initial rates nevertheless, laboratory instruments only measure substrate or product concentration versus reaction time (progress curves). To overcome this problem we present a methodology which uses integrated models based on Michaelis-Menten equation. The most severe practical limitation of progress curve analysis occurs when the enzyme shows a loss of activity under the chosen assay conditions. To avoid this problem it is possible to work with the same experimental points utilized for initial rates determination. This methodology is illustrated by the use of integrated kinetic equations with the well-known reaction catalyzed by alkaline phosphatase enzyme. In this work nonlinear regression was performed with the Solver supplement (Microsoft Office Excel). It is easy to work with and track graphically the convergence of SSE (sum of square errors). The diagnosis of enzyme inhibition was performed according to Akaike information criterion. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  18. Determination of the Michaelis-Menten kinetics and the genes expression involved in phyto-degradation of cyanide and ferri-cyanide.

    Science.gov (United States)

    Yu, Xiao-Zhang; Zhang, Xue-Hong

    2016-07-01

    Hydroponic experiments were conducted with different species of plants (rice, maize, soybean and willow) exposed to ferri-cyanide to investigate the half-saturation constant (K M ) and the maximal metabolic capacity (v max ) involved in phyto-assimilation. Three varieties for each testing species were collected from different origins. Measured concentrations show that the uptake rates responded biphasically to ferri-cyanide treatments by showing increases linearly at low and almost constant at high concentrations from all treatments, indicating that phyto-assimilation of ferri-cyanide followed the Michaelis-Menten kinetics. Using non-linear regression, the highest v max was by rice, followed by willows. The lowest v max was found for soybean. All plants, except maize (DY26) and rice (XJ12), had a similar K M value, suggesting the same enzyme was active in phyto-assimilation of ferri-cyanide. Transcript level, by real-time quantitative PCR, of enzymes involved in degradation of cyanides showed that the analyzed genes were differently expressed during different cyanides exposure. The expression of CAS and ST genes responded positively to KCN exposure, suggesting that β-CAS and ST pathways were two possible pathways for cyanide detoxification in rice. The transcript level of NIT and ASPNASE genes also showed a remarkable up-regulation to KCN, implying the contribution to the pool of amino acid aspartate, which is an end product of CN metabolism. Up-regulation of GS genes suggests that acquisition of ammonium released from cyanide degradation may be an additional nitrogen source for plant nutrition. Results also revealed that the expressions of these genes, except for GS, were relatively constant during iron cyanide exposure, suggesting that they are likely metabolized by plants through a non-defined pathway rather than the β-CAS pathway.

  19. Microscale Measurements of Michaelis-Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage.

    Science.gov (United States)

    Gattu, Srikanth; Crihfield, Cassandra L; Holland, Lisa A

    2017-01-03

    Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis-Menten constants (K M ) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A K M value of 3.3 ± 0.8 mM (V max , 2100 ± 200 μM/min) was obtained for 3'-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a K M of 2 ± 1 mM (V max , 400 ± 100 μM/min) was obtained for the 6'-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a K M value of 3 ± 2 mM (V max , 900 ± 300 μM/min) for 3'-sialyllactose. With a knowledge of V max , the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3' and 6' sialic acid linkages.

  20. New types of experimental data shape the use of enzyme kinetics for dynamic network modeling.

    Science.gov (United States)

    Tummler, Katja; Lubitz, Timo; Schelker, Max; Klipp, Edda

    2014-01-01

    Since the publication of Leonor Michaelis and Maude Menten's paper on the reaction kinetics of the enzyme invertase in 1913, molecular biology has evolved tremendously. New measurement techniques allow in vivo characterization of the whole genome, proteome or transcriptome of cells, whereas the classical enzyme essay only allows determination of the two Michaelis-Menten parameters V and K(m). Nevertheless, Michaelis-Menten kinetics are still commonly used, not only in the in vitro context of enzyme characterization but also as a rate law for enzymatic reactions in larger biochemical reaction networks. In this review, we give an overview of the historical development of kinetic rate laws originating from Michaelis-Menten kinetics over the past 100 years. Furthermore, we briefly summarize the experimental techniques used for the characterization of enzymes, and discuss web resources that systematically store kinetic parameters and related information. Finally, describe the novel opportunities that arise from using these data in dynamic mathematical modeling. In this framework, traditional in vitro approaches may be combined with modern genome-scale measurements to foster thorough understanding of the underlying complex mechanisms. © 2013 FEBS.

  1. Steady-state cerebral glucose concentrations and transport in the human brain

    OpenAIRE

    Gruetter, R.; Ugurbil, K.; Seaquist, E. R.

    1998-01-01

    Understanding the mechanism of brain glucose transport across the blood- brain barrier is of importance to understanding brain energy metabolism. The specific kinetics of glucose transport nave been generally described using standard Michaelis-Menten kinetics. These models predict that the steady- state glucose concentration approaches an upper limit in the human brain when the plasma glucose level is well above the Michaelis-Menten constant for half-maximal transport, K(t). In experiments wh...

  2. Kinetics and mechanism of oxidation of aliphatic alcohols by ...

    Indian Academy of Sciences (India)

    TBATB) in aqueous acetic acid leads to the formation of the corresponding aldehydes. The reaction is first order with respect to TBATB. Michaelis-Menten type kinetics is observed with respect to alcohols. The reaction failed to induce the ...

  3. Transient competitive complexation in biological kinetic isotope fractionation explains nonsteady isotopic effects: Theory and application to denitrification in soils

    Science.gov (United States)

    Maggi, Federico; Riley, William J.

    2009-12-01

    The theoretical formulation of biological kinetic isotope fractionation often assumes first-order or Michaelis-Menten kinetics, the latter solved under the quasi-steady state assumption. Both formulations lead to a constant isotope fractionation factor, therefore they may return incorrect estimations of isotopic effects and misleading interpretations of isotopic signatures when fractionation is not a steady process. We have analyzed the isotopic signature of denitrification in biogeochemical soil systems by Menyailo and Hungate (2006) in which high and variable 15N-N2O enrichment during N2O production and inverse isotope fractionation during N2O consumption could not be explained with first-order kinetics and the Rayleigh equation, or with Michaelis-Menten kinetics. When Michaelis-Menten kinetics were coupled to Monod kinetics to describe biomass and enzyme dynamics, and the quasi-steady state assumption was relaxed, transient Michaelis-Menten-Monod kinetics accurately reproduced the observed concentrations, and variable and inverse isotope fractionations. These results imply a substantial revision in modeling isotopic effects, suggesting that steady state kinetics such as first-order, Rayleigh, and classic Michaelis-Menten kinetics should be superseded by transient kinetics in conjunction with biomass and enzyme dynamics.

  4. Michaelis - Menten equation for degradation of insoluble substrate

    DEFF Research Database (Denmark)

    Andersen, Morten; Kari, Jeppe; Borch, Kim

    2017-01-01

    substrate it is difficult to assess whether the requirement of the MM equation is met. In this paper we study a simple kinetic model, where removal of attack sites expose new ones which preserve the total accessible substrate, and denote this approach the substrate conserving model. The kinetic equations...... are solved in closed form, both steady states and progress curves, for any admissible values of initial conditions and rate constants. The model is shown to merge with the MM equation and the reverse MM equation when these are valid. The relation between available molar concentration of attack sites and mass...

  5. Transient competitive complexation in biological kinetic isotope fractionation explains non-steady isotopic effects: Theory and application to denitrification in soils

    Energy Technology Data Exchange (ETDEWEB)

    Maggi, F.M.; Riley, W.J.

    2009-06-01

    The theoretical formulation of biological kinetic reactions in isotopic applications often assume first-order or Michaelis-Menten-Monod kinetics under the quasi-steady-state assumption to simplify the system kinetics. However, isotopic e ects have the same order of magnitude as the potential error introduced by these simpli cations. Both formulations lead to a constant fractionation factor which may yield incorrect estimations of the isotopic effect and a misleading interpretation of the isotopic signature of a reaction. We have analyzed the isotopic signature of denitri cation in biogeochemical soil systems by Menyailo and Hungate [2006], where high {sup 15}N{sub 2}O enrichment during N{sub 2}O production and inverse isotope fractionation during N{sub 2}O consumption could not be explained with first-order kinetics and the Rayleigh equation, or with the quasi-steady-state Michaelis-Menten-Monod kinetics. When the quasi-steady-state assumption was relaxed, transient Michaelis-Menten-Monod kinetics accurately reproduced the observations and aided in interpretation of experimental isotopic signatures. These results may imply a substantial revision in using the Rayleigh equation for interpretation of isotopic signatures and in modeling biological kinetic isotope fractionation with first-order kinetics or quasi-steady-state Michaelis-Menten-Monod kinetics.

  6. Evaluation of rate law approximations in bottom-up kinetic models of metabolism

    DEFF Research Database (Denmark)

    Du, Bin; Zielinski, Daniel C.; Kavvas, Erol S.

    2016-01-01

    mass action rate law that removes the role of the enzyme from the reaction kinetics. We utilized in vivo data for the human red blood cell to compare the effect of rate law choices against the backdrop of physiological flux and concentration differences. We found that the Michaelis-Menten rate law......Background: The mechanistic description of enzyme kinetics in a dynamic model of metabolism requires specifying the numerical values of a large number of kinetic parameters. The parameterization challenge is often addressed through the use of simplifying approximations to form reaction rate laws....... These approximate rate laws were: 1) a Michaelis-Menten rate law with measured enzyme parameters, 2) a Michaelis-Menten rate law with approximated parameters, using the convenience kinetics convention, 3) a thermodynamic rate law resulting from a metabolite saturation assumption, and 4) a pure chemical reaction...

  7. A stochastic model of enzyme kinetics

    Science.gov (United States)

    Stefanini, Marianne; Newman, Timothy; McKane, Alan

    2003-10-01

    Enzyme kinetics is generally modeled by deterministic rate equations, and in the simplest case leads to the well-known Michaelis-Menten equation. It is plausible that stochastic effects will play an important role at low enzyme concentrations. We have addressed this by constructing a simple stochastic model which can be exactly solved in the steady-state. Throughout a wide range of parameter values Michaelis-Menten dynamics is replaced by a new and simple theoretical result.

  8. Journal of Chemical Sciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    Kinetics and mechanism of oxidation of formic and oxalic acids by quinolinium fluorochromate (QFC) have been studied in dimethylsulphoxide. The main product of oxidation is carbon dioxide. The reaction is first-order with respect to QFC. Michaelis-Menten type of kinetics were observed with respect to the reductants.

  9. Elimination of intermediate species in multiscale stochastic reaction networks

    DEFF Research Database (Denmark)

    Cappelletti, Daniele; Wiuf, Carsten

    2016-01-01

    such as the substrate-enzyme complex in the Michaelis-Menten mechanism. Such species are virtually in all real-world networks, they are typically short-lived, degraded at a fast rate and hard to observe experimentally. We provide conditions under which the Markov process of a multiscale reaction network...

  10. Picolinic acid promoted oxidative decarboxylation of ...

    African Journals Online (AJOL)

    The kinetics and mechanism of picolinic acid promoted reaction of phenylsulfinylacetic acid (PSAA) with Cr(VI) was carried out in aqueous acetonitrile medium under pseudo first order conditions. The reaction follows Michaelis-Menten type of kinetics with respect to PSAA. The catalytic activity by picolinic acid can be ...

  11. More Nuts and Bolts of Michaelis-Menten Enzyme Kinetics

    Science.gov (United States)

    Lechner, Joseph H.

    2011-01-01

    Several additions to a classroom activity are proposed in which an "enzyme" (the student) converts "substrates" (nut-bolt assemblies) into "products" (separated nuts and bolts) by unscrewing them. (Contains 1 table.)

  12. New insights into the catalytic mechanism of human glycine N-acyltransferase.

    Science.gov (United States)

    van der Sluis, Rencia; Ungerer, Vida; Nortje, Carla; A van Dijk, Alberdina; Erasmus, Elardus

    2017-11-01

    Even though the glycine conjugation pathway was one of the first metabolic pathways to be discovered, this pathway remains very poorly characterized. The bi-substrate kinetic parameters of a recombinant human glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13) were determined using the traditional colorimetric method and a newly developed HPLC-ESI-MS/MS method. Previous studies analyzing the kinetic parameters of GLYAT, indicated a random Bi-Bi and/or ping-pong mechanism. In this study, the hippuric acid concentrations produced by the GLYAT enzyme reaction were analyzed using the allosteric sigmoidal enzyme kinetic module. Analyses of the initial rate (v) against substrate concentration plots, produced a sigmoidal curve (substrate activation) when the benzoyl-CoA concentrations was kept constant, whereas the plot with glycine concentrations kept constant, passed through a maximum (substrate inhibition). Thus, human GLYAT exhibits mechanistic kinetic cooperativity as described by the Ferdinand enzyme mechanism rather than the previously assumed Michaelis-Menten reaction mechanism. © 2017 Wiley Periodicals, Inc.

  13. Response to a temperature modulation as a signature of chemical mechanisms.

    Science.gov (United States)

    Berthoumieux, H; Jullien, L; Lemarchand, A

    2007-11-01

    We consider n reactive species involved in unimolecular reactions and submitted to a temperature modulation of small amplitude. We determine the conditions on the rate constants for which the deviations from the equilibrium concentrations of each species can be optimized and find the analytical expression of the frequency associated with an extremum of concentration shift in the case n=3. We prove that the frequency dependence of the displacement of equilibrium gives access to the number n of species involved in the mechanism. We apply the results to the case of the transformation of a reactant into a product through a possible reactive intermediate and find the order relation obeyed by the activation energies of the different barriers. The results typically apply to enzymatic catalysis with kinetics of Michaelis-Menten type.

  14. An Inverse Michaelis–Menten Approach for Interfacial Enzyme Kinetics

    DEFF Research Database (Denmark)

    Kari, Jeppe; Andersen, Morten; Borch, Kim

    2017-01-01

    Interfacial enzyme reactions are ubiquitous both in vivo and in technical applications, but analysis of their kinetics remains controversial. In particular, it is unclear whether conventional Michaelis–Menten theory, which requires a large excess of substrate, can be applied. Here, an extensive...... experimental study of the enzymatic hydrolysis of insoluble cellulose indeed showed that the conventional approach had a limited applicability. Instead we argue that, unlike bulk reactions, interfacial enzyme catalysis may reach a steady-state condition in the opposite experimental limit, where...... for kinetic analyses of interfacial enzyme reactions and that its analogy to established theory provides a bridge to the accumulated understanding of steady-state enzyme kinetics. Finally, we show that the ratio of parameters from conventional and inverted Michaelis–Menten analysis reveals the density...

  15. The mechanism distinguishability problem in biochemical kinetics: the single-enzyme, single-substrate reaction as a case study.

    Science.gov (United States)

    Schnell, Santiago; Chappell, Michael J; Evans, Neil D; Roussel, Marc R

    2006-01-01

    A theoretical analysis of the distinguishability problem of two rival models of the single enzyme-single substrate reaction, the Michaelis-Menten and Henri mechanisms, is presented. We also outline a general approach for analysing the structural indistinguishability between two mechanisms. The approach involves constructing, if possible, a smooth mapping between the two candidate models. Evans et al. [N.D. Evans, M.J. Chappell, M.J. Chapman, K.R. Godfrey, Structural indistinguishability between uncontrolled (autonomous) nonlinear analytic systems, Automatica 40 (2004) 1947-1953] have shown that if, in addition, either of the mechanisms satisfies a particular criterion then such a transformation always exists when the models are indistinguishable from their experimentally observable outputs. The approach is applied to the single enzyme-single substrate reaction mechanism. In principle, mechanisms can be distinguished using this analysis, but we show that our ability to distinguish mechanistic models depends both on the precise measurements made, and on our knowledge of the system prior to performing the kinetics experiments.

  16. At the centennial of Michaelis and Menten, competing Michaelis-Menten steps explain effect of GLP-1 on blood-brain transfer and metabolism of glucose

    DEFF Research Database (Denmark)

    Jensen, Michael Gejl; Rungby, Jørgen; Brock, Birgitte

    2014-01-01

    Glucagon-like peptide-1 (GLP-1) is a potent insulinotropic incretin hormone with pancreatic and extrapancreatic effects. Studies reveal significant effects in regions of brain tissue that regulate appetite and satiety. The effects cause that mimetics of GLP-1 serves as treatment of type 2 diabete...

  17. Quantitative Evaluation of the Mechanism Underlying the Biotransportation of the Active Ingredients in Puerariae lobatae Radix and Chuanxiong rhizoma.

    Science.gov (United States)

    Liang, Xin-Li; Zhang, Jing; Liao, Zheng-Gen; Zhao, Guo-Wei; Luo, Yun; Li, Zhe; Satterlee, Andrew

    2015-06-15

    The objective of this study was to establish a quantitative method to evaluate the biotransportation of a drug across the cell membrane. Through the application of the law of mass conservation, the drug transportation rate was calculated based on Fick's law of passive diffusion and the Michaelis-Menten equation. The overall membrane-transportation rate was the sum of the passive diffusion rate and the carrier-mediated diffusion rate, which were calculated as the transportation mass divided by the respective rate. The active ingredients of Puerariae lobatae Radix and Chuanxiong rhizoma, namely, puerarin and ferulic acid, respectively, were used as two model drugs. The transportation rates of puerarin and ferulic acid were obtained by fitting a model that includes both Fick's law of diffusion and the Michaelis-Menten equation. Compared with the overall transportation, the carrier-mediated transport and passive diffusion of 1.59 mmol/L puerarin were -35.07% and 64.93%, respectively, whereas the respective transportation modes of 0.1 mmol/L ferulic acid were -35.40% and 64.60%, respectively. Verapamil and MK-571 increased the overall transport rate and ratio, and MK-571 treatment changed the carrier-mediated transport from negative to positive. However, the transport rate and ratio of ferulic acid did not change significantly. The cell transportation mechanisms of puerarin and ferulic acid primarily involve simple passive diffusion and carrier-mediated transportation. Moreover, P-glycoprotein and multidrug resistance-associated protein efflux proteins, and other transportation proteins were found to be involved in the transportation of puerarin. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  18. Michaelis' hundred Questions and the Royal Instructions

    DEFF Research Database (Denmark)

    Friis, Ib

    2017-01-01

    Michaelis' 100 questions for the expedition is a remarkable document. It provides insight into the sources and methods of biblical research anno 1762, at the same time as highlighting the challenges the members of the expedition faced. As the scholarly foundation of the expedition, the questions ...

  19. Michaelis' Hundred Questions and the Royal Instruction

    DEFF Research Database (Denmark)

    Friis, Ib

    2015-01-01

    Michaelis' 100 questions for the expedition is a remarkable document. It provides insight into the sources and methods of biblical research anno 1762, at the same time as highlighting the challenges the members of the expedition faced. As the scholarly foundation of the expedition, the questions ...

  20. Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes

    International Nuclear Information System (INIS)

    Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu; Dua, Arti

    2016-01-01

    Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kinetics resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.

  1. Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu; Dua, Arti, E-mail: arti@iitm.ac.in [Department of Chemistry, Indian Institute of Technology, Madras, Chennai 600036 (India)

    2016-08-28

    Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kinetics resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.

  2. Emergence of dynamic cooperativity in the stochastic kinetics of fluctuating enzymes

    Science.gov (United States)

    Kumar, Ashutosh; Chatterjee, Sambarta; Nandi, Mintu; Dua, Arti

    2016-08-01

    Dynamic co-operativity in monomeric enzymes is characterized in terms of a non-Michaelis-Menten kinetic behaviour. The latter is believed to be associated with mechanisms that include multiple reaction pathways due to enzymatic conformational fluctuations. Recent advances in single-molecule fluorescence spectroscopy have provided new fundamental insights on the possible mechanisms underlying reactions catalyzed by fluctuating enzymes. Here, we present a bottom-up approach to understand enzyme turnover kinetics at physiologically relevant mesoscopic concentrations informed by mechanisms extracted from single-molecule stochastic trajectories. The stochastic approach, presented here, shows the emergence of dynamic co-operativity in terms of a slowing down of the Michaelis-Menten (MM) kinetics resulting in negative co-operativity. For fewer enzymes, dynamic co-operativity emerges due to the combined effects of enzymatic conformational fluctuations and molecular discreteness. The increase in the number of enzymes, however, suppresses the effect of enzymatic conformational fluctuations such that dynamic co-operativity emerges solely due to the discrete changes in the number of reacting species. These results confirm that the turnover kinetics of fluctuating enzyme based on the parallel-pathway MM mechanism switches over to the single-pathway MM mechanism with the increase in the number of enzymes. For large enzyme numbers, convergence to the exact MM equation occurs in the limit of very high substrate concentration as the stochastic kinetics approaches the deterministic behaviour.

  3. Competition between roots and microorganisms for nitrogen: mechanisms and ecological relevance

    Science.gov (United States)

    Kuzyakov, Yakov; Xu, Xingliang

    2014-05-01

    Demand of all living organisms on the same nutrients forms the basis for interspecific competition between plants and microorganisms in soils. This competition is especially strong in the rhizosphere. To evaluate competitive and mutualistic interactions between plants and microorganisms and to analyse ecological consequences of these interactions, we analysed 424 data pairs from 41 15N-labelling studies that investigated 15N redistribution between roots and microorganisms. Calculated Michaelis-Menten kinetics based on Km (Michaelis constant) and Vmax (maximum uptake capacity) values from 77 studies on the uptake of nitrate, ammonia, and amino acids by roots and microorganisms clearly showed that, shortly after nitrogen (N) mobilization from soil organic matter and litter, microorganisms take up most N. Lower Km values of microorganisms suggest that they are especially efficient at low N concentrations, but can also acquire more N at higher N concentrations (Vmax) compared with roots. Because of the unidirectional flow of nutrients from soil to roots, plants are the winners for N acquisition in the long run. Therefore, despite strong competition between roots and microorganisms for N, a temporal niche differentiation reflecting their generation times leads to mutualistic relationships in the rhizosphere. This temporal niche differentiation is highly relevant ecologically because it: protects ecosystems from N losses by leaching during periods of slow or no root uptake; continuously provides roots with available N according to plant demand; and contributes to the evolutionary development of mutualistic interactions between roots and microorganisms.

  4. Transport and phosphorylation of choline in higher plant cells. Phosphorus-31 nuclear magnetic resonance studies

    Energy Technology Data Exchange (ETDEWEB)

    Bligny, R.; Foray, M.F.; Roby, C.; Douce, R.

    1989-03-25

    When sycamore cells were suspended in basal medium containing choline, the latter was taken up by the cells very rapidly. A facilitated diffusion system appertained at low concentrations of choline and exhibited Michaelis-Menten kinetics. At higher choline concentrations simple diffusion appeared to be the principal mode of uptake. Addition of choline to the perfusate of compressed sycamore cells monitored by /sup 31/P NMR spectroscopy resulted in a dramatic accumulation of P-choline in the cytoplasmic compartment containing choline kinase and not in the vacuole. The total accumulation of P-choline over a 10-h period exhibited Michaelis-Menten kinetics. During this period, in the absence of Pi in the perfusion medium there was a marked depletion of glucose-6-P, and the cytoplasmic Pi resonance disappeared almost completely. When a threshold of cytoplasmic Pi was attained, the phosphorylation of choline was sustained by the continuous release of Pi from the vacuole although at a much lower rate. However, when 100 microM inorganic phosphate was present in the perfusion medium, externally added Pi was preferentially used to sustain P-choline synthesis. It is clear, therefore, that cytosolic choline kinase associated with a carrier-mediated transport system for choline uptake appeared as effective systems for continuously trapping cytoplasmic Pi including vacuolar Pi entering the cytoplasm.

  5. A century of enzyme kinetic analysis, 1913 to 2013.

    Science.gov (United States)

    Johnson, Kenneth A

    2013-09-02

    This review traces the history and logical progression of methods for quantitative analysis of enzyme kinetics from the 1913 Michaelis and Menten paper to the application of modern computational methods today. Following a brief review of methods for fitting steady state kinetic data, modern methods are highlighted for fitting full progress curve kinetics based upon numerical integration of rate equations, including a re-analysis of the original Michaelis-Menten full time course kinetic data. Finally, several illustrations of modern transient state kinetic methods of analysis are shown which enable the elucidation of reactions occurring at the active sites of enzymes in order to relate structure and function. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. Una carta de doña Carolina Michaelis

    Directory of Open Access Journals (Sweden)

    José Fradejas Lebrero

    2003-06-01

    Full Text Available Uno de los tres filólogos o críticos literarios más importantes de la Literatura peninsular del siglo XX es doña Carolina Michaelis de Vasconcellos. Cuando se crea la Gesellschaf für Romanische Literatur (1902, los tres van juntos: el socio num. 4 es don Marcelino Menendez Pelayo, el num. 5 don Ramón Menendez Pidal y el num. 6 doña Carolina Michaelis y conste que era más vieja, pues había nacido en 1851 y aún no era doctora, lo sería en Friburgo en 1904, con una obra monumental y maestra: la edición y estudio del Cancionero de Ajuda. Había ya colaborado en el Homenaje a Menendez Pelayo (1899, con quien se carteaba de cuando en cuando y en 1903 proporcionó el texto más interesante de la Leyenda del abad don Juan de Montemayor a don Ramón Menendez Pidal…

  7. Cooperativity in CYP2E1 Metabolism of Acetaminophen and Styrene Mixtures

    OpenAIRE

    Hartman, Jessica H.; Letzig, Lynda G.; Robertsc, Dean W.; James, Laura P.; Fifer, E. Kim; Miller, Grover P.

    2015-01-01

    Risk assessment for exposure to mixtures of drugs and pollutants relies heavily on in vitro characterization of their bioactivation and/or metabolism individually and extrapolation to mixtures assuming no interaction. Herein, we demonstrated that in vitro CYP2E1 metabolic activation of acetaminophen and styrene mixtures could not be explained through the Michaelis-Menten mechanism or any models relying on that premise. As a baseline for mixture studies with styrene, steady-state analysis of a...

  8. Kinetic evaluation of an anaerobic fluidised-bed reactor treating slaughterhouse wastewater

    Energy Technology Data Exchange (ETDEWEB)

    Borja, R. [Consejo Superior de Investigaciones Cientificas, Seville (Spain). Inst. de la Grasa; Banks, C.J.; Zhengjian Wang [Manchester Univ. (United Kingdom). Inst. of Science and Technology

    1995-09-01

    An anaerobic fluidised-bed reactor for purification of slaughterhouse wastewater was modelled as a continuous-flow, completely-mixed homogeneous microbial system, with the feed COD as the limiting-substrate concentration. The average microbial residence time in the reactor was defined in terms of conventional sludge-retention-time. The experimental data obtained indicated that the Michaelis-Menten expression was applicable to a description of substrate utilisation (i.e. COD removal) in the anaerobic fluidised-bed system. The maximum substrate utilisation rate, k, and the Michaelis constant, K{sub s}, were determined to be 1.2/day and 0.039 g/l. The observed biomass yield in the reactor decreased with increasing sludge-retention-time. The specific methane production rate observed was a linear function of the specific substrate-utilisation rate. (Author)

  9. Kinetics and Mechanism of Oxidation of Diethyl Ether by Chloramine-T in Acidic Medium

    Directory of Open Access Journals (Sweden)

    Y. I. Hassan

    2012-01-01

    Full Text Available The kinetics of oxidation of diethyl ether (DE with sodium N-chloro-p-toluenesulphonamide (CAT in hydrochloric acid solution has been studied at (313°K.The reaction rate show a first order dependence on [CAT] and fractional order dependence on each [DE] and [H+] .The variation of ionic strength of the medium has no significant effect on the reaction rate , addition of p-toluenesulphonamide (p-TSA affects the reaction rate marginally the rate increased with decreasing dielectric constant of the medium , the stochiometry of the reaction was found to be 1:2 and oxidation products were identified , A MichaelisMenten type mechanism has been suggested to explain the results.The equilibrium and the decomposition constants of CAT – diethyl ether complex have been evaluated. Thermodynamic parameters were computed by studying reaction at temperatures range ( 308 – 323°K for the rate limiting step and for the observed first order constants by the linear Arrhenius plot. The mechanism proposed and the derived rate law are consistent with observed kinetics.

  10. Continuous ammonium enrichment of a woodland stream: uptake kinetics, leaf decomposition, and nitrification

    Energy Technology Data Exchange (ETDEWEB)

    Newbold, J D; Elwood, J W; Schulze, M S; Stark, R W; Barmeier, J C

    1983-01-01

    In order to test for nitrogen limitation and examine ammonium uptake by stream sediments, ammonium hydroxide was added continuously at concentrations averaging 100 /sup +/gl/sup -1/ for 70 days to a second-order reach of Walker Branch, an undisturbed woodland stream in Tennessee. Ammonium uptake during the first 4 h of addition corresponded to adsorption kinetics rather than to first-order uptake or to Michaelis-Menten kinetics. However, the calculated adsorption partition coefficient was two to four orders of magnitude greater than values reported for physical adsorption of ammonium, suggesting that the uptake was largely biotic. Mass balance indicated that the uptake of ammonium from the water could be accounted for by increased nitrogen content in benthic organic detritus. Nitrification, inferred from longitudinal gradients in NO/sub 3/, began soon after enrichment and increased dramatically near the end of the experiment. Both ammonium and nitrate concentrations dropped quickly to near background levels when input ceased, indicating little desorption or nitrification of excess nitrogen stored in the reach. There was no evidence of nitrogen limitation as measured by weight loss, oxygen consumption, phosphorus content, and macroinvertebrate density of red oak leaf packs, or by chlorophyll content and aufwuchs biomass on plexiglass slides. A continuous phosphorus enrichment 1 year earlier had demonstrated phosphorus limitation in Walker Branch. 38 references, 6 figures, 3 tables.

  11. Competition between roots and microorganisms for nitrogen: mechanisms and ecological relevance.

    Science.gov (United States)

    Kuzyakov, Yakov; Xu, Xingliang

    2013-05-01

    Demand of all living organisms on the same nutrients forms the basis for interspecific competition between plants and microorganisms in soils. This competition is especially strong in the rhizosphere. To evaluate competitive and mutualistic interactions between plants and microorganisms and to analyse ecological consequences of these interactions, we analysed 424 data pairs from 41 (15)N-labelling studies that investigated (15)N redistribution between roots and microorganisms. Calculated Michaelis-Menten kinetics based on K(m) (Michaelis constant) and V(max) (maximum uptake capacity) values from 77 studies on the uptake of nitrate, ammonia, and amino acids by roots and microorganisms clearly showed that, shortly after nitrogen (N) mobilization from soil organic matter and litter, microorganisms take up most N. Lower K(m) values of microorganisms suggest that they are especially efficient at low N concentrations, but can also acquire more N at higher N concentrations (V(max)) compared with roots. Because of the unidirectional flow of nutrients from soil to roots, plants are the winners for N acquisition in the long run. Therefore, despite strong competition between roots and microorganisms for N, a temporal niche differentiation reflecting their generation times leads to mutualistic relationships in the rhizosphere. This temporal niche differentiation is highly relevant ecologically because it: protects ecosystems from N losses by leaching during periods of slow or no root uptake; continuously provides roots with available N according to plant demand; and contributes to the evolutionary development of mutualistic interactions between roots and microorganisms. © 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.

  12. application of ascorbic acid 2-phosphate as a new voltammetric

    African Journals Online (AJOL)

    a

    acid 2-phosphate (AAP) as a new voltammetric substrate has been described in this paper. In the alkaline buffer .... ALP labeled goat anti-rabbit ..... Classical Michaelis-Menten kinetic experiments were carried out to measure the maximum.

  13. Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

    Science.gov (United States)

    Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

    2017-07-01

    How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme-substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor-acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor-substrate complex.

  14. Cyclodextrin Aldehydes are Oxidase Mimics

    DEFF Research Database (Denmark)

    Fenger, Thomas Hauch; Bjerre, Jeannette; Bols, Mikael

    2009-01-01

    Cyclodextrins containing 6-aldehyde groups were found to catalyse oxidation of aminophenols in the presence of hydrogen peroxide. The catalysis followed Michaelis-Menten kinetics and is related to the catalysis previously observed with cyclodextrin ketones. A range of different cyclodextrin aldeh...

  15. Enzymatic oxidation of rutin by horseradish peroxidase: kinetic mechanism and identification of a dimeric product by LC-Orbitrap mass spectrometry.

    Science.gov (United States)

    Savic, Sasa; Vojinovic, Katarina; Milenkovic, Sanja; Smelcerovic, Andrija; Lamshoeft, Marc; Petronijevic, Zivomir

    2013-12-15

    Flavonoid oxidation is important issue in food processing and quality. The kinetic mechanism of enzymatic oxidation of rutin by horseradish peroxidase (HRP) was studied. Rutin oxidation reaction was followed by recording of spectral changes over the time at 360 nm. The studied oxidation is mostly enzymatic and less part non-enzymatic. The reaction with HRP has a higher rate compared with the reaction without of HRP, whereby is part of non-enzymatic reaction about 10% of the total reaction. Kinetic parameters were determined from graphics of linear Michaelis-Menten equation, and it was found that investigated reactions of rutin oxidation by HRP take place in a ping-pong kinetic mechanism. High resolution HPLC-MS analysis of the mixture of oxidized products of rutin revealed the presence of rutin dimer. Because of widely distribution of rutin as well as presence of peroxidases and hydrogen peroxide in fresh foods identification of this enzymatic modification product can be beneficial for foods quality and safety. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Stability in a diffusive food chain model with Michaelis-Menten functional response

    DEFF Research Database (Denmark)

    Lin, Zhigui; Pedersen, Michael

    2004-01-01

    This paper deals with the behavior of positive solutions to a reaction-diffusion system with homogeneous Neumann boundary conditions describing a three species food chain. A sufficient condition for the local asymptotical stability is given by linearization and also a sufficient condition...... for the global asymptotical stability is given by a Lyapunov function. Our result shows that the equilibrium solution is globally asymptotically stable if the net birth rate of the first species is big enough and the net death rate of the third species is neither too big nor too small. (C) 2004 Elsevier Ltd. All...

  17. Complex Reaction Kinetics in Chemistry: A Unified Picture Suggested by Mechanics in Physics

    Directory of Open Access Journals (Sweden)

    Elena Agliari

    2018-01-01

    Full Text Available Complex biochemical pathways can be reduced to chains of elementary reactions, which can be described in terms of chemical kinetics. Among the elementary reactions so far extensively investigated, we recall the Michaelis-Menten and the Hill positive-cooperative kinetics, which apply to molecular binding and are characterized by the absence and the presence, respectively, of cooperative interactions between binding sites. However, there is evidence of reactions displaying a more complex pattern: these follow the positive-cooperative scenario at small substrate concentration, yet negative-cooperative effects emerge as the substrate concentration is increased. Here, we analyze the formal analogy between the mathematical backbone of (classical reaction kinetics in Chemistry and that of (classical mechanics in Physics. We first show that standard cooperative kinetics can be framed in terms of classical mechanics, where the emerging phenomenology can be obtained by applying the principle of least action of classical mechanics. Further, since the saturation function plays in Chemistry the same role played by velocity in Physics, we show that a relativistic scaffold naturally accounts for the kinetics of the above-mentioned complex reactions. The proposed formalism yields to a unique, consistent picture for cooperative-like reactions and to a stronger mathematical control.

  18. Enzyme-like catalysis via ternary complex mechanism: alkoxy-bridged dinuclear cobalt complex mediates chemoselective O-esterification over N-amidation.

    Science.gov (United States)

    Hayashi, Yukiko; Santoro, Stefano; Azuma, Yuki; Himo, Fahmi; Ohshima, Takashi; Mashima, Kazushi

    2013-04-24

    Hydroxy group-selective acylation in the presence of more nucleophilic amines was achieved using acetates of first-row late transition metals, such as Mn, Fe, Co, Cu, and Zn. Among them, cobalt(II) acetate was the best catalyst in terms of reactivity and selectivity. The combination of an octanuclear cobalt carboxylate cluster [Co4(OCOR)6O]2 (2a: R = CF3, 2b: R = CH3, 2c: R = (t)Bu) with nitrogen-containing ligands, such as 2,2'-bipyridine, provided an efficient catalytic system for transesterification, in which an alkoxide-bridged dinuclear complex, Co2(OCO(t)Bu)2(bpy)2(μ2-OCH2-C6H4-4-CH3)2 (10), was successfully isolated as a key intermediate. Kinetic studies and density functional theory calculations revealed Michaelis-Menten behavior of the complex 10 through an ordered ternary complex mechanism similar to dinuclear metallo-enzymes, suggesting the formation of alkoxides followed by coordination of the ester.

  19. MODELING OF MIXED CHEMOSTAT CULTURES OF AN AEROBIC BACTERIUM, COMAMONAS-TESTOSTERONI, AND AN ANAEROBIC BACTERIUM, VEILLONELLA-ALCALESCENS - COMPARISON WITH EXPERIMENTAL-DATA

    NARCIS (Netherlands)

    GERRITSE, J; SCHUT, F; GOTTSCHAL, JC

    A mathematical model of mixed chemostat cultures of the obligately aerobic bacterium Comamonas testosteroni and the anaerobic bacterium Veillonella alcalescens grown under dual limitation Of L-lactate and oxygen was constructed. The model was based on Michaelis-Menten-type kinetics for the

  20. Immobilization of urease on copper chelated EC-Tri beads and ...

    African Journals Online (AJOL)

    Maximum reaction rate (Vmax) and Michaelis-Menten constant (km) were determined for the free and immobilized enzymes. Various characteristics of immobilized urease such as the temperature activity curve, thermal stability, operational stability and storage stability were evaluated. The results demonstrated that triazole ...

  1. Rethinking fundamentals of enzyme action.

    Science.gov (United States)

    Northrop, D B

    1999-01-01

    Despite certain limitations, investigators continue to gainfully employ concepts rooted in steady-state kinetics in efforts to draw mechanistically relevant inferences about enzyme catalysis. By reconsidering steady-state enzyme kinetic behavior, this review develops ideas that allow one to arrive at the following new definitions: (a) V/K, the ratio of the maximal initial velocity divided by the Michaelis-Menten constant, is the apparent rate constant for the capture of substrate into enzyme complexes that are destined to yield product(s) at some later point in time; (b) the maximal velocity V is the apparent rate constant for the release of substrate from captured complexes in the form of free product(s); and (c) the Michaelis-Menten constant K is the ratio of the apparent rate constants for release and capture. The physiologic significance of V/K is also explored to illuminate aspects of antibiotic resistance, the concept of "perfection" in enzyme catalysis, and catalytic proficiency. The conceptual basis of congruent thermodynamic cycles is also considered in an attempt to achieve an unambiguous way for comparing an enzyme-catalyzed reaction with its uncatalyzed reference reaction. Such efforts promise a deeper understanding of the origins of catalytic power, as it relates to stabilization of the reactant ground state, stabilization of the transition state, and reciprocal stabilizations of ground and transition states.

  2. General chemistry

    International Nuclear Information System (INIS)

    2009-01-01

    This book explains chemical equilibrium with nature and characteristic of chemical equilibrium, law of mass action and direction of chemical equilibrium, acid-base equilibrium with principle of acid and base and amino acid, solubility and precipitation equilibrium with equilibrium of solubility, complex ion and solubility, electrochemistry on oxidation-reduction reaction, battery and fuel cell, decay and electrolysis, chemical reaction speed and nuclear reaction with Michaelis-Menten mechanism safety of nuclear, transition elements and coordination compound with introduction, name, structure and ligand EDTA and solid structure with categorization of solid and unit cell.

  3. Michaelis' hundrede spørgsmål og Den Kongelige Instruks

    DEFF Research Database (Denmark)

    Friis, Ib

    2017-01-01

    Michaelis' Fragen med de 100 spørgsmål til rejseselskabet er et forunderligt dokument. Det giver et bredt indblik i bibelforkningens kilder og metoder anno 1762, og tegner samtidig klart de udfordringer, som de rejsende stod overfor. Som ekspeditionens videnskabelige fundament er Fragen afgørende...

  4. (CSTR) and 1-plug flow reactor (PFR)

    African Journals Online (AJOL)

    Administrator

    2010-12-27

    Dec 27, 2010 ... 2Department of Chemical Engineering, Obafemi Awolowo University, Ile Ife, Osun State, Nigeria. ... modeled as PFR and described by Michaelis. Menten equation. Designed equations derived from the two equations are used for the reactor sizing of ... Herbivores make a living on cellulose by possessing.

  5. Signaling Cascades: Consequences of Varying Substrate and Phosphatase Levels

    DEFF Research Database (Denmark)

    Feliu, Elisenda; Knudsen, Michael; Wiuf, Carsten Henrik

    2012-01-01

    We study signaling cascades with an arbitrary number of layers of one-site phosphorylation cycles. Such cascades are abundant in nature and integrated parts of many pathways. Based on the Michaelis-Menten model of enzyme kinetics and the law of mass-action, we derive explicit analytic expressions...

  6. Cyclodextrin-based artificial oxidases with high rate accelerations and selectivity

    DEFF Research Database (Denmark)

    Zhou, You; Lindbäck, Emil Anders; Pedersen, Christian Marcus

    2014-01-01

    Three cyclodextrin derivatives with one to four 2-O-formylmethyl groups attached to the secondary rim were prepared and investigated as catalysts for the oxidation of aminophenols in buffered dilute hydrogen peroxide. The derivatives were found to be Michaelis-Menten catalysts and to give rate ac...

  7. Purification and characterization of a chlorite dismutase from Pseudomonas chloritidismutans

    NARCIS (Netherlands)

    Mehboob, F.; Wolterink, A.F.W.M.; Vermeulen, A.J.; Jiang, B.; Hagedoorn, P.L.; Stams, A.J.M.; Kengen, S.W.M.

    2009-01-01

    The chlorite dismutase (Cld) of Pseudomonas chloritidismutans was purified from the periplasmic fraction in one step by hydroxyapatite chromatography. The enzyme has a molecular mass of 110 kDa and consists of four 31-kDa subunits. Enzyme catalysis followed Michaelis-Menten kinetics, with Vmax and

  8. Wang and Li Afr J Tradit Complement Altern Med. (2016) 13(1):99 ...

    African Journals Online (AJOL)

    PROF ADEWUNMI

    It possesses antiseptic, anti-inflammatory, analgesic, anti-cancer and antioxidant ... All experiments on animals were conducted in accordance with and after approval by the ... With the content of gallic acid control as horizontal coordinate and the ... The kinetic constants were calculated based on Michaelis-Menten equation ...

  9. Modelling the extra and intracellular uptake and discharge of heavy metals in Fontinalis antipyretica transplanted along a heavy metal and pH contamination gradient

    International Nuclear Information System (INIS)

    Fernandez, J.A.; Vazquez, M.D.; Lopez, J.; Carballeira, A.

    2006-01-01

    Samples of the aquatic bryophyte Fontinalis antipyretica Hedw. were transplanted to different sites with the aim of characterizing the kinetics of the uptake and discharge of heavy metals in the extra and intracellular compartments. The accumulation of metals in extracellular compartments, characterized by an initial rapid accumulation, then a gradual slowing down over time, fitted perfectly to a Michaelis-Menten model. The discharge of metals from the same compartment followed an inverse linear model or an inverse Michaelis-Menten model, depending on the metal. In intracellular sites both uptake and discharge occurred more slowly and progressively, following a linear model. We also observed that the acidity of the environment greatly affected metal accumulation in extracellular sites, even when the metals were present at relatively high concentrations, whereas the uptake of metals within cells was much less affected by pH. - The kinetics of uptake and discharge of heavy metals, in different cellular locations, were studied in transplanted aquatic mosses

  10. A path-independent integral for the characterization of solute concentration and flux at biofilm detachments

    Science.gov (United States)

    Moran, B.; Kulkarni, S.S.; Reeves, H.W.

    2007-01-01

    A path-independent (conservation) integral is developed for the characterization of solute concentration and flux in a biofilm in the vicinity of a detachment or other flux limiting boundary condition. Steady state conditions of solute diffusion are considered and biofilm kinetics are described by an uptake term which can be expressed in terms of a potential (Michaelis-Menten kinetics). An asymptotic solution for solute concentration at the tip of the detachment is obtained and shown to be analogous to that of antiplane crack problems in linear elasticity. It is shown that the amplitude of the asymptotic solution can be calculated by evaluating a path-independent integral. The special case of a semi-infinite detachment in an infinite strip is considered and the amplitude of the asymptotic field is related to the boundary conditions and problem parameters in closed form for zeroth and first order kinetics and numerically for Michaelis-Menten kinetics. ?? Springer Science+Business Media, Inc. 2007.

  11. Chemical Continuous Time Random Walks

    Science.gov (United States)

    Aquino, T.; Dentz, M.

    2017-12-01

    Traditional methods for modeling solute transport through heterogeneous media employ Eulerian schemes to solve for solute concentration. More recently, Lagrangian methods have removed the need for spatial discretization through the use of Monte Carlo implementations of Langevin equations for solute particle motions. While there have been recent advances in modeling chemically reactive transport with recourse to Lagrangian methods, these remain less developed than their Eulerian counterparts, and many open problems such as efficient convergence and reconstruction of the concentration field remain. We explore a different avenue and consider the question: In heterogeneous chemically reactive systems, is it possible to describe the evolution of macroscopic reactant concentrations without explicitly resolving the spatial transport? Traditional Kinetic Monte Carlo methods, such as the Gillespie algorithm, model chemical reactions as random walks in particle number space, without the introduction of spatial coordinates. The inter-reaction times are exponentially distributed under the assumption that the system is well mixed. In real systems, transport limitations lead to incomplete mixing and decreased reaction efficiency. We introduce an arbitrary inter-reaction time distribution, which may account for the impact of incomplete mixing. This process defines an inhomogeneous continuous time random walk in particle number space, from which we derive a generalized chemical Master equation and formulate a generalized Gillespie algorithm. We then determine the modified chemical rate laws for different inter-reaction time distributions. We trace Michaelis-Menten-type kinetics back to finite-mean delay times, and predict time-nonlocal macroscopic reaction kinetics as a consequence of broadly distributed delays. Non-Markovian kinetics exhibit weak ergodicity breaking and show key features of reactions under local non-equilibrium.

  12. DETERMINATION OF THE SPECIFIC GROWTH RATE ON ...

    African Journals Online (AJOL)

    Sewage generation is one of the dense problems Nigerians encounter on daily bases, mostly at the urbanized area where factories and industries are located. This paper is aimed at determining the specific growth rate “K” of biological activities on cassava wastewater during degradation using Michaelis-Menten Equation.

  13. Nonthermal effect of microwave irradiation on nitrite uptake in Chlamydomonas reinhardtii

    International Nuclear Information System (INIS)

    Pedrajas, C.; Cotrino, J.

    1989-01-01

    When cells of the unicellular green alga Chlamydomonas reinhardtii were subjected to microwave irradiation at 2.45 GHz, nitrite uptake kinetics still obeyed the Michaelis-Menten equation, the Km of the process remaining constant, whereas V max increased, which indicates an enhanced nonthermal permeability in irradiated cells. (author)

  14. Journal of Chemical Sciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    The oxidation of eleven amino acids by tetrabutylammonium tribromide (TBATB) in aqueous acetic acid results in the formation of the corresponding carbonyl compounds and ammonia. The reaction is first order with respect to TBATB. Michaelis-Menten type kinetics is observed with some of the amino acids while others ...

  15. International Meeting on Cholinesterases (5th) Held in Madras, India on 24-28 September, 1994.

    Science.gov (United States)

    1994-09-01

    found that hydrolysis of thioesters deviated from simple Michaelis-Menten model. Kinetics was triphasic , displaying complexities of both BuChE and ACHE...of recombinant human acetylcholinesterase (rHuAChE) produced by human embryonic kidney cell line (293) in a fixed-bed reactor (1) was investigated at

  16. Bringing metabolic networks to life: convenience rate law and thermodynamic constraints

    Directory of Open Access Journals (Sweden)

    Klipp Edda

    2006-12-01

    Full Text Available Abstract Background Translating a known metabolic network into a dynamic model requires rate laws for all chemical reactions. The mathematical expressions depend on the underlying enzymatic mechanism; they can become quite involved and may contain a large number of parameters. Rate laws and enzyme parameters are still unknown for most enzymes. Results We introduce a simple and general rate law called "convenience kinetics". It can be derived from a simple random-order enzyme mechanism. Thermodynamic laws can impose dependencies on the kinetic parameters. Hence, to facilitate model fitting and parameter optimisation for large networks, we introduce thermodynamically independent system parameters: their values can be varied independently, without violating thermodynamical constraints. We achieve this by expressing the equilibrium constants either by Gibbs free energies of formation or by a set of independent equilibrium constants. The remaining system parameters are mean turnover rates, generalised Michaelis-Menten constants, and constants for inhibition and activation. All parameters correspond to molecular energies, for instance, binding energies between reactants and enzyme. Conclusion Convenience kinetics can be used to translate a biochemical network – manually or automatically - into a dynamical model with plausible biological properties. It implements enzyme saturation and regulation by activators and inhibitors, covers all possible reaction stoichiometries, and can be specified by a small number of parameters. Its mathematical form makes it especially suitable for parameter estimation and optimisation. Parameter estimates can be easily computed from a least-squares fit to Michaelis-Menten values, turnover rates, equilibrium constants, and other quantities that are routinely measured in enzyme assays and stored in kinetic databases.

  17. Aarne Michaël Tallgren and the International Discussion on the Bronze Age of Russia

    Directory of Open Access Journals (Sweden)

    Timo Salminen

    2017-04-01

    Full Text Available This paper is on international scholarly discussion on the Bronze Age of Russia from 1908 until 1939, and in particular on the related role of the internationally renowned Finnish archaeologist Aarne Michaël Tallgren (1885–1945. How did a social network of researchers produce new interpretations and what were the key factors that distinguished the participants in the discussion? Was it a continuous process or a series of sudden changes? How did different ideological backgrounds influence the interpretations? In Western Europe, Tallgren’s most important interlocutors were Gero von Merhart, V Gordon Childe and Ellis H Minns, and in Russia V A Gorodcov and A A Spicyn. The paper is mainly based on correspondence between Tallgren and his colleagues.

  18. In memory of Professor Leonor Michaelis in Nagoya: great contributions to biochemistry in Japan in the first half of the 20th century.

    Science.gov (United States)

    Nagatsu, Toshiharu Toshi

    2013-09-02

    Leonor Michaelis spent the years of 1922-1926 as Professor of Biochemistry of the Aichi Medical College (now Graduate School of Medicine, Nagoya University) in Nagoya, Japan. Michaelis succeeded in gathering many bright young biochemists from all over Japan into his laboratory, and made tremendous contributions to the promotion of biochemistry in Japan. Michaelis was invited to many places in Japan to present lectures over those years. Kunio Yagi, who was Professor of Biochemistry at Nagoya University in the second half of the 20th century, succeeded in crystallizing the "Michaelis" enzyme-substrate complex. Historically, Michelis has had an enormous impact on biochemistry in Japan. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  19. Michaël Jackson, un people précurseur

    OpenAIRE

    Dubied, Annik; Gorin, Valérie

    2009-01-01

    En parcourant la presse people parue au moment du décès du King of Pop, on observe à quel point Michaël Jackson a été et restera un personnage people précurseur. La titraille, les photographies et la mise en page d'une série de magazines francophones et anglo-saxons présentent un certain nombre de particularités qui résonnent à l'unisson avec la définition du genre people.

  20. A Qualitative Approach to Enzyme Inhibition

    Science.gov (United States)

    Waldrop, Grover L.

    2009-01-01

    Most general biochemistry textbooks present enzyme inhibition by showing how the basic Michaelis-Menten parameters K[subscript m] and V[subscript max] are affected mathematically by a particular type of inhibitor. This approach, while mathematically rigorous, does not lend itself to understanding how inhibition patterns are used to determine the…

  1. Benefits of a clinical pharmacokinetic service in optimising ...

    African Journals Online (AJOL)

    before the termination of the study (test period). Patients kept a seizure diary throughout the study. The MichaelisMenten model was used to calculate doses and predict steady-state serum concentrations. Setting. ine epilepsy clinics. Subjects. One hundred and ninety-five (113 black and 82 coloured) compliant people with ...

  2. Use of Mushroom Tyrosinase to Introduce Michaelis-Menten Enzyme Kinetics to Biochemistry Students

    Science.gov (United States)

    Flurkey, William H.; Inlow, Jennifer K.

    2017-01-01

    An inexpensive enzyme kinetics laboratory exercise for undergraduate biochemistry students is described utilizing tyrosinase from white button mushrooms. The exercise can be completed in one or two three-hour lab sessions. The optimal amounts of enzyme, substrate (catechol), and inhibitor (kojic acid) are first determined, and then kinetic data is…

  3. Michaëlis--Menten kinetics of phenazone elimination in the perfused pig liver

    DEFF Research Database (Denmark)

    Andreasen, B; Tonnesen, K; Rabol, A

    1977-01-01

    The purpose of the present study was to define the elimination kinetics of phenazone (NFN) in the isolated perfused pig liver. In five experiments phenazone was administered as constant infusion to obtain steady-state periods over a wide range of concentrations. The elimination of phenazone follo...

  4. New functions to estimate 305-days milk production of Gir cows : Novas Funções para Estimar a Produção de Leite, em 305 Dias de Lactação, de Vacas da Raça Gir

    NARCIS (Netherlands)

    Reboucas, G.F.; Moraes Goncalves, de T.; Martines, M.L.; Azevedo Junior, J.; Koops, W.J.

    2008-01-01

    This study aimed to calculate new accumulated and daily functions based on the Michaelis-Menten equation to estimate the 305-days production of Gir cows using test day milk yields. Data consisted of 7,412 lactation records of 3,416 Gir cows (Bos indicus) collected from 1987 to 2004 in 51 herds

  5. Diatomite Modified Immobilized Delftia sp. for the Bio-Abiotic Removal of Antibiotics Amoxicillin in the Aqueous System

    Science.gov (United States)

    Gao, Lijuan; Sun, Jing; Guan, Kai; Shen, Tingting; Wang, Xikui

    2017-05-01

    Diatomite modified sodium alginate (Si/SA) immobilized Delftia sp. A2(2011) (STT01) was applied to degrade amoxicillin. The immobilized pellets provided a direct and visual probe for the degradation process due to their intrinsic bright colour. The results demonstrated that 100% of amoxicillin and 68.5% of CODcr removal were achieved after 72 h, comparing with the cases of sodium alginate (SA) system (81.2%, 46.9%) and the free cells system (60.5%, 35.5%). The degradation kinetics was in good agreement with Michaelis-Menten equation. The maximum rate (Vm ) and Michaelis constant (Km ) were calculated as 9.09 mg L-1 h-1 and 228 mg L-1, respectively. The results further revealed that diatomite not only acted as immobilization support to improve the mechanical strength and lifetime of the pellets but also as absorbent to promote the treatment efficiency. Therefore, both enzymatic catalysis and chemisorption were responsible for the removal of amoxicillin.

  6. Fock space, symbolic algebra, and analytical solutions for small stochastic systems.

    Science.gov (United States)

    Santos, Fernando A N; Gadêlha, Hermes; Gaffney, Eamonn A

    2015-12-01

    Randomness is ubiquitous in nature. From single-molecule biochemical reactions to macroscale biological systems, stochasticity permeates individual interactions and often regulates emergent properties of the system. While such systems are regularly studied from a modeling viewpoint using stochastic simulation algorithms, numerous potential analytical tools can be inherited from statistical and quantum physics, replacing randomness due to quantum fluctuations with low-copy-number stochasticity. Nevertheless, classical studies remained limited to the abstract level, demonstrating a more general applicability and equivalence between systems in physics and biology rather than exploiting the physics tools to study biological systems. Here the Fock space representation, used in quantum mechanics, is combined with the symbolic algebra of creation and annihilation operators to consider explicit solutions for the chemical master equations describing small, well-mixed, biochemical, or biological systems. This is illustrated with an exact solution for a Michaelis-Menten single enzyme interacting with limited substrate, including a consideration of very short time scales, which emphasizes when stiffness is present even for small copy numbers. Furthermore, we present a general matrix representation for Michaelis-Menten kinetics with an arbitrary number of enzymes and substrates that, following diagonalization, leads to the solution of this ubiquitous, nonlinear enzyme kinetics problem. For this, a flexible symbolic maple code is provided, demonstrating the prospective advantages of this framework compared to stochastic simulation algorithms. This further highlights the possibilities for analytically based studies of stochastic systems in biology and chemistry using tools from theoretical quantum physics.

  7. Journal of Chemical Sciences | Indian Academy of Sciences

    Indian Academy of Sciences (India)

    ... behaves as an effective catalyst towards oxidation of 3,5-ditertiarybutyl catechol (3,5-DTBC) in acetonitrile to its corresponding quinone derivative in air. The reaction follows first-order reaction kinetics with rate constant 4.28 × 10−5 min-1. The reaction follows Michaelis-Menten enzymatic kinetics with a turnover number of ...

  8. Metabolic stereoselectivity of cytochrome P450 3A4 towards deoxypodophyllotoxin : In silico predictions and experimental validation

    NARCIS (Netherlands)

    Julsing, Mattijs K.; Vasilev, Nikolay P.; Schneidman-Duhovny, Dina; Muntendarn, Remco; Woerdenbag, Herman J.; Quax, Wim J.; Wolfson, Haim J.; Ionkova, Iliana; Kayser, Oliver

    Deoxypodophyllotoxin is stereoselectively converted into epipodophyllotoxin by recombinant human cytochrome P450 3A4 (CY-P3A4). Further kinetic analysis revealed that the Michaelis-Menten K(m) and V(max) for hydroxylation of deoxypodophyllotoxin by CYP3A4 at C7 position were 1.93 mu M and 1.48

  9. Interactions of NH4+ and L-glutamate with NO3- transport processes of non-mycorrhizal Fagus sylvatica roots

    NARCIS (Netherlands)

    Kreuzwieser, J; Herschbach, C; Stulen, [No Value; Wiersema, P; Vaalburg, W; Rennenberg, H

    The processes of NO3- uptake and transport and the effects of NH4+ or L-glutamate on these processes were investigated with excised non-mycorrhizal beech (Fagus sylvatica L,) roots, NO3- net uptake followed uniphasic Michaelis-Menten kinetics in a concentration range of 10 mu M to 1 mM with an

  10. Modeling uptake kinetics of cadmium by field-grown lettuce

    Energy Technology Data Exchange (ETDEWEB)

    Chen Weiping [Department of Environmental Sciences, University of California, 900 University Avenue, Riverside, CA 92521 (United States)], E-mail: chenweip@yahoo.com.cn; Li Lianqing [Institute of Resources, Ecosystem and Environment of Agriculture, Nanjing Agricultural University, Nanjing 210095 (China); Chang, Andrew C.; Wu Laosheng [Department of Environmental Sciences, University of California, 900 University Avenue, Riverside, CA 92521 (United States); Kwon, Soon-Ik [Agricultural Environmental and Ecology Division, National Institute of Agricultural Science and Technology, Suwon 441-707 (Korea, Republic of); Bottoms, Rick [Desert Research and Extension Center, 1004 East Holton Road, El Centro, CA 92243 (United States)

    2008-03-15

    Cadmium uptake by field grown Romaine lettuce treated with P-fertilizers of different Cd levels was investigated over an entire growing season. Results indicated that the rate of Cd uptake at a given time of the season can be satisfactorily described by the Michaelis-Menten kinetics, that is, plant uptake increases as the Cd concentration in soil solution increases, and it gradually approaches a saturation level. However, the rate constant of the Michaelis-Menten kinetics changes over the growing season. Under a given soil Cd level, the cadmium content in plant tissue decreases exponentially with time. To account for the dynamic nature of Cd uptake, a kinetic model integrating the time factor was developed to simulate Cd plant uptake over the growing season: C{sub Plant} = C{sub Solution} . PUF{sub max} . exp[-b . t], where C{sub Plant} and C{sub Solution} refer to the Cd content in plant tissue and soil solution, respectively, PUF{sub max} and b are kinetic constants. - A kinetic model was developed to evaluate the uptake of Cd under field conditions.

  11. Modeling uptake kinetics of cadmium by field-grown lettuce

    International Nuclear Information System (INIS)

    Chen Weiping; Li Lianqing; Chang, Andrew C.; Wu Laosheng; Kwon, Soon-Ik; Bottoms, Rick

    2008-01-01

    Cadmium uptake by field grown Romaine lettuce treated with P-fertilizers of different Cd levels was investigated over an entire growing season. Results indicated that the rate of Cd uptake at a given time of the season can be satisfactorily described by the Michaelis-Menten kinetics, that is, plant uptake increases as the Cd concentration in soil solution increases, and it gradually approaches a saturation level. However, the rate constant of the Michaelis-Menten kinetics changes over the growing season. Under a given soil Cd level, the cadmium content in plant tissue decreases exponentially with time. To account for the dynamic nature of Cd uptake, a kinetic model integrating the time factor was developed to simulate Cd plant uptake over the growing season: C Plant = C Solution . PUF max . exp[-b . t], where C Plant and C Solution refer to the Cd content in plant tissue and soil solution, respectively, PUF max and b are kinetic constants. - A kinetic model was developed to evaluate the uptake of Cd under field conditions

  12. Inhibition of serotonin transport by (+)McN5652 is noncompetitive

    Energy Technology Data Exchange (ETDEWEB)

    Hummerich, Rene [Biochemical Laboratory, Central Institute of Mental Health, 68159 Mannheim (Germany); Schulze, Oliver [Department of Nuclear Medicine, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg (Germany); Raedler, Thomas [Department of Psychiatry and Psychotherapy, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg (Germany); Mikecz, Pal [Department of Nuclear Medicine, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg (Germany); Reimold, Matthias [Department of Nuclear Medicine, University Hospital Tuebingen, D-72076 Tuebingen (Germany); Brenner, Winfried [Department of Nuclear Medicine, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg (Germany); Clausen, Malte [Department of Nuclear Medicine, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg (Germany); Schloss, Patrick [Biochemical Laboratory, Central Institute of Mental Health, 68159 Mannheim (Germany); Buchert, Ralph [Department of Nuclear Medicine, University Medical Center Hamburg-Eppendorf, D-20246 Hamburg (Germany)]. E-mail: buchert@uke.uni-hamburg.de

    2006-04-15

    Introduction: Imaging of the serotonergic innervation of the brain using positron emission tomography (PET) with the serotonin transporter (SERT) ligand [{sup 11C}] (+)McN5652 might be affected by serotonin in the synaptic cleft if there is relevant interaction between [{sup 11}C] (+)McN5652 and serotonin at the SERT. The aim of the present study therefore was to pharmacologically characterize the interaction of [{sup 11}C] (+)McN5652 and serotonin at the SERT. Methods: In vitro saturation analyses of [{sup 3}H]serotonin uptake into HEK293 cells stably expressing the human SERT were performed in the absence and presence of unlabelled (+)McN5652. Data were evaluated assuming Michaelis-Menten kinetics. Results: Unlabelled (+)McN5652 significantly reduced the maximal rate of serotonin transport V {sub max} of SERT without affecting the Michaelis-Menten constant K {sub M}. Conclusions: This finding indicates that (+)McN5652 inhibits serotonin transport through the SERT in a noncompetitive manner. This might suggest that [{sup 11}C] (+)McN5652 PET is not significantly affected by endogenous serotonin.

  13. Inhibition of serotonin transport by (+)McN5652 is noncompetitive

    International Nuclear Information System (INIS)

    Hummerich, Rene; Schulze, Oliver; Raedler, Thomas; Mikecz, Pal; Reimold, Matthias; Brenner, Winfried; Clausen, Malte; Schloss, Patrick; Buchert, Ralph

    2006-01-01

    Introduction: Imaging of the serotonergic innervation of the brain using positron emission tomography (PET) with the serotonin transporter (SERT) ligand [ 11C ] (+)McN5652 might be affected by serotonin in the synaptic cleft if there is relevant interaction between [ 11 C] (+)McN5652 and serotonin at the SERT. The aim of the present study therefore was to pharmacologically characterize the interaction of [ 11 C] (+)McN5652 and serotonin at the SERT. Methods: In vitro saturation analyses of [ 3 H]serotonin uptake into HEK293 cells stably expressing the human SERT were performed in the absence and presence of unlabelled (+)McN5652. Data were evaluated assuming Michaelis-Menten kinetics. Results: Unlabelled (+)McN5652 significantly reduced the maximal rate of serotonin transport V max of SERT without affecting the Michaelis-Menten constant K M . Conclusions: This finding indicates that (+)McN5652 inhibits serotonin transport through the SERT in a noncompetitive manner. This might suggest that [ 11 C] (+)McN5652 PET is not significantly affected by endogenous serotonin

  14. An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon

    International Nuclear Information System (INIS)

    Shenouda, Josephine; Green, Paula; Sultatos, Lester

    2009-01-01

    Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of α/β-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005-3.0 mM, with 317 pM human butyrylcholinesterase in vitro, resulted in rates of production of thiocholine that were accurately described by simple Michaelis-Menten kinetics, with a K m of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis-Menten kinetics, with a k i of 3048 nM -1 h -1 , and a K D of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k i increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 and 0.3 nM gave k i s of 1.2 and 19.3 nM -1 h -1 , respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.

  15. Nitrate transport processes in Fagus-Laccaria-mycorrhizae

    NARCIS (Netherlands)

    Kreuzwieser, J; Stulen, [No Value; Wiersema, P; Vaalburg, W; Rennenberg, H

    2000-01-01

    The contribution of influx and efflux of NO3- on NO3- net uptake has been studied in excised mycorrhizae of 18-20 week old beech (Fagus sylvatica L.) trees. Net uptake rates of NO3- followed uniphasic Michaelis-Menten kinetics in the concentration range between 10 mu M and 1.0 mM external NO3-, with

  16. Efficient and 'green' microwave-assisted synthesis of haloalkylphosphonates via the Michaelis-Arbuzov reaction

    Czech Academy of Sciences Publication Activity Database

    Jansa, Petr; Holý, Antonín; Dračínský, Martin; Baszczyňski, Ondřej; Česnek, Michal; Janeba, Zlatko

    2011-01-01

    Roč. 13, č. 4 (2011), s. 882-888 ISSN 1463-9262 R&D Projects: GA AV ČR KJB400550903; GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : microwave-assisted synthesis * haloalkylphosphonates * Michaelis-Arbuzov reaction Subject RIV: CC - Organic Chemistry Impact factor: 6.320, year: 2011

  17. Carolina Michaëlis o los saberes de la Filología

    Directory of Open Access Journals (Sweden)

    Joaquín Rubio Tovar

    2005-06-01

    Full Text Available Two important international conferences (Berlin, Santiago on the work of Carolina Michaëlis have been held in recent years. The papers collected value her work within the historical background when it was carried out. Besides, they review the validity of her approach and analysis and present some modem contributions to the fields which she studied, particularly the Cancioneiro da Ajuda.

  18. Catalytic monolayer voltammetry and in situ scanning tunneling microscopy of copper nitrite reductase on cysteamine-modified Au(111) electrodes

    DEFF Research Database (Denmark)

    Zhang, Jingdong; Welinder, A.C.; Hansen, Allan Glargaard

    2003-01-01

    electrochemical scanning tunneling microscopy (in situ STM) directly in aqueous acetate buffer, pH 6.0 has been used. High-resolution in situ STM shows that cysteamine packs into ordered domains with strip features of a periodic distance of 11.7 +/- 0.3 Angstrom. No voltammetric signals of the nitrite substrate...... on this surface could be detected. A strong cathodic catalytic wave appears in the presence of nitrite. The catalytic current follows a Michaelis-Menten pattern with a Michaelis constant of K-m approximate to 44 muM, which is close to the value for AxCuNiR in homogeneous solution. The apparent catalytic rate...

  19. Thermodynamic Activity-Based Progress Curve Analysis in Enzyme Kinetics.

    Science.gov (United States)

    Pleiss, Jürgen

    2018-03-01

    Macrokinetic Michaelis-Menten models based on thermodynamic activity provide insights into enzyme kinetics because they separate substrate-enzyme from substrate-solvent interactions. Kinetic parameters are estimated from experimental progress curves of enzyme-catalyzed reactions. Three pitfalls are discussed: deviations between thermodynamic and concentration-based models, product effects on the substrate activity coefficient, and product inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Concentration-dependent interactions of the organophosphates chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase

    International Nuclear Information System (INIS)

    Kaushik, R.; Rosenfeld, Clint A.; Sultatos, L.G.

    2007-01-01

    For many decades it has been thought that oxygen analogs (oxons) of organophosphorus insecticides phosphorylate the catalytic site of acetylcholinesterase by a mechanism that follows simple Michaelis-Menten kinetics. More recently, the interactions of at least some oxons have been shown to be far more complex and likely involve binding of oxons to a second site on acetylcholinesterase that modulates the inhibitory capacity of other oxon molecules at the catalytic site. The current study has investigated the interactions of chlorpyrifos oxon and methyl paraoxon with human recombinant acetylcholinesterase. Both chlorpyrifos oxon and methyl paraoxon were found to have k i 's that change as a function of oxon concentration. Furthermore, 10 nM chlorpyrifos oxon resulted in a transient increase in acetylthiocholine hydrolysis, followed by inhibition. Moreover, in the presence of 100 nM chlorpyrifos oxon, acetylthiocholine was found to influence both the K d (binding affinity) and k 2 (phosphorylation constant) of this oxon. Collectively, these results demonstrate that the interactions of chlorpyrifos oxon and methyl paraoxon with acetylcholinesterase cannot be described by simple Michaelis-Menten kinetics but instead support the hypothesis that these oxons bind to a secondary site on acetylcholinesterase, leading to activation/inhibition of the catalytic site, depending on the nature of the substrate and inhibitor. Additionally, these data raise questions regarding the adequacy of estimating risk of low levels of insecticide exposure from direct extrapolation of insecticide dose-response curves since the capacity of individual oxon molecules at low oxon levels could be greater than individual oxon molecules in vivo associated with the dose-response curve

  1. Biodegradation of BTEX (Benzene, Toluene, Ethylbenzene and Xylenes) composites present in the petrochemical effluents industries; Biodegradacao dos compostos BTX (Benzeno, Tolueno e Xilenos) presentes em efluentes petroquimicos

    Energy Technology Data Exchange (ETDEWEB)

    Minatti, Gheise; Mello, Josiane M.M. de; Souza, Selene M.A. Guelli Ulson de; Ulson de, Antonio Augusto [Universidade Federal de Santa Catarina (UFSC), Florianopolis, SC (Brazil)

    2008-07-01

    The compounds BTX inside of the petrochemical effluent have presented a high potential of pollution, representing a serious risk to the environment and to the human. The great improvements in the field of biological treatment of liquid effluent were reached through the process using biofilm capable of degrading toxic compounds. The objective of this paper is to determine the degradation kinetics of BTX using biofilm. The experimental data were compared with two kinetic models, kinetic of first order and model of Michaelis-Menten. The kinetic parameters of BTX compounds were experimentally obtained in a bioreactor in batch with biomass immobilized in activated-carbon, being fed daily with solution of nutrients and BTX. For the kinetic models studied in this paper, the best performance was achieved with the model of Michaelis-Menten showing a good correlation coefficient for the three compounds. The biomass amount in these bioreactors was 49.18, 28.35 and 5.15 mg of SSV per gram of support for the toluene, benzene and o-xylene, respectively. The experimental tests showed that the biomass inside of bioreactor is capable to degrade all compounds in a time of approximately 300 minutes. (author)

  2. Quantification of in vivo metabolic kinetics of hyperpolarized pyruvate in rat kidneys using dynamic 13C MRSI.

    Science.gov (United States)

    Xu, Tao; Mayer, Dirk; Gu, Meng; Yen, Yi-Fen; Josan, Sonal; Tropp, James; Pfefferbaum, Adolf; Hurd, Ralph; Spielman, Daniel

    2011-10-01

    With signal-to-noise ratio enhancements on the order of 10,000-fold, hyperpolarized MRSI of metabolically active substrates allows the study of both the injected substrate and downstream metabolic products in vivo. Although hyperpolarized [1-(13)C]pyruvate, in particular, has been used to demonstrate metabolic activities in various animal models, robust quantification and metabolic modeling remain important areas of investigation. Enzyme saturation effects are routinely seen with commonly used doses of hyperpolarized [1-(13)C]pyruvate; however, most metrics proposed to date, including metabolite ratios, time-to-peak of metabolic products and single exchange rate constants, fail to capture these saturation effects. In addition, the widely used small-flip-angle excitation approach does not correctly model the inflow of fresh downstream metabolites generated proximal to the target slice, which is often a significant factor in vivo. In this work, we developed an efficient quantification framework employing a spiral-based dynamic spectroscopic imaging approach. The approach overcomes the aforementioned limitations and demonstrates that the in vivo (13)C labeling of lactate and alanine after a bolus injection of [1-(13)C]pyruvate is well approximated by saturatable kinetics, which can be mathematically modeled using a Michaelis-Menten-like formulation, with the resulting estimated apparent maximal reaction velocity V(max) and apparent Michaelis constant K(M) being unbiased with respect to critical experimental parameters, including the substrate dose, bolus shape and duration. Although the proposed saturatable model has a similar mathematical formulation to the original Michaelis-Menten kinetics, it is conceptually different. In this study, we focus on the (13)C labeling of lactate and alanine and do not differentiate the labeling mechanism (net flux or isotopic exchange) or the respective contribution of various factors (organ perfusion rate, substrate transport

  3. Kinetics of Butyrate, Acetate, and Hydrogen Metabolism in a Thermophilic, Anaerobic, Butyrate-Degrading Triculture

    OpenAIRE

    Ahring, Birgitte K.; Westermann, Peter

    1987-01-01

    Kinetics of butyrate, acetate, and hydrogen metabolism were determined with butyrate-limited, chemostat-grown tricultures of a thermophilic butyrate-utilizing bacterium together with Methanobacterium thermoautotrophicum and the TAM organism, a thermophilic acetate-utilizing methanogenic rod. Kinetic parameters were determined from progress curves fitted to the integrated form of the Michaelis-Menten equation. The apparent half-saturation constants, Km, for butyrate, acetate, and dissolved hyd...

