An optimal strategy to model microbial growth in a multiple substrate environment

A comprehensive model is developed based on an optimal strategy describing varied microbial growth phenomenon involving sequential and simultaneous utilization of substrate. The model mimics the complex regulatory process of a cell which results in diverse growth process with the help of simple multi-variable constrained optimization, which aims at maximizing the specific cell growth. The metabolic processes of a cell are represented by simple flux balance equations. The different growth phenomenon exhibited by a microorganism are attributed to different levels of control present inside the cell. Provision is made in the model for these controls, in the form of constraints in the optimization formulation. The model prediction matches well with the experimental data of simultaneous growth of E. coli K12 on a mixture of glucose and organic acids like lactate, pyruvate, and acetate. Moreover, the model predictions are well in agreement with earlier published experimental data for the growth of E. coli K12 on other organic acids like fumarate, alpha-ketoglutarate, and succinate. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 635-644, 1997.     

Temporal nitric oxide dynamics in the paranasal sinuses during humming.

In this study, the temporal shape of voice-induced nitric oxide (NO) signals in exhaled air has been investigated in eight healthy individuals by means of laser magnetic resonance spectroscopy. The results of the experimental part have been compared with calculated signals obtained by using a simple one-compartment model of the paranasal sinuses. In the experimental part, a rapidly increasing NO concentration has been found when the subjects started humming. After reaching a maximum, the emission starts to decrease with the shape of an exponential decay and finally reaches a constant level. The time constant of this decay (NO washout) is 3.0 +/- 1.2 s. The peak height of the NO emission during humming increases when the time between two humming processes increases. When no voice-induced NO emission takes place, the NO concentration in the paranasal sinuses rebuilds again to a maximum concentration. The typical time constant for the NO recovery is 4.5 +/- 3.2 min. A three-compartment model defining exactly the geometry and anatomy of the paranasal sinuses has been developed that is based on three main assumptions of the NO dynamics: 1) constant NO production of the epithelium in the sinuses; 2) the rate of the chemical reaction of NO with the epithelium of the paranasal sinuses is proportional to the NO concentration; and 3) the emission of NO from the sinuses (volume/s) is proportional to the NO concentration. It is shown that the three-compartment model under the experimental conditions can be reduced to a one-compartment model, which describes the complete temporal behavior of the NO exchange.     

Thiolate coordination to Fe(II)-porphyrin NO centers

The interaction of the Fe(II)-porphyrin NO model complex [Fe(TPP)(NO)] (1, TPP=tetraphenylporphyrin) with thiophenolate ligands and tetrahydrothiophene is explored both computationally and experimentally. Complex 1 is reacted with substituted thiophenolates and the obtained six-coordinate adducts of type [Fe(TPP)(SR)(NO)](-) are investigated in solution using electron paramagnetic resonance (EPR) spectroscopy. From the obtained g values and (14)N hyperfine pattern of the NO ligand it is concluded that the interaction of the thiophenolates with the Fe(II) center is weak in comparison to the corresponding 1-methylimidazole adduct. The strength of the Fe-S bond is increased when alkylthiolates are used as evidenced by comparison with the published EPR spectra of ferrous NO adducts in cytochromes P450 and P450nor, which have an axial cysteinate ligand. These results are further evaluated by density functional (DFT) calculations. The six-coordinate model complex [Fe(P)(SMe)(NO)](-) (1-SMe; P=porphine ligand used for the calculations) has an interesting electronic structure where NO acts as a medium strong sigma donor and pi acceptor ligand. Compared to the N-donor adducts with 1-methylimidazole (1-MeIm), etc., donation from the pi(h)( *) orbital of NO to Fe(II) is reduced due to the stronger trans effect of the alkylthiolate ligand. This is reflected by the predicted longer Fe-NO bond length and smaller Fe-NO force constant for 1-SMe compared to the 1-MeIm adduct. Therefore, the Fe(II)-porphyrin NO adducts with trans alkylthiolate coordination have to be described as Fe(II)-NO(radical) systems. The N-O stretching frequency of these complexes is predicted below 1600cm(-1) in agreement with the available experimental data. In addition, 1-SMe has a unique spin density distribution where Fe has a negative spin density of -0.26 from the calculations. The implications of this unusual electronic structure for the reactivity of the Fe(II)-NO alkylthiolate adducts as they occur in cytochrome P450nor are discussed.     

Kinetic analysis of substrate inhibition in nitric oxide reductase of Paracoccus denitrificans.

The current kinetic model for the nitric oxide reductase reaction (Girsch, P., and de Vries, S. (1997) Biochim. Biophys. Acta 1318, 202-216) does not involve the concentration of an electron donor. Here we introduce this variable and show, both theoretically and experimentally, its role in determining the extent of substrate inhibition by the excess of nitric oxide. NO is found to inhibit competitively with the electron donor, possibly by binding to the oxidized form of the enzyme. The observed partial character of the inhibition is tentatively explained by a slow reduction of the non-productive NO complex.     

A new and more accurate technique to characterize airway nitric oxide using different breath-hold times.

Exhaled nitric oxide (NO) arises from both airway and alveolar regions of the lungs, which provides an opportunity to characterize region-specific inflammation. Current methodologies rely on vital capacity breathing maneuvers and controlled exhalation flow rates, which can be difficult to perform, especially for young children and individuals with compromised lung function. In addition, recent theoretical and experimental studies demonstrate that gas-phase axial diffusion of NO has a significant impact on the exhaled NO signal. We have developed a new technique to characterize airway NO, which requires a series of progressively increasing breath-hold times followed by exhalation of only the airway compartment. Using our new technique, we determined values (means +/- SE) in healthy adults (20-38 yr, n = 8) for the airway diffusing capacity [4.5 +/- 1.6 pl.s(-1).parts per billion (ppb)(-1)], the airway wall concentration (1,340 +/- 213 ppb), and the maximum airway wall flux (4,350 +/- 811 pl/s). The new technique is simple to perform, and application of this data to simpler models with cylindrical airways and no axial diffusion yields parameters consistent with previous methods. Inclusion of axial diffusion as well as an anatomically correct trumpet-shaped airway geometry results in significant loss of NO from the airways to the alveolar region, profoundly impacting airway NO characterization. In particular, the airway wall concentration is more than an order of magnitude larger than previous estimates in healthy adults and may approach concentrations (approximately 5 nM) that can influence physiological processes such as smooth muscle tone in disease states such as asthma.     

A dynamic model of nitric oxide inhibition of mitochondrial cytochrome c oxidase

Nitric oxide can inhibit mitochondrial cytochrome oxidase in both oxygen competitive and uncompetitive modes. A previous model described these interactions assuming equilibrium binding to the reduced and oxidised enzyme respectively (Mason, et al. Proc. Natl. Acad. Sci. U S A 103 (2006) 708-713). Here we demonstrate that the equilibrium assumption is inappropriate as it requires unfeasibly high association constants for NO to the oxidised enzyme. Instead we develop a model which explicitly includes NO binding and its enzyme-bound conversion to nitrite. Removal of the nitrite complex requires electron transfer to the binuclear centre from haem a. This revised model fits the inhibition constants at any value of substrate concentration (ferrocytochrome c or oxygen). It predicts that the inhibited steady state should be a mixture of the reduced haem nitrosyl complex and the oxidized-nitrite complex. Unlike the previous model, binding to the oxidase is always proportional to the degree of inhibition of oxygen consumption. The model is consistent with data and models from a recent paper suggesting that the primary effect of NO binding to the oxidised enzyme is to convert NO to nitrite, rather than to inhibit enzyme activity (Antunes et al. Antioxid. Redox Signal. 9 (2007) 1569-1579).     

Interplay between ferritin metabolism, reactive oxygen species and nitric oxide

 The biological relevance of each of the three inorganic species – iron, oxygen, and nitric oxide (NO) – is crucial. Moreover, their metabolic pathways cross each other and thus create a complex network of connections responsible for the regulation of many essential biological processes. The iron storage protein ferritin, one of the main regulators of iron homeostasis, influences oxygen and NO metabolism. Here, examples are given of the biological interactions of the ferritin molecule (ferritin iron and ferritin shell) with reactive oxygen species (ROS) and NO. The focus is the regulation of ferritin expression by ROS and NO. From these data, ferritin emerges as an important cytoprotective component of the cellular response to ROS and NO. Also, by its ability to alter the amount of intracellular "free" iron, ferritin may affect the metabolism of ROS and NO. It is proposed that this putative activity of ferritin may constitute a missing link in the regulatory loop between iron, ROS, and NO. Received: 2 January 1997 / Accepted: 9 June 1997     

Modeling the growth and proteinase A production in continuous cultures of recombinant Saccharomyces cerevisiae

Overexpression of the homologous protein proteinase A (PrA) in Saccharomyces cerevisiae has been achieved by inserting the PrA gene (PEP4) with its own promoter on a 2mu multicopy plasmid. With this system the specific PrA production rate was found to be described well by a linear function of the oxidative glucose metabolism, the reductive glucose metabolism, and the oxidative ethanol metabolism, with a significant lower yield resulting from the reductive glucose metabolism compared with the oxidative glucose metabolism. To describe the experimental data, a simple mathematical model has been set up. The model is based on an assumption of a limited respiratory capacity as suggested by Sonnleitner and K?ppeli but extended to describe production of an extracellular protein. The model predicts correctly the critical dilution rate to be between 0.15 and 0.16 h(-1), the decrease in the biomass yield above the critical dilution rate, and the production of proteinase A at different dilution rates. Both the experimental data and model simulations suggest that the optimum operating conditions for protein production is just at the critical dilution rate. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 447-454, 1997.     

Nitric oxide-forming reaction between the iron-N-methyl-D-glucamine dithiocarbamate complex and nitrite

The objective of this study was to elucidate the origin of the nitric oxide-forming reactions from nitrite in the presence of the iron-N-methyl-D-glucamine dithiocarbamate complex ((MGD)(2)Fe(2+)). The (MGD)(2)Fe(2+) complex is commonly used in electron paramagnetic resonance (EPR) spectroscopic detection of NO both in vivo and in vitro. Although it is widely believed that only NO can react with (MGD)(2)Fe(2+) complex to form the (MGD)(2)Fe(2+).NO complex, a recent article reported that the (MGD)(2)Fe(2+) complex can react not only with NO, but also with nitrite to produce the characteristic triplet EPR signal of (MGD)(2)Fe(2+).NO (Hiramoto, K., Tomiyama, S., and Kikugawa, K. (1997) Free Radical Res. 27, 505-509). However, no detailed reaction mechanisms were given. Alternatively, nitrite is considered to be a spontaneous NO donor, especially at acidic pH values (Samouilov, A., Kuppusamy, P., and Zweier, J. L. (1998) Arch Biochem. Biophys. 357, 1-7). However, its production of nitric oxide at physiological pH is unclear. In this report, we demonstrate that the (MGD)(2)Fe(2+) complex and nitrite reacted to form NO as follows: 1) (MGD)(2)Fe(2).NO complex was produced at pH 7.4; 2) concomitantly, the (MGD)(3)Fe(3+) complex, which is the oxidized form of (MGD)(2)Fe(2+), was formed; 3) the rate of formation of the (MGD)(2)Fe(2+).NO complex was a function of the concentration of [Fe(2+)](2), [MGD], [H(+)] and [nitrite].     

Simulation of citric acid production by rotating disk contactor

A simple model was presented to describe the time courses of citric acid production by a rotating disc contactor (RDC) using Aspergillus niger. The model is expressed by Monod-type cell growth, Luedeking-Piret-type citric acid production rate equations, and the diffusion equation for oxygen in the biofilm. The model contains five parameters which were determined by the nonlinear least squares method by fitting the numerical solution to the experimental data. In solving the equations, the cell density of the biofilm was estimated from the value of cellular mass per unit of biofilm area using an empirical equation. The experimental time courses in citric acid production period were well simulated with this model. The relation between the specific biofilm surface area and the rate of citric acid production was also explained by the simulation using the average values of five parameters of twelve runs. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 689-696, 1997.     

Theoretical analysis of biochemical pathways of nitric oxide release from vascular endothelial cells

Vascular endothelium expressing endothelial nitric oxide synthase (eNOS) produces nitric oxide (NO), which has a number of important physiological functions in the microvasculature. The rate of NO production by the endothelium is a critical determinant of NO distribution in the vascular wall. We have analyzed the biochemical pathways of NO synthesis and formulated a model to estimate NO production by the microvascular endothelium under physiological conditions. The model quantifies the NO produced by eNOS based on the kinetics of NO synthesis and the availability of eNOS and its intracellular substrates. The predicted NO production from microvessels was in the range of 0.005-0.1 microM/s. This range of predicted values is in agreement with some experimental values but is much lower than other rates previously measured or estimated from experimental data with the help of mathematical modeling. Paradoxical discrepancies between the model predictions and previously reported results based on experimental measurements of NO concentration in the vicinity of the arteriolar wall suggest that NO can also be released through eNOS-independent mechanisms, such as catalysis by neuronal NOS (nNOS). We also used our model to test the sensitivity of NO production to substrate availability, eNOS concentration, and potential rate-limiting factors. The results indicated that the predicted low level of NO production can be attributed primarily to a low expression of eNOS in the microvascular endothelial cells.     

Requirement of argininosuccinate lyase for systemic nitric oxide production

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases.     

Dinuclear copper(II) complex as nitric oxide scavenger in a stimulated murine macrophage model

Nitric oxide is a gaseous, short-living free radical which behaves as an important signaling molecule with pleiotropic capacities including vasodilatation, neurotransmission, and microbial and tumor cell killing, as well as in tissue damage and organ-specific autoimmune disorders. Here, a synthesized, dinuclear copper complex system in vitro obtained by the simple aza-phenolic ligand 2,6-bis[[bis-(2-aminoethyl)amino]methyl]phenol (L) and Cu(II) ion has been used. The stability constants of ligand L with Cu(II) ion were determined through potentiometric measurements in aqueous solution (37.1 +/- 0.1 degrees C, I = 0.15 M of NaCl) to mimic the biological medium. The measurements demonstrated that [Cu(2)H(-1)L(OH)](2+) (DCu) is the predominant species present in solution at pH 7.4. The molecular structure of the ligand in this species permits the cooperation of the two copper ions in assembling the substrate, thus the complex can be used as a receptor for small molecules such as NO. As a biological model, we chose the production of NO catalyzed by inducible nitric oxide synthase obtained from RAW 264.7 murine macrophage cell line stimulated with LPS, which enabled us to prove that NO is coordinated by the DCu complex, modifying its EPR spectra. The coordination of NO with DCu reduces the level of nitrite in the culture medium of stimulated RAW 264.7 macrophages without any inhibition in the expression of iNOS.     

Modeling pulmonary nitric oxide exchange.

Nitric oxide (NO) was first detected in the exhaled breath more than a decade ago and has since been investigated as a noninvasive means of assessing lung inflammation. Exhaled NO arises from the airway and alveolar compartments, and new analytical methods have been developed to characterize these sources. A simple two-compartment model can adequately represent many of the observed experimental observations of exhaled concentration, including the marked dependence on exhalation flow rate. The model characterizes NO exchange by using three flow-independent exchange parameters. Two of the parameters describe the airway compartment (airway NO diffusing capacity and either the maximum airway wall NO flux or the airway wall NO concentration), and the third parameter describes the alveolar region (steady-state alveolar NO concentration). A potential advantage of the two-compartment model is the ability to partition exhaled NO into an airway and alveolar source and thus improve the specificity of detecting altered NO exchange dynamics that differentially impact these regions of the lungs. Several analytical techniques have been developed to estimate the flow-independent parameters in both health and disease. Future studies will focus on improving our fundamental understanding of NO exchange dynamics, the analytical techniques used to characterize NO exchange dynamics, as well as the physiological interpretation and the clinical relevance of the flow-independent parameters.     

Regulation of inducible nitric oxide synthase by self-generated NO

A ferric heme-nitric oxide (NO) complex can build up in mouse inducible nitric oxide synthase (iNOS) during NO synthesis from L-arginine. We investigated its formation kinetics, effect on catalytic activity, dependence on solution NO concentration, and effect on enzyme oxygen response (apparent KmO2). Heme-NO complex formation was biphasic and was linked kinetically to an inhibition of electron flux and catalysis in iNOS. Experiments that utilized a superoxide generating system to scavenge NO showed that the magnitude of heme-NO complex formation directly depended on the NO concentration achieved in the reaction solution. However, a minor portion of heme-NO complex (20%) still formed during NO synthesis even when solution NO was completely scavenged. Formation of the intrinsic heme-NO complex, and the heme-NO complex related to buildup of solution NO, increased the apparent KmO2 of iNOS by 10- and 4-fold, respectively. Together, the data show heme-NO complex buildup in iNOS is due to both intrinsic NO binding and to equilibrium binding of solution NO, with the latter predominating when NO reaches high nanomolar to low micromolar concentrations. This behavior distinguishes iNOS from the other NOS isoforms and indicates a more complex regulation is possible for its activity and oxygen response in biologic settings.     

Kinetics of NO3(-) net fluxes in Pinus pinaster, Rhizopogon roseolus and their ectomycorrhizal association, as affected by the presence of NO3(-) and NH4+

The impact of mineral N supply, N-free or NO3(-) with or without NH4+, on the subsequent uptake of NO3(-) by maritime pine seedlings associated with the ectomycorrhizal fungus Rhizopogon roseolus was studied using ion-selective microelectrodes. NO3(-) net fluxes into N-starved non-mycorrhizal short roots (NMSRs) were low and measurable only over the NO3(-) concentration range of 0-70 microM. The simple kinetics observed in those roots may reflect the dominant operation of a high-affinity NO3(-) transport system (HATS) which is constitutive. NO3(-) pretreatment increased the NO3(-) net fluxes and led to a complex kinetics that may reflect the operation of other HATS. A simple kinetics was observed in plants pre-incubated at high NH4+ concentration. In contrast, NO3(-) uptake kinetics presented only one saturation phase in the fungus, whether associated with the plant or not. NO3(-) uptake was greater after a pretreatment in N-free or NO3 (-) solution, but NH4+ pretreatment led to a threefold reduction in NO3 (-) uptake. These results suggest that the regulation of NO3(-) transport systems varies between the host and the fungal partner. This variation is likely to contribute to the positive effect of mycorrhizal association on N uptake in plants when the N supply is low and fluctuating.     

Analysis of the effects of nitric oxide and oxygen on nitric oxide production by macrophages

The interactions between NO and O(2) in activated macrophages were analysed by incorporating previous cell culture and enzyme kinetic results into a novel reaction-diffusion model for plate cultures. The kinetic factors considered were: (i) the effect of O(2) on NO production by inducible NO synthase (iNOS); (ii) the effect of NO on NO synthesis by iNOS; (iii) the effect of NO on respiratory and other O(2) consumption; and (iv) the effects of NO and O(2) on NO consumption by a possible NO dioxygenase (NOD). Published data obtained by varying the liquid depth in macrophage cultures provided a revealing test of the model, because varying the depth should perturb both the O(2) and the NO concentrations at the level of the cells. The model predicted that the rate of NO(2)(-) production should be nearly constant, and that the net rate of NO production should decline sharply with increases in liquid depth, in excellent agreement with the experimental findings. In further agreement with available results for macrophage cultures, the model predicted that net NO synthesis should be more sensitive to liquid depth than to the O(2) concentration in the headspace. The main reason for the decrease in NO production with increasing liquid depth was the modulation of NO synthesis by NO, with O(2) availability playing only a minor role. The model suggests that it is the ability of iNOS to consume NO, as well as to synthesize it, that creates very sensitive feedback control, setting an upper bound on the NO concentration of approximately 1 microM. The effect of NO consumption by other possible pathways (e.g., NOD) would be similar to that of iNOS, in that it would help limit net NO production. The O(2) utilized during enzymatic NO consumption is predicted to make the O(2) demands of activated macrophages much larger than those of unactivated ones (where iNOS is absent); this remains to be tested experimentally.     

Nitric oxide red blood cell membrane permeability at high and low oxygen tension.

Red blood cell (RBC) encapsulated hemoglobin in the blood scavenges nitric oxide (NO) much more slowly than cell-free hemoglobin would. Part of this reduced NO scavenging has been attributed to an intrinsic membrane barrier to diffusion of NO through the RBC membrane. Published values for the permeability of RBCs to NO vary over several orders of magnitude. Recently, the rate that RBCs scavenge NO has been shown to depend on the hematocrit (percentage volume of RBCs) and oxygen tension. The difference in rate constants was hypothesized to be due to oxygen modulation of the RBC membrane permeability, but also could have been due to the difference in bimolecular rate constants for the reaction of NO and oxygenated vs deoxygenated hemoglobin. Here, we model NO scavenging by RBCs under previously published experimental conditions. A finite-element based computer program model is constrained by published values for the reaction rates of NO with oxygenated and deoxygenated hemoglobin as well as RBC NO scavenging rates. We find that the permeability of RBCs to NO under oxygenated conditions is between 4400 and 5100 microm s(-1) while the permeability under deoxygenated conditions is greater than 64,000 microm s(-1). The permeability changes by a factor of 10 or more upon oxygenation of anoxic RBCs. These results may have important implications with respect to NO import or export in physiology.     

Probing subtle coordination changes in the iron-quinone complex of photosystem II during charge separation,by the use of NO

The terminal electron acceptor of Photosystem II, PSII, is a linear complex consisting of a primary quinone, a non-heme iron(II), and a secondary quinone, Q(A)Fe(2+)Q(B). The complex is a sensitive site of PSII, where electron transfer is modulated by environmental factors and notably by bicarbonate. Earlier studies showed that NO and other small molecules (CN(-), F(-), carboxylate anions) bind reversibly on the non-heme iron in competition with bicarbonate. In the present study, we report on an unusual new mode of transient binding of NO, which is favored in the light-reduced state (Q(A)(-)Fe(2+)Q(B)) of the complex. The related observations are summarized as follows: (i) Incubation with NO at -30 degrees C, following light-induced charge separation, results in the evolution of a new EPR signal at g = 2.016. The signal correlates with the reduced state Q(A)(-)Fe(2+) of the iron-quinone complex. (ii) Cyanide, at low concentrations, converts the signal to a more rhombic form with g values at 2.027 (peak) and 1.976 (valley), while at high concentrations it inhibits formation of the signals. (iii) Electron spin-echo envelope modulation (ESEEM) experiments show the existence of two protein (14)N nuclei coupled to electron spin. These two nitrogens have been detected consistently in the environment of the semiquinone Q(A)(-) in a number of PSII preparations. (iv) NO does not directly contribute to the signals, as indicated by the absence of a detectable isotopic effect ((15)NO vs (14)NO) in cw EPR. (v) A third signal with g values (2.05, 2.03, 2.01) identical to those of an Fe(NO)(2)(imidazole) synthetic complex develops slowly in the dark, or faster following illumination. (vi) In comparison with the untreated Q(A)(-)Fe(2+) complex, the present signals not only are confined to a narrow spectral region but also saturate at low microwave power. At 11 K the g = 2.016 signal saturates with a P(1/2) of 110 microW and the g = 2.027/1.976 signal with a P(1/2) of 10 microW. (vii) The spectral shape and spin concentration of these signals is successfully reproduced, assuming a weak magnetic interaction (J values in the range 0.025-0.05 cm(-)(1)) between an iron-NO complex with total spin of (1)/(2) and the spin, (1)/(2), of the semiquinone, Q(A)(-). The different modes of binding of NO to the non-heme iron are examined in the context of a molecular model. An important aspect of the model is a trans influence of Q(A) reduction on the bicarbonate ligation to the iron, transmitted via H-bonding of Q(A) with an imidazole ligand to the iron.