Activation of electroneutral K flux in Amphiuma red blood cells by N- ethylmaleimide. Distinction between K/H exchange and KCl cotransport

Exposure of Amphiuma red blood cells to millimolar concentrations of N-ethylmaleimide (NEM) resulted in net K loss. In order to determine whether net K loss was conductive or was by electroneutral K/H exchange or KCl cotransport, studies were performed evaluating K flux in terms of the thermodynamic forces to which K flux by the above pathways should couple. The direction and magnitude of the NEM-induced net K flux did not correspond with the direction and magnitude of the forces relevant to K conductance or electroneutral KCl cotransport. Both the magnitude and direction of the NEM-activated K flux responded to the driving force for K/H exchange. We therefore conclude that NEM-induced K loss, like that by osmotically swollen Amphiuma red blood cells, is by an electroneutral K/H exchanger. In addition to the above studies, we evaluated the kinetic behavior of the volume- and NEM-induced K/H exchange flux pathways in media where Cl was replaced by SCN, NO3, para-aminohippurate (PAH), or gluconate. The anion replacement studies did not permit a distinction between K/H exchange and KCl cotransport, since, depending upon the anion used as a Cl replacement, partial inhibition or stimulation of volume-activated K/H exchange fluxes was observed. In contrast, all anions used were stimulatory to the NEM-induced K loss. Since, on the basis of force-flow analysis, both volume-and NEM-induced K loss are K/H exchange, it was necessary to reevaluate assumptions (i.e., anions serve as substrates and therefore probe the translocation step) associated with the use of anion replacement as a means of flux route identification. When viewed together with the force-flow studies, the Cl replacement studies suggest that anion effects upon K/H exchange are indirect. The different anions appear to alter mechanisms that couple NEM exposure and cell swelling to the activation of K/H exchange, as opposed to exerting direct effects upon K and H translocation.     

p-Chloromercuribenzenesulfonic acid stimulation of chloride-dependent sodium and potassium transport in human red blood cells

The organic mercurial p-chloromercuribenzensulfonic acid (PCMBS) reversibly increases fluxes of sodium and potassium across the human red blood cell membrane. We examined the effect of different monovalent anions on cation fluxes stimulated by PCMBS. A substantial portion of the fluxes of both cations was found to have a specific anion requirement for chloride or bromide, and was not observed when chloride was replaced by nitrate, acetate or methylsulfate. The chloride-dependent component of the cation fluxes was only observed when the cells were exposed to PCMBS concentrations of 0.5 mM or greater. Furosemide (1 mM) did not inhibit the PCMBS-stimulated cation fluxes. The observed anion specificity is directly associated with the transport process rather than PCMBS binding to the membrane. A portion of the potassium transport stimulated by PCMBS appears to involve K+-K+ exchange; however, Na+ + K+ cotransport is not stimulated by this sulfhydryl reagent.     

An analytical model of ionic movements in airway epithelial cells.

A new mathematical model of ion movements in airway epithelia is presented, which allows predictions of ion fluxes, membrane potentials and ion concentrations. The model includes sodium and chloride channels in the apical membrane, a Na/K pump and a cotransport system for Cl- with stoichiometry Na+:K+:2Cl- in the basolateral membrane. Potassium channels in the basolateral membrane are used to regulate cell volume. Membrane potentials, ion fluxes and intracellular ion concentration are calculated as functions of apical ion permeabilities, the maximum pump current and the cotransport parameters. The major predictions of the model are: (1) Cl- concentration in the cell is determined entirely by the intracellular concentration of negatively charged impermeable ions and the osmotic conditions; (2) changes in intracellular Na+ and K+ concentrations are inversely related; (3) cotransport provides the major driving force for Cl- flux, increases intracellular Na+ concentration, decreases intracellular K+ concentration and hyperpolarizes the cell interior; (4) the maximum rate of the Na/K pump, by contrast, has little effect on Na+ or Cl- transepithelial fluxes and a much less pronounced effect on cell membrane polarization; (5) an increase in apical Na+ permeability causes an increase in intracellular Na+ concentration and a significant increase in Na+ flux; (6) an increase in apical Cl- permeability decreases intracellular Na+ concentration and Na+ flux; (7) assuming Na+ and Cl- permeabilities equal to those measured in human nasal epithelia, the model predicts that under short circuit conditions, Na+ absorption is much higher than Cl- secretion, in agreement with experimental measurements.     

Anion exchange in oxyntic cell apical membrane: Relationship to thiocyanate inhibition of acid secretion

The effects of SCN- on H+-accumulation by inside-out gastric vesicles derived from the apical membrane of secreting oxyntic cells are reported. SCN- inhibited the formation of pH gradients in Cl- and isethionate media. In Cl-, the concentration of SCN- required to achieve a certain degree of inhibition of H+ uptake (or dissipation of performed gradients) was increased with the increase in Cl- concentration, indicating some competitive phenomena between these anions. Comparison of the rates of dissipation of similar pH gradients achieved in Cl- vs. isethionate suggested the existence of a fast Cl-/SCN- exchange. In addition, direct isotopic fluxes confirmed the existence of rapid anion exchange and K-salt transport for both Cl- and SCN-. The rates of anion-exchange and K-salt transport were of similar magnitude, and the rates for SCN- in either countertransport against Cl- or cotransport with K+ were twice as fast as the equivalent values for Cl-. These mediated pathways in the apical membrane provide the possible means for rapid access of SCN- to the acidic canalicular spaces of the oxyntic cell that is implicit in recent proposals to explain SCN- inhibition of gastric HCl secretion.     

Anion transport systems in the plasma membrane of vertebrate cells

In the case of the red blood cell, anion transport is a highly specific one-for-one exchange catalyzed by a major membrane protein known as band 3 or as capnophorin. This red cell anion-exchange system mediates the Cl-(-)HCO3- exchange responsible for most of the bicarbonate transport capacity of the blood. The rapidly expanding knowledge of the molecular biology and the transport kinetics of this specialized transport system is very briefly reviewed in Section III. Exchange diffusion mechanisms for anions are found in many cells other than erythrocytes. The exchange diffusion system in Ehrlich cells has several similarities to that in red cells. In several cell types (subsection IV-B), there is evidence that intracellular pH regulation depends on Cl-(-)HCO3- exchange processes. Anion exchange in other single cells is described in Section IV, and its role in pH regulation is described in Section VII. Anion exchange mechanism operating in parallel with, and only functionally linked to Na+-H+ or K+-H+ exchange mechanisms can also play a role in cell volume regulation as described in Section VII. In the Ehrlich ascites cell and other vertebrate cells, electroneutral anion transfer has been found to occur also by a cotransport system for cations and chloride operating in parallel with the exchange diffusion system. The cotransport system is capable of mediating secondary active chloride influx. In avian red cells, the cotransport system has been shown to be activated by adrenergic agonists and by cyclic AMP, suggesting that the cotransport is involved in regulatory processes (see subsection V-A.). In several cell types, cotransport systems are activated and play a role during volume regulation, as described in Section V and in Section VII. It is also likely that this secondary active cotransport of chloride plays a significant role for the apparently active extrusion of acid equivalents from certain cells. If a continuous influx of chloride against an electrochemical gradient is maintained by a cotransport system, the chloride disequilibrium can drive an influx of bicarbonate through the anion exchange mechanism, as described in Section VII. Finally, even the electrodiffusion of anions is shown to be regulated, and in Ehrlich cells and human lymphocytes an activation of the anion diffusion pathway plays a major role in cell volume regulation as described in Section VI and subsection VII-B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular hypertonicity is responsible for water flux associated with Na+/glucose cotransport

Detection of a significant transmembrane water flux immediately after cotransporter stimulation is the experimental basis for the controversial hypothesis of secondary active water transport involving a proposed stoichiometry for the human Na(+)/glucose cotransporter (SGLT1) of two Na(+), one glucose, and 264 water molecules. Volumetric measurements of Xenopus laevis oocytes coexpressing human SGLT1 and aquaporin can be used to detect osmotic gradients with high sensitivity. Adding 2 mM of the substrate alpha-methyl-glucose (alphaMG) created mild extracellular hypertonicity and generated a large cotransport current with minimal cell volume changes. After 20, 40, and 60 s of cotransport, the return to sugar-free, isotonic conditions was accompanied by measurable cell swelling averaging 0.051, 0.061, and 0.077 nl/s, respectively. These water fluxes are consistent with internal hypertonicities of 1.5, 1.7, and 2.2 mOsm for these cotransport periods. In the absence of aquaporin, the measured hypertonicites were 4.6, 5.0, and 5.3 mOsm for the same cotransport periods Cotransport-dependent water fluxes, previously assumed to be water cotransport, could be largely explained by hypertonicities of such amplitudes. Using intracellular Na(+) injection and Na(+)-selective electrode, the intracellular diffusion coefficient for Na(+) was estimated at 0.29 +/- 0.03 x 10(-5) cm(2) s(-1). Using the effect of intracellular alphaMG injection on the SGLT1-mediated outward current, the intracellular diffusion coefficient of alphaMG was estimated at 0.15 +/- 0.01 x 10(-5) cm(2) s(-1). Although these intracellular diffusion coefficients are much lower than in free aqueous solution, a diffusion model for a single solute in an oocyte would require a diffusion coefficient three times lower than estimated to explain the local osmolyte accumulation that was experimentally detected. This suggests that either the diffusion coefficients were overestimated, possibly due to the presence of convection, or the diffusion in cytosol of an oocyte is more complex than depicted by a simple model.     

Human placental brush-border membrane Na(+)-biotin cotransport.

Membrane transport pathways for transplacental transfer of the water-soluble vitamin biotin were investigated by assessing the possible presence of a Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells. The presence of Na(+)-biotin cotransport was determined from radiolabeled tracer flux measurements of biotin uptake using preparations of purified brush-border membrane vesicles. The imposition of an inwardly directed Na+ gradient stimulated vesicle uptake of biotin to levels approximately 25-fold greater than those observed at equilibrium. The voltage sensitivity of Na+ gradient-driven biotin uptake suggested Na(+)-biotin cotransport is electrogenic occurring with net transfer of positive charge. A kinetic analysis of the activation of biotin uptake by increasing Na+ was most consistent with an interaction of Na+ at 2 sites in the transport protein. Static head determinations used to identify the magnitude of opposing driving forces bringing flux through the cotransport mechanism to equilibrium indicated a coupling ratio of 2 Na+ per biotin. Substrate specificity studies using chemical analogues of biotin suggested both the terminal carboxylic acid of the valeric acid side chain and a second nucleus of anionic charge were important determinants for substrate interaction with the cotransport protein. Initial rate determinations of biotin uptake indicate biotin interacts with a single saturable site (Km = 21 microM) with a maximal transport rate of 4.5 nmol/mg/min. The results of this study provide evidence for an electrogenic Na(+)-biotin cotransport mechanism in the maternal-facing membrane of human placental epithelial cells.     

Calculations of K+, Na+, and Cl? fluxes across cell membrane with Na+/K+ pump, NKCC, NC cotransport and ionic channels with non-Goldman rectification in K+-channels: Normal and apoptotic cells

The balance of K⁺, Na⁺, and Cl⁻ fluxes across the cell membrane with the Na⁺/K⁺ pump, ion channels, and Na⁺K⁺2Cl⁻ (NKCC) and Na⁺-Cl⁻ (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K⁺, Na⁺, and Cl⁻ fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K⁺, Na⁺, and Cl⁻ concentrations in cell water; total Cl⁻ flux; total K⁺ influx; and the ouabain-inhibited pump component of the Rb⁺(K⁺) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na⁺ channels, whereas K⁺ and Cl⁻ channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K⁺, Na⁺, and Cl⁻ account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl⁻, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.     

Reconstruction and flux analysis of coupling between metabolic pathways of astrocytes and neurons: application to cerebral hypoxia

Background

It is a daunting task to identify all the metabolic pathways of brain energy metabolism and develop a dynamic simulation environment that will cover a time scale ranging from seconds to hours. To simplify this task and make it more practicable, we undertook stoichiometric modeling of brain energy metabolism with the major aim of including the main interacting pathways in and between astrocytes and neurons.

Model

The constructed model includes central metabolism (glycolysis, pentose phosphate pathway, TCA cycle), lipid metabolism, reactive oxygen species (ROS) detoxification, amino acid metabolism (synthesis and catabolism), the well-known glutamate-glutamine cycle, other coupling reactions between astrocytes and neurons, and neurotransmitter metabolism. This is, to our knowledge, the most comprehensive attempt at stoichiometric modeling of brain metabolism to date in terms of its coverage of a wide range of metabolic pathways. We then attempted to model the basal physiological behaviour and hypoxic behaviour of the brain cells where astrocytes and neurons are tightly coupled.

Results

The reconstructed stoichiometric reaction model included 217 reactions (184 internal, 33 exchange) and 216 metabolites (183 internal, 33 external) distributed in and between astrocytes and neurons. Flux balance analysis (FBA) techniques were applied to the reconstructed model to elucidate the underlying cellular principles of neuron-astrocyte coupling. Simulation of resting conditions under the constraints of maximization of glutamate/glutamine/GABA cycle fluxes between the two cell types with subsequent minimization of Euclidean norm of fluxes resulted in a flux distribution in accordance with literature-based findings. As a further validation of our model, the effect of oxygen deprivation (hypoxia) on fluxes was simulated using an FBA-derivative approach, known as minimization of metabolic adjustment (MOMA). The results show the power of the constructed model to simulate disease behaviour on the flux level, and its potential to analyze cellular metabolic behaviour in silico.

Conclusion

The predictive power of the constructed model for the key flux distributions, especially central carbon metabolism and glutamate-glutamine cycle fluxes, and its application to hypoxia is promising. The resultant acceptable predictions strengthen the power of such stoichiometric models in the analysis of mammalian cell metabolism.     

Glucose Accumulation Can Account for the Initial Water Flux Triggered by Na+/Glucose Cotransport

Over the last decade, several cotransport studies have led to the proposal of secondary active transport of water, challenging the dogma that all water transport is passive. The major observation leading to this interpretation was that a Na⁺ influx failed to reproduce the large and rapid cell swelling induced by Na⁺/solute cotransport. We have investigated this phenomenon by comparing a Na⁺/glucose (hSGLT1) induced water flux to water fluxes triggered either by a cationic inward current (using ROMK2 K⁺ channels) or by a glucose influx (using GLUT2, a passive glucose transporter). These proteins were overexpressed in Xenopus oocytes and assayed through volumetric measurements combined with double-electrode electrophysiology or radioactive uptake measurements. The osmotic gradients driving the observed water fluxes were estimated by comparison with the swelling induced by osmotic shocks of known amplitude. We found that, for equivalent cation or glucose uptakes, the combination of substrate accumulations observed with ROMK2 and GLUT2 are sufficient to provide the osmotic gradient necessary to account for a passive water flux through SGLT1. Despite the fact that the Na⁺/glucose stoichiometry of SGLT1 is 2:1, glucose accumulation accounts for two-thirds of the osmotic gradient responsible for the water flux observed at t = 30 s. It is concluded that the different accumulation processes for neutral versus charged solutes can quantitatively account for the fast water flux associated with Na⁺/glucose cotransport activation without having to propose the presence of secondary active water transport.     

Metabolic flux distributions in Penicillium chrysogenum during fed-batch cultivations

Based on a review of the Penicillium chrysogenum biochemistry a stoichiometric model has been set up. The model considers 61 internal fluxes and there are 49 intracellular metabolites which are assumed to be in pseudo-steady state. In addition to the intracellular fluxes the model considers the uptake of 21 amino acids. From the stoichiometric model the maximum theoretical yield of penicillin V is calculated to 0.43 mol/mol glucose. If biosynthesis of cysteine is by direct sulfhydrylation rather than by transsulfuration, the maximum theoretical yield is about 20% higher, i.e., 0.50 mol/mol glucose. The theoretical yield decreases substantially if alpha-aminoadipate is converted to 6-oxo-piperidine-2-carboxylic acid (OPC). If only 40% of the alpha-aminoadipate is recycled, the maximum theoretical yield is 0.31 mol/mol glucose. The uptake rates of glucose, lactate, gamma-aminobutyrate, and 21 amino acids were measured during fed-batch cultivations. The rates of formation of penicillin V, delta-(L-alpha)-aminoadipyl-L-cysteinyl-D-valine (ACV), OPC, and the pool of isopenicillin N, 6-APA, and 8-HPA were also measured. Finally the synthesis rates of the biomass constituents RNA/DNA, protein, lipid, carbohydrate, and amino carbohydrate were measured. From these measured rates and the stoichiometric model the metabolic fluxes through the different intracellular pathways are calculated. The calculations show that penicillin formation is accompanied by a large flux through the pentose phosphate (PP) pathway due to a large requirement for nicotinamide-adenine dinucleotide phosphate (NADPH) used in the biosynthesis of cysteine. If cysteine is added to the medium, the flux through the PP pathway decreases. From the stoichiometric model Y(xATP) is calculated to 87 mmol adenosine triphosphate (ATP)/g dry weight (DW), and from the flux calculations m(ATP) is found to 3 mmol ATP/g DW/h. (c) 1995 John Wiley & Sons, Inc.     

Self-inhibition of chloride transport in Ehrlich ascites tumor cells

Previous studies have shown that mediated Cl- transport which occurs by at least two processes (Cl- -dependent cation cotransport and Cl- self-exchange) becomes progressively inhibited when extracellular Cl- exceeds about 60 mM (Hoffmann et al., 1979). To account for this type of kinetic behavior, that is, self-inhibition, an anion transport system possessing two sites, a high affinity transport site and a lower affinity modifier site is suggested (Dalmark, 1976). In the present experiments we have attempted to determine which of the mediated transport pathways is susceptible to self-inhibition by studying the dependence of the steady state Cl- flux on the extracellular Cl- concentration and how DIDS, an inhibitor of Cl- self-exchange, and H + affect this relationship. Addition of DIDS to Ehrlich cells results in inhibition of Cl- transport at every Cl- concentration tested (40-150 mM). Moreover, the Cl- flux/Cl- concentration relationship no longer exhibits self-inhibition, suggesting that this phenomenon is a characteristic of the Cl- self-exchanger rather than of the Cl- -dependent cation cotransport system. Lowering the extracellular pH (pHo) from 7.35 to 5.30 stimulates Cl- transport by a process that saturates with respect to [H +]. Half-maximal stimulation occurs at pHo 6.34. A comparison of the kinetic parameters, Ks and Jmax, calculated from the ascending limb of the Cl- flux/Cl- concentration curve at pHo 7.30 to those at pHo 5.50 show that the values for Ks are almost identical (23.6 mM and 21.3 mM, respectively), while the values for Jmax [22.2 mEq/Kg dry wt) X min] differ by only 15%. This finding along with the observation that DIDS completely blocks H + stimulation of Cl- transport is compatible with the suggestion that H + interact at the modifer site of the Cl- self-exchanger and thereby prevents self-inhibition.     

Identification and reconstitution of a Na+/K+/Cl- cotransporter and K+ channel from luminal membranes of renal red outer medulla

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na+/K+/Cl- cotransport system and a K+ channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na+/K+/Cl- cotransporter and K+ channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of 86Rb+ uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two 86Rb+ fluxes. (A) A furosemide-inhibited 86Rb+ flux in the absence of Na+ (K+-K+ exchange). This flux is stimulated by an inward Na+ gradient (Na+/K+ cotransport) and is inhibited also by bumetanide. (B) A Ba2+-inhibited 86Rb+ flux, through the K+ channel. Luminal membranes containing the Na+/K+/Cl- cotransporter and K+ channels, and basolateral membranes containing the Na+/K+ pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na+/K+/Cl- cotransporter and K+ channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.     

Dynamic flux cartography of hairy roots primary metabolism

A dynamic model for plant cell and hairy root primary metabolism is presented. The model includes nutrient uptake (Pi, sugars, nitrogen sources), the glycolysis and pentose phosphate pathways, the TCA cycle, amino acid biosynthesis, respiratory chain, biosynthesis of cell building blocks (structural hexoses, organic acids, lipids, and organic phosphated molecules). The energy shuttles (ATP, ADP) and cofactors (NAD/H, NADP/H) are also included. The model describes the kinetics of 44 biochemical reactions (fluxes) of the primary metabolism of plant cells and includes 41 biochemical species (metabolites, nutrients, biomass components). Multiple Michaelis-Menten type kinetics are used to describe biochemical reaction rates. Known regulatory phenomena on metabolic pathways are included using sigmoid switch functions. A visualization framework showing fluxes and metabolite concentrations over time is presented. The visualization of fluxes and metabolites is used to analyze simulation results from Catharanthus roseus hairy root 50 d batch cultures. The visualization of the metabolic system allows analyzing split ratios between pathways and flux time-variations. For carbon metabolism, the cells were observed to have relatively high and stable fluxes for the central carbon metabolism and low and variable fluxes for anabolic pathways. For phosphate metabolism, a very high free intracellular Pi turnover rate was observed with higher flux variations than for the carbon metabolism. Nitrogen metabolism also exhibited large flux variations. The potential uses of the model are also discussed.     

Application of metabolic flux analysis for the identification of metabolic bottlenecks in the biosynthesis of penicillin-G

A detailed stoichiometric model was developed for growth and penicillin-G production in Penicillium chrysogenum. From an a priori metabolic flux analysis using this model it appeared that penicillin production requires significant changes in fluxes through the primary metabolic pathways. This is brought about by the biosynthesis of carbon precursors for the beta-lactan nucleus and an increased demand for NADPH, mainly for sulfate reduction. As a result, significant changes in flux partitioning occur around four principal nodes in primary metabolism. These are located at: (1) glucose-6-phosphate; (2) 3-phosphoglycerate; (3) mitochondrial pyruvate; and (4) mitochondrial isocitrate. These nodes should be regarded as potential bottlenecks for increased productivity. The flexibility of these principal nodes was investigated by experimental manipulation of the fluxes through the central metabolic pathways using a high-producing strain of P. chrysogenum. Metabolic fluxes were manipulated through growth of the cells on different substrates in carbon-limited chemostat culture. Metabolic flux analysis, based on measured input and output fluxes, was used to calculate the fluxes around the principal nodes. It was found that, for growth on glucose, ethanol, and acetate, the flux partitioning around these nodes differed significantly. However, this had hardly any effect on penicillin productivity, showing that primary carbon metabolism is not likely to contain potential bottlenecks. Further experiments were performed to manipulate the total metabolic demand for the cofactor nicotinamide adenine dinucleotide phosphate (NADPH). NADPH demand was increased stepwise by cultivating the cells on glucose or xylose as the carbon source combined with either ammonia or nitrate as the nitrogen source, which resulted in a stepwise decrease of penicillin production. This clearly shows that, in penicillin fermentation, possible limitations in primary metabolism reside in the supply/regeneration of cofactors (NADPH) rather than in the supply of carbon precursors.     

Sodium-dependent lysine flux across bullfrog alveolar epithelium

Amino acid transport across the alveolar epithelial barrier was studied by measuring radiolabeled lysine fluxes across bullfrog lungs in an Ussing chamber. In the absence of a transmural electrical gradient, L-[14C]lysine was instilled into the upstream reservoir and the rate of appearance of the radiolabel in the downstream reservoir was determined. Two lungs from the same animal were used simultaneously to determine tracer fluxes both into and out of the alveolar bath. Results showed that the radiolabel flux measured in the alveolar to the pleural direction was greater than that measured in the opposite direction in the presence of sodium in the bathing fluids. The net flux of L-[14C]lysine was saturable with [Na+], with an apparent transport coefficient (Kt) of 28 mM for Na+. Hill analysis of [14C]lysine flux vs. [Na+] indicated a coupling ratio of 1:1 between sodium and radiolabeled L-lysine. Total L-lysine flux as a function of [L-lysine] was also saturable, with Kt of 7.3 mM for L-lysine. Ouabain significantly decreased absorptive (alveolar-to-pleural) radiolabel flux, while slightly increasing the flux observed in the opposite direction. L-leucine completely inhibited absorptive net flux of L-[14C]lysine. alpha-Methylaminoisobutyric acid (MeAIB), on the other hand, only slightly reduced net flux of L-[14C]lysine from the control value. The presence of a net absorptive, Na+-dependent amino acid flux across the alveolar epithelial barrier indicates that the tissue is capable of removing amino acids and sodium from the alveolar fluid by a coupled cotransport mechanism, which may be important for both protein metabolism and fluid balance by alveolar epithelium.     

Proton and hydrogen currents in photosynthetic water oxidation

The photosynthetic processes that lead to water oxidation involve an evolution in time from photon dynamics to photochemically-driven electron transfer to coupled electron/proton chemistry. The redox-active tyrosine, Y(Z), is the component at which the proton currents necessary for water oxidation are switched on. The thermodynamic and kinetic implications of this function for Y(Z) are discussed. These considerations also provide insight into the related roles of Y(Z) in preserving the high photochemical quantum efficiency in Photosystem II (PSII) and of conserving the highly oxidizing conditions generated by the photochemistry in the PSII reaction center. The oxidation of Y(Z) by P(680)(+) can be described well by a treatment that invokes proton coupling within the context of non-adiabatic electron transfer. The reduction of Y(.)(Z), however, appears to proceed by an adiabatic process that may have hydrogen-atom transfer character.     

Genome-scale modeling and in silico analysis of ethanologenic bacteria Zymomonas mobilis

Bioethanol has been recognized as a potential alternative energy source. Among various ethanol-producing microbes, Zymomonas mobilis has acquired special attention due to its higher ethanol yield and tolerance. However, cellular metabolism in Z. mobilis remains unclear, hindering its practical application for bioethanol production. To elucidate such physiological characteristics, we reconstructed and validated a genome-scale metabolic network (iZM363) of Z. mobilis ATCC31821 (ZM4) based on its annotated genome and biochemical information. The phenotypic behaviors and metabolic states predicted by our genome-scale model were highly consistent with the experimental observations of Z. mobilis ZM4 strain growing on glucose as well as NMR-measured intracellular fluxes of an engineered strain utilizing glucose, fructose, and xylose. Subsequent comparative analysis with Escherichia coli and Saccharomyces cerevisiae as well as gene essentiality and flux coupling analyses have also confirmed the functional role of pdc and adh genes in the ethanologenic activity of Z. mobilis, thus leading to better understanding of this natural ethanol producer. In future, the current model could be employed to identify potential cell engineering targets, thereby enhancing the productivity of ethanol in Z. mobilis.     

Hypolimnetic phosphorus and nitrogen dynamics in a small,eutrophic lake with a seasonally anoxic hypolimnion

In situ estimates of sediment nutrient flux are necessary to understand seasonal variations in internal loading in lakes. We investigated the sources and sinks of nutrients in the hypolimnion of a small (0.33 km²), relatively shallow (18 m max. depth), eutrophic lake (Lake Okaro, New Zealand) in order to determine changes in sediment nutrient fluxes resulting from a whole lake sediment capping trial using a modified zeolite phosphorus inactivation agent (Z2G1). Sediment nutrient fluxes in the hypolimnion were estimated as the residual term in a nutrient budget model that accounted for mineralisation of organic nutrients, nutrient uptake by phytoplankton and mixing, nitrification, adsorption/desorption and diffusion of dissolved nutrients at the thermocline. Of the total hypolimnetic phosphate and ammonium fluxes during one period of seasonal stratification (2007–08), up to 60 and 50%, respectively, were derived from the bottom sediments, 18 and 24% were due to mineralisation of organic species, 36 and 28% were due to phytoplankton uptake and 9 and 6% were from diffusion across the thermocline. Adsorption/desorption of phosphate to suspended solids and nitrification were of minor (<8%) importance to the total fluxes. Any reduction in sediment nutrient release by Z2G1 was small compared with both the total sediment nutrient flux and the sum of other hypolimnetic fluxes. Uneven sediment coverage of Z2G1 may have been responsible for the limited effect of the sediment capping layer formed by Z2G1.     

The use of HgCl2 to evaluate the cosubstrate: amino acid transport stoichiometry in Ehrlich ascites tumor cells

Recent investigations have indicated that cellular rheogenic properties may interfere with the correct estimation of Na+ and amino transport stoichiometry. We have reevaluated the stoichiometry of Na+ and alpha-aminoisobutyric acid (alpha-AIB) cotransport in Ehrlich ascites tumor cells depleted of Na+ and ATP by incubation in Na+-free HEPES-buffered medium (pH 7.2) containing 160 mM K+ and 2.5 microM valinomycin. Transfer of the cells to a medium with 10 mM 22Na+, 10 mM 3H-AIB, and 150 mM K+ resulted in an enhancement of Na+ flux above basal levels, which represents 0.6 of the AIB uptake. Under these conditions the membrane potential, -7.0 +/- 0.1 mV (SEM), does not change with the addition of AIB, -7