  4. Inhibition of basolateral branchial Na{sup +}/K{sup +}-ATPase may be the key mechanism for silver toxicity in fish

    Energy Technology Data Exchange (ETDEWEB)

    Ferguson, E.; Hogstrand, C. [Univ. of Kentucky, Lexington, KY (United States). T.H. Morgan School of Biological Science

    1995-12-31

    Recent studies of freshwater fish have indicated that the mechanism of silver toxicity may lie in the ability of the metal to compromise bronchial transport of Na{sup +} and Cl (Wood et al, 1994). As part of a study, investigating the physiological mechanisms of silver toxicity to marine fish, the effect of Ag{sup +} on Na{sup +}/K{sup +}-ATPase in isolated basolateral membranes (BLM) from gills of sea-water acclimated rainbow trout was analyzed. Silver inhibition of purified dog kidney Na{sup +}/K{sup +}-ATPase was studied in a pilot experiment and the results from this experiment were compared to those obtained for rainbow trout BLMS. Michaelis-Menten kinetics of Na{sup +}/K{sup +}-ATPase activity was performed during control conditions by varying the concentrations of Na+ and K+ in the assay medium while keeping the total salt concentration constant. Inhibition of Na{sup +}/K{sup +}-ATPase activities was investigated by adding 1 nM--1 {micro}M of Ag{sup +} to the medium. Assay conditions were optimized separately for dog and rainbow trout Na{sup +}/K{sup +}-ATPase. Results from both series indicate effect of Ag{sup +} at a concentration as low as 1 nM escalating to complete quenching at a Ag{sup +} activity of 1 {micro}M. These results suggest the key mechanism of silver toxicity in marine fish involves blockage of the Na{sup +}/K{sup +}-ATPase activity in the gill epithelium.

  5. Subcellular distribution and uptake mechanism of di-n-butyl phthalate in roots of pumpkin (Cucurbita moschata) seedlings.

    Science.gov (United States)

    Lin, Qingqi; Yang, Xiuhong; Huang, Xiongfei; Wang, Shizhong; Chao, Yuanqing; Qiu, Rongliang

    2016-01-01

    Phthalate acid esters (PAEs) are of particular concern due to their potential environmental risk to human and nonhuman organisms. Although uptake of PAEs by plants has been reported by several researchers, information about the intracellular distribution and uptake mechanisms of PAEs is still lacking. In this study, a series of hydroponic experiments using intact pumpkin (Cucurbita moschata) seedlings was conducted to investigate how di-n-butyl phthalate (DnBP), one of the most frequently identified PAEs in the environment, enters and is distributed in roots. DnBP was transported into subcellular tissues rapidly in the initial uptake period (<12 h). More than 80% of DnBP was detected in the cell walls and organelles, which suggests that DnBP is primarily accumulated in these two fractions due to their high affinity to DnBP. The kinetics of DnBP uptake were fitted well with the Michaelis-Menten equation, suggesting that a carrier-mediated process was involved. The application of 2,4-dinitrophenol and sodium vanadate reduced the uptake of DnBP by 37 and 26%, respectively, while aquaporin inhibitors, silver and glycerol, had no effect on DnBP uptake. These data demonstrated that the uptake of DnBP included a carrier-mediated and energy-dependent process without the participation of aquaporins.

  6. Heterotrophic and mixotrophic growth of Micractinium pusillum Fresenius in the presence of acetate and glucose: effect of light and acetate gradient concentration.

    Science.gov (United States)

    Bouarab, L; Dauta, A; Loudiki, M

    2004-06-01

    The main objective of this study was to determine the importance of secondary mechanism of organic carbon utilization (mixotrophic and heterotrophic modes) in addition to CO2 fixation (photoautotrophic mode) in the green alga, Micractinium pusillum Fresenius (chlorophyta), isolated from a waste stabilization pond. The growth was studied in the presence of acetate and glucose. The incorporation rate of 14C- acetate was measured in the light and in the dark at different concentrations. Finally, in order to underline the role of photosynthesis and respiration processes in the acetate assimilation, the effect of two specific metabolic inhibitors, a specific inhibitor of photosystem II (DCMU) and an uncoupler respiratory (DNP), has been studied. The obtained results showed that M. pusillum grows in the presence of organic substrates, i.e., glucose and acetate, in the light (mixotrophic growth) as well as in the dark (Heterotrophic growth). The growth was much more important in the light than in the dark and more in the presence of glucose than of acetate. In the light, the presence of acetate led to a variation of growth parameters mumax, iotaopt, and beta. The effect of acetate gradient on the growth of the microalga was severe as soon as its concentration in the medium was higher. The acetate uptake followed a Michaelis-Menten kinetic in the light as well as in the dark. The capacity of assimilation was slightly higher in the dark. The utilization of DNP and DCMU indicates that acetate incorporation is an active process depending on both anabolic (photosynthesis) and catabolic (respiration) metabolisms, corroborating the model of the Michaelis-Menten kinetic.

  7. The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.

    Science.gov (United States)

    Jackson, R H; Cole, J A; Cornish-Bowden, A

    1981-01-01

    The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a mechanism in which hydroxylamine binds during catalysis to a different enzyme form from that generated when NAD+ is released. The apparent maximum velocity with NADH as varied substrate increases as the NAD+ concentration increases from 0.05 to 0.7 mM with 1 mM-NO2- or 100 mM-hydroxylamine as oxidized substrate. This increase is more marked for hydroxylamine reduction than for NO2- reduction. Models incorporating only one binding site for NAD can account for the variation in the Michaelis-Menten parameters for both NADH and hydroxylamine with [NAD+] for hydroxylamine reduction. According to these models, activation of the reaction occurs by reversal of an over-reduction of the enzyme by NADH. If the observed activation of the enzyme by NAD+ derives both from activation of the generation of the enzyme-hydroxylamine complex from the enzyme-NO2- complex during NO2- reduction and from activation of the reduction of the enzyme-hydroxylamine complex to form NH4+, then the variation of Vapp. for NO2- or hydroxylamine with [NAD+] is consistent with the occurrence of the same enzyme-hydroxylamine complex as an intermediate in both reactions. PMID:6279095

  8. Optimization of enzyme parameters for fermentative production of biorenewable fuels and chemicals

    Directory of Open Access Journals (Sweden)

    Ping Liu

    2012-10-01

    Full Text Available Microbial biocatalysts such as Escherichia coli and Saccharomyces cerevisiae have been extensively subjected to Metabolic Engineering for the fermentative production of biorenewable fuels and chemicals. This often entails the introduction of new enzymes, deletion of unwanted enzymes and efforts to fine-tune enzyme abundance in order to attain the desired strain performance. Enzyme performance can be quantitatively described in terms of the Michaelis-Menten type parameters Km, turnover number kcat and Ki, which roughly describe the affinity of an enzyme for its substrate, the speed of a reaction and the enzyme sensitivity to inhibition by regulatory molecules. Here we describe examples of where knowledge of these parameters have been used to select, evolve or engineer enzymes for the desired performance and enabled increased production of biorenewable fuels and chemicals. Examples include production of ethanol, isobutanol, 1-butanol and tyrosine and furfural tolerance. The Michaelis-Menten parameters can also be used to judge the cofactor dependence of enzymes and quantify their preference for NADH or NADPH. Similarly, enzymes can be selected, evolved or engineered for the preferred cofactor preference. Examples of exporter engineering and selection are also discussed in the context of production of malate, valine and limonene.

  9. A novel H(2)O(2) amperometric biosensor based on gold nanoparticles/self-doped polyaniline nanofibers.

    Science.gov (United States)

    Chen, Xiaojun; Chen, Zixuan; Zhu, Jinwei; Xu, Chenbin; Yan, Wei; Yao, Cheng

    2011-10-01

    A new kind of gold nanoparticles/self-doped polyaniline nanofibers (Au/SPAN) with grooves has been prepared for the immobilization of horseradish peroxidase (HRP) on the surface of glassy carbon electrode (GCE). The ratio of gold in the composite nanofibers was up to 64%, which could promote the conductivity and biocompatibility of SPAN and increase the immobilized amount of HRP molecules greatly. The electrode exhibits enhanced electrocatalytic activity in the reduction of H(2)O(2) in the presence of the mediator hydroquinone (HQ). The effects of concentration of HQ, solution pH and the working potential on the current response of the modified electrode toward H(2)O(2) were optimized to obtain the maximal sensitivity. The proposed biosensor exhibited a good linear response in the range from 10 to 2000 μM with a detection limit of 1.6 μM (S/N=3) under the optimum conditions. The response showed Michaelis-Menten behavior at larger H(2)O(2) concentrations, and the apparent Michaelis-Menten constant K(m) was estimated to be 2.21 mM. The detection of H(2)O(2) concentration in real sample showed acceptable accuracy with the traditional potassium permanganate titration. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. OPTIMIZATION OF ENZYME PARAMETERS FOR FERMENTATIVE PRODUCTION OF BIORENEWABLE FUELS AND CHEMICALS

    Directory of Open Access Journals (Sweden)

    Laura R. Jarboe

    2012-10-01

    Full Text Available Microbial biocatalysts such as Escherichia coli and Saccharomyces cerevisiae have been extensively subjected to Metabolic Engineering for the fermentative production of biorenewable fuels and chemicals. This often entails the introduction of new enzymes, deletion of unwanted enzymes and efforts to fine-tune enzyme abundance in order to attain the desired strain performance. Enzyme performance can be quantitatively described in terms of the Michaelis-Menten type parameters Km, turnover number kcat and Ki, which roughly describe the affinity of an enzyme for its substrate, the speed of a reaction and the enzyme sensitivity to inhibition by regulatory molecules. Here we describe examples of where knowledge of these parameters have been used to select, evolve or engineer enzymes for the desired performance and enabled increased production of biorenewable fuels and chemicals. Examples include production of ethanol, isobutanol, 1-butanol and tyrosine and furfural tolerance. The Michaelis-Menten parameters can also be used to judge the cofactor dependence of enzymes and quantify their preference for NADH or NADPH. Similarly, enzymes can be selected, evolved or engineered for the preferred cofactor preference. Examples of exporter engineering and selection are also discussed in the context of production of malate, valine and limonene.

  11. Use of an uncertainty analysis for genome-scale models as a prediction tool for microbial growth processes in subsurface environments.

    Science.gov (United States)

    Klier, Christine

    2012-03-06

    The integration of genome-scale, constraint-based models of microbial cell function into simulations of contaminant transport and fate in complex groundwater systems is a promising approach to help characterize the metabolic activities of microorganisms in natural environments. In constraint-based modeling, the specific uptake flux rates of external metabolites are usually determined by Michaelis-Menten kinetic theory. However, extensive data sets based on experimentally measured values are not always available. In this study, a genome-scale model of Pseudomonas putida was used to study the key issue of uncertainty arising from the parametrization of the influx of two growth-limiting substrates: oxygen and toluene. The results showed that simulated growth rates are highly sensitive to substrate affinity constants and that uncertainties in specific substrate uptake rates have a significant influence on the variability of simulated microbial growth. Michaelis-Menten kinetic theory does not, therefore, seem to be appropriate for descriptions of substrate uptake processes in the genome-scale model of P. putida. Microbial growth rates of P. putida in subsurface environments can only be accurately predicted if the processes of complex substrate transport and microbial uptake regulation are sufficiently understood in natural environments and if data-driven uptake flux constraints can be applied.

  12. A Finite-Difference Solution of Solute Transport through a Membrane Bioreactor

    Directory of Open Access Journals (Sweden)

    B. Godongwana

    2015-01-01

    Full Text Available The current paper presents a theoretical analysis of the transport of solutes through a fixed-film membrane bioreactor (MBR, immobilised with an active biocatalyst. The dimensionless convection-diffusion equation with variable coefficients was solved analytically and numerically for concentration profiles of the solutes through the MBR. The analytical solution makes use of regular perturbation and accounts for radial convective flow as well as axial diffusion of the substrate species. The Michaelis-Menten (or Monod rate equation was assumed for the sink term, and the perturbation was extended up to second-order. In the analytical solution only the first-order limit of the Michaelis-Menten equation was considered; hence the linearized equation was solved. In the numerical solution, however, this restriction was lifted. The solution of the nonlinear, elliptic, partial differential equation was based on an implicit finite-difference method (FDM. An upwind scheme was employed for numerical stability. The resulting algebraic equations were solved simultaneously using the multivariate Newton-Raphson iteration method. The solution allows for the evaluation of the effect on the concentration profiles of (i the radial and axial convective velocity, (ii the convective mass transfer rates, (iii the reaction rates, (iv the fraction retentate, and (v the aspect ratio.

  13. Synthetic, structural mimetics of the β-hairpin flap of HIV-1 protease inhibit enzyme function.

    Science.gov (United States)

    Chauhan, Jay; Chen, Shen-En; Fenstermacher, Katherine J; Naser-Tavakolian, Aurash; Reingewertz, Tali; Salmo, Rosene; Lee, Christian; Williams, Emori; Raje, Mithun; Sundberg, Eric; DeStefano, Jeffrey J; Freire, Ernesto; Fletcher, Steven

    2015-11-01

    Small-molecule mimetics of the β-hairpin flap of HIV-1 protease (HIV-1 PR) were designed based on a 1,4-benzodiazepine scaffold as a strategy to interfere with the flap-flap protein-protein interaction, which functions as a gated mechanism to control access to the active site. Michaelis-Menten kinetics suggested our small-molecules are competitive inhibitors, which indicates the mode of inhibition is through binding the active site or sterically blocking access to the active site and preventing flap closure, as designed. More generally, a new bioactive scaffold for HIV-1PR inhibition has been discovered, with the most potent compound inhibiting the protease with a modest K(i) of 11 μM. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Thymidine kinase 2 enzyme kinetics elucidate the mechanism of thymidine-induced mitochondrial DNA depletion.

    Science.gov (United States)

    Sun, Ren; Wang, Liya

    2014-10-07

    Mitochondrial thymidine kinase 2 (TK2) is a nuclear gene-encoded protein, synthesized in the cytosol and subsequently translocated into the mitochondrial matrix, where it catalyzes the phosphorylation of thymidine (dT) and deoxycytidine (dC). The kinetics of dT phosphorylation exhibits negative cooperativity, but dC phosphorylation follows hyperbolic Michaelis-Menten kinetics. The two substrates compete with each other in that dT is a competitive inhibitor of dC phosphorylation, while dC acts as a noncompetitive inhibitor of dT phosphorylation. In addition, TK2 is feedback inhibited by dTTP and dCTP. TK2 also phosphorylates a number of pyrimidine nucleoside analogues used in antiviral and anticancer therapy and thus plays an important role in mitochondrial toxicities caused by nucleoside analogues. Deficiency in TK2 activity due to genetic alterations causes devastating mitochondrial diseases, which are characterized by mitochondrial DNA (mtDNA) depletion or multiple deletions in the affected tissues. Severe TK2 deficiency is associated with early-onset fatal mitochondrial DNA depletion syndrome, while less severe deficiencies result in late-onset phenotypes. In this review, studies of the enzyme kinetic behavior of TK2 enzyme variants are used to explain the mechanism of mtDNA depletion caused by TK2 mutations, thymidine overload due to thymidine phosphorylase deficiency, and mitochondrial toxicity caused by antiviral thymidine analogues.

  15. A Biochemist's View of Ecosystem Rates and their Response to Changing Temperature

    Science.gov (United States)

    Arcus, V. L.

    2017-12-01

    Enzyme kinetics lie at the heart of biochemistry and the Michaelis-Menten equation that defines the relationship between substrate and rate is over 100 years old. About 80 years ago Eyring and Polyani formulated Transistion State Theory (TST) which describes the temperature-dependence of chemical reaction rates and the precise relationship between activation energy and the rate. TST provided a robust theoretical foundation for the Arrhenius equation and together, these equations are the foundation equations for the biochemist. Can these equations provide any insights into rates at larger scales, such as organism growth rates and those rates that interest ecosystem scientists (e.g. heterotrophic respiration, gross primary production)? Let us begin by considering a microbial cell. Microbial growth (i.e. cell division) requires the coordinated kinetics of thousands of enzymes including DNA/RNA polymerases, ribosomes, biosynthetic enzymes - all under a regime of highly complex regulatory effects. There is no a priori reason to expect that Michaelis-Menten kinetics and TST will adequately describe this vastly complex process. Indeed, Lloyd and Taylor showed 23 years ago that soil respiration is not well described by the Arrhenius function. More recently, Heskel and colleagues showed that leaf respiration is also not well described by the Arrhenius function. It is the same case for rates of photosynthesis. Despite this failure of the basic equations of biochemistry to map to biological rates at greater scales, what insights can biochemistry provide to ecosystem science? As nearly all of biological metabolism is mediated through enzyme kinetics, I will begin with the Michaelis-Menten equation under regimes of low and high substrate concentrations. This simplified view can provide surprising insights into processes at larger scales. I will also consider the relationship between the activation energy and the reaction rate. Many, many ecosystem-rate papers focus on the

  16. Low birth rates and reproductive skew limit the viability of Europe’s captive eastern black rhinoceros, Diceros bicornis michaeli

    NARCIS (Netherlands)

    Edwards, K.L.; Walker, S.L.; Durham, A.E.; Pilgrim, M.; Ouma, B.O.; Shultz, S.

    2015-01-01

    Ex situ populations play a critical role for the conservation of endangered species, especially where in situ populations face imminent threats. For such populations to act as vital reserves, they must be viable and sustainable. Eastern black rhinoceros (Diceros bicornis michaeli) epitomise the

  17. Collective behaviours: from biochemical kinetics to electronic circuits

    Science.gov (United States)

    Agliari, Elena; Barra, Adriano; Burioni, Raffaella; di Biasio, Aldo; Uguzzoni, Guido

    2013-12-01

    In this work we aim to highlight a close analogy between cooperative behaviors in chemical kinetics and cybernetics; this is realized by using a common language for their description, that is mean-field statistical mechanics. First, we perform a one-to-one mapping between paradigmatic behaviors in chemical kinetics (i.e., non-cooperative, cooperative, ultra-sensitive, anti-cooperative) and in mean-field statistical mechanics (i.e., paramagnetic, high and low temperature ferromagnetic, anti-ferromagnetic). Interestingly, the statistical mechanics approach allows a unified, broad theory for all scenarios and, in particular, Michaelis-Menten, Hill and Adair equations are consistently recovered. This framework is then tested against experimental biological data with an overall excellent agreement. One step forward, we consistently read the whole mapping from a cybernetic perspective, highlighting deep structural analogies between the above-mentioned kinetics and fundamental bricks in electronics (i.e. operational amplifiers, flashes, flip-flops), so to build a clear bridge linking biochemical kinetics and cybernetics.

  18. Hybrid dynamic modeling of Escherichia coli central metabolic network combining Michaelis–Menten and approximate kinetic equations

    DEFF Research Database (Denmark)

    Costa, Rafael S.; Machado, Daniel; Rocha, Isabel

    2010-01-01

    , represent nowadays the limiting factor in the construction of such models. In this study, we compare four alternative modeling approaches based on Michaelis–Menten kinetics for the bi-molecular reactions and different types of simplified rate equations for the remaining reactions (generalized mass action......The construction of dynamic metabolic models at reaction network level requires the use of mechanistic enzymatic rate equations that comprise a large number of parameters. The lack of knowledge on these equations and the difficulty in the experimental identification of their associated parameters...

  19. The steady-state kinetics of the NADH-dependent nitrite reductase from Escherichia coli K 12. Nitrite and hydroxylamine reduction.

    OpenAIRE

    Jackson, R H; Cole, J A; Cornish-Bowden, A

    1981-01-01

    The reduction of both NO2- and hydroxylamine by the NADH-dependent nitrite reductase of Escherichia coli K 12 (EC 1.6.6.4) appears to follow Michaelis-Menten kinetics over a wide range of NADH concentrations. Substrate inhibition can, however, be detected at low concentrations of the product NAD+. In addition, NAD+ displays mixed product inhibition with respect to NADH and mixed or uncompetitive inhibition with respect to hydroxylamine. These inhibition characteristics are consistent with a m...

  20. An Assessment of Factors Affecting Reactive Transport of Biodegradable BTEX in an Unconfined Aquifer System, Tehran Oil Refinery, Iran

    Directory of Open Access Journals (Sweden)

    A. Agah

    2012-12-01

    Full Text Available Risk-based assessment methods are commonly used at the contaminated sites by hydrocarbon pollutants. This paper presents the results of a two-dimensional finite volume model of reactive transport of biodegradable BTEX which have been developed for the saturated zone of an unconfined aquifer in the Pump station area of Tehran oil refinery, Iran. The model governing equations were numerically solved by modification of a general commercial software called PHOENICS. To reduce costs in general, many input parameters of a model are often approximated based on the used values in the contaminated sites with same conditions. It was not fully recognised the effect of errors in these inputs on modelling outputs. Thus, a sensitivity analysis was carried out to determine the influence of parameters variability on the results of model. For this analysis, the sensitivity of the model to changes in the dispersivity, distribution coefficient, parameters of Monod, Michaelis-Menten, first- and zero- order kinetics modes on the BTEX contaminant plume were examined by performing several simulations. It was found that the model is sensitive to changes in dispersivity and parameters of Michaelis-Menten, first- and zero- order kinetics model. On the other hand, the predictions for plumes assuming Monod kinetics are similar, even if different values for parameterization are chosen. The reason for this insensibility is that degradation is not limited by microbial kinetics in the simulation, but by dispersive mixing. Quantifying the effect of changes in model input parameters on the modelling results is essential when it is desired to recognise which model parameters are more vital on the fate and transport of reactive pollutants. Furthermore, this process can provide an insight into understanding pollutant transportation mechanisms.

  1. On the precision of quasi steady state assumptions in stochastic dynamics

    Science.gov (United States)

    Agarwal, Animesh; Adams, Rhys; Castellani, Gastone C.; Shouval, Harel Z.

    2012-07-01

    Many biochemical networks have complex multidimensional dynamics and there is a long history of methods that have been used for dimensionality reduction for such reaction networks. Usually a deterministic mass action approach is used; however, in small volumes, there are significant fluctuations from the mean which the mass action approach cannot capture. In such cases stochastic simulation methods should be used. In this paper, we evaluate the applicability of one such dimensionality reduction method, the quasi-steady state approximation (QSSA) [L. Menten and M. Michaelis, "Die kinetik der invertinwirkung," Biochem. Z 49, 333369 (1913)] for dimensionality reduction in case of stochastic dynamics. First, the applicability of QSSA approach is evaluated for a canonical system of enzyme reactions. Application of QSSA to such a reaction system in a deterministic setting leads to Michaelis-Menten reduced kinetics which can be used to derive the equilibrium concentrations of the reaction species. In the case of stochastic simulations, however, the steady state is characterized by fluctuations around the mean equilibrium concentration. Our analysis shows that a QSSA based approach for dimensionality reduction captures well the mean of the distribution as obtained from a full dimensional simulation but fails to accurately capture the distribution around that mean. Moreover, the QSSA approximation is not unique. We have then extended the analysis to a simple bistable biochemical network model proposed to account for the stability of synaptic efficacies; the substrate of learning and memory [J. E. Lisman, "A mechanism of memory storage insensitive to molecular turnover: A bistable autophosphorylating kinase," Proc. Natl. Acad. Sci. U.S.A. 82, 3055-3057 (1985)], 10.1073/pnas.82.9.3055. Our analysis shows that a QSSA based dimensionality reduction method results in errors as big as two orders of magnitude in predicting the residence times in the two stable states.

  2. Kinetics of the norepinephrine analog [76Br]-meta-bromobenzylguanidine in isolated working rat heart

    International Nuclear Information System (INIS)

    Raffel, David; Loc'h, Christian; Mardon, Karine; Maziere, Bernard; Syrota, Andre

    1998-01-01

    A related set of kinetic studies of the norepinephrine analog [ 76 Br]-meta-bromobenzylguanidine (MBBG) were performed with an isolated working rat heart preparation. A series of constant infusion studies over a wide range of MBBG concentrations allowed estimation of the Michaelis-Menten constants for transport by the neuronal norepinephrine transporter (uptake 1 ) and the extraneuronal uptake system (uptake 2 ). Pharmacological blocking studies with inhibitors of uptake 1 , uptake 2 and vesicular uptake were performed to delineate the relative importance of these norepinephrine handling mechanisms on the kinetics of MBBG in the rat heart. Bolus injection studies were done to assess the ability of compartmental modeling techniques to characterize the kinetics of MBBG. These studies demonstrate that MBBG shares many of the same uptake mechanisms as norepinephrine in the rat heart. PET imaging studies with MBBG would be useful for assessing sympathetic nerve status in the living human heart

  3. Correlation analysis of reactivity in the oxidation of some organic diols by tripropylammonium fluorochromate in non-aqueous media

    Directory of Open Access Journals (Sweden)

    S. Sheik Mansoor

    2016-09-01

    Full Text Available The kinetics of oxidation of some organic diols by tripropylammonium fluorochromate (TriPAFC have been studied in dimethylsulfoxide (DMSO. The main product of oxidation is the corresponding hydroxy aldehydes. The reaction is first order with respect to TriPAFC and exhibited Michaelis-Menten type kinetics with respect to organic diols. The reaction is catalyzed by hydrogen ions. The hydrogen ion dependence has the form: kobs = a + b[H+]. Various thermodynamic parameters for the oxidation have been reported and discussed along with the validity of isokinetic relationship. Oxidation of diols was studied in 18 different organic solvents. The rate data are showing satisfactory correlation with Kamlet–Taft solvotochromic parameters (α, β and π∗. A suitable mechanism of oxidation has been proposed.

  4. Catalytic Activity of μ-Carbido-Dimeric Iron(IV) Octapropylporphyrazinate in the 3,5,7,2',4'-Pentahydroxyflavone Oxidation Reaction with tert-Butyl Hydroperoxide

    Science.gov (United States)

    Tyurin, D. V.; Zaitseva, S. V.; Kudrik, E. V.

    2018-05-01

    It is found for the first time that μ-carbido-dimeric iron(IV) octapropylporphyrazinate displays catalytic activity in the oxidation reaction of natural flavonol morin with tert-butyl hydroperoxide, with the catalyst being stable under conditions of the reaction. The kinetics of this reaction are studied. It is shown the reaction proceeds via tentative formation of a complex between the catalyst and the oxidant, followed by O‒O bond homolytic cleavage. The kinetics of the reaction is described in the coordinates of the Michaelis-Menten equation. A linear dependence of the apparent reaction rate constant on the concentration of the catalyst is observed, testifying to its participation in the limiting reaction step. The equilibrium constants and rates of interaction are found. A mechanism is proposed for the reaction on the basis of the experimental data.

  5. Differential potassium influx influences growth of two cotton varieties in hydroponics

    International Nuclear Information System (INIS)

    Ali, L.; Maqsood, M.A.; Kanwal, S.; Aziz, T.

    2010-01-01

    Potassium uptake rate of two cotton (Gossypium hirsutum L.) varieties viz., NIBGE-2 and MNH-786 was investigated in nutrient solution culture having deficient K at the rate 0.3 mM and deficient K+ Na at the rate 0.3 +2.7 mM. Depletion of K from solution was monitored over a period of 24 h at regular time intervals after 0, 0.5, 1.0, 1.5, 2, 3, 4, 5, 6, 8, 10, 12 and 24 h to estimate K uptake kinetics of the roots i.e. maximum influx, I/sub max/ and the Michaelis-Menten constant, Km. NIBGE-2 had about 2-fold higher (2.0 mg g rdw-1 hr-1) I/sub max/ value for K uptake rate at deficient K+Na than that (1.207 mg g rdw-1 hr-1) for MNH-786. Higher, Michaelis-Menten constant, Km (12.82 ppm) for K uptake rate was observed in both cultivars NIBGE-2 and MNH-786 at deficient K+Na than that at deficient K. Main effects of treatments and varieties had significant (p< 0.05) effect on shoot dry matter, root dry matter, total dry matter and leaf area per plant. Maximum K influx in NIBGE-2 at deficient K and deficient K +Na was attributed to enhanced growth response as compared to that in MNH-786. (author)

  6. Use of CdSe/ZnS luminescent quantum dots incorporated within sol-gel matrix for urea detection

    International Nuclear Information System (INIS)

    Duong, Hong Dinh; Rhee, Jong Il

    2008-01-01

    In this work, urea detection techniques based on the pH sensitivity of CdSe/ZnS QDs were developed using three types of sol-gel membranes: a QD-entrapped membrane, urease-immobilized membrane and double layer consisting of a QD-entrapped membrane and urease-immobilized membrane. The surface morphology of the sol-gel membranes deposited on the wells in a 24-well microtiter plate was investigated. The linear detection range of urea was in the range of 0-10 mM with the three types of sol-gel membranes. The urea detection technique based on the double layer consisting of the QD-entrapped membrane and urease-immobilized membrane resulted in the highest sensitivity to urea due to the Michaelis-Menten kinetic parameters. That is, the Michaelis-Menten constant (K m =2.0745 mM) of the free urease in the QD-entrapped membrane was about 4-fold higher than that (K m =0.549 mM) of the immobilized urease in the urease-immobilized membrane and about 12-fold higher than that (K m =0.1698 mM) of the immobilized urease in the double layer. The good stability of the three sol-gel membranes for urea sensing over 2 months showed that the use of sol-gel membranes immobilized with QDs or an enzyme is suitable for biomedical and environmental applications

  7. Use of CdSe/ZnS luminescent quantum dots incorporated within sol-gel matrix for urea detection.

    Science.gov (United States)

    Duong, Hong Dinh; Rhee, Jong Il

    2008-09-19

    In this work, urea detection techniques based on the pH sensitivity of CdSe/ZnS QDs were developed using three types of sol-gel membranes: a QD-entrapped membrane, urease-immobilized membrane and double layer consisting of a QD-entrapped membrane and urease-immobilized membrane. The surface morphology of the sol-gel membranes deposited on the wells in a 24-well microtiter plate was investigated. The linear detection range of urea was in the range of 0-10mM with the three types of sol-gel membranes. The urea detection technique based on the double layer consisting of the QD-entrapped membrane and urease-immobilized membrane resulted in the highest sensitivity to urea due to the Michaelis-Menten kinetic parameters. That is, the Michaelis-Menten constant (K(m)=2.0745mM) of the free urease in the QD-entrapped membrane was about 4-fold higher than that (K(m)=0.549mM) of the immobilized urease in the urease-immobilized membrane and about 12-fold higher than that (K(m)=0.1698mM) of the immobilized urease in the double layer. The good stability of the three sol-gel membranes for urea sensing over 2 months showed that the use of sol-gel membranes immobilized with QDs or an enzyme is suitable for biomedical and environmental applications.

  8. ZnO nanowire-based glucose biosensors with different coupling agents

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Juneui [Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of); Lim, Sangwoo, E-mail: swlim@yonsei.ac.kr [Department of Chemical and Biomolecular Engineering, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-749 (Korea, Republic of)

    2013-01-15

    Highlights: Black-Right-Pointing-Pointer Fabrication of ZnO nanowire-based glucose biosensors using different coupling agents. Black-Right-Pointing-Pointer Highest sensitivity for (3-aminopropyl)methyldiethoxysilane-treated biosensor. Black-Right-Pointing-Pointer Larger amount of glucose oxidase and lower electron transfer resistance for (3-aminopropyl)methyldiethoxysilane-treated biosensor. - Abstract: ZnO-nanowire-based glucose biosensors were fabricated by immobilizing glucose oxidase (GOx) onto a linker attached to ZnO nanowires. Different coupling agents were used, namely (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), and (3-aminopropyl)methyldiethoxysilane (APS), to increase the affinity of GOx binding to ZnO nanowires. The amount of GOx immobilized on the ZnO nanowires, the performance, sensitivity, and Michaelis-Menten constant of each biosensor, and the electron transfer resistance through the biosensor were all measured in order to investigate the effect of the coupling agent on the ZnO nanowire-based biosensor. Among the different biosensors, the APS-treated biosensor had the highest sensitivity (17.72 {mu}A cm{sup -2} mM{sup -1}) and the lowest Michaelis-Menten constant (1.37 mM). Since APS-treated ZnO nanowires showed the largest number of C-N groups and the lowest electron transfer resistance through the biosensor, we concluded that these properties were the key factors in the performance of APS-treated glucose biosensors.

  9. Crude Aloe vera Gel Shows Antioxidant Propensities and Inhibits Pancreatic Lipase and Glucose Movement In Vitro

    Science.gov (United States)

    Taukoorah, Urmeela; Mahomoodally, M. Fawzi

    2016-01-01

    Aloe vera gel (AVG) is traditionally used in the management of diabetes, obesity, and infectious diseases. The present study aimed to investigate the inhibitory potential of AVG against α-amylase, α-glucosidase, and pancreatic lipase activity in vitro. Enzyme kinetic studies using Michaelis-Menten (K m) and Lineweaver-Burk equations were used to establish the type of inhibition. The antioxidant capacity of AVG was evaluated for its ferric reducing power, 2-diphenyl-2-picrylhydrazyl hydrate scavenging ability, nitric oxide scavenging power, and xanthine oxidase inhibitory activity. The glucose entrapment ability, antimicrobial activity, and total phenolic, flavonoid, tannin, and anthocyanin content were also determined. AVG showed a significantly higher percentage inhibition (85.56 ± 0.91) of pancreatic lipase compared to Orlistat. AVG was found to increase the Michaelis-Menten constant and decreased the maximal velocity (V max) of lipase, indicating mixed inhibition. AVG considerably inhibits glucose movement across dialysis tubes and was comparable to Arabic gum. AVG was ineffective against the tested microorganisms. Total phenolic and flavonoid contents were 66.06 ± 1.14 (GAE)/mg and 60.95 ± 0.97 (RE)/mg, respectively. AVG also showed interesting antioxidant properties. The biological activity observed in this study tends to validate some of the traditional claims of AVG as a functional food. PMID:26880905

  10. nitrogen saturation in stream ecosystems

    OpenAIRE

    Earl, S. R.; Valett, H. M.; Webster, J. R.

    2006-01-01

    The concept of nitrogen (N) saturation has organized the assessment of N loading in terrestrial ecosystems. Here we extend the concept to lotic ecosystems by coupling Michaelis-Menten kinetics and nutrient spiraling. We propose a series of saturation response types, which may be used to characterize the proximity of streams to N saturation. We conducted a series of short-term N releases using a tracer ((NO3)-N-15-N) to measure uptake. Experiments were conducted in streams spanning a gradient ...

  11. Metabolomics on integrated circuit

    OpenAIRE

    Cheah, Boon Chong; MacDonald, Alasdair I.; Barrett, Michael P.; Cumming, David R.S.

    2017-01-01

    We have demonstrated a chip-based diagnostics tool for the quantification of metabolites, using specific enzymes, to study enzyme kinetics and calculate the Michaelis-Menten constant. An array of 256×256 ion-sensitive field effect transistors (ISFETs) fabricated in a complementary metal oxide semiconductor (CMOS) process is used for this prototype. We have used hexokinase enzyme reaction on the ISFET CMOS chip with glucose concentration in the physiological range of 0.05 mM – 231 mM and succe...

  12. Extraction of Crude Chitinase from Higher Plants and their Chitin-Hydrolysis Activities; Kotosyokubutu yurai kichinaze no chusyutu to kichin bunkai kassei

    Energy Technology Data Exchange (ETDEWEB)

    Kondo, K.; Harada, K.; Shibata, M.; Maeda, R. [Doshisha Univ., Kyoto (Japan). Faculty of Engineering

    1997-07-10

    To prepare a purified chitinase from higher plants, firstly, crude enzymes were extracted from six higher plants, namely, radish seeds, sunflower seeds, watermelon seeds, bamboo leaves, orange skin, and persimmon skin. Using these crude enzymes, pH dependencies of hydrolysis reaction of colloidal chitin are investigated. For radish seeds and bamboo leaves, which have relatively high activities, the kinetics of enzymatic reaction are studies. It is clear that these reactions obey Michaelis-Menten kinetics. 7 refs., 3 figs., 2 tabs.

  13. Effect and Modeling of Glucose Inhibition and In Situ Glucose Removal During Enzymatic Hydrolysis of Pretreated Wheat Straw

    DEFF Research Database (Denmark)

    Andric, Pavle; Meyer, Anne S.; Jensen, Peter Arendt

    2010-01-01

    The enzymatic hydrolysis of lignocellulosic biomass is known to be product-inhibited by glucose. In this study, the effects on cellulolytic glucose yields of glucose inhibition and in situ glucose removal were examined and modeled during extended treatment of heat-pretreated wheat straw......, during 96 h of reaction. When glucose was removed by dialysis during the enzymatic hydrolysis, the cellulose conversion rates and glucose yields increased. In fact, with dialytic in situ glucose removal, the rate of enzyme-catalyzed glucose release during 48-72 h of reaction recovered from 20......-40% to become approximate to 70% of the rate recorded during 6-24 h of reaction. Although Michaelis-Menten kinetics do not suffice to model the kinetics of the complex multi-enzymatic degradation of cellulose, the data for the glucose inhibition were surprisingly well described by simple Michaelis...

  14. Enzyme activity and kinetics in substrate-amended river sediments

    Energy Technology Data Exchange (ETDEWEB)

    Duddridge, J E; Wainwright, M

    1982-01-01

    In determining the effects of heavy metals in microbial activity and litter degradation in river sediments, one approach is to determine the effects of these pollutants on sediment enzyme activity and synthesis. Methods to assay amylase, cellulase and urease activity in diverse river sediments are reported. Enzyme activity was low in non-amended sediments, but increased markedly when the appropriate substrate was added, paralleling both athropogenic and natural amendment. Linear relationships between enzyme activity, length of incubation, sample size and substrate concentration were established. Sediment enzyme activity generally obeyed Michaelis-Menton kinetics, but of the three enzymes, urease gave least significant correlation coefficients when the data for substrate concentration versus activity was applied to the Eadie-Hofstee transformation of the Michaelis-Menten equation. K/sub m/ and V/sub max/ for amylase, cellulase and urease in sediments are reported. (JMT)

  15. Characterization of lipase in reversed micelles formulated by Cibacron Blue F-3GA modified Span 85

    DEFF Research Database (Denmark)

    Zhang, Dong Hao; Guo, Zheng; Sun, Yan

    2007-01-01

    Sorbitan trioleate (Span 85) modified by Cibacron Blue F-3GA (CB) was prepared and used as an affinity surfactant to formulate a reversed micellar system for Candida rugosa lipase (CRL) solubilization. The system was characterized and evaluated by employing CRL-catalyzed hydrolysis of olive oil...... of the encapsulated lipase remained unchanged, but the apparent activity was significantly higher than that of the native enzyme in bulk solution. Kinetic studies indicated that the encapsulated lipase in the reversed micelles of CB-formulated Span 85 followed the Michaelis-Menten equation. The Michaelis constant...... was found to decrease with increasing surfactant concentration, suggesting an increase of the enzyme affinity for the substrate. Stability of the lipase in the reversed micelles was negatively correlated to W0. Introduction Reversed micelles are nanometer-scale transparent aggregates of water and surfactant...

  16. No-threshold dose-response curves for nongenotoxic chemicals: Findings and applications for risk assessment

    International Nuclear Information System (INIS)

    Sheehan, Daniel M.

    2006-01-01

    We tested the hypothesis that no threshold exists when estradiol acts through the same mechanism as an active endogenous estrogen. A Michaelis-Menten (MM) equation accounting for response saturation, background effects, and endogenous estrogen level fit a turtle sex-reversal data set with no threshold and estimated the endogenous dose. Additionally, 31 diverse literature dose-response data sets were analyzed by adding a term for nonhormonal background; good fits were obtained but endogenous dose estimations were not significant due to low resolving power. No thresholds were observed. Data sets were plotted using a normalized MM equation; all 178 data points were accommodated on a single graph. Response rates from ∼1% to >95% were well fit. The findings contradict the threshold assumption and low-dose safety. Calculating risk and assuming additivity of effects from multiple chemicals acting through the same mechanism rather than assuming a safe dose for nonthresholded curves is appropriate

  17. Passage of delta sleep-inducing peptide (DSIP) across the blood-cerebrospinal fluid barrier

    International Nuclear Information System (INIS)

    Zlokovic, B.V.; Segal, M.B.; Davson, H.; Jankov, R.M.

    1988-01-01

    Unidirectional flux of 125 I-labeled DSIP at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier was studied in the perfused in situ choroid plexuses of the lateral ventricles of the sheep. Arterio-venous loss of 125 I-radioactivity suggested a low-to-moderate permeability of the choroid epithelium to the intact peptide from the blood side. A saturable mechanism with Michaelis-Menten type kinetics with high affinity and very low capacity (approximate values: Kt = 5.0 +/- 0.4 nM; Vmax = 272 +/- 10 fmol.min-1) was demonstrated at the blood-tissue interface of the choroid plexus. The clearance of DSIP from the ventricles during ventriculo-cisternal perfusion in the rabbit indicated no significant flux of the intact peptide out of the CSF. The results suggest that DSIP crosses the blood-CSF barrier, while the system lacks the specific mechanisms for removal from the CSF found with most, if not all, amino acids and several peptides

  18. Spectral Quasi-Equilibrium Manifold for Chemical Kinetics.

    Science.gov (United States)

    Kooshkbaghi, Mahdi; Frouzakis, Christos E; Boulouchos, Konstantinos; Karlin, Iliya V

    2016-05-26

    The Spectral Quasi-Equilibrium Manifold (SQEM) method is a model reduction technique for chemical kinetics based on entropy maximization under constraints built by the slowest eigenvectors at equilibrium. The method is revisited here and discussed and validated through the Michaelis-Menten kinetic scheme, and the quality of the reduction is related to the temporal evolution and the gap between eigenvalues. SQEM is then applied to detailed reaction mechanisms for the homogeneous combustion of hydrogen, syngas, and methane mixtures with air in adiabatic constant pressure reactors. The system states computed using SQEM are compared with those obtained by direct integration of the detailed mechanism, and good agreement between the reduced and the detailed descriptions is demonstrated. The SQEM reduced model of hydrogen/air combustion is also compared with another similar technique, the Rate-Controlled Constrained-Equilibrium (RCCE). For the same number of representative variables, SQEM is found to provide a more accurate description.

  19. A kinetic model and simulation of starch saccharification and simultaneous ethanol fermentation by amyloglucosidase and Zymomonas mobilis

    Energy Technology Data Exchange (ETDEWEB)

    Lee, C G [Michigan Univ., Ann Arbor, MI (United States). Dept. of Chemical Engineering; Kim, C H; Rhee, S K [Korea Inst. of Science and Technology, Taejon (Korea, Republic of). Genetic Engineering Research Inst.

    1992-07-01

    A mathematical model is described for the simultaneous saccharification and ethanol fermentation (SSF) of sago starch using amyloglycosidase (AMG) and Zymomonas mobilis. By introducing the degree of polymerization (DP) of oligosaccharides produced from sago starch treated with {alpha}-amylase, a series of Michaelis-Menten equations was obtained. After determining kinetic parameters from the results of simple experiments and from the subsite mapping theory, this model was adapted to simulate the SSF process. The results of simulation for SSF are in good agreement with experimental results. (orig.).

  20. The sociology of language in Johann David Michaelis's dissertation of 1760.

    Science.gov (United States)

    Smith, R N

    1976-10-01

    In 1759 Johann David Michaelis won a prize from the Prussian Royal Academy for his essay Beantwortung der Frage von dem Einfluss der Meinungen in die Sprache, und der sprache in die Meinungen. The essay was published in the following year and translated into French in 1762, into English in 1769, and into Dutch in 1771. The work has two major themes--linguistic relativity and language change--with ancillary discussions of language in general and of homonymy. Its most significant contribution to the theory of language is its discussion of linguistic relativity, especially in its manifestations in the influence of language on thought. Given the intellectual milieu of the work where inquiry was centered on the origin of language and language universals, it stands as one of the few discussions of this topic and it is also one of the most fruitful discussions of linguistic relativity for any period of history.

  1. Nutrient Removal from Wastewater using Microalgae: A Kinetic Evaluation and Lipid Analysis.

    Science.gov (United States)

    2017-09-15

    The objective of this study was to examine the performance of mixed microalgal bioreactors in treating three differenttypes of wastewaters - kitchen wastewater (KWW), palm oil mill effluent (POME), and pharmaceutical wastewater (PWW) in semi-continuous mode and to analyze the lipid content in the harvested algal biomass. The reactors were monitored for total nitrogen and phosphate removal at eight solid retention times (SRTs) - 2, 4, 6, 8, 10, 12, 14, and 16 days. The nutrient uptake kinetic parameters were quantified using linearized Michaelis-Menten and Monod models at steady-state conditions. The nutrient removal efficiency and lipid production were found to be higher in KWW when compared with the other wastewaters. Saturated fatty acids (C16:0, C18:0, and C18:1) accounted for more than 60% of the algal fatty acids for all the wastewaters. The lipid is, therefore, considered suitable for synthesizing biodiesel.

  2. DICER-ARGONAUTE2 complex in continuous fluorogenic assays of RNA interference enzymes.

    Directory of Open Access Journals (Sweden)

    Mark A Bernard

    Full Text Available Mechanistic studies of RNA processing in the RNA-Induced Silencing Complex (RISC have been hindered by lack of methods for continuous monitoring of enzymatic activity. "Quencherless" fluorogenic substrates of RNAi enzymes enable continuous monitoring of enzymatic reactions for detailed kinetics studies. Recombinant RISC enzymes cleave the fluorogenic substrates targeting human thymidylate synthase (TYMS and hypoxia-inducible factor 1-α subunit (HIF1A. Using fluorogenic dsRNA DICER substrates and fluorogenic siRNA, DICER+ARGONAUTE2 mixtures exhibit synergistic enzymatic activity relative to either enzyme alone, and addition of TRBP does not enhance the apparent activity. Titration of AGO2 and DICER in enzyme assays suggests that AGO2 and DICER form a functional high-affinity complex in equimolar ratio. DICER and DICER+AGO2 exhibit Michaelis-Menten kinetics with DICER substrates. However, AGO2 cannot process the fluorogenic siRNA without DICER enzyme, suggesting that AGO2 cannot self-load siRNA into its active site. The DICER+AGO2 combination processes the fluorogenic siRNA substrate (Km=74 nM with substrate inhibition kinetics (Ki=105 nM, demonstrating experimentally that siRNA binds two different sites that affect Dicing and AGO2-loading reactions in RISC. This result suggests that siRNA (product of DICER bound in the active site of DICER may undergo direct transfer (as AGO2 substrate to the active site of AGO2 in the DICER+AGO2 complex. Competitive substrate assays indicate that DICER+AGO2 cleavage of fluorogenic siRNA is specific, since unlabeled siRNA and DICER substrates serve as competing substrates that cause a concentration-dependent decrease in fluorescent rates. Competitive substrate assays of a series of DICER substrates in vitro were correlated with cell-based assays of HIF1A mRNA knockdown (log-log slope=0.29, suggesting that improved DICER substrate designs with 10-fold greater processing by the DICER+AGO2 complex can provide a

  3. Nitrogen saturation in stream ecosystems.

    Science.gov (United States)

    Earl, Stevan R; Valett, H Maurice; Webster, Jackson R

    2006-12-01

    The concept of nitrogen (N) saturation has organized the assessment of N loading in terrestrial ecosystems. Here we extend the concept to lotic ecosystems by coupling Michaelis-Menten kinetics and nutrient spiraling. We propose a series of saturation response types, which may be used to characterize the proximity of streams to N saturation. We conducted a series of short-term N releases using a tracer (15NO3-N) to measure uptake. Experiments were conducted in streams spanning a gradient of background N concentration. Uptake increased in four of six streams as NO3-N was incrementally elevated, indicating that these streams were not saturated. Uptake generally corresponded to Michaelis-Menten kinetics but deviated from the model in two streams where some other growth-critical factor may have been limiting. Proximity to saturation was correlated to background N concentration but was better predicted by the ratio of dissolved inorganic N (DIN) to soluble reactive phosphorus (SRP), suggesting phosphorus limitation in several high-N streams. Uptake velocity, a reflection of uptake efficiency, declined nonlinearly with increasing N amendment in all streams. At the same time, uptake velocity was highest in the low-N streams. Our conceptual model of N transport, uptake, and uptake efficiency suggests that, while streams may be active sites of N uptake on the landscape, N saturation contributes to nonlinear changes in stream N dynamics that correspond to decreased uptake efficiency.

  4. Quantifying stream nutrient uptake from ambient to saturation with instantaneous tracer additions

    Science.gov (United States)

    Covino, T. P.; McGlynn, B. L.; McNamara, R.

    2009-12-01

    Stream nutrient tracer additions and spiraling metrics are frequently used to quantify stream ecosystem behavior. However, standard approaches limit our understanding of aquatic biogeochemistry. Specifically, the relationship between in-stream nutrient concentration and stream nutrient spiraling has not been characterized. The standard constant rate (steady-state) approach to stream spiraling parameter estimation, either through elevating nutrient concentration or adding isotopically labeled tracers (e.g. 15N), provides little information regarding the stream kinetic curve that represents the uptake-concentration relationship analogous to the Michaelis-Menten curve. These standard approaches provide single or a few data points and often focus on estimating ambient uptake under the conditions at the time of the experiment. Here we outline and demonstrate a new method using instantaneous nutrient additions and dynamic analyses of breakthrough curve (BTC) data to characterize the full relationship between spiraling metrics and nutrient concentration. We compare the results from these dynamic analyses to BTC-integrated, and standard steady-state approaches. Our results indicate good agreement between these three approaches but we highlight the advantages of our dynamic method. Specifically, our new dynamic method provides a cost-effective and efficient approach to: 1) characterize full concentration-spiraling metric curves; 2) estimate ambient spiraling metrics; 3) estimate Michaelis-Menten parameters maximum uptake (Umax) and the half-saturation constant (Km) from developed uptake-concentration kinetic curves, and; 4) measure dynamic nutrient spiraling in larger rivers where steady-state approaches are impractical.

  5. A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics.

    Science.gov (United States)

    Saaby, Lasse; Brodin, Birger

    2017-09-01

    Transport proteins expressed in the different barriers of the human body can have great implications on absorption, distribution, and excretion of drug compounds. Inhibition or saturation of a transporter can potentially alter these absorbtion, distribution, metabolism and elimination properties and thereby also the pharmacokinetic profile and bioavailability of drug compounds. P-glycoprotein (P-gp, ABCB1) is an efflux transporter which is present in most of the barriers of the body, including the small intestine, the blood-brain barrier, the liver, and the kidney. In all these tissues, P-gp may mediate efflux of drug compounds and may also be a potential site for drug-drug interactions. Consequently, there is a need to be able to predict the saturation and inhibition of P-gp and other transporters in vivo. For this purpose, Michaelis-Menten steady-state analysis has been applied to estimate kinetic parameters, such as K m and V max , for carrier-mediated transport, whereas half-maximal inhibitor concentration (IC 50 ) and the disassociation constant for an inhibitor/P-gp complex (K i ) have been determined to estimate P-gp inhibition. This review addresses in vitro methods commonly used to study P-gp transport kinetics and aims at providing a critical evaluation of the application of steady-state Michaelis-Menten analysis of kinetic parameters for substrate/P-gp interactions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  6. Depuración aerobia de los efluentes resultantes del proceso de biometanización del alpechín

    Directory of Open Access Journals (Sweden)

    Borja Padilla, R.

    1992-02-01

    Full Text Available A study of aerobic treatment in batch regime of the effluents produced in the olive mill wastewater biomethanation process was carried out. An 83% of effluents organic substances was removal after the third day of fermentation. The substrate removal rate follows a zero-order kinetic for high concentrations, and a first-order kinetic for low organic matter concentration, during the last days of fermentation. The kinetic parameters (qmáx and K were obtained from Michaelis- Menten model.

    Se ha efectuado un estudio del proceso de depuración aerobia, en régimen discontinuo, de los efluentes procedentes del proceso de depuración anaerobia o biometanización del alpechín. Se comprueba que el 83% de la materia orgánica presente en este efluente se elimina a partir del tercer día de fermentación. La eliminación de sustrato sigue una cinética de orden cero para altas concentraciones del mismo y una cinética de primer orden para bajas concentraciones de materia orgánica, es decir, durante los últimos días de fermentación. Se aplica el modelo de Michaelis-Menten de eliminación de sustrato para la obtención de los parámetros cinéticos qmáx y Ks que rigen este proceso.

  7. Comparison of biological H2S removal characteristics between a composite packing material with and without functional microorganisms

    Science.gov (United States)

    Zhu, Rencheng; Li, Shunyi; Bao, Xiaofeng; Dumont, Éric

    2017-02-01

    The performances of two identical biofilters, filled with a new composite packing material (named CM-5) embedded with functional microorganisms or sterilized CM-5 without microorganisms, were investigated for H2S treatment. Running parameters in terms of microbial counts, pressure drops, and inlet and outlet H2S concentrations were measured. The results show that the microbial count of the CM-5 was approximately ×105 CFU/g before being filled into the biofilter, while that of the sterilized CM-5 was negligible. The functional microorganisms embedded in CM-5 adapted to the environment containing H2S quickly. In most cases, pressure drops of the CM-5 biofilter were slightly higher than those of the sterilized CM-5 biofilter when the gas flow rate was 0.6-2.5 m3/h. The maximum elimination capacity (EC) of the CM-5 biofilter in treating H2S could reach up to 65 g/(m3·h) when the loading rate (LR) was approximately 80 g/(m3·h). If the LR was much higher, the measured EC showed a slight downward trend. The experimental ECs of biofilters were fitted by two typical dynamic models: the Michaelis-Menten model and the Haldane model. Compared with the Michaelis-Menten model, the Haldane model fit the experimental ECs better for the two biofilters because of the presence of the substrate inhibition behaviour.

  8. Ammonium and nitrite oxidation at nanomolar oxygen concentrations in oxygen minimum zone waters.

    Science.gov (United States)

    Bristow, Laura A; Dalsgaard, Tage; Tiano, Laura; Mills, Daniel B; Bertagnolli, Anthony D; Wright, Jody J; Hallam, Steven J; Ulloa, Osvaldo; Canfield, Donald E; Revsbech, Niels Peter; Thamdrup, Bo

    2016-09-20

    A major percentage of fixed nitrogen (N) loss in the oceans occurs within nitrite-rich oxygen minimum zones (OMZs) via denitrification and anammox. It remains unclear to what extent ammonium and nitrite oxidation co-occur, either supplying or competing for substrates involved in nitrogen loss in the OMZ core. Assessment of the oxygen (O2) sensitivity of these processes down to the O2 concentrations present in the OMZ core (Chile at manipulated O2 levels between 5 nmol⋅L(-1) and 20 μmol⋅L(-1) Rates of both processes were detectable in the low nanomolar range (5-33 nmol⋅L(-1) O2), but demonstrated a strong dependence on O2 concentrations with apparent half-saturation constants (Kms) of 333 ± 130 nmol⋅L(-1) O2 for ammonium oxidation and 778 ± 168 nmol⋅L(-1) O2 for nitrite oxidation assuming one-component Michaelis-Menten kinetics. Nitrite oxidation rates, however, were better described with a two-component Michaelis-Menten model, indicating a high-affinity component with a Km of just a few nanomolar. As the communities of ammonium and nitrite oxidizers were similar to other OMZs, these kinetics should apply across OMZ systems. The high O2 affinities imply that ammonium and nitrite oxidation can occur within the OMZ core whenever O2 is supplied, for example, by episodic intrusions. These processes therefore compete with anammox and denitrification for ammonium and nitrite, thereby exerting an important control over nitrogen loss.

  9. Modeling networks of coupled enzymatic reactions using the total quasi-steady state approximation.

    Directory of Open Access Journals (Sweden)

    Andrea Ciliberto

    2007-03-01

    Full Text Available In metabolic networks, metabolites are usually present in great excess over the enzymes that catalyze their interconversion, and describing the rates of these reactions by using the Michaelis-Menten rate law is perfectly valid. This rate law assumes that the concentration of enzyme-substrate complex (C is much less than the free substrate concentration (S0. However, in protein interaction networks, the enzymes and substrates are all proteins in comparable concentrations, and neglecting C with respect to S0 is not valid. Borghans, DeBoer, and Segel developed an alternative description of enzyme kinetics that is valid when C is comparable to S0. We extend this description, which Borghans et al. call the total quasi-steady state approximation, to networks of coupled enzymatic reactions. First, we analyze an isolated Goldbeter-Koshland switch when enzymes and substrates are present in comparable concentrations. Then, on the basis of a real example of the molecular network governing cell cycle progression, we couple two and three Goldbeter-Koshland switches together to study the effects of feedback in networks of protein kinases and phosphatases. Our analysis shows that the total quasi-steady state approximation provides an excellent kinetic formalism for protein interaction networks, because (1 it unveils the modular structure of the enzymatic reactions, (2 it suggests a simple algorithm to formulate correct kinetic equations, and (3 contrary to classical Michaelis-Menten kinetics, it succeeds in faithfully reproducing the dynamics of the network both qualitatively and quantitatively.

  10. Phosphotyrosine as a substrate of acid and alkaline phosphatases.

    Science.gov (United States)

    Apostoł, I; Kuciel, R; Wasylewska, E; Ostrowski, W S

    1985-01-01

    A new spectrophotometric method for following dephosphorylation of phosphotyrosine has been described. The absorption spectra of phosphotyrosine and tyrosine were plotted over the pH range from 3 to 9. The change in absorbance accompanying the conversion of phosphotyrosine to tyrosine was the greatest at 286 nm. The difference absorption coefficients were calculated for several pH values. Dephosphorylation of phosphotyrosine by acid phosphatases from human prostate gland, from wheat germ and potatoes obeys the Michaelis-Menten equation, whereas alkaline phosphatases calf intestine and E. coli are inhibited by excess of substrate.

  11. A kinetic model for the penicillin biosynthetic pathway in

    DEFF Research Database (Denmark)

    Nielsen, Jens; Jørgensen, Henrik

    1996-01-01

    A kinetic model for the first two steps in the penicillin biosynthetic pathway, i.e. the ACV synthetase (ACVS) and the isopenicillin N synthetase (IPNS) is proposed. The model is based on Michaelis-Menten type kinetics with non-competitive inhibition of the ACVS by ACV, and competitive inhibition...... of the IPNS by glutathione. The model predicted flux through the pathway corresponds well with the measured rate of penicillin biosynthesis. From the kinetic model the elasticity coefficients and the flux control coefficients are calculated throughout a fed-batch cultivation, and it is found...

  12. AIR POLLUTION FROM ANIMAL AND MUNICIPAL WASTEWATER: ASSESSMENT OF PRODUCTION AND RELEASE OF NOXIOUS GASES

    DEFF Research Database (Denmark)

    Dai, Xiaorong

    from animal manure (mixture of urine and feces) by hydrolysis of urinary urea catalyzed by microbial urease present in feces. To better understand the enzymatic process of ammonia formation in manure, experiments based on Michaelis-Menten kinetics were conducted to obtain accurate estimates...... of the kinetic parameters of urease activity of feces and manure from pig and cattle, and to investigate the effects of pH on animal fecal urease by individual ammonium generation rate determination at five pH levels. Investigating the gas production and release mechanisms is important not only for estimating...... characteristics of different types of wastes (e.g., the total nitrogen, total ammoniacal nitrogen, dry matter, and pH) had great influence on the releases of NH3, CO2, H2S, and SO2. The investigation of kinetic parameter showed that the maximum urease activity for pig feces is at around pH 7, while...

  13. Synthesis, Surface Modification and Optical Properties of Thioglycolic Acid-Capped ZnS Quantum Dots for Starch Recognition at Ultralow Concentration

    Science.gov (United States)

    Tayebi, Mahnoush; Tavakkoli Yaraki, Mohammad; Ahmadieh, Mahnaz; Mogharei, Azadeh; Tahriri, Mohammadreza; Vashaee, Daryoosh; Tayebi, Lobat

    2016-11-01

    In this research, water-soluble thioglycolic acid-capped ZnS quantum dots (QDs) are synthesized by the chemical precipitation method. The prepared QDs are characterized using x-ray diffraction and transmission electron microscopy. Results revealed that ZnS QDs have a 2.73 nm crystallite size, cubic zinc blende structure, and spherical morphology with a diameter less than 10 nm. Photoluminescence (PL) spectroscopy is performed to determine the presence of low concentrations of starch. Four emission peaks are observed at 348 nm, 387 nm, 422 nm, and 486 nm and their intensities are quenched by increasing concentration of starch. PL intensity variations in the studied concentrations range (0-100 ppm) are best described by a Michaelis-Menten model. The Michaelis constant ( K m) for immobilized α-amylase in this system is about 101.07 ppm. This implies a great tendency for the enzyme to hydrolyze the starch as substrate. Finally, the limit of detection is found to be about 6.64 ppm.

  14. Is oral absorption of vigabatrin carrier-mediated?

    DEFF Research Database (Denmark)

    Nøhr, M. K.; Juul, R. V.; Thale, Z. I.

    2015-01-01

    by mechanistic non-linear mixed effects modelling, evaluating PAT1-ligands as covariates on the PK parameters with a full covariate modelling approach. The oral absorption of vigabatrin was adequately described by a Michaelis-Menten type saturable absorption. Using a Michaelis constant of 32.8 mM, the model......-mediated and if the proton-coupled amino acid transporter 1 (PAT1) was involved in the absorption processes. Vigabatrin (0.3-300 mg/kg) was administered orally or intravenously to Sprague Dawley rats in the absence or presence of PAT1-ligands l-proline, l-tryptophan or sarcosine. The PK profiles of vigabatrin were described...... estimated a maximal oral absorption rate (Vmax) of 64.6 mmol/min and dose-dependent bioavailability with a maximum of 60.9%. Bioavailability was 58.5-60.8% at 0.3-30 mg/kg doses, but decreased to 46.8% at 300 mg/kg. Changes in oral vigabatrin PK after co-administration with PAT1-ligands was explained...

  15. Sufficient conditions for optimality for a mathematical model of drug treatment with pharmacodynamics

    Directory of Open Access Journals (Sweden)

    Maciej Leszczyński

    2017-01-01

    Full Text Available We consider an optimal control problem for a general mathematical model of drug treatment with a single agent. The control represents the concentration of the agent and its effect (pharmacodynamics is modelled by a Hill function (i.e., Michaelis-Menten type kinetics. The aim is to minimize a cost functional consisting of a weighted average related to the state of the system (both at the end and during a fixed therapy horizon and to the total amount of drugs given. The latter is an indirect measure for the side effects of treatment. It is shown that optimal controls are continuous functions of time that change between full or no dose segments with connecting pieces that take values in the interior of the control set. Sufficient conditions for the strong local optimality of an extremal controlled trajectory in terms of the existence of a solution to a piecewise defined Riccati differential equation are given.

  16. Hidrólisis enzimática de residuos de la cosecha de caña de azúcar

    Directory of Open Access Journals (Sweden)

    Jairo G. Salcedo Mendoza

    2012-01-01

    Full Text Available Título en inglés: Hydrolysis Enzymatic of crop residues sugar cane Resumen: En esta investigación, se hidrolizó un sustrato deslignificado proveniente de residuos de la cosecha caña de azúcar  (hojas y cogollos usando un  preparado enzimático con 27.53 unidades de papel filtro (FPU, obtenido a partir de enzimas comerciales. La hidrólisis se llevó a cabo a un pH de 4.2 y una temperatura de 50 oC. Fueron analizados  modelos de inhibición por sustrato, glucosa e inhibición total por producto. Los resultados mostraron que los modelos que mejor se ajustan a los datos experimentales, son los modelos de inhibición competitiva por glucosa, con una constante de Michaelis (Km de 20.37 g/L,  velocidad máxima (Vmax 39 g/L h y una constante de inhibición  (ki de 0.442. En el caso que las relaciones  enzima – Sustrato (E/S sean mayores  de 0.5, se puede aplicar el modelo cinético de Michaelis-Menten. Palabras clave: Inhibición; modelos cinéticos; hojas y cogollos; coctel de enzimas Abstract: In this research, a delignified substrate from crops residues sugar cane residues (leaves and top cane was hydrolyzed using an enzyme preparation with 27.53 FPU. This enzyme was obtained from trade. Hydrolysis was carried out to pH of 4.2 and a temperature of 50 oC. Models of inhibition models substrate, glucose and total inhibition product was analyzed. The results showed that models that best fit the data experimental was the models competitive glucose inhibition (Km= 20.37, Vmax=39 and ki= 0.442. In the event that E/S is above 0.5, can applied kinetic models of MichaelisMenten. Keywords: Inhibition; kinetic models; leaves and tops cane; enzyme cocktail.

  17. KINETIKA FERMENTASI SELULOSA MURNI OLEH Trichoderma reesi QM 9414 MENJADI GLUKOSA DAN PENERAPANNYA PADA JERAMI PADI BEBAS LIGNIN [Kinetics of Pure Cellulose Fermentation by Trichoderma Reesei QM 9414 to Glucose and Its Application of on Lignin Free Rice Straw

    Directory of Open Access Journals (Sweden)

    M Iyan Sofyan

    2004-12-01

    Full Text Available The objectives of this research were: 1 to determine aeration rate and substrate concentration of pure cellulose to produce maximum glucose by Trichoderma reesei QM 9414 at 30 oC, and agitation 150 rpm; 2 to study the kinetics of pure cellulose fermentation by Trichoderma reesei QM 9414 to glucose and its implication upon fermentation of the lignin free rice straw. The experiment was arranged in factorial randomized complete design in three times replication. Treatments consisted of three levels of aeration (1,00 vvm; 1,5 vvm; 2,0 vvm and three levels of substrate concentration (0,75 ; 1,00 ; 1,25 % w/v. The results showed that at the exponential phase the average specific growth of Trichoderma reesei QM 9414 was 0,05374 hour-1, the maximum glucose product concentration of pure cellulose was 0.1644 gL-1,and the oxygen transfer was 0,0328 mg L-1 hour-1. According to t-test, the kinetics of pure cellulose fermentation model just the same as the lignin free rice straw fermentation.The enzymes produced by Trichoderma reesei QM 9414 in pure cellulose fermentation media followed the Michaelis-Menten model. The enzyme kinetic parameters were the maximum growth rate was 37x10-3 hour-1 and Michaelis-Menten constant was ½ maximum μ =17,5x10-3 hour-1. The volumetric oxygen transfer (KLa using rice straw was 0,0337 mg.hour-1. The value of KLa could be used for conversion from bioreactor at laboratory scale to commercial scale design.

  18. A highly efficient nano-cluster artificial peroxidase and its direct electrochemistry on a nano complex modified glassy carbon electrode.

    Science.gov (United States)

    Hong, Jun; Wang, Wei; Huang, Kun; Yang, Wei-Yun; Zhao, Ying-Xue; Xiao, Bao-Lin; Gao, Yun-Fei; Moosavi-Movahedi, Zainab; Ghourchian, Hedayatollah; Moosavi-Movahedi, Ali Akbar

    2012-01-01

    A nano-cluster with highly efficient peroxide activity was constructed based on nafion (NF) and cytochrome c (Cyt c). UV-Vis spectrometry and transmission electron microscopy (TEM) methods were utilized for characterization of the nano-structured enzyme or artificial peroxidase (AP). The nano-cluster was composed of a Chain-Ball structure, with an average ball size of about 40 nm. The Michaelis-Menten (K(m)) and catalytic rate (k(cat)) constants of the AP were determined to be 2.5 ± 0.4 µM and 0.069 ± 0.001 s(-1), respectively, in 50 mM PBS at pH 7.0. The catalytic efficiency of the AP was evaluated to be 0.028 ± 0.005 µM(-1) s(-1), which was 39 ± 5% as efficient as the native horseradish peroxidase (HRP). The AP was also immobilized on a functional multi-wall carbon nanotube (MWNCTs)-gold colloid nanoparticles (AuNPs) nano-complex modified glassy carbon (GC) electrode. The cyclic voltammetry of AP on the nano complex modified GC electrode showed a pair of well-defined redox peaks with a formal potential (E°') of -45 ± 2 mV (vs. Ag/AgCl) at a scan rate of 0.05 V/s. The heterogeneous electron transfer rate constant (k(s)) was evaluated to be 0.65 s(-1). The surface concentration of electroactive AP on GC electrode (Γ) was 7 × 10(-10) mol cm(-2). The apparent Michaelis-Menten constant (K(m)(app)) was 0.23 nM.

  19. Solvent 1H/2H isotopic effects in the reaction of the L-Tyrosine oxidation catalyzed by Tyrosinase

    International Nuclear Information System (INIS)

    Kozlowska, M.; Kanska, M.

    2006-01-01

    Tyrosinase is well known catalyst in the oxidation of L-Tyrosine to L-DOPA and following oxidation of L-DOPA to dopachinone. The aim of communication is to present the results of studies on the solvent isotopic effects (SIE) in the above reactions for the 1 H/ 2 H in the 3',5' and 2',6' substituted tyrosine. Obtained dependence of the reaction rate on the substrate concentration were applied for optimization of the kinetic parameters, k cat and k cat /K m , in the Michaelis-Menten equation. As a result - better understanding of the L-DOPA creation can be achieved

  20. Glucose biosensing using glassy carbon electrode modified with polyhydroxy-C60, glucose oxidase and ionic-liquid.

    Science.gov (United States)

    Yang, Tian; Yang, Xiao-Lu; Zhang, Yu-Shuai; Xiao, BaoLin; Hong, Jun

    2014-01-01

    Direct electrochemistry of glucose oxidase (GOD) was achieved when an ionic liquid/GOD-Polyhydroxy-C60 functional membrane was confined on a glassy carbon electrode (GCE). The cyclic voltammograms (CVs) of the modified GCE showed a pair of redox peaks with a formal potential (E°') of - 329 ± 2 mV. The heterogeneous electron transfer constant (k(s)) was 1.43 s-1. The modified GCE response to glucose was linear in the range from 0.02 to 2.0 mM. The detection limit was 1 μM. The apparent Michaelis-Menten constant (K(m)(app)) was 1.45 mM.

  1. Estudio de la cinética de la hidrólisis enzimática del bagazo pretratado

    OpenAIRE

    Albernas Carvajal, Yailet; Corsano, Gabriela; Mesa Garriga, Leyanis; Santos Herrero, Ronaldo; Gonzalez Suarez, Erenio

    2016-01-01

    En el estudio se determinan los parámetros fundamentales de la cinética enzimática a partir de un modelo cinético pseudo-homogéneo de Michaelis Menten, previamente estudiado con otros materiales lignocelulósicos, para determinar la velocidad de producción de Azúcares Reductores Totales (ART) en el tiempo. Para ello se emplearon los resultados obtenidos a nivel de laboratorio, en el cual se llevó a cabo la hidrólisis enzimática del bagazo previamente pretratado de forma ácida y básica. Se empl...

  2. Estudo da dissolução oxidativa microbiológica de uma complexa amostra mineral contendo pirita (FeS2, Pirrotita (Fe1-xS e Molibdenita (MoS2 Microbiological oxidative dissolution of a complex mineral sample containing pyrite (FeS2, pyrrotite (Fe1-xS and molybdenite (MoS2

    Directory of Open Access Journals (Sweden)

    Wilmo E. Francisco Jr

    2007-10-01

    Full Text Available This work aims to study the oxidation of a complex molybdenite mineral which contains pyrite and pyrrotite, by Acidithiobacillus ferrooxidans. This study was performed by respirometric essays and bioleaching in shake flasks. Respirometric essays yielded the kinetics of mineral oxidation. The findings showed that sulfide oxidation followed classical Michaelis-Menten kinetics. Bioleaching in shake flasks allowed evaluation of chemical and mineralogical changes resulting from sulfide oxidation. The results demonstrated that pyrrotite and pyrite were completely oxidized in A. ferrooxidans cultures whereas molybdenite was not consumed. These data indicated that molybdenite was the most recalcitrant sulfide in the sample.

  3. Microbiological oxidative dissolution of a complex mineral sample containing pyrite (FeS2), pyrrotite (Fe1-xS) and molybdenite (MoS2)

    International Nuclear Information System (INIS)

    Francisco Junior, Wilmo E.; Bevilaqua, Denise; Garcia Junior, Oswaldo

    2007-01-01

    This work aims to study the oxidation of a complex molybdenite mineral which contains pyrite and pyrrotite, by Acidithiobacillus ferroxidans. This study was performed by respirometric essays and bioleaching in shake flasks. Respirometric essays yielded the kinetics of mineral oxidation. The findings showed that sulfide oxidation followed classical Michaelis-Menten kinetics. Bioleaching in shake flasks allowed evaluation of chemical and mineralogical changes resulting from sulfide oxidation. The results demonstrated that pyrrotite and pyrite were completely oxidized in A. ferrooxidans cultures whereas molybdenite was not consumed. These data indicated that molybdenite was the most recalcitrant sulfide in the sample. (author)

  4. Covalent immobilization of invertase on PAMAM-dendrimer modified superparamagnetic iron oxide nanoparticles

    International Nuclear Information System (INIS)

    Uzun, K.; Cevik, E.; Senel, M.; Soezeri, H.; Baykal, A.; Abasiyanik, M. F.; Toprak, M. S.

    2010-01-01

    In this study, polyamidoamine (PAMAM) dendrimer was synthesized on the surface of superparamagnetite nanoparticles to enhance invertase immobilization. The amount of immobilized enzyme on the surface-hyperbranched magnetite nanoparticle was up to 2.5 times (i.e., 250%) as much as that of magnetite nanoparticle modified with only amino silane. Maximum reaction rate (V max ) and Michaelis-Menten constant (K m ) were determined for the free and immobilized enzymes. Various characteristics of immobilized invertase such as; the temperature activity, thermal stability, operational stability, and storage stability were evaluated and results revealed that stability of the enzyme is improved upon immobilization.

  5. Microbiological oxidative dissolution of a complex mineral sample containing pyrite (FeS{sub 2}), pyrrotite (Fe{sub 1-x}S) and molybdenite (MoS{sub 2}); Estudo da dissolucao oxidativa microbiologica de uma complexa amostra mineral contendo pirita (FeS{sub 2}), Pirrotita (Fe{sub 1-x}S) e Molibdenita (MoS{sub 2})

    Energy Technology Data Exchange (ETDEWEB)

    Francisco Junior, Wilmo E.; Bevilaqua, Denise; Garcia Junior, Oswaldo [UNESP, Araraquara, SP (Brazil). Inst. de Quimica. Dept. de Bioquimica e Tecnologia Quimica]. E-mail: wilmojr@bol.com.br

    2007-09-15

    This work aims to study the oxidation of a complex molybdenite mineral which contains pyrite and pyrrotite, by Acidithiobacillus ferroxidans. This study was performed by respirometric essays and bioleaching in shake flasks. Respirometric essays yielded the kinetics of mineral oxidation. The findings showed that sulfide oxidation followed classical Michaelis-Menten kinetics. Bioleaching in shake flasks allowed evaluation of chemical and mineralogical changes resulting from sulfide oxidation. The results demonstrated that pyrrotite and pyrite were completely oxidized in A. ferrooxidans cultures whereas molybdenite was not consumed. These data indicated that molybdenite was the most recalcitrant sulfide in the sample. (author)

  6. Kinetics of alcoholic fermentation during the culturing of bakers' yeast

    Energy Technology Data Exchange (ETDEWEB)

    Franz, B

    1961-01-01

    A synthesis was made of the effects of various factors on the rate of fermentation by Saccharomyces cerevisiae. The rate obeyed the Michaelis-Menten equation, was independent of the concentration of yeast, was maximal at 20/sup 0/ (0.61 ml ethanol/g dry yeast/h), was not significantly affected between pH 6.5 and 3.0 but declined at 3.0, was inhibited by ethanol at a rate proportional to the concentration squared (at ethanol = 12 volume %, the fermentation rate was practically zero), and was enhanced by the addition of phosphorus when a P-poor yeast was employed.

  7. Monitoring the enzymatic conversion of urea to ammonium by conventional or microchip capillary electrophoresis with contactless conductivity detection.

    Science.gov (United States)

    Schuchert-Shi, Aiping; Hauser, Peter C

    2008-05-15

    Capillary electrophoresis with contactless conductivity detection was used to directly quantify the ammonium produced in the enzymatic conversion of urea with urease. This allowed the characterization of the reaction without having to use more elaborate indirect optical methods for quantification. The maximum rate of reaction, V(max), was determined as 5.1 mmol x mL(-1) x min(-1), and the Michaelis-Menten constant, K(m), was determined as 16 mM. Furthermore, the method was successfully applied to the determination of urea in clinical samples of human blood by using a conventional capillary and a microchip device.

  8. Solution of non-steady-state substrate concentration in the action of biosensor response at mixed enzyme kinetics

    Science.gov (United States)

    Senthamarai, R.; Jana Ranjani, R.

    2018-04-01

    In this paper, a mathematical model of an amperometric biosensor at mixed enzyme kinetics and diffusion limitation in the case of substrate inhibition has been developed. The model is based on time dependent reaction diffusion equation containing a non -linear term related to non -Michaelis - Menten kinetics of the enzymatic reaction. Solution for the concentration of the substrate has been derived for all values of parameters using the homotopy perturbation method. All the approximate analytic expressions of substrate concentration are compared with simulation results using Scilab/Matlab program. Finally, we have given a satisfactory agreement between them.

  9. Estudio de la cinética de la hidrólisis enzimática del bagazo pretratado

    OpenAIRE

    Albernas-Carvajal, Yailet; Corsano, Gabriela; Mesa Garriga, Layanis; Santos Herrero, Ronaldo; González Suárez, Erenio

    2015-01-01

    En el estudio se determinan los parámetros fundamentales de la cinética enzimática a partir de un modelo cinético pseudo-homogéneo de Michaelis Menten, previamente estudiado con otros materiales lignocelulósicos, para determinar la velocidad de producción de Azúcares Reductores Totales (ART) en el tiempo. Para ello se emplearon los resultados obtenidos a nivel de laboratorio, en el cual se llevó a cabo la hidrólisis enzimática del bagazo previamente pretratado de forma ácida y básica. Se empl...

  10. Emergence of a code in the polymerization of amino acids along RNA templates.

    Directory of Open Access Journals (Sweden)

    Jean Lehmann

    2009-06-01

    Full Text Available The origin of the genetic code in the context of an RNA world is a major problem in the field of biophysical chemistry. In this paper, we describe how the polymerization of amino acids along RNA templates can be affected by the properties of both molecules. Considering a system without enzymes, in which the tRNAs (the translation adaptors are not loaded selectively with amino acids, we show that an elementary translation governed by a Michaelis-Menten type of kinetics can follow different polymerization regimes: random polymerization, homopolymerization and coded polymerization. The regime under which the system is running is set by the relative concentrations of the amino acids and the kinetic constants involved. We point out that the coding regime can naturally occur under prebiotic conditions. It generates partially coded proteins through a mechanism which is remarkably robust against non-specific interactions (mismatches between the adaptors and the RNA template. Features of the genetic code support the existence of this early translation system.

  11. Modeling of an immobilized lipase tubular reactor for the production of glycerol and fatty acids from oils; Modelado de un reactor tubular de lipasas inmovilizadas para la produccion de glicerol y acidos grasos a partir de aceites

    Energy Technology Data Exchange (ETDEWEB)

    Oddone, S.; Grasselli, M.; Cuellas, A.

    2010-07-01

    Advances in the design of a bioreactor in the fats and oils industry have permitted the hydrolysis of triglycerides in mild conditions and improved productivity while avoiding the formation of unwanted byproducts. The present work develops a mathematical model that describes the hydrolytic activity of a tubular reactor with immobilized lipases for the production of glycerol and fatty acids from the oil trade. Runge Kuttas numerical method of high order has been applied, considering that there is no accumulation of the substratum in the surface of the membrane, where the enzyme is. At the same time, different equations based on the kinetic model of Michaelis Mentens and the Ping-Pong bi-bi mechanism were examined. Experimental data in discontinuous systems are the basis for the development of the quantitative mathematical model that was used to simulate the process computationally. The obtained results allow for optimizing both the operative variables and the economic aspects of industrial processes. (Author)

  12. Kinetics of Oxidation of Metochlopramide withChloramine-T in HClO4 Medium

    Directory of Open Access Journals (Sweden)

    K. M. Meenakshi

    2009-01-01

    Full Text Available The kinetics of oxidation of metochlopramide hydrochloride (MCP with sodium N-chloro p-toluenesulfonamide (CAT in perchloric acid solution has been studied at 313K. The reaction rate shows a first order dependence on [CAT], fractional order on [MCP] and inverse fractional order on [H+]. There is a negative effect of dielectric constant of the solvent. The addition of the reduction product of CAT has no significant effect on the rate. The rate remained unchanged with the variation in the ionic strength of the medium. The reaction fails to induce the polymerization of acrylonitrile. Thermodynamic parameters have been computed by Arrhenius plot. The stoichiometry of the reaction was found to be 1:2 and oxidation products were identified. The Michaelis-Menten type of kinetics has been proposed. CH3C6H4SO2NHCl have been assumed to be the reactive oxidizing species. Thermodynamic parameters were computed by studying reactions at different temperatures. A mechanism consistent with observed kinetics is proposed.

  13. Mechanical property characterization of polymeric composites reinforced by continuous microfibers

    Science.gov (United States)

    Zubayar, Ali

    Innumerable experimental works have been conducted to study the effect of polymerization on the potential properties of the composites. Experimental techniques are employed to understand the effects of various fibers, their volume fractions and matrix properties in polymer composites. However, these experiments require fabrication of various composites which are time consuming and cost prohibitive. Advances in computational micromechanics allow us to study the various polymer based composites by using finite element simulations. The mechanical properties of continuous fiber composite strands are directional. In traditional continuous fiber laminated composites, all fibers lie in the same plane. This provides very desirable increases in the in-plane mechanical properties, but little in the transverse mechanical properties. The effect of different fiber/matrix combinations with various orientations is also available. Overall mechanical properties of different micro continuous fiber reinforced composites with orthogonal geometry are still unavailable in the contemporary research field. In this research, the mechanical properties of advanced polymeric composite reinforced by continuous micro fiber will be characterized based on analytical investigation and FE computational modeling. Initially, we have chosen IM7/PEEK, Carbon Fiber/Nylon 6, and Carbon Fiber/Epoxy as three different case study materials for analysis. To obtain the equivalent properties of the micro-hetero structures, a concept of micro-scale representative volume elements (RVEs) is introduced. Five types of micro scale RVEs (3 square and 2 hexagonal) containing a continuous micro fiber in the polymer matrix were designed. Uniaxial tensile, lateral expansion and transverse shear tests on each RVE were designed and conducted by the finite element computer modeling software ANSYS. The formulae based on elasticity theory were derived for extracting the equivalent mechanical properties (Young's moduli, shear

  14. Design of Electro-Mechanical Dual-Acting Pulley Continuously Variable Transmission

    Directory of Open Access Journals (Sweden)

    K.B. Tawi

    2015-06-01

    Full Text Available Pulley-based continuously variable transmission (CVT with a metal pushing V-belt is fast becoming the preferred choice for global carmakers due to its potential particularly in terms of fuel efficiency thanks to its continuous and wide ratio range. Nevertheless, the existing CVTs still face the issues of high power consumption from the engine because of the application of an electro-hydro-mechanical (EHM actuation system for its ratio changing process and clamping force mechanism. To address this issue, researchers from Universiti Teknologi Malaysia have successfully developed the prototype of an electro-mechanical dual-acting pulley continuously variable transmission (EMDAP CVT for automotive applications. The prototype of EMDAP CVT is developed for a maximum input torque of 160 Nm with the application of a metal pushing V-belt. The results from the testing prove that the prototype can vary its ratio from 2.8 to 0.6 and no continuous power is required to maintain a constant CVT ratio. These results suggest that the prototype is workable and future testing in a real car is possible.

  15. 'In-Crystallo' Capture of a Michaelis Complex And Product Binding Modes of a Bacterial Phosphotriesterase

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, C.J.; Foo, J.-L.; Kim, H.-K.; Carr, P.D.; Liu, J.-W.; Salem, G.; Ollis, D.L.

    2009-05-18

    The mechanism by which the binuclear metallophosphotriesterases (PTEs, E.C. 3.1.8.1) catalyse substrate hydrolysis has been extensively studied. The {mu}-hydroxo bridge between the metal ions has been proposed to be the initiating nucleophile in the hydrolytic reaction. In contrast, analysis of some biomimetic systems has indicated that {mu}-hydroxo bridges are often not themselves nucleophiles, but act as general bases for freely exchangeable nucleophilic water molecules. Herein, we present crystallographic analyses of a bacterial PTE from Agrobacterium radiobacter, OpdA, capturing the enzyme-substrate complex during hydrolysis. This model of the Michaelis complex suggests the alignment of the substrate will favor attack from a solvent molecule terminally coordinated to the {alpha}-metal ion. The bridging of both metal ions by the product, without disruption of the {mu}-hydroxo bridge, is also consistent with nucleophilic attack occurring from the terminal position. When phosphodiesters are soaked into crystals of OpdA, they coordinate bidentately to the {beta}-metal ion, displacing the {mu}-hydroxo bridge. Thus, alternative product-binding modes exist for the PTEs, and it is the bridging mode that appears to result from phosphotriester hydrolysis. Kinetic analysis of the PTE and promiscuous phosphodiesterase activities confirms that the presence of a {mu}-hydroxo bridge during phosphotriester hydrolysis is correlated with a lower pK{sub a} for the nucleophile, consistent with a general base function during catalysis.

  16. In vitro modulation of cytochrome P450 reductase supported indoleamine 2,3-dioxygenase activity by allosteric effectors cytochrome b(5) and methylene blue.

    Science.gov (United States)

    Pearson, Josh T; Siu, Sophia; Meininger, David P; Wienkers, Larry C; Rock, Dan A

    2010-03-30

    Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of l-Trp follows substrate inhibition kinetics (k(cat) = 0.89 +/- 0.04 s(-1), K(m) = 0.72 +/- 0.15 microM, and K(i) = 9.4 +/- 2.0 microM). Addition of cytochrome b(5) to CPR-supported l-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (k(cat) = 1.7 +/- 0.3 s(-1), K(m) = 1.5 +/- 0.9 microM, and K(i) = 1.9 +/- 0.3). CPR-supported d-Trp oxidations (+/-cytochrome b(5)) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of d-Trp turnover and modulation of l-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b(5).

  17. Phenytoin pharmacokinetics in critically ill trauma patients.

    Science.gov (United States)

    Boucher, B A; Rodman, J H; Jaresko, G S; Rasmussen, S N; Watridge, C B; Fabian, T C

    1988-12-01

    Preliminary data have suggested that phenytoin systemic clearance may increase during initial therapy in critically ill patients. The objectives for this study were to model the time-variant phenytoin clearance and evaluate concomitant changes in protein binding and urinary metabolite elimination. Phenytoin was given as an intravenous loading dose of 15 mg/kg followed by an initial maintenance dose of 6 mg/kg/day in 10 adult critically ill trauma patients. Phenytoin bound and unbound plasma concentrations were determined in 10 patients and urinary excretion of the metabolite p-hydroxyphenyl phenylhydantoin (p-HPPH) was measured in seven patients for 7 to 14 days. A Michaelis-Menten one-compartment model incorporating a time-variant maximal velocity (Vmax) was sufficient to describe the data and superior to a conventional time-invariant Michaelis-Menten model. Vmax for the time-variant model was defined as V'max + Vmax delta (1 - e(-kindt)). Vmax infinity is the value for Vmax when t is large. The median values (ranges) for the parameters were Km = 4.8 (2.6 to 20) mg/L, Vmax infinity = 1348 (372 to 4741) mg/day, and kind = 0.0115 (0.0045 to 0.132) hr-1. Phenytoin free fraction increased in a majority of patients during the study period, with a binding ratio inversely related to albumin. Measured urinary p-HPPH data were consistent with the proposed model. A loading and constant maintenance dose of phenytoin frequently yielded a substantial, clinically significant fall in plasma concentrations with a pattern of apparently increasing clearance that may be a consequence of changes in protein binding, induction of metabolism, or the influence of stress on hepatic metabolic capacity.

  18. Substrate specificity and copper loading of the manganese-oxidizing multicopper oxidase Mnx from Bacillus sp. PL-12.

    Science.gov (United States)

    Butterfield, Cristina N; Tebo, Bradley M

    2017-02-22

    Manganese(ii) oxidation in the environment is thought to be driven by bacteria because enzymatic catalysis is many orders of magnitude faster than the abiotic processes. The heterologously purified Mn oxidase (Mnx) from marine Bacillus sp. PL-12 is made up of the multicopper oxidase (MCO) MnxG and two small Cu and heme-binding proteins of unknown function, MnxE and MnxF. Mnx binds Cu and oxidizes both Mn(ii) and Mn(iii), generating Mn(iv) oxide minerals that resemble those found on the Bacillus spore surface. Spectroscopic techniques have illuminated details about the metallo-cofactors of Mnx, but very little is known about their requirement for catalytic activity, and even less is known about the substrate specificity of Mnx. Here we quantify the canonical MCO Cu and persistent peripheral Cu bound to Mnx, and test Mnx oxidizing ability toward different substrates at varying pH. Mn(ii) appears to be the best substrate in terms of k cat , but its oxidation does not follow Michaelis-Menten kinetics, instead showing a sigmoidal cooperative behavior. Mnx also oxidizes Fe(ii) substrate, but in a Michaelis-Menten manner and with a decreased activity, as well as organic substrates. The reduced metals are more rapidly consumed than the larger organic substrates, suggesting the hypothesis that the Mnx substrate site is small and tuned for metal oxidation. Of biological relevance is the result that Mnx has the highest catalytic efficiency for Mn(ii) at the pH of sea water, especially when the protein is loaded with greater than the requisite four MCO copper atoms, suggesting that the protein has evolved specifically for Mn oxidation.

  19. Communication: Limitations of the stochastic quasi-steady-state approximation in open biochemical reaction networks

    Science.gov (United States)

    Thomas, Philipp; Straube, Arthur V.; Grima, Ramon

    2011-11-01

    It is commonly believed that, whenever timescale separation holds, the predictions of reduced chemical master equations obtained using the stochastic quasi-steady-state approximation are in very good agreement with the predictions of the full master equations. We use the linear noise approximation to obtain a simple formula for the relative error between the predictions of the two master equations for the Michaelis-Menten reaction with substrate input. The reduced approach is predicted to overestimate the variance of the substrate concentration fluctuations by as much as 30%. The theoretical results are validated by stochastic simulations using experimental parameter values for enzymes involved in proteolysis, gluconeogenesis, and fermentation.

  20. The repair-fixation model: general aspects and the influence of radiation quality

    International Nuclear Information System (INIS)

    Kiefer, J.; Loebrich, M.

    1992-01-01

    To explain the shape of cell survival curves after radiation action it is assumed that initial lesions are transient in nature and subject to repair or fixation. Since the underlying processes are controlled by enzymes, Michaelis-Menten kinetics are assumed. No qualitative differences between repair and fixation are postulated, the only differences being the kinetic parameters. This model yields a mathematical expression which is formally equivalent to the ''lethal-potentially-lethal'' (LPL) model. It is demonstrated that both mammalian as well as microbial survival data can be fitted. The inclusion of linear energy transfer (LET) effects is shown to be possible and is discussed qualitatively. (author)

  1. Oxidation study of the synthetic sulfides molybdenite (MoS2) and covellite (CuS) by acidithiobacillus ferrooxidants using respirometric experiments

    International Nuclear Information System (INIS)

    Francisco Junior, Wilmo E.; Universidade Estadual Paulista; Bevilaqua, Denise; Garcia Junior, Oswaldo

    2009-01-01

    This paper analyses the oxidation of covellite and molybdenite by Acidithiobacillus ferrooxidans strain LR using respirometric experiments. The results showed that both sulfides were oxidized by A. ferrooxidans, however, the covellite oxidation was much higher than molybdenite. Regarding the kinetic oxidation, the findings revealed that just molybdenite oxidation followed the classical Michaelis-Menten kinetic. It is probably associated with the pathway which these sulfides react to chemistry-bacterial attack, what is influenced by its electronic structures. Besides, experiments conducted in the presence of Fe 3+ did not indicate alterations in molybdenite oxidation. Thus, ferric ions seem not to be essential to the sulfide oxidations. (author)

  2. Oxidation study of the synthetic sulfides molybdenite (MoS{sub 2}) and covellite (CuS) by acidithiobacillus ferrooxidants using respirometric experiments; Estudo da oxidacao dos sulfetos sinteticos molibdenita (MoS2) e covelita (CuS) por Acidithiobacillus ferrooxidans via respirometria celular

    Energy Technology Data Exchange (ETDEWEB)

    Francisco Junior, Wilmo E. [Universidade Federal de Rondonia (UFRO), Porto Velho, RO (Brazil). Dept. de Quimica; Universidade Estadual Paulista (UNESP), Araraquara, SP (Brazil). Inst. de Quimica. Dept. de Bioquimica e Tecnologia Quimica], e-mail: wilmojr@bol.com.br; Bevilaqua, Denise; Garcia Junior, Oswaldo [Universidade Estadual Paulista (UNESP), Araraquara, SP (Brazil). Inst. de Quimica. Dept. de Bioquimica e Tecnologia Quimica

    2009-07-01

    This paper analyses the oxidation of covellite and molybdenite by Acidithiobacillus ferrooxidans strain LR using respirometric experiments. The results showed that both sulfides were oxidized by A. ferrooxidans, however, the covellite oxidation was much higher than molybdenite. Regarding the kinetic oxidation, the findings revealed that just molybdenite oxidation followed the classical Michaelis-Menten kinetic. It is probably associated with the pathway which these sulfides react to chemistry-bacterial attack, what is influenced by its electronic structures. Besides, experiments conducted in the presence of Fe{sup 3+} did not indicate alterations in molybdenite oxidation. Thus, ferric ions seem not to be essential to the sulfide oxidations. (author)

  3. Estudo da oxidação dos sulfetos sintéticos molibdenita (MoS2 e covelita (CuS por Acidithiobacillus ferrooxidans via respirometria celular Oxidation study of the synthetic sulfides molybdenite (MoS2 and covellite (CuS by Acidithiobacillus ferrooxidans using respirometric experiments

    Directory of Open Access Journals (Sweden)

    Wilmo E. Francisco Junior

    2009-01-01

    Full Text Available This paper analyses the oxidation of covellite and molybdenite by Acidithiobacillus ferrooxidans strain LR using respirometric experiments. The results showed that both sulfides were oxidized by A. ferrooxidans, however, the covellite oxidation was much higher than molybdenite. Regarding the kinetic oxidation, the findings revealed that just molybdenite oxidation followed the classical Michaelis-Menten kinetic. It is probably associated with the pathway which these sulfides react to chemistry-bacterial attack, what is influenced by its electronic structures. Besides, experiments conducted in the presence of Fe3+ did not indicate alterations in molybdenite oxidation. Thus, ferric ions seem not to be essential to the sulfide oxidations.

  4. Study on kinetics of glucose uptake by some species of plankton

    Science.gov (United States)

    Li, Wenquan; Wang, Xian; Zhang, Yaohua

    1993-03-01

    The rates of glucose uptake by some species of plankton were determined by3H-glucose tracer method. Experimental results indicated that the observed glucose uptake at natural seawater concentrations by Platymonas subcordiformis and Brachionus plicatilis was principally a metabolic process fitted with the Michaelis-Menten equation in the range of adaptive temperatures. Heterotrophic uptake by Platymonas subcordiformis was mainly dependent on diffusion at high glucose levels. The uptake by Brachionus plicatilis showed active transport even at high glucose levels, indicating its high heterotrophic activity. The uptake rate by Artemia salina was lower, and its V m/K ratio was lower than those of the other two species of plankton.

  5. Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

    Science.gov (United States)

    Guerra, Nelson P.; Pastrana Castro, Lorenzo

    2012-01-01

    The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, v max decreased significantly (P enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G]. PMID:22666116

  6. Kinetics of glucose transport in rat muscle

    DEFF Research Database (Denmark)

    Ploug, Thorkil; Galbo, Henrik; Vinten, Jørgen

    1987-01-01

    The effects of insulin and prior muscle contractions, respectively, on 3-O-methylglucose (3-O-MG) transport in skeletal muscle were studied in the perfused rat hindquarter. Initial rates of entry of 3-O-MG in red gastrocnemius, soleus, and white gastrocnemius muscles as a function of perfusate 3-O-MG...... concentration exhibited Michaelis-Menten kinetics. Uptake by simple diffusion could not be detected. The maximum 3-O-MG transport velocity (Vmax) was increased more by maximum isometric contractions (10- to 40-fold, depending on fiber type) than by insulin (20,000 microU/ml; 3- to 20-fold) in both red and white...

  7. Using a Mechanistic Reactive Transport Model to Represent Soil Organic Matter Dynamics and Climate Sensitivity

    Science.gov (United States)

    Guerry, N.; Riley, W. J.; Maggi, F.; Torn, M. S.; Kleber, M.

    2011-12-01

    The nature of long term Soil Organic Matter (SOM) dynamics is uncertain and the mechanisms involved are crudely represented in site, regional, and global models. Recent work challenging the paradigm that SOM is stabilized because of its sequential transformations to more intrinsically recalcitrant compounds motivated us to develop a mechanistic modeling framework that can be used to test hypotheses of SOM dynamics. We developed our C cycling model in TOUGHREACT, an established 3-dimensional reactive transport solver that accounts for multiple phases (aqueous, gaseous, sorbed), multiple species, advection and diffusion, and multiple microbial populations. Energy and mass exchange through the soil boundaries are accounted for via ground heat flux, rainfall, C sources (e.g., exudation, woody, leaf, root litter) and C losses (e.g., CO2 emissions and DOC deep percolation). SOM is categorized according to the various types of compounds commonly found in the above mentioned C sources and microbial byproducts, including poly- and monosaccharides, lignin, amino compounds, organic acids, nucleic acids, lipids, and phenols. Each of these compounds is accounted for by one or more representative species in the model. A reaction network was developed to describe the microbially-mediated processes and chemical interactions of these species, including depolymerization, microbial assimilation, respiration and deposition of byproducts, and incorporation of dead biomass into SOM stocks. Enzymatic reactions are characterized by Michaelis-Menten kinetics, with maximum reaction rates determined by the species' O/C ratio. Microbial activity is further regulated by soil moisture content, O2 availability, pH, and temperature. For the initial set of simulations, literature values were used to constrain microbial Monod parameters, Michaelis-Menten parameters, sorption parameters, physical protection, partitioning of microbial byproducts, and partitioning of litter inputs, although there is

  8. Preliminary Study on the Kinetics and Mechanism of the Oxidation of Naphthol Green B by Dichromate Ion in Aqueous Hydrochloric Acid Medium

    Directory of Open Access Journals (Sweden)

    Bako Myek

    2014-04-01

    Full Text Available The kinetics of the oxidation of naphthol green B (NGB3- by Cr2O7 2- has been studied in aqueous hydrochloric acid medium at an ionic strength, I = 0.50 mol dm-3 (NaCl, H+ =1.0  10-4 mol dm-3 (HCl and T = 25  1C. The redox reaction displayed a stoichiometry of 1:1 and obeys the rate law: -dNGB3-/dt = k2NGB3-]Cr2O7 2- . The second order rate constant increases with increase in acid concentration and in the ionic strength of reaction medium. The rates of reaction displayed a positive salt effect. Addition of acrylonitrile to a partially reacted mixture in the presence of excess methanol did not lead to gel formation. Added cations and anions inhibited the naphthol green B - Cr2O7 2- reaction. Results of the MichaelisMenten plot gave no evidence of intermediate complex formation during the course of the reaction. Based on the results obtained, the reaction is believed to proceed through the outersphere mechanistic pathway.

  9. Distinct timing mechanisms produce discrete and continuous movements.

    Directory of Open Access Journals (Sweden)

    Raoul Huys

    2008-04-01

    Full Text Available The differentiation of discrete and continuous movement is one of the pillars of motor behavior classification. Discrete movements have a definite beginning and end, whereas continuous movements do not have such discriminable end points. In the past decade there has been vigorous debate whether this classification implies different control processes. This debate up until the present has been empirically based. Here, we present an unambiguous non-empirical classification based on theorems in dynamical system theory that sets discrete and continuous movements apart. Through computational simulations of representative modes of each class and topological analysis of the flow in state space, we show that distinct control mechanisms underwrite discrete and fast rhythmic movements. In particular, we demonstrate that discrete movements require a time keeper while fast rhythmic movements do not. We validate our computational findings experimentally using a behavioral paradigm in which human participants performed finger flexion-extension movements at various movement paces and under different instructions. Our results demonstrate that the human motor system employs different timing control mechanisms (presumably via differential recruitment of neural subsystems to accomplish varying behavioral functions such as speed constraints.

  10. A Study on Pharmacokinetics of Bosentan with Systems Modeling, Part 2: Prospectively Predicting Systemic and Liver Exposure in Healthy Subjects.

    Science.gov (United States)

    Li, Rui; Kimoto, Emi; Niosi, Mark; Tess, David A; Lin, Jian; Tremaine, Larry M; Di, Li

    2018-04-01

    Predicting human pharmacokinetics of novel compounds is a critical step in drug discovery and clinical study design but continues to be a challenging task for hepatic transporter substrates, particularly in predicting their liver exposures. In this study, using bosentan as an example, we prospectively predicted systemic exposure and the (pseudo) steady-state unbound liver-to-unbound plasma ratio ( K p uu ) in healthy subjects using 1) a mechanistic approach solely based on in vitro hepatocyte assays and 2) an approach based on hepatic process rates from monkey in vivo data but Michaelis-Menten constants from in vitro data. Both methods reasonably match the observed human systemic time course data, but the second method leads to better prediction accuracy. In addition, the second method can predict a human K p uu value that is close to the value deduced using clinical data. We also generated rat and monkey liver K p uu values in terminal studies. However, these directly measured animal values are different from the deduced human value. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  11. "THE EVALUATION OF THE POSSIBLE EFFECT OF POSITIVE END EXPIRATORY PRESSURE (PEEP ON PHARMACOKINETICS OF PHENYTOIN IN PATIENTS WITH ACUTE BRAIN INJURY UNDER MECHANICAL VENTILATION."

    Directory of Open Access Journals (Sweden)

    "Elham Hadidi

    2005-04-01

    Full Text Available Positive ventilation has shown to have an influence on pharmacokinetic and disposition of some drugs.Beacause phenytoin with a narrow therapautic range, is the most commonly used drug for prophylaxis and treatment of early seizures after acute brain injuries, in the present study the effect of short term PEEP (5-10 cm H2O for at least 8 hours on phenytoin serum concentration and pharmacokinetic parameters such as Vmax and clearance in brain injured patients under mechanical ventilation was examined. Ten patients with moderate to severe acute brain injury who were placed on mechanical ventilation with an initial PEEP level of 0-5 cm H2O were included in the study. Patients received phenytoin loading dose of 15 mg/kg followed by a maintenance daily dose of 3-7 mg/kg initiated within 12 hours of loading dose. Sampels were taken on two different occasions before and after PEEP elevation. Total phenytoin serum concentrations were determined by HPLC method. A time invarient Michaelis-Menten pharmacokinetic model was used to calculate Vmax and clearance for each patient.Derrived variables were calculated as follows: Vmax, 3.5-6.8 and 3.7-8.2 mg/kg/day; Clearance, 0.1-0.7 and 0.1-1.2 l/kg/day (before and after PEEP elevation, respectively. Our data have shown a wide range of variability (2.6-32.5 mg/l in phenytoin serum concentrations. There were no statistically significant differences in the measured total concentrations (p=0.721 and calculated Vmax and clearance (p=0.285before and after PEEP elevation. Administration of fluid and inotropic agents, limitation in application of higher levels of PEEP and drug interactions, shall be considered as possible explanations for these findings.

  12. Comparison of dopamine kinetics in the larval Drosophila ventral nerve cord and protocerebrum with improved optogenetic stimulation.

    Science.gov (United States)

    Privman, Eve; Venton, B Jill

    2015-11-01

    Dopamine release and uptake have been studied in the Drosophila larval ventral nerve cord (VNC) using optogenetics to stimulate endogenous release. However, other areas of the central nervous system remain uncharacterized. Here, we compare dopamine release in the VNC and protocerebrum of larval Drosophila. Stimulations were performed with CsChrimson, a new, improved, red light-activated channelrhodopsin. In both regions, dopamine release was observed after only a single, 4 ms duration light pulse. Michaelis-Menten modeling was used to understand release and uptake parameters for dopamine. The amount of dopamine released ([DA]p ) on the first stimulation pulse is higher than the average [DA]p released from subsequent pulses. The initial and average amount of dopamine released per stimulation pulse is smaller in the protocerebrum than in the VNC. The average Vmax of 0.08 μM/s in the protocerebrum was significantly higher than the Vmax of 0.05 μM/s in the VNC. The average Km of 0.11 μM in the protocerebrum was not significantly different from the Km of 0.10 μM in the VNC. When the competitive dopamine transporter (DAT) inhibitor nisoxetine was applied, the Km increased significantly in both regions while Vmax stayed the same. This work demonstrates regional differences in dopamine release and uptake kinetics, indicating important variation in the amount of dopamine available for neurotransmission and neuromodulation. We use a new optogenetic tool, red light activated CsChrimson, to stimulate the release of dopamine in the ventral nerve cord and medial protocerebrum of the larval Drosophila central nervous system. We monitored extracellular dopamine by fast scan cyclic voltammetry and used Michaelis-Menten modeling to probe the regulation of extracellular dopamine, discovering important similarities and differences in these two regions. © 2015 International Society for Neurochemistry.

  13. Direct measurement of catalase activity in living cells and tissue biopsies

    International Nuclear Information System (INIS)

    Scaglione, Christine N.; Xu, Qijin; Ramanujan, V. Krishnan

    2016-01-01

    Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear

  14. Direct measurement of catalase activity in living cells and tissue biopsies

    Energy Technology Data Exchange (ETDEWEB)

    Scaglione, Christine N.; Xu, Qijin; Ramanujan, V. Krishnan, E-mail: Ramanujanv@csmc.edu

    2016-01-29

    Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear

  15. Characterization of the human cytochrome P450 enzymes involved in the metabolism of dihydrocodeine

    Science.gov (United States)

    Kirkwood, L. C.; Nation, R. L.; Somogyi, A. A.

    1997-01-01

    Aims Using human liver microsomes from donors of the CYP2D6 poor and extensive metabolizer genotypes, the role of individual cytochromes P-450 in the oxidative metabolism of dihydrocodeine was investigated. Methods The kinetics of formation of N- and O-demethylated metabolites, nordihydrocodeine and dihydromorphine, were determined using microsomes from six extensive and one poor metabolizer and the effects of chemical inhibitors selective for individual P-450 enzymes of the 1A, 2A, 2C, 2D, 2E and 3A families and of LKM1 (anti-CYP2D6) antibodies were studied. Results Nordihydrocodeine was the major metabolite in both poor and extensive metabolizers. Kinetic constants for N-demethylation derived from the single enzyme Michaelis-Menten model did not differ between the two groups. Troleandomycin and erythromycin selectively inhibited N-demethylation in both extensive and poor metabolizers. The CYP3A inducer, α-naphthoflavone, increased N-demethylation rates. The kinetics of formation of dihydromorphine in both groups were best described by a single enzyme Michaelis-Menten model although inhibition studies in extensive metabolizers suggested involvement of two enzymes with similar Km values. The kinetic constants for O-demethylation were significantly different in extensive and poor metabolizers. The extensive metabolizers had a mean intrinsic clearance to dihydromorphine more than ten times greater than the poor metabolizer. The CYP2D6 chemical inhibitors, quinidine and quinine, and LKM1 antibodies inhibited O-demethylation in extensive metabolizers; no effect was observed in microsomes from a poor metabolizer. Conclusions CYP2D6 is the major enzyme mediating O-demethylation of dihydrocodeine to dihydromorphine. In contrast, nordihydrocodeine formation is predominantly catalysed by CYP3A. PMID:9431830

  16. Efecto de la sacarosa en la producción de celulosa por Gluconacetobacter xylinus en cultivo estático

    Directory of Open Access Journals (Sweden)

    Rubén Jaramillo L.

    2012-08-01

    Full Text Available Objetivo. Determinar el efecto de sacarosa en la productividad de BC por Gluconacetobacter xylinus IFO 13693 en condición estática. Materiales y métodos. La síntesis de celulosa bacteriana (BC por Gluconacetobacter xylinus se llevo a cabo en un cultivo estático discontinuo a temperatura ambiente, en presencia de sacarosa como la principal fuente de carbono a concentraciones iniciales de 0.8 a 7.6 % (p/v. Las concentraciones remanentes de BC, sacarosa, glucosa y fructosa se determinaron cada semana. Para la cinética de la hidrólisis de la sacarosa y formación de celulosa y el coeficiente de rendimiento del producto se utilizo el software Microcal Origin 6.0®. Resultados. En la cuarta semana los valores de BC se encontraron entre 32.5 a 39.5 g/L para las diferentes concentraciones de sacarosa. La cinética para la hidrólisis de sacarosa se ajusta al modelo de Michaelis-Menten, con una Vmax de 0.0002 mol L-1 h-1 y Km de 0.018 M. La producción de BC se ajusta al modelo propuesto por Marx-Figini y Pion, con un valor de la pendiente (kc, entre 0.0018 y 0.0024 h-1 para las diferentes concentraciones iniciales de sacarosa. Los coeficientes de rendimiento tienen valores de 0.8 a 2.4 g de BC producida/g de sacarosa consumida. Conclusiones. La hidrólisis de sacarosa, el consumo de glucosa y fructosa se refleja en la síntesis de celulosa. La hidrólisis de sacarosa y la producción de BC se ajustan a los modelos de Michaelis-Menten y al propuesto por Marx-Figini y Pion, respectivamente. Finalmente, el rendimiento depende de la concentración de sacarosa.

  17. Hepatic and intestinal glucuronidation of mono(2-ethylhexyl) phthalate, an active metabolite of di(2-ethylhexyl) phthalate, in humans, dogs, rats, and mice: an in vitro analysis using microsomal fractions.

    Science.gov (United States)

    Hanioka, Nobumitsu; Isobe, Takashi; Kinashi, Yu; Tanaka-Kagawa, Toshiko; Jinno, Hideto

    2016-07-01

    Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP) and has endocrine-disrupting effects. MEHP is metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the hepatic and intestinal glucuronidation of MEHP in humans, dogs, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis-Menten model for humans and dogs, and the biphasic model for rats and mice. The K m and V max values of human liver microsomes were 110 µM and 5.8 nmol/min/mg protein, respectively. The kinetics of intestinal microsomes followed the biphasic model for humans, dogs, and mice, and the Michaelis-Menten model for rats. The K m and V max values of human intestinal microsomes were 5.6 µM and 0.40 nmol/min/mg protein, respectively, for the high-affinity phase, and 430 µM and 0.70 nmol/min/mg protein, respectively, for the low-affinity phase. The relative levels of V max estimated by Eadie-Hofstee plots were dogs (2.0) > mice (1.4) > rats (1.0) ≈ humans (1.0) for liver microsomes, and mice (8.5) > dogs (4.1) > rats (3.1) > humans (1.0) for intestinal microsomes. The percentages of the V max values of intestinal microsomes to liver microsomes were mice (120 %) > rats (57 %) > dogs (39 %) > humans (19 %). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward MEHP markedly differed among species, and imply that these species differences are strongly associated with the toxicity of DEHP.

  18. Primary Structure and Mechanical Properties of AlSi2 Alloy Continuous Ingots

    Directory of Open Access Journals (Sweden)

    Wróbel T.

    2017-06-01

    Full Text Available The paper presents the research results of horizontal continuous casting of ingots of aluminium alloy containing 2% wt. silicon (AlSi2. Together with the casting velocity (velocity of ingot movement we considered the influence of electromagnetic stirring in the area of the continuous casting mould on refinement of the ingot’s primary structure and their selected mechanical properties, i.e. tensile strength, yield strength, hardness and elongation. The effect of primary structure refinement and mechanical properties obtained by electromagnetic stirring was compared with refinement obtained by using traditional inoculation, which consists in introducing additives, i.e. Ti, B and Sr, to the metal bath. On the basis of the obtained results we confirmed that inoculation done by electromagnetic stirring in the range of the continuous casting mould guarantees improved mechanical properties and also decreases the negative influence of casting velocity, thus increasing the structure of AlSi2 continuous ingots.

  19. Threshold for extinction and survival in stochastic tumor immune system

    Science.gov (United States)

    Li, Dongxi; Cheng, Fangjuan

    2017-10-01

    This paper mainly investigates the stochastic character of tumor growth and extinction in the presence of immune response of a host organism. Firstly, the mathematical model describing the interaction and competition between the tumor cells and immune system is established based on the Michaelis-Menten enzyme kinetics. Then, the threshold conditions for extinction, weak persistence and stochastic persistence of tumor cells are derived by the rigorous theoretical proofs. Finally, stochastic simulation are taken to substantiate and illustrate the conclusion we have derived. The modeling results will be beneficial to understand to concept of immunoediting, and develop the cancer immunotherapy. Besides, our simple theoretical model can help to obtain new insight into the complexity of tumor growth.

  20. Kinetic characterisation of primer mismatches in allele-specific PCR: a quantitative assessment.

    Science.gov (United States)

    Waterfall, Christy M; Eisenthal, Robert; Cobb, Benjamin D

    2002-12-20

    A novel method of estimating the kinetic parameters of Taq DNA polymerase during rapid cycle PCR is presented. A model was constructed using a simplified sigmoid function to represent substrate accumulation during PCR in combination with the general equation describing high substrate inhibition for Michaelis-Menten enzymes. The PCR progress curve was viewed as a series of independent reactions where initial rates were accurately measured for each cycle. Kinetic parameters were obtained for allele-specific PCR (AS-PCR) amplification to examine the effect of mismatches on amplification. A high degree of correlation was obtained providing evidence of substrate inhibition as a major cause of the plateau phase that occurs in the later cycles of PCR.

  1. Cholesterol biosensor based on rf sputtered zinc oxide nanoporous thin film

    International Nuclear Information System (INIS)

    Singh, S. P.; Arya, Sunil K.; Pandey, Pratibha; Malhotra, B. D.; Saha, Shibu; Sreenivas, K.; Gupta, Vinay

    2007-01-01

    Cholesterol oxidase (ChOx) has been immobilized onto zinc oxide (ZnO) nanoporous thin films grown on gold surface. A preferred c-axis oriented ZnO thin film with porous surface morphology has been fabricated by rf sputtering under high pressure. Optical studies and cyclic voltammetric measurements show that the ChOx/ZnO/Au bioelectrode is sensitive to the detection of cholesterol in 25-400 mg/dl range. A relatively low value of enzyme's kinetic parameter (Michaelis-Menten constant) ∼2.1 mM indicates enhanced enzyme affinity of ChOx to cholesterol. The observed results show promising application of nanoporous ZnO thin film for biosensing application without any functionalization

  2. Lipase-catalyzed ring-opening polymerization of lactones to polyesters and its mechanistic aspects.

    Science.gov (United States)

    Namekawa, S; Suda, S; Uyama, H; Kobayashi, S

    1999-01-01

    Lipase catalysis induced a ring-opening polymerization of lactones with different ring-sizes. Small-size (four-membered) and medium-size lactones (six- and seven-membered) as well as macrolides (12-, 13-, 16-, and 17-membered) were subjected to lipase-catalyzed polymerization. The polymerization behaviors depended primarily on the lipase origin and the monomer structure. The macrolides showing much lower anionic polymerizability were enzymatically polymerized faster than epsilon-caprolactone. The granular immobilized lipase derived from Candida antartica showed extremely efficient catalysis in the polymerization of epsilon-caprolactone. Single-step terminal functionalization of the polyester was achieved by initiator and terminator methods. The enzymatic polymerizability of lactones was quantitatively evaluated by Michaelis-Menten kinetics.

  3. Design and application of a fluorogenic assay for monitoring inflammatory caspase activity.

    Science.gov (United States)

    Ranganathan, Raj; Lenti, Gena; Tassone, Nicholas M; Scannell, Brian J; Southern, Cathrine A; Karver, Caitlin E

    2018-02-15

    Various fluorogenic assays exist for monitoring the activity of inflammatory caspases. However, there are no continuous assays that provide C-terminal substrate sequence specificity for inflammatory caspases. As a first step towards this, we have developed a continuous in vitro assay that relies on monitoring emission from tryptophan after cleavage of a quenching coumarin chromophore. The coumarin can be attached as an amino acid side chain or capping the C-terminus of the peptide. When the coumarin is a side chain, it allows for C-terminal and N-terminal sequence specificities to be explored. Using this assay, we obtained Michaelis-Menten kinetic data for four proof-of-principle peptides: WEHD-AMC (K M  = 15 ± 2 μM), WEHD-MCA (K M  = 93 ± 19 μM), WEHDG-MCA (K M  = 21 ± 6 μM) and WEHDA-MCA (K M  = 151 ± 37 μM), where AMC is 7-amino-4-methylcoumarin and MCA is β-(7-methoxy-coumarin-4-yl)-Ala. The results indicate the viability of this new assay approach in the design of effective fluorogenic substrates for inflammatory caspases. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Functional and Structural Characterization of a (+)-Limonene Synthase from Citrus sinensis.

    Science.gov (United States)

    Morehouse, Benjamin R; Kumar, Ramasamy P; Matos, Jason O; Olsen, Sarah Naomi; Entova, Sonya; Oprian, Daniel D

    2017-03-28

    Terpenes make up the largest and most diverse class of natural compounds and have important commercial and medical applications. Limonene is a cyclic monoterpene (C 10 ) present in nature as two enantiomers, (+) and (-), which are produced by different enzymes. The mechanism of production of the (-)-enantiomer has been studied in great detail, but to understand how enantiomeric selectivity is achieved in this class of enzymes, it is important to develop a thorough biochemical description of enzymes that generate (+)-limonene, as well. Here we report the first cloning and biochemical characterization of a (+)-limonene synthase from navel orange (Citrus sinensis). The enzyme obeys classical Michaelis-Menten kinetics and produces exclusively the (+)-enantiomer. We have determined the crystal structure of the apoprotein in an "open" conformation at 2.3 Å resolution. Comparison with the structure of (-)-limonene synthase (Mentha spicata), which is representative of a fully closed conformation (Protein Data Bank entry 2ONG ), reveals that the short H-α1 helix moves nearly 5 Å inward upon substrate binding, and a conserved Tyr flips to point its hydroxyl group into the active site.

  5. Victor Henri: 111 years of his equation.

    Science.gov (United States)

    Cornish-Bowden, Athel; Mazat, Jean-Pierre; Nicolas, Serge

    2014-12-01

    Victor Henri's great contribution to the understanding of enzyme kinetics and mechanism is not always given the credit that it deserves. In addition, his earlier work in experimental psychology is totally unknown to biochemists, and his later work in spectroscopy and photobiology almost equally so. Applying great rigour to his analysis he succeeded in obtaining a model of enzyme action that explained all of the observations available to him, and he showed why the considerable amount of work done in the preceding decade had not led to understanding. His view was that only physical chemistry could explain the behaviour of enzymes, and that models should be judged in accordance with their capacity not only to explain previously known facts but also to predict new observations against which they could be tested. The kinetic equation usually attributed to Michaelis and Menten was in reality due to him. His thesis of 1903 is now available in English. Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

  6. Uptake of acidic and basic sugar derivatives in Lemna gibba G1

    International Nuclear Information System (INIS)

    Sanz, A.; Ullrich, C.I.

    1989-01-01

    The uptake of acidic and basic sugar derivatives in Lemna gibba L. was studied. Uronic acids applied to the experimental solution induced a small decrease of the membrane potential. After incubation of the plants in a 0.1 millimolar solution of these substrates, no decrease in the concentration of reducing groups in the external solution was detected. Respiration increased by 31% with 50 millimolar galacturonic acid, whereas no effect was found with the same concentration of glucuronic acid. Glucosamine caused a considerable concentration-dependent membrane depolarization. ( 14 C)glucosamine uptake followed Michaelis-Menten kinetics together with a linear component. Influx of this substrate was inhibited by glucose but the type of competition could not be clearly distinguished. Glucosamine, 50 millimolar, inhibited the respiration rate by 30%. The glucosamine uptake was pH-dependent, with maximum uptake at around pH 7. Lack of enhancement of uptake by low pH as well as the permanent membrane depolarization suggest a uniport mechanism for the charged species of the substrate and an electroneutral diffusion of the uncharged species

  7. The on-line synthesis of enzyme functionalized silica nanoparticles in a microfluidic reactor using polyethylenimine polymer and R5 peptide

    International Nuclear Information System (INIS)

    He Ping; Greenway, Gillian; Haswell, Stephen J

    2008-01-01

    A simple microfluidic reactor system is described for the effective synthesis of enzyme functionalized nanoparticles which offers many advantages over batch reactions, including excellent enzyme efficiencies. Better control of the process parameters in the microfluidic reactor system over batch based methodology enables the production of silica nanoparticles with the optimum size for efficient enzyme immobilization with long-term stability. The synthetic approach is demonstrated with glucose oxidase (GOD) and two different nucleation catalysts of similar molecular mass: the natural R5 peptide, and polyethylenimine (PEI) polymer. Near-quantitative immobilization of GOD in the nanoparticles is obtained using PEI; the immobilization is attributed to electrostatic interaction between PEI and GOD. This interaction, however, limits the mobility of the immobilized enzyme, producing orientation hindrance of the enzyme's active sites as compared to free GOD in solution. In contrast, when the GOD is immobilized inside the silica nanoparticles using R5, lower enzyme immobilization efficiencies are obtained compared to using PEI polymers; however, similar Michaelis-Menten kinetic parameters (i.e. Michaelis constant and turnover number) to those of free GOD are observed. Reactions were monitored in situ using simple, rapid, separation-free amperometric detection

  8. Real-Time Enzyme Kinetics by Quantitative NMR Spectroscopy and Determination of the Michaelis-Menten Constant Using the Lambert-W Function

    Science.gov (United States)

    Her, Cheenou; Alonzo, Aaron P.; Vang, Justin Y.; Torres, Ernesto; Krishnan, V. V.

    2015-01-01

    Enzyme kinetics is an essential part of a chemistry curriculum, especially for students interested in biomedical research or in health care fields. Though the concept is routinely performed in undergraduate chemistry/biochemistry classrooms using other spectroscopic methods, we provide an optimized approach that uses a real-time monitoring of the…

  9. Hydrodynamic chronoamperometry for probing kinetics of anaerobic microbial metabolism - case study of Faecalibacterium prausnitzii

    Science.gov (United States)

    Prévoteau, Antonin; Geirnaert, Annelies; Arends, Jan B. A.; Lannebère, Sylvain; van de Wiele, Tom; Rabaey, Korneel

    2015-07-01

    Monitoring in vitro the metabolic activity of microorganisms aids bioprocesses and enables better understanding of microbial metabolism. Redox mediators can be used for this purpose via different electrochemical techniques that are either complex or only provide non-continuous data. Hydrodynamic chronoamperometry using a rotating disc electrode (RDE) can alleviate these issues but was seldom used and is poorly characterized. The kinetics of Faecalibacterium prausnitzii A2-165, a beneficial gut microbe, were determined using a RDE with riboflavin as redox probe. This butyrate producer anaerobically ferments glucose and reduces riboflavin whose continuous monitoring on a RDE provided highly accurate kinetic measurements of its metabolism, even at low cell densities. The metabolic reaction rate increased linearly over a broad range of cell concentrations (9 × 104 to 5 × 107 cells.mL-1). Apparent Michaelis-Menten kinetics was observed with respect to riboflavin (KM = 6 μM kcat = 5.3×105 s-1, at 37 °C) and glucose (KM = 6 μM kcat = 2.4 × 105 s-1). The short temporal resolution allows continuous monitoring of fast cellular events such as kinetics inhibition with butyrate. Furthermore, we detected for the first time riboflavin reduction by another potential probiotic, Butyricicoccus pullicaecorum. The ability of the RDE for fast, accurate, simple and continuous measurements makes it an ad hoc tool for assessing bioprocesses at high resolution.

  10. Hydrodynamic chronoamperometry for probing kinetics of anaerobic microbial metabolism--case study of Faecalibacterium prausnitzii.

    Science.gov (United States)

    Prévoteau, Antonin; Geirnaert, Annelies; Arends, Jan B A; Lannebère, Sylvain; Van de Wiele, Tom; Rabaey, Korneel

    2015-07-01

    Monitoring in vitro the metabolic activity of microorganisms aids bioprocesses and enables better understanding of microbial metabolism. Redox mediators can be used for this purpose via different electrochemical techniques that are either complex or only provide non-continuous data. Hydrodynamic chronoamperometry using a rotating disc electrode (RDE) can alleviate these issues but was seldom used and is poorly characterized. The kinetics of Faecalibacterium prausnitzii A2-165, a beneficial gut microbe, were determined using a RDE with riboflavin as redox probe. This butyrate producer anaerobically ferments glucose and reduces riboflavin whose continuous monitoring on a RDE provided highly accurate kinetic measurements of its metabolism, even at low cell densities. The metabolic reaction rate increased linearly over a broad range of cell concentrations (9 × 10(4) to 5 × 10(7) cells.mL(-1)). Apparent Michaelis-Menten kinetics was observed with respect to riboflavin (KM = 6 μM; kcat = 5.3 × 10(5) s(-1), at 37 °C) and glucose (KM = 6 μM; kcat = 2.4 × 10(5) s(-1)). The short temporal resolution allows continuous monitoring of fast cellular events such as kinetics inhibition with butyrate. Furthermore, we detected for the first time riboflavin reduction by another potential probiotic, Butyricicoccus pullicaecorum. The ability of the RDE for fast, accurate, simple and continuous measurements makes it an ad hoc tool for assessing bioprocesses at high resolution.

  11. Bohmian mechanics, open quantum systems and continuous measurements

    CERN Document Server

    Nassar, Antonio B

    2017-01-01

    This book shows how Bohmian mechanics overcomes the need for a measurement postulate involving wave function collapse. The measuring process plays a very important role in quantum mechanics. It has been widely analyzed within the Copenhagen approach through the Born and von Neumann postulates, with later extension due to Lüders. In contrast, much less effort has been invested in the measurement theory within the Bohmian mechanics framework. The continuous measurement (sharp and fuzzy, or strong and weak) problem is considered here in this framework. The authors begin by generalizing the so-called Mensky approach, which is based on restricted path integral through quantum corridors. The measuring system is then considered to be an open quantum system following a stochastic Schrödinger equation. Quantum stochastic trajectories (in the Bohmian sense) and their role in basic quantum processes are discussed in detail. The decoherence process is thereby described in terms of classical trajectories issuing from th...

  12. Ion pump as Brownian motor: theory of electroconformational coupling and proof of ratchet mechanism for Na,K-ATPase action

    Science.gov (United States)

    Tsong, Tian Yow; Chang, Cheng-Hung

    2003-04-01

    This article reviews some concepts of the Brownian Ratchet which are relevant to our discussion of mechanisms of action of Na,K-ATPase, a universal ion pump and an elemental motor protein of the biological cell. Under wide ranges of ionic compositions it can hydrolyze an ATP and use the γ-phosphorous bond energy of ATP to pump 3 Na + out of, and 2 K + into the cell, both being uphill transport. During the ATP-dependent pump cycle, the enzyme oscillates between E1 and E2 states. Our experiment replaces ATP with externally applied electric field of various waveforms, amplitudes, and frequencies. The field enforced-oscillation, or fluctuation of E1 and E2 states enables the enzyme to harvest energy from the applied field and convert it to the chemical gradient energy of cations. A theory of electroconformational coupling (TEC), which embodies all the essential features of the Brownian Ratchet, successfully simulates these experimental results. Our analysis based on a four-state TEC model indicates that the equilibrium and the rate constants of the transport system define the frequency and the amplitude of the field for the optimal activation. Waveform, frequency, and amplitude are three elements of signal. Thus, electric signal of the ion pump is found by TEC analysis of the experimental data. Electric noise (white) superimposed on an electric signal changes the pump efficiency and produces effects similar to the stochastic resonance reported in other biological systems. The TEC concept is compared with the most commonly used Michaelis-Menten enzyme mechanism (MME) for similarities and differences. Both MME and TEC are catalytic wheels, which recycle the catalyst in each turnover. However, a MME can only catalyze reaction of descending free energy while a TEC enzyme can catalyze reaction of ascending free energy by harvesting needed energy from an off-equilibrium electric noise. The TEC mechanism is shown to be applicable to other biological motors and engines, as

  13. SACCHARIFICATION OF NATIVE CASSAVA STARCH AT HIGH DRY SOLIDS IN AN ENZYMATIC MEMBRANE REACTOR

    Directory of Open Access Journals (Sweden)

    I Nyoman Widiasa

    2012-02-01

    Full Text Available This study is aimed to develop a novel process scheme for hydrolysis of native cassava starch at high dry solids using an enzymatic membrane reactor (EMR. Firstly, liquefied cassava starch having solids content up to 50% by weight was prepared by three stage liquefactions in a conventional equipment using a commercially available heat stable a-amylase (Termamyl 120L. The liquefied cassava starch was further saccharified in an EMR using glucoamylase (AMG E. By using the developed process scheme, a highly clear hydrolysate with dextrose equivalent (DE approximately 97 could be produced, provided the increase of solution viscosity during the liquefaction was precisely controlled. The excessive space time could result in reduction in conversion degree of starch. Moreover, a residence time distribution study confirmed that the EMR could be modelled as a simple continuous stirred tank reactor (CSTR. Using Lineweaver-Burk analysis, the apparent Michaelis-Menten constant (Km and glucose production rate constant (k2 were 552 (g/l and 4.04 (min-1, respectively. Application of simple CSTR model with those kinetic parameters was quietly appropriate to predict the reactor’s performance at low space time.

  14. Fabrication of a sulfite biosensor by the use of conducting polymer

    International Nuclear Information System (INIS)

    Hosseini, M.; Bahmani, B; Moztarzadeh, F.; Rabiee, M.

    2008-01-01

    In this research, an enzyme modified electrode has been produced during the electro polymerization of aniline through incorporation of Sulfite oxidase into a conducting polymer. Then the bioelectrochemical response of resulted sulfite biosensor was investigated at different experimental conditions. Study of the stability of the resulted sulfite biosensor revealed that formation of a passive film on the aluminum surface causes improved stability of the electro active films formed on the electrode surface. The bioelectrochemical response of the enzyme-modified electrode as a sulfite biosensor was investigated at different experimental conditions. The optimum p H and temperature were 8.5 and 35 d eg C , respectively. The apparent Michaelis-Menten constant and the activation energy of the enzyme catalyzed reaction were calculated

  15. A glassy carbon electrode modified with a composite consisting of reduced graphene oxide, zinc oxide and silver nanoparticles in a chitosan matrix for studying the direct electron transfer of glucose oxidase and for enzymatic sensing of glucose

    International Nuclear Information System (INIS)

    Li, Zhenjiang; Sheng, Liying; Xie, Cuicui; Meng, Alan; Zhao, Kun

    2016-01-01

    The authors describe the fabrication of a nanocomposite consisting of reduced graphene oxide, zinc oxide and silver nanoparticles by microwave-assisted synthesis. The composite was further reduced in-situ with hydrazine hydrate and then placed, along with the enzyme glucose oxidase, on a glassy carbon electrode. The synergistic effect of the materials employed in the nanocomposite result in excellent electrocatalytic activity. The Michaelis-Menten constant of the adsorbed GOx is 0.25 mM, implying a remarkable affinity of the GOx for glucose. The amperometric response of the modified GCE is linearly proportional to the concentration of glucose in 0.1 to 12.0 mM concentration range, and the detection limit is 10.6 µM. The biosensor is highly selective, well reproducible and stable. (author)

  16. NUMERICAL SOLUTION OF STEADY STATE DISPERSION FLOW MODEL FOR LACTOSE-LACTASE HYDROLYSIS WITH DIFFERENT KINETICS IN FIXED BED

    Directory of Open Access Journals (Sweden)

    OLAOSEBIKAN ABIDOYE OLAFADEHAN

    2010-06-01

    Full Text Available A detailed computational procedure for evaluating lactose hydrolysis with immobilized enzyme in a packed bed tubular reactor under dispersion flow conditions is presented. The dispersion flow model for lactose hydrolysis using different kinetics, taking cognizance of external mass transfer resistances, was solved by the method of orthogonal collocation. The reliability of model simulations was tested using experimental data from a laboratory packed bed column, where the -galactosidase of Kluyveromyces fragilis was immobilized on spherical chitosan beads. Comparison of the simulated results with experimental exit conversion shows that the dispersion flow model and using Michaelis-Menten kinetics with competitive product (galactose inhibition are appropriate to interpret the experimental results and simulate the process of lactose hydrolysis in a fixed bed.

  17. Nanoporous cerium oxide thin film for glucose biosensor.

    Science.gov (United States)

    Saha, Shibu; Arya, Sunil K; Singh, S P; Sreenivas, K; Malhotra, B D; Gupta, Vinay

    2009-03-15

    Nanoporous cerium oxide (CeO(2)) thin film deposited onto platinum (Pt) coated glass plate using pulsed laser deposition (PLD) has been utilized for immobilization of glucose oxidase (GOx). Atomic force microscopy studies reveal the formation of nanoporous surface morphology of CeO(2) thin film. Response studies carried out using differential pulsed voltammetry (DPV) and optical measurements show that the GOx/CeO(2)/Pt bio-electrode shows linearity in the range of 25-300 mg/dl of glucose concentration. The low value of Michaelis-Menten constant (1.01 mM) indicates enhanced enzyme affinity of GOx to glucose. The observed results show promising application of the nanoporous CeO(2) thin film for glucose sensing application without any surface functionalization or mediator.

  18. Determining "small parameters" for quasi-steady state

    Science.gov (United States)

    Goeke, Alexandra; Walcher, Sebastian; Zerz, Eva

    2015-08-01

    For a parameter-dependent system of ordinary differential equations we present a systematic approach to the determination of parameter values near which singular perturbation scenarios (in the sense of Tikhonov and Fenichel) arise. We call these special values Tikhonov-Fenichel parameter values. The principal application we intend is to equations that describe chemical reactions, in the context of quasi-steady state (or partial equilibrium) settings. Such equations have rational (or even polynomial) right-hand side. We determine the structure of the set of Tikhonov-Fenichel parameter values as a semi-algebraic set, and present an algorithmic approach to their explicit determination, using Groebner bases. Examples and applications (which include the irreversible and reversible Michaelis-Menten systems) illustrate that the approach is rather easy to implement.

  19. Role of carboxydobacteria in consumption of atmospheric carbon monoxide by soil

    Energy Technology Data Exchange (ETDEWEB)

    Conrad, R. (Max-Planck-Institut fuer Chemie, Mainz, Germany); Meyer, O.; Seiler, W.

    1981-08-01

    The carbon monoxide consumption rates of the carboxydobacteria Pseudomonas (Seliberia) carboxydohydrogena, P. carboxydovorans, and P. carboxydoflava were measured at high (50%) and low (0.5 ..mu..l liter/sup -1/) mixing ratios of CO in air. CO was only consumed when the bacteria had been grown under CO-autotrophic conditions. At low cell densities the CO comsumption rates measured at low CO mixing ratios were similar in cell suspensions and in mixtures of bacteria in soil. CO consumption observed in natural soil (loess, eolian sand, chernozem) as well as in suspensions or soil mixtures of carboxydobacteria showed Michaelis-Menten kinetics. Considering the difference of the K/sub m/, values and the observed V/sub max/ values, carboxydobacteria cannot contribute significantly to the consumption of atmospheric CO.

  20. Fluctuations induced extinction and stochastic resonance effect in a model of tumor growth with periodic treatment

    Energy Technology Data Exchange (ETDEWEB)

    Li Dongxi, E-mail: lidongxi@mail.nwpu.edu.c [Department of Applied Mathematics, Northwestern Polytechnical University, Xi' an 710072 (China); Xu Wei; Guo, Yongfeng; Xu Yong [Department of Applied Mathematics, Northwestern Polytechnical University, Xi' an 710072 (China)

    2011-01-31

    We investigate a stochastic model of tumor growth derived from the catalytic Michaelis-Menten reaction with positional and environmental fluctuations under subthreshold periodic treatment. Firstly, the influences of environmental fluctuations on the treatable stage are analyzed numerically. Applying the standard theory of stochastic resonance derived from the two-state approach, we derive the signal-to-noise ratio (SNR) analytically, which is used to measure the stochastic resonance phenomenon. It is found that the weak environmental fluctuations could induce the extinction of tumor cells in the subthreshold periodic treatment. The positional stability is better in favor of the treatment of the tumor cells. Besides, the appropriate and feasible treatment intensity and the treatment cycle should be highlighted considered in the treatment of tumor cells.

  1. Fluctuations induced extinction and stochastic resonance effect in a model of tumor growth with periodic treatment

    International Nuclear Information System (INIS)

    Li Dongxi; Xu Wei; Guo, Yongfeng; Xu Yong

    2011-01-01

    We investigate a stochastic model of tumor growth derived from the catalytic Michaelis-Menten reaction with positional and environmental fluctuations under subthreshold periodic treatment. Firstly, the influences of environmental fluctuations on the treatable stage are analyzed numerically. Applying the standard theory of stochastic resonance derived from the two-state approach, we derive the signal-to-noise ratio (SNR) analytically, which is used to measure the stochastic resonance phenomenon. It is found that the weak environmental fluctuations could induce the extinction of tumor cells in the subthreshold periodic treatment. The positional stability is better in favor of the treatment of the tumor cells. Besides, the appropriate and feasible treatment intensity and the treatment cycle should be highlighted considered in the treatment of tumor cells.

  2. A long-lived lunar dynamo driven by continuous mechanical stirring.

    Science.gov (United States)

    Dwyer, C A; Stevenson, D J; Nimmo, F

    2011-11-09

    Lunar rocks contain a record of an ancient magnetic field that seems to have persisted for more than 400 million years and which has been attributed to a lunar dynamo. Models of conventional dynamos driven by thermal or compositional convection have had difficulty reproducing the existence and apparently long duration of the lunar dynamo. Here we investigate an alternative mechanism of dynamo generation: continuous mechanical stirring arising from the differential motion, due to Earth-driven precession of the lunar spin axis, between the solid silicate mantle and the liquid core beneath. We show that the fluid motions and the power required to drive a dynamo operating continuously for more than one billion years and generating a magnetic field that had an intensity of more than one microtesla 4.2 billion years ago are readily obtained by mechanical stirring. The magnetic field is predicted to decrease with time and to shut off naturally when the Moon recedes far enough from Earth that the dissipated power is insufficient to drive a dynamo; in our nominal model, this occurred at about 48 Earth radii (2.7 billion years ago). Thus, lunar palaeomagnetic measurements may be able to constrain the poorly known early orbital evolution of the Moon. This mechanism may also be applicable to dynamos in other bodies, such as large asteroids.

  3. Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose

    DEFF Research Database (Denmark)

    Olsen, Johan P.; Alasepp, Kadri; Kari, Jeppe

    2016-01-01

    The cellobiohydrolase cellulase Cel7A is extensively utilized in industrial treatment of lignocellulosic biomass under conditions of high product concentrations, and better understanding of inhibition mechanisms appears central in attempts to improve the efficiency of this process. We have...... the lines of conventional enzyme kinetic theory. We found that the product cellobiose lowered the maximal rate without affecting the Michaelis constant, and this kinetic pattern could be rationalized by two fundamentally distinct molecular mechanisms. One was simple reversibility, that is, an increasing...

  4. Curaua and eucalyptus nanofibers films by continuous casting: Mechanical and thermal properties.

    Science.gov (United States)

    Claro, Pedro Ivo Cunha; Corrêa, Ana Carolina; de Campos, Adriana; Rodrigues, Vanessa Bolzan; Luchesi, Bruno Ribeiro; Silva, Luiz Eduardo; Mattoso, Luiz Henrique Capparelli; Marconcini, José Manoel

    2018-02-01

    A wide variety of new green materials such as curaua leaf fibers (CLFs) has potential applications in nanotechnology. This study aims to investigate the thermomechanical properties and morphological structure of cellulose nanocrystals (CNCs) and cellulose nanofibrils (CNFs) films obtained by continuous casting. The CNCs were obtained by acid hydrolysis and CNFs by mechanical shearing from bleached CLFs and eucalyptus pulp. The morphology after continuous casting resulted in oriented nanofibers, and as a consequence there was mechanical anisotropy. CNCs films showed the greatest values of tensile strength (36±4MPa) and the more effective fibrillation provided better mechanical strength of eucalyptus CNFs films than curaua CNFs films. Sulfur groups and mechanical shear degradation affected the stability of CNCs and CNFs films, respectively. Thus, the type of nanostructure, the way they interact to each other, the cellulose source and the process interfere significantly on the properties of the films. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Insight into the mechanism revealing the peroxidase mimetic catalytic activity of quaternary CuZnFeS nanocrystals: colorimetric biosensing of hydrogen peroxide and glucose

    Science.gov (United States)

    Dalui, Amit; Pradhan, Bapi; Thupakula, Umamahesh; Khan, Ali Hossain; Kumar, Gundam Sandeep; Ghosh, Tanmay; Satpati, Biswarup; Acharya, Somobrata

    2015-05-01

    Artificial enzyme mimetics have attracted immense interest recently because natural enzymes undergo easy denaturation under environmental conditions restricting practical usefulness. We report for the first time chalcopyrite CuZnFeS (CZIS) alloyed nanocrystals (NCs) as novel biomimetic catalysts with efficient intrinsic peroxidase-like activity. Novel peroxidase activities of CZIS NCs have been evaluated by catalytic oxidation of the peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). CZIS NCs demonstrate the synergistic effect of elemental composition and photoactivity towards peroxidase-like activity. The quaternary CZIS NCs show enhanced intrinsic peroxidase-like activity compared to the binary NCs with the same constituent elements. Intrinsic peroxidase-like activity has been correlated with the energy band position of CZIS NCs extracted using scanning tunneling spectroscopy and ultraviolet photoelectron spectroscopy. Kinetic analyses indicate Michaelis-Menten enzyme kinetic model catalytic behavior describing the rate of the enzymatic reaction by correlating the reaction rate with substrate concentration. Typical color reactions arising from the catalytic oxidation of TMB over CZIS NCs with H2O2 have been utilized to establish a simple and sensitive colorimetric assay for detection of H2O2 and glucose. CZIS NCs are recyclable catalysts showing high efficiency in multiple uses. Our study may open up the possibility of designing new photoactive multi-component alloyed NCs as enzyme mimetics in biotechnology applications.Artificial enzyme mimetics have attracted immense interest recently because natural enzymes undergo easy denaturation under environmental conditions restricting practical usefulness. We report for the first time chalcopyrite CuZnFeS (CZIS) alloyed nanocrystals (NCs) as novel biomimetic catalysts with efficient intrinsic peroxidase-like activity. Novel peroxidase activities of CZIS NCs have been

  6. Continuous three dimensional analysis of running mechanics during a marathon by means of inertial magnetic measurement units to objectify changes in running mechanics

    NARCIS (Netherlands)

    Reenalda, Jasper; Maartens, Erik; Maartens, Erik; Homan, Lotte; Buurke, Jaap

    2016-01-01

    Recent developments in wearable and wireless sensor technology allow for a continuous three dimensional analysis of running mechanics in the sport specific setting. The present study is the first to demonstrate the possibility of analyzing three dimensional (3D) running mechanics continuously, by

  7. FEATURES OF LONG-TERM MECHANICAL CIRCULATORY SUPPORT WITH CONTINUOUS-FLOW PUMP

    Directory of Open Access Journals (Sweden)

    G. P. Itkin

    2012-01-01

    Full Text Available In a review of the comparative analysis of methods and tools for long-term mechanical circulatory support with continuous flow and pulsatile flow implantable pumps. Particular attention is paid to the choice of the optimal modes of the operation of pumps based on the physical principles of the interaction between a the steady flow of blood to the pulsatile mechanics of the heart chambers. 

  8. Pharmacokinetic Modeling of Voriconazole To Develop an Alternative Dosing Regimen in Children.

    Science.gov (United States)

    Gastine, Silke; Lehrnbecher, Thomas; Müller, Carsten; Farowski, Fedja; Bader, Peter; Ullmann-Moskovits, Judith; Cornely, Oliver A; Groll, Andreas H; Hempel, Georg

    2018-01-01

    The pharmacokinetic variability of voriconazole (VCZ) in immunocompromised children is high, and adequate exposure, particularly in the first days of therapy, is uncertain. A population pharmacokinetic model was developed to explore VCZ exposure in plasma after alternative dosing regimens. Concentration data were obtained from a pediatric phase II study. Nonlinear mixed effects modeling was used to develop the model. Monte Carlo simulations were performed to test an array of three-times-daily (TID) intravenous dosing regimens in children 2 to 12 years of age. A two-compartment model with first-order absorption, nonlinear Michaelis-Menten elimination, and allometric scaling best described the data (maximal kinetic velocity for nonlinear Michaelis-Menten clearance [ V max ] = 51.5 mg/h/70 kg, central volume of distribution [ V 1 ] = 228 liters/70 kg, intercompartmental clearance [ Q ] = 21.9 liters/h/70 kg, peripheral volume of distribution [ V 2 ] = 1,430 liters/70 kg, bioavailability [ F ] = 59.4%, K m = fixed value of 1.15 mg/liter, absorption rate constant = fixed value of 1.19 h -1 ). Interindividual variabilities for V max , V 1 , Q , and F were 63.6%, 45.4%, 67%, and 1.34% on a logit scale, respectively, and residual variability was 37.8% (proportional error) and 0.0049 mg/liter (additive error). Monte Carlo simulations of a regimen of 9 mg/kg of body weight TID simulated for 24, 48, and 72 h followed by 8 mg/kg two times daily (BID) resulted in improved early target attainment relative to that with the currently recommended BID dosing regimen but no increased rate of accumulation thereafter. Pharmacokinetic modeling suggests that intravenous TID dosing at 9 mg/kg per dose for up to 3 days may result in a substantially higher percentage of children 2 to 12 years of age with adequate exposure to VCZ early during treatment. Before implementation of this regimen in patients, however, validation of exposure, safety, and tolerability in a carefully designed

  9. Structural Basis of Binding and Rationale for the Potent Urease Inhibitory Activity of Biscoumarins

    Science.gov (United States)

    Lodhi, Muhammad Arif; Choudhary, Muhammad Iqbal; Lodhi, Atif; Ul-Haq, Zaheer; Jalil, Saima; Nawaz, Sarfraz Ahmad; Khan, Khalid Mohammed; Iqbal, Sajid; Rahman, Atta-ur

    2014-01-01

    Urease belongs to a family of highly conserved urea-hydrolyzing enzymes. A common feature of these enzymes is the presence of two Lewis acid nickel ions and reactive cysteine residue in the active sites. In the current study we examined a series of biscoumarins 1–10 for their mechanisms of inhibition with the nickel containing active sites of Jack bean and Bacillus pasteurii ureases. All these compounds competitively inhibited Jack bean urease through interaction with the nickel metallocentre, as deduced from Michaelis-Menten kinetics, UV-visible absorbance spectroscopic, and molecular docking simulation studies. Some of the compounds behaved differently in case of Bacillus pasteurii urease. We conducted the enzyme kinetics, UV-visible spectroscopy, and molecular docking results in terms of the known protein structure of the enzyme. We also evaluated possible molecular interpretations for the site of biscoumarins binding and found that phenyl ring is the major active pharmacophore. The excellent in vitro potency and selectivity profile of the several compounds described combined with their nontoxicity against the human cells and plants suggest that these compounds may represent a viable lead series for the treatment of urease associated problems. PMID:25295281

  10. The effects of carbon nanotube addition and oxyfluorination on the glucose-sensing capabilities of glucose oxidase-coated carbon fiber electrodes

    Energy Technology Data Exchange (ETDEWEB)

    Im, Ji Sun; Yun, Jumi; Kim, Jong Gu [Department of Fine Chemical Engineering and Applied Chemistry, BK21-E2 M, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Bae, Tae-Sung [Department of Fine Chemical Engineering and Applied Chemistry, BK21-E2 M, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Korea Basic Science Institute (KBSI), Jeonju 561-756 (Korea, Republic of); Lee, Young-Seak, E-mail: youngslee@cnu.ac.kr [Department of Fine Chemical Engineering and Applied Chemistry, BK21-E2 M, Chungnam National University, Daejeon 305-764 (Korea, Republic of)

    2012-01-15

    Glucose-sensing electrodes were constructed from carbon fibers by electrospinning and heat treatment. By controlling the pore size, the specific surface area and pore volume of the electrospun carbon fibers were increased for efficient immobilization of the glucose oxidase. Carbon nanotubes were embedded as an electrically conductive additive to improve the electrical property of the porous carbon fibers. In addition, the surface of the porous carbon fibers was modified with hydrophilic functional groups by direct oxyfluorination to increase the affinity between the hydrophobic carbon surface and the hydrophilic glucose oxidase molecules. The porosity of the carbon fibers was improved significantly with approximately 28- and 35-fold increases in the specific surface area and pore volume, respectively. The number of chemical bonds between carbon and oxygen were increased with higher oxygen content during oxyfluorination based on the X-ray photoelectron spectroscopy results. Glucose sensing was carried out by current voltagram and amperometric methods. A high-performance glucose sensor was obtained with high sensitivity and rapid response time as a result of carbon nanotube addition, physical activation and surface modification. The mechanism of the highly sensitive prepared glucose sensor was modeled by an enzyme kinetics study using the Michaelis-Menten equation.

  11. Lipase-catalyzed synthesis of palmitanilide: Kinetic model and antimicrobial activity study.

    Science.gov (United States)

    Liu, Kuan-Miao; Liu, Kuan-Ju

    2016-01-01

    Enzymatic syntheses of fatty acid anilides are important owing to their wide range of industrial applications in detergents, shampoo, cosmetics, and surfactant formulations. The amidation reaction of Mucor miehei lipase Lipozyme IM20 was investigated for direct amidation of triacylglycerol in organic solvents. The process parameters (reaction temperature, substrate molar ratio, enzyme amount) were optimized to achieve the highest yield of anilide. The maximum yield of palmitanilide (88.9%) was achieved after 24 h of reaction at 40 °C at an enzyme concentration of 1.4% (70 mg). Kinetics of lipase-catalyzed amidation of aniline with tripalmitin has been investigated. The reaction rate could be described in terms of the Michaelis-Menten equation with a Ping-Pong Bi-Bi mechanism and competitive inhibition by both the substrates. The kinetic constants were estimated by using non-linear regression method using enzyme kinetic modules. The enzyme operational stability study showed that Lipozyme IM20 retained 38.1% of the initial activity for the synthesis of palmitanilide (even after repeated use for 48 h). Palmitanilide, a fatty acid amide, exhibited potent antimicrobial activity toward Bacillus cereus. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. On enzyme kinetic parameters modification of gamma irradiation

    International Nuclear Information System (INIS)

    Ferdes, O.S.; Ferdes, M.; Turcu, G.R.

    1993-01-01

    To elucidate the molecular mechanisms of gamma-ray action on biomolecules there were investigated the modifications in activity and other kinetic parameters for some enzymes irradiated in pure dry state at relative high doses. There were considered bacterial and fungal α-amylases, glucoamylase and Mucor sp. protease irradiated by a 60 Co gamma-ray source in the dose range 1.0-30.0 kGy, at different dose-rates between 0.5-2.0 kGy/h, at room temperature. Considering the enzyme inactivation in this dose range, the dose-effect relationships have an expected form and depend on the irradiation conditions but not significantly on the dose rate. The catalytic properties of enzymes were modified by irradiation. By usual methods it is evidenced a direct correlation between the enzymatic activities, Michaelis-Menten constant, K m , reaction velocities, v, and the irradiation dose. These experimental findings can support a self-consistent theoretical approach on biophysical radiation action on biological active molecules like enzymes. At the same time, some enzyme behaviour to irradiation could be considered like a good biological indicator of radiation response. (Author) 4 Figs., 19 Refs

  13. Simple method for preparing glucose biosensor based on in-situ polypyrrole cross-linked chitosan/glucose oxidase/gold bionanocomposite film.

    Science.gov (United States)

    Şenel, Mehmet

    2015-03-01

    A film of chitosan-polypyrrole-gold nanoparticles was fabricated by in-situ chemical synthesis method and its application in glucose biosensor was investigated. The obtained biosensor exhibited a high and reproducible sensitivity of 0.58μA/mM, response time ~4s, linear dynamic range from 1 to 20mM, correlation coefficient of R(2)=0.9981, and limit of detection (LOD), based on S/N ratio (S/N=3) of 0.068mM. A value of 1.83mM for the apparent Michaelis-Menten constant was obtained. The resulting bio-nanocomposite provided a suitable environment for the enzyme to retain its bioactivity at considerably extreme conditions, and the decorated gold nanoparticles in the bio-nanocomposite offer good affinity to enzyme. Copyright © 2014. Published by Elsevier B.V.

  14. Tomato root growth and phosphorus absorption kinetics by tomato plants as affected by phosphorus concentration in nutrient solution

    International Nuclear Information System (INIS)

    Fontes, P.C.R.; Barber, S.A.

    1984-01-01

    To evaluate the effects P concentrations in nutrient solution on root growth and on root physiological characteristics involved in P uptake by tomato Lycopersicon esculentum Mill plants, six seedlings were grown in nutrient solution at initial concentrations of 48.5, 97, 194 and 388 μMP until one day before harvest. They were then transferred to solutions with P at 20 μM and 30 μM, and the depletion curves and Michaelis-Menten parameters were determined. The conclusions were that as P supply increased and as the plant P contents are sufficient for maximum growth, the rate of P uptake tends to be lower. The results also indicate that total P uptake by tomato seedlings depends on the amount of root surface area exposed to P. (M.A.C.) [pt

  15. A novel hydrogen peroxide biosensor based on hemoglobin-collagen-CNTs composite nanofibers.

    Science.gov (United States)

    Li, J; Mei, H; Zheng, W; Pan, P; Sun, X J; Li, F; Guo, F; Zhou, H M; Ma, J Y; Xu, X X; Zheng, Y F

    2014-06-01

    In this paper, carbon nanotubes (CNTs) were successfully incorporated in the composite composed of hemoglobin (Hb) and collagen using co-electrospinning technology. The formed Hb-collagen-CNTs composite nanofibers possessed distinct advantage of three-dimensional porous structure, biocompatibility and excellent stability. The Hb immobilized in the electrospun nanofibers retained its natural structure and the heterogeneous electron transfer rate constant (ks) of the direct electron transfer between Hb and electrodes was 5.3s(-1). In addition, the electrospun Hb-collagen-CNTs nanofibers modified electrodes showed good electrocatalytic properties toward H2O2 with a detection limit of 0.91μM (signal-to-noise ratio of 3) and the apparent Michaelis-Menten constant (Km(app)) of 32.6μM. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Relationship between soil cellulolytic activity and suppression of seedling blight of barley in arable soils

    DEFF Research Database (Denmark)

    Rasmussen, Peter Have; Knudsen, I.; Elmholt, S.

    2002-01-01

    the Hanes-Wolf transformation of the Michaelis-Menten equation. Soil samples from 6 to 13 cm depth were collected in the early spring as undisturbed blocks from 10 arable soils with different physico-chemical properties and cultivation history. Significant correlations were found between soil suppresiveness......The objective was to investigate the relationship between soil suppression of seedling blight of barley caused by Fusarium culmorum (W.G. Smith) Sacc. and the soil cellulolytic activity of beta-glucosidase, cellobiohydrolase and endocellulase. Disease suppression was investigated in bioassays...... with test soils mixed with sand, and barley seeds inoculated with F. culmorum. After 19 days, disease severity was evaluated on the barley seedlings. Soil cellulolytic activities were measured using 4-methylumbelliferyl-labelled fluorogenic substrates, and were expressed as V-max values obtained by using...

  17. Characterization of the anion sensitive ATPase in intact vacuoles of Kalanchoe diagremontiana

    Energy Technology Data Exchange (ETDEWEB)

    Kobza, J.; Uribe, E.G.

    1986-04-01

    A method for the isolation of intact vacuoles from K. daigremontiana was developed which produced high yields of relatively pure vacuoles as determined by marker enzyme contamination. Upon isolation, the vacuoles were stabilized by the inclusion of 5% (w/v) ficoll. Enzyme activity was insensitive to vanadate and azide but was strongly inhibited by DCCD. Enzyme activity was strictly dependent on the inclusion of Mg/sup 2 +/ and was stimulated by anions as depicted by the series, NO/sub 3//sup -/ < Br/sup -/ < SO/sub 4//sup -/ < HCO/sub 3//sup -/ < Cl/sup -/. It was found that in intact vacuoles the ATPase activity was stimulated by phosphate to a level equivalent to that found with the chloride. The enzyme exhibited Michaelis-Menten kinetics with a Km for Mg-ATP complex of 0.51 mM.

  18. Biodegradation of Benzene, Toluene, Ethylbenzene, and o-, m-, and p-Xylenes by the Newly Isolated Bacterium Comamonas sp. JB.

    Science.gov (United States)

    Jiang, Bei; Zhou, Zunchun; Dong, Ying; Tao, Wei; Wang, Bai; Jiang, Jingwei; Guan, Xiaoyan

    2015-07-01

    A bacterium designated strain JB, able to degrade six benzene, toluene, ethylbenzene, and o-, m-, and p-xylene (BTEX) compounds, was isolated from petroleum-contaminated soil. Taxonomic analyses showed that the isolate belonged to Comamonas, and until now, the genus Comamonas has not included any known BTEX degraders. The BTEX biodegradation rate was slightly low on the mineral salt medium (MSM), but adding a small amount of yeast extract greatly enhanced the biodegradation. The relationship between specific degradation rate and individual BTEX was described well by Michaelis-Menten kinetics. The treatment of petrochemical wastewater containing BTEX mixture and phenol was shown to be highly efficient by BTEX-grown JB. In addition, toxicity assessment indicated the treatment of the petrochemical wastewater by BTEX-grown JB led to less toxicity than untreated wastewater.

  19. Michał Kalecki i problem racjonalnej alokacji zasobów w socjalizmie

    Directory of Open Access Journals (Sweden)

    Damian Winczewski

    2015-06-01

    Full Text Available Celem artykułu jest rekonstrukcja poglądów Michała Kaleckiego i jego zwolenników na temat racjonalnej kalkulacji i alokacji zasobów w gospodarce socjalistycznej, a także próba zestawienia tych poglądów z poglądami zwolenników zarówno kapitalizmu, jak i alternatywnych modeli socjalizmu. Pod tym kątem przeanalizowano artykuły polskiego ekonomisty dotyczące schematu tworzenia się cen w kapitalizmie i socjalizmie, roli klasy robotniczej oraz systemu bodźców i sposobu zarządzania gospodarką. W artykule omówiono też poglądy autorów, którzy bazując na pracach Kaleckiego, podjęli się polemiki z obiegową narracją wyjaśniającą porażkę realnego socjalizmu. Zarówno realny kapitalizm, jak i realny socjalizm zmagają się z problemami niedoskonałej informacji i miękkich budżetów, nie istnieje również doskonały model zarządzania gospodarką, w związku z tym nie są to bezpośrednie przyczyny niepowodzenia projektu socjalistycznego. Oprócz problemu innowacji i optymalnych nakładów inwestycyjnych z perspektywy kaleckiańskiej zasadniczym problemem socjalizmu wydaje się ustanowienie właściwych stosunków produkcji w marksistowskim sensie, rozumianych jako zdemokratyzowanie relacji pomiędzy klasą robotniczą a warstwą zarządzającą produkcją.

  20. Michaelis kinetic analysis of extracellular cellulase and amylase excreted by Lactobacillus plantarum during cassava fermentation

    Science.gov (United States)

    Frediansyah, Andri; Kurniadi, Muhamad

    2017-01-01

    Our previous study reveal that single culture of Lactobacillus plantarum has ability to ferment cassava tuber in relation to produce modified cassava flour (mocaf). It was used to accelerate a fermentation process. L. plantarum grow well and produce some extracellular enzymes i.e. cellulase to change the structure and breakdown the cell wall of cassava tuber. Then, the starchy materials will be hydrolyzed by i.e. amylase into simple sugar and convert to organic acid. All of these process will give new characteristic of cassava i.e. lower fiber content, good flavor, taste, aroma and texture and the amount of cyanide acid is lower. Therefore this present study was to analyze Michaelis kinetics of extracellular carboxymethyl cellulase and amylase production by L. plantarum during cassava fermentation. The maximum carboxymethyl cellulase and amylase activity of 8.60 U/ml and 14.07 U/ml, respectively, were obtained from filtrate which has been incubated at 37°C for 18 h under stationary conditions. The Vmax and Km of CMCase were 0.8506 × 10-3 U/ml and 0.9594 × 10-3 g/mL, respectively. For amylase were 9.291 × 10-3 U/ml and 0.9163 × 10-3 g/ml, respectively.

  1. A highly sensitive electrochemical glucose sensor structuring with nickel hydroxide and enzyme glucose oxidase

    International Nuclear Information System (INIS)

    Mathew, Manjusha; Sandhyarani, N.

    2013-01-01

    Graphical abstract: A combination of Ni 2+ /Ni 3+ redox couple and glucose oxidase has successfully been exploited for the realization of a highly sensitive glucose sensor for the first time. -- Highlights: • A multilayered glucose biosensor with enhanced sensitivity was fabricated. • Combination of Ni 2+ /Ni 3+ redox couple and glucose oxidase has been exploited for the first time. • Exhibits a lower detection limit of 100 nM with a high sensitivity of 16,840 μA mM −1 cm −2 . • The surface shows a low Michaelis–Menten constant value of 2.4 μM. • Detailed mechanism of sensing was proposed and justified. -- Abstract: A multilayered glucose biosensor with enhanced electron transport was fabricated via the sequential electrodeposition of chitosan gold nanocomposite (CGNC) and nickel hydroxide (Ni(OH) 2 ) on a bare gold electrode and subsequent immobilization of glucose oxidase. A thin film of Ni(OH) 2 deposited on CGNC modified gold electrode serves as an electrochemical redox probe as well as a matrix for the immobilization of glucose oxidase retaining its activity. Electron transport property of CGNC has been exploited to enhance the electron transport between the analyte and electrode. Electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Under optimal conditions the biosensor exhibits a linear range from 1 μM to 100 μM with a limit of detection (lod) down to 100 nM. The sensor shows a low Michaelis-Menten constant value of 2.4 μM indicates the high affinity of enzyme to the analyte points to the retained activity of enzyme after immobilization. The present glucose sensor with the high selectivity, sensitivity and stability is promising for practical clinical applications

  2. Estudio cinético del proceso de digestión anaerobia de aguas de lavado de aceitunas de almazara en reactores de mezcla completa con microorganismos inmovilizados

    Directory of Open Access Journals (Sweden)

    Borja, Rafael

    1999-04-01

    Full Text Available A kinetic study of the continuous anaerobic digestion process of wastewaters from the washing of olives prior to the oil production process was carried out in a completely mixed reactor with sepiolite immobilised biomass at 35° C. Four influent COD concentrations, 4,5, 3,5, 2,5 y 1,5 g COD/I were used as substrate operating for a hydraulic retention time range of 4,5 to 1,25 days. The Michaelis-Menten model has been applied, obtaining the values of the maximum substrate utilization rate, k( (g COD/g VSS day, and the kinetic constant of the process, Ks (g COD/I, for each influent substrate concentration studied. The values of these parameters significantly decreased with decreasing concentration of influent wastewater, showing the highest values for the greatest influent substrate concentration used as feed. The microbial yield coefficient, Yx, and sludge decay rate coefficient, kd, were also determined to be 0,14 g VSS/g COD and 0,003 days-1 respectively.

    Se ha efectuado un estudio cinético del proceso de digestión anaerobia en régimen continuo de aguas de lavado de aceitunas de almazara en un reactor de mezcla completa utilizando sepiolita como soporte para la inmovilización de los microorganismos responsables del proceso, a la temperatura de 35° C. Se utilizan cuatro concentraciones de sustrato como alimento, 4,5, 3,5, 2,5 y 1,5g DQO/I, operando con cada una de ellas en un rango de tiempos de retención hidráulicos de 4,5 a 1,25 días. Para ello, se aplica el método cinético de Michaelis-Menten, obteniéndose los valores de la velocidad máxima de utilización de sustrato, k(g DQO/g SSV día, y de la constante cinética del proceso, Ks (g DQO/I, para cada concentración de sustrato objeto de estudio. Se observa que estas constantes cinéticas disminuyen de forma significativa al disminuir la concentración de sustrato en el alimento

  3. Canceling effect: a natural mechanism to reduce the effects of global warming

    Science.gov (United States)

    Razavi, Bahar S.; Blagodatskaya, Evgenia; kuzyakov, Yakov

    2016-04-01

    , and incorporated into Earth system models to improve the predictions at regional and global levels. Key words: carbon cycle, Michaelis-Menten kinetics, Arrhenius function, soil enzymes, temperature sensitivity, canceling effect, activation energy.

  4. Physiological Response of Plants Grown on Porous Ceramic Tubes

    Science.gov (United States)

    Tsao, David; Okos, Martin

    1997-01-01

    This research involves the manipulation of the root-zone water potential for the purposes of discriminating the rate limiting step in the inorganic nutrient uptake mechanism utilized by higher plants. This reaction sequence includes the pathways controlled by the root-zone conditions such as water tension and gradient concentrations. Furthermore, plant based control mechanisms dictated by various protein productions are differentiated as well. For the nutrients limited by the environmental availability, the kinetics were modeled using convection and diffusion equations. Alternatively, for the nutrients dependent upon enzyme manipulations, the uptakes are modeled using Michaelis-Menten kinetics. In order to differentiate between these various mechanistic steps, an experimental apparatus known as the Porous Ceramic Tube - Nutrient Delivery System (PCT-NDS) was used. Manipulation of the applied suction pressure circulating a nutrient solution through this system imposes a change in the matric component of the water potential. This compensates for the different osmotic components of water potential dictated by nutrient concentration. By maintaining this control over the root-zone conditions, the rate limiting steps in the uptake of the essential nutrients into tomato plants (Lycopersicon esculentum cv. Cherry Elite) were differentiated. Results showed that the uptake of some nutrients were mass transfer limited while others were limited by the enzyme kinetics. Each of these were adequately modeled with calculations and discussions of the parameter estimations provided.

  5. Biochemical characteristics and modulation by external and internal factors of aminopeptidase-N activity in the hepatopancreas of a euryhaline burrowing crab.

    Science.gov (United States)

    Michiels, M S; del Valle, J C; López Mañanes, A A

    2015-07-01

    Strikingly, in spite of its physiological importance, information about occurrence, biochemical characteristics and mechanisms of regulation of aminopeptidase-N (APN) in the hepatopancreas of intertidal euryhaline crabs is still lacking. In this work, we determined the occurrence, biochemical characteristics, response to environmental salinity and dopamine of APN in the hepatopancreas of the euryhaline crab Neohelice granulata (Dana 1851) from the open mudflat of Mar Chiquita coastal lagoon (Buenos Aires province, Argentina). APN activity was maximal at pH and temperature range of 7.6-9.0 and 37-45 °C, respectively. APN activity exhibited Michaelis-Menten kinetics (apparent Km = 0.19 ± 0.10 mM) (pH 7.6, 37 °C) and appeared to be sensitive to bestatin (I 50 = 15 mM) and EDTA (I 50 = 9 mM). In crabs acclimated to 10 psu (hyper-regulation conditions) and 37 psu (hypo-regulation conditions), APN activity was about 45 and 160% higher, respectively, than in 35 psu (osmoconformation). APN activity in the hepatopancreas was stimulated in vitro (about 137%) by 10(-4) M dopamine. Higher dopamine concentrations produced a similar extent of increase. The responses of APN activity to salinity and dopamine in vitro suggest the role of APN in digestive adjustments upon hyper and hypo-regulatory conditions and its modulation via direct mechanisms on hepatopancreas by dopamine.

  6. Eugenol nanocapsule for enhanced therapeutic activity against periodontal infections.

    Science.gov (United States)

    Pramod, Kannissery; Aji Alex, M R; Singh, Manisha; Dang, Shweta; Ansari, Shahid H; Ali, Javed

    2016-01-01

    Eugenol is a godsend to dental care due to its analgesic, local anesthetic, and anti-inflammatory and antibacterial effects. The aim of the present research work was to prepare, characterize and evaluate eugenol-loaded nanocapsules (NCs) against periodontal infections. Eugenol-loaded polycaprolactone (PCL) NCs were prepared by solvent displacement method. The nanometric size of the prepared NCs was confirmed by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The in vitro drug release was found to follow a biphasic pattern and followed Michaelis-Menten like model. The percentage cell viability values near to 100 in the cell viability assay indicated that the NCs are not cytotoxic. In the in vivo studies, the eugenol NC group displayed significant difference in the continuity of epithelium of the interdental papilla in comparison to the untreated, pure eugenol and placebo groups. The in vivo performance of the eugenol-loaded NCs using ligature-induced periodontitis model in rats indicated that eugenol-loaded NCs could prevent septal bone resorption in periodontitis. On the basis of our research findings it could be concluded that eugenol-loaded PCL NCs could serve as a novel colloidal drug delivery system for enhanced therapeutic activity of eugenol in the treatment of periodontal infections.

  7. Steady-state oxidation of cholesterol catalyzed by cholesterol oxidase in lipid bilayer membranes on platinum electrodes

    International Nuclear Information System (INIS)

    Bokoch, Michael P.; Devadoss, Anando; Palencsar, Mariela S.; Burgess, James D.

    2004-01-01

    Cholesterol oxidase is immobilized in electrode-supported lipid bilayer membranes. Platinum electrodes are initially modified with a self-assembled monolayer of thiolipid. A vesicle fusion method is used to deposit an outer leaflet of phospholipids onto the thiolipid monolayer forming a thiolipid/lipid bilayer membrane on the electrode surface. Cholesterol oxidase spontaneously inserts into the electrode-supported lipid bilayer membrane from solution and is consequently immobilized to the electrode surface. Cholesterol partitions into the membrane from buffer solutions containing cyclodextrin. Cholesterol oxidase catalyzes the oxidation of cholesterol by molecular oxygen, forming hydrogen peroxide as a product. Amperometric detection of hydrogen peroxide for continuous solution flow experiments are presented, where flow was alternated between cholesterol solution and buffer containing no cholesterol. Steady-state anodic currents were observed during exposures of cholesterol solutions ranging in concentration from 10 to 1000 μM. These data are consistent with the Michaelis-Menten kinetic model for oxidation of cholesterol as catalyzed by cholesterol oxidase immobilized in the lipid bilayer membrane. The cholesterol detection limit is below 1 μM for cholesterol solution prepared in buffered cyclodextrin. The response of the electrodes to low density lipoprotein solutions is increased upon addition of cyclodextrin. Evidence for adsorption of low density lipoprotein to the electrode surface is presented

  8. Evaluation of sources of variation on in vitro fermentation kinetics of feedstuffs in a gas production system.

    Science.gov (United States)

    Keim, Juan P; Alvarado-Gilis, Christian; Arias, Rodrigo A; Gandarillas, Mónica; Cabanilla, Jaime

    2017-10-01

    The aim of this study was to evaluate the effect of different sources of variation in gas production technique on the in vitro gas production kinetics of feedstuffs. Triplicates of commercial concentrate, grass silage, grass hay and grass pasture were incubated in three experiments: experiment 1 assessed two agitation methods; experiment 2 evaluated different rumen inocula (pooled or different donor cows for each incubation run); and experiment 3 used Goering-Van Soest or Mould buffers for media preparation. Gas production data were fitted into the Michaelis-Menten model and then subjected to analysis of variance. Gas production (GP) at 48 h and asymptote gas production (A) were lower when bottles were continuously under horizontal movement. Time to produce half and 75% of A, and A were affected by rumen inocula, while buffer type affected time to produce half and 25% of A and GP. No interactions between substrates and sources of variation were observed, suggesting that the effects of substrates on GP parameters were not modified. It is concluded that comparison of numerical data from in vitro experiments that follow different protocols must be done carefully. However, the ranking of different substrates is more robust and less affected by the sources of variation. © 2017 Japanese Society of Animal Science.

  9. Kinetic analysis of enzyme systems with suicide substrate in the presence of a reversible competitive inhibitor, tested by simulated progress curves.

    Science.gov (United States)

    Moruno-Dávila, M A; Garrido-del Solo, C; García-Moreno, M; Havsteen, B H; Garcia-Sevilla, F; Garcia-Cánovas, F; Varón, R

    2001-02-01

    The use of suicide substrates remains a very important and useful method in enzymology for studying enzyme mechanisms and designing potential drugs. Suicide substrates act as modified substrates for the target enzymes and bind to the active site. Therefore the presence of a competitive reversible inhibitor decreases the rate of substrate-induced inactivation and protects the enzyme from this inactivation. This lowering on the inactivation rate has evident physiological advantages, since it allows the easy acquisition of experimental data and facilitates kinetic data analysis by providing another variable (inhibitor concentration). However despite the importance of the simultaneous action of a suicide substrate and a competitive reversible inhibition, to date no corresponding kinetic analysis has been carried out. Therefore we present a general kinetic analysis of a Michaelis-Menten reaction mechanism with double inhibition caused by both, a suicide substrate and a competitive reversible inhibitor. We assume rapid equilibrium of the reversible reaction steps involved, while the time course equations for the reaction product have been derived with the assumption of a limiting enzyme. The goodness of the analytical solutions has been tested by comparison with the simulated curves obtained by numerical integration. A kinetic data analysis to determine the corresponding kinetic parameters from the time progress curve of the product is suggested. In conclusion, we present a complete kinetic analysis of an enzyme reaction mechanism as described above in an attempt to fill a gap in the theoretical treatment of this type of system.

  10. Enantiomeric fractioning, degradation and metabolite formation of Mecoprop in subsoils with a phenoxy acid contamination history

    DEFF Research Database (Denmark)

    Frkova, Zuzana; Johansen, Anders; Karlson, Ulrich G.

    2015-01-01

    for their ability to degrade mecoprop under natural and amended conditions. Degradation of mecoprop was studied at elevated and environmentally relevant mecoprop concentrations as affected by nitrate and glucose at nitrate-reducing conditions and at a presence of oxygen (mimicking purging the soil with air. Results......As persistence and toxicity of the enantiomers of chiral pesticides are different a more comprehensive understanding of the fate of enantiomers of agrochemicals in the environment is necessary. Subsoils sampled vertically (2.5-6 m) at a site with a history of phenoxy acid contamination were used...... and enantioselectivity. Glucose hinders mecoprop degradation and changes the EF. Changing EF confirmed enzymatic dgradation of mecoprop in soils, which was well interpreted using the Michaelis-Menten kinetic model. The highest mecoprop degradation rate was measured in soils incubated at nitrate-reducing conditions...

  11. Model-Based Optimization of Scaffold Geometry and Operating Conditions of Radial Flow Packed-Bed Bioreactors for Therapeutic Applications

    Directory of Open Access Journals (Sweden)

    Danilo Donato

    2014-01-01

    Full Text Available Radial flow perfusion of cell-seeded hollow cylindrical porous scaffolds may overcome the transport limitations of pure diffusion and direct axial perfusion in the realization of bioengineered substitutes of failing or missing tissues. Little has been reported on the optimization criteria of such bioreactors. A steady-state model was developed, combining convective and dispersive transport of dissolved oxygen with Michaelis-Menten cellular consumption kinetics. Dimensional analysis was used to combine more effectively geometric and operational variables in the dimensionless groups determining bioreactor performance. The effectiveness of cell oxygenation was expressed in terms of non-hypoxic fractional construct volume. The model permits the optimization of the geometry of hollow cylindrical constructs, and direction and magnitude of perfusion flow, to ensure cell oxygenation and culture at controlled oxygen concentration profiles. This may help engineer tissues suitable for therapeutic and drug screening purposes.

  12. Transformation frequency of γ irradiated plasmid DNA and the enzymatic double strand break formation by incubation in a protein extract of Escherichia coli

    International Nuclear Information System (INIS)

    Schulte-Frohlinde, D.; Mark, F.; Ventur, Y.

    1994-01-01

    It was found that incubation of γ-irradiated or DNaseI-treated plasmid DNA in a protein extract of Escherichia coli leads to enzyme-induced formation of double strand breaks (dsb) in competition with repair of precursors of these dsb. A survival curve of the plasmid DNA (as determined by transformation of E. coli) was calculated on the basis of enzyme-induced dsb as well as those produced by irradiation assuming that they are lethal. The calculated D O value was the same as that measured directly by transformation of irradiated plasmid DNA. Two models are presented that fit the experimental survival data as a function of dose. One is based on damage formation in the plasmid DNA including enzymatic conversion of single strand damage into dsb (U-model), the other is an enzymatic repair saturation model based on Michaelis-Menten kinetics. (Author)

  13. Aza Cope Rearrangement of Propargyl Enammonium Cations Catalyzed By a Self-Assembled `Nanozyme

    Energy Technology Data Exchange (ETDEWEB)

    Hastings, Courntey J.; Fiedler, Dorothea; Bergman, Robert G.; Raymond, Kenneth N.

    2008-02-27

    The tetrahedral [Ga{sub 4}L{sub 6}]{sup 12-} assembly (L = N,N-bis(2,3-dihydroxybenzoyl)-1,5-diaminonaphthalene) encapsulates a variety of cations, including propargyl enammonium cations capable of undergoing the aza Cope rearrangement. For propargyl enammonium substrates that are encapsulated in the [Ga{sub 4}L{sub 6}]{sup 12-} assembly, rate accelerations of up to 184 are observed when compared to the background reaction. After rearrangement, the product iminium ion is released into solution and hydrolyzed allowing for catalytic turnover. The activation parameters for the catalyzed and uncatalyzed reaction were determined, revealing that a lowered entropy of activation is responsible for the observed rate enhancements. The catalyzed reaction exhibits saturation kinetics; the rate data obey the Michaelis-Menten model of enzyme kinetics, and competitive inhibition using a non-reactive guest has been demonstrated.

  14. Parallel synthesis of libraries of anodic and cathodic functionalized electrodeposition paints as immobilization matrix for amperometric biosensors.

    Science.gov (United States)

    Ngounou, Bertrand; Aliyev, Elchin H; Guschin, Dmitrii A; Sultanov, Yusif M; Efendiev, Ayaz A; Schuhmann, Wolfgang

    2007-09-01

    The integration of flexible anchoring groups bearing imidazolyl or pyridyl substituents into the structure of electrodeposition paints (EDP) is the basis for the parallel synthesis of a library containing 107 members of different cathodic and anodic EDPs with a high variation in polymer properties. The obtained EDPs were used as immobilization matrix for biosensor fabrication using glucose oxidase as a model enzyme. Amperometric glucose sensors based on the different EDPs showed a wide variation in their sensor characteristics with respect to the apparent Michaelis-Menten constant (KM(app)) representing the linear measuring range and the maximum current (Imax(app)). Based on these results first assumptions concerning the impact of different side chains in the EDP on the expected biosensor properties could be obtained allowing for an improved rational optimization of EDPs used as immobilization matrix in amperometric biosensors.

  15. Characteristics of 36C103- influx into nitrate reductase deficient mutant E1 pisum sativum seedlings: evidence for restricted ''induction'' by nitrate compared with wild type

    International Nuclear Information System (INIS)

    Deane-Drummond, C.E.; Jacobsen, E.

    1986-01-01

    The characteristics of nitrate uptake into seedlings of Pisum sativum L. cv. Rondo mutant E 1 defective for nitrate reductase (NR) and of its parent variety Rondo have been investigated using 36 C10 3 - as an analogue for nitrate. The apparent Michaelis Menten constants (K m ) for 36 ClO 3 - influx measured over 10 min were similar for mutant E 1 and the wild type (Wt). There was a 28% increase in 36 C10 3 - into Wt seedlings following nitrate pretreatment but this was not found when mutant seedlings were used. N starvation increased 36 C10 3 - influx into both mutant and Wt seedlings, and the rate of cycling E/I was also enhanced to a similar extent. The results are discussed in terms of current ideas on the regulation of nitrate uptake and assimilation. (author)

  16. A Critical View on In Vitro Analysis of P-glycoprotein (P-gp) Transport Kinetics

    DEFF Research Database (Denmark)

    Saaby, Lasse; Brodin, Birger

    2017-01-01

    Transport proteins expressed in the different barriers of the human body can have great implications on absorption, distribution, and excretion of drug compounds. Inhibition or saturation of a transporter can potentially alter these absorbtion, distribution, metabolism and elimination properties...... and thereby also the pharmacokinetic profile and bioavailability of drug compounds. P-glycoprotein (P-gp, ABCB1) is an efflux transporter which is present in most of the barriers of the body, including the small intestine, the blood-brain barrier, the liver, and the kidney. In all these tissues, P-gp may...... mediate efflux of drug compounds and may also be a potential site for drug-drug interactions. Consequently, there is a need to be able to predict the saturation and inhibition of P-gp and other transporters in vivo. For this purpose, Michaelis-Menten steady-state analysis has been applied to estimate...

  17. A Solution of the Convective-Diffusion Equation for Solute Mass Transfer inside a Capillary Membrane Bioreactor

    Directory of Open Access Journals (Sweden)

    B. Godongwana

    2010-01-01

    Full Text Available This paper presents an analytical model of substrate mass transfer through the lumen of a membrane bioreactor. The model is a solution of the convective-diffusion equation in two dimensions using a regular perturbation technique. The analysis accounts for radial-convective flow as well as axial diffusion of the substrate specie. The model is applicable to the different modes of operation of membrane bioreactor (MBR systems (e.g., dead-end, open-shell, or closed-shell mode, as well as the vertical or horizontal orientation. The first-order limit of the Michaelis-Menten equation for substrate consumption was used to test the developed model against available analytical results. The results obtained from the application of this model, along with a biofilm growth kinetic model, will be useful in the derivation of an efficiency expression for enzyme production in an MBR.

  18. The kinetics of denitrification in permeable sediments

    DEFF Research Database (Denmark)

    Evrard, Victor; Glud, Ronnie N.; Cook, Perran L. M.

    2013-01-01

    Permeable sediments comprise the majority of shelf sediments, yet the rates of denitrification remain highly uncertain in these environments. Computational models are increasingly being used to understand the dynamics of denitrification in permeable sediments, which are complex environments...... on sediments taken from six shallow coastal sites in Port Phillip Bay, Victoria, Australia. The results showed that denitrification commenced rapidly (within 30 min) after the onset of anoxia and the kinetics could be well described by Michaelis-Menten kinetics with half saturation constants (apparent K...... in cohesive sediments despite organic carbon contents one order of magnitude lower for the sediments studied here. The ratio of sediment O-2 consumption to V-max was in the range of 0.02-0.09, and was on average much lower than the theoretical ratio of 0.8. As a consequence, models implemented...

  19. Development of a highly efficient indigo dyeing method using indican with an immobilized beta-glucosidase from Aspergillus niger.

    Science.gov (United States)

    Song, Jingyuan; Imanaka, Hiroyuki; Imamura, Koreyoshi; Kajitani, Kouichi; Nakanishi, Kazuhiro

    2010-09-01

    A highly efficient method for dyeing textiles with indigo is described. In this method, the substrate, indican is first hydrolyzed at an acidic pH of 3 using an immobilized beta-glucosidase to produce indoxyl, under which conditions indigo formation is substantially repressed. The textile sample is then dipped in the prepared indoxyl solution and the textile is finally exposed to ammonia vapor for a short time, resulting in rapid indigo dyeing. As an enzyme, we selected a beta-glucosidase from Aspergillus niger, which shows a high hydrolytic activity towards indican and was thermally stable at temperatures up to 50-60 degrees C, in an acidic pH region. The A. niger beta-glucosidase, when immobilized on Chitopearl BCW-3001 by treatment with glutaraldehyde, showed an optimum reaction pH similar to that of the free enzyme with a slightly higher thermal stability. The kinetics for the hydrolysis of indican at pH 3, using the purified free and immobilized enzymes was found to follow Michaelis-Menten type kinetics with weak competitive inhibition by glucose. Using the immobilized enzyme, we successfully carried out repeated-batch and continuous hydrolyses of indican at pH 3 when nitrogen gas was continuously supplied to the substrate solution. Various types of model textiles were dyed using the proposed method although the color yield varied, depending on the type of textile used. Copyright 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  20. Graphene as a Prototypical Model for Two-Dimensional Continuous Mechanics

    Directory of Open Access Journals (Sweden)

    Philippe Lambin

    2017-08-01

    Full Text Available This paper reviews a few problems where continuous-medium theory specialized to two-dimensional media provides a qualitatively correct picture of the mechanical behavior of graphene. A critical analysis of the parameters involved is given. Among other results, a simple mathematical description of a folded graphene sheet is proposed. It is also shown how the graphene–graphene adhesion interaction is related to the cleavage energy of graphite and its C 33 bulk elastic constant.

  1. Processing-structure-mechanical property relationship in Ti-Nb microalloyed steel: Continuous cooling versus interrupted cooling

    Energy Technology Data Exchange (ETDEWEB)

    Natarajan, V.V. [Laboratory for Excellence in Advanced Steel Research, Materials Science and Engineering Program, Department of Metallurgical, Materials and Biomedical Engineering, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968 (United States); Liu, S. [Laboratory for Excellence in Advanced Steel Research, Materials Science and Engineering Program, Department of Metallurgical, Materials and Biomedical Engineering, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968 (United States); School of Materials Science and Engineering, University of Science and Technology, Beijing (China); Challa, V.S.A. [Laboratory for Excellence in Advanced Steel Research, Materials Science and Engineering Program, Department of Metallurgical, Materials and Biomedical Engineering, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968 (United States); Misra, R.D.K., E-mail: dmisra2@utep.edu [Laboratory for Excellence in Advanced Steel Research, Materials Science and Engineering Program, Department of Metallurgical, Materials and Biomedical Engineering, University of Texas at El Paso, 500 W. University Avenue, El Paso, TX 79968 (United States); Sidorenko, D.M.; Mulholland, M.D.; Manohar, M.; Hartmann, J.E. [ArcelorMittal Global R& D Center, 3001 East Columbus Drive, East Chicago, IN 46312 (United States)

    2016-08-01

    The process parameters associated with thermo-mechanical controlled processing (TMCP) of steels play an important role in influencing the ultimate mechanical properties. The study of TMCP parameters have not received the required attention. In this regard, we elucidate here the impact of finish cooling temperature on interrupted cooling and compare with continuous cooling on microstructural evolution and precipitation behavior and associated mechanical properties in Ti-Nb microalloyed steels. The microstructural evolution was studied via transmission electron microscopy and electron back scattered diffraction (EBSD). The microstructure of continuously cooled and interrupted cooled steels with different finish exit temperatures consisted of polygonal ferrite, bainite and martensite/austenite constituent. However, the fraction of different microstructural constituents was different in each of the experimental steels. Similarly, there were differences in the distribution and average size of (Nb, Ti)C precipitates. The aforementioned differences in the microstructure and precipitation introduced differences in tensile properties. Furthermore, electron back scattered diffraction studies indicated distinct variation in average grain area and high angle boundaries between continuously cooled and interrupted cooled steels.

  2. Annealing helicase HARP closes RPA-stabilized DNA bubbles non-processively.

    Science.gov (United States)

    Burnham, Daniel R; Nijholt, Bas; De Vlaminck, Iwijn; Quan, Jinhua; Yusufzai, Timur; Dekker, Cees

    2017-05-05

    We investigate the mechanistic nature of the Snf2 family protein HARP, mutations of which are responsible for Schimke immuno-osseous dysplasia. Using a single-molecule magnetic tweezers assay, we construct RPA-stabilized DNA bubbles within torsionally constrained DNA to investigate the annealing action of HARP on a physiologically relevant substrate. We find that HARP closes RPA-stabilized bubbles in a slow reaction, taking on the order of tens of minutes for ∼600 bp of DNA to be re-annealed. The data indicate that DNA re-anneals through the removal of RPA, which is observed as clear steps in the bubble-closing traces. The dependence of the closing rate on both ionic strength and HARP concentration indicates that removal of RPA occurs via an association-dissociation mechanism where HARP does not remain associated with the DNA. The enzyme exhibits classical Michaelis-Menten kinetics and acts cooperatively with a Hill coefficient of 3 ± 1. Our work also allows the determination of some important features of RPA-bubble structures at low supercoiling, including the existence of multiple bubbles and that RPA molecules are mis-registered on the two strands. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Relation of cardiac energy metabolism to workload studied with 31P NMR

    International Nuclear Information System (INIS)

    Ligeti, L.; Osbakken, M.; Clark, B.J.; Schnall, M.; Bolinger, L.; Subramanian, H.; Leigh, J.S.; Chance, B.

    1986-01-01

    The relationship between myocardial work and concentration of mediators of oxidative phosphorylation (ATP, PCr, Pi) was studied in vivo in 6 dogs and 4 cats with 31 P NMR. The heart was exposed via thoracotomy for placement of surface coils and pacing electrodes. Work (defined as heart rate X blood pressure product) was increased by pacing the heart from 2Hz (rest) to 5Hz. In cats, an increase in work from 1.9 x 10 4 +/- .32 to 2.4 x 10 4 +/- .08 was associated with an increase in Pi/PCr ratio from .29 +/- .08 to .68 +/- .33; in dogs, work increase from 1.74 x 10 4 .82 to 3.4 x 10 4 +/- .84 did not cause significant change in Pi/PCr (.29 +/- .15 to .30 +/- .17). If these data are analyzed via the Michaelis-Menten algorithm, the cat heart can be considered to be set closer to V/sub max/ and the dog heart set closer to V/sub o/ (i.e. having a larger metabolic reserve). The difference between the 2 species could be due to microvascular and/or metabolic control mechanisms. Delineation of the different metabolic responses to work in these animal models may be helpful in understanding the physiological basis of heart disease

  4. In vivo measurements of brain glucose transport using the reversible michaelis-menten model and simultaneous measurements of cerebral blood flow changes during hypoglycemia

    OpenAIRE

    Choi, I.-Y.; Lee, S.-P.; Kim, S.-G.; Gruetter, R.

    2001-01-01

    Glucose is the major substrate that sustains normal brain function. When the brain glucose concentration approaches zero, glucose transport across the blood-brain barrier becomes rate limiting for metabolism during, for example, increased metabolic activity and hypoglycemia. Steady-state brain glucose concentrations in α-chloralose anesthetized rats were measured noninvasively as a function of plasma glucose. The relation between brain and plasma glucose was linear at 4.5 to 30 mmol/L plasma ...

  5. Seeing diabetes: visual detection of glucose based on the intrinsic peroxidase-like activity of MoS2 nanosheets

    Science.gov (United States)

    Lin, Tianran; Zhong, Liangshuang; Guo, Liangqia; Fu, Fengfu; Chen, Guonan

    2014-09-01

    Molybdenum disulfide (MoS2) has attracted increasing research interest recently due to its unique physical, optical and electrical properties, correlated with its 2D ultrathin atomic-layered structure. Until now, however, great efforts have focused on its applications such as lithium ion batteries, transistors, and hydrogen evolution reactions. Herein, for the first time, MoS2 nanosheets are discovered to possess an intrinsic peroxidase-like activity and can catalytically oxidize 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to produce a color reaction. The catalytic activity follows the typical Michaelis-Menten kinetics and is dependent on temperature, pH, H2O2 concentration, and reaction time. Based on this finding, a highly sensitive and selective colorimetric method for H2O2 and glucose detection is developed and applied to detect glucose in serum samples. Moreover, a simple, inexpensive, instrument-free and portable test kit for the visual detection of glucose in normal and diabetic serum samples is constructed by utilizing agarose hydrogel as a visual detection platform.Molybdenum disulfide (MoS2) has attracted increasing research interest recently due to its unique physical, optical and electrical properties, correlated with its 2D ultrathin atomic-layered structure. Until now, however, great efforts have focused on its applications such as lithium ion batteries, transistors, and hydrogen evolution reactions. Herein, for the first time, MoS2 nanosheets are discovered to possess an intrinsic peroxidase-like activity and can catalytically oxidize 3,3',5,5'-tetramethylbenzidine (TMB) by H2O2 to produce a color reaction. The catalytic activity follows the typical Michaelis-Menten kinetics and is dependent on temperature, pH, H2O2 concentration, and reaction time. Based on this finding, a highly sensitive and selective colorimetric method for H2O2 and glucose detection is developed and applied to detect glucose in serum samples. Moreover, a simple, inexpensive

  6. Species Differences in the Oxidative Desulfurization of a Thiouracil-Based Irreversible Myeloperoxidase Inactivator by Flavin-Containing Monooxygenase Enzymes.

    Science.gov (United States)

    Eng, Heather; Sharma, Raman; Wolford, Angela; Di, Li; Ruggeri, Roger B; Buckbinder, Leonard; Conn, Edward L; Dalvie, Deepak K; Kalgutkar, Amit S

    2016-08-01

    N1-Substituted-6-arylthiouracils, represented by compound 1 [6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one], are a novel class of selective irreversible inhibitors of human myeloperoxidase. The present account is a summary of our in vitro studies on the facile oxidative desulfurization in compound 1 to a cyclic ether metabolite M1 [5-(2,4-dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one] in NADPH-supplemented rats (t1/2 [half-life = mean ± S.D.] = 8.6 ± 0.4 minutes) and dog liver microsomes (t1/2 = 11.2 ± 0.4 minutes), but not in human liver microsomes (t1/2 > 120 minutes). The in vitro metabolic instability also manifested in moderate-to-high plasma clearances of the parent compound in rats and dogs with significant concentrations of M1 detected in circulation. Mild heat deactivation of liver microsomes or coincubation with the flavin-containing monooxygenase (FMO) inhibitor imipramine significantly diminished M1 formation. In contrast, oxidative metabolism of compound 1 to M1 was not inhibited by the pan cytochrome P450 inactivator 1-aminobenzotriazole. Incubations with recombinant FMO isoforms (FMO1, FMO3, and FMO5) revealed that FMO1 principally catalyzed the conversion of compound 1 to M1. FMO1 is not expressed in adult human liver, which rationalizes the species difference in oxidative desulfurization. Oxidation by FMO1 followed Michaelis-Menten kinetics with Michaelis-Menten constant, maximum rate of oxidative desulfurization, and intrinsic clearance values of 209 μM, 20.4 nmol/min/mg protein, and 82.7 μl/min/mg protein, respectively. Addition of excess glutathione essentially eliminated the conversion of compound 1 to M1 in NADPH-supplemented rat and dog liver microsomes, which suggests that the initial FMO1-mediated S-oxygenation of compound 1 yields a sulfenic acid intermediate capable of redox cycling to the parent compound in a glutathione-dependent fashion or undergoing further oxidation to a more

  7. Modeling metabolic networks in C. glutamicum: a comparison of rate laws in combination with various parameter optimization strategies

    Directory of Open Access Journals (Sweden)

    Oldiges Marco

    2009-01-01

    Full Text Available Abstract Background To understand the dynamic behavior of cellular systems, mathematical modeling is often necessary and comprises three steps: (1 experimental measurement of participating molecules, (2 assignment of rate laws to each reaction, and (3 parameter calibration with respect to the measurements. In each of these steps the modeler is confronted with a plethora of alternative approaches, e. g., the selection of approximative rate laws in step two as specific equations are often unknown, or the choice of an estimation procedure with its specific settings in step three. This overall process with its numerous choices and the mutual influence between them makes it hard to single out the best modeling approach for a given problem. Results We investigate the modeling process using multiple kinetic equations together with various parameter optimization methods for a well-characterized example network, the biosynthesis of valine and leucine in C. glutamicum. For this purpose, we derive seven dynamic models based on generalized mass action, Michaelis-Menten and convenience kinetics as well as the stochastic Langevin equation. In addition, we introduce two modeling approaches for feedback inhibition to the mass action kinetics. The parameters of each model are estimated using eight optimization strategies. To determine the most promising modeling approaches together with the best optimization algorithms, we carry out a two-step benchmark: (1 coarse-grained comparison of the algorithms on all models and (2 fine-grained tuning of the best optimization algorithms and models. To analyze the space of the best parameters found for each model, we apply clustering, variance, and correlation analysis. Conclusion A mixed model based on the convenience rate law and the Michaelis-Menten equation, in which all reactions are assumed to be reversible, is the most suitable deterministic modeling approach followed by a reversible generalized mass action kinetics

  8. Thermo-mechanical simulation and parameters optimization for beam blank continuous casting

    International Nuclear Information System (INIS)

    Chen, W.; Zhang, Y.Z.; Zhang, C.J.; Zhu, L.G.; Lu, W.G.; Wang, B.X.; Ma, J.H.

    2009-01-01

    The objective of this work is to optimize the process parameters of beam blank continuous casting in order to ensure high quality and productivity. A transient thermo-mechanical finite element model is developed to compute the temperature and stress profile in beam blank continuous casting. By comparing the calculated data with the metallurgical constraints, the key factors causing defects of beam blank can be found out. Then based on the subproblem approximation method, an optimization program is developed to search out the optimum cooling parameters. Those optimum parameters can make it possible to run the caster at its maximum productivity, minimum cost and to reduce the defects. Now, online verifying of this optimization project has been put in practice, which can prove that it is very useful to control the actual production

  9. Modelling and finite-time stability analysis of psoriasis pathogenesis

    Science.gov (United States)

    Oza, Harshal B.; Pandey, Rakesh; Roper, Daniel; Al-Nuaimi, Yusur; Spurgeon, Sarah K.; Goodfellow, Marc

    2017-08-01

    A new systems model of psoriasis is presented and analysed from the perspective of control theory. Cytokines are treated as actuators to the plant model that govern the cell population under the reasonable assumption that cytokine dynamics are faster than the cell population dynamics. The analysis of various equilibria is undertaken based on singular perturbation theory. Finite-time stability and stabilisation have been studied in various engineering applications where the principal paradigm uses non-Lipschitz functions of the states. A comprehensive study of the finite-time stability properties of the proposed psoriasis dynamics is carried out. It is demonstrated that the dynamics are finite-time convergent to certain equilibrium points rather than asymptotically or exponentially convergent. This feature of finite-time convergence motivates the development of a modified version of the Michaelis-Menten function, frequently used in biology. This framework is used to model cytokines as fast finite-time actuators.

  10. Statistical identifiability and convergence evaluation for nonlinear pharmacokinetic models with particle swarm optimization.

    Science.gov (United States)

    Kim, Seongho; Li, Lang

    2014-02-01

    The statistical identifiability of nonlinear pharmacokinetic (PK) models with the Michaelis-Menten (MM) kinetic equation is considered using a global optimization approach, which is particle swarm optimization (PSO). If a model is statistically non-identifiable, the conventional derivative-based estimation approach is often terminated earlier without converging, due to the singularity. To circumvent this difficulty, we develop a derivative-free global optimization algorithm by combining PSO with a derivative-free local optimization algorithm to improve the rate of convergence of PSO. We further propose an efficient approach to not only checking the convergence of estimation but also detecting the identifiability of nonlinear PK models. PK simulation studies demonstrate that the convergence and identifiability of the PK model can be detected efficiently through the proposed approach. The proposed approach is then applied to clinical PK data along with a two-compartmental model. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  11. Preparation of biosensors by immobilization of polyphenol oxidase in conducting copolymers and their use in determination of phenolic compounds in red wine.

    Science.gov (United States)

    Böyükbayram, A Elif; Kiralp, Senem; Toppare, Levent; Yağci, Yusuf

    2006-10-01

    Electrochemically produced graft copolymers of thiophene capped polytetrahydofuran (TPTHF1 and TPTHF2) and pyrrole were achieved by constant potential electrolysis using sodium dodecylsulfate (SDS) as the supporting electrolyte. Characterizations were based on Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Electrical conductivities were measured by the four-probe technique. Novel biosensors for phenolic compounds were constructed by immobilizing polyphenol oxidase (PPO) into conducting copolymers prepared by electropolymerization of pyrrole with thiophene capped polytetrahydrofuran. Kinetic parameters, maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) and optimum conditions regarding temperature and pH were determined for the immobilized enzyme. Operational stability and shelf-life of the enzyme electrodes were investigated. Enzyme electrodes of polyphenol oxidase were used to determine the amount of phenolic compounds in two brands of Turkish red wines and found very useful owing to their high kinetic parameters and wide pH working range.

  12. Kinetic Studies on Trichoderna Viride Cellulase

    International Nuclear Information System (INIS)

    Saw Aung; Oo Aung; Aung Myint

    2002-02-01

    Studies on cellulase enzyme (EC 3.2.1.4), which catalyzes the hydrolysis of. cellulose to yield glucose, were made. Cellulase from a fungus source, Trichoderma viride was cultivated on Czapek's agar medium and enzyme production broth medium was employed for parameter tests. The microscopic examination and cellulase hydrolysis test on subcultured fungi were applied to confirm the T. viride species. A calibration curve for standard glucose was plotted by using visible spectroscopy. Dinitrosalicylic acid was used as enzyme reaction inhibitor and the colour intensity was measured in a UV-visible spectrophotometer at a λ max of 570 nm. The parameters such as optimum pH, optimum temperature, effect of substrate concentration, effect, of enzyme concentration, enzyme unit (EU), reaction order (n), maximum velocity (V max ), Michaelis-Menten constant (K m ) using various substrates, viz., carboxy methylcellulose, cotton fibre and filter paper determined. (author)

  13. Evidence of enzymatic catalysis of oxygen reduction on stainless steels under marine biofilm.

    Science.gov (United States)

    Faimali, Marco; Benedetti, Alessandro; Pavanello, Giovanni; Chelossi, Elisabetta; Wrubl, Federico; Mollica, Alfonso

    2011-04-01

    Cathodic current trends on stainless steel samples with different surface percentages covered by biofilm and potentiostatically polarized in natural seawater were studied under oxygen concentration changes, temperature increases, and additions of enzymic inhibitors to the solution. The results showed that on each surface fraction covered by biofilm the oxygen reduction kinetics resembled a reaction catalyzed by an immobilised enzyme with high oxygen affinity (apparent Michaelis-Menten dissociation constant close to K(O(2))(M)  ≈ 10 μM) and low activation energy (W ≈ 20 KJ mole(-1)). The proposed enzyme rapidly degraded when the temperature was increased above the ambient (half-life time of ∼1 day at 25°C, and of a few minutes at 50°C). Furthermore, when reversible enzymic inhibitors (eg sodium azide and cyanide) were added, the cathodic current induced by biofilm growth was inhibited.

  14. Dimension reduction for stochastic dynamical systems forced onto a manifold by large drift: a constructive approach with examples from theoretical biology

    Science.gov (United States)

    Parsons, Todd L.; Rogers, Tim

    2017-10-01

    Systems composed of large numbers of interacting agents often admit an effective coarse-grained description in terms of a multidimensional stochastic dynamical system, driven by small-amplitude intrinsic noise. In applications to biological, ecological, chemical and social dynamics it is common for these models to posses quantities that are approximately conserved on short timescales, in which case system trajectories are observed to remain close to some lower-dimensional subspace. Here, we derive explicit and general formulae for a reduced-dimension description of such processes that is exact in the limit of small noise and well-separated slow and fast dynamics. The Michaelis-Menten law of enzyme-catalysed reactions, and the link between the Lotka-Volterra and Wright-Fisher processes are explored as a simple worked examples. Extensions of the method are presented for infinite dimensional systems and processes coupled to non-Gaussian noise sources.

  15. Tracer kinetics: Modelling of tracer behaviour in nonlinear and nonsteady state systems exemplified by the evaluation of protein turnover in plant organs

    International Nuclear Information System (INIS)

    Winkler, E.

    1991-01-01

    If nonlinear biological processes are investigated by means of tracer experiments they can be modelled with linear kinetic equations (compartment equations) as long as the total system is in a stationary state. But if nonstationary behaviour is included considerations on the kinetics of the individual processes are necessary. Within the range of biological and agricultural investigations especially first order reactions (constant fraction processes), zero order reactions (constant amount process) and saturation reactions (Michaelis-Menten-kinetics) are to be taken into account. A rigorous treatment of data based on system theory can be preceeded by graphic-algebraic procedure which may be more or less uncertain in its results but which can easily be handled. An example is given of methodological considerations concerning the combination of evaluation procedures and the discrimination between different reaction mechanisms. It treats protein turnover in 2 different parts of growing wheat plants investigated by means of an 15 N-tracer experiment. Whereas in a stationary system (upper stalk section) linear tracer equations were sufficient irrespective of the true reaction mechanism, for protein synthesis in the upper leaf as a nonstationary system it was necessary to decide between the hypotheses of a zero order and a first order reaction. In accordance with statements in the literature the unambiguous result was a combination of protein synthesis as a zero order process and of protein degradation as a first order process. (orig.) [de

  16. Energetics of glucose metabolism: a phenomenological approach to metabolic network modeling.

    Science.gov (United States)

    Diederichs, Frank

    2010-08-12

    A new formalism to describe metabolic fluxes as well as membrane transport processes was developed. The new flux equations are comparable to other phenomenological laws. Michaelis-Menten like expressions, as well as flux equations of nonequilibrium thermodynamics, can be regarded as special cases of these new equations. For metabolic network modeling, variable conductances and driving forces are required to enable pathway control and to allow a rapid response to perturbations. When applied to oxidative phosphorylation, results of simulations show that whole oxidative phosphorylation cannot be described as a two-flux-system according to nonequilibrium thermodynamics, although all coupled reactions per se fulfill the equations of this theory. Simulations show that activation of ATP-coupled load reactions plus glucose oxidation is brought about by an increase of only two different conductances: a [Ca(2+)] dependent increase of cytosolic load conductances, and an increase of phosphofructokinase conductance by [AMP], which in turn becomes increased through [ADP] generation by those load reactions. In ventricular myocytes, this feedback mechanism is sufficient to increase cellular power output and O(2) consumption several fold, without any appreciable impairment of energetic parameters. Glucose oxidation proceeds near maximal power output, since transformed input and output conductances are nearly equal, yielding an efficiency of about 0.5. This conductance matching is fulfilled also by glucose oxidation of β-cells. But, as a price for the metabolic mechanism of glucose recognition, β-cells have only a limited capability to increase their power output.

  17. Routes to chaos in continuous mechanical systems: Part 2. Modelling transitions from regular to chaotic dynamics

    International Nuclear Information System (INIS)

    Krysko, A.V.; Awrejcewicz, J.; Papkova, I.V.; Krysko, V.A.

    2012-01-01

    In second part of the paper both classical and novel scenarios of transition from regular to chaotic dynamics of dissipative continuous mechanical systems are studied. A detailed analysis allowed us to detect the already known classical scenarios of transition from periodic to chaotic dynamics, and in particular the Feigenbaum scenario. The Feigenbaum constant was computed for all continuous mechanical objects studied in the first part of the paper. In addition, we illustrate and discuss different and novel scenarios of transition of the analysed systems from regular to chaotic dynamics, and we show that the type of scenario depends essentially on excitation parameters.

  18. Flux continuity and probability conservation in complexified Bohmian mechanics

    International Nuclear Information System (INIS)

    Poirier, Bill

    2008-01-01

    Recent years have seen increased interest in complexified Bohmian mechanical trajectory calculations for quantum systems as both a pedagogical and computational tool. In the latter context, it is essential that trajectories satisfy probability conservation to ensure they are always guided to where they are most needed. We consider probability conservation for complexified Bohmian trajectories. The analysis relies on time-reversal symmetry considerations, leading to a generalized expression for the conjugation of wave functions of complexified variables. This in turn enables meaningful discussion of complexified flux continuity, which turns out not to be satisfied in general, though a related property is found to be true. The main conclusion, though, is that even under a weak interpretation, probability is not conserved along complex Bohmian trajectories

  19. Growth, chamber building rate and reproduction time of Palaeonummulites venosus (Foraminifera) under natural conditions

    Science.gov (United States)

    Kinoshita, Shunichi; Eder, Wolfgang; Wöger, Julia; Hohenegger, Johann; Briguglio, Antonino

    2017-12-01

    We investigated the symbiont-bearing benthic foraminifer Palaeonummulites venosus to determine the chamber building rate (CBR), test diameter increase rate (DIR), reproduction time and longevity using the `natural laboratory' approach. This is based on the decomposition of monthly obtained frequency distributions of chamber number and test diameter into normally distributed components. Test measurements were taken using MicroCT. The shift of the mean and standard deviation of component parameters during the 15-month investigation period was used to calculate Michaelis-Menten functions applied to estimate the averaged CBR and DIR under natural conditions. The individual dates of birth were estimated using the inverse averaged CBR and the inverse DIR fitted by the individual chamber number or the individual test diameter at the sampling date. Distributions of frequencies and densities (i.e., frequency divided by sediment weight) based on both CBR and DIR revealed continuous reproduction throughout the year with two peaks, a stronger one in June determined as the onset of the summer generation (generation 1) and a weaker one in November determined as the onset of the winter generation (generation 2). This reproduction scheme explains the presence of small and large specimens in the same sample. Longevity, calculated as the maximum difference in days between the individual's birth date and the sampling date, is approximately 1.5 yr, an estimation obtained by using both CBR and DIR.

  20. Kinetics of the removal of mono-chlorobenzene vapour from waste gases using a trickle bed air biofilter.

    Science.gov (United States)

    Mathur, Anil K; Sundaramurthy, J; Balomajumder, C

    2006-10-11

    The performance of a trickle bed air biofilter (TBAB) in the removal of mono-chlorobenzene (MCB) was evaluated in concentrations varying from 0.133 to 7.187 g m(-3) and at empty bed residence time (EBRT) varying from 37.7 to 188.52 s. More than 90% removal efficiency in the trickle bed air biofilter was achieved for the inlet MCB concentration up to 1.069 g m(-3) and EBRT less than 94.26 s. The trickle bed air biofilter was constructed with coal packing material, inoculated with a mixed consortium of activated sludge obtained from sewage treatment plant. The continuous performance of the removal of MCB in the trickle bed air biofilter was monitored for various gas concentrations, gas flow rates, and empty bed residence time. The experiment was conducted for a period of 75 days. The trickle bed air biofilter degrading MCB with an average elimination capacity of 80 g m(-3) h(-1) was obtained. The effect of starvation was also studied. After starvation period of 8 days, the degradation was low but recovered within a short period of time. Using macrokinetic determination method, the Michaelis-Menten kinetic constant K(m) and maximum reaction rate, r(max) evaluated as 0.121 g m(-3) s(-1) and 7.45 g m(-3), respectively.

  1. Hysteresis response of daytime net ecosystem exchange during drought

    Directory of Open Access Journals (Sweden)

    N. Pingintha

    2010-03-01

    Full Text Available Continuous measurements of net ecosystem CO2 exchange (NEE using the eddy-covariance method were made over an agricultural ecosystem in the southeastern US. During optimum environmental conditions, photosynthetically active radiation (PAR was the primary driver controlling daytime NEE, accounting for as much as 67 to 89% of the variation in NEE. However, soil water content became the dominant factor limiting the NEE-PAR response during the peak growth stage. NEE was significantly depressed when high PAR values coincided with very low soil water content. The presence of a counter-clockwise hysteresis of daytime NEE with PAR was observed during periods of water stress. This is a result of the stomatal closure control of photosynthesis at high vapor pressure deficit and enhanced respiration at high temperature. This result is significant since this hysteresis effect limits the range of applicability of the Michaelis-Menten equation and other related expressions in the determination of daytime NEE as a function of PAR. The systematic presence of hysteresis in the response of NEE to PAR suggests that the gap-filling technique based on a non-linear regression approach should take into account the presence of water-limited field conditions. Including this step is therefore likely to improve current evaluation of ecosystem response to increased precipitation variability arising from climatic changes.

  2. The Flavin-Containing Reductase Domain of Cytochrome P450 BM3 Acts as a Surrogate for Mammalian NADPH-P450 Reductase.

    Science.gov (United States)

    Park, Seon-Ha; Kang, Ji-Yeon; Kim, Dong-Hyun; Ahn, Taeho; Yun, Chul-Ho

    2012-11-01

    Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is a self-sufficient monooxygenase that consists of a heme domain and FAD/FMN-containing reductase domain (BMR). In this report, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by BMR was evaluated as a method for monitoring BMR activity. The electron transfer proceeds from NADPH to BMR and then to BMR substrates, MTT and CTC. MTT and CTC are monotetrazolium salts that form formazans upon reduction. The reduction of MTT and CTC followed classical Michaelis-Menten kinetics (kcat =4120 min(-1), Km =77 μM for MTT and kcat =6580 min(-1), Km =51 μM for CTC). Our continuous assay using MTT and CTC allows the simple, rapid measurement of BMR activity. The BMR was able to metabolize mitomycin C and doxorubicin, which are anticancer drug substrates for CPR, producing the same metabolites as those produced by CPR. Moreover, the BMR was able to interact with CYP1A2 and transfer electrons to promote the oxidation reactions of substrates by CYP1A2 and CYP2E1 in humans. The results of this study suggest the possibility of the utilization of BMR as a surrogate for mammalian CPR.

  3. Preparation of reusable bioreactors using reversible immobilization of enzyme on monolithic porous polymer support with attached gold nanoparticles.

    Science.gov (United States)

    Lv, Yongqin; Lin, Zhixing; Tan, Tianwei; Svec, Frantisek

    2014-01-01

    Porcine lipase has been reversibly immobilized on a monolithic polymer support containing thiol functionalities prepared within confines of a fused silica capillary and functionalized with gold nanoparticles. Use of gold nanoparticles enabled rejuvenation of the activity of the deactivated reactor simply by stripping the inactive enzyme from the nanoparticles using 2-mercaptoethanol and subsequent immobilization of fresh lipase. This flow through enzymatic reactor was then used to catalyze the hydrolysis of glyceryl tributyrate (tributyrin). The highest activity was found within a temperature range of 37-40°C. The reaction kinetics is characterized by Michaelis-Menten constant, Km  = 10.9 mmol/L, and maximum reaction rate, Vmax  = 5.0 mmol/L min. The maximum reaction rate for the immobilized enzyme is 1,000 times faster compared to lipase in solution. The fast reaction rate enabled to achieve 86.7% conversion of tributyrin in mere 2.5 min and an almost complete conversion in 10 min. The reactor lost only less than 10% of its activity even after continuous pumping through it a solution of substrate equaling 1,760 reactor volumes. Finally, potential application of this enzymatic reactor was demonstrated with the transesterification of triacylglycerides from kitchen oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel. © 2013 Wiley Periodicals, Inc.

  4. Kinetics of butyrate, acetate, and hydrogen metabolism in a thermophilic, anaerobic, butyrate-degrading triculture.

    Science.gov (United States)

    Ahring, B K; Westermann, P

    1987-02-01

    Kinetics of butyrate, acetate, and hydrogen metabolism were determined with butyrate-limited, chemostat-grown tricultures of a thermophilic butyrate-utilizing bacterium together with Methanobacterium thermoautotrophicum and the TAM organism, a thermophilic acetate-utilizing methanogenic rod. Kinetic parameters were determined from progress curves fitted to the integrated form of the Michaelis-Menten equation. The apparent half-saturation constants, K(m), for butyrate, acetate, and dissolved hydrogen were 76 muM, 0.4 mM, and 8.5 muM, respectively. Butyrate and hydrogen were metabolized to a concentration of less than 1 muM, whereas acetate uptake usually ceased at a concentration of 25 to 75 muM, indicating a threshold level for acetate uptake. No significant differences in K(m) values for butyrate degradation were found between chemostat- and batch-grown tricultures, although the maximum growth rate was somewhat higher in the batch cultures in which the medium was supplemented with yeast extract. Acetate utilization was found to be the rate-limiting reaction for complete degradation of butyrate to methane and carbon dioxide in continuous culture. Increasing the dilution rate resulted in a gradual accumulation of acetate. The results explain the low concentrations of butyrate and hydrogen normally found during anaerobic digestion and the observation that acetate is the first volatile fatty acid to accumulate upon a decrease in retention time or increase in organic loading of a digestor.

  5. Catalytic properties of IgMs with amylolytic activity isolated from patients with multiple sclerosis.

    Science.gov (United States)

    Ivanen, Dina R; Kulminskaya, Anna A; Shabalin, Konstantin A; Isaeva-Ivanova, Luydmila V; Ershova, Nadezhda A; Saveliev, Andrew N; Nevinsky, Gregory A; Neustroev, Kirill N

    2004-08-01

    Recently, amylolytic activity was detected in IgMs isolated from the sera of the patients with multiple sclerosis. All purified samples of IgM were electrophoretically homogenous and did not contain any co-purified a-amylase and a-glucosidase activities, in accordance with a set of criteria developed for abzymes. The amylolytic activity of abzymes was studied in the hydrolysis of p-nitrophenyl a-D-maltooligosaccharides with different degrees of polymerization from 1 to 8 by TLC and reverse-phase HPLC techniques. All IgM samples isolated from 54 patients with clinically definite multiple sclerosis demonstrated hydrolytic activity towards the above artificial substrates. The Michaelis constant values (Km) in the hydrolysis of p-nitrophenyl a-D-maltoheptaoside were in the range of 10 p-nitrophenyl or p-nitrophenyl a-D-glucosides, thus indicating the presence of an a-D-glucosidase activity. For a number of the investigated samples, specific amylolytic activity increased depending on the length of substrates (from p-nitrophenyl maltopentaoside to p-nitrophenyl maltohexaoside); for other IgMs, the opposite dependence was observed. All IgMs studied did not exhibit any other glycoside hydrolase activities toward p-nitrophenyl glycoside substrates. Abzyme fractions from different donors demonstrated catalytic heterogeneity in Michaelis-Menten parameters and different modes of action in the hydrolysis of p-nitrophenyl maltooligosaccharides. Enzymatic properties of the IgMs tested varied from human a-amylases. All investigated abzyme samples did not show transglycosylating ability.

  6. [Investigations on the physiology of the glands of carnivorous plants : IV. The kinetics of chloride secretion by the gland tissue of Nepenthes].

    Science.gov (United States)

    Lüttge, U

    1966-03-01

    The transport of chloride in isolated tissue from Nepenthes pitchers was investigated using (36)Cl(-), an Aminco-Cotlove chloride-titrator for the determinations of Cl(-) concentrations, and KCN and AsO 4 (-) -as metabolic inhibitors.The tissue was brought in contact with different experimental solutions (=medium). The surface corresponding to the outside of the pitchers was cut with a razor blade to remove the cutinized epidermal layer. At this surface the Cl(-) uptake from the medium is a metabolic process which depends on the Cl(-)-concentration of the medium in a manner that corresponds to the MICHAELIS-MENTEN kinetics. The Michaelis-constant of this transport step was 3×10(-2)M. The Cl(-)-efflux into the medium, however, is a passive process.The opposite surface of the tissue slices (corresponding to the inside of the pitchers) carries the glands. The chloride secretion taking place here is also dependent on metabolism. In vitro it occurs even when a high gradient of chloride concentration has been set up between the medium and the solution which is in contact with the glands. In vivo the Cl(-)-concentration of the pitcher fluid and the amount of Cl(-) per gram of tissue water are almost equal.The rôle of chloride in the physiology of Nepenthes is still under investigation, A correlation between the chloride content of the pitcher fluid and its enzymatic activity (Casein-test), however, could already be demonstrated.

  7. THE KINETICS OF THE REACTIONS CATALYZED BY AN ENZYMATIC PREPARATION PRODUCED BY A BACILLUS LICHENIFORMIS STRAIN

    Directory of Open Access Journals (Sweden)

    MONICA DRAGOMIRESCU

    2007-05-01

    Full Text Available Robust immobilization techniques that preserve the activity of biomolecules have manypotential applications. In recent years, a number of new bioimobilisation methods in solgel-derived materials were reported. The interactions between the biomolecule and theinorganic material determine the degree to which the biomolecule retains its nativeproperties. The newer technological developments in the field of immobilizedbiocatalysts can offer the possibility of a wider and more economical exploitation ofbiocatalysts in biological applications, food and feed industry, medicine, and in thedevelopment of bioprocess monitoring devices, like the biosensors.The aim of this study was to obtain immobilized enzymatic preparations by methodswhich affect enzyme conformations and kinetic parameters as less as possible. Weimmobilized the enzymatic preparation with protease activity produced by a Bacilluslicheniformis B 40 local strain by physical bonding on ceramics and entrapment into solgel-derived glasses obtained from tetraethyl orthosilicate (TEOS, deposited in thin layeron a ceramic support (entrapment/deposition. Both physically adsorbed andentrapped/deposited enzymes follow Michaelis-Menten kinetics, similar with the solubleenzyme. In the case of immobilized enzymes, the apparent Michaelis constant, Km, wasgreater than that of the native one, as it was expected. The kinetic parameters indicatethat the enzymatic preparations adsorbed on ceramic support and entrapped/depositedshow less affinity for the substrate, Km being 1.3 and 2.1 times higher than that of thenative enzyme, respectively. The maximum velocity increased also by 3.5 and 7.9 timesrespectively, compared with the free counterpart (according to Lineweaver-Burklinearization.

  8. Variance decomposition in stochastic simulators.

    Science.gov (United States)

    Le Maître, O P; Knio, O M; Moraes, A

    2015-06-28

    This work aims at the development of a mathematical and computational approach that enables quantification of the inherent sources of stochasticity and of the corresponding sensitivities in stochastic simulations of chemical reaction networks. The approach is based on reformulating the system dynamics as being generated by independent standardized Poisson processes. This reformulation affords a straightforward identification of individual realizations for the stochastic dynamics of each reaction channel, and consequently a quantitative characterization of the inherent sources of stochasticity in the system. By relying on the Sobol-Hoeffding decomposition, the reformulation enables us to perform an orthogonal decomposition of the solution variance. Thus, by judiciously exploiting the inherent stochasticity of the system, one is able to quantify the variance-based sensitivities associated with individual reaction channels, as well as the importance of channel interactions. Implementation of the algorithms is illustrated in light of simulations of simplified systems, including the birth-death, Schlögl, and Michaelis-Menten models.

  9. Variance decomposition in stochastic simulators

    Science.gov (United States)

    Le Maître, O. P.; Knio, O. M.; Moraes, A.

    2015-06-01

    This work aims at the development of a mathematical and computational approach that enables quantification of the inherent sources of stochasticity and of the corresponding sensitivities in stochastic simulations of chemical reaction networks. The approach is based on reformulating the system dynamics as being generated by independent standardized Poisson processes. This reformulation affords a straightforward identification of individual realizations for the stochastic dynamics of each reaction channel, and consequently a quantitative characterization of the inherent sources of stochasticity in the system. By relying on the Sobol-Hoeffding decomposition, the reformulation enables us to perform an orthogonal decomposition of the solution variance. Thus, by judiciously exploiting the inherent stochasticity of the system, one is able to quantify the variance-based sensitivities associated with individual reaction channels, as well as the importance of channel interactions. Implementation of the algorithms is illustrated in light of simulations of simplified systems, including the birth-death, Schlögl, and Michaelis-Menten models.

  10. Analysis of mathematical modelling on potentiometric biosensors.

    Science.gov (United States)

    Mehala, N; Rajendran, L

    2014-01-01

    A mathematical model of potentiometric enzyme electrodes for a nonsteady condition has been developed. The model is based on the system of two coupled nonlinear time-dependent reaction diffusion equations for Michaelis-Menten formalism that describes the concentrations of substrate and product within the enzymatic layer. Analytical expressions for the concentration of substrate and product and the corresponding flux response have been derived for all values of parameters using the new homotopy perturbation method. Furthermore, the complex inversion formula is employed in this work to solve the boundary value problem. The analytical solutions obtained allow a full description of the response curves for only two kinetic parameters (unsaturation/saturation parameter and reaction/diffusion parameter). Theoretical descriptions are given for the two limiting cases (zero and first order kinetics) and relatively simple approaches for general cases are presented. All the analytical results are compared with simulation results using Scilab/Matlab program. The numerical results agree with the appropriate theories.

  11. Nonlinearities and transit times in soil organic matter models: new developments in the SoilR package

    Science.gov (United States)

    Sierra, Carlos; Müller, Markus

    2016-04-01

    SoilR is an R package for implementing diverse models representing soil organic matter dynamics. In previous releases of this package, we presented the implementation of linear first-order models with any number of pools as well as radiocarbon dynamics. We present here new improvements of the package regarding the possibility to implement models with nonlinear interactions among state variables and the possibility to calculate ages and transit times for nonlinear models with time dependencies. We show here examples on how to implement model structures with Michaelis-Menten terms for explicit microbial growth and resource use efficiency, and Langmuir isotherms for representing adsorption of organic matter to mineral surfaces. These nonlinear terms can be implemented for any number of organic matter pools, microbial functional groups, or mineralogy, depending on user's requirements. Through a simple example, we also show how transit times of organic matter in soils are controlled by the time-dependencies of the input terms.

  12. Dependence of nitrite oxidation on nitrite and oxygen in low-oxygen seawater

    Science.gov (United States)

    Sun, Xin; Ji, Qixing; Jayakumar, Amal; Ward, Bess B.

    2017-08-01

    Nitrite oxidation is an essential step in transformations of fixed nitrogen. The physiology of nitrite oxidizing bacteria (NOB) implies that the rates of nitrite oxidation should be controlled by concentration of their substrate, nitrite, and the terminal electron acceptor, oxygen. The sensitivities of nitrite oxidation to oxygen and nitrite concentrations were investigated using 15N tracer incubations in the Eastern Tropical North Pacific. Nitrite stimulated nitrite oxidation under low in situ nitrite conditions, following Michaelis-Menten kinetics, indicating that nitrite was the limiting substrate. The nitrite half-saturation constant (Ks = 0.254 ± 0.161 μM) was 1-3 orders of magnitude lower than in cultivated NOB, indicating higher affinity of marine NOB for nitrite. The highest rates of nitrite oxidation were measured in the oxygen depleted zone (ODZ), and were partially inhibited by additions of oxygen. This oxygen sensitivity suggests that ODZ specialist NOB, adapted to low-oxygen conditions, are responsible for apparently anaerobic nitrite oxidation.

  13. A metabolic and pharmacokinetic comparison of theophylline and aminophylline (theophylline ethylenediamine).

    Science.gov (United States)

    Monks, T J; Smith, R L; Caldwell, J

    1981-02-01

    The metabolism and pharmacokinetics of intravenously administered theophylline and aminophylline (theophylline ethylenediamine) have been studied in 3 volunteers, using 14C-labelled theophylline. Both compounds were metabolized extensively and 1,3-dimethyluric acid, 1-methyluric acid, 3-methylxanthine and two unknown minor metabolites were excreted in the urine, in addition to theophylline. The elimination of theophylline, 1,3-dimethyluric acid, 1-methyluric acid and the unknown metabolites followed first-order kinetics, but that of 3-methylxanthine followed Michaelis-Menten kinetics. When given as aminophylline, theophylline was metabolized more rapidly and extensively than when given alone. The recovery of 14C in the urine was significantly higher after aminophylline than after theophylline. Abstention from intake of dietary methylxanthines for 7 days resulted in more rapid and extensive metabolism of aminophylline compared with results from the same subjects on their usual diets. The results indicate that, from a metabolic and pharmacokinetic viewpoint, aminophylline and theophylline are not equivalent.

  14. Measurement and Modeling of Respiration Rate of Tomato (Cultivar Roma) for Modified Atmosphere Storage.

    Science.gov (United States)

    Kandasamy, Palani; Moitra, Ranabir; Mukherjee, Souti

    2015-01-01

    Experiments were conducted to determine the respiration rate of tomato at 10, 20 and 30 °C using closed respiration system. Oxygen depletion and carbon dioxide accumulation in the system containing tomato was monitored. Respiration rate was found to decrease with increasing CO2 and decreasing O2 concentration. Michaelis-Menten type model based on enzyme kinetics was evaluated using experimental data generated for predicting the respiration rate. The model parameters that obtained from the respiration rate at different O2 and CO2 concentration levels were used to fit the model against the storage temperatures. The fitting was fair (R2 = 0.923 to 0.970) when the respiration rate was expressed as O2 concentation. Since inhibition constant for CO2 concentration tended towards negetive, the model was modified as a function of O2 concentration only. The modified model was fitted to the experimental data and showed good agreement (R2 = 0.998) with experimentally estimated respiration rate.

  15. Enhancing Activity and Stability of Uricase from Lactobacillus plantarum by Zeolite immobilization

    Science.gov (United States)

    Iswantini, D.; Nurhidayat, N.; Sarah

    2017-03-01

    Lactobacillus plantarum has been known be able to produce uricase for uric acid biosensor. Durability and stability of L. plantarum in generating uricase enzyme was low. Hence, we tried to enhance its durability and stability by immobilizing it onto activated 250 mg zeolite at room temperature using 100 μL L.plantarum suspension and 2.87 mM uric acid, while Michaelis-Menten constant (KM) and Vmax were obtained at 6.7431 mM and 0.9171 µA consecutively, and the linearity range was 0.1-3.3 mM (R2 = 0.9667). Limit of detection (LOD) and limit of quantification (LOQ) value of the measurement were 0.4827 mM and 1.6092 mM respectively. Biosensor stability treatment was carried out in two different treatments, using the same electrode and using disposable electrode. The disposable electrode stability showed better result based on repeated measurements, but stability was still need improvement.

  16. Scopadulcic acid B, a new tetracyclic diterpenoid from Scoparia dulcis L. Its structure, H+, K(+)-adenosine triphosphatase inhibitory activity and pharmacokinetic behaviour in rats.

    Science.gov (United States)

    Hayashi, T; Okamura, K; Kakemi, M; Asano, S; Mizutani, M; Takeguchi, N; Kawasaki, M; Tezuka, Y; Kikuchi, T; Morita, N

    1990-10-01

    The structure of scopadulcic acid B (2, SDB), a major ingredient of the Paraguayan herb "Typychá kuratũ" (Scoparia dulcis L.), was elucidated mainly by comparison of its spectral data with that of scopadulcic acid A (1). SDB inhibited both the K(+)-dependent adenosine triphosphatase (ATPase) activity of a hog gastric proton pump (H+, K(+)-ATPase) with a value of 20-30 microM for IC50 and proton transport into gastric vesicles. Pharmacokinetic studies of SDB in rats indicated that plasma SDB concentrations after i.v. injection of the sodium salt of SDB (SDB-Na) were described reasonably well by a two-compartment open model with Michaelis-Menten elimination kinetics. Plasma concentrations after oral administration of SDB-Na or SDB showed a much slower decline than what was expected following the i. v. study. It was suggested that the sustained plasma level of SDB after oral administration of SDB-Na or SDB was accounted for by relatively slow but efficient gastro-intestinal absorption in rats.

  17. Electrochemical quantification of the antioxidant capacity of medicinal plants using biosensors.

    Science.gov (United States)

    Rodríguez-Sevilla, Erika; Ramírez-Silva, María-Teresa; Romero-Romo, Mario; Ibarra-Escutia, Pedro; Palomar-Pardavé, Manuel

    2014-08-08

    The working area of a screen-printed electrode, SPE, was modified with the enzyme tyrosinase (Tyr) using different immobilization methods, namely entrapment with water-soluble polyvinyl alcohol (PVA), cross-linking using glutaraldehyde (GA), and cross-linking using GA and human serum albumin (HSA); the resulting electrodes were termed SPE/Tyr/PVA, SPE/Tyr/GA and SPE/Tyr/HSA/GA, respectively. These biosensors were characterized by means of amperometry and EIS techniques. From amperometric evaluations, the apparent Michaelis-Menten constant, Km', of each biosensor was evaluated while the respective charge transfer resistance, Rct, was assessed from impedance measurements. It was found that the SPE/Tyr/GA had the smallest Km' (57 ± 7) µM and Rct values. This electrode also displayed both the lowest detection and quantification limits for catechol quantification. Using the SPE/Tyr/GA, the Trolox Equivalent Antioxidant Capacity (TEAC) was determined from infusions prepared with "mirto" (Salvia microphylla), "hHierba dulce" (Lippia dulcis) and "salve real" (Lippia alba), medicinal plants commonly used in Mexico.

  18. Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains.

    Science.gov (United States)

    Karow, Anne R; Theissen, Bettina; Klostermeier, Dagmar

    2007-01-01

    RNA helicases mediate structural rearrangements of RNA or RNA-protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis-Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication.

  19. Thermo-kinetics of lipase-catalyzed synthesis of 6-O-glucosyldecanoate.

    Science.gov (United States)

    Gumel, A M; Annuar, M S M; Heidelberg, T; Chisti, Y

    2011-10-01

    Lipase-catalyzed synthesis of 6-O-glucosyldecanoate from d-glucose and decanoic acid was performed in dimethyl sulfoxide (DMSO), a mixture of DMSO and tert-butanol and tert-butanol alone with a decreasing order of polarity. The highest conversion yield (> 65%) of decanoic acid was obtained in the blended solvent of intermediate polarity mainly because it could dissolve relatively large amounts of both the reactants. The reaction obeyed Michaelis-Menten type of kinetics. The affinity of the enzyme towards the limiting substrate (decanoic acid) was not affected by the polarity of the solvent, but increased significantly with temperature. The esterification reaction was endothermic with activation energy in the range of 60-67 kJ mol⁻¹. Based on the Gibbs energy values, in the solvent blend of DMSO and tert-butanol the position of the equilibrium was shifted more towards the products compared to the position in pure solvents. Monoester of glucose was the main product of the reaction. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Direct electron transfer of horseradish peroxidase on Nafion-cysteine modified gold electrode

    International Nuclear Information System (INIS)

    Hong, Jun; Moosavi-Movahedi, Ali Akbar; Ghourchian, Hedayatollah; Rad, Ahmad Molaei; Rezaei-Zarchi, Saeed

    2007-01-01

    Direct electron transfer of horseradish peroxidase, immobilized on a functional membrane-modified gold electrode, was studied. The electrode showed a quasi-reversible electrochemical redox behavior with a formal potential of 60mV (versus Ag/AgCl) in 20mM potassium phosphate buffer solution at pH 7.0 and temperature 25 o C. The cathodic transfer coefficient was 0.42 and electron transfer rate constant was evaluated to be 1.6s -1 . Furthermore, the modified electrode was used as a biosensor and exhibited a satisfactory stability and sensitivity to H 2 O 2 . The linear range of this biosensor for H 2 O 2 determination was from 5.0x10 -6 to 1.5x10 -4 M while its detection limit, based on a signal-to-noise ratio of 3, was 1.3x10 -6 M. The apparent Michaelis-Menten constant (K m app ) for immobilized HRP was calculated to be 1.6x10 -4 M

  1. Variance decomposition in stochastic simulators

    Energy Technology Data Exchange (ETDEWEB)

    Le Maître, O. P., E-mail: olm@limsi.fr [LIMSI-CNRS, UPR 3251, Orsay (France); Knio, O. M., E-mail: knio@duke.edu [Department of Mechanical Engineering and Materials Science, Duke University, Durham, North Carolina 27708 (United States); Moraes, A., E-mail: alvaro.moraesgutierrez@kaust.edu.sa [King Abdullah University of Science and Technology, Thuwal (Saudi Arabia)

    2015-06-28

    This work aims at the development of a mathematical and computational approach that enables quantification of the inherent sources of stochasticity and of the corresponding sensitivities in stochastic simulations of chemical reaction networks. The approach is based on reformulating the system dynamics as being generated by independent standardized Poisson processes. This reformulation affords a straightforward identification of individual realizations for the stochastic dynamics of each reaction channel, and consequently a quantitative characterization of the inherent sources of stochasticity in the system. By relying on the Sobol-Hoeffding decomposition, the reformulation enables us to perform an orthogonal decomposition of the solution variance. Thus, by judiciously exploiting the inherent stochasticity of the system, one is able to quantify the variance-based sensitivities associated with individual reaction channels, as well as the importance of channel interactions. Implementation of the algorithms is illustrated in light of simulations of simplified systems, including the birth-death, Schlögl, and Michaelis-Menten models.

  2. Uptake of oxytetracycline and its phytotoxicity to alfalfa (Medicago sativa L.)

    International Nuclear Information System (INIS)

    Kong, W.D.; Zhu, Y.G.; Liang, Y.C.; Zhang, J.; Smith, F.A.; Yang, M.

    2007-01-01

    A series of experiments were conducted in a hydroponic system to investigate the uptake of oxytetracycline (OTC) and its toxicity to alfalfa (Medicago sativa L.). OTC inhibited alfalfa shoot and root growth by up to 61% and 85%, respectively. The kinetics of OTC uptake could be well described by Michaelis-Menten equation with V max of 2.25 μmol g -1 fresh weight h -1 , and K m of 0.036 mM. The uptake of OTC by alfalfa was strongly inhibited by the metabolic inhibitor, 2,4-DNP (2,4-dinitrophenol), at pH 3.5 and 6.0, but not by the aquaporin competitors, glycerol and Ag + . OTC uptake, however, was significantly inhibited by Hg 2+ , suggesting that the inhibition of influx was due to general cellular stress rather than the specific action of Hg 2+ on aquaporins. Results from the present study suggested that OTC uptake into alfalfa is an energy-dependent process. - Plant uptake of antibiotic oxytetracycline is energy-dependent

  3. Comparison of indocyanine green clearance with Child's-Pugh score and hepatic histology: a multivariate analysis.

    Science.gov (United States)

    Mukherjee, Sandeep; Rogers, Mary A M; Buniak, Borys

    2006-01-01

    Indocyanine green clearance, measured by percentage disappearance rate, detects alterations in liver function and may be used as a non-invasive determinant of hepatic reserve. The aims of this study were to compare liver histology and Child's-Pugh score with percentage disappearance rate and determine which variables correlated with PDR. Child's-Pugh score, liver function tests, liver biopsies and indocyanine green testing (0.5mg/kg) were performed in 102 consecutive patients with cirrhosis of diverse etiologies. Indocyanine green concentration was determined using spectrophotometric analysis (806nm) and plotted logarithmically with Michaelis-Menten kinetics to calculate the percentage disappearance rate. Liver biopsies were graded using the modified Knodell score to obtain a histological activity index. In bivariable analysis, percentage disappearance rate significantly correlated with Child's-Pugh score, albumin, bilirubin, prothrombin time and histological activity index. Albumin, prothrombin time and histological activity index were independent predictors of percentage disappearance rate in the final model (albumin ptime ptime and histological activity index.

  4. Phenols removal by immobilized tyrosinase reactor in on-line high performance liquid chromatography

    International Nuclear Information System (INIS)

    Girelli, Anna Maria; Mattei, Enrico; Messina, Antonella

    2006-01-01

    The development of an immobilized enzyme reactor (IMER) based on tyrosinase immobilized on aminopropyl-controlled pore glass (AP-CPG) for the removal of phenols from model aqueous solutions was reported. To elucidate the influence of the substrate nature, the apparent (V ' max , K ' m ) and the inherent (V max , K m ) Michaelis-Menten constants were determined by Lineweaver-Burk method and the external diffusional contributions on measured enzyme activities were removed by a graphical method. The dephenolization process was realized by recycling the phenol solutions through the bioreactor connected to a chitosan trap in order to remove the colored quinone-type products of the tyrosinase reactions. The results indicated that a complete removal of phenol derivatives in the range of 150-300min, with the exception of 60% removal for phenol reached in 400min, was obtained. The observed sequence: cresol>4-methylcathecol>catechol>4-Cl-phenol-bar phenol was in accordance to the V ' max /K ' m values

  5. Purification and characterization of broccoli (Brassica oleracea var. italica) myrosinase (β-thioglucosidase glucohydrolase).

    Science.gov (United States)

    Mahn, Andrea; Angulo, Alejandro; Cabañas, Fernanda

    2014-12-03

    Myrosinase (β-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.

  6. Practical steady-state enzyme kinetics.

    Science.gov (United States)

    Lorsch, Jon R

    2014-01-01

    Enzymes are key components of most biological processes. Characterization of enzymes is therefore frequently required during the study of biological systems. Steady-state kinetics provides a simple and rapid means of assessing the substrate specificity of an enzyme. When combined with site-directed mutagenesis (see Site-Directed Mutagenesis), it can be used to probe the roles of particular amino acids in the enzyme in substrate recognition and catalysis. Effects of interaction partners and posttranslational modifications can also be assessed using steady-state kinetics. This overview explains the general principles of steady-state enzyme kinetics experiments in a practical, rather than theoretical, way. Any biochemistry textbook will have a section on the theory of Michaelis-Menten kinetics, including derivations of the relevant equations. No specific enzymatic assay is described here, although a method for monitoring product formation or substrate consumption over time (an assay) is required to perform the experiments described. © 2014 Elsevier Inc. All rights reserved.

  7. Uptake, accumulation and metabolic response of ferricyanide in weeping willows.

    Science.gov (United States)

    Yu, Xiao-Zhang; Gu, Ji-Dong

    2009-01-01

    The remediation potential and metabolic responses of plants to ferricyanide were investigated using pre-rooted weeping willows (Salix babylonica L.) grown hydroponically in growth chambers and treated with potassium ferricyanide. Positive responses were observed for the plants exposed to cyanide recovered in plant biomass was constant in all treatments, indicating that transport is a major limiting step for the uptake of ferricyanide by plants. The majority of the ferricyanide taken up from the growth media was possibly assimilated during transport through plants. The velocity of the removal processes can be described by Michaelis-Menten kinetics, and the half-saturation constant (K(M)) and the maximum removal capacity (v(max)) were estimated to be 228.1 mg CN L(-1) and 36.43 mg CN kg(-1) d(-1), respectively, using non-linear regression methods. These results suggest that weeping willows can take up, transport and assimilate ferricyanide; and phytoremediation is an option for cleaning up the environmental sites contaminated with cyanide complexes.

  8. Variance decomposition in stochastic simulators

    KAUST Repository

    Le Maî tre, O. P.; Knio, O. M.; Moraes, Alvaro

    2015-01-01

    This work aims at the development of a mathematical and computational approach that enables quantification of the inherent sources of stochasticity and of the corresponding sensitivities in stochastic simulations of chemical reaction networks. The approach is based on reformulating the system dynamics as being generated by independent standardized Poisson processes. This reformulation affords a straightforward identification of individual realizations for the stochastic dynamics of each reaction channel, and consequently a quantitative characterization of the inherent sources of stochasticity in the system. By relying on the Sobol-Hoeffding decomposition, the reformulation enables us to perform an orthogonal decomposition of the solution variance. Thus, by judiciously exploiting the inherent stochasticity of the system, one is able to quantify the variance-based sensitivities associated with individual reaction channels, as well as the importance of channel interactions. Implementation of the algorithms is illustrated in light of simulations of simplified systems, including the birth-death, Schlögl, and Michaelis-Menten models.

  9. The Investigation of Electrochemistry Behaviors of Tyrosinase Based on Directly-Electrodeposited Grapheneon Choline-Gold Nanoparticles.

    Science.gov (United States)

    He, Yaping; Yang, Xiaohui; Han, Quan; Zheng, Jianbin

    2017-06-23

    A novel catechol (CA) biosensor was developed by embedding tyrosinase (Tyr) onto in situ electrochemical reduction graphene (EGR) on choline-functionalized gold nanoparticle (AuNPs-Ch) film. The results of UV-Vis spectra indicated that Tyr retained its original structure in the film, and an electrochemical investigation of the biosensor showed a pair of well-defined, quasi-reversible redox peaks with E pa = -0.0744 V and E pc = -0.114 V (vs. SCE) in 0.1 M, pH 7.0 sodium phosphate-buffered saline at a scan rate of 100 mV/s. The transfer rate constant k s is 0.66 s -1 . The Tyr-EGR/AuNPs-Ch showed a good electrochemical catalytic response for the reduction of CA, with the linear range from 0.2 to 270 μM and a detection limit of 0.1 μM (S/N = 3). The apparent Michaelis-Menten constant was estimated to be 109 μM.

  10. Continuous depth-sensing nano-mechanical characterization of living, fixed and dehydrated cells attached on a glass substrate

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Yun-Ta; Liao, Jiunn-Der; Chang, Chia-Wei [Department of Materials Science and Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Lin, Chou-Ching K [Department of Neurology, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China); Ju, Ming-Shaung, E-mail: jdliao@mail.ncku.edu.tw [Department of Mechanical Engineering, National Cheng Kung University, No. 1, University Road, Tainan 70101, Taiwan (China)

    2010-07-16

    Continuous depth-sensing nano-indentation on living, fixed and dehydrated fibroblast cells was performed using a dynamic contact module and vertically measured from a pre-contact state to the glass substrate. The nano-indentation tip-on-cell approaches took advantage of finding a contact surface, followed by obtaining a continuous nano-mechanical profile along the nano-indentation depths. In the experiment, serial indentations from the leading edge, i.e., the lamellipodium to nucleus regions of living, fixed and dehydrated fibroblast cells were examined. Nano-indentations on a living cell anchored upon glass substrate were competent in finding the tip-on-cell contact surfaces and cell heights. For the result on the fixed and the dehydrated cells, cellular nano-mechanical properties were clearly characterized by continuous harmonic contact stiffness (HCS) measurements. The relations of HCS versus measured displacement, varied from the initial tip-on-cell contact to the glass substrate, were presumably divided into three stages, respectively induced by cellular intrinsic behavior, the substrate-dominant property, and the substrate property. This manifestation is beneficial to elucidate how the underlying substrate influences the interpretation of the nano-mechanical property of thin soft matter on a hard substrate. These findings, based upon continuous depth-sensing nano-indentations, are presumably valuable as a reference to related work, e.g., accomplished by atomic force microscopy.

  11. Continuous depth-sensing nano-mechanical characterization of living, fixed and dehydrated cells attached on a glass substrate

    International Nuclear Information System (INIS)

    Yang, Yun-Ta; Liao, Jiunn-Der; Chang, Chia-Wei; Lin, Chou-Ching K; Ju, Ming-Shaung

    2010-01-01

    Continuous depth-sensing nano-indentation on living, fixed and dehydrated fibroblast cells was performed using a dynamic contact module and vertically measured from a pre-contact state to the glass substrate. The nano-indentation tip-on-cell approaches took advantage of finding a contact surface, followed by obtaining a continuous nano-mechanical profile along the nano-indentation depths. In the experiment, serial indentations from the leading edge, i.e., the lamellipodium to nucleus regions of living, fixed and dehydrated fibroblast cells were examined. Nano-indentations on a living cell anchored upon glass substrate were competent in finding the tip-on-cell contact surfaces and cell heights. For the result on the fixed and the dehydrated cells, cellular nano-mechanical properties were clearly characterized by continuous harmonic contact stiffness (HCS) measurements. The relations of HCS versus measured displacement, varied from the initial tip-on-cell contact to the glass substrate, were presumably divided into three stages, respectively induced by cellular intrinsic behavior, the substrate-dominant property, and the substrate property. This manifestation is beneficial to elucidate how the underlying substrate influences the interpretation of the nano-mechanical property of thin soft matter on a hard substrate. These findings, based upon continuous depth-sensing nano-indentations, are presumably valuable as a reference to related work, e.g., accomplished by atomic force microscopy.

  12. Nitrate decreases xanthine oxidoreductase-mediated nitrite reductase activity and attenuates vascular and blood pressure responses to nitrite.

    Science.gov (United States)

    Damacena-Angelis, Célio; Oliveira-Paula, Gustavo H; Pinheiro, Lucas C; Crevelin, Eduardo J; Portella, Rafael L; Moraes, Luiz Alberto B; Tanus-Santos, Jose E

    2017-08-01

    Nitrite and nitrate restore deficient endogenous nitric oxide (NO) production as they are converted back to NO, and therefore complement the classic enzymatic NO synthesis. Circulating nitrate and nitrite must cross membrane barriers to produce their effects and increased nitrate concentrations may attenuate the nitrite influx into cells, decreasing NO generation from nitrite. Moreover, xanthine oxidoreductase (XOR) mediates NO formation from nitrite and nitrate. However, no study has examined whether nitrate attenuates XOR-mediated NO generation from nitrite. We hypothesized that nitrate attenuates the vascular and blood pressure responses to nitrite either by interfering with nitrite influx into vascular tissue, or by competing with nitrite for XOR, thus inhibiting XOR-mediated NO generation. We used two independent vascular function assays in rats (aortic ring preparations and isolated mesenteric arterial bed perfusion) to examine the effects of sodium nitrate on the concentration-dependent responses to sodium nitrite. Both assays showed that nitrate attenuated the vascular responses to nitrite. Conversely, the aortic responses to the NO donor DETANONOate were not affected by sodium nitrate. Further confirming these results, we found that nitrate attenuated the acute blood pressure lowering effects of increasing doses of nitrite infused intravenously in freely moving rats. The possibility that nitrate could compete with nitrite and decrease nitrite influx into cells was tested by measuring the accumulation of nitrogen-15-labeled nitrite ( 15 N-nitrite) by aortic rings using ultra-performance liquid chromatography tandem mass-spectrometry (UPLC-MS/MS). Nitrate exerted no effect on aortic accumulation of 15 N-nitrite. Next, we used chemiluminescence-based NO detection to examine whether nitrate attenuates XOR-mediated nitrite reductase activity. Nitrate significantly shifted the Michaelis Menten saturation curve to the right, with a 3-fold increase in the

  13. A new two-photon mechanism of the formation of a continuous spectrum of photons emitted by secondary emission products of atomic particles

    International Nuclear Information System (INIS)

    Veksler, V.I.

    1986-01-01

    A two-photon mechanism of the formation of a continuous spectrum of photons emitted by products of metal sputtering is considered. The following process of the two-photon mechanism is considered: the continuous spectrum is formed under quadrupole two-photon transitions in sputtered excited atoms having vacancies at the d level in atoms of transition metals or at the of level in lanthanides found against the filled conduction band. It is shown that the suggested mechanism should play an essential role in the formation of the continuous spectrum of optical radiation

  14. Oleamide synthesizing activity from rat kidney: identification as cytochrome c.

    Science.gov (United States)

    Driscoll, William J; Chaturvedi, Shalini; Mueller, Gregory P

    2007-08-03

    Oleamide (cis-9-octadecenamide) is the prototype member of an emerging class of lipid signaling molecules collectively known as the primary fatty acid amides. Current evidence suggests that oleamide participates in the biochemical mechanisms underlying the drive to sleep, thermoregulation, and antinociception. Despite the potential importance of oleamide in these physiologic processes, the biochemical pathway for its synthesis in vivo has not been established. We report here the discovery of an oleamide synthetase found in rat tissues using [(14)C]oleoyl-CoA and ammonium ion. Hydrogen peroxide was subsequently found to be a required cofactor. The enzyme displayed temperature and pH optima in the physiologic range, a remarkable resistance to proteolysis, and specificity for long-chain acyl-CoA substrates. The reaction demonstrated Michaelis-Menten kinetics with a K(m) for oleoyl-CoA of 21 microm. Proteomic, biochemical, and immunologic analyses were used to identify the source of the oleamide synthesizing activity as cytochrome c. This identification was based upon peptide mass fingerprinting of isolated synthase protein, a tight correlation between enzymatic activity and immunoreactivity for cytochrome c, and identical functional properties shared by the tissue-derived synthetase and commercially obtained cytochrome c. The ability of cytochrome c to catalyze the formation of oleamide experimentally raises the possibility that cytochrome c may mediate oleamide biosynthesis in vivo.

  15. Brain glucose transport and phosphorylation under acute insulin-induced hypoglycemia in mice: an 18F-FDG PET study.

    Science.gov (United States)

    Alf, Malte F; Duarte, João M N; Schibli, Roger; Gruetter, Rolf; Krämer, Stefanie D

    2013-12-01

    We addressed the questions of how cerebral glucose transport and phosphorylation change under acute hypoglycemia and what the underlying mechanisms of adaptation are. Quantitative (18)F-FDG PET combined with the acquisition of real-time arterial input function was performed on mice. Hypoglycemia was induced and maintained by insulin infusion. PET data were analyzed with the 2-tissue-compartment model for (18)F-FDG, and the results were evaluated with Michaelis-Menten saturation kinetics. Glucose clearance from plasma to brain (K1,glc) and the phosphorylation rate constant increased with decreasing plasma glucose (Gp), in particular at a Gp of less than 2.5 mmol/L. Estimated cerebral glucose extraction ratios taking into account an increased cerebral blood flow (CBF) at a Gp of less than 2 mmol/L were between 0.14 and 0.79. CBF-normalized K1,glc values were in agreement with saturation kinetics. Phosphorylation rate constants indicated intracellular glucose depletion at a Gp of less than 2-3 mmol/L. When brain regions were compared, glucose transport under hypoglycemia was lowest in the hypothalamus. Alterations in glucose transport and phosphorylation, as well as intracellular glucose depletion, under acute hypoglycemia can be modeled by saturation kinetics taking into account an increase in CBF. Distinct transport kinetics in the hypothalamus may be involved in its glucose-sensing function.

  16. Characterization of inorganic phosphate transport in the triple-negative breast cancer cell line, MDA-MB-231.

    Science.gov (United States)

    Russo-Abrahão, Thais; Lacerda-Abreu, Marco Antônio; Gomes, Tainá; Cosentino-Gomes, Daniela; Carvalho-de-Araújo, Ayra Diandra; Rodrigues, Mariana Figueiredo; Oliveira, Ana Carolina Leal de; Rumjanek, Franklin David; Monteiro, Robson de Queiroz; Meyer-Fernandes, José Roberto

    2018-01-01

    Recent studies demonstrate that interstitial inorganic phosphate is significantly elevated in the breast cancer microenvironment as compared to normal tissue. In addition it has been shown that breast cancer cells express high levels of the NaPi-IIb carrier (SLC34A2), suggesting that this carrier may play a role in breast cancer progression. However, the biochemical behavior of inorganic phosphate (Pi) transporter in this cancer type remains elusive. In this work, we characterize the kinetic parameters of Pi transport in the aggressive human breast cancer cell line, MDA-MB-231, and correlated Pi transport with cell migration and adhesion. We determined the influence of sodium concentration, pH, metabolic inhibitors, as well as the affinity for inorganic phosphate in Pi transport. We observed that the inorganic phosphate is dependent on sodium transport (K0,5 value = 21.98 mM for NaCl). Furthermore, the transport is modulated by different pH values and increasing concentrations of Pi, following the Michaelis-Menten kinetics (K0,5 = 0.08 mM Pi). PFA, monensin, furosemide and ouabain inhibited Pi transport, cell migration and adhesion. Taken together, these results showed that the uptake of Pi in MDA-MB-231 cells is modulated by sodium and by regulatory mechanisms of intracellular sodium gradient. General Significance: Pi transport might be regarded as a potential target for therapy against tumor progression.

  17. A physiologically based kinetic model for bacterial sulfide oxidation.

    Science.gov (United States)

    Klok, Johannes B M; de Graaff, Marco; van den Bosch, Pim L F; Boelee, Nadine C; Keesman, Karel J; Janssen, Albert J H

    2013-02-01

    In the biotechnological process for hydrogen sulfide removal from gas streams, a variety of oxidation products can be formed. Under natron-alkaline conditions, sulfide is oxidized by haloalkaliphilic sulfide oxidizing bacteria via flavocytochrome c oxidoreductase. From previous studies, it was concluded that the oxidation-reduction state of cytochrome c is a direct measure for the bacterial end-product formation. Given this physiological feature, incorporation of the oxidation state of cytochrome c in a mathematical model for the bacterial oxidation kinetics will yield a physiologically based model structure. This paper presents a physiologically based model, describing the dynamic formation of the various end-products in the biodesulfurization process. It consists of three elements: 1) Michaelis-Menten kinetics combined with 2) a cytochrome c driven mechanism describing 3) the rate determining enzymes of the respiratory system of haloalkaliphilic sulfide oxidizing bacteria. The proposed model is successfully validated against independent data obtained from biological respiration tests and bench scale gas-lift reactor experiments. The results demonstrate that the model is a powerful tool to describe product formation for haloalkaliphilic biomass under dynamic conditions. The model predicts a maximum S⁰ formation of about 98 mol%. A future challenge is the optimization of this bioprocess by improving the dissolved oxygen control strategy and reactor design. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Direct Visualization of Barrier Crossing Dynamics in a Driven Optical Matter System.

    Science.gov (United States)

    Figliozzi, Patrick; Peterson, Curtis W; Rice, Stuart A; Scherer, Norbert F

    2018-04-25

    A major impediment to a more complete understanding of barrier crossing and other single-molecule processes is the inability to directly visualize the trajectories and dynamics of atoms and molecules in reactions. Rather, the kinetics are inferred from ensemble measurements or the position of a transducer ( e. g., an AFM cantilever) as a surrogate variable. Direct visualization is highly desirable. Here, we achieve the direct measurement of barrier crossing trajectories by using optical microscopy to observe position and orientation changes of pairs of Ag nanoparticles, i. e. passing events, in an optical ring trap. A two-step mechanism similar to a bimolecular exchange reaction or the Michaelis-Menten scheme is revealed by analysis that combines detailed knowledge of each trajectory, a statistically significant number of repetitions of the passing events, and the driving force dependence of the process. We find that while the total event rate increases with driving force, this increase is due to an increase in the rate of encounters. There is no drive force dependence on the rate of barrier crossing because the key motion for the process involves a random (thermal) radial fluctuation of one particle allowing the other to pass. This simple experiment can readily be extended to study more complex barrier crossing processes by replacing the spherical metal nanoparticles with anisotropic ones or by creating more intricate optical trapping potentials.

  19. Enzymatic Synthesis of Furfuryl Alcohol Ester with Oleic Acid by Candida antarctica Lipase B and Its Kinetic Study

    Science.gov (United States)

    Sengupta, Avery; Dey, Tanmoy; Ghosh, Mahua; Ghosh, Jaydip; Ghosh, Santinath

    2012-08-01

    This study investigated the successful enzymatic production of furfuryl oleate and its detailed kinetic study by Michaelis-Menten model. Esterification of oleic acid and furfuryl alcohol by Candida antarctica lipase B (Novozym 435 preparation) in a solvent free system was studied in the present work at 1:1 molar ratio of furfuryl alcohol and oleic acid. About 99 % conversion (on the basis of oleic acid) has been achieved within 6 h at 5 % enzyme concentration. Ping-pong bi-bi mechanism (inhibition phenomenon taken into account) was applied to describe the ratios as a complex kinetic model. The kinetic parameters were determined using MATLAB language programme. The two initial rate constants KA and KB respectively were found out by different progress curves plotted with the help of MATLAB language programme. It was concluded from the results that furfuryl alcohol considerably inhibited the enzymatic reaction while oleic acid had negligible inhibitory effect. It was clearly seen that the initial rate was increased with the increase in the furfuryl alcohol concentration until 2 M/L after which there was a drop in the initial rate depicting the inhibitory effect of furfuryl alcohol. Surprisingly, it has been observed that addition of 0.1 mol of product activated the esterification reaction. Finally, the model was found to be statistically fitting well with the experimental data.

  20. A graphical user interface for a method to infer kinetics and network architecture (MIKANA).

    Science.gov (United States)

    Mourão, Márcio A; Srividhya, Jeyaraman; McSharry, Patrick E; Crampin, Edmund J; Schnell, Santiago

    2011-01-01

    One of the main challenges in the biomedical sciences is the determination of reaction mechanisms that constitute a biochemical pathway. During the last decades, advances have been made in building complex diagrams showing the static interactions of proteins. The challenge for systems biologists is to build realistic models of the dynamical behavior of reactants, intermediates and products. For this purpose, several methods have been recently proposed to deduce the reaction mechanisms or to estimate the kinetic parameters of the elementary reactions that constitute the pathway. One such method is MIKANA: Method to Infer Kinetics And Network Architecture. MIKANA is a computational method to infer both reaction mechanisms and estimate the kinetic parameters of biochemical pathways from time course data. To make it available to the scientific community, we developed a Graphical User Interface (GUI) for MIKANA. Among other features, the GUI validates and processes an input time course data, displays the inferred reactions, generates the differential equations for the chemical species in the pathway and plots the prediction curves on top of the input time course data. We also added a new feature to MIKANA that allows the user to exclude a priori known reactions from the inferred mechanism. This addition improves the performance of the method. In this article, we illustrate the GUI for MIKANA with three examples: an irreversible Michaelis-Menten reaction mechanism; the interaction map of chemical species of the muscle glycolytic pathway; and the glycolytic pathway of Lactococcus lactis. We also describe the code and methods in sufficient detail to allow researchers to further develop the code or reproduce the experiments described. The code for MIKANA is open source, free for academic and non-academic use and is available for download (Information S1).

  1. Mechanical properties of permeable materials with an organized structure on the base of continuous metal fibers

    International Nuclear Information System (INIS)

    Karpinos, D.M.; Rutkovskij, A.E.; Zorin, V.A.; Ivanchuk, A.A.

    1979-01-01

    The mechanical properties were studied for permeable fibrous materials with an organized structure on the base of continuous metal fibers (from Kh18N9T steel) subjected to preliminary reprocessing volumetric net half-finished products. The effect of geometrical parameters of the net half-finished products and of their orientation in packing are shown to affect the mechanical properties within a wide range of porosities

  2. Microstructures and mechanical properties evolution of an Al–Fe–Cu alloy processed by repetitive continuous extrusion forming

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Xiangxin [College of Materials Science and Engineering, Hunan University, Changsha 410082 (China); Zhang, Hui, E-mail: zhanghui63hunu@163.com [College of Materials Science and Engineering, Hunan University, Changsha 410082 (China); Hunan Province Key Laboratory for Spray Deposition Technology and Application, Hunan University, Changsha 410082 (China); Ji, Xiankun [College of Materials Science and Engineering, Hunan University, Changsha 410082 (China)

    2014-08-26

    Repetitive continuous extrusion forming process (R-Conform process), as a continuous severe plastic deformation method, was performed on a horizontal continuous casting Al–0.74Fe–0.23Cu alloy. The microstructural evolution and mechanical properties were studied by optical microscope, X-ray diffraction, scanning electron microscope, transmission electron microscope, and tensile testing. The results show that tensile ductility of the Al–0.74Fe–0.23Cu alloy is greatly improved but tensile strength is gradually decreased after repetitive Conform processing. The necking is more intense and the size of dimples becomes bigger with increasing Conform passes. The first pass Conform process induces obviously grains refining, dissolution of AlFe, AlFeSi and AlSi primary phases, strain-induced precipitation and transformation of crystal orientation distributions, but further Conform deformation only changes the redistribution of precipitates. The changes of mechanical properties may be attributed to a complex progress of recovery, recrystallization and redistribution of precipitates during repetitive Conform process.

  3. A fundamental trade-off in covalent switching and its circumvention by enzyme bifunctionality in glucose homeostasis.

    Science.gov (United States)

    Dasgupta, Tathagata; Croll, David H; Owen, Jeremy A; Vander Heiden, Matthew G; Locasale, Jason W; Alon, Uri; Cantley, Lewis C; Gunawardena, Jeremy

    2014-05-09

    Covalent modification provides a mechanism for modulating molecular state and regulating physiology. A cycle of competing enzymes that add and remove a single modification can act as a molecular switch between "on" and "off" and has been widely studied as a core motif in systems biology. Here, we exploit the recently developed "linear framework" for time scale separation to determine the general principles of such switches. These methods are not limited to Michaelis-Menten assumptions, and our conclusions hold for enzymes whose mechanisms may be arbitrarily complicated. We show that switching efficiency improves with increasing irreversibility of the enzymes and that the on/off transition occurs when the ratio of enzyme levels reaches a value that depends only on the rate constants. Fluctuations in enzyme levels, which habitually occur due to cellular heterogeneity, can cause flipping back and forth between on and off, leading to incoherent mosaic behavior in tissues, that worsens as switching becomes sharper. This trade-off can be circumvented if enzyme levels are correlated. In particular, if the competing catalytic domains are on the same protein but do not influence each other, the resulting bifunctional enzyme can switch sharply while remaining coherent. In the mammalian liver, the switch between glycolysis and gluconeogenesis is regulated by the bifunctional 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). We suggest that bifunctionality of PFK-2/FBPase-2 complements the metabolic zonation of the liver by ensuring coherent switching in response to insulin and glucagon.

  4. Partial characterization and response under hyperregulating conditions of Na+-K+ ATPase and levamisole-sensitive alkaline phosphatase activities in chela muscle of the euryhaline crab Cyrtograpsus angulatus

    Directory of Open Access Journals (Sweden)

    Silvina Andrea Pinoni

    2008-03-01

    Full Text Available The occurrence, characteristics and response to changes in environmental salinity of Na+-K+ ATPase and levamisole-sensitive alkaline phosphatase (AP activities were studied in chela muscle of the euryhaline crab Cyrtograpsus angulatus. Chela muscle exhibited an Na+-K+ ATPase activity which was strongly dependent on ATP concentration, pH and temperature of the reaction mixture. Maximal activity was found at 1 mM ATP, 30-37°C and pH 7.4. Levamisole-sensitive AP activity was characterised at physiological pH 7.4 and at pH 8.0. I50 for levamisole-sensitive AP activity was 8.8 mM and 8.0 mM at pH 7.4 and 8.0, respectively. At both pH levels, levamisole-sensitive AP activity exhibited Michaelis-Menten kinetics (Km=3.451 mM and 6.906 mM at pH 7.4 and 8.0, respectively. Levamisole-sensitive AP activities were strongly affected by temperature, exhibiting a peak at 37ºC. In crabs acclimated to low salinity (10; hyperegulating conditions, Na+-K+ ATPase activity and levamisole-sensitive AP activity at the physiological pH were higher than in 35 psu (osmoconforming conditions. The response to low salinity suggests that both activities could be components of muscle regulatory mechanisms at the biochemical level secondary to hyperegulation of C. angulatus. The study of these activities under hyperegulating conditions contributes to a better understanding of the complexity of biochemical mechanisms underlying the adaptive process of euryhaline crabs.

  5. One-dimensional Brownian motion of charged nanoparticles along microtubules: a model system for weak binding interactions.

    Science.gov (United States)

    Minoura, Itsushi; Katayama, Eisaku; Sekimoto, Ken; Muto, Etsuko

    2010-04-21

    Various proteins are known to exhibit one-dimensional Brownian motion along charged rodlike polymers, such as microtubules (MTs), actin, and DNA. The electrostatic interaction between the proteins and the rodlike polymers appears to be crucial for one-dimensional Brownian motion, although the underlying mechanism has not been fully clarified. We examined the interactions of positively-charged nanoparticles composed of polyacrylamide gels with MTs. These hydrophilic nanoparticles bound to MTs and displayed one-dimensional Brownian motion in a charge-dependent manner, which indicates that nonspecific electrostatic interaction is sufficient for one-dimensional Brownian motion. The diffusion coefficient decreased exponentially with an increasing particle charge (with the exponent being 0.10 kBT per charge), whereas the duration of the interaction increased exponentially (exponent of 0.22 kBT per charge). These results can be explained semiquantitatively if one assumes that a particle repeats a cycle of binding to and movement along an MT until it finally dissociates from the MT. During the movement, a particle is still electrostatically constrained in the potential valley surrounding the MT. This entire process can be described by a three-state model analogous to the Michaelis-Menten scheme, in which the two parameters of the equilibrium constant between binding and movement, and the rate of dissociation from the MT, are derived as a function of the particle charge density. This study highlights the possibility that the weak binding interactions between proteins and rodlike polymers, e.g., MTs, are mediated by a similar, nonspecific charge-dependent mechanism. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  6. Michaël Oustinoff et Christine Raguet-Bouvard, eds. Contraintes syntaxiques et liberté stylistique : le déplacement dans les éléments de la phrase. Palimpsestes n° 14.

    Directory of Open Access Journals (Sweden)

    Monique DeMattia

    2006-04-01

    Full Text Available Le numéro 14 de la revue Palimpsestes, comprend sept articles de recherche, suivis d’une table ronde contenant six interventions, ainsi que quelques extraits de débats. Michaël Oustinoff signe l’avant-propos de ce numéro, ainsi que la présentation de la table ronde.Ce volume se propose d’approfondir notre connaissance de ce domaine si vaste et complexe qu’est la traduction. Il aborde les difficultés rencontrées par tout traducteur, qui se doit de naviguer entre deux écueils majeurs, pour repr...

  7. Analysis of Äspö Pillar Stability Experiment: Continuous thermo-mechanical model development and calibration

    Czech Academy of Sciences Publication Activity Database

    Blaheta, Radim; Byczanski, Petr; Čermák, M.; Hrtus, Rostislav; Kohut, Roman; Kolcun, Alexej; Malík, Josef; Sysala, Stanislav

    2013-01-01

    Roč. 5, č. 2 (2013), s. 124-135 ISSN 1674-7755 Institutional support: RVO:68145535 Keywords : in situ pillar stability experiment * model calibration by back analysis * continuous mechanics * damage of granite rocks * Finite element method (FEM) Subject RIV: BA - General Mathematics http://www.sciencedirect.com/science/article/pii/S1674775513000103

  8. CYP2E1 Metabolism of Styrene Involves Allostery

    Science.gov (United States)

    Hartman, Jessica H.; Boysen, Gunnar

    2012-01-01

    We are the first to report allosterism during styrene oxidation by recombinant CYP2E1 and human liver microsomes. At low styrene concentrations, oxidation is inefficient because of weak binding to CYP2E1 (Ks = 830 μM). A second styrene molecule then binds CYP2E1 with higher affinity (Kss = 110 μM) and significantly improves oxidation to achieve a kcat of 6.3 nmol · min−1 · nmol CYP2E1−1. The transition between these metabolic cycles coincides with reported styrene concentrations in blood from exposed workers; thus, this CYP2E1 mechanism may be relevant in vivo. Scaled modeling of the in vitro-positive allosteric mechanism for styrene metabolism to its in vivo clearance led to significant deviations from the traditional model based on Michaelis-Menten kinetics. Low styrene levels were notably much less toxic than generally assumed. We interrogated the allosteric mechanism using the CYP2E1-specific inhibitor and drug 4-methylpyrazole, which we have shown binds two CYP2E1 sites. From the current studies, styrene was a positive allosteric effector on 4-methylpyrazole binding, based on a 10-fold increase in 4-methylpyrazole binding affinity from Ki 0.51 to Ksi 0.043 μM. The inhibitor was a negative allosteric effector on styrene oxidation, because kcat decreased 6-fold to 0.98 nmol · min−1 · nmol CYP2E1−1. Consequently, mixtures of styrene and other molecules can induce allosteric effects on binding and metabolism by CYP2E1 and thus mitigate the efficiency of their metabolism and corresponding effects on human health. Taken together, our elucidation of mechanisms for these allosteric reactions provides a powerful tool for further investigating the complexities of CYP2E1 metabolism of drugs and pollutants. PMID:22807108

  9. Growth, chamber building rate and reproduction time of Palaeonummulites venosus under natural conditions.

    Science.gov (United States)

    Kinoshita, Shunichi; Eder, Wolfgang; Wöger, Julia; Hohenegger, Johann; Briguglio, Antonino

    2017-04-01

    Investigations on Palaeonummulites venosus using the natural laboratory approach for determining chamber building rate, test diameter increase rate, reproduction time and longevity is based on the decomposition of monthly obtained frequency distributions based on chamber number and test diameter into normal-distributed components. The shift of the component parameters 'mean' and 'standard deviation' during the investigation period of 15 months was used to calculate Michaelis-Menten functions applied to estimate the averaged chamber building rate and diameter increase rate under natural conditions. The individual dates of birth were estimated using the inverse averaged chamber building rate and the inverse diameter increase rate fitted by the individual chamber number or the individual test diameter at the sampling date. Distributions of frequencies and densities (i.e. frequency divided by sediment weight) based on chamber building rate and diameter increase rate resulted both in a continuous reproduction through the year with two peaks, the stronger in May /June determined as the beginning of the summer generation (generation1) and the weaker in November determined as the beginning of the winter generation (generation 2). This reproduction scheme explains the existence of small and large specimens in the same sample. Longevity, calculated as the maximum difference in days between the individual's birth date and the sampling date seems to be round about one year, obtained by both estimations based on the chamber building rate and the diameter increase rate.

  10. Comparing of mechanical behavior and microstructure of continuous cast and hot worked Cu Zn 40 Al 1 alloy

    International Nuclear Information System (INIS)

    Ebrahimzadeh, I.; Akbari, G. H.

    2007-01-01

    The performance of components produced by conventional route of a thermo mechanical process and those produced by continuous casting is interesting from different aspects of economy and technology. The performance of products in their service depends on their properties which are strongly influenced by production routes. In the present work the hardness, tensile and tensile-impact behaviors of Cu Zn 40 Al 1 alloys produced by continuous casting and extrusion were investigated. Micro structural features and fracture surfaces were studied by optical and scanning electron microscopy. Results showed that wrought samples exhibited higher absorbed energy than those of continuous cast samples. Reduction of impact velocity led to a higher absorbed energy in all samples. A systematic and meaningful relationship was observed between micro structural features and mechanical properties such as hardness, yield stress and ultimate tensile strength. Fractography investigations showed that fracture occurred with dimple formation in all cases

  11. Improvement of the mechanical and frictional properties of steels by continuous and pulsed ion irradiation

    International Nuclear Information System (INIS)

    Romanov, I.G.

    1992-01-01

    Effect of continuous and powerful pulsed ion beams (PIB) on structural, mechanical, tribological properties and surface morphology of steels were investigated. The results obtained demonstrate the significant influence of ion irradiation type on microhardness, friction coefficient, wear resistance and surface roughness characteristics. Friction coefficient variation in irradiated steels is interpreted within the framework of an adhesion-deformation model

  12. Continuous positive airway pressure and conventional mechanical ventilation in the treatment of meconium aspiration syndrome.

    Science.gov (United States)

    Goldsmith, J P

    2008-12-01

    Meconium aspiration syndrome (MAS) is a complex syndrome that ranges in severity from mild respiratory distress to severe respiratory failure, persistent pulmonary hypertension of the newborn and sometimes death. Understanding of the syndrome's complicated pathophysiology will help determine the appropriate treatment strategy, including the use of continuous positive airway pressure (CPAP), conventional mechanical ventilation (CMV) and other therapies. Approximately 30 to 50% of infants diagnosed with MAS will require CPAP or mechanical ventilation. The optimum modes of ventilation for MAS are not known. Very few studies have been conducted to determine 'best' ventilatory strategies. Despite the introduction, over the last two decades, of innovative ventilatory treatments for this disease (for example, surfactant, high-frequency ventilation, inhaled nitric oxide, extracorporeal membrane oxygenation), the majority of infants can be successfully managed with CPAP or mechanical ventilation alone.

  13. Mechanisms that contribute to the tendency to continue chemotherapy in patients with advanced cancer. Qualitative observations in the clinical setting.

    Science.gov (United States)

    Brom, Linda; Onwuteaka-Philipsen, Bregje D; Widdershoven, Guy A M; Pasman, H Roeline W

    2016-03-01

    The study aims to describe mechanisms that contribute to the tendency towards continuing chemotherapy in patients with advanced cancer. The study conducted qualitative observations of outpatient clinic visits of 28 patients with advanced cancer (glioblastoma and metastatic colorectal cancer). We uncovered four mechanisms in daily oncology practice that can contribute to the tendency towards continuing chemotherapy in patients with advanced cancer: (1) "presenting the full therapy sets the standard"--patients seemed to base their justification for continuing chemotherapy on the "standard" therapy with the maximum number of cycles as presented by the physician at the start of the treatment; (2) "focus on standard evaluation moments hampers evaluation of care goals"--whether or not to continue the treatment was mostly only considered at standard evaluation moments; (3) "opening question guides towards focus on symptoms"--most patients gave an update of their physical symptoms in answer to the opening question of "How are you doing?" Physicians consequently discussed how to deal with this at length, which often took up most of the visit; (4) "treatment is perceived as the only option"--patients mostly wanted to continue with chemotherapy because they felt that they had to try every available option the physician offered. Physicians also often seemed to focus on treatment as the only option. Discussing care goals more regularly with the patient, facilitated for instance by implementing early palliative care, might help counter the mechanisms and enable a more well-considered decision. This could be either stopping or continuing chemotherapy.

  14. A new system for continuous and remote monitoring of patients receiving home mechanical ventilation.

    Science.gov (United States)

    Battista, L

    2016-09-01

    Home mechanical ventilation is the treatment of patients with respiratory failure or insufficiency by means of a mechanical ventilator at a patient's home. In order to allow remote patient monitoring, several tele-monitoring systems have been introduced in the last few years. However, most of them usually do not allow real-time services, as they have their own proprietary communication protocol implemented and some ventilation parameters are not always measured. Moreover, they monitor only some breaths during the whole day, despite the fact that a patient's respiratory state may change continuously during the day. In order to reduce the above drawbacks, this work reports the development of a novel remote monitoring system for long-term, home-based ventilation therapy; the proposed system allows for continuous monitoring of the main physical quantities involved during home-care ventilation (e.g., differential pressure, volume, and air flow rate) and is developed in order to allow observations of different remote therapy units located in different places of a city, region, or country. The developed remote patient monitoring system is able to detect various clinical events (e.g., events of tube disconnection and sleep apnea events) and has been successfully tested by means of experimental tests carried out with pulmonary ventilators typically used to support sick patients.

  15. Efficacy of continuous versus intermittent subglottic secretion drainage in preventing ventilator-associated pneumonia in patients requiring mechanical ventilation: A single-center randomized controlled trial.

    Science.gov (United States)

    Fujimoto, Hiroko; Yamaguchi, Osamu; Hayami, Hajime; Shimosaka, Mika; Tsuboi, Sayaka; Sato, Mitsunori; Takebayashi, Shigeo; Morita, Satoshi; Saito, Mari; Goto, Takahisa; Kurahashi, Kiyoyasu

    2018-03-23

    Aspiration of subglottic secretion is a widely used intervention to prevent ventilator-associated pneumonia (VAP). This study aimed to compare the efficacy of continuous and intermittent subglottic secretion drainage (SSD) in preventing VAP. A single-center randomized controlled trial was conducted on adult postoperative patients who were expected to undergo mechanical ventilation for more than 48 hours. Primary outcome measure was incidence of VAP and secondary outcome measures were length of mechanical ventilation and intensive-care unit (ICU) stay. Fifty-nine patients received continuous SSD, while 60 patients received intermittent SSD. Of these 119 patients, 88 (74%) were excluded and 15 and 16 patients were allocated to receive continuous and intermittent SSD, respectively. VAP was detected in 4 (26.7%) and 7 (43.8%) patients in the continuous and intermittent groups, respectively, (p=0.320). The length of mechanical ventilation was significantly shorter (p=0.034) in the continuous group (99.5±47.1 h) than in the intermittent group (159.9±94.5 h). The length of ICU stay was also shorter (p=0.0097) in the continuous group (6.3±2.1 days) than the intermittent group (9.8±4.8 days). Although continuous SSD did not reduce the incidence of VAP, it reduced the length of mechanical ventilation and ICU stay when compared to intermittent SSD.

  16. The mechanical properties, deformation and thermomechanical properties of alkali treated and untreated Agave continuous fibre reinforced epoxy composites

    International Nuclear Information System (INIS)

    Mylsamy, K.; Rajendran, I.

    2011-01-01

    Research highlights: → New renewable and biodegradable Agave americana fibre. → Environmentally free materials. → Good mechanical properties of Agave fibre reinforced epoxy composite materials. → Surface modification of the fibre (Alkali treatment) imported good mechanical properties. → Future scope in light weight materials manufacture. -- Abstract: The mechanical properties such as tensile, compressive, flexural, impact strength and water absorption of the alkali treated continuous Agave fibre reinforced epoxy composite (TCEC) and untreated continuous Agave fibre reinforced epoxy composite (UTCEC) were analysed. A comparison of the surfaces of TCEC and UTCEC composites was carried out by dynamic mechanical analysis (DMA), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The thermomechanical properties of the composite reinforced with sodium hydroxide (NaOH) treated Agave fibres were considerably good as the shrinkage of the fibre during alkali treatment had facilitated more points of fibre resin interface. The SEM micrograph and FTIR spectra of the impact fracture surfaces of TCEC clearly demonstrate the better interfacial adhesion between fibre and the matrix. In both analyses the TCEC gave good performance than UTCEC and, thus, there is a scope for its application in light weight manufacture in future.

  17. Effect of wine inhibitors on the proteolytic activity of papain from Carica papaya L. latex.

    Science.gov (United States)

    Benucci, Ilaria; Esti, Marco; Liburdi, Katia

    2015-01-01

    The influence of potential inhibitors naturally present in wine on the proteolytic activity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine-like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO2 ), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO2 , which acted as a mixed-type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis-Menten equation, was determined to be strongly affected by free SO2 and total phenol level. © 2014 American Institute of Chemical Engineers.

  18. Artificial Enzymes, "Chemzymes"

    DEFF Research Database (Denmark)

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M

    2008-01-01

    Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successf......Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...... that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well...

  19. Kinetic comparison of microbial assemblages for the anaerobic treatment of wastewater with high sulfate and heavy metal contents.

    Science.gov (United States)

    Sinbuathong, Nusara; Sirirote, Pramote; Liengcharernsit, Winai; Khaodhiar, Sutha; Watts, Daniel J

    2009-01-01

    Mixed-microbial assemblages enriched from a septic tank, coastal sediment samples, the digester sludge of a brewery wastewater treatment plant and acidic sulfate soil samples were compared on the basis of growth rate, waste and sulfate reduction rate under sulfate reducing conditions at 30 degrees C. The specific growth rate of various cultures was in the range 0.0013-0.0022 hr(-1). Estimates of waste and sulfate reduction rate were obtained by fitting substrate depletion and sulfate reduction data with the Michaelis-Menten equation. The waste reduction rates were in the range 4x10(-8)-1x10(-7) I mg(-1) hr(-1) and generally increased in the presence of copper, likely by copper sulfide precipitation that reduced sulfide and copper toxicity and thus protected the anaerobic microbes. Anaerobic microorganisms from a brewery digester sludge were found to be the most appropriate culture for the treatment of wastewater with high sulfate and heavy metal content due to their growth rate, and waste and sulfate reduction rate.

  20. Stoichiometry and kinetics of single and mixed substrate uptake in Aspergillus niger.

    Science.gov (United States)

    Lameiras, Francisca; Ras, Cor; Ten Pierick, Angela; Heijnen, Joseph J; van Gulik, Walter M

    2018-02-01

    In its natural environment, the filamentous fungus Aspergillus niger grows on decaying fruits and plant material, thereby enzymatically degrading the lignocellulosic constituents (lignin, cellulose, hemicellulose, and pectin) into a mixture of mono- and oligosaccharides. To investigate the kinetics and stoichiometry of growth of this fungus on lignocellulosic sugars, we carried out batch cultivations on six representative monosaccharides (glucose, xylose, mannose, rhamnose, arabinose, and galacturonic acid) and a mixture of these. Growth on these substrates was characterized in terms of biomass yields, oxygen/biomass ratios, and specific conversion rates. Interestingly, in combination, some of the carbon sources were consumed simultaneously and some sequentially. With a previously developed protocol, a sequential chemostat cultivation experiment was performed on a feed mixture of the six substrates. We found that the uptake of glucose, xylose, and mannose could be described with a Michaelis-Menten-type kinetics; however, these carbon sources seem to be competing for the same transport systems, while the uptake of arabinose, galacturonic acid, and rhamnose appeared to be repressed by the presence of other substrates.

  1. ISOLASI DAN KARAKTERISASI BAKTERI DENITRIFIKASI SEBAGAI AGEN BIOREMEDIASI NITROGEN ANORGANIK

    Directory of Open Access Journals (Sweden)

    Khairul Syahputra

    2011-08-01

    Full Text Available Denitrifikasi merupakan salah satu proses utama yang mengurangi kandungan senyawa nitrogen anorganik di lingkungan. Proses ini dapat digunakan untuk mengatasi kelebihan senyawa nitrogen anorganik yang tinggi di kolam budidaya perikanan. Penelitian ini bertujuan untuk mengisolasi dan mengkarakterisasi isolat bakteri denitrifikasi sebagai agen bioremediasi senyawa nitrogen anorganik. Sebanyak 21 isolat bakteri pereduksi nitrat berhasil diisolasi dari medium pengkayaan dengan konsentrasi nitrat 100 µM dan 1500 µM. Sebanyak 6 isolat merupakan kelompok bakteri denitrifikasi (fermentatif negatif dan 15 isolat termasuk kelompok bakteri fermentatif. Berdasarkan hasil seleksi didapatkan isolat HNF5 dan LNF mempunyai kemampuan reduksi nitrat yang tinggi. Aktivitas reduksi nitrat terjadi dari awal inkubasi, di mana aktivitas paling cepat terjadi pada fase eksponensial pertumbuhan bakteri. Isolat HNF5 dan LNF memiliki kecepatan maksimum reduksi nitrat (Vmaks 0,17 mM.h-1 dan 0,16 mM.h-1 dengan nilai konstanta Michaelis-Menten (Km 0,40 mM dan 0,28 mM. Identifikasi dengan sekuen 16S-rRNA memperlihatkan bahwa isolat HNF5 dan LNF mempunyai kemiripan dengan Pseudomonas aeruginosa.

  2. The effect of increased caffeine intake on the metabolism and pharmacokinetics of theophylline in man.

    Science.gov (United States)

    Monks, T J; Lawrie, C A; Caldwell, J

    1981-01-01

    The metabolism and pharmacokinetics of intravenously administered theophylline (100 mg) have been investigated in three healthy male volunteers who consumed 6 bottles/day of a cola beverage, in addition to their usual intake of methylxanthines, for 7 days prior to and during the study. Five urinary metabolites were detected in addition to unchanged theophylline, that is 3-methylxanthine, 1,3-dimethyluric acid, 1-methyluric acid, and two minor unknown metabolites. The elimination of theophylline, 1,3-dimethyluric acid, 1-methyluric acid, and the two unknowns was described by first-order kinetics, whereas that of 3-methylxanthine was described by Michaelis-Menten kinetics. The results have been compared with those previously obtained in the same volunteers while consuming their usual intake of methylxanthine-containing foods and beverages, and this shows that the addition of extra methylxanthines to the diet does not influence the disposition of theophylline. This is in marked contrast to the effect of deprivation of dietary methylxanthines on theophylline metabolism. The results are discussed in terms of the influence of methylxanthines on theophylline metabolism, and of its possible dose-dependency.

  3. Electrochemical Quantification of the Antioxidant Capacity of Medicinal Plants Using Biosensors

    Directory of Open Access Journals (Sweden)

    Erika Rodríguez-Sevilla

    2014-08-01

    Full Text Available The working area of a screen-printed electrode, SPE, was modified with the enzyme tyrosinase (Tyr using different immobilization methods, namely entrapment with water-soluble polyvinyl alcohol (PVA, cross-linking using glutaraldehyde (GA, and cross-linking using GA and human serum albumin (HSA; the resulting electrodes were termed SPE/Tyr/PVA, SPE/Tyr/GA and SPE/Tyr/HSA/GA, respectively. These biosensors were characterized by means of amperometry and EIS techniques. From amperometric evaluations, the apparent Michaelis-Menten constant, Km′, of each biosensor was evaluated while the respective charge transfer resistance, Rct, was assessed from impedance measurements. It was found that the SPE/Tyr/GA had the smallest Km′ (57 ± 7 µM and Rct values. This electrode also displayed both the lowest detection and quantification limits for catechol quantification. Using the SPE/Tyr/GA, the Trolox Equivalent Antioxidant Capacity (TEAC was determined from infusions prepared with “mirto” (Salvia microphylla, “hHierba dulce” (Lippia dulcis and “salve real” (Lippia alba, medicinal plants commonly used in Mexico.

  4. Electrochemical Quantification of the Antioxidant Capacity of Medicinal Plants Using Biosensors

    Science.gov (United States)

    Rodríguez-Sevilla, Erika; Ramírez-Silva, María-Teresa; Romero-Romo, Mario; Ibarra-Escutia, Pedro; Palomar-Pardavé, Manuel

    2014-01-01

    The working area of a screen-printed electrode, SPE, was modified with the enzyme tyrosinase (Tyr) using different immobilization methods, namely entrapment with water-soluble polyvinyl alcohol (PVA), cross-linking using glutaraldehyde (GA), and cross-linking using GA and human serum albumin (HSA); the resulting electrodes were termed SPE/Tyr/PVA, SPE/Tyr/GA and SPE/Tyr/HSA/GA, respectively. These biosensors were characterized by means of amperometry and EIS techniques. From amperometric evaluations, the apparent Michaelis-Menten constant, Km′, of each biosensor was evaluated while the respective charge transfer resistance, Rct, was assessed from impedance measurements. It was found that the SPE/Tyr/GA had the smallest Km′ (57 ± 7) μM and Rct values. This electrode also displayed both the lowest detection and quantification limits for catechol quantification. Using the SPE/Tyr/GA, the Trolox Equivalent Antioxidant Capacity (TEAC) was determined from infusions prepared with “mirto” (Salvia microphylla), “hHierba dulce” (Lippia dulcis) and “salve real” (Lippia alba), medicinal plants commonly used in Mexico. PMID:25111237

  5. Conversion of Cassava Starch to Produce Glucose and Fructose by Enzymatic Process Using Microwave Heating

    Directory of Open Access Journals (Sweden)

    Sumardiono Siswo

    2018-01-01

    Full Text Available In this study, variation of glycosidase enzyme concentration and saccharification time on enzymatic hydrolysis using microwave have been investigated. Concentration and kinetic parameters rate of glucose and fructose were analyzed. Cassava starch was liquefied and gelatinized by microwave at 80°C. The gelatinized starch was saccharified at 60°C using (0.2;0.4;0.6;0.8;1% (w/v glycosidase enzyme for 24, 48 and 72 hours. The glucose which has been saccharified with 1% glycosidase enzyme for 72 hours gave highest conversion 66.23 %. The optimization process by multilevel reaction gave the highest conversion at enzyme concentrations 0.88 %and saccharification time 29 hours that 68.82%. The highest conversion of glucose was isomerized to fructose. The fructose which has been isomerized for 180 minutes gave highest conversion 20.05 %. The kinetics enzymatic reaction was approached and determined by Michaelis - Menten equation, Km and Vmax of reaction for glucose 22.94 g/L; 2.70 g/L hours and for fructose 3.39 g/L; 0.38 g/L. min respectively.

  6. Determinação das propriedades catalíticas em meio aquoso e orgânico da lipase de Candida rugosa imobilizada em celulignina quimicamente modificada por carbonildiimidazol Assessment of catalytic properties in aqueous and organic media of lipase from Candida rugosa immobilized on wood cellulignin activated with carbonyldiimidazole

    Directory of Open Access Journals (Sweden)

    Fabrício M. Gomes

    2006-07-01

    Full Text Available Microbial lipase from Candida rugosa was immobilized by covalent binding on wood cellulignin (Eucaliptus grandis chemically modified with carbonyldiimidazole. The immobilized system was fully evaluated in aqueous (olive oil hydrolysis and organic (ester synthesis media. A comparative study between free and immobilized lipase was carried out in terms of pH, temperature and thermal stability. A higher pH value (8.0 was found optimal for the immobilized lipase. The optimal reaction temperature shifted from 37 °C for the free lipase to 45 °C for the immobilized lipase. The pattern of heat stability indicated that the immobilization process tends to stabilize the enzyme. Kinetics tests at 37 °C following the hydrolysis of olive oil obeyed the Michaelis-Menten rate equation. Values for Km = 924.9 mM and Vmax = 198.3 U/mg were lower than for free lipase, suggesting that the affinity towards the substrate changed and the activity of the immobilized lipase decreased during the course of immobilization. The immobilized derivative was also tested in the ester synthesis from several alcohols and carboxylic acids.

  7. Permeability, transport, and metabolism of solutes in Caco-2 cell monolayers: a theoretical study.

    Science.gov (United States)

    Sun, Huadong; Pang, K Sandy

    2008-01-01

    We explored the properties of a catenary model that includes the basolateral (B), apical (A), and cellular compartments via simulations under linear and nonlinear conditions to understand the asymmetric observations arising from transporters, enzymes, and permeability in Caco-2 cells. The efflux ratio (EfR; P(app,B-->A)/P(app,A-->B)), obtained from the effective permeability from the A-->B and B-->A direction under linear conditions, was unity for passively permeable drugs whose transport does not involve transporters; the value was unaffected by cellular binding or metabolism, but increased with apical efflux. Metabolism was asymmetric, showing lesser metabolite accrual for the B-->A than A-->B direction because of inherent differences in the volumes for A and B. Moreover, the net flux (total - passive permeation) due to saturable apical efflux, absorption, or metabolism showed nonconformity to simple Michaelis-Menten kinetics against C(D,0), the loading donor concentration. EfR values differed with saturable apical efflux and metabolism (>1), as well as apical absorption (EfRs transport and metabolic data in Caco-2 cells.

  8. Lipase from liver of seabass (Lates calcarifer): Characteristics and the use for defatting of fish skin.

    Science.gov (United States)

    Sae-Leaw, Thanasak; Benjakul, Soottawat

    2018-02-01

    Lipase from liver of seabass (Lates calcarifer), with a molecular weight of 60kDa, was purified to homogeneity using ammonium sulfate precipitation and a series of chromatographies, including diethylaminoethyl sepharose (DEAE) and Sephadex G-75 size exclusion columns. The optimal pH and temperature were 8.0 and 50°C, respectively. Purified lipase had Michaelis-Menten constant (K m ) and catalytic constant (k cat ) of 0.30mM and 2.16s -1 , respectively, when p-nitrophenyl palmitate (p-NPP) was used as the substrate. When seabass skin was treated with crude lipase from seabass liver at various levels (0.15 and 0.30units/g dry skin) for 1-3h at 30°C, the skin treated with lipase at 0.30 units/g dry skin for 3h had the highest lipid removal (84.57%) with lower lipid distribution in skin. Efficacy in defatting was higher than when isopropanol was used. Thus, lipase from liver of seabass could be used to remove fat in fish skin. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Nitrile hydratase of Rhodococcus erythropolis: characterization of the enzyme and the use of whole cells for biotransformation of nitriles.

    Science.gov (United States)

    Kamble, Ashwini L; Banoth, Linga; Meena, Vachan Singh; Singh, Amit; Chisti, Yusuf; Banerjee, U C

    2013-08-01

    The intracellular cobalt-type nitrile hydratase was purified from the bacterium Rhodococcuserythropolis. The pure enzyme consisted of two subunits of 29 and 30 kDa. The molecular weight of the native enzyme was estimated to be 65 kDa. At 25 °C the enzyme had a half-life of 25 h. The Michaelis-Menten constants K m and v max for the enzyme were 0.624 mM and 5.12 μmol/min/mg, respectively, using 3-cyanopyridine as the substrate. The enzyme-containing freely-suspended bacterial cells and the cells immobilized within alginate beads were evaluated for converting the various nitriles to amides. In a packed bed reactor, alginate beads (2 % alginate; 3 mm bead diameter) containing 200 mg/mL of cells, achieved a conversion of >90 % for benzonitrile and 4-cyanopyridine in 38 h (25 °C, pH 7.0) at a feed substrate concentration of 100 mM. The beads could be reused for up to six reaction cycles.

  10. HYDROLYSIS OF CHEESEWHEY PROTEINSWITH TRYPSIN, CHYMOTRYPSINAND CARBOXYPEPTIDASEA

    Directory of Open Access Journals (Sweden)

    M. F. CUSTÓDIO

    2009-01-01

    Full Text Available

    This work presents a method for adding value to cheese whey residues by whey proteins hydrolysis, using trypsin, chymotrypsin and carboxypeptidase A as catalysts. Sweet cheese whey was dialyzed and filtered in kaolin. Lactose and protein contents were analyzed after each step. The activities of bovine pancreas trypsin and chymotrypsin were measured at different pHs and temperatures. The optimal pH for the hydrolysis of whey proteins was 9.0 for both enzymes. Optima temperatures were 60ºC for trypsin, and 50ºC for chymotrypsin. Trypsin exhibited typical Michaelis-Menten behavior, but chymotrypsin did not. Electrophoretic analysis showed that neither trypsin nor chymotrypsin alone hydrolyzed whey proteins in less than three hours. Hydrolysis rates of -lactalbumin by trypsin, and of bovine serum albumin by chymotrypsin were low. When these enzymes were combined, however, all protein fractions were attacked and rates of hydrolysis were enhanced by one order of magnitude. The addition of carboxypeptidase A to the others enzymes did not improve the process yield.

  11. Study on biofiltration capacity and kinetics of nutrient uptake by Gracilaria cervicornis (Turner J. Agardh (Rhodophyta, Gracilariaceae

    Directory of Open Access Journals (Sweden)

    Marcella A. A. Carneiro

    2011-04-01

    Full Text Available The absorption efficiency and kinetic parameters (Vmax, Ks and Vmax:Ks of the seaweed Gracilaria cervicornis for the nutrients NH4+, NO3- and PO4(3- were evaluated. Absorption efficiency was measured by monitoring nutrient concentrations for 5 h in culture media with initial concentrations of 5, 10, 20 and 30µM. Kinetic parameters were determined by using the Michaelis-Menten formula. Absorption efficiencies for this algae were greater in treatments with lower concentrations, as evidenced by a reduction of 85.3, 97.5 and 81.2% for NH4+, NO3- and PO4(3-, respectively. Kinetic parameters show that G. cervicornis exhibits greater ability to take up high concentrations of NH4+ (Vmax=158.5µM g dw-1 h-1 and low concentrations of PO4(3- (Ks=5µM and Vmax:Ks=10.3. These results suggest that this algal species has good absorption capacity for the nutrients tested and may be a promising candidate as a bioremediator of eutrophized environments.

  12. Recovery of Whey Proteins and Enzymatic Hydrolysis of Lactose Derived from Casein Whey Using a Tangential Flow Ultrafiltration Module

    Science.gov (United States)

    Das, Bipasha; Bhattacharjee, Sangita; Bhattacharjee, Chiranjib

    2013-09-01

    In this study, ultrafiltration (UF) of pretreated casein whey was carried out in a cross-flow module fitted with 5 kDa molecular weight cut-off polyethersulfone membrane to recover whey proteins in the retentate and lactose in the permeate. Effects of processing conditions, like transmembrane pressure and pH on permeate flux and rejection were investigated and reported. The polarised layer resistance was found to increase with time during UF even in this high shear device. The lactose concentration in the permeate was measured using dinitro salicylic acid method. Enzymatic kinetic study for lactose hydrolysis was carried out at three different temperatures ranging from 30 to 50 °C using β-galactosidase enzyme. The glucose formed during lactose hydrolysis was analyzed using glucose oxidase-peroxidase method. Kinetics of enzymatic hydrolysis of lactose solution was found to follow Michaelis-Menten model and the model parameters were estimated by Lineweaver-Burk plot. The hydrolysis rate was found to be maximum (with Vmax = 5.5091 mmol/L/min) at 30 °C.

  13. Uptake of oxytetracycline and its phytotoxicity to alfalfa (Medicago sativa L.)

    Energy Technology Data Exchange (ETDEWEB)

    Kong, W D [Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Zhu, Y G [Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Liang, Y C [Ministry of Agriculture Key Laboratory of Plant Nutrition and Nutrient Cycling, Institute of Soils and Fertilizers, Chinese Academy of Agricultural Sciences, Beijing 100081 (China); Zhang, J [Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Smith, F A [Soil and Land Systems, School of Earth and Environmental Sciences, University of Adelaide, DP 636, Adelaide, SA 5005 (Australia); Yang, M [Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China)

    2007-05-15

    A series of experiments were conducted in a hydroponic system to investigate the uptake of oxytetracycline (OTC) and its toxicity to alfalfa (Medicago sativa L.). OTC inhibited alfalfa shoot and root growth by up to 61% and 85%, respectively. The kinetics of OTC uptake could be well described by Michaelis-Menten equation with V {sub max} of 2.25 {mu}mol g{sup -1} fresh weight h{sup -1}, and K {sub m} of 0.036 mM. The uptake of OTC by alfalfa was strongly inhibited by the metabolic inhibitor, 2,4-DNP (2,4-dinitrophenol), at pH 3.5 and 6.0, but not by the aquaporin competitors, glycerol and Ag{sup +}. OTC uptake, however, was significantly inhibited by Hg{sup 2+}, suggesting that the inhibition of influx was due to general cellular stress rather than the specific action of Hg{sup 2+} on aquaporins. Results from the present study suggested that OTC uptake into alfalfa is an energy-dependent process. - Plant uptake of antibiotic oxytetracycline is energy-dependent.

  14. Phenols removal by immobilized tyrosinase reactor in on-line high performance liquid chromatography

    Energy Technology Data Exchange (ETDEWEB)

    Girelli, Anna Maria [Dipartimento di Chimica, Universita degli Studi di Roma ' La Sapienza' , P.le Aldo Moro 5, 00185 Rome (Italy)]. E-mail: annamaria.girelli@uniroma1.it; Mattei, Enrico [Dipartimento di Chimica, Universita degli Studi di Roma ' La Sapienza' , P.le Aldo Moro 5, 00185 Rome (Italy); Messina, Antonella [Dipartimento di Chimica, Universita degli Studi di Roma ' La Sapienza' , P.le Aldo Moro 5, 00185 Rome (Italy)

    2006-11-24

    The development of an immobilized enzyme reactor (IMER) based on tyrosinase immobilized on aminopropyl-controlled pore glass (AP-CPG) for the removal of phenols from model aqueous solutions was reported. To elucidate the influence of the substrate nature, the apparent (V{sup '}{sub max}, K{sup '}{sub m}) and the inherent (V{sub max}, K{sub m}) Michaelis-Menten constants were determined by Lineweaver-Burk method and the external diffusional contributions on measured enzyme activities were removed by a graphical method. The dephenolization process was realized by recycling the phenol solutions through the bioreactor connected to a chitosan trap in order to remove the colored quinone-type products of the tyrosinase reactions. The results indicated that a complete removal of phenol derivatives in the range of 150-300min, with the exception of 60% removal for phenol reached in 400min, was obtained. The observed sequence: cresol>4-methylcathecol>catechol>4-Cl-phenol-bar phenol was in accordance to the V{sup '}{sub max}/K{sup '}{sub m} values.

  15. Amperometric bienzyme glucose biosensor based on carbon nanotube modified electrode with electropolymerized poly(toluidine blue O) film

    International Nuclear Information System (INIS)

    Wang Wenju; Wang Fang; Yao Yanli; Hu Shengshui; Shiu, Kwok-Keung

    2010-01-01

    The amperometric bienzyme glucose biosensor utilizing horseradish peroxidase (HRP) and glucose oxidase (GOx) immobilized in poly(toluidine blue O) (PTBO) film was constructed on multi-walled carbon nanotube (MWNT) modified glassy carbon electrode. The HRP layer could be used to analyze hydrogen peroxide with toluidine blue O (TBO) mediators, while the bienzyme system (HRP + GOx) could be utilized for glucose determination. Glucose underwent biocatalytic oxidation by GOx in the presence of oxygen to yield H 2 O 2 which was further reduced by HRP at the MWNT-modified electrode with TBO mediators. In the absence of oxygen, glucose oxidation proceeded with electron transfer between GOx and the electrode mediated by TBO moieties without H 2 O 2 production. The bienzyme electrode offered high sensitivity for amperometric determination of glucose at low potential, displaying Michaelis-Menten kinetics. The bienzyme glucose biosensor displayed linear response from 0.1 to 1.2 mM with a sensitivity of 113 mA M -1 cm -2 at an applied potential of -0.10 V in air-saturated electrolytes.

  16. Purification and characterization of a platelet aggregation inhibitor and anticoagulant Cc 5_NTase, CD 73-like, from Cerastes cerastes venom.

    Science.gov (United States)

    Saoud, Samah; Chérifi, Fatah; Benhassine, Traki; Laraba-Djebari, Fatima

    2017-05-01

    The present study is the first attempt to report the characterization of a nucleotidase from Cerastes cerastes venom. A 70 kDa 5'-nucleotidase (Cc-5'NTase) was purified to homogeneity. The amino acid sequence of Cc-5'NTase displayed high homology with many nucleotidases. Its activity was optimal at pH 7 with a specific hydrolytic activity toward mono-, di-, and triphosphate adenylated nucleotides. Cc-5'NTase preferentially hydrolyzed ADP and obeyed Michaelis-Menten kinetics. Among the metals and inhibitors tested, Ni 2+ and Mg 2+ completely potentiated enzyme activity, whereas EGTA, PMSF, iodoacetamide, vanillic acid, vanillyl mandelic acid, and 1,10-phenanthroline partially abolished its activity. Cc-5'NTase was not lethal for mice at 5 mg/kg and exhibited in vivo anticoagulant effect. It also dose-dependently inhibited adenosine diphosphate-induced platelet aggregation by converting adenosine diphosphate to adenosine and prohibited arachidonic acid-induced aggregation but was not effective on fibrinogen-induced aggregation. Cc-5'NTase could be a good tool as pharmacological molecule in thrombosis diagnostic and/or therapy. © 2016 Wiley Periodicals, Inc.

  17. Valorisation of food and beverage waste via saccharification for sugars recovery.

    Science.gov (United States)

    Kwan, Tsz Him; Ong, Khai Lun; Haque, Md Ariful; Kwan, Wing Hei; Kulkarni, Sandeep; Lin, Carol Sze Ki

    2018-05-01

    Valorisation of mixed food and beverage (F&B) waste was studied for the recovery of sugars via saccharification. Glucoamylase and sucrase were employed to hydrolyse the starch and sucrose present in the mixed F&B waste because of the high cost-effectiveness for such recovery. The Michaelis-Menten kinetics model suggests that preservatives and additives in beverages did not inhibit glucoamylase and sucrase during saccharification. High levels of glucose (228.1 g L -1 ) and fructose (55.7 g L -1 ) were efficiently produced within 12 h at a solid-to-liquid ratio of 37.5% (w/v) in 2.5 L bioreactors. An overall conversion yield of 0.17 g sugars per g of mixed F&B waste was obtained in mass balance analysis. Lastly, possible industrial applications of the sugar-rich hydrolysate and by-products are discussed. This study is believed to cast insights into F&B waste recycling via biotechnology to produce high-value added products to promote the establishment of a circular bio-economy. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Enhanced amperometric response of a glucose oxidase and horseradish peroxidase based bienzyme glucose biosensor modified with a film of polymerized toluidine blue containing reduced graphene oxide

    International Nuclear Information System (INIS)

    Wang, Fang; Gong, Wencheng; Wang, Lili; Chen, Zilin

    2015-01-01

    Reduced graphene oxide (RGO) was used to construct a bienzyme biosensor containing horseradish peroxidase (HRP) and glucose oxidase (GOx). A poly(toluidine blue) (pTB) film containing RGO acted as both enzyme immobilization matrix and electron transfer mediator. The bienzyme biosensor was characterized by electrochemical techniques and displays a highly sensitive amperometric response to glucose and hydrogen peroxide (H 2 O 2 ) at a potential as low as −0.1 V (vs. SCE). It is shown that use of RGO causes a strong enhancement on the amperometric responses. H 2 O 2 formed by the action of GOx in the presence of oxygen can be further reduced by HRP in the pTB film contacting the RGO modified electrode. In the absence of oxygen, glucose oxidation proceeds by another mechanism in which electron transfer occurs from GOx to the electrode and with pTB acting as the mediator. Amperometric responses to glucose and H2O2 follow Michaelis-Menten kinetics. The experimental conditions were optimized, and under these conditions glucose can be determined in the 80 μM to 3.0 mM range with a detection limit of 50 μM. H 2 O 2 , in turn, can be quantified in up to 30.0 μM concentration with a detection limit of 0.2 μM. The bienzyme biosensor is reproducible, repeatable and stable. Finally, it has been successfully applied to the determination of glucose in plasma samples. (author)

  19. Selective inhibition of CYP2C8 by fisetin and its methylated metabolite, geraldol, in human liver microsomes.

    Science.gov (United States)

    Shrestha, Riya; Kim, Ju-Hyun; Nam, Wongshik; Lee, Hye Suk; Lee, Jae-Mok; Lee, Sangkyu

    2018-04-01

    Fisetin is a flavonol compound commonly found in edible vegetables and fruits. It has anti-tumor, antioxidant, and anti-inflammatory effects. Geraldol, the O-methyl metabolite of fisetin in mice, is reported to suppress endothelial cell migration and proliferation. Although the in vivo and in vitro effects of fisetin and its metabolites are frequently reported, studies on herb-drug interactions have not yet been performed. This study was designed to investigate the inhibitory effect of fisetin and geraldol on eight isoforms of human cytochrome P450 (CYP) by using cocktail assay and LC-MS/MS analysis. The selective inhibition of CYP2C8-catalyzed paclitaxel hydroxylation by fisetin and geraldol were confirmed in pooled human liver microsomes (HLMs). In addition, an IC 50 shift assay under different pre-incubation conditions confirmed that fisetin and geraldol shows a reversible concentration-dependent, but not mechanism-based, inhibition of CYP2C8. Moreover, Michaelis-Menten, Lineweaver-burk plots, Dixon and Eadie-Hofstee showed a non-competitive inhibition mode with an equilibrium dissociation constant of 4.1 μM for fisetin and 11.5 μM for geraldol, determined from secondary plot of the Lineweaver-Burk plot. In conclusion, our results indicate that fisetin showed selective reversible and non-competitive inhibition of CYP2C8 more than its main metabolite, geraldol, in HLMs. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  20. Hepatic taurine transport: a Na+-dependent carrier on the basolateral plasma membrane

    International Nuclear Information System (INIS)

    Bucuvalas, J.C.; Goodrich, A.L.; Suchy, F.J.

    1987-01-01

    Highly purified rat basolateral liver plasma membrane vesicles were used examine the mechanism and the driving forces for hepatic uptake of the β-amino acid, taurine. An inwardly directed 100 mM NaCl gradient stimulated the initial rate of taurine uptake and energized a transient twofold accumulation of taurine above equilibrium (overshoot). In contrast, uptake was slower and no overshoot was detected in the presence of a KCl gradient. A negative intravesicular electrical potential generated by the presence of permeant anions or an outwardly directed K + gradient with valinomycin increased Na + -stimulated taurine uptake. External Cl - stimulated Na + -dependent taurine uptake independent of effects on the transmembrane electrical potential difference. Na + -dependent taurine uptake showed a sigmoidal dependence on extravesicular Na + concentration, suggesting multiple Na + ions are involved in the translocation of each taurine molecule. Na + -dependent taurine uptake demonstrated Michaelis-Menten kinetics with a maximum velocity of 0.537 nmol x mg protein -1 x min -1 and an apparent K/sub m/ of 174 μM. [ 3 H]taurine uptake was inhibited by the presence of excess unlabeled taurine, β-alanine, or hypotaurine but not by L-glutamine or L-alanine. In summary, using basolateral liver plasma membrane vesicles, the authors have shown that hepatic uptake of taurine occurs by a carrier-mediated, secondary active transport process specific for β-amino acids. Uptake is electrogenic, stimulated by external Cl - , and requires multiple Na + ions for the translocation of each taurine molecule

  1. Process Integration for the Disruption of Candida guilliermondii Cultivated in Rice Straw Hydrolysate and Recovery of Glucose-6-Phosphate Dehydrogenase by Aqueous Two-Phase Systems.

    Science.gov (United States)

    Gurpilhares, Daniela B; Pessoa, Adalberto; Roberto, Inês C

    2015-07-01

    Remaining cells of Candida guilliermondii cultivated in hemicellulose-based fermentation medium were used as intracellular protein source. Recovery of glucose-6-phosphate dehydrogenase (G6PD) was attained in conventional aqueous two-phase systems (ATPS) was compared with integrated process involving mechanical disruption of cells followed by ATPS. Influences of polyethylene glycol molar mass (M PEG) and tie line lengths (TLL) on purification factor (PF), yields in top (Y T ) and bottom (Y B ) phases and partition coefficient (K) were evaluated. First scheme resulted in 65.9 % enzyme yield and PF of 2.16 in salt-enriched phase with clarified homogenate (M PEG 1500 g mol(-1), TLL 40 %); Y B of 75.2 % and PF B of 2.9 with unclarified homogenate (M PEG 1000 g mol(-1), TLL 35 %). The highest PF value of integrated process was 2.26 in bottom phase (M PEG 1500 g mol(-1), TLL 40 %). In order to optimize this response, a quadratic model was predicted for the response PFB for process integration. Maximum response achieved was PFB = 3.3 (M PEG 1500 g mol(-1), TLL 40 %). Enzyme characterization showed G6P Michaelis-Menten constant (K M ) equal 0.07-0.05, NADP(+) K M 0.02-1.98 and optimum temperature 70 °C, before and after recovery. Overall, our data confirmed feasibility of disruption/extraction integration for single-step purification of intracellular proteins from remaining yeast cells.

  2. A simple theory of motor protein kinetics and energetics. II.

    Science.gov (United States)

    Qian, H

    2000-01-10

    A three-state stochastic model of motor protein [Qian, Biophys. Chem. 67 (1997) pp. 263-267] is further developed to illustrate the relationship between the external load on an individual motor protein in aqueous solution with various ATP concentrations and its steady-state velocity. A wide variety of dynamic motor behavior are obtained from this simple model. For the particular case of free-load translocation being the most unfavorable step within the hydrolysis cycle, the load-velocity curve is quasi-linear, V/Vmax = (cF/Fmax-c)/(1-c), in contrast to the hyperbolic relationship proposed by A.V. Hill for macroscopic muscle. Significant deviation from the linearity is expected when the velocity is less than 10% of its maximal (free-load) value--a situation under which the processivity of motor diminishes and experimental observations are less certain. We then investigate the dependence of load-velocity curve on ATP (ADP) concentration. It is shown that the free load Vmax exhibits a Michaelis-Menten like behavior, and the isometric Fmax increases linearly with ln([ATP]/[ADP]). However, the quasi-linear region is independent of the ATP concentration, yielding an apparently ATP-independent maximal force below the true isometric force. Finally, the heat production as a function of ATP concentration and external load are calculated. In simple terms and solved with elementary algebra, the present model provides an integrated picture of biochemical kinetics and mechanical energetics of motor proteins.

  3. Base sequence effects on DNA replication influenced by bulky adducts. Final report, March 1, 1995--February 28, 1997

    Energy Technology Data Exchange (ETDEWEB)

    Geacintov, N.E.

    1997-05-31

    Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants that are present in air, food, and water. While PAH compounds are chemically inert and are sparingly soluble in aqueous solutions, in living cells they are metabolized to a variety of oxygenated derivatives, including the high mutagenic and tumorigenic diol epoxide derivatives. The diol epoxides of the sterically hindered fjord region compound benzo[c]phenanthrene (B[c]PhDE) are among the most powerful tumorigenic compounds in animal model test systems. In this project, site-specifically modified oligonucleotides containing single B[c]PhDE-N{sup 6}-dA lesions derived from the reactions of the 1S,2R,3R,4S and 1R,2S,3S,4R diol epoxides of B[c]PhDE with dA residues were synthesized. The replication of DNA catalyzed by a prokaryotic DNA polymerase (the exonuclease-free Klenow fragment E. Coli Po1 I) in the vicinity of the lesion at base-specific sites on B[c]PhDE-modified template strands was investigated in detail. The Michaelis-Menten parameters for the insertion of single deoxynucleotide triphosphates into growing DNA (primer) strands using the modified dA* and the bases just before and after the dA* residue as templates, depend markedly on the stereochemistry of the B[c]PhDE-modified dA residues. These observations provide novel insights into the mechanisms by which bulky PAH-DNA adducts affect normal DNA replication.

  4. Noncompetitive inhibition of indolethylamine-N-methyltransferase by N,N-dimethyltryptamine and N,N-dimethylaminopropyltryptamine.

    Science.gov (United States)

    Chu, Uyen B; Vorperian, Sevahn K; Satyshur, Kenneth; Eickstaedt, Kelsey; Cozzi, Nicholas V; Mavlyutov, Timur; Hajipour, Abdol R; Ruoho, Arnold E

    2014-05-13

    Indolethylamine-N-methyltransferase (INMT) is a Class 1 transmethylation enzyme known for its production of N,N-dimethyltryptamine (DMT), a hallucinogen with affinity for various serotonergic, adrenergic, histaminergic, dopaminergic, and sigma-1 receptors. DMT is produced via the action of INMT on the endogenous substrates tryptamine and S-adenosyl-l-methionine (SAM). The biological, biochemical, and selective small molecule regulation of INMT enzyme activity remain largely unknown. Kinetic mechanisms for inhibition of rabbit lung INMT (rabINMT) by the product, DMT, and by a new novel tryptamine derivative were determined. After Michaelis-Menten and Lineweaver-Burk analyses had been applied to study inhibition, DMT was found to be a mixed competitive and noncompetitive inhibitor when measured against tryptamine. The novel tryptamine derivative, N-[2-(1H-indol-3-yl)ethyl]-N',N'-dimethylpropane-1,3-diamine (propyl dimethyl amino tryptamine or PDAT), was shown to inhibit rabINMT by a pure noncompetitive mechanism when measured against tryptamine with a Ki of 84 μM. No inhibition by PDAT was observed at 2 mM when it was tested against structurally similar Class 1 methyltransferases, such as human phenylethanolamine-N-methyltransferase (hPNMT) and human nicotinamide-N-methyltransferase (hNNMT), indicating selectivity for INMT. The demonstration of noncompetitive mechanisms for INMT inhibition implies the presence of an inhibitory allosteric site. In silico analyses using the computer modeling software Autodock and the rabINMT sequence threaded onto the human INMT (hINMT) structure (Protein Data Bank entry 2A14 ) identified an N-terminal helix-loop-helix non-active site binding region of the enzyme. The energies for binding of DMT and PDAT to this region of rabINMT, as determined by Autodock, were -6.34 and -7.58 kcal/mol, respectively. Assessment of the allosteric control of INMT may illuminate new biochemical pathway(s) underlying the biology of INMT.

  5. Building New Bridges between In Vitro and In Vivo in Early Drug Discovery: Where Molecular Modeling Meets Systems Biology.

    Science.gov (United States)

    Pearlstein, Robert A; McKay, Daniel J J; Hornak, Viktor; Dickson, Callum; Golosov, Andrei; Harrison, Tyler; Velez-Vega, Camilo; Duca, José

    2017-01-01

    Cellular drug targets exist within networked function-generating systems whose constituent molecular species undergo dynamic interdependent non-equilibrium state transitions in response to specific perturbations (i.e.. inputs). Cellular phenotypic behaviors are manifested through the integrated behaviors of such networks. However, in vitro data are frequently measured and/or interpreted with empirical equilibrium or steady state models (e.g. Hill, Michaelis-Menten, Briggs-Haldane) relevant to isolated target populations. We propose that cells act as analog computers, "solving" sets of coupled "molecular differential equations" (i.e. represented by populations of interacting species)via "integration" of the dynamic state probability distributions among those populations. Disconnects between biochemical and functional/phenotypic assays (cellular/in vivo) may arise with targetcontaining systems that operate far from equilibrium, and/or when coupled contributions (including target-cognate partner binding and drug pharmacokinetics) are neglected in the analysis of biochemical results. The transformation of drug discovery from a trial-and-error endeavor to one based on reliable design criteria depends on improved understanding of the dynamic mechanisms powering cellular function/dysfunction at the systems level. Here, we address the general mechanisms of molecular and cellular function and pharmacological modulation thereof. We outline a first principles theory on the mechanisms by which free energy is stored and transduced into biological function, and by which biological function is modulated by drug-target binding. We propose that cellular function depends on dynamic counter-balanced molecular systems necessitated by the exponential behavior of molecular state transitions under non-equilibrium conditions, including positive versus negative mass action kinetics and solute-induced perturbations to the hydrogen bonds of solvating water versus kT. Copyright© Bentham

  6. Strong negative self regulation of Prokaryotic transcription factors increases the intrinsic noise of protein expression

    Directory of Open Access Journals (Sweden)

    Jenkins Dafyd J

    2008-01-01

    Full Text Available Abstract Background Many prokaryotic transcription factors repress their own transcription. It is often asserted that such regulation enables a cell to homeostatically maintain protein abundance. We explore the role of negative self regulation of transcription in regulating the variability of protein abundance using a variety of stochastic modeling techniques. Results We undertake a novel analysis of a classic model for negative self regulation. We demonstrate that, with standard approximations, protein variance relative to its mean should be independent of repressor strength in a physiological range. Consequently, in that range, the coefficient of variation would increase with repressor strength. However, stochastic computer simulations demonstrate that there is a greater increase in noise associated with strong repressors than predicted by theory. The discrepancies between the mathematical analysis and computer simulations arise because with strong repressors the approximation that leads to Michaelis-Menten-like hyperbolic repression terms ceases to be valid. Because we observe that strong negative feedback increases variability and so is unlikely to be a mechanism for noise control, we suggest instead that negative feedback is evolutionarily favoured because it allows the cell to minimize mRNA usage. To test this, we used in silico evolution to demonstrate that while negative feedback can achieve only a modest improvement in protein noise reduction compared with the unregulated system, it can achieve good improvement in protein response times and very substantial improvement in reducing mRNA levels. Conclusion Strong negative self regulation of transcription may not always be a mechanism for homeostatic control of protein abundance, but instead might be evolutionarily favoured as a mechanism to limit the use of mRNA. The use of hyperbolic terms derived from quasi-steady-state approximation should also be avoided in the analysis of stochastic

  7. Virtual powers and virtual works in mechanics of micropolar continuous media

    International Nuclear Information System (INIS)

    Grossetie, J.C.

    1981-01-01

    The expression of virtual work and virtual powers of internal actions is established for a continuous medium where the actions are defined by fields of forces and moments. A basic formulation of the mechanical problem is thus obtained and can serve as the starting point for variational and quasi-variational formulations of these problems. This expression may be used in thermodynamic developments relating to research or constitutive laws of materials within the context of so-called hidden internal variable formulations. The principle of virtual works is given in general applied then to the classic case through a demonstration consistent with this principle and finally applied to the general case in which distributions of moments are included. The viewpoint adopted is that Euler-Cauchy. This expression will have to be accounted for in establishing thermodynamic relations regarding materials subjected to nuclear effects (irradiated materials)

  8. Operculina from the northwestern Pacific (Sesoko Island, Japan) Species Differentiation, Population Dynamics, Growth and Development

    Science.gov (United States)

    Woeger, Julia; Eder, Wolfgang; Kinoshita, Shunichi; Briguglio, Antonino; Hohenegger, Johann

    2017-04-01

    During the last decades larger benthic foraminifera have gained importance as indicator species and are used in a variety of applications, from ecological monitoring, studying the effects of ocean acidification, or reconstructing paleoenvironments. They significantly contribute to the carbonate budget of costal areas and are invaluable tools in biostratigraphy. Even before their advancement as bioindicators, laboratory experiments have been conducted to investigate the effects of various ecological parameters on community composition, biology of single species, or investigating the effects of salinity and temperature on stable isotope composition of the foraminiferal test, to name only a few. The natural laboratory approach (continuous sampling over a period of more than one year) was conducted at the island of Sesoko (Okinawa, Japan). in combination with µ-CT scanning was used to reveal population dynamics of 3 different morphotypes of Operculina. The clarification of reproductive cycles as well as generation and size abundances were used to calculate natural growth models. Best fit was achieved using Bertalanffy and Michaelis-Menten functions. Exponential-, logistic-, generalized logistic-, Gompertz-function yielded weaker fits, when compared by coefficient of determination as well as Akaike Information criterion. The resulting growth curves and inferred growth rates were in turn used to evaluate the quality of a laboratory cultivation experiment carried out simultaneously over a period of 15 months. Culturing parameters such as temperature, light intensities, salinity and pH and light-dark duration were continuously adapted to measurements in the field. The average investigation time in culture was 77days. 13 Individuals lived more than 200 days, 3 reproduced asexually and one sexually. 14% of 186 Individuals were lost, while 22% could not be kept alive for more than one month. Growth curves also represent an instrumental source of information for the various

  9. Reconciling semiclassical and Bohmian mechanics. III. Scattering states for continuous potentials

    International Nuclear Information System (INIS)

    Trahan, Corey; Poirier, Bill

    2006-01-01

    In a previous paper [B. Poirier, J. Chem. Phys. 121, 4501 (2004)] a unique bipolar decomposition Ψ=Ψ 1 +Ψ 2 was presented for stationary bound states Ψ of the one-dimensional Schroedinger equation, such that the components Ψ 1 and Ψ 2 approach their semiclassical WKB analogs in the large-action limit. The corresponding bipolar quantum trajectories, as defined in the usual Bohmian mechanical formulation, are classical-like and well behaved, even when Ψ has many nodes or is wildly oscillatory. A modification for discontinuous potential stationary scattering states was presented in a second, companion paper [C. Trahan and B. Poirier, J. Chem. Phys.124, 034115 (2006), previous paper], whose generalization for continuous potentials is given here. The result is an exact quantum scattering methodology using classical trajectories. For additional convenience in handling the tunneling case, a constant-velocity-trajectory version is also developed

  10. Treatment of ferrous-NTA-based NO x scrubber solution by an up-flow anaerobic packed bed bioreactor.

    Science.gov (United States)

    Chandrashekhar, B; Sahu, Nidhi; Tabassum, Heena; Pai, Padmaraj; Morone, Amruta; Pandey, R A

    2015-06-01

    A bench scale system consisting of an up-flow packed bed bioreactor (UAPBR) made of polyurethane foam was used for the treatment and regeneration of aqueous solution of ferrous-NTA scrubbed with nitric oxide (NO). The biomass in the UAPBR was sequentially acclimatized under denitrifying and iron reducing conditions using ethanol as electron donor, after which nitric oxide (NO) gas was loaded continuously to the system by absorption. The system was investigated for different parameters viz. pH, removal efficiency of nitric oxide, biological reduction efficiency of Fe(II)NTA-NO and COD utilization. The Fe(II)NTA-NO reduction efficiency reached 87.8 % at a loading rate of 0.24 mmol L(-1) h(-1), while the scrubber efficiency reached more than 75 % with 250 ppm NO. Stover-Kincannon and a Plug-flow kinetic model based on Michaelis-Menten equation were used to describe the UAPBR performance with respect to Fe(II)NTA-NO and COD removal. The Stover-Kincannon model was found capable of describing the Fe(II)NTA-NO reduction (R m = 8.92 mM h(-1) and K NO = 11.46 mM h(-1)) while plug-flow model provided better fit to the COD utilization (U m = 66.62 mg L(-1) h(-1), K COD = 7.28 mg L(-1)). Analyses for pH, Fe(III)NTA, ammonium, nitrite concentration, and FTIR analysis of the medium samples indicated degradation of NTA, which leads to ammonium and nitrite accumulation in the medium, and affect the regeneration process.

  11. Mechanism of inactivation of human leukocyte elastase by a chloromethyl ketone: kinetic and solvent isotope effect studies

    International Nuclear Information System (INIS)

    Stein, R.L.; Trainor, D.A.

    1986-01-01

    The mechanism of inactivation of human leukocyte elastase (HLE) by the chloromethyl ketone MeOSuc-Ala-Ala-Pro-Val-CH 2 Cl was investigated. The dependence of the first-order rate constant for inactivation on concentration of chloromethyl ketone is hyperbolic and suggests formation of a reversible Michaelis complex prior to covalent interaction between the enzyme and inhibitor. However, the observed Ki value is 10 microM, at least 10-fold lower than dissociation constants for complexes formed from interaction of HLE with structurally related substrates or reversible inhibitors, and suggests that Ki is a complex kinetic constant, reflecting the formation and accumulation of both the Michaelis complex and a second complex. It is proposed that this second complex is a hemiketal formed from attack of the active site serine on the carbonyl carbon of the inhibitor. The accumulation of this intermediate may be a general feature of reactions of serine proteases and chloromethyl ketones derived from specific peptides and accounts for the very low Ki values observed for these reactions. The solvent deuterium isotope effect (SIE) on the inactivation step (ki) is 1.58 +/- 0.07 and is consistent with rate-limiting, general-catalyzed attack of the active site His on the methylene carbon of the inhibitor with displacement of chloride anion. The general catalyst is thought to be the active site Asp. In contrast, the SIE on the second-order rate constant for HLE inactivation, ki/Ki, is inverse and equals 0.64 +/- 0.05

  12. Different Mechanisms of Soil Microbial Response to Global Change Result in Different Outcomes in the MIMICS-CN Model

    Science.gov (United States)

    Kyker-Snowman, E.; Wieder, W. R.; Grandy, S.

    2017-12-01

    Microbial-explicit models of soil carbon (C) and nitrogen (N) cycling have improved upon simulations of C and N stocks and flows at site-to-global scales relative to traditional first-order linear models. However, the response of microbial-explicit soil models to global change factors depends upon which parameters and processes in a model are altered by those factors. We used the MIcrobial-MIneral Carbon Stabilization Model with coupled N cycling (MIMICS-CN) to compare modeled responses to changes in temperature and plant inputs at two previously-modeled sites (Harvard Forest and Kellogg Biological Station). We spun the model up to equilibrium, applied each perturbation, and evaluated 15 years of post-perturbation C and N pools and fluxes. To model the effect of increasing temperatures, we independently examined the impact of decreasing microbial C use efficiency (CUE), increasing the rate of microbial turnover, and increasing Michaelis-Menten kinetic rates of litter decomposition, plus several combinations of the three. For plant inputs, we ran simulations with stepwise increases in metabolic litter, structural litter, whole litter (structural and metabolic), or labile soil C. The cumulative change in soil C or N varied in both sign and magnitude across simulations. For example, increasing kinetic rates of litter decomposition resulted in net releases of both C and N from soil pools, while decreasing CUE produced short-term increases in respiration but long-term accumulation of C in litter pools and shifts in soil C:N as microbial demand for C increased and biomass declined. Given that soil N cycling constrains the response of plant productivity to global change and that soils generate a large amount of uncertainty in current earth system models, microbial-explicit models are a critical opportunity to advance the modeled representation of soils. However, microbial-explicit models must be improved by experiments to isolate the physiological and stoichiometric

  13. Continuity and completeness in physical theory: Schroedinger's return to the wave interpretation of quantum mechanics in the 1950's

    International Nuclear Information System (INIS)

    D'Agostino, S.

    1992-01-01

    In the 50s, Schroedinger proposed a new conception of a continuous theory of Quantum Mechanics, which remarkably modified his 1926 ideas on ondulatory mechanics. The lack of individuality of the atomic particles presented in the new statistics, and in Heisenberg's Indeterminacy Relations, was by him considered as an aspect of a more general crisis in the anthology itself of classical atomism. Unlike his 1926 ideas, he proposed now to represent the wave equation in an n-dimensional space and he considered second-quantization technique as the proper mathematical tool for his new physical conception. Although he accepted that space-time discontinuities and casual gaps may appear here and there on the observational level (e.g. in the Indeterminacy Relations), he was convinced that they could be made compatible with a continuous pure theory, provided one accepted a suitable conception of the theory's epistemiological status. For him, only a continuous theory satisfied the conditions for a complete theory. On these matters, he thought he was somehow orthodox to the ideas of Hertz and Boltzmann, which were also reflected in the teaching of Exner. (author). 69 refs

  14. Kinetic investigation of the catalytic mechanism for bovine liver mitochondrial monoamine oxidase

    International Nuclear Information System (INIS)

    Walker, M.C.

    1988-01-01

    The kinetic behavior of the oxidative deamination reaction catalyzed by bovine liver mitochondrial monoamine oxidase was investigated with a series of ring-substituted benzylamines. Oxidation rates were fastest with the meta isomers. Dalziel coefficients were consistent with a mechanism involving a ternary complex for all substrates tested. Alterations in the Michaelis constant for oxygen were similar in magnitude to those for the rate of catalysis. Deuterium and tritium isotope effects were determined to obtain more detailed information on the mechanism of catalysis. Large deuterium isotope effects expressed on k cat were obtained for all substrates. Determination of the tritium isotope effect for benzylamine allowed the calculation of an intrinsic isotope effect of 6.5 and a secondary isotope effect of 1.17. Steady-state experiments were supplemented with pre-steady-state kinetic techniques. Rates of flavin reduction were faster than that of turnover. The deuterium isotope effect obtained for the rate of flavin reduction was 7-15 for the various substrates. The observed isotope effect was found to be an appropriate estimate for the intrinsic isotope effect

  15. The fluid mechanics of continuous flow electrophoresis

    Science.gov (United States)

    Saville, D. A.

    1990-01-01

    The overall objective is to establish theoretically and confirm experimentally the ultimate capabilities of continuous flow electrophoresis chambers operating in an environment essentially free of particle sedimentation and buoyancy. The efforts are devoted to: (1) studying the effects of particle concentration on sample conductivity and dielectric constant. The dielectric constant and conductivity were identified as playing crucial roles in the behavior of the sample and on the resolving power and throughput of continuous flow devices; and (2) improving the extant mathematical models to predict flow fields and particle trajectories in continuous flow electrophoresis. A dielectric spectrometer was designed and built to measure the complex dielectric constant of a colloidal dispersion as a function of frequency between 500 Hz and 200 kHz. The real part of the signal can be related to the sample's conductivity and the imaginary part to its dielectric constant. Measurements of the dielectric constants of several different dispersions disclosed that the dielectric constants of dilute systems of the sort encountered in particle electrophoresis are much larger than would be expected based on the extant theory. Experiments were carried out to show that, in many cases, this behavior is due to the presence of a filamentary structure of small hairs on the particle surface. A technique for producing electrokinetically ideal synthetic latex particles by heat treating was developed. Given the ubiquitous nature of hairy surfaces with both cells and synthetic particles, it was deemed necessary to develop a theory to explain their behavior. A theory for electrophoretic mobility of hairy particles was developed. Finally, the extant computer programs for predicting the structure of electro-osmotically driven flows were extended to encompass flow channels with variable wall mobilities.

  16. Continuous recrystallization and mechanical properties of a C45 steel after high pressure torsion

    Energy Technology Data Exchange (ETDEWEB)

    Haddad, Mike; Fecht, Hans [Institute of Micro and Nanomaterials, University of Ulm, Ulm, Gemany (Germany); Institute of Nanotechnology, Karlsruhe Institut of Technology, Karlsruhe (Germany); Ning, Jiang Li; Ivanisenko, Yulia [Institute of Nanotechnology, Karlsruhe Institut of Technology, Karlsruhe (Germany); Hoermann, Ute [Central Facility for Electron Microscopy, University of Ulm, Ulm (Germany); Murashkin, Maxim [IPAM, Ufa State Aviation Technical University, Ufa (Russian Federation)

    2011-07-01

    An initial microstructure of C45 steel (Fe, 0.42-0.5% C, 0.5-0.8% Mn, <0.4% Si, <0.045% S, 0.045% P * All in wt%) was modified from ferritic-pearlitic to bainitic through heating at 900 C for 1 hour and quenching in a lead bath at 500 C for 30 min. The steel was processed by High Pressure Torsion (HPT) at room temperature for 3 and 5 rotations, which led to an ultrafine or nano-scale grained structure. Then the material was annealed at 400 C and 450 C for two hours, respectively. TEM observation showed that continuous recrystallization occurred during the annealing. The yield strength and elongation after annealing at 400 C and 450 C were 1277 MPa, 3%, and 1100 MPa, 11%, respectively. Compared with the mechanical properties before annealing, it was revealed that the continuous recrystallization during annealing led to an enhancement of the ductility, but without sacrificing much of the strength. Funding by the Deutsche Forschungsgemeinschaft and Russian Foundation for basic research is gratefully aknowledged.

  17. Prolonged lateral steep position impairs respiratory mechanics during continuous lateral rotation therapy in respiratory failure.

    Science.gov (United States)

    Schellongowski, Peter; Losert, Heidrun; Locker, Gottfried J; Laczika, Klaus; Frass, Michael; Holzinger, Ulrike; Bojic, Andja; Staudinger, Thomas

    2007-04-01

    To establish whether prolonged lateral steep position during continuous rotation therapy leads to improvement on pulmonary gas exchange, respiratory mechanics and hemodynamics. Prospective observational study. Intensive care unit of a university hospital. Twelve consecutive patients suffering from acute lung injury or adult respiratory distress syndrome undergoing continuous rotation therapy. Blood gas analysis, static lung compliance, blood pressure, cardiac index and pulmonary shunt fraction were measured in supine as well as in left and right lateral steep position at 62 degrees during continuous rotation therapy (phase I). Rotation was then stopped for 30 min with the patients in supine position, left and right lateral steep position, and the same measurements were performed every 10 min (phase II). Phase I and II revealed no significant changes in PaO(2)/FiO(2) ratio, mean arterial blood pressure, pulmonary shunt fraction, or cardiac index. Significantly lower static compliance was observed in lateral steep position than in supine position (pposition than in left and right lateral steep position (ppositioning impairs the compliance of the respiratory system. Prolonged lateral steep position does not lead to benefits with respect to oxygenation or hemodynamics. Individual response to the different positions is unpredictable. The pauses in "extreme" positions should be as short as possible.

  18. Allosteric Inhibition of Factor XIIIa. Non-Saccharide Glycosaminoglycan Mimetics, but Not Glycosaminoglycans, Exhibit Promising Inhibition Profile.

    Directory of Open Access Journals (Sweden)

    Rami A Al-Horani

    Full Text Available Factor XIIIa (FXIIIa is a transglutaminase that catalyzes the last step in the coagulation process. Orthostery is the only approach that has been exploited to design FXIIIa inhibitors. Yet, allosteric inhibition of FXIIIa is a paradigm that may offer a key advantage of controlled inhibition over orthosteric inhibition. Such an approach is likely to lead to novel FXIIIa inhibitors that do not carry bleeding risks. We reasoned that targeting a collection of basic amino acid residues distant from FXIIIa's active site by using sulfated glycosaminoglycans (GAGs or non-saccharide GAG mimetics (NSGMs would lead to the discovery of the first allosteric FXIIIa inhibitors. We tested a library of 22 variably sulfated GAGs and NSGMs against human FXIIIa to discover promising hits. Interestingly, although some GAGs bound to FXIIIa better than NSGMs, no GAG displayed any inhibition. An undecasulfated quercetin analog was found to inhibit FXIIIa with reasonable potency (efficacy of 98%. Michaelis-Menten kinetic studies revealed an allosteric mechanism of inhibition. Fluorescence studies confirmed close correspondence between binding affinity and inhibition potency, as expected for an allosteric process. The inhibitor was reversible and at least 9-fold- and 26-fold selective over two GAG-binding proteins factor Xa (efficacy of 71% and thrombin, respectively, and at least 27-fold selective over a cysteine protease papain. The inhibitor also inhibited the FXIIIa-mediated polymerization of fibrin in vitro. Overall, our work presents the proof-of-principle that FXIIIa can be allosterically modulated by sulfated non-saccharide agents much smaller than GAGs, which should enable the design of selective and safe anticoagulants.

  19. Distribution of nutrients and antinutrients in milled fractions of chickpea and horse gram: seed coat phenolics and their distinct modes of enzyme inhibition.

    Science.gov (United States)

    Sreerama, Yadahally N; Neelam, Dennis A; Sashikala, Vadakkoot B; Pratape, Vishwas M

    2010-04-14

    Milled fractions of chickpea ( Cicer arietinum L.) and horse gram ( Macrotyloma uniflorum L. Verdc.) were evaluated for their nutritional and antinutritional characteristics. Crude protein content of these fractions ranged from 22.6-23.8 g 100(-1) g in cotyledon to 7.3-9.1 g 100(-1) g in seed coat fractions. The fat content of chickpea fractions (1.6-7.8 g 100(-1) g) was higher than that of horse gram fractions (0.6-2.6 g 100(-1) g). Crude fiber content was higher in seed coat fractions of both legumes than embryonic axe and cotyledon fractions. Seed coat fractions had high dietary fiber content (28.2-36.4 g 100(-1) g), made up of mainly insoluble dietary fiber. Most of the phytic acid and oligosaccharides were located in the cotyledon fractions, whereas phenolic compounds in higher concentrations were found in seed coats. Significantly higher concentrations of proteinaceous and phenolic inhibitors of digestive enzymes were found in cotyledon and seed coat fractions, respectively. The kinetic studies, using Michaelis-Menten and Lineweaver-Burk derivations, revealed that seed coat phenolics inhibit alpha-amylase activity by mixed noncompetitive (chickpea) and noncompetitive (horse gram) inhibition mechanisms. In the case of trypsin, chickpea and horse gram seed coat phenolics showed noncompetitive and uncompetitive modes of inhibition, respectively. These results suggest the wide variability in the nutrient and antinutrient composition in different milled fractions of legumes and potential utility of these fractions as ingredients in functional food product development.

  20. A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases.

    Science.gov (United States)

    Lisa, María-Natalia; Palacios, Antonela R; Aitha, Mahesh; González, Mariano M; Moreno, Diego M; Crowder, Michael W; Bonomo, Robert A; Spencer, James; Tierney, David L; Llarrull, Leticia I; Vila, Alejandro J

    2017-09-14

    Carbapenem-resistant Enterobacteriaceae threaten human health, since carbapenems are last resort drugs for infections by such organisms. Metallo-β-lactamases (MβLs) are the main mechanism of resistance against carbapenems. Clinically approved inhibitors of MBLs are currently unavailable as design has been limited by the incomplete knowledge of their mechanism. Here, we report a biochemical and biophysical study of carbapenem hydrolysis by the B1 enzymes NDM-1 and BcII in the bi-Zn(II) form, the mono-Zn(II) B2 Sfh-I and the mono-Zn(II) B3 GOB-18. These MβLs hydrolyse carbapenems via a similar mechanism, with accumulation of the same anionic intermediates. We characterize the Michaelis complex formed by mono-Zn(II) enzymes, and we identify all intermediate species, enabling us to propose a chemical mechanism for mono and binuclear MβLs. This common mechanism open avenues for rationally designed inhibitors of all MβLs, notwithstanding the profound differences between these enzymes' active site structure, β-lactam specificity and metal content.Carbapenem-resistant bacteria pose a major health threat by expressing metallo-β-lactamases (MβLs), enzymes able to hydrolyse these life-saving drugs. Here the authors use biophysical and computational methods and show that different MβLs share the same reaction mechanism, suggesting new strategies for drug design.

  1. Insight into the recognition, binding, and reactivity of catalytic metallodrugs targeting stem loop IIb of hepatitis C IRES RNA.

    Science.gov (United States)

    Bradford, Seth S; Ross, Martin James; Fidai, Insiya; Cowan, James A

    2014-06-01

    The complex Cu-GGHYrFK-amide (1-Cu) was previously reported as a novel metallotherapeutic that catalytically inactivates stem loop IIb (SLIIb) of the hepatitis C virus (HCV) internal ribosomal entry site (IRES) RNA and demonstrates significant antiviral activity in a cellular HCV replicon assay. Herein we describe additional studies focused on understanding the cleavage mechanism as well as the relationship of catalyst configuration to structural recognition and site-selective cleavage of the structured RNA motif. These are advanced by use of a combination of MALDI-TOF mass spectrometry, melting temperature determinations, and computational analysis to develop a structural model for binding and reactivity toward SLIIb of the IRES RNA. In addition, the binding, reactivity, and structural chemistry of the all-D-amino acid form of this metallopeptide, complex 2-Cu, are reported and compared with those of complex 1-Cu. In vitro RNA binding and cleavage assays for complex 2-Cu show a KD value of 76 ± 3 nM, and Michaelis-Menten parameters of kcat =0.14 ± 0.01 min(-1) and KM =7.9 ± 1.2 μM, with a turnover number exceeding 40. In a luciferase-based cellular replicon assay Cu-GGhyrfk-amide shows activity similar to that of the 1-Cu parent peptide, with an IC50 value of 1.9 ± 0.4 μM and cytotoxicity exceeding 100 μM. RT-PCR experiments confirm a significant decrease in HCV RNA levels in replicon assays for up to nine days when treated with complex 1-Cu in three-day dosing increments. This study shows the influence that the α-carbon stereocenter has for this new class of compounds, while detailed mass spectrometry and computational analyses provide new insight into the mechanisms of recognition, binding, and reactivity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Metabolic dynamics in skeletal muscle during acute reduction in blood flow and oxygen supply to mitochondria: in-silico studies using a multi-scale, top-down integrated model.

    Science.gov (United States)

    Dash, Ranjan K; Li, Yanjun; Kim, Jaeyeon; Beard, Daniel A; Saidel, Gerald M; Cabrera, Marco E

    2008-09-09

    Control mechanisms of cellular metabolism and energetics in skeletal muscle that may become evident in response to physiological stresses such as reduction in blood flow and oxygen supply to mitochondria can be quantitatively understood using a multi-scale computational model. The analysis of dynamic responses from such a model can provide insights into mechanisms of metabolic regulation that may not be evident from experimental studies. For the purpose, a physiologically-based, multi-scale computational model of skeletal muscle cellular metabolism and energetics was developed to describe dynamic responses of key chemical species and reaction fluxes to muscle ischemia. The model, which incorporates key transport and metabolic processes and subcellular compartmentalization, is based on dynamic mass balances of 30 chemical species in both capillary blood and tissue cells (cytosol and mitochondria) domains. The reaction fluxes in cytosol and mitochondria are expressed in terms of a general phenomenological Michaelis-Menten equation involving the compartmentalized energy controller ratios ATP/ADP and NADH/NAD(+). The unknown transport and reaction parameters in the model are estimated simultaneously by minimizing the differences between available in vivo experimental data on muscle ischemia and corresponding model outputs in coupled with the resting linear flux balance constraints using a robust, nonlinear, constrained-based, reduced gradient optimization algorithm. With the optimal parameter values, the model is able to simulate dynamic responses to reduced blood flow and oxygen supply to mitochondria associated with muscle ischemia of several key metabolite concentrations and metabolic fluxes in the subcellular cytosolic and mitochondrial compartments, some that can be measured and others that can not be measured with the current experimental techniques. The model can be applied to test complex hypotheses involving dynamic regulation of cellular metabolism and

  3. Kinetics of adsorption and uptake of Cu2+ by Chlorella vulgaris: influence of pH, temperature, culture age, and cations.

    Science.gov (United States)

    Mehta, S K; Singh, Alpana; Gaur, J P

    2002-03-01

    Adsorption and uptake of Cu2+ by Chlorella vulgaris were distinguished by extracting the surface-bound Cu2+ with EDTA. The uptake of Cu2+ followed Michaelis Menten kinetics. The maximum rate of Cu2+ uptake (0.362fmolcell(-1) h(-1)) was obtained at pH 6.0. The rate of Cu2+ uptake was greater for cultures in the exponential phase of growth, and increased with a rise in temperature from 6 to 25 degrees C, thus pointing towards an active mechanism. The maximum number of Cu2+ binding sites was 3.245 fmol cell(-1) at pH 4.5. Adsorption of Cu2+ was strongly pH-dependent thereby indicating that the number and nature of metal binding sites on the cell surface change with changing chemistry of the solution. Unlike uptake, the adsorption remained unaffected by small changes in temperature. Older cultures displayed a higher Cu2+ adsorption capacity than the exponentially growing ones thus suggesting generation of new and/or additional Cu2+ binding sites on older cells of C. vulgaris. By pH titration, the cation-exchange capacity of Chlorella, measured in terms of H+/ Na+ exchange, was about 17 fmol cell(-1) at pH 10.5. Negligible cation exchange capacity at and below pH 5.0 indicated that ion exchange was not the sole mechanism of Cu2+ adsorption by Chlorella. The uptake and adsorption of Cu2+ were inhibited by 100 microM of various cations including other heavy metal ions. The general concept that cations competitively inhibit accumulation of metals in living organisms does not hold for C. vulgaris. Non-competitive, uncompetitive and mixed inhibition of Cu2+ uptake and adsorption by various cations were more common than competitive inhibition.

  4. Studies on the kinetics of absorption of phosphorus by rice (Orysa sativa L.) and beans (Phaseolus vulgaris L.)

    International Nuclear Information System (INIS)

    Baraibar, A.; Villamil, J.; Fiore, M.F.; Marcondes, R.F.; Muraoka, T.; Cabral, C.P.; Malavolta, M.L.; Malavolta, E.

    1987-01-01

    Three experiments were conducted under controlled conditions with the objectives of evaluating the effect of different concentrations of phosphorus and on the presence of other ions on the kinetic of absorption. Excised roots of rice and bean were placed in aereated solutions containing increasing concentrations of NaH 2 PO 4 (10 -7 M to 5x10 -2 M) during 90 minutes. The rate of absorption (v = umols P/g dry matter) and the kinetic constants Vmax and Km were determined. Similar procedure was used to to evaluate the interaction of Mg +2 , Al +3 , K + , N-NH 4 + , N-NO 3 - and N-ureia in the uptake of phosphorus during 120 minutes. In another experiment, the effect of the presence of Mg +2 and/for Al +3 in the uptake and redistribution of phosphorus, was evaluated by varying the external concentration (1 ppm, 5 ppm, 10 ppm and 20 ppm) during a period of 17 hours, and utilizing whole rice plants. It was observed a dual mechanism, with two phases following the Michaelis-Menten kinetics and with transition phase 1 - 50 x 10 -5 M. The best explanation of the experimental data was obtained, by transforming the data in accordance with HOFSTEE (1952). Bean was more efficient than rice in the first phase of uptake (higher Vmax). Al 3 had a clear stimulatory effect on the uptake of phosphorus, promoting, however, the anion fixation in the root at lower concentrations. At the highest concentrations (20 ppm) of phosphorus this effect was not evident. No effect on the uptake was observed with Mg +2 , K + and different forms of nitrogen. Urea could have a depressive effect although, not significant. Possible mechanisms involved are discussed. (author) [pt

  5. Self-similar pattern formation and continuous mechanics of self-similar systems

    Directory of Open Access Journals (Sweden)

    A. V. Dyskin

    2007-01-01

    Full Text Available In many cases, the critical state of systems that reached the threshold is characterised by self-similar pattern formation. We produce an example of pattern formation of this kind – formation of self-similar distribution of interacting fractures. Their formation starts with the crack growth due to the action of stress fluctuations. It is shown that even when the fluctuations have zero average the cracks generated by them could grow far beyond the scale of stress fluctuations. Further development of the fracture system is controlled by crack interaction leading to the emergence of self-similar crack distributions. As a result, the medium with fractures becomes discontinuous at any scale. We develop a continuum fractal mechanics to model its physical behaviour. We introduce a continuous sequence of continua of increasing scales covering this range of scales. The continuum of each scale is specified by the representative averaging volume elements of the corresponding size. These elements determine the resolution of the continuum. Each continuum hides the cracks of scales smaller than the volume element size while larger fractures are modelled explicitly. Using the developed formalism we investigate the stability of self-similar crack distributions with respect to crack growth and show that while the self-similar distribution of isotropically oriented cracks is stable, the distribution of parallel cracks is not. For the isotropically oriented cracks scaling of permeability is determined. For permeable materials (rocks with self-similar crack distributions permeability scales as cube of crack radius. This property could be used for detecting this specific mechanism of formation of self-similar crack distributions.

  6. Continuity and completeness in physical theory: Schroedinger`s return to the wave interpretation of quantum mechanics in the 1950`s

    Energy Technology Data Exchange (ETDEWEB)

    D`Agostino, S. [Rome Univ. (Italy)

    1992-12-31

    In the 50s, Schroedinger proposed a new conception of a continuous theory of Quantum Mechanics, which remarkably modified his 1926 ideas on ondulatory mechanics. The lack of individuality of the atomic particles presented in the new statistics, and in Heisenberg`s Indeterminacy Relations, was by him considered as an aspect of a more general crisis in the anthology itself of classical atomism. Unlike his 1926 ideas, he proposed now to represent the wave equation in an n-dimensional space and he considered second-quantization technique as the proper mathematical tool for his new physical conception. Although he accepted that space-time discontinuities and casual gaps may appear here and there on the observational level (e.g. in the Indeterminacy Relations), he was convinced that they could be made compatible with a continuous pure theory, provided one accepted a suitable conception of the theory`s epistemiological status. For him, only a continuous theory satisfied the conditions for a complete theory. On these matters, he thought he was somehow orthodox to the ideas of Hertz and Boltzmann, which were also reflected in the teaching of Exner. (author). 69 refs.

  7. Reversible control of kinesin activity and microtubule gliding speeds by switching the doping states of a conducting polymer support

    Energy Technology Data Exchange (ETDEWEB)

    Martin, Brett D [US Naval Research Laboratory, Code 6930, Washington, DC 20375 (United States); Velea, Luminita M [US Naval Research Laboratory, Code 6930, Washington, DC 20375 (United States); Soto, Carissa M [US Naval Research Laboratory, Code 6930, Washington, DC 20375 (United States); Whitaker, Craig M [US Naval Academy, Department of Chemistry, Annapolis, MD 21402 (United States); Gaber, Bruce P [US Naval Research Laboratory, Code 6930, Washington, DC 20375 (United States); Ratna, Banahalli [US Naval Research Laboratory, Code 6930, Washington, DC 20375 (United States)

    2007-02-07

    We describe a method for reversibly controlling the ATPase activity of streptavidin-linked kinesin by changing the doping states of a conducting polymer support. When the polymer (poly(CH{sub 2}OH-EDOT)) was electrochemically switched from its dedoped (semiconducting) state to its doped (conducting) state, the ATPase activity of the adsorbed kinesin complex decreased by 35% with a concomitant decrease in the gliding speeds of kinesin-driven microtubules. When the polymer was switched back to its original dedoped state, nearly identical increases were observed in the kinesin ATPase activity and microtubule speeds. Use of a fluorescent ATP substrate analogue showed that the total amount of kinesin adsorbed on the poly(CH{sub 2}OH-EDOT) surface remained constant as the doping state of the polymer was switched. The microtubules exhibited nearly identical speed differences on the doped and dedoped surfaces for both chemical and electrochemical doping methods. Michaelis-Menten modelling suggests that the doped surface acts as an 'uncompetitive inhibitor' of kinesin. This work represents an investigation into the phenomenon of an electrically switchable surface exerting a moderating effect on the activity of an adsorbed protein that does not contain a bound, electroactive metal ion.

  8. Modeling cereal starch hydrolysis during simultaneous saccharification and lactic acid fermentation; case of a sorghum-based fermented beverage, gowé.

    Science.gov (United States)

    Mestres, Christian; Bettencourt, Munanga de J C; Loiseau, Gérard; Matignon, Brigitte; Grabulos, Joël; Achir, Nawel

    2017-10-01

    Gowé is an acidic beverage obtained after simultaneous saccharification and fermentation (SSF) of sorghum. A previous paper focused on modeling the growth of lactic acid bacteria during gowé processing. This paper focuses on modeling starch amylolysis to build an aggregated SSF model. The activity of α-amylase was modeled as a function of temperature and pH, and the hydrolysis rates of both native and soluble starch were modeled via a Michaelis-Menten equation taking into account the maltose and glucose inhibition constants. The robustness of the parameter estimators was ensured by step by step identification in sets of experiments conducted with different proportions of native and gelatinized starch by modifying the pre-cooking temperature. The aggregated model was validated on experimental data and showed that both the pre-cooking and fermentation parameters, particularly temperature, are significant levers for controlling not only acid and sugar contents but also the expected viscosity of the final product. This generic approach could be used as a tool to optimize the sanitary and sensory quality of fermentation of other starchy products. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Ultrasound assisted intensification of enzyme activity and its properties: a mini-review.

    Science.gov (United States)

    Nadar, Shamraja S; Rathod, Virendra K

    2017-08-22

    Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (k m and V max ) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

  10. [Kinetics of uptake of phosphates and nitrates by marine multicellular algae Gelidium latifolium (Grev.) Born. et Thur].

    Science.gov (United States)

    Silkin, V A; Chubchikova, I N

    2007-01-01

    We studied nonstationary kinetics of the uptake of phosphates and nitrates by the red marine algae Gelidium latifolium (Grev.) Born et Thur. and calculated constants of the Michaelis-Menten equation for these elements. In the area of 0-3 microM, the kinetics of phosphate consumption had the following coefficients: maximum rate of uptake 0.8 micromol/(g x h), constant of half-saturation 1.745 microM. For nitrate nitrogen at 0-30 microM, an adaptive strategy of uptake kinetics was noted with change of the equation parameters with time: after 1 h, the maximum rate of uptake was 5.1 micromol/(g x h) and constant of half-saturation 19 gM, while within 2 h, the maximum rate of uptake significantly increased. This could be related to the synthesis of nitrate reductase. Coupled with the uptake of nitrates, nonstationary kinetics of the release of nitrates in the surrounding medium had a one-peak pattern: the maximum concentration of nitrites in the medium and the time of its achievement increased with the initial concentration of nitrates. The maximum concentration of nitrites was 6 to 14% of the initial concentration in the medium.

  11. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    Science.gov (United States)

    Adi, Y. A.; Kusumo, F. A.; Aryati, L.; Hardianti, M. S.

    2016-04-01

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  12. Bi-enzyme L-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode.

    Science.gov (United States)

    Stasyuk, Nataliya; Smutok, Oleh; Gayda, Galina; Vus, Bohdan; Koval'chuk, Yevgen; Gonchar, Mykhailo

    2012-01-01

    A novel L-arginine-selective amperometric bi-enzyme biosensor based on recombinant human arginase I isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer was placed onto a polyaniline-Nafion composite platinum electrode and covered with a calcium alginate gel. The developed sensor revealed a good selectivity to L-arginine. The sensitivity of the biosensor was 110 ± 1.3 nA/(mM mm(2)) with the apparent Michaelis-Menten constant (K(M)(app)) derived from an L-arginine (L-Arg) calibration curve of 1.27 ± 0.29 mM. A linear concentration range was observed from 0.07 to 0.6mM, a limit of detection being 0.038 mM and a response time - 10s. The developed biosensor demonstrated good storage stability. A laboratory prototype of the proposed amperometric biosensor was applied to the samples of three commercial pharmaceuticals ("Tivortin", "Cytrarginine", "Aminoplazmal 10% E") for L-Arg testing. The obtained L-Arg-content values correlated well with those declared by producers. Copyright © 2012 Elsevier B.V. All rights reserved.

  13. A highly efficient microfluidic nano biochip based on nanostructured nickel oxide.

    Science.gov (United States)

    Ali, Md Azahar; Solanki, Pratima R; Patel, Manoj K; Dhayani, Hemant; Agrawal, Ved Varun; John, Renu; Malhotra, Bansi D

    2013-04-07

    We present results of the studies relating to fabrication of a microfluidic biosensor chip based on nickel oxide nanorods (NRs-NiO) that is capable of directly measuring the concentration of total cholesterol in human blood through electrochemical detection. Using this chip we demonstrate, with high reliability and in a time efficient manner, the detection of cholesterol present in buffer solutions at clinically relevant concentrations. The microfluidic channel has been fabricated onto a nickel oxide nanorod-based electrode co-immobilized with cholesterol esterase (ChEt) and cholesterol oxidase (ChOx) that serves as the working electrode. Bare indium tin oxide served as the counter electrode. A Ag/AgCl wire introduced to the outlet of the microchannel acts as a reference electrode. The fabricated NiO nanorod-based electrode has been characterized using X-ray diffraction, Raman spectroscopy, HR-TEM, FT-IR, UV-visible spectroscopy and electrochemical techniques. The presented NRs-NiO based microfluidic sensor exhibits linearity in the range of 1.5-10.3 mM, a high sensitivity of 0.12 mA mM(-1) cm(-2) and a low value of 0.16 mM of the Michaelis-Menten constant (Km).

  14. Exploring between the extremes: conversion-dependent kinetics of phosphite-modified hydroformylation catalysis.

    Science.gov (United States)

    Kubis, Christoph; Selent, Detlef; Sawall, Mathias; Ludwig, Ralf; Neymeyr, Klaus; Baumann, Wolfgang; Franke, Robert; Börner, Armin

    2012-07-09

    The kinetics of the hydroformylation of 3,3-dimethyl-1-butene with a rhodium monophosphite catalyst has been studied in detail. Time-dependent concentration profiles covering the entire olefin conversion range were derived from in situ high-pressure FTIR spectroscopic data for both, pure organic components and catalytic intermediates. These profiles fit to Michaelis-Menten-type kinetics with competitive and uncompetitive side reactions involved. The characteristics found for the influence of the hydrogen concentration verify that the pre-equilibrium towards the catalyst substrate complex is not established. It has been proven experimentally that the hydrogenolysis of the intermediate acyl complex remains rate limiting even at high conversions when the rhodium hydride is the predominant resting state and the reaction is nearly of first order with respect to the olefin. Results from in situ FTIR and high-pressure (HP) NMR spectroscopy and from DFT calculations support the coordination of only one phosphite ligand in the dominating intermediates and a preferred axial position of the phosphite in the electronically saturated, trigonal bipyramidal (tbp)-structured acyl rhodium complex. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. A new amperometric enzyme electrode for alcohol determination.

    Science.gov (United States)

    Gülce, H; Gülce, A; Kavanoz, M; Coşkun, H; Yildiz, A

    2002-06-01

    A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.

  16. Target-mediated drug disposition model and its approximations for antibody-drug conjugates.

    Science.gov (United States)

    Gibiansky, Leonid; Gibiansky, Ekaterina

    2014-02-01

    Antibody-drug conjugate (ADC) is a complex structure composed of an antibody linked to several molecules of a biologically active cytotoxic drug. The number of ADC compounds in clinical development now exceeds 30, with two of them already on the market. However, there is no rigorous mechanistic model that describes pharmacokinetic (PK) properties of these compounds. PK modeling of ADCs is even more complicated than that of other biologics as the model should describe distribution, binding, and elimination of antibodies with different toxin load, and also the deconjugation process and PK of the released toxin. This work extends the target-mediated drug disposition (TMDD) model to describe ADCs, derives the rapid binding (quasi-equilibrium), quasi-steady-state, and Michaelis-Menten approximations of the TMDD model as applied to ADCs, derives the TMDD model and its approximations for ADCs with load-independent properties, and discusses further simplifications of the system under various assumptions. The developed models are shown to describe data simulated from the available clinical population PK models of trastuzumab emtansine (T-DM1), one of the two currently approved ADCs. Identifiability of model parameters is also discussed and illustrated on the simulated T-DM1 examples.

  17. Effective immobilization of glucose oxidase on chitosan submicron particles from gladius of Todarodes pacificus for glucose sensing.

    Science.gov (United States)

    Anusha, J R; Fleming, Albin T; Kim, Hee-Je; Kim, Byung Chul; Yu, Kook-Hyun; Raj, C Justin

    2015-08-01

    An effective enzymatic glucose biosensor was developed by immobilizing glucose oxidase on chitosan submicron particles synthesized from the gladius of Todarodes pacificus (GCSP). The chemically synthesized chitosan from gladius was pulverized to submicron particles by ball milling technique, which was further characterized and compared with the standard chitosan (SCS). The degree of deacetylation of GCSP was determined using FTIR spectroscopy which was comparable to the value of standard chitosan. The glucose oxidase (GOx) was immobilized over GCSP on porous zinc oxide/platinum nanoparticle (ZnO/Pt) based electrode. The morphological and structural properties of the electrodes were analyzed using scanning electron microscopy and X-ray diffraction analysis. The glucose sensing behavior of electrode was estimated using electrochemical analysis and showed an excellent analytical performance. The electrode ZnO/Pt/GCSP conjugated with GOx displayed high sensitivity (88.76 μA mM(-1) cm(-2)) with low detection limit in short response time. In addition, the very low value of Michaelis-Menten constant for GCSP based electrode contributes a better affinity of the electrode surface towards glucose oxidase. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. A deterministic method for estimating free energy genetic network landscapes with applications to cell commitment and reprogramming paths.

    Science.gov (United States)

    Olariu, Victor; Manesso, Erica; Peterson, Carsten

    2017-06-01

    Depicting developmental processes as movements in free energy genetic landscapes is an illustrative tool. However, exploring such landscapes to obtain quantitative or even qualitative predictions is hampered by the lack of free energy functions corresponding to the biochemical Michaelis-Menten or Hill rate equations for the dynamics. Being armed with energy landscapes defined by a network and its interactions would open up the possibility of swiftly identifying cell states and computing optimal paths, including those of cell reprogramming, thereby avoiding exhaustive trial-and-error simulations with rate equations for different parameter sets. It turns out that sigmoidal rate equations do have approximate free energy associations. With this replacement of rate equations, we develop a deterministic method for estimating the free energy surfaces of systems of interacting genes at different noise levels or temperatures. Once such free energy landscape estimates have been established, we adapt a shortest path algorithm to determine optimal routes in the landscapes. We explore the method on three circuits for haematopoiesis and embryonic stem cell development for commitment and reprogramming scenarios and illustrate how the method can be used to determine sequential steps for onsets of external factors, essential for efficient reprogramming.

  19. Biotransformation of tetracycline by a novel bacterial strain Stenotrophomonas maltophilia DT1.

    Science.gov (United States)

    Leng, Yifei; Bao, Jianguo; Chang, Gaofeng; Zheng, Han; Li, Xingxing; Du, Jiangkun; Snow, Daniel; Li, Xu

    2016-11-15

    Although several abiotic processes have been reported that can transform antibiotics, little is known about whether and how microbiological processes may degrade antibiotics in the environment. This work isolated one tetracycline degrading bacterial strain, Stenotrophomonas maltophilia strain DT1, and characterized the biotransformation of tetracycline by DT1 under various environmental conditions. The biotransformation rate was the highest when the initial pH was 9 and the reaction temperature was at 30°C, and can be described using the Michaelis-Menten model under different initial tetracycline concentrations. When additional substrate was present, the substrate that caused increased biomass resulted in a decreased biotransformation rate of tetracycline. According to disk diffusion tests, the biotransformation products of tetracycline had lower antibiotic potency than the parent compound. Six possible biotransformation products were identified, and a potential biotransformation pathway was proposed that included sequential removal of N-methyl, carbonyl, and amine function groups. Results from this study can lead to better estimation of the fate and transport of antibiotics in the environment and has the potential to be utilized in designing engineering processes to remove tetracycline from water and soil. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Increase of a Calcium Independent Transglutaminase Activity in the Erythrocyte during the Infection with Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Wasserman Moisés

    1999-01-01

    Full Text Available We have studied the activity of a calcium dependent transglutaminase (EC 2.3.2.13 during the growth of the parasite Plasmodium falciparum inside the infected human erythrocyte. There is only one detectable transglutaminase in the two-cell-system, and its origin is erythrocytic. No activity was detected in preparations of the parasite devoid of erythrocyte cytoplasm. The Michaelis Menten constants (Km of the enzyme for the substrates N'N'dimethylcaseine and putrescine were undistinguishable whether the cell extracts used in their determination were obtained from normal or from infected red cells. The total activity of transglutaminase in stringently synchronized cultures, measured at 0.5mM Ca2+, decreased with the maturation of the parasite. However, a fraction which became irreversibly activated and independent of calcium concentration was detected. The proportion of this fraction grew with maturation; it represented only 20% of the activity in 20 hr-old-trophozoites while in 48-hr-schizonts it was more than 85% of the total activity. The activation of this fraction of transglutaminase did not depend on an increase in the erythrocyte cytoplasmic calcium, since most of the calcium was shown to be located in the parasite.

  1. Biodegradation of organic solid wastes from market places

    Directory of Open Access Journals (Sweden)

    Carlos Ariel Cardona Alzate

    2004-07-01

    Full Text Available In this research, the organic wastes from local market place were characterized, classified and conditioned. Feasible conversion treatments into added value products were analyzed. The transformation of starch and cellulose contained in wastes was chosen. The best conditions of temperature, pH and enzyme doses were established in order to transform the polysaccharides into reducing sugars. Commercial glucoamylase and cellulases for converting starch and cellulose were used. Starch conversion reached 60% at 50 °C and pH of 6.0. Cellulose conversion was of 4% at 60 °C and pH of 4,0. The kinetic research of starch hydrolysis based on Michaelis-Menten model was carried out. Ethanol was obtained from new-formed raw material (reducing sugars. In the same way, biogas and compost production was evaluated. It was determined that from each kilogram of treated wastes can be produced approximately 4 L of biogas at mesophilic range of temperature. It was recognized the possibility to carry out a composting process of plant wastes, despite their relatively low values of C/N ratio. Key words: Enzyme hydrolysis, ethanol, biogas, composting, starch.

  2. Effect of mass transfer in a recirculation batch reactor system for immobilized penicillin amidase.

    Science.gov (United States)

    Park, J M; Choi, C Y; Seong, B L; Han, M H

    1982-10-01

    The effect of external mass transfer resistance on the overall reaction rate of the immobilized whole cell penicillin amidase of E. coli in a recirculation batch reactor was investigated. The internal diffusional resistance was found negligible as indicated by the value of effectiveness factor, 0.95. The local environmental change in a column due to the pH drop was successfully overcome by employing buffer solution. The reaction rate was measured by pH-stat method and was found to follow the simple Michaelis-Menten law at the initial stage of the reaction. The values of the net reaction rate experimentally determined were used to calculate the substrate concentration at the external surface of the catalyst pellet and then to calculate the mass transfer coefficient, k(L), at various flow rates and substrate concentrations. The correlation proposed by Chilton and Colburn represented adequately the experimental data. The linear change of log j(D) at low log N(Re) with negative slope was ascribed to the fact that the external mass transfer approached the state of pure diffusion in the limit of zero superficial velocity.

  3. Studies on dosage of HIDANTOL-F by simultaneous determination of serum phenobarbital and diphenylhydantoin

    International Nuclear Information System (INIS)

    Yamada, Hideo; Tanno, Munehiko; Muraki, Toshio; Fuse, Masaaki.

    1987-01-01

    In order to investigate causes of untoward effects frequently observed in elderly patients on HIDANTOL-F which contains 300 mg of phenobarbital(PB), 100 mg of diphenylhydantoin(DPH) and 200 mg of Na benzoate-caffein, blood levels of both PB and DPH were measured by radioimmunoassay. About half of the patients showed blood levels of PB distributed in therapeutic range, some of which were very cross to toxic range. None of the blood samples except one, however, was in therapeutic range in regard to DPH. There was some degree of correlation between blood levels of these two drugs (r = 0.53). Absolute doses of PB and blood levels showed linear relationship, while DPH was curvelinear in shape in regard to doseblood level relation, suggestive of MichaelisMenten pharmacokinetics. Taking account of blood levels of these two drugs in the present study, phenobarbital must be responsible for untoward effects of HIDANTOL-F as well as anticonvulsive effect. Synergistic effect and mutual influences over pharmacokinetics of PB and DPH were discussed. Ratio of several drugs in case of combination therapy should be determined on the basis of study on pharmacokinetics and interaction of drugs. (author)

  4. Studies on dosage of HIDANTOL-F by simultaneous determination of serum phenobarbital and diphenylhydantoin

    Energy Technology Data Exchange (ETDEWEB)

    Yamada, Hideo; Tanno, Munehiko; Muraki, Toshio; Fuse, Masaaki.

    1987-10-01

    In order to investigate causes of untoward effects frequently observed in elderly patients on HIDANTOL-F which contains 300 mg of phenobarbital(PB), 100 mg of diphenylhydantoin(DPH) and 200 mg of Na benzoate-caffein, blood levels of both PB and DPH were measured by radioimmunoassay. About half of the patients showed blood levels of PB distributed in therapeutic range, some of which were very cross to toxic range. None of the blood samples except one, however, was in therapeutic range in regard to DPH. There was some degree of correlation between blood levels of these two drugs (r = 0.53). Absolute doses of PB and blood levels showed linear relationship, while DPH was curvelinear in shape in regard to doseblood level relation, suggestive of MichaelisMenten pharmacokinetics. Taking account of blood levels of these two drugs in the present study, phenobarbital must be responsible for untoward effects of HIDANTOL-F as well as anticonvulsive effect. Synergistic effect and mutual influences over pharmacokinetics of PB and DPH were discussed. Ratio of several drugs in case of combination therapy should be determined on the basis of study on pharmacokinetics and interaction of drugs.

  5. Nonlinear Growth Models as Measurement Models: A Second-Order Growth Curve Model for Measuring Potential.

    Science.gov (United States)

    McNeish, Daniel; Dumas, Denis

    2017-01-01

    Recent methodological work has highlighted the promise of nonlinear growth models for addressing substantive questions in the behavioral sciences. In this article, we outline a second-order nonlinear growth model in order to measure a critical notion in development and education: potential. Here, potential is conceptualized as having three components-ability, capacity, and availability-where ability is the amount of skill a student is estimated to have at a given timepoint, capacity is the maximum amount of ability a student is predicted to be able to develop asymptotically, and availability is the difference between capacity and ability at any particular timepoint. We argue that single timepoint measures are typically insufficient for discerning information about potential, and we therefore describe a general framework that incorporates a growth model into the measurement model to capture these three components. Then, we provide an illustrative example using the public-use Early Childhood Longitudinal Study-Kindergarten data set using a Michaelis-Menten growth function (reparameterized from its common application in biochemistry) to demonstrate our proposed model as applied to measuring potential within an educational context. The advantage of this approach compared to currently utilized methods is discussed as are future directions and limitations.

  6. Relationship among reaction rate, release rate and efficiency of nanomachine-based targeted drug delivery.

    Science.gov (United States)

    Zhao, Qingying; Li, Min; Luo, Jun

    2017-12-04

    In nanomachine applications towards targeted drug delivery, drug molecules released by nanomachines propagate and chemically react with tumor cells in aqueous environment. If the nanomachines release drug molecules faster than the tumor cells react, it will result in loss and waste of drug molecules. It is a potential issue associated with the relationship among reaction rate, release rate and efficiency. This paper aims to investigate the relationship among reaction rate, release rate and efficiency based on two drug reception models. We expect to pave a way for designing a control method of drug release. We adopted two analytical methods that one is drug reception process based on collision with tumors and another is based on Michaelis Menten enzymatic kinetics. To evaluate the analytical formulations, we used the well-known simulation framework N3Sim to establish simulations. The analytical results of the relationship among reaction rate, release rate and efficiency is obtained, which match well with the numerical simulation results in a 3-D environment. Based upon two drug reception models, the results of this paper would be beneficial for designing a control method of nanomahine-based drug release.

  7. In vitro characterization of the NAD+ synthetase NadE1 from Herbaspirillum seropedicae.

    Science.gov (United States)

    Laskoski, Kerly; Santos, Adrian R S; Bonatto, Ana C; Pedrosa, Fábio O; Souza, Emanuel M; Huergo, Luciano F

    2016-05-01

    Nicotinamide adenine dinucleotide synthetase enzyme (NadE) catalyzes the amination of nicotinic acid adenine dinucleotide (NaAD) to form NAD(+). This reaction represents the last step in the majority of the NAD(+) biosynthetic routes described to date. NadE enzymes typically use either glutamine or ammonium as amine nitrogen donor, and the reaction is energetically driven by ATP hydrolysis. Given the key role of NAD(+) in bacterial metabolism, NadE has attracted considerable interest as a potential target for the development of novel antibiotics. The plant-associative nitrogen-fixing bacteria Herbaspirillum seropedicae encodes two putative NadE, namely nadE1 and nadE2. The nadE1 gene is linked to glnB encoding the signal transduction protein GlnB. Here we report the purification and in vitro characterization of H. seropedicae NadE1. Gel filtration chromatography analysis suggests that NadE1 is an octamer. The NadE1 activity was assayed in vitro, and the Michaelis-Menten constants for substrates NaAD, ATP, glutamine and ammonium were determined. Enzyme kinetic and in vitro substrate competition assays indicate that H. seropedicae NadE1 uses glutamine as a preferential nitrogen donor.

  8. Solution Process Synthesis of High Aspect Ratio ZnO Nanorods on Electrode Surface for Sensitive Electrochemical Detection of Uric Acid

    Science.gov (United States)

    Ahmad, Rafiq; Tripathy, Nirmalya; Ahn, Min-Sang; Hahn, Yoon-Bong

    2017-04-01

    This study demonstrates a highly stable, selective and sensitive uric acid (UA) biosensor based on high aspect ratio zinc oxide nanorods (ZNRs) vertical grown on electrode surface via a simple one-step low temperature solution route. Uricase enzyme was immobilized on the ZNRs followed by Nafion covering to fabricate UA sensing electrodes (Nafion/Uricase-ZNRs/Ag). The fabricated electrodes showed enhanced performance with attractive analytical response, such as a high sensitivity of 239.67 μA cm-2 mM-1 in wide-linear range (0.01-4.56 mM), rapid response time (~3 s), low detection limit (5 nM), and low value of apparent Michaelis-Menten constant (Kmapp, 0.025 mM). In addition, selectivity, reproducibility and long-term storage stability of biosensor was also demonstrated. These results can be attributed to the high aspect ratio of vertically grown ZNRs which provides high surface area leading to enhanced enzyme immobilization, high electrocatalytic activity, and direct electron transfer during electrochemical detection of UA. We expect that this biosensor platform will be advantageous to fabricate ultrasensitive, robust, low-cost sensing device for numerous analyte detection.

  9. Modelling the removal of p-TSA (para-toluenesulfonamide) during rapid sand filtration used for drinking water treatment.

    Science.gov (United States)

    Meffe, Raffaella; Kohfahl, Claus; Holzbecher, Ekkehard; Massmann, Gudrun; Richter, Doreen; Dünnbier, Uwe; Pekdeger, Asaf

    2010-01-01

    A finite element model was set-up to determine degradation rate constants for p-TSA during rapid sand filtration (RSF). Data used for the model originated from a column experiment carried out in the filter hall of a drinking water treatment plant in Berlin (Germany). Aerated abstracted groundwater was passed through a 1.6m long column-shaped experimental sand filter applying infiltration rates from 2 to 6mh(-1). Model results were fitted to measured profiles and breakthrough curves of p-TSA for different infiltration rates using both first-order reaction kinetics and Michaelis-Menten kinetics. Both approaches showed that degradation rates varied both in space and time. Higher degradation rates were observed in the upper part of the column, probably related to higher microbial activity in this zone. Measured and simulated breakthrough curves revealed an adaption phase with lower degradation rates after infiltration rates were changed, followed by an adapted phase with more elevated degradation rates. Irrespective of the mathematical approach and the infiltration rate, degradation rates were very high, probably owing to the fact that filter sands have been in operation for decades, receiving high p-TSA concentrations with the raw water.

  10. Concentration profiles near an activated enzyme.

    Science.gov (United States)

    Park, Soohyung; Agmon, Noam

    2008-09-25

    When a resting enzyme is activated, substrate concentration profile evolves in its vicinity, ultimately tending to steady state. We use modern theories for many-body effects on diffusion-influenced reactions to derive approximate analytical expressions for the steady-state profile and the Laplace transform of the transient concentration profiles. These show excellent agreement with accurate many-particle Brownian-dynamics simulations for the Michaelis-Menten kinetics. The steady-state profile has a hyperbolic dependence on the distance of the substrate from the enzyme, albeit with a prefactor containing the complexity of the many-body effects. These are most conspicuous for the substrate concentration at the surface of the enzyme. It shows an interesting transition as a function of the enzyme turnover rate. When it is high, the contact concentration decays monotonically to steady state. However, for slow turnover it is nonmonotonic, showing a minimum due to reversible substrate binding, then a maximum due to diffusion of new substrate toward the enzyme, and finally decay to steady state. Under certain conditions one can obtain a good estimate for the critical value of the turnover rate constant at the transition.

  11. Role of conformational dynamics in kinetics of an enzymatic cycle in a nonequilibrium steady state

    Science.gov (United States)

    Min, Wei; Xie, X. Sunney; Bagchi, Biman

    2009-08-01

    Enzyme is a dynamic entity with diverse time scales, ranging from picoseconds to seconds or even longer. Here we develop a rate theory for enzyme catalysis that includes conformational dynamics as cycling on a two-dimensional (2D) reaction free energy surface involving an intrinsic reaction coordinate (X) and an enzyme conformational coordinate (Q). The validity of Michaelis-Menten (MM) equation, i.e., substrate concentration dependence of enzymatic velocity, is examined under a nonequilibrium steady state. Under certain conditions, the classic MM equation holds but with generalized microscopic interpretations of kinetic parameters. However, under other conditions, our rate theory predicts either positive (sigmoidal-like) or negative (biphasic-like) kinetic cooperativity due to the modified effective 2D reaction pathway on X-Q surface, which can explain non-MM dependence previously observed on many monomeric enzymes that involve slow or hysteretic conformational transitions. Furthermore, we find that a slow conformational relaxation during product release could retain the enzyme in a favorable configuration, such that enzymatic turnover is dynamically accelerated at high substrate concentrations. The effect of such conformation retainment in a nonequilibrium steady state is evaluated.

  12. Rapid Determination of Enzyme Kinetics from Fluorescence: Overcoming the Inner Filter Effect

    Science.gov (United States)

    Palmier, Mark O.; Van Doren, Steven R.

    2007-01-01

    Fluorescence change is convenient for monitoring enzyme kinetics. Unfortunately, it looses linearity as the absorbance of the fluorescent substrate increases with concentration. When the sum of absorbance at excitation and emission wavelengths exceeds 0.08, this inner filtering effect (IFE) alters apparent initial velocities, Km, and kcat. The IFE distortion of apparent initial velocities can be corrected without doing fluorophore dilution assays. Using the substrate’s extinction coefficients at excitation and emission wavelengths, the inner filter effect can be modeled during curve fitting for more accurate Michaelis-Menten parameters. A faster and simpler approach is to derive kcat and Km from progress curves. Strategies to obtain reliable and reproducible estimates of kcat and Km from only two or three progress curves are illustrated using matrix metalloproteinase-12 and alkaline phosphatase. Accurate estimates of concentration of enzyme active sites and specificity constant kcat/Km (from one progress curve with [S] ≪ Km) confer accuracy, freedom of choices of [S], and robustness to kcat and Km globally fitted to a few progress curves. The economies of the progress curve approach make accurate kcat and Km more accessible from fluorescence measurements. PMID:17706587

  13. The Non-Linear Child: Ontogeny, Isoniazid Concentration, and NAT2 Genotype Modulate Enzyme Reaction Kinetics and Metabolism

    Directory of Open Access Journals (Sweden)

    Zoe Rogers

    2016-09-01

    Full Text Available N-acetyltransferase 2 (NAT2 catalyzes the acetylation of isoniazid to N-acetylisoniazid. NAT2 polymorphism explains 88% of isoniazid clearance variability in adults. We examined the effects of clinical and genetic factors on Michaelis-Menten reaction kinetic constants of maximum velocity (Vmax and affinity (Km in children 0–10 years old. We measured the rates of isoniazid elimination and N-acetylisoniazid production in the blood of 30 children. Since maturation effects could be non-linear, we utilized a pharmacometric approach and the artificial intelligence method, multivariate adaptive regression splines (MARS, to identify factors predicting NAT2 Vmax and Km by examining clinical, genetic, and laboratory factors in toto. Isoniazid concentration predicted both Vmax and Km and superseded the contribution of NAT2 genotype. Age non-linearly modified the NAT2 genotype contribution until maturation at ≥5.3 years. Thus, enzyme efficiency was constrained by substrate concentration, genes, and age. Since MARS output is in the form of basis functions and equations, it allows multiscale systems modeling from the level of cellular chemical reactions to whole body physiological parameters, by automatic selection of significant predictors by the algorithm.

  14. A Novel ZnO-Methylene Blue Nanocomposite Matrix for Biosensing Application

    Directory of Open Access Journals (Sweden)

    Shibu Saha

    2011-01-01

    Full Text Available A novel hybrid matrix of zinc oxide-methylene blue (ZnO-MB has been successfully developed for biosensing application. The introduction of methylene blue into the ZnO thin film leads to reduction in the charge transfer resistance and suggests an increase in the electron transfer capacity of the composite. Glucose oxidase (GOx was chosen as the model enzyme and effectively immobilized on the surface of hybrid ZnO-MB nanocomposite matrix. Electrochemical measurements were employed to study biosensing response of the GOx/ZnO-MB/ITO bioelectrode as a function of glucose concentration. The low oxidation potential (−0.23 V of the hybrid bioelectrode, in a mediatorless electrolyte, makes it resistant against interference from other bio-molecules. The low value of Michaelis-Menten constant (2.65 mM indicates that immobilized GOx retains its enzymatic activity significantly on the surface of nanocomposite hybrid matrix that results in an enhanced affinity towards its substrate (glucose. The ZnO-MB nanocomposite hybrid matrix, exhibiting enhanced sensing response (0.2 μAmM−1cm−2 with long shelf-life (>10 weeks, has potential for the realization of an integrated biosensing device.

  15. Amperometric cholesterol biosensor based on the direct electrochemistry of cholesterol oxidase and catalase on a graphene/ionic liquid-modified glassy carbon electrode.

    Science.gov (United States)

    Gholivand, Mohammad Bagher; Khodadadian, Mehdi

    2014-03-15

    Cholesterol oxidase (ChOx) and catalase (CAT) were co-immobilized on a graphene/ionic liquid-modified glassy carbon electrode (GR-IL/GCE) to develop a highly sensitive amperometric cholesterol biosensor. The H2O2 generated during the enzymatic reaction of ChOx with cholesterol could be reduced electrocatalytically by immobilized CAT to obtain a sensitive amperometric response to cholesterol. The direct electron transfer between enzymes and electrode surface was investigated by cyclic voltammetry. Both enzymes showed well-defined redox peaks with quasi-reversible behaviors. An excellent sensitivity of 4.163 mA mM(-1)cm(-2), a response time less than 6s, and a linear range of 0.25-215 μM (R(2)>0.99) have been observed for cholesterol determination using the proposed biosensor. The apparent Michaelis-Menten constant (KM(app)) was calculated to be 2.32 mM. The bienzymatic cholesterol biosensor showed good reproducibility (RSDsascorbic acid and uric acid. The CAT/ChOx/GR-IL/GCE showed excellent analytical performance for the determination of free cholesterol in human serum samples. © 2013 Elsevier B.V. All rights reserved.

  16. Direct Electrochemistry of Horseradish Peroxidase-Gold Nanoparticles Conjugate

    Directory of Open Access Journals (Sweden)

    Chanchal K. Mitra

    2009-02-01

    Full Text Available We have studied the direct electrochemistry of horseradish peroxidase (HRP coupled to gold nanoparticles (AuNP using electrochemical techniques, which provide some insight in the application of biosensors as tools for diagnostics because HRP is widely used in clinical diagnostics kits. AuNP capped with (i glutathione and (ii lipoic acid was covalently linked to HRP. The immobilized HRP/AuNP conjugate showed characteristic redox peaks at a gold electrode. It displayed good electrocatalytic response to the reduction of H2O2, with good sensitivity and without any electron mediator. The covalent linking of HRP and AuNP did not affect the activity of the enzyme significantly. The response of the electrode towards the different concentrations of H2O2 showed the characteristics of Michaelis Menten enzyme kinetics with an optimum pH between 7.0 to 8.0. The preparation of the sensor involves single layer of enzyme, which can be carried out efficiently and is also highly reproducible when compared to other systems involving the layer-by-layer assembly, adsorption or encapsulation of the enzyme. The immobilized AuNP-HRP can be used for immunosensor applications

  17. Biophysical characterization of Vpu from HIV-1 suggests a channel-pore dualism.

    Science.gov (United States)

    Mehnert, T; Routh, A; Judge, P J; Lam, Y H; Fischer, D; Watts, A; Fischer, W B

    2008-03-01

    Vpu from HIV-1 is an 81 amino acid type I integral membrane protein which consists of a cytoplasmic and a transmembrane (TM) domain. The TM domain is known to alter membrane permeability for ions and substrates when inserted into artificial membranes. Peptides corresponding to the TM domain of Vpu (Vpu(1-32)) and mutant peptides (Vpu(1-32)-W23L, Vpu(1-32)-R31V, Vpu(1-32)-S24L) have been synthesized and reconstituted into artificial lipid bilayers. All peptides show channel activity with a main conductance level of around 20 pS. Vpu(1-32)-W23L has a considerable flickering pattern in the recordings and longer open times than Vpu(1-32). Whilst recordings for Vpu(1-32)-R31V are almost indistinguishable from those of the WT peptide, recordings for Vpu(1-32)-S24L do not exhibit any noticeable channel activity. Recordings of WT peptide and Vpu(1-32)-W23L indicate Michaelis-Menten behavior when the salt concentration is increased. Both peptide channels follow the Eisenman series I, indicative for a weak ion channel with almost pore like characteristics. 2007 Wiley-Liss, Inc.

  18. Estimate of respiration rate and physicochemical changes of fresh-cut apples stored under different temperatures

    Directory of Open Access Journals (Sweden)

    Cristiane Fagundes

    2013-03-01

    Full Text Available In this study, the influence of storage temperature and passive modified packaging (PMP on the respiration rate and physicochemical properties of fresh-cut Gala apples (Malus domestica B. was investigated. The samples were packed in flexible multilayer bags and stored at 2 °C, 5 °C, and 7 °C for eleven days. Respiration rate as a function of CO2 and O2 concentrations was determined using gas chromatography. The inhibition parameters were estimated using a mathematical model based on Michaelis-Menten equation. The following physicochemical properties were evaluated: total soluble solids, pH, titratable acidity, and reducing sugars. At 2 °C, the maximum respiration rate was observed after 150 hours. At 5 °C and 7 °C the maximum respiration rates were observed after 100 and 50 hours of storage, respectively. The inhibition model results obtained showed a clear effect of CO2 on O2 consumption. The soluble solids decreased, although not significantly, during storage at the three temperatures studied. Reducing sugars and titratable acidity decreased during storage and the pH increased. These results indicate that the respiration rate influenced the physicochemical properties.

  19. Metastable α-AgVO3 microrods as peroxidase mimetics for colorimetric determination of H2O2.

    Science.gov (United States)

    Wang, Yi; Zhang, Dun; Wang, Jin

    2017-12-01

    Single phase metastable α-AgVO 3 microrods with high crystallinity, tetragonal rod-like microstructure, uniform particle size distribution, and good dispersion were synthesized by direct coprecipitation at room temperature. They are shown to be viable peroxidase mimics that catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of H 2 O 2 . Kinetic analysis indicated typical Michaelis-Menten catalytic behavior. The findings were used to design a colorimetric assay for H 2 O 2 , best measured at 652 nm. The method has a linear response in the 60 to 200 μM H 2 O 2 concentration range, with a 2 μM detection limit. Benefitting from the chemical stability of the microrods, the method is well reproducible. It also is easily performed and highly specific. Graphic abstract Single phase metastable α-AgVO 3 microrods with high crystallinity, tetragonal rod-like microstructure, uniform particle size distribution, and good dispersion can efficiently catalyze the oxidation reaction of peroxidase substrate 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of H 2 O 2 to produce a blue color change.

  20. Immobilization of glucose oxidase using CoFe2O4/SiO2 nanoparticles as carrier

    Science.gov (United States)

    Wang, Hai; Huang, Jun; Wang, Chao; Li, Dapeng; Ding, Liyun; Han, Yun

    2011-04-01

    Aminated-CoFe2O4/SiO2 magnetic nanoparticles (NPs) were prepared from primary silica particles using modified StÖber method. Glucose oxidase (GOD) was immobilized on CoFe2O4/SiO2 NPs via cross-linking with glutaraldehyde (GA). The optimal immobilization condition was achieved with 1% (v/v) GA, cross-linking time of 3 h, solution pH of 7.0 and 0.4 mg GOD (in 3.0 mg carrier). The immobilized GOD showed maximal catalytic activity at pH 6.5 and 40 °C. After immobilization, the GOD exhibited improved thermal, storage and operation stability. The immobilized GOD still maintained 80% of its initial activity after the incubation at 50 °C for 25 min, whereas free enzyme had only 20% of initial activity after the same incubation. After kept at 4 °C for 28 days, the immobilized and free enzyme retained 87% and 40% of initial activity, respectively. The immobilized GOD maintained approximately 57% of initial activity after reused 7 times. The KM (Michaelis-Menten constant) values for immobilized GOD and free GOD were 14.6 mM and 27.1 mM, respectively.

  1. A mathematical model of phosphorylation AKT in Acute Myeloid Leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Adi, Y. A., E-mail: yudi.adi@math.uad.ac.id [Department of Mathematic Faculty of MIPA Universitas Ahmad Dahlan (Indonesia); Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Kusumo, F. A.; Aryati, L. [Department of Mathematic Faculty of MIPA Universitas Gadjah Mada (Indonesia); Hardianti, M. S. [Department of Internal Medicine, Faculty of Medicine, Universitas Gadjah Mada (Indonesia)

    2016-04-06

    In this paper we consider a mathematical model of PI3K/AKT signaling pathways in phosphorylation AKT. PI3K/AKT pathway is an important mediator of cytokine signaling implicated in regulation of hematopoiesis. Constitutive activation of PI3K/AKT signaling pathway has been observed in Acute Meyloid Leukemia (AML) it caused by the mutation of Fms-like Tyrosine Kinase 3 in internal tandem duplication (FLT3-ITD), the most common molecular abnormality associated with AML. Depending upon its phosphorylation status, protein interaction, substrate availability, and localization, AKT can phosphorylate or inhibite numerous substrates in its downstream pathways that promote protein synthesis, survival, proliferation, and metabolism. Firstly, we present a mass action ordinary differential equation model describing AKT double phosphorylation (AKTpp) in a system with 11 equations. Finally, under the asumtion enzyme catalyst constant and steady state equilibrium, we reduce the system in 4 equation included Michaelis Menten constant. Simulation result suggested that a high concentration of PI3K and/or a low concentration of phospatase increased AKTpp activation. This result also indicates that PI3K is a potential target theraphy in AML.

  2. Microscale Measurements of Michaelis–Menten Constants of Neuraminidase with Nanogel Capillary Electrophoresis for the Determination of the Sialic Acid Linkage

    Science.gov (United States)

    2016-01-01

    Phospholipid nanogels enhance the stability and performance of the exoglycosidase enzyme neuraminidase and are used to create a fixed zone of enzyme within a capillary. With nanogels, there is no need to covalently immobilize the enzyme, as it is physically constrained. This enables rapid quantification of Michaelis–Menten constants (KM) for different substrates and ultimately provides a means to quantify the linkage (i.e., 2-3 versus 2-6) of sialic acids. The fixed zone of enzyme is inexpensive and easily positioned in the capillary to support electrophoresis mediated microanalysis using neuraminidase to analyze sialic acid linkages. To circumvent the limitations of diffusion during static incubation, the incubation period is reproducibly achieved by varying the number of forward and reverse passes the substrate makes through the stationary fixed zone using in-capillary electrophoretic mixing. A KM value of 3.3 ± 0.8 mM (Vmax, 2100 ± 200 μM/min) was obtained for 3′-sialyllactose labeled with 2-aminobenzoic acid using neuraminidase from Clostridium perfringens that cleaves sialic acid monomers with an α2-3,6,8,9 linkage, which is similar to values reported in the literature that required benchtop analyses. The enzyme cleaves the 2-3 linkage faster than the 2-6, and a KM of 2 ± 1 mM (Vmax, 400 ± 100 μM/min) was obtained for the 6′-sialyllactose substrate. An alternative neuraminidase selective for 2-3 sialic acid linkages generated a KM value of 3 ± 2 mM (Vmax, 900 ± 300 μM/min) for 3′-sialyllactose. With a knowledge of Vmax, the method was applied to a mixture of 2-3 and 2-6 sialyllactose as well as 2-3 and 2-6 sialylated triantennary glycan. Nanogel electrophoresis is an inexpensive, rapid, and simple alternative to current technologies used to distinguish the composition of 3′ and 6′ sialic acid linkages. PMID:27936604

  3. Mercury transfer from soil to olive trees. A comparison of three different contaminated sites.

    Science.gov (United States)

    Higueras, Pablo L; Amorós, José Á; Esbrí, José Maria; Pérez-de-los-Reyes, Caridad; López-Berdonces, Miguel A; García-Navarro, Francisco J

    2016-04-01

    Mercury contents in soil and olive tree leaves have been studied in 69 plots around three different source areas of this element in Spain: Almadén (Ciudad Real), Flix (Tarragona) and Jódar (Jaén). Almadén was the world's largest cinnabar (HgS) mining district and was active until 2003, Flix is the oldest Spanish chlor-alkali plant (CAP) and has been active from 1898 to the present day and Jódar is a decommissioned CAP that was active for 14 years (1977-1991). Total mercury contents have been measured by high-frequency modulation atomic absorption spectrometry with Zeeman effect (ZAAS-HFM) in the soils and olive tree leaves from the three studied areas. The average soil contents range from 182 μg kg(-1) in Flix to 23,488 μg kg(-1) in Almadén, while the average leaf content ranges from 161 μg kg(-1) in Jódar to 1213 μg kg(-1) in Almadén. Despite the wide range of data, a relationship between soil-leaf contents has been identified: in Almadén and Jódar, multiplicative (bilogarithmic) models show significant correlations (R = 0.769 and R = 0.484, respectively). Significant correlations were not identified between soil and leaf contents in Flix. The continuous activity of the Flix CAP, which remains open today, can explain the different uptake patterns for mercury, which is mainly atmospheric in origin, in comparison to the other two sites, where activity ceased more than 10 years ago and only soil uptake patterns based on the Michaelis-Menten enzymatic model curve are observed.

  4. 21 CFR 868.5895 - Continuous ventilator.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Continuous ventilator. 868.5895 Section 868.5895...) MEDICAL DEVICES ANESTHESIOLOGY DEVICES Therapeutic Devices § 868.5895 Continuous ventilator. (a) Identification. A continuous ventilator (respirator) is a device intended to mechanically control or assist...

  5. Thermodynamically consistent coarse graining of biocatalysts beyond Michaelis–Menten

    Science.gov (United States)

    Wachtel, Artur; Rao, Riccardo; Esposito, Massimiliano

    2018-04-01

    Starting from the detailed catalytic mechanism of a biocatalyst we provide a coarse-graining procedure which, by construction, is thermodynamically consistent. This procedure provides stoichiometries, reaction fluxes (rate laws), and reaction forces (Gibbs energies of reaction) for the coarse-grained level. It can treat active transporters and molecular machines, and thus extends the applicability of ideas that originated in enzyme kinetics. Our results lay the foundations for systematic studies of the thermodynamics of large-scale biochemical reaction networks. Moreover, we identify the conditions under which a relation between one-way fluxes and forces holds at the coarse-grained level as it holds at the detailed level. In doing so, we clarify the speculations and broad claims made in the literature about such a general flux–force relation. As a further consequence we show that, in contrast to common belief, the second law of thermodynamics does not require the currents and the forces of biochemical reaction networks to be always aligned.

  6. Function of mechanically lengthened jejunum after restoration into continuity.

    Science.gov (United States)

    Sullins, Veronica F; Wagner, Justin P; Walthers, Christopher M; Chiang, Elvin K; Lee, Steven L; Wu, Benjamin M; Dunn, James C Y

    2014-06-01

    Distraction enterogenesis is a potential treatment for patients with short bowel syndrome. We previously demonstrated successful lengthening of jejunum using a degradable spring device in rats. Absorptive function of the lengthened jejunum after restoration into intestinal continuity needs to be determined. Encapsulated polycaprolactone springs were placed into isolated jejunal segments in rats for four weeks. Lengthened segments of jejunum were subsequently restored into intestinal continuity. Absorption studies were performed by placing a mixture of a non-absorbable substrate and glucose into the lumen of the restored jejunum. Restored jejunal segments demonstrated visible peristalsis at specimen retrieval. Compared to normal jejunal controls, restored segments demonstrated equal water absorption and greater glucose absorption. Restored segments had thicker smooth muscle, increased villus height, increased crypt depth, and decreased sucrase activity compared to normal jejunum. The density of enteric ganglia increased after restoration to near normal levels in the submucosa and to normal levels in the myenteric plexus. Jejunum lengthened with a degradable device demonstrates peristaltic and enzymatic activity as well as glucose and water absorption after restoration into intestinal continuity. Our findings further demonstrate the therapeutic potential of a degradable device. Copyright © 2014 Elsevier Inc. All rights reserved.

  7. 15 CFR 923.57 - Continuing consultation.

    Science.gov (United States)

    2010-01-01

    ... § 923.57 Continuing consultation. (a) As required by subsection 306(d)(3)(B) of the Act, a State must establish an effective mechanism for continuing consultation and coordination between the management agency... 15 Commerce and Foreign Trade 3 2010-01-01 2010-01-01 false Continuing consultation. 923.57...

  8. The effect of microstructure and texture evolution on mechanical properties of low-carbon steel processed by the continuous hybrid process

    International Nuclear Information System (INIS)

    Hwang, Sun Kwang; Baek, Hyun Moo; Son, Il-Heon; Im, Yong-Taek; Bae, Chul Min

    2013-01-01

    In this paper, the continuous hybrid process is newly designed and applied for producing grain-refined long and large cross-section wires of low-carbon steel at high speed at room temperature. The initial specimen, with a diameter of 13 mm, continuously passes through the rolls, equal channel angular pressing (ECAP) dies, and wire-drawing dies in sequence during the process. The specimens deformed by the continuous hybrid process without and with the wire-drawing dies were obtained to investigate the role in the deformation separately. Their microstructures, textures, and mechanical properties were investigated by optical microscopy (OM), electron backscattering diffraction (EBSD), X-ray diffraction (XRD), tension, and Vickers micro-hardness tests and were compared with those for the case processed by the conventional wire-drawing process. According to the present investigation, the continuous hybrid process can more efficiently manufacture fine-grained wires with a strong shear texture in a continuous way than the conventional wire-drawing process can. In addition, the ultimate tensile strength value of the specimen processed by the continuous hybrid process was 23.9% higher, although the elongation was slightly lower than the one produced by the conventional wire-drawing process. The plastic deformation was mainly imposed by the ECAP dies, and the wire-drawing dies improve the dimensional accuracy and increase the local strain homogeneity in the continuous hybrid process. It is demonstrated that the continuous hybrid process might be beneficial in commercializing a continuous application of the severe plastic deformation process for producing grain-refined wires for industrial applications

  9. Investigation on the Mechanical and Electrical Behavior of a Tuning Fork-Shaped Ionic Polymer Metal Composite Actuator with a Continuous Water Supply Mechanism.

    Science.gov (United States)

    Feng, Guo-Hua; Huang, Wei-Lun

    2016-03-25

    This paper presents an innovative tuning fork-shaped ionic polymer metal composite (IPMC) actuator. With an integrated soft strain gauge and water supply mechanism (WSM), the surface strain of the actuator can be sensed in situ, and providing a continuous water supply maintains the water content inside the IPMC for long-term operation in air. The actuator was fabricated using a micromachining technique and plated with a nickel electrode. The device performance was experimentally characterized and compared with an actuator without a WSM. A large displacement of 1.5 mm was achieved for a 6 mm-long prong with 7-V dc actuation applied for 30 s. The measured current was analyzed using an electrochemical model. The results revealed that the faradaic current plays a crucial role during operation, particularly after 10 s. The measured strain confirms both the bending and axial strain generation during the open-and-close motion of the actuator prongs. Most of the water loss during device operation was due to evaporation rather than hydrolysis. The constructed WSM effectively maintained the water content inside the IPMC for long-term continuous operation.

  10. Arbuscular mycorrhiza and kinetic parameters of phosphorus absorption by bean plants Micorriza arbuscular e os parâmetros cinéticos de absorção de fósforo pelo feijoeiro

    Directory of Open Access Journals (Sweden)

    Adriana Parada Dias da Silveira

    2004-04-01

    Full Text Available The mechanisms that determine greater P absorption by mycorrhizal plants are still not completely clear, and are attributed, in part, to an increase in the number of absorption sites promoted by the hyphae, and/or to a greater affinity of the colonized hypha or root carriers to P. The effect of mycorrhizae formed by Glomus etunicatum on the kinetic parameters of P absorption by the roots and on P influx in bean plants of the IAC-Carioca cultivar was evaluated, in two distinct plant development periods: at the onset of flowering and at the pod-filling stage (35 and 50 days after sowing, respectively. A mixture of sand and silica (9:1 was utilized as substrate and irrigated with nutrient solution. The kinetics assay was performed by the method of 32P depletion from the solution (depletion curve, using intact plants. Mycorrhization promoted greater growth and P absorption by bean plants, which was more conspicuously observed at the pod-filling stage. Mycorrhizal plants showed higher values of maximum ion uptake rate (Vmax and net P influx at the flowering stage. Lower minimum ion concentration (Cmin and Michaelis-Menten constant (Km values were verified in mycorrhizal plants at the pod-filling stage. Mycorrhizal plants also presented higher net P influx per plant, in both stages. Cmin was the kinetic parameter more intimately related to P absorption, and a significant correlation was obtained between this parameter and shoot P content and accumulation in bean plants.Os mecanismos envolvidos na maior absorção de P pela planta micorrizada ainda não estão totalmente esclarecidos, atribuindo-se, em parte, ao aumento no número de sítios de absorção promovido pela hifa e/ou maior afinidade dos carregadores da hifa ou da raiz colonizada ao P. Avaliou-se o efeito da micorriza formada por Glomus etunicatum nos parâmetros cinéticos da absorção radicular de P e no influxo de P em feijoeiro, cultivar IAC-Carioca, em duas épocas do ciclo da planta

  11. Understanding transporter specificity and the discrete appearance of channel-like gating domains in transporters

    Directory of Open Access Journals (Sweden)

    GEORGE eDIALLINAS

    2014-09-01

    Full Text Available Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open towards the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action.

  12. Kinetic characterization of a novel acid ectophosphatase from Enterobacter asburiae.

    Science.gov (United States)

    Sato, Vanessa Sayuri; Galdiano Júnior, Renato F; Rodrigues, Gisele Regina; Lemos, Eliana G M; Pizauro Junior, João Martins

    2016-02-01

    Expression of acid ectophosphatase by Enterobacter asburiae, isolated from Cattleya walkeriana (Orchidaceae) roots and identified by the 16S rRNA gene sequencing analysis, was strictly regulated by phosphorus ions, with its optimal activity being observed at an inorganic phosphate concentration of 7 mM. At the optimum pH 3.5, intact cells released p-nitrophenol at a rate of 350.76 ± 13.53 nmol of p-nitrophenolate (pNP)/min/10(8) cells. The membrane-bound enzyme was obtained by centrifugation at 100,000 × g for 1 h at 4 °C. p-Nitrophenylphosphate (pNPP) hydrolysis by the enzyme follows "Michaelis-Menten" kinetics with V = 61.2 U/mg and K0.5 = 60 μM, while ATP hydrolysis showed V = 19.7 U/mg, K0.5 = 110 μM, and nH = 1.6 and pyrophosphate hydrolysis showed V = 29.7 U/mg, K0.5 = 84 μM, and nH = 2.3. Arsenate and phosphate were competitive inhibitors with K i = 0.6 mM and K i = 1.8 mM, respectively. p-Nitrophenyl phosphatase (pNPPase) activity was inhibited by vanadate, while p-hydroxymercuribenzoate, EDTA, calcium, copper, and cobalt had no inhibitory effects. Magnesium ions were stimulatory (K0.5 = 2.2 mM and nH = 0.5). Production of an acid ectophosphatase can be a mechanism for the solubilization of mineral phosphates by microorganisms such as Enterobacter asburiae that are versatile in the solubilization of insoluble minerals, which, in turn, increases the availability of nutrients for plants, particularly in soils that are poor in phosphorus.

  13. Catalytic Properties of the Isolated Diaphorase Fragment of the NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha

    Science.gov (United States)

    Lauterbach, Lars; Idris, Zulkifli; Vincent, Kylie A.; Lenz, Oliver

    2011-01-01

    The NAD+-reducing soluble hydrogenase (SH) from Ralstonia eutropha H16 catalyzes the H2-driven reduction of NAD+, as well as reverse electron transfer from NADH to H+, in the presence of O2. It comprises six subunits, HoxHYFUI2, and incorporates a [NiFe] H+/H2 cycling catalytic centre, two non-covalently bound flavin mononucleotide (FMN) groups and an iron-sulfur cluster relay for electron transfer. This study provides the first characterization of the diaphorase sub-complex made up of HoxF and HoxU. Sequence comparisons with the closely related peripheral subunits of Complex I in combination with UV/Vis spectroscopy and the quantification of the metal and FMN content revealed that HoxFU accommodates a [2Fe2S] cluster, FMN and a series of [4Fe4S] clusters. Protein film electrochemistry (PFE) experiments show clear electrocatalytic activity for both NAD+ reduction and NADH oxidation with minimal overpotential relative to the potential of the NAD+/NADH couple. Michaelis-Menten constants of 56 µM and 197 µM were determined for NADH and NAD+, respectively. Catalysis in both directions is product inhibited with K I values of around 0.2 mM. In PFE experiments, the electrocatalytic current was unaffected by O2, however in aerobic solution assays, a moderate superoxide production rate of 54 nmol per mg of protein was observed, meaning that the formation of reactive oxygen species (ROS) observed for the native SH can be attributed mainly to HoxFU. The results are discussed in terms of their implications for aerobic functioning of the SH and possible control mechanism for the direction of catalysis. PMID:22016788

  14. Environmental Factors Modulating the Stability and Enzymatic Activity of the Petrotoga mobilis Esterase (PmEst.

    Directory of Open Access Journals (Sweden)

    Jose L S Lopes

    Full Text Available Enzymes isolated from thermophilic organisms found in oil reservoirs can find applications in many fields, including the oleochemical, pharmaceutical, bioenergy, and food/dairy industries. In this study, in silico identification and recombinant production of an esterase from the extremophile bacteria Petrotoga mobilis (designated PmEst were performed. Then biochemical, bioinformatics and structural characterizations were undertaken using a combination of synchrotron radiation circular dichroism (SRCD and fluorescence spectroscopies to correlate PmEst stability and hydrolytic activity on different substrates. The enzyme presented a high Michaelis-Menten constant (KM 0.16 mM and optimum activity at ~55°C for p-nitrophenyl butyrate. The secondary structure of PmEst was preserved at acid pH, but not under alkaline conditions. PmEst was unfolded at high concentrations of urea or guanidine through apparently different mechanisms. The esterase activity of PmEst was preserved in the presence of ethanol or propanol and its melting temperature increased ~8°C in the presence of these organic solvents. PmEst is a mesophilic esterase with substrate preference towards short-to medium-length acyl chains. The SRCD data of PmEst is in agreement with the prediction of an α/β protein, which leads us to assume that it displays a typical fold of esterases from this family. The increased enzyme stability in organic solvents may enable novel applications for its use in synthetic biology. Taken together, our results demonstrate features of the PmEst enzyme that indicate it may be suitable for applications in industrial processes, particularly, when the use of polar organic solvents is required.

  15. Digestive enzyme activities and gastrointestinal fermentation in wood-eating catfishes.

    Science.gov (United States)

    German, Donovan P; Bittong, Rosalie A

    2009-11-01

    To determine what capabilities wood-eating and detritivorous catfishes have for the digestion of refractory polysaccharides with the aid of an endosymbiotic microbial community, the pH, redox potentials, concentrations of short-chain fatty acids (SCFAs), and the activity levels of 14 digestive enzymes were measured along the gastrointestinal (GI) tracts of three wood-eating taxa (Panaque cf. nigrolineatus "Marañon", Panaque nocturnus, and Hypostomus pyrineusi) and one detritivorous species (Pterygoplichthys disjunctivus) from the family Loricariidae. Negative redox potentials (-600 mV) were observed in the intestinal fluids of the fish, suggesting that fermentative digestion was possible. However, SCFA concentrations were low (<3 mM in any intestinal region), indicating that little GI fermentation occurs in the fishes' GI tracts. Cellulase and xylanase activities were low (<0.03 U g(-1)), and generally decreased distally in the intestine, whereas amylolytic and laminarinase activities were five and two orders of magnitude greater, respectively, than cellulase and xylanase activities, suggesting that the fish more readily digest soluble polysaccharides. Furthermore, the Michaelis-Menten constants (K(m)) of the fishes' beta-glucosidase and N-acetyl-beta-D-glucosaminidase enzymes were significantly lower than the K(m) values of microbial enzymes ingested with their food, further suggesting that the fish efficiently digest soluble components of their detrital diet rather than refractory polysaccharides. Coupled with rapid gut transit and poor cellulose digestibility, the wood-eating catfishes appear to be detritivores reliant on endogenous digestive mechanisms, as are other loricariid catfishes. This stands in contrast to truly "xylivorous" taxa (e.g., beavers, termites), which are reliant on an endosymbiotic community of microorganisms to digest refractory polysaccharides.

  16. How energetic and environmental constraints of microorganisms determine the carbon turnover in soils

    Science.gov (United States)

    Don, A.; Rödenbeck, C.; Gleixner, G.

    2012-04-01

    Microorganisms are the main catalysts driving carbon fluxes from soils. Traditional concepts of soil carbon stabilization failed to account for environmental and energy constraints of microorganisms. The distribution and density of organic carbon in the soil profile maybe a key factor determining the carbon stability and carbon flux. Decomposition is a two-step process following the Michaelis Menten kinetics: In a first step enzyme and substrate form a joint complex and then the decomposition reaction is catalyzed. Thus, biological decomposition relies on the encounter of substrate and the degradation catalyst, the microorganisms. Lower substrate concentration decreases the likelihood of an enzyme to hit a substrate molecule, to form an enzyme-substrate complex, and thus to catalyze the reaction. However, it was unproofen if this concept can be appliued to soils also. A long-term lab experiment revealed that the soil carbon turnover decreased with increasing carbon dilution due to mixture with soil minerals. The ability of microorganisms to move towards substrate in soils seems to be limited. To elucidate the effect of concentration-controlled carbon turnover, we devised the simple simulation model SCAMP based on the two-step kinetic with microorganism and carbon particles been simulated explicitly. The SCAMP model was able to simulate soil carbon profiles and age profiles in a realistic manner. The only carbon stabilization mechanism implemented in the model is the distribution of microorganisms and carbon particles in the soil and thus the availability of carbon for microorganism, which is especially important for subsoil carbon dynamics. The experiments and the model help to explain why large fractions of soil carbon have been stabilized for millennia and decoupled from the global carbon cycle.

  17. Aspergillus ficuum phytase activity is inhibited by cereal grain components.

    Science.gov (United States)

    Bekalu, Zelalem Eshetu; Madsen, Claus Krogh; Dionisio, Giuseppe; Brinch-Pedersen, Henrik

    2017-01-01

    In the current study, we report for the first time that grain components of barley, rice, wheat and maize can inhibit the activity of Aspergillus ficuum phytase. The phytase inhibition is dose dependent and varies significantly between cereal species, between cultivars of barley and cultivars of wheat and between Fusarium graminearum infected and non-infected wheat grains. The highest endpoint level of phytase activity inhibition was 90%, observed with grain protein extracts (GPE) from F. graminearum infected wheat. Wheat GPE from grains infected with F. graminearum inhibits phytase activity significantly more than GPE from non-infected grains. For four barley cultivars studied, the IC50 value ranged from 0.978 ± 0.271 to 3.616 ± 0.087 mg×ml-1. For two non-infected wheat cultivars investigated, the IC50 values were varying from 2.478 ± 0.114 to 3.038 ± 0.097 mg×ml-1. The maize and rice cultivars tested gaveIC50 values on 0.983 ± 0.205 and 1.972 ± 0.019 mg×ml-1, respectively. After purifying the inhibitor from barley grains via Superdex G200, an approximately 30-35 kDa protein was identified. No clear trend for the mechanism of inhibition could be identified via Michaelis-Menten kinetics and Lineweaver-Burk plots. However, testing of the purified phytase inhibitor together with the A. ficuum phytase and the specific protease inhibitors pepstatin A, E64, EDTA and PMSF revealed that pepstatin A repealed the phytase inhibition. This indicates that the observed inhibition of A. ficuum phytase by cereal grain extracts is caused by protease activity of the aspartic proteinase type.

  18. Continuous Modeling Technique of Fiber Pullout from a Cement Matrix with Different Interface Mechanical Properties Using Finite Element Program

    Directory of Open Access Journals (Sweden)

    Leandro Ferreira Friedrich

    Full Text Available Abstract Fiber-matrix interface performance has a great influence on the mechanical properties of fiber reinforced composite. This influence is mainly presented during fiber pullout from the matrix. As fiber pullout process consists of fiber debonding stage and pullout stage which involve complex contact problem, numerical modeling is a best way to investigate the interface influence. Although many numerical research works have been conducted, practical and effective technique suitable for continuous modeling of fiber pullout process is still scarce. The reason is in that numerical divergence frequently happens, leading to the modeling interruption. By interacting the popular finite element program ANSYS with the MATLAB, we proposed continuous modeling technique and realized modeling of fiber pullout from cement matrix with desired interface mechanical performance. For debonding process, we used interface elements with cohesive surface traction and exponential failure behavior. For pullout process, we switched interface elements to spring elements with variable stiffness, which is related to the interface shear stress as a function of the interface slip displacement. For both processes, the results obtained are very good in comparison with other numerical or analytical models and experimental tests. We suggest using the present technique to model toughening achieved by randomly distributed fibers.

  19. Electrochemistry and biosensing activity of cytochrome c immobilized on a mesoporous interface assembled from carbon nanospheres

    International Nuclear Information System (INIS)

    Wang, Y.; Bian, X.; Liao, L.; Zhu, J.; Guo, K.; Kong, J.; Liu, B.

    2012-01-01

    We report on an amperometric biosensor for hydrogen peroxide. It is obtained via layer-by-layer assembly of ordered mesoporous carbon nanospheres and poly(diallyldimethylammonium) on the surface of an indium tin oxide (ITO) glass electrode and subsequent adsorption of cytochrome c. UV-vis absorption spectroscopy was applied to characterize the process of forming the assembled layers. Cyclic voltammetry revealed a direct and quasi-reversible electron transfer between cytochrome c and the surface of the modified ITO electrode. The surface-controlled electron transfer has an apparent heterogeneous electron-transfer rate constant (k s ) of 5.9 ± 0.2 s -1 in case of the 5-layer electrode. The biosensor displays good electrocatalytic response to the reduction of H 2O 2, and the amperometric signal increase steadily with the concentration of H 2 O 2 in the range from 5 μM to 1.5 mM. The detection limit is 1 μM at pH 7.4. The apparent Michaelis-Menten constant (K m ) of the sensor is 0.53 mM. We assume that the observation of a direct electron transfer of cytochrome c on mesoporous carbon nanospheres may form the basis for a feasible approach for durable and reliable detection of H 2 O 2 . (author)

  20. A novel tyrosinase biosensor based on hydroxyapatite-chitosan nanocomposite for the detection of phenolic compounds

    International Nuclear Information System (INIS)

    Lu Limin; Zhang Li; Zhang Xiaobing; Huan Shuangyan; Shen Guoli; Yu Ruqin

    2010-01-01

    A novel tyrosinase biosensor based on hydroxyapatite nanoparticles (nano-HA)-chitosan nanocomposite has been developed for the detection of phenolic compounds. The uniform and size controlled nano-HA was synthesized by hydrothermal method, and its morphological characterization was examined by transmission electron microscope (TEM). Tyrosinase was then immobilized on a nano-HA-chitosan nanocomposite-modified gold electrode. Electrochemical impedance spectroscopy and cyclic voltammetry were used to characterize the sensing film. The prepared biosensor was applied to determine phenolic compounds by monitoring the reduction signal of the biocatalytically produced quinone species at -0.2 V (vs. saturated calomel electrode). The effects of the pH, temperature and applied potential on the biosensor performance were investigated, and experimental conditions were optimized. The biosensor exhibited a linear response to catechol over a wide concentration range from 10 nM to 7 μM, with a high sensitivity of 2.11 x 10 3 μA mM -1 cm -2 , and a limit of detection down to 5 nM (based on S/N = 3). The apparent Michaelis-Menten constants of the enzyme electrode were estimated to be 3.16, 1.31 and 3.52 μM for catechol, phenol and m-cresol, respectively. Moreover, the stability and reproducibility of this biosensor were evaluated with satisfactory results.

  1. Amperometric biosensor for hydrogen peroxide based on Hemoglobin/DNA/Poly-2,6-pyridinediamine modified gold electrode

    International Nuclear Information System (INIS)

    Tong Zhongqiang; Yuan Ruo; Chai Yaqin; Chen Shihong; Xie Yi

    2007-01-01

    An amperometric biosensor for hydrogen peroxide (H 2 O 2 ) was fabricated based on immobilization of hemoglobin (Hb) on DNA/Poly-2,6-pyridinediamine (PPD) modified Au electrode. PPD thin films were firstly electro-deposited on Au electrode surface which provide a template to attach negatively charged DNA molecules by electrostatic attraction. The adsorbed DNA network provides a good microenvironment for the immobilization of biomolecules and promotes electron transfer between the immobilized Hb and the electrode surface. The fabrication process of the biosensor was characterized by electrochemical impedance spectroscopy. Experimental conditions influencing the biosensor performance such as pH, potential and temperature were assessed and optimized. The proposed biosensor displayed a good electrocatalytic response to the reduction of H 2 O 2 , its linear range is 1.7 μM to 3 mM with a detection limit of 1.0 μM based on the signal-to-noise ratio of 3 (S/N = 3) under the optimized conditions. The Michaelis-Menten constant K m app of Hb immobilized on the electrode surface was found to be 0.8 mM. The biosensor shows high sensitivity and stability. Importantly, this deposition methodology could be further developed for the immobilization of other proteins and biocompounds

  2. In situ measurement of methane oxidation in groundwater by using natural-gradient tracer tests

    International Nuclear Information System (INIS)

    Smith, R.L.; Howes, B.L.; Garabedian, S.P.

    1991-01-01

    Methane oxidation was measured in an unconfined sand and gravel aquifer (Cape Cod, Mass.) by using in situ natural-gradient tracer tests at both a pristine, oxygenated site and an anoxic, sewage-contaminated site. The tracer sites were equipped with multilevel sampling devices to create target grids of sampling points; the injectate was prepared with groundwater from the tracer site to maintain the same geochemical conditions. Methane oxidation was calculated from breakthrough curves of methane relative to halide and inert gas (hexafluoroethane) tracers and was confirmed by the appearance of 13 C-enriched carbon dioxide in experiments in which 13 C-enriched methane was used as the tracer. A V max for methane oxidation could be calculated when the methane concentration was sufficiently high to result in zero-order kinetics throughout the entire transport interval. Methane breakthrough curves could be simulated by modifying a one-dimensional advection-dispersion transport model to include a Michaelis-Menten-based consumption term for methane oxidation. The K m values for methane oxidation that gave the best match for the breakthrough curve peaks were 6.0 and 9.0 μM for the uncontaminated and contaminated sites, respectively. Natural-gradient tracer tests are a promising approach for assessing microbial processes and for testing in situ bioremediation potential in groundwater systems

  3. Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing.

    Science.gov (United States)

    Wan, Dongjin; Liu, Yongde; Niu, Zhenhua; Xiao, Shuhu; Li, Daorong

    2016-02-01

    Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis-Menten model described the perchlorate reduction kinetics well. Model parameters q(max) and K(s) were 2.521-3.245 (mg ClO4(-)/gVSS h) and 5.44-8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0-11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO3(-) > ClO4(-) > SO4(2-). Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.

  4. Carbon monoxide photoproduction: implications for photoreactivity of Arctic permafrost-derived soil dissolved organic matter.

    Science.gov (United States)

    Hong, Jun; Xie, Huixiang; Guo, Laodong; Song, Guisheng

    2014-08-19

    Apparent quantum yields of carbon monoxide (CO) photoproduction (AQY(CO)) for permafrost-derived soil dissolved organic matter (SDOM) from the Yukon River Basin and Alaska coast were determined to examine the dependences of AQY(CO) on temperature, ionic strength, pH, and SDOM concentration. SDOM from different locations and soil depths all exhibited similar AQY(CO) spectra irrespective of soil age. AQY(CO) increased by 68% for a 20 °C warming, decreased by 25% from ionic strength 0 to 0.7 mol L(-1), and dropped by 25-38% from pH 4 to 8. These effects combined together could reduce AQY(CO) by up to 72% when SDOM transits from terrestrial environemnts to open-ocean conditions during summer in the Arctic. A Michaelis-Menten kinetics characterized the influence of SDOM dilution on AQY(CO) with a very low substrate half-saturation concentration. Generalized global-scale relationships between AQY(CO) and salinity and absorbance demostrate that the CO-based photoreactivity of ancient permaforst SDOM is comparable to that of modern riverine DOM and that the effects of the physicochemical variables revealed here alone could account for the seaward decline of AQY(CO) observed in diverse estuarine and coastal water bodies.

  5. High-dynamic range imaging techniques based on both color-separation algorithms used in conventional graphic arts and the human visual perception modeling

    Science.gov (United States)

    Lo, Mei-Chun; Hsieh, Tsung-Hsien; Perng, Ruey-Kuen; Chen, Jiong-Qiao

    2010-01-01

    The aim of this research is to derive illuminant-independent type of HDR imaging modules which can optimally multispectrally reconstruct of every color concerned in high-dynamic-range of original images for preferable cross-media color reproduction applications. Each module, based on either of broadband and multispectral approach, would be incorporated models of perceptual HDR tone-mapping, device characterization. In this study, an xvYCC format of HDR digital camera was used to capture HDR scene images for test. A tone-mapping module was derived based on a multiscale representation of the human visual system and used equations similar to a photoreceptor adaptation equation, proposed by Michaelis-Menten. Additionally, an adaptive bilateral type of gamut mapping algorithm, using approach of a multiple conversing-points (previously derived), was incorporated with or without adaptive Un-sharp Masking (USM) to carry out the optimization of HDR image rendering. An LCD with standard color space of Adobe RGB (D65) was used as a soft-proofing platform to display/represent HDR original RGB images, and also evaluate both renditionquality and prediction-performance of modules derived. Also, another LCD with standard color space of sRGB was used to test gamut-mapping algorithms, used to be integrated with tone-mapping module derived.

  6. Amperometric biosensor for hydrogen peroxide based on hemoglobin entrapped in titania sol-gel film

    International Nuclear Information System (INIS)

    Yu Jiuhong; Ju Huangxian

    2003-01-01

    Hemoglobin (Hb) was entrapped in a titania sol-gel matrix and used as a mimetic peroxidase to construct a novel amperometric biosensor for hydrogen peroxide. The Hb entrapped titania sol-gel film was obtained with a vapor deposition method, which simplified the traditional sol-gel process for protein immobilization. The morphologies of both titania sol-gel and the Hb films were characterized using scanning electron microscopy (SEM) and proved to be chemically clean, porous, homogeneous. This matrix provided a biocompatible microenvironment for retaining the native structure and activity of the entrapped Hb and a very low mass transport barrier to the substrates. H 2 O 2 could be reduced by the catalysis of the entrapped hemoglobin at -300 mV without any mediator. The reagentless H 2 O 2 sensor exhibited a fast response (less than 5 s) and sensitivity as high as 1.29 mA mM -1 cm -2 . The linear range for H 2 O 2 determination was from 5.0x10 -7 to 5.4x10 -5 M with a detection limit of 1.2x10 -7 M. The apparent Michaelis-Menten constant of the encapsulated hemoglobin was calculated to be 0.18±0.02 mM. The stability of the biosensor was also evaluated

  7. Colorimetric Glucose Assay Based on Magnetic Particles Having Pseudo-peroxidase Activity and Immobilized Glucose Oxidase.

    Science.gov (United States)

    Martinkova, Pavla; Opatrilova, Radka; Kruzliak, Peter; Styriak, Igor; Pohanka, Miroslav

    2016-05-01

    Magnetic particles (MPs) are currently used as a suitable alternative for peroxidase in the construction of novel biosensors, analytic and diagnostic methods. Their better chemical and thermal stabilities predestine them as appropriate pseudo-enzymatic catalysts. In this point of view, our research was focused on preparation of simply and fast method for immobilization of glucose oxidase onto surface of MPs with peroxidase-like activity. Spectrophotometric method (wavelength 450 nm) optimized for glucose determination using modified MPs has been successfully developed. Concentration curve for optimization of method was assayed, and Michaelis-Menten constant (K m) calculated, maximum reaction rate (V max), limit of detection, and correlation coefficient were determined to be 0.13 mmol/l (2.34 mg/dl), 1.79 pkat, 3.74 µmol/l (0.067 mg/dl), and 0.996, respectively. Interferences of other sugars such as sucrose, sorbitol, deoxyribose, maltose, and fructose were determined as well as effect of substances presenting in plasma (ascorbic acid, reduced glutathione, trolox, and urea). Results in comparison with positive and negative controls showed no interferences of the other sugars and no influence of plasma substances to measuring of glucose. The constructed method showed corresponding results with linear dependence and a correlation coefficient of 0.997. Possibility of repeated use of modified MPs was successfully proved.

  8. Direct electrochemistry of glucose oxidase and glucose biosensing on a hydroxyl fullerenes modified glassy carbon electrode.

    Science.gov (United States)

    Gao, Yun-Fei; Yang, Tian; Yang, Xiao-Lu; Zhang, Yu-Shuai; Xiao, Bao-Lin; Hong, Jun; Sheibani, Nader; Ghourchian, Hedayatollah; Hong, Tao; Moosavi-Movahedi, Ali Akbar

    2014-10-15

    Direct electrochemistry of glucose oxidase (GOD) was achieved when GOD-hydroxyl fullerenes (HFs) nano-complex was immobilized on a glassy carbon (GC) electrode and protected with a chitosan (Chit) membrane. The ultraviolet-visible absorption spectrometry (UV-vis), transmission electron microscopy (TEM), and circular dichroism spectropolarimeter (CD) methods were utilized for additional characterization of the GOD, GOD-HFs and Chit/GOD-HFs. Chit/HFs may preserve the secondary structure and catalytic properties of GOD. The cyclic voltammograms (CVs) of the modified GC electrode showed a pair of well-defined quasi-reversible redox peaks with the formal potential (E°') of 353 ± 2 mV versus Ag/AgCl at a scan rate of 0.05 V/s. The heterogeneous electron transfer constant (ks) was calculated to be 2.7 ± 0.2s(-1). The modified electrode response to glucose was linear in the concentrations ranging from 0.05 to 1.0mM, with a detection limit of 5 ± 1 μM. The apparent Michaelis-Menten constant (Km(app)) was 694 ± 8 μM. Thus, the modified electrode could be applied as a third generation biosensor for glucose with high sensitivity, selectivity and low detection limit. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Direct electron transfer of glucose oxidase and biosensing for glucose based on PDDA-capped gold nanoparticle modified graphene/multi-walled carbon nanotubes electrode.

    Science.gov (United States)

    Yu, Yanyan; Chen, Zuanguang; He, Sijing; Zhang, Beibei; Li, Xinchun; Yao, Meicun

    2014-02-15

    In this work, poly (diallyldimethylammonium chloride) (PDDA)-capped gold nanoparticles (AuNPs) functionalized graphene (G)/multi-walled carbon nanotubes (MWCNTs) nanocomposites were fabricated. Based on the electrostatic attraction, the G/MWCNTs hybrid material can be decorated with AuNPs uniformly and densely. The new hierarchical nanostructure can provide a larger surface area and a more favorable microenvironment for electron transfer. The AuNPs/G/MWCNTs nanocomposite was used as a novel immobilization platform for glucose oxidase (GOD). Direct electron transfer (DET) was achieved between GOD and the electrode. Field emission scanning electron microscopy (FESEM), UV-vis spectroscopy and cyclic voltammetry (CV) were used to characterize the electrochemical biosensor. The glucose biosensor fabricated based on GOD electrode modified with AuNPs/G/MWCNTs demonstrated satisfactory analytical performance with high sensitivity (29.72mAM(-1)cm(-2)) and low limit of detection (4.8 µM). The heterogeneous electron transfer rate constant (ΚS) and the apparent Michaelis-Menten constant (Km) of GOD were calculated to be 11.18s(-1) and 2.09 mM, respectively. With satisfactory selectivity, reproducibility, and stability, the nanostructure we proposed offered an alternative for electrode fabricating and glucose biosensing. © 2013 Elsevier B.V. All rights reserved.

  10. Fabrication and characterization of junctionless carbon nanotube field effect transistor for cholesterol detection

    Energy Technology Data Exchange (ETDEWEB)

    Barik, Md. Abdul, E-mail: abdulnpl@gmail.com; Dutta, Jiten Ch. [Department of Electronics and Communication Engineering, Tezpur University, Napaam, Tezpur, Assam 784028 (India)

    2014-08-04

    We have reported fabrication and characterization of polyaniline (PANI)/zinc oxide (ZnO) membrane-based junctionless carbon nanotube field effect transistor deposited on indium tin oxide glass plate for the detection of cholesterol (0.5–22.2 mM). Cholesterol oxidase (ChOx) has been immobilized on the PANI/ZnO membrane by physical adsorption technique. Electrical response has been recorded using digital multimeter (Agilent 3458A) in the presence of phosphate buffer saline of 50 mM, pH 7.0, and 0.9% NaCl contained in a glass pot. The results of response studies for cholesterol reveal linearity as 0.5–16.6 mM and improved sensitivity of 60 mV/decade in good agreement with Nernstian limit ∼59.2 mV/decade. The life time of this sensor has been found up to 5 months and response time of 1 s. The limit of detection with regression coefficient (r) ∼ 0.998 and Michaelis-Menten constant (K{sub m}) were found to be ∼0.25 and 1.4 mM, respectively, indicating high affinity of ChOx to cholesterol. The results obtained in this work show negligible interference with glucose and urea.

  11. Stochastic simulation of biological reactions, and its applications for studying actin polymerization.

    Science.gov (United States)

    Ichikawa, Kazuhisa; Suzuki, Takashi; Murata, Noboru

    2010-11-30

    Molecular events in biological cells occur in local subregions, where the molecules tend to be small in number. The cytoskeleton, which is important for both the structural changes of cells and their functions, is also a countable entity because of its long fibrous shape. To simulate the local environment using a computer, stochastic simulations should be run. We herein report a new method of stochastic simulation based on random walk and reaction by the collision of all molecules. The microscopic reaction rate P(r) is calculated from the macroscopic rate constant k. The formula involves only local parameters embedded for each molecule. The results of the stochastic simulations of simple second-order, polymerization, Michaelis-Menten-type and other reactions agreed quite well with those of deterministic simulations when the number of molecules was sufficiently large. An analysis of the theory indicated a relationship between variance and the number of molecules in the system, and results of multiple stochastic simulation runs confirmed this relationship. We simulated Ca²(+) dynamics in a cell by inward flow from a point on the cell surface and the polymerization of G-actin forming F-actin. Our results showed that this theory and method can be used to simulate spatially inhomogeneous events.

  12. A new thermophilic nitrilase from an antarctic hyperthermophilic microorganism.

    Directory of Open Access Journals (Sweden)

    Geraldine V. Dennett

    2016-02-01

    Full Text Available Several environmental samples from Antarctica were collected and enriched to search for microorganisms with nitrilase activity. A new thermostable nitrilase from a novel hyperthermophilic archaea Pyrococcus sp. M24D13 was purified and characterized. The activity of this enzyme increased as the temperatures rise from 70 up to 85 °C. Its optimal activity occurred at 85 °C and pH 7.5. This new enzyme shows a remarkable resistance to thermal inactivation retaining more than 50% of its activity even after 8 h of incubation at 85 °C.In addition, this nitrilase is highly versatile demonstrating activity towards different substrates such as benzonitrile (60 mM, aromatic nitrile and butyronitrile (60 mM, aliphatic nitrile, with a specific activity of 3286.7 U mg-1 of protein and 4008.2 U mg-1 of protein respectively. Moreover the enzyme NitM24D13 also presents cyanidase activity.The apparent Michaelis-Menten constant (Km and Vmáx of this Nitrilase for benzonitrile were 0.3 mM and 333.3 µM min-1, respectively, and the specificity constant (kcat/Km for benzonitrile was 2.05×105 s-1 M-1.

  13. Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis.

    Science.gov (United States)

    de Sousa, Marylane; Manzo, Ricardo M; García, José L; Mammarella, Enrique J; Gonçalves, Luciana R B; Pessela, Benevides C

    2017-12-06

    l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N -His-l-AI and C -His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C -His-l-AI was preferentially hexameric in solution, whereas N -His-l-AI was mainly monomeric. The specific activity of the N -His-l-AI at acidic pH was higher than that of C -His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg -1 , respectively. However, C -His-l-AI was more active and stable at alkaline pH than N -His-l-AI. N -His-l-AI follows a Michaelis-Menten kinetic, whereas C -His-l-AI fitted to a sigmoidal saturation curve.

  14. Enzymatic conversion of D-galactose to D-tagatose: heterologous expression and characterisation of a thermostable L-arabinose isomerase from Thermoanaerobacter mathranii.

    Science.gov (United States)

    Jørgensen, F; Hansen, O C; Stougaard, P

    2004-06-01

    The ability to convert D-galactose into D-tagatose was compared among a number of bacterial L-arabinose isomerases ( araA). One of the most efficient enzymes, from the anaerobic thermophilic bacterium Thermoanaerobacter mathranii, was produced heterologously in Escherichia coli and characterised. Amino acid sequence comparisons indicated that this enzyme is only distantly related to the group of previously known araA sequences in which the sequence similarity is evident. The substrate specificity and the Michaelis-Menten constants of the enzyme determined with L-arabinose, D-galactose and D-fucose also indicated that this enzyme is an unusual, versatile L-arabinose isomerase which is able to isomerise structurally related sugars. The enzyme was immobilised and used for production of D-tagatose at 65 degrees C. Starting from a 30% solution of D-galactose, the yield of D-tagatose was 42% and no sugars other than D-tagatose and D-galactose were detected. Direct conversion of lactose to D-tagatose in a single reactor was demonstrated using a thermostable beta-galactosidase together with the thermostable L-arabinose isomerase. The two enzymes were also successfully combined with a commercially available glucose isomerase for conversion of lactose into a sweetening mixture comprising lactose, glucose, galactose, fructose and tagatose.

  15. Simulation Models of Leaf Area Index and Yield for Cotton Grown with Different Soil Conditioners.

    Directory of Open Access Journals (Sweden)

    Lijun Su

    Full Text Available Simulation models of leaf area index (LAI and yield for cotton can provide a theoretical foundation for predicting future variations in yield. This paper analyses the increase in LAI and the relationships between LAI, dry matter, and yield for cotton under three soil conditioners near Korla, Xinjiang, China. Dynamic changes in cotton LAI were evaluated using modified logistic, Gaussian, modified Gaussian, log normal, and cubic polynomial models. Universal models for simulating the relative leaf area index (RLAI were established in which the application rate of soil conditioner was used to estimate the maximum LAI (LAIm. In addition, the relationships between LAIm and dry matter mass, yield, and the harvest index were investigated, and a simulation model for yield is proposed. A feasibility analysis of the models indicated that the cubic polynomial and Gaussian models were less accurate than the other three models for simulating increases in RLAI. Despite significant differences in LAIs under the type and amount of soil conditioner applied, LAIm could be described by aboveground dry matter using Michaelis-Menten kinetics. Moreover, the simulation model for cotton yield based on LAIm and the harvest index presented in this work provided important theoretical insights for improving water use efficiency in cotton cultivation and for identifying optimal application rates of soil conditioners.

  16. Fractionation and Characterization of Tannin Acyl Hydrolase from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    YUNITA ARIAN SANI ANWAR

    2009-09-01

    Full Text Available We previously produced tannin acyl hydrolase (tannase from Aspergillus niger isolated from cacao pod. In the present study the enzyme was subjected to fractionation by ammonium sulphate followed by dialysis process. The saturation level of ammonium sulphate used was 30-80% where the best enzyme activity was obtained at the saturation level of 60%. Compared to that of crude enzyme, specific activity of tannase after dialysis was four folds. Characterization results showed that optimum activity was at 35-50 oC and pH 6. Tannase was activated by K+ and Na+ at concentration of 0.01 and 0.05 M respectively. Mg2+ was found activate tannase only at 0.01 M. Addition of metal ions like Zn2+, Cu2+, Ca2+, Mn2+ and Fe2+ inhibited the enzyme activity. Kinetics analysis of various substrates tested showed that the Km value of tannic acid and gallotannin was 0.401 and 6.611 mM respectively. Vmax value of tannic acid was 10.804 U/ml and of gallotannin was 12.406 U/ml. Based on Michaelis-Menten constant (Km, the tannase obtained in the present study was more active in hydrolysing depside bonds rather than ester bonds.

  17. Fractionation and Characterization of Tannin Acyl Hydrolase from Aspergillus niger

    Directory of Open Access Journals (Sweden)

    YUNITA ARIAN SANI ANWAR

    2009-09-01

    Full Text Available We previously produced tannin acyl hydrolase (tannase from Aspergillus niger isolated from cacao pod. In the present study the enzyme was subjected to fractionation by ammonium sulphate followed by dialysis process. The saturation level of ammonium sulphate used was 30–80% where the best enzyme activity was obtained at the saturation level of 60%. Compared to that of crude enzyme, specific activity of tannase after dialysis was four folds. Characterization results showed that optimum activity was at 35–50 °C and pH 6. Tannase was activated by K+ and Na+ at concentration of 0.01 and 0.05 M respectively. Mg2+ was found activate tannase only at 0.01 M. Addition of metal ions like Zn2+, Cu2+, Ca2+, Mn2+ and Fe2+ inhibited the enzyme activity. Kinetics analysis of various substrates tested showed that the Km value of tannic acid and gallotannin was 0.401 and 6.611 mM respectively. Vmax value of tannic acid was 10.804 U/ml and of gallotannin was 12.406 U/ml. Based on Michaelis-Menten constant (Km, the tannase obtained in the present study was more active in hydrolysing depside bonds rather than ester bonds.

  18. Facile and controllable preparation of glucose biosensor based on Prussian blue nanoparticles hybrid composites.

    Science.gov (United States)

    Li, Lei; Sheng, Qinglin; Zheng, Jianbin; Zhang, Hongfang

    2008-11-01

    A glucose biosensor based on polyvinylpyrrolidone (PVP) protected Prussian blue nanoparticles (PBNPs)-polyaniline/multi-walled carbon nanotubes hybrid composites was fabricated by electrochemical method. A novel route for PBNPs preparation was applied in the fabrication with the help of PVP, and from scanning electron microscope images, Prussian blue particles on the electrode were found nanoscaled. The biosensor exhibits fast current response (<6 s) and a linearity in the range from 6.7x10(-6) to 1.9x10(-3) M with a high sensitivity of 6.28 microA mM(-1) and a detection limit of 6x10(-7) M (S/N=3) for the detection of glucose. The apparent activation energy of enzyme-catalyzed reaction and the apparent Michaelis-Menten constant are 23.9 kJ mol(-1) and 1.9 mM respectively, which suggests a high affinity of the enzyme-substrate. This easy and controllable construction method of glucose biosensor combines the characteristics of the components of the hybrid composites, which favors the fast and sensitive detection of glucose with improved analytical capabilities. In addition, the biosensor was examined in human serum samples for glucose determination with a recovery between 95.0 and 104.5%.

  19. Zinc oxide-potassium ferricyanide composite thin film matrix for biosensing applications

    Energy Technology Data Exchange (ETDEWEB)

    Saha, Shibu [Department of Physics and Astrophysics, University of Delhi, Delhi 110007 (India); Arya, Sunil K. [Department of Science and Technology Centre on Biomolecular Electronics, National Physical Laboratory, New Delhi 110012 (India); Singh, S.P. [Department of Engineering Science and Materials, University of Puerto Rico, Mayaguez, PR 00680 (United States); Sreenivas, K. [Department of Physics and Astrophysics, University of Delhi, Delhi 110007 (India); Malhotra, B.D. [Department of Science and Technology Centre on Biomolecular Electronics, National Physical Laboratory, New Delhi 110012 (India); Gupta, Vinay, E-mail: vgupta@physics.du.ac.in [Department of Physics and Astrophysics, University of Delhi, Delhi 110007 (India)

    2009-10-27

    Thin film of zinc oxide-potassium ferricyanide (ZnO-KFCN) composite has been deposited on indium tin oxide (ITO) coated corning glass using pulsed laser deposition (PLD). The composite thin film electrode has been exploited for amperometric biosensing in a mediator-free electrolyte. The composite matrix has the advantages of high iso-electric point of ZnO along with enhanced electron communication due to the presence of a redox species in the matrix itself. Glucose oxidase (GOx) has been chosen as the model enzyme for studying the application of the developed matrix to biosensing. The sensing response of the bio-electrode, GOx/ZnO-KFCN/ITO/glass, towards glucose was studied using cylic voltammetry (CV) and photometric assay. The bio-electrode exhibits good linearity from 2.78 mM to 11.11 mM glucose concentration. The low value of Michaelis-Menten constant (1.69 mM) indicates an enhanced affinity of the immobilized enzyme towards its substrate. A quassireversible system is obtained with the composite matrix. The results confirm promising application of the ZnO-KFCN composite matrix for amperometric biosensing applications in a mediator-less electrolyte that could lead to the realization of an integrated lab-on-chip device.

  20. Zinc oxide-potassium ferricyanide composite thin film matrix for biosensing applications

    International Nuclear Information System (INIS)

    Saha, Shibu; Arya, Sunil K.; Singh, S.P.; Sreenivas, K.; Malhotra, B.D.; Gupta, Vinay

    2009-01-01

    Thin film of zinc oxide-potassium ferricyanide (ZnO-KFCN) composite has been deposited on indium tin oxide (ITO) coated corning glass using pulsed laser deposition (PLD). The composite thin film electrode has been exploited for amperometric biosensing in a mediator-free electrolyte. The composite matrix has the advantages of high iso-electric point of ZnO along with enhanced electron communication due to the presence of a redox species in the matrix itself. Glucose oxidase (GOx) has been chosen as the model enzyme for studying the application of the developed matrix to biosensing. The sensing response of the bio-electrode, GOx/ZnO-KFCN/ITO/glass, towards glucose was studied using cylic voltammetry (CV) and photometric assay. The bio-electrode exhibits good linearity from 2.78 mM to 11.11 mM glucose concentration. The low value of Michaelis-Menten constant (1.69 mM) indicates an enhanced affinity of the immobilized enzyme towards its substrate. A quassireversible system is obtained with the composite matrix. The results confirm promising application of the ZnO-KFCN composite matrix for amperometric biosensing applications in a mediator-less electrolyte that could lead to the realization of an integrated lab-on-chip device.

  1. Pectin methylesterase of Datura species, purification, and characterization from Datura stramonium and its application.

    Science.gov (United States)

    Dixit, Sameer; Upadhyay, Santosh Kumar; Singh, Harpal; Pandey, Bindu; Chandrashekar, Krishnappa; Verma, Praveen Chandra

    2013-10-01

    Pectin methylesterases (PME; EC 3.1.1.11) involved in de-esterification of pectin and have applicability in food, textiles, wines, pulp, and paper industries. In the present study, we compared PME activity of different parts of 3 Datura species and found that fruit coat showed maximum PME activity followed by leaf and seed. PME from leaves of D. stramonium (DsPME) was purified and characterized. DsPME showed optimum activity at 60 °C and pH 9 in the presence of 0.3 M NaCl. DsPME was stable at 70 °C and retained more than 40% activity after 60 min of incubation. However, enzyme activity completely abolished at 80 after 5 min of incubation. It follows Michaelis-Menten enzyme kinetics. Km and Vmax with citrus pectin were 0.008 mg/ml and 16.96 µmol/min, respectively. DsPME in combination with polygalactourenase (PGA) increased the clarity of orange, apple, pomegranate and pineapple juices by 2.9, 2.6, 2.3, and 3.6 fold, respectively in comparison to PGA alone. Due to very high de-esterification activity, easy denaturation and significant efficacy in incrementing clarification of fruit juice makes DsPME useful for industrial application.

  2. Immobilization of Aspergillus awamori β-glucosidase on commercial gelatin: An inexpensive and efficient process.

    Science.gov (United States)

    Nishida, Verônica S; de Oliveira, Roselene F; Brugnari, Tatiane; Correa, Rúbia Carvalho G; Peralta, Rosely A; Castoldi, Rafael; de Souza, Cristina G M; Bracht, Adelar; Peralta, Rosane M

    2018-05-01

    In this work, a β-glucosidase of Aspergillus awamori with a molecular weight of 180 kDa was produced in solid-state cultures using a mixture of pineapple crown leaves and wheat bran. Maximum production of the enzyme (820 ± 30 U/g substrate) was obtained after 8 days of culture at 28 °C and initial moisture of 80%. The crude enzyme was efficiently immobilized on glutaraldehyde cross-linked commercial gelatin. Immobilization changed the kinetics of the enzyme, whose behavior could no longer be described by a saturation function of the Michaelis-Menten type. Comparative evaluation of the free and immobilized enzyme showed that the immobilized enzyme was more thermostable and less inhibited by glucose than the free form. In consequence of these properties, the immobilized enzyme was able to hydrolyze cellobiose more extensively. In association with Trichoderma reesei cellulase, the free and immobilized β-glucosidase increased the liberation of glucose from cellulose 3- and 5-fold, respectively. Immobilization of the A. awamori β-glucosidase on glutaraldehyde cross-linked commercial gelatin is an efficient and cheap method allowing the reuse of the enzyme by at least 10 times. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. Rapid Ammonia Gas Transport Accounts for Futile Transmembrane Cycling under NH3/NH4+ Toxicity in Plant Roots1[C][W

    Science.gov (United States)

    Coskun, Devrim; Britto, Dev T.; Li, Mingyuan; Becker, Alexander; Kronzucker, Herbert J.

    2013-01-01

    Futile transmembrane NH3/NH4+ cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH3/NH4+ toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH3 or NH4+) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope 13N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH3 species, rather than the NH4+ ion. Influx of 13NH3/13NH4+, which exceeded 200 µmol g–1 h–1, was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH3 transport. Influx followed Michaelis-Menten kinetics for NH3 (but not NH4+), as a function of external concentration (Km = 152 µm, Vmax = 205 µmol g–1 h–1). Efflux of 13NH3/13NH4+ responded with a nearly identical Km. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH3 is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions. PMID:24134887

  4. Rapid ammonia gas transport accounts for futile transmembrane cycling under NH3/NH4+ toxicity in plant roots.

    Science.gov (United States)

    Coskun, Devrim; Britto, Dev T; Li, Mingyuan; Becker, Alexander; Kronzucker, Herbert J

    2013-12-01

    Futile transmembrane NH3/NH4(+) cycling in plant root cells, characterized by extremely rapid fluxes and high efflux to influx ratios, has been successfully linked to NH3/NH4(+) toxicity. Surprisingly, the fundamental question of which species of the conjugate pair (NH3 or NH4(+)) participates in such fluxes is unresolved. Using flux analyses with the short-lived radioisotope (13)N and electrophysiological, respiratory, and histochemical measurements, we show that futile cycling in roots of barley (Hordeum vulgare) seedlings is predominately of the gaseous NH3 species, rather than the NH4(+) ion. Influx of (13)NH3/(13)NH4(+), which exceeded 200 µmol g(-1) h(-1), was not commensurate with membrane depolarization or increases in root respiration, suggesting electroneutral NH3 transport. Influx followed Michaelis-Menten kinetics for NH3 (but not NH4(+)), as a function of external concentration (Km = 152 µm, Vmax = 205 µmol g(-1) h(-1)). Efflux of (13)NH3/(13)NH4(+) responded with a nearly identical Km. Pharmacological characterization of influx and efflux suggests mediation by aquaporins. Our study fundamentally revises the futile-cycling model by demonstrating that NH3 is the major permeating species across both plasmalemma and tonoplast of root cells under toxicity conditions.

  5. Enzymatic and biochemical characterization of Bungarus sindanus snake venom acetylcholinesterase

    Directory of Open Access Journals (Sweden)

    M Ahmed

    2012-01-01

    Full Text Available This study analyses venom from the elapid krait snake Bungarus sindanus, which contains a high level of acetylcholinesterase (AChE activity. The enzyme showed optimum activity at alkaline pH (8.5 and 45ºC. Krait venom AChE was inhibited by substrate. Inhibition was significantly reduced by using a high ionic strength buffer; low ionic strength buffer (10 mM PO4 pH 7.5 inhibited the enzyme by 1. 5mM AcSCh, while high ionic strength buffer (62 mM PO4 pH 7.5 inhibited it by 1 mM AcSCh. Venom acetylcholinesterase was also found to be thermally stable at 45ºC; it only lost 5% of its activity after incubation at 45ºC for 40 minutes. The Michaelis-Menten constant (Km for acetylthiocholine iodide hydrolysis was found to be 0.068 mM. Krait venom acetylcholinesterase was also inhibited by ZnCl2, CdCl2, and HgCl2 in a concentrationdependent manner. Due to the elevated levels of AChE with high catalytic activity and because it is more stable than any other sources, Bungarus sindanus venom is highly valuable for biochemical studies of this enzyme.

  6. Lipase-nanoporous gold biocomposite modified electrode for reliable detection of triglycerides.

    Science.gov (United States)

    Wu, Chao; Liu, Xueying; Li, Yufei; Du, Xiaoyu; Wang, Xia; Xu, Ping

    2014-03-15

    For triglycerides biosensor design, protein immobilization is necessary to create the interface between the enzyme and the electrode. In this study, a glassy carbon electrode (GCE) was modified with lipase-nanoporous gold (NPG) biocomposite (denoted as lipase/NPG/GCE). Due to highly conductive, porous, and biocompatible three-dimensional structure, NPG is suitable for enzyme immobilization. In cyclic voltammetry experiments, the lipase/NPG/GCE bioelectrode displayed surface-confined reaction in a phosphate buffer solution. Linear responses were obtained for tributyrin concentrations ranging from 50 to 250 mg dl(-1) and olive oil concentrations ranging from 10 to 200 mg dl(-1). The value of apparent Michaelis-Menten constant for tributyrin was 10.67 mg dl(-1) and the detection limit was 2.68 mg dl(-1). Further, the lipase/NPG/GCE bioelectrode had strong anti-interference ability against urea, glucose, cholesterol, and uric acid as well as a long shelf-life. For the detection of triglycerides in human serum, the values given by the lipase/NPG/GCE bioelectrode were in good agreement with those of an automatic biochemical analyzer. These properties along with a long self-life make the lipase/NPG/GCE bioelectrode an excellent choice for the construction of triglycerides biosensor. © 2013 Elsevier B.V. All rights reserved.

  7. Effects of 3G cell phone exposure on the structure and function of the human cytochrome P450 reductase.

    Science.gov (United States)

    Tanvir, Shazia; Thuróczy, György; Selmaoui, Brahim; Silva Pires Antonietti, Viviane; Sonnet, Pascal; Arnaud-Cormos, Delia; Lévêque, Philippe; Pulvin, Sylviane; de Seze, René

    2016-10-01

    Cell phones increase exposure to radiofrequency (RF) electromagnetic fields (EMFs). Whether EMFs exert specific effects on biological systems remains debatable. This study investigated the effect of cell phone exposure on the structure and function of human NADPH-cytochrome P450 reductase (CPR). CPR plays a key role in the electron transfer to cytochrome P450, which takes part in a wide range of oxidative metabolic reactions in various organisms from microbes to humans. Human CPR was exposed for 60min to 1966-MHz RF inside a transverse electromagnetic cell (TEM-cell) placed in an incubator. The specific absorption rate (SAR) was 5W·kg(-1). Conformation changes have been detected through fluorescent spectroscopy of flavin and tryptophan residues, and investigated through circular dichroism, dynamic light scattering and microelectrophoresis. These showed that CPR was narrowed. By using cytochrome C reductase activity to assess the electron flux through the CPR, the Michaelis Menten constant (Km) and the maximum initial velocity (Vmax) decreased by 22% as compared with controls. This change was due to small changes in the tertiary and secondary structures of the protein at 37°C. The relevance of these findings to an actual RF exposure scenario demands further biochemical and in-vivo confirmation. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Direct electrochemistry of myoglobin in a layer-by-layer film on an ionic liquid modified electrode containing CeO2 nanoparticles and hyaluronic acid

    International Nuclear Information System (INIS)

    Gao, R.; Zheng, J.; Zheng, X.

    2011-01-01

    We describe an ionic liquid modified electrode (CPE-IL) for sensing hydrogen peroxide (HP) that was modified by the layer-by-layer technique with myoglobin (Mb). In addition, the surface of the electrode was modified with CeO 2 nanoparticles (nano-CeO 2 ) and hyaluronic acid. UV-vis and FTIR spectroscopy confirmed that Mb retains its native structure in the composite film. Scanning electron microscopy showed that the nano-CeO 2 closely interact with Mb to form an inhomogeneously distributed film. Cyclic voltammetry reveals a pair of quasi-reversible redox peaks of Mb, with the cathodic peak at -0. 357 V and the anodic peak at -0. 269 V. The peak separation (ΔE p ) and the formal potential (E σ ) are 88 mV and -0. 313 V (vs. Ag/AgCl), respectively. The Mb immobilized in the modified electrode displays an excellent electrocatalytic activity towards HP in the 0. 6 to 78. 0 μM concentration range. The limit of detection is 50 nM (S/N = 3), and then the Michaelis-Menten constant is 71. 8 μM. We believe that such a composite film has potential to further investigate other redox proteins and in the fabrication of third-generation biosensors. (author)

  9. Kinetics of Single-Enzyme Reactions on Vesicles: Role of Substrate Aggregation

    Science.gov (United States)

    Zhdanov, Vladimir P.

    2015-03-01

    Enzymatic reactions occurring in vivo on lipid membranes can be influenced by various factors including macromolecular crowding in general and substrate aggregation in particular. In academic studies, the role of these factors can experimentally be clarified by tracking single-enzyme kinetics occurring on individual lipid vesicles. To extend the conceptual basis for such experiments, we analyze herein the corresponding kinetics mathematically with emphasis on the role of substrate aggregation. In general, the aggregation may occur on different length scales. Small aggregates may e.g. contain a few proteins or peptides while large aggregates may be mesoscopic as in the case of lipid domains which can be formed in the membranes composed of different lipids. We present a kinetic model describing comprehensively the effect of aggregation of the former type on the dependence of the reaction rate on substrate membrane concentration. The results obtained with physically reasonable parameters indicate that the aggregation-related deviations from the conventional Michaelis-Menten kinetics may be appreciable. Special Issue Comments: This theoretical article is focused on single-enzyme reactions occurring in parallel with substrate aggregation on individual vesicles. This subject is related to a few Special Issue articles concerning enzyme dynamics6,7 and function8 and mathematical aspects of stochastic kinetics.9

  10. The Non-Linear Child: Ontogeny, Isoniazid Concentration, and NAT2 Genotype Modulate Enzyme Reaction Kinetics and Metabolism.

    Science.gov (United States)

    Rogers, Zoe; Hiruy, Hiwot; Pasipanodya, Jotam G; Mbowane, Chris; Adamson, John; Ngotho, Lihle; Karim, Farina; Jeena, Prakash; Bishai, William; Gumbo, Tawanda

    2016-09-01

    N-acetyltransferase 2 (NAT2) catalyzes the acetylation of isoniazid to N-acetylisoniazid. NAT2 polymorphism explains 88% of isoniazid clearance variability in adults. We examined the effects of clinical and genetic factors on Michaelis-Menten reaction kinetic constants of maximum velocity (V max ) and affinity (K m ) in children 0-10years old. We measured the rates of isoniazid elimination and N-acetylisoniazid production in the blood of 30 children. Since maturation effects could be non-linear, we utilized a pharmacometric approach and the artificial intelligence method, multivariate adaptive regression splines (MARS), to identify factors predicting NAT2 V max and K m by examining clinical, genetic, and laboratory factors in toto. Isoniazid concentration predicted both V max and K m and superseded the contribution of NAT2 genotype. Age non-linearly modified the NAT2 genotype contribution until maturation at ≥5.3years. Thus, enzyme efficiency was constrained by substrate concentration, genes, and age. Since MARS output is in the form of basis functions and equations, it allows multiscale systems modeling from the level of cellular chemical reactions to whole body physiological parameters, by automatic selection of significant predictors by the algorithm. Copyright © 2016 Forschungsgesellschaft für Arbeitsphysiologie und Arbeitschutz e.V. Published by Elsevier B.V. All rights reserved.

  11. Some Investigations on Protease Enzyme Production Kinetics Using Bacillus licheniformis BBRC 100053 and Effects of Inhibitors on Protease Activity

    Directory of Open Access Journals (Sweden)

    Zahra Ghobadi Nejad

    2014-01-01

    Full Text Available Due to great commercial application of protease, it is necessary to study kinetic characterization of this enzyme in order to improve design of enzymatic reactors. In this study, mathematical modeling of protease enzyme production kinetics which is derived from Bacillus licheniformis BBRC 100053 was studied (at 37°C, pH 10 after 73 h in stationary phase, and 150 rpm. The aim of the present paper was to determine the best kinetic model and kinetic parameters for production of protease and calculating Ki (inhibition constant of different inhibitors to find the most effective one. The kinetic parameters Km (Michaelis-Menten constant and Vm (maximum rate were calculated 0.626 mM and 0.0523 mM/min. According to the experimental results, using DFP (diisopropyl fluorophosphate and PMSF (phenylmethanesulfonyl fluoride as inhibitors almost 50% of the enzyme activity could be inhibited when their concentrations were 0.525 and 0.541 mM, respectively. Ki for DFP and PMSF were 0.46 and 0.56 mM, respectively. Kinetic analysis showed that the Lineweaver-Burk model was the best fitting model for protease production kinetics DFP was more effective than PMSF and both of them should be covered in the group of noncompetitive inhibitors.

  12. Recognition of acidic phospholipase A2 activity in plasma membranes of resident peritoneal macrophages

    International Nuclear Information System (INIS)

    Shibata, Y.; Abiko, Y.; Ohno, H.; Araki, T.; Takiguchi, H.

    1988-01-01

    Phospholipase (PLase) activities in the plasma membrane of guinea pig peritoneal macrophages were studied, as these enzymes having such activity may be candidates for the release of arachidonic acid (AA) from phosphatidylcholine (PC). An AA release system operating at acidic pH was identified in the macrophage plasma membrane and characterized. This membrane-bound acidic PLase A 2 had an optimum pH at 4.5, and enzyme activation was observed in Ca ++ -free medium; but the maximum activity was found at 0.5 mM Ca ++ concentration. The Km value for PC of acidic PLase A 2 was 4.2 μM, and a Michaelis-Menten relationship was evident. Calcium might act as a cofactor at some intermediate step during the activation of acidic PLase A 2 in light of the uncompetitive manner of Ca ++ action. Furthermore, the release of [ 3 H]-AA from preradiolabelled macrophage plasma membranes occurred with the addition of Ca ++ at pH 4.5. These data suggest that the acid PLase A 2 is a component of the plasma membrane and is not due to lysosomal contamination since membrane-bound acidic PLase A 2 properties are opposite to those found for lysosomal PLase A 2

  13. Nonresonant Double Hopf Bifurcation in Toxic Phytoplankton-Zooplankton Model with Delay

    Science.gov (United States)

    Yuan, Rui; Jiang, Weihua; Wang, Yong

    This paper investigates a toxic phytoplankton-zooplankton model with Michaelis-Menten type phytoplankton harvesting. The model has rich dynamical behaviors. It undergoes transcritical, saddle-node, fold, Hopf, fold-Hopf and double Hopf bifurcation, when the parameters change and go through some of the critical values, the dynamical properties of the system will change also, such as the stability, equilibrium points and the periodic orbit. We first study the stability of the equilibria, and analyze the critical conditions for the above bifurcations at each equilibrium. In addition, the stability and direction of local Hopf bifurcations, and the completion bifurcation set by calculating the universal unfoldings near the double Hopf bifurcation point are given by the normal form theory and center manifold theorem. We obtained that the stable coexistent equilibrium point and stable periodic orbit alternate regularly when the digestion time delay is within some finite value. That is, we derived the pattern for the occurrence, and disappearance of a stable periodic orbit. Furthermore, we calculated the approximation expression of the critical bifurcation curve using the digestion time delay and the harvesting rate as parameters, and determined a large range in terms of the harvesting rate for the phytoplankton and zooplankton to coexist in a long term.

  14. Further In-vitro Characterization of an Implantable Biosensor for Ethanol Monitoring in the Brain

    Directory of Open Access Journals (Sweden)

    Gaia Rocchitta

    2013-07-01

    Full Text Available Ethyl alcohol may be considered one of the most widespread central nervous system (CNS depressants in Western countries. Because of its toxicological and neurobiological implications, the detection of ethanol in brain extracellular fluid (ECF is of great importance. In a previous study, we described the development and characterization of an implantable biosensor successfully used for the real-time detection of ethanol in the brain of freely-moving rats. The implanted biosensor, integrated in a low-cost telemetry system, was demonstrated to be a reliable device for the short-time monitoring of exogenous ethanol in brain ECF. In this paper we describe a further in-vitro characterization of the above-mentioned biosensor in terms of oxygen, pH and temperature dependence in order to complete its validation. With the aim of enhancing ethanol biosensor performance, different enzyme loadings were investigated in terms of apparent ethanol Michaelis-Menten kinetic parameters, viz. IMAX, KM and linear region slope, as well as ascorbic acid interference shielding. The responses of biosensors were studied over a period of 28 days. The overall findings of the present study confirm the original biosensor configuration to be the best of those investigated for in-vivo applications up to one week after implantation.

  15. Mechanistic kinetic modeling generates system-independent P-glycoprotein mediated transport elementary rate constants for inhibition and, in combination with 3D SIM microscopy, elucidates the importance of microvilli morphology on P-glycoprotein mediated efflux activity.

    Science.gov (United States)

    Ellens, Harma; Meng, Zhou; Le Marchand, Sylvain J; Bentz, Joe

    2018-06-01

    In vitro transporter kinetics are typically analyzed by steady-state Michaelis-Menten approximations. However, no clear evidence exists that these approximations, applied to multiple transporters in biological membranes, yield system-independent mechanistic parameters needed for reliable in vivo hypothesis generation and testing. Areas covered: The classical mass action model has been developed for P-glycoprotein (P-gp) mediated transport across confluent polarized cell monolayers. Numerical integration of the mass action equations for transport using a stable global optimization program yields fitted elementary rate constants that are system-independent. The efflux active P-gp was defined by the rate at which P-gp delivers drugs to the apical chamber, since as much as 90% of drugs effluxed by P-gp partition back into nearby microvilli prior to reaching the apical chamber. The efflux active P-gp concentration was 10-fold smaller than the total expressed P-gp for Caco-2 cells, due to their microvilli membrane morphology. The mechanistic insights from this analysis are readily extrapolated to P-gp mediated transport in vivo. Expert opinion: In vitro system-independent elementary rate constants for transporters are essential for the generation and validation of robust mechanistic PBPK models. Our modeling approach and programs have broad application potential. They can be used for any drug transporter with minor adaptations.

  16. Continuous culture apparatus and methodology

    International Nuclear Information System (INIS)

    Conway, H.L.

    1975-01-01

    At present, we are investigating the sorption of potentially toxic trace elements by phytoplankton under controlled laboratory conditions. Continuous culture techniques were used to study the mechanism of the sorption of the trace elements by unialgal diatom populations and the factors influencing this sorption. Continuous culture methodology has been used extensively to study bacterial kinetics. It is an excellent technique for obtaining a known physiological state of phytoplankton populations. An automated method for the synthesis of continuous culture medium for use in these experiments is described

  17. Blood cholinesterases as human biomarkers of organophosphorus pesticide exposure.

    Science.gov (United States)

    Nigg, H N; Knaak, J B

    2000-01-01

    The organophosphorus pesticides of this review were discovered in 1936 during the search for a replacement for nicotine for cockroach control. The basic biochemical characteristics of RBC AChE and BChE were determined in the 1940s. The mechanism of inhibition of both enzymes and other serine esterases was known in the 1940s and, in general, defined in the 1950s. In 1949, the death of a parathion mixer-loader dictated blood enzyme monitoring to prevent acute illness from organophosphorus pesticide intoxication. However, many of the chemical and biochemical steps for serine enzyme inhibition by OP compounds remain unknown today. The possible mechanisms of this inhibition are presented kinetically beginning with simple (by comparison) Michaelis-Menten substrate enzyme interaction kinetics. As complicated as the inhibition kinetics appear here, PBPK model kinetics will be more complex. The determination of inter- and intraindividual variation in RBC ChE and BChE was recognized early as critical knowledge for a blood esterase monitoring program. Because of the relatively constant production of RBCs, variation in RBC AChE was determined by about 1970. The source of plasma (or serum) BChE was shown to be the liver in the 1960s with the change in BChE phenotype to the donor in liver transplant patients. BChE activity was more variable than RBC AChE, and only in the 1990s have BChE individual variation questions been answered. We have reviewed the chemistry, metabolism, and toxicity of organophosphorus insecticides along with their inhibitory action toward tissue acetyl- and butyrylcholinesterases. On the basis of the review, a monitoring program for individuals mixing-loading and applying OP pesticides for commercial applicators was recommended. Approximately 41 OPs are currently registered for use by USEPA in the United States. Under agricultural working conditions, OPs primarily are absorbed through the skin. Liver P-450 isozymes catalyze the desulfurization of

  18. The metabolic network of Clostridium acetobutylicum: Comparison of the approximate Bayesian computation via sequential Monte Carlo (ABC-SMC) and profile likelihood estimation (PLE) methods for determinability analysis.

    Science.gov (United States)

    Thorn, Graeme J; King, John R

    2016-01-01

    The Gram-positive bacterium Clostridium acetobutylicum is an anaerobic endospore-forming species which produces acetone, butanol and ethanol via the acetone-butanol (AB) fermentation process, leading to biofuels including butanol. In previous work we looked to estimate the parameters in an ordinary differential equation model of the glucose metabolism network using data from pH-controlled continuous culture experiments. Here we combine two approaches, namely the approximate Bayesian computation via an existing sequential Monte Carlo (ABC-SMC) method (to compute credible intervals for the parameters), and the profile likelihood estimation (PLE) (to improve the calculation of confidence intervals for the same parameters), the parameters in both cases being derived from experimental data from forward shift experiments. We also apply the ABC-SMC method to investigate which of the models introduced previously (one non-sporulation and four sporulation models) have the greatest strength of evidence. We find that the joint approximate posterior distribution of the parameters determines the same parameters as previously, including all of the basal and increased enzyme production rates and enzyme reaction activity parameters, as well as the Michaelis-Menten kinetic parameters for glucose ingestion, while other parameters are not as well-determined, particularly those connected with the internal metabolites acetyl-CoA, acetoacetyl-CoA and butyryl-CoA. We also find that the approximate posterior is strongly non-Gaussian, indicating that our previous assumption of elliptical contours of the distribution is not valid, which has the effect of reducing the numbers of pairs of parameters that are (linearly) correlated with each other. Calculations of confidence intervals using the PLE method back this up. Finally, we find that all five of our models are equally likely, given the data available at present. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Phosphorus Uptake Kinetics of Different Types of Duckweed.%不同品种浮萍磷素吸收动力学特征

    Institute of Scientific and Technical Information of China (English)

    蔡树美; 刘文桃; 张震; 柏彦超; 钱晓晴

    2011-01-01

    以长江三角洲地区常见的3种浮萍--稀脉浮萍(Lemna aequinoctialis)、少根紫萍(Spirodela oligorrhiza)和紫萍(Spriodela polyrrhiza)为对象,研究不同品种浮萍对HPO42-的吸收动力学影响.结果表明,3种浮萍对无机磷的吸收动力学特征基本可采用Michaelis-Menten方程描述,3种浮萍无机磷吸收速率V对磷浓度S双倒数曲线的拟合关系均在α=0.01水平上显著.稀脉浮萍对无机磷的亲和力较强,适宜应用于对低磷浓度污水的净化;紫萍对无机磷的最大吸收速率Vmax较高,适宜应用于对高磷浓度污水的净化.%An increasing attention has been paid to the use of duckweed in treating eutrophicated water as a tool of bio-remediation of domestic wastewater.Phosphorus uptake kinetics was studied,of different species of duckweeds, i.e.Lemna aequinoctialis, Spirodela oligorrhiza and Spriodela polyrrhiza, which are common in the Yangtze River Delta area.Results show that the phosphorus uptake kinetics of the duckweeds could be expressed with the Michaelis-Menten equation; and the relation between phosphorus uptake rate (V) of the duckweeds and inorganic phosphorus(Pi) concentration (S), was well fitted with a double-reciprocal curve with at =0.01; the Km value for Pi uptake of Lemna aequinoctialis was lower than that of Spirodela oligorrhiza and Spriodela polyrrhiza, making it a better candidate for treating wastewater low in Pi concentration.The maximum phosphorus uptake rate of Spriodela polyrrhiza was higher than that of the other two species, which allows Spriodela polyrrhiza better performance in purifying wastewater higher in Pi concentration.

  20. Effect of doxorubicin and daunorubicin on the activity of acetylcholinesterase in acute lymphoblastic leukamia

    International Nuclear Information System (INIS)

    Din, I.U.; Ali, A.

    2011-01-01

    Background: Our study was based on the alteration in the Michaelis Mentin parameters Apparent Michaelis Constant (aKm) and Apparent Maximum Velocity (aVm), which reflects activity of actyl cholinesterase (AChE). This activity decreases in Acute Lymphoblastic Leukaemia (ALL). This decrease in aKm and aVm values shows bad prognosis. Similarly the anticancer drugs like Daunorubicin and Doxorubicin further decreases the aKm and aVm values which worsen the prognosis. The objective of this study was to determine and compare the extent of inhibition of Acetylcholine Esterase by Daunorubicin and Doxorubicin in ALL. Methods: Study of 100 patients including both male and female children who's age ranged from 4 to 8 years and were advised doxorubicin and daunorubicin separately were tested by Ellman's method using acetylcholine iodide as substrate and 5,5-dithiobis 2-nitrobenzine as a colour reagent regardless of dose regimen i.e. (once in 3 week, small dose per week or a continuous infusion for 72 to 96 hours. Results: In this study the Michaelis Mentin parameters Apparent Michaelis Constant (aKm) and Apparent Maximum Velocity (aVm) of the enzyme were estimated both in normal individuals and in the patients and also during treatment with daunorubicin and doxorubicin. The value of Michaelis Mentin parameters, aKm, aVm and percentage activity of the enzyme in normal individual are 23, 70, and 100 respectively. The values of aKm, aVm and percentage activity of the enzyme were also estimated in the patients before and after treatment. The values of aKm and aVm in patients of acute lymphoblastic leukaemia and percentage activity of enzyme is decreased. After the treatment with daunorubicin and doxorubicin the values and activity is further decreased. Conclusion: We conclude that the drugs under study both decrease the enzyme activity but daunorubicin inhibits the enzyme more than doxorubicin. (author)

  1. Vibration parameters assessment to develop a continuous lateral canopy shaker for mechanical harvesting of traditional olive trees

    Energy Technology Data Exchange (ETDEWEB)

    Sola-Guirado, R.R.; Jimenez-Jimenez, F.; Blanco-Roldan, G.L.; Castro-Garcia, S.; Castillo-Ruiz, F.J.; Gil Ribes, J.A.

    2016-11-01

    The fruit harvesting is a key factor involving both product quality and profitability. Particularly, mechanical harvesting of traditional oil olive orchards is hint by tree training system for manual harvesting, tree size and several and slanted trunks which makes difficult trunk shaker work. Therefore, canopy shaker technology could be a feasible alternative to develop an integral harvester able to work on irregular canopies. The aim of this research was to determine vibration parameters applied to the olive tree for efficient mechanical harvesting by canopy shaker measuring fruit removal efficiency and debris. In this work, a continuous lateral canopy shaker harvester has been developed and tested on large olive trees in order to analyse the operating harvester parameters and tree properties to improve mutual adaptation. Vibration amplitude and frequency, rod density and ground speed were assessed. Vibration amplitude and frequency beside ground speed were decisive factors on fruit removal efficiency. Increasing rod density has not influenced on removal efficiency although it increased significantly debris. Promising results has been reached with 77.3% of removal efficiency, applying a 28 s shaking duration, 0.17 m amplitude vibration and 12 rod drum. This result was obtained reporting 0.26 s of accumulative shaking time over 200 m/s2 resultant acceleration. The canopy shaker mechanism enabled more than 65% of detached fruits to fall vertically, facilitating catch fruit. In order to improve removal efficiency it is advisable to adapt trees, set high amplitude in the shaker mechanism, and enhance the contact time between rods and tree. (Author)

  2. Estimate of respiration rate and physicochemical changes of fresh-cut apples stored under different temperatures Estimativa da taxa de respiração e das mudanças físico-químicas de maçãs minimamente processadas e estocadas sob diferentes temperaturas

    Directory of Open Access Journals (Sweden)

    Cristiane Fagundes

    2013-03-01

    Full Text Available In this study, the influence of storage temperature and passive modified packaging (PMP on the respiration rate and physicochemical properties of fresh-cut Gala apples (Malus domestica B. was investigated. The samples were packed in flexible multilayer bags and stored at 2 °C, 5 °C, and 7 °C for eleven days. Respiration rate as a function of CO2 and O2 concentrations was determined using gas chromatography. The inhibition parameters were estimated using a mathematical model based on Michaelis-Menten equation. The following physicochemical properties were evaluated: total soluble solids, pH, titratable acidity, and reducing sugars. At 2 °C, the maximum respiration rate was observed after 150 hours. At 5 °C and 7 °C the maximum respiration rates were observed after 100 and 50 hours of storage, respectively. The inhibition model results obtained showed a clear effect of CO2 on O2 consumption. The soluble solids decreased, although not significantly, during storage at the three temperatures studied. Reducing sugars and titratable acidity decreased during storage and the pH increased. These results indicate that the respiration rate influenced the physicochemical properties.Neste estudo, a influência da temperatura de armazenamento e da embalagem com atmosfera modificada passiva (AMP na taxa respiratória e nas propriedades físico-químicas de maçãs da variedade Gala (Malus domestica, B. minimamente processadas foi investigada. As amostras foram acondicionadas em embalagens flexíveis e armazenados a 2 °C, 5 °C e 7 °C, durante 11 dias. A taxa de respiração foi gerada para diferentes concentrações de O2 e CO2, obtidas por cromatografia gasosa. Os parâmetros de inibição foram estimados por um modelo matemático baseado na equação de Michaelis-Menten. Foram avaliadas as seguintes propriedades físico-químicas: sólidos solúveis totais, pH, acidez titulável e açúcares redutores. A 2 °C, a taxa de respiração máxima foi

  3. Postictal apnea as an important mechanism for SUDEP: A near-SUDEP with continuous EEG-ECG-EMG recording.

    Science.gov (United States)

    Jin, Lang; Zhang, Ying; Wang, Xiao-Li; Zhang, Wen-Juan; Liu, Yong-Hong; Jiang, Zhao

    2017-09-01

    Sudden unexpected death in epilepsy (SUDEP) is one of the most frequent causes of death among patients with epilepsy. Most SUDEP or near-SUDEP are unwitnessed and not observed or recorded during video-EEG recording in epilepsy monitoring units. This report describes a young woman with post ictal apnea and generalized EEG suppression (PGES) after a secondary generalized tonic-clonic seizure (sGTCS). This was accompanied by bradycardia and then ventricular tachycardia (VT). But at the end of VT, the patient's breath recovered without any intervention, such as cardio-respiratory resuscitation. This case report with continuous EEG, EKG, EMG during near SUDEP may provide insights into the mechanism of action. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Integrating plant-microbe interactions to understand soil C stabilization with the MIcrobial-MIneral Carbon Stabilization model (MIMICS)

    Science.gov (United States)

    Grandy, Stuart; Wieder, Will; Kallenbach, Cynthia; Tiemann, Lisa

    2014-05-01

    If soil organic matter is predominantly microbial biomass, plant inputs that build biomass should also increase SOM. This seems obvious, but the implications fundamentally change how we think about the relationships between plants, microbes and SOM. Plant residues that build microbial biomass are typically characterized by low C/N ratios and high lignin contents. However, plants with high lignin contents and high C/N ratios are believed to increase SOM, an entrenched idea that still strongly motivates agricultural soil management practices. Here we use a combination of meta-analysis with a new microbial-explicit soil biogeochemistry model to explore the relationships between plant litter chemistry, microbial communities, and SOM stabilization in different soil types. We use the MIcrobial-MIneral Carbon Stabilization (MIMICS) model, newly built upon the Community Land Model (CLM) platform, to enhance our understanding of biology in earth system processes. The turnover of litter and SOM in MIMICS are governed by the activity of r- and k-selected microbial groups and temperature sensitive Michaelis-Menten kinetics. Plant and microbial residues are stabilized short-term by chemical recalcitrance or long-term by physical protection. Fast-turnover litter inputs increase SOM by >10% depending on temperature in clay soils, and it's only in sandy soils devoid of physical protection mechanisms that recalcitrant inputs build SOM. These results challenge centuries of lay knowledge as well as conventional ideas of SOM formation, but are they realistic? To test this, we conducted a meta-analysis of the relationships between the chemistry of plant liter inputs and SOM concentrations. We find globally that the highest SOM concentrations are associated with plant inputs containing low C/N ratios. These results are confirmed by individual tracer studies pointing to greater stabilization of low C/N ratio inputs, particularly in clay soils. Our model and meta-analysis results suggest

  5. Exploring C-water-temperature interactions and non-linearities in soils through developments in process-based models

    Science.gov (United States)

    Esteban Moyano, Fernando; Vasilyeva, Nadezda; Menichetti, Lorenzo

    2016-04-01

    Soil carbon models developed over the last couple of decades are limited in their capacity to accurately predict the magnitudes and temporal variations in observed carbon fluxes and stocks. New process-based models are now emerging that attempt to address the shortcomings of their more simple, empirical counterparts. While a spectrum of ideas and hypothetical mechanisms are finding their way into new models, the addition of only a few processes known to significantly affect soil carbon (e.g. enzymatic decomposition, adsorption, Michaelis-Menten kinetics) has shown the potential to resolve a number of previous model-data discrepancies (e.g. priming, Birch effects). Through model-data validation, such models are a means of testing hypothetical mechanisms. In addition, they can lead to new insights into what soil carbon pools are and how they respond to external drivers. In this study we develop a model of soil carbon dynamics based on enzymatic decomposition and other key features of process based models, i.e. simulation of carbon in particulate, soluble and adsorbed states, as well as enzyme and microbial components. Here we focus on understanding how moisture affects C decomposition at different levels, both directly (e.g. by limiting diffusion) or through interactions with other components. As the medium where most reactions and transport take place, water is central en every aspect of soil C dynamics. We compare results from a number of alternative models with experimental data in order to test different processes and parameterizations. Among other observations, we try to understand: 1. typical moisture response curves and associated temporal changes, 2. moisture-temperature interactions, and 3. diffusion effects under changing C concentrations. While the model aims at being a process based approach and at simulating fluxes at short time scales, it remains a simplified representation using the same inputs as classical soil C models, and is thus potentially

  6. Anomalous stress diffusion, Omori's law and Continuous Time Random Walk in the 2010 Efpalion aftershock sequence (Corinth rift, Greece)

    Science.gov (United States)

    Michas, Georgios; Vallianatos, Filippos; Karakostas, Vassilios; Papadimitriou, Eleftheria; Sammonds, Peter

    2014-05-01

    Efpalion aftershock sequence occurred in January 2010, when an M=5.5 earthquake was followed four days later by another strong event (M=5.4) and numerous aftershocks (Karakostas et al., 2012). This activity interrupted a 15 years period of low to moderate earthquake occurrence in Corinth rift, where the last major event was the 1995 Aigion earthquake (M=6.2). Coulomb stress analysis performed in previous studies (Karakostas et al., 2012; Sokos et al., 2012; Ganas et al., 2013) indicated that the second major event and most of the aftershocks were triggered due to stress transfer. The aftershocks production rate decays as a power-law with time according to the modified Omori law (Utsu et al., 1995) with an exponent larger than one for the first four days, while after the occurrence of the second strong event the exponent turns to unity. We consider the earthquake sequence as a point process in time and space and study its spatiotemporal evolution considering a Continuous Time Random Walk (CTRW) model with a joint probability density function of inter-event times and jumps between the successive earthquakes (Metzler and Klafter, 2000). Jump length distribution exhibits finite variance, whereas inter-event times scale as a q-generalized gamma distribution (Michas et al., 2013) with a long power-law tail. These properties are indicative of a subdiffusive process in terms of CTRW. Additionally, the mean square displacement of aftershocks is constant with time after the occurrence of the first event, while it changes to a power-law with exponent close to 0.15 after the second major event, illustrating a slow diffusive process. During the first four days aftershocks cluster around the epicentral area of the second major event, while after that and taking as a reference the second event, the aftershock zone is migrating slowly with time to the west near the epicentral area of the first event. This process is much slower from what would be expected from normal diffusion, a

  7. Microcontroller Based Proportional Derivative Plus Conditional Integral Controller for Electro-Mechanical Dual Acting Pulley Continuously Variable Transmission Ratio Control

    International Nuclear Information System (INIS)

    Budianto, A; Tawi, K B; Hussein, M; Supriyo, B; Kob, M S Che; Zulkifli, Mohd Ezlamy; Khairuldean A K; Daraoh, Aishah; Ariyono, S

    2012-01-01

    Electro-Mechanical Dual Acting Pulley (EMDAP) Continuously Variable Transmission (CVT) is a transmission utilized by electro-mechanical actuated system. It has a potential to reduce energy consumption because it only needs power during changing CVT ratio and no power is needed to maintain CVT ratio due to self lock mechanism design. This paper proposed simple proportional derivative plus conditional integral (PDCI) controller to control EMDAP CVT ratio which can be simply implemented on a microcontroller. This proposed controller used Astrom-Hagglund method and Ziegler-Nichols formula to tune PDCI gain. The Proportional Derivative controller is directly activated from the start but Integral controller is only activated when the error value reaches error value setting point. Simulation using Matlab/Simulink software was conducted to evaluate PDCI system performance. The simulation results showed PDCI controller has ability to perform maximum overshoot 0.1%, 0.001 steady state error and 0.5s settling time. For clamping condition, settling time is about 11.46s during changing ratio from 2.0 to 0.7, while for release condition, settling time is about 8.33s during changing ratio from 0.7 to 2.0.

  8. In vitro interactions of malachite green and leucomalachite green with hepatic drug-metabolizing enzyme systems in the rainbow trout (Onchorhyncus mykiss).

    Science.gov (United States)

    Nebbia, Carlo; Girolami, Flavia; Carletti, Monica; Gasco, Laura; Zoccarato, Ivo; Giuliano Albo, Alessandra

    2017-10-05

    Malachite green (MG) has been widely used in aquaculture to treat a number of microbial and parasitic diseases. It is currently banned in the EU because of the high cytotoxicity and carcinogenic activity, which is also shared by leucomalachite green (LMG), a reduced MG metabolite that can persist in fish tissues for months. There is scant information about the ability of either compound to interact with drug metabolizing enzymes in fish. Therefore we evaluated the in vitro effects of MG and LMG (25, 50 and 100μM) on some DMEs and glutathione (GSH) content in rainbow trout liver subfractions. LMG did not affect any of the examined parameters. In contrast, MG proved to deplete GSH and to depress to a various extent the activities of NAD(P)H cytochrome c reductase, 7-ethoxycoumarin O-deethylase, 1-naphthol uridindiphosphoglucuronyl-transferase and maximally those of 7-ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) accepting 1-chloro2,4-dinitrobenzene (CDNB) as substrate. The inhibition mechanisms of EROD and GST were investigated by means of non-linear Michaelis-Menten kinetics and Lineweaver-Burk plots using 0.175-8μM MG. The calculated IC 50 for EROD was 7.1μM, and the inhibition appeared to be competitive (K i 2.78±0.24μM). In the case of GST, the calculated IC 50 was 0.53μM. The inhibition was best described as competitive toward GSH (Ki 0.39±0.02μM) and of mixed-type toward CDNB (Ki 0.64±0.06μM). Our findings indicate that, contrary to LMG, MG behaves as a relatively strong inhibitor of certain liver DMEs and can reversibly bind GSH. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. The conserved Lysine69 residue plays a catalytic role in Mycobacterium tuberculosis shikimate dehydrogenase

    Directory of Open Access Journals (Sweden)

    Rodrigues Valnês

    2009-01-01

    Full Text Available Abstract Background The shikimate pathway is an attractive target for the development of antitubercular agents because it is essential in Mycobacterium tuberculosis, the causative agent of tuberculosis, but absent in humans. M. tuberculosis aroE-encoded shikimate dehydrogenase catalyzes the forth reaction in the shikimate pathway. Structural and functional studies indicate that Lysine69 may be involved in catalysis and/or substrate binding in M. tuberculosis shikimate dehydrogenase. Investigation of the kinetic properties of mutant enzymes can bring important insights about the role of amino acid residues for M. tuberculosis shikimate dehydrogenase. Findings We have performed site-directed mutagenesis, steady-state kinetics, equilibrium binding measurements and molecular modeling for both the wild-type M. tuberculosis shikimate dehydrogenase and the K69A mutant enzymes. The apparent steady-state kinetic parameters for the M. tuberculosis shikimate dehydrogenase were determined; the catalytic constant value for the wild-type enzyme (50 s-1 is 68-fold larger than that for the mutant K69A (0.73 s-1. There was a modest increase in the Michaelis-Menten constant for DHS (K69A = 76 μM; wild-type = 29 μM and NADPH (K69A = 30 μM; wild-type = 11 μM. The equilibrium dissociation constants for wild-type and K69A mutant enzymes are 32 (± 4 μM and 134 (± 21, respectively. Conclusion Our results show that the residue Lysine69 plays a catalytic role and is not involved in substrate binding for the M. tuberculosis shikimate dehydrogenase. These efforts on M. tuberculosis shikimate dehydrogenase catalytic mechanism determination should help the rational design of specific inhibitors, aiming at the development of antitubercular drugs.

  10. A disposable biosensor based on immobilization of laccase with silica spheres on the MWCNTs-doped screen-printed electrode

    Directory of Open Access Journals (Sweden)

    Li Yuanting

    2012-09-01

    Full Text Available Abstract Background Biosensors have attracted increasing attention as reliable analytical instruments in in situ monitoring of public health and environmental pollution. For enzyme-based biosensors, the stabilization of enzymatic activity on the biological recognition element is of great importance. It is generally acknowledged that an effective immobilization technique is a key step to achieve the construction quality of biosensors. Results A novel disposable biosensor was constructed by immobilizing laccase (Lac with silica spheres on the surface of multi-walled carbon nanotubes (MWCNTs-doped screen-printed electrode (SPE. Then, it was characterized in morphology and electrochemical properties by scanning electron microscopy (SEM and cyclic voltammetry (CV. The characterization results indicated that a high loading of Lac and a good electrocatalytic activity could be obtained, attributing to the porous structure, large specific area and good biocompatibility of silica spheres and MWCNTs. Furthermore, the electrochemical sensing properties of the constructed biosensor were investigated by choosing dopamine (DA as the typical model of phenolic compounds. It was shown that the biosensor displays a good linearity in the range from 1.3 to 85.5 μM with a detection limit of 0.42 μM (S/N = 3, and the Michaelis-Menten constant (Kmapp was calculated to be 3.78 μM. Conclusion The immobilization of Lac was successfully achieved with silica spheres to construct a disposable biosensor on the MWCNTs-doped SPE (MWCNTs/SPE. This biosensor could determine DA based on a non-oxidative mechanism in a rapid, selective and sensitive way. Besides, the developed biosensor could retain high enzymatic activity and possess good stability without cross-linking reagents. The proposed immobilization approach and the constructed biosensor offer a great potential for the fabrication of the enzyme-based biosensors and the analysis of phenolic compounds.

  11. A novel functionalisation process for glucose oxidase immobilisation in poly(methyl methacrylate) microchannels in a flow system for amperometric determinations.

    Science.gov (United States)

    Cerqueira, Marcos Rodrigues Facchini; Grasseschi, Daniel; Matos, Renato Camargo; Angnes, Lucio

    2014-08-01

    Different materials like glass, silicon and poly(methyl methacrylate) (PMMA) are being used to immobilise enzymes in microchannels. PMMA shows advantages such as its low price, biocompatibility and attractive mechanical and chemical properties. Despite this, the introduction of reactive functional groups on PMMA is still problematic, either because of the complex chemistry or extended reaction time involved. In this paper, a new methodology was developed to immobilise glucose oxidase (GOx) in PMMA microchannels, with the benefit of a rapid immobilisation process and a very simple route. The new procedure involves only two steps, based on the reaction of 5.0% (w/w) polyethyleneimine (PEI) with PMMA in a dimethyl sulphoxide medium, followed by the immobilisation of glucose oxidase using a solution containing 100U enzymes and 1.0% (v/v) glutaraldehyde. The reactors prepared in this way were evaluated by a flowing system with amperometric detection (+0.60V) based on the oxidation of the H2O2 produced by the reactor. The microreactor proposed here was able to work with high bioconversion and a frequency of 60 samples h(-1), with detection and quantification limits of 0.50 and 1.66µmol L(-1), respectively. Michaelis-Menten parameters (Vmax and KM) were calculated as 449±47.7nmol min(-1) and 7.79±0.98mmol. Statistical evaluations were done to validate the proposed methodology. The content of glucose in natural and commercial coconut water samples was evaluated using the developed method. Comparison with spectrophotometric measurements showed that both methodologies have a very good correlation (tcalculated, 0.05, 4=1.35

  12. Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa.

    Science.gov (United States)

    Fellner, Matthias; Aloi, Sekotilani; Tchesnokov, Egor P; Wilbanks, Sigurd M; Jameson, Guy N L

    2016-03-08

    Thiol dioxygenases catalyze the synthesis of sulfinic acids in a range of organisms from bacteria to mammals. A thiol dioxygenase from the bacterium Pseudomonas aeruginosa oxidizes both 3-mercaptopropionic acid and cysteine, with a ∼70 fold preference for 3-mercaptopropionic acid over all pHs. This substrate reactivity is widened compared to other thiol dioxygenases and was exploited in this investigation of the residues important for activity. A simple model incorporating two protonation events was used to fit profiles of the Michaelis-Menten parameters determined at different pH values for both substrates. The pKs determined using plots of k(cat)/Km differ at low pH, but not in a way easily attributable to protonation of the substrate alone and share a common value at higher pH. Plots of k(cat) versus pH are also quite different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs. At higher pH, k(cat) decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked, the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs reactivity of the crosslinked form.

  13. Intracellular accumulation of potent amiloride analogues by human neutrophils

    International Nuclear Information System (INIS)

    Simchowitz, L.; Woltersdorf, O.W. Jr.; Cragoe, E.J. Jr.

    1987-01-01

    The mechanism of uptake of a series of amiloride derivatives by human neutrophils was investigated using [ 14 C]amiloride and the 14 C-labeled 5-(1-hexahydroazepinyl)-6-bromo analogue (BrMM) which is approximately 500-fold more potent than the parent compound at inhibiting Na+/H+ exchange. At an external concentration of 2 microM, the influx of BrMM at 37 degrees C was rapid, reaching a steady state by approximately 20 min. The rate of BrMM uptake (approximately 25 mumol/liter.min) was approximately 90-fold faster than for the same concentration of amiloride, a finding which correlates with differences in lipid partitioning of the two compounds. Uptake was unrelated to specific binding to Na+/H+ exchange transport sites: influx of either drug was nonsaturable whereas amiloride- and BrMM-mediated inhibition of Na+/H+ countertransport obeyed Michaelis-Menten kinetics with apparent Ki values of approximately 75 and approximately 0.2 microM. Entry occurred exclusively via the neutral (uncharged) forms (pK'a 8.40-8.55). Influx was markedly pH-dependent: it was enhanced by extracellular alkalinization and reduced by acidification. Influx was, however, insensitive to large changes in membrane voltage, thereby implying the protonated (charged) species to be impermeant. About 75% of the total intracellular pool of amiloride, but only approximately 25% of BrMM, is contained within the lysosomes, an expected consequence of the partitioning and subsequent trapping of a weak base within this strongly acidic subcellular compartment. With BrMM, there was a relative approximately 60-fold enrichment in the internal/external water concentration ratio of the drug; the value for amiloride was much less, approximately 4. This disparity is consistent with substantial binding of BrMM to internal constituents, presumably to proteins and/or nucleic acids

  14. Sided functions of an arginine-agmatine antiporter oriented in liposomes.

    Science.gov (United States)

    Tsai, Ming-Feng; Fang, Yiling; Miller, Christopher

    2012-02-28

    The arginine-dependent extreme acid resistance system helps enteric bacteria survive the harsh gastric environment. At the center of this multiprotein system is an arginine-agmatine antiporter, AdiC. To maintain cytoplasmic pH, AdiC imports arginine and exports its decarboxylated product, agmatine, resulting in a net extrusion of one "virtual proton" in each turnover. The random orientation of AdiC in reconstituted liposomes throws up an obstacle to quantifying its transport mechanism. To overcome this problem, we introduced a mutation, S26C, near the substrate-binding site. This mutant exhibits substrate recognition and pH-dependent activity similar to those of the wild-type protein but loses function completely upon reaction with thiol reagents. The membrane-impermeant MTSES reagent can then be used as a cleanly sided inhibitor to silence those S26C-AdiC proteins whose extracellular portion projects from the external side of the liposome. Alternatively, the membrane-permeant MTSEA and membrane-impermeant reducing reagent, TCEP, can be used together to inhibit proteins in the opposite orientation. This approach allows steady-state kinetic analysis of AdiC in a sided fashion. Arginine and agmatine have similar Michaelis-Menten parameters for both sides of the protein, while the extracellular side selects arginine over argininamide, a mimic of the carboxylate-protonated form of arginine, more effectively than does the cytoplasmic side. Moreover, the two sides of AdiC have different pH sensitivities. AdiC activity increases to a plateau at pH 4 as the extracellular side is acidified, while the cytoplasmic side shows an optimal pH of 5.5, with further acidification inhibiting transport. This oriented system allows more precise analysis of AdiC-mediated substrate transport than has been previously available and permits comparison to the situation experienced by the bacterial membrane under acid stress.

  15. Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity.

    Science.gov (United States)

    Samuni, Amram; Maimon, Eric; Goldstein, Sara

    2017-08-01

    Horseradish peroxidase (HRP) catalyzes H 2 O 2 dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H 2 O 2 -induced inactivation, have been investigated. HRP reaction with H 2 O 2 was studied by following H 2 O 2 depletion, O 2 evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow. Nitroxide protects HRP against H 2 O 2 -induced inactivation. The rate of H 2 O 2 dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H 2 O 2 . The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO)>4-OH-TPO>3-carbamoyl proxyl>4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III. Nitroxide catalytically protects HRP against inactivation induced by H 2 O 2 while modulating its catalase-like activity. The protective role of nitroxide at μM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex. Nitroxides catalytically protect heme proteins against inactivation induced by H 2 O 2 revealing an additional role played by nitroxide antioxidants in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. A conserved cysteine motif is critical for rice ceramide kinase activity and function.

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    Fang-Cheng Bi

    Full Text Available Ceramide kinase (CERK is a key regulator of cell survival in dicotyledonous plants and animals. Much less is known about the roles of CERK and ceramides in mediating cellular processes in monocot plants. Here, we report the characterization of a ceramide kinase, OsCERK, from rice (Oryza sativa spp. Japonica cv. Nipponbare and investigate the effects of ceramides on rice cell viability.OsCERK can complement the Arabidopsis CERK mutant acd5. Recombinant OsCERK has ceramide kinase activity with Michaelis-Menten kinetics and optimal activity at 7.0 pH and 40°C. Mg2+ activates OsCERK in a concentration-dependent manner. Importantly, a CXXXCXXC motif, conserved in all ceramide kinases and important for the activity of the human enzyme, is critical for OsCERK enzyme activity and in planta function. In a rice protoplast system, inhibition of CERK leads to cell death and the ratio of added ceramide and ceramide-1-phosphate, CERK's substrate and product, respectively, influences cell survival. Ceramide-induced rice cell death has apoptotic features and is an active process that requires both de novo protein synthesis and phosphorylation, respectively. Finally, mitochondria membrane potential loss previously associated with ceramide-induced cell death in Arabidopsis was also found in rice, but it occurred with different timing.OsCERK is a bona fide ceramide kinase with a functionally and evolutionarily conserved Cys-rich motif that plays an important role in modulating cell fate in plants. The vital function of the conserved motif in both human and rice CERKs suggests that the biochemical mechanism of CERKs is similar in animals and plants. Furthermore, ceramides induce cell death with similar features in monocot and dicot plants.

  17. Understanding the sub-cellular dynamics of silicon transportation and synthesis in diatoms using population-level data and computational optimization.

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    Narjes Javaheri

    2014-06-01

    Full Text Available Controlled synthesis of silicon is a major challenge in nanotechnology and material science. Diatoms, the unicellular algae, are an inspiring example of silica biosynthesis, producing complex and delicate nano-structures. This happens in several cell compartments, including cytoplasm and silica deposition vesicle (SDV. Considering the low concentration of silicic acid in oceans, cells have developed silicon transporter proteins (SIT. Moreover, cells change the level of active SITs during one cell cycle, likely as a response to the level of external nutrients and internal deposition rates. Despite this topic being of fundamental interest, the intracellular dynamics of nutrients and cell regulation strategies remain poorly understood. One reason is the difficulties in measurements and manipulation of these mechanisms at such small scales, and even when possible, data often contain large errors. Therefore, using computational techniques seems inevitable. We have constructed a mathematical model for silicon dynamics in the diatom Thalassiosira pseudonana in four compartments: external environment, cytoplasm, SDV and deposited silica. The model builds on mass conservation and Michaelis-Menten kinetics as mass transport equations. In order to find the free parameters of the model from sparse, noisy experimental data, an optimization technique (global and local search, together with enzyme related penalty terms, has been applied. We have connected population-level data to individual-cell-level quantities including the effect of early division of non-synchronized cells. Our model is robust, proven by sensitivity and perturbation analysis, and predicts dynamics of intracellular nutrients and enzymes in different compartments. The model produces different uptake regimes, previously recognized as surge, externally-controlled and internally-controlled uptakes. Finally, we imposed a flux of SITs to the model and compared it with previous classical kinetics

  18. A monomeric variant of insulin degrading enzyme (IDE loses its regulatory properties.

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    Eun Suk Song

    2010-03-01

    Full Text Available Insulin degrading enzyme (IDE is a key enzyme in the metabolism of both insulin and amyloid beta peptides. IDE is unique in that it is subject to allosteric activation which is hypothesized to occur through an oligomeric structure.IDE is known to exist as an equilibrium mixture of monomers, dimers, and higher oligomers, with the dimer being the predominant form. Based on the crystal structure of IDE we deleted the putative dimer interface in the C-terminal region, which resulted in a monomeric variant. Monomeric IDE retained enzymatic activity, however instead of the allosteric behavior seen with wild type enzyme it displayed Michaelis-Menten kinetic behavior. With the substrate Abz-GGFLRKHGQ-EDDnp, monomeric IDE retained approximately 25% of the wild type activity. In contrast with the larger peptide substrates beta-endorphin and amyloid beta peptide 1-40, monomeric IDE retained only 1 to 0.25% of wild type activity. Unlike wild type IDE neither bradykinin nor dynorphin B-9 activated the monomeric variant of the enzyme. Similarly, monomeric IDE was not activated by polyphosphates under conditions in which the activity of wild type enzyme was increased more than 50 fold.These findings serve to establish the dimer interface in IDE and demonstrate the requirement for an oligomeric form of the enzyme for its regulatory properties. The data support a mechanism where the binding of activators to oligomeric IDE induces a conformational change that cannot occur in the monomeric variant. Since a conformational change from a closed to a more open structure is likely the rate-determining step in the IDE reaction, the subunit induced conformational change likely shifts the structure of the oligomeric enzyme to a more open conformation.

  19. Rough case-based reasoning system for continues casting

    Science.gov (United States)

    Su, Wenbin; Lei, Zhufeng

    2018-04-01

    The continuous casting occupies a pivotal position in the iron and steel industry. The rough set theory and the CBR (case based reasoning, CBR) were combined in the research and implementation for the quality assurance of continuous casting billet to improve the efficiency and accuracy in determining the processing parameters. According to the continuous casting case, the object-oriented method was applied to express the continuous casting cases. The weights of the attributes were calculated by the algorithm which was based on the rough set theory and the retrieval mechanism for the continuous casting cases was designed. Some cases were adopted to test the retrieval mechanism, by analyzing the results, the law of the influence of the retrieval attributes on determining the processing parameters was revealed. A comprehensive evaluation model was established by using the attribute recognition theory. According to the features of the defects, different methods were adopted to describe the quality condition of the continuous casting billet. By using the system, the knowledge was not only inherited but also applied to adjust the processing parameters through the case based reasoning method as to assure the quality of the continuous casting and improve the intelligent level of the continuous casting.

  20. Noninvasive Positive Pressure Ventilation or Conventional Mechanical Ventilation for Neonatal Continuous Positive Airway Pressure Failure

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    Zohreh Badiee

    2014-01-01

    Full Text Available Background: The aim of this study was to assess the success rate of nasal intermittent positive pressure ventilation (NIPPV for treatment of continuous positive airway pressure (CPAP failure and prevention of conventional ventilation (CV in preterm neonates. Methods: Since November 2012 to April 2013, a total number of 55 consecutive newborns with gestational ages of 26-35 weeks who had CPAP failure were randomly assigned to one of the two groups. The NIPPV group received NIPPV with the initial peak inspiratory pressure (PIP of 16-20 cmH 2 O and frequency of 40-60 breaths/min. The CV group received PIP of 12-20 cmH 2 O and frequency of 40-60 breaths/min. Results: About 74% of newborns who received NIPPV for management of CPAP failure responded to NIPPV and did not need intubation and mechanical ventilation. Newborns with lower postnatal age at entry to the study and lower 5 min Apgar score more likely had NIPPV failure. In addition, treatment failure was higher in newborns who needed more frequent doses of surfactant. Duration of oxygen therapy was 9.28 days in CV group and 7.77 days in NIPPV group (P = 0.050. Length of hospital stay in CV group and NIPPV groups were 48.7 and 41.7 days, respectively (P = 0.097. Conclusions: NIPPV could decrease the need for intubation and mechanical ventilation in preterm infants with CPAP failure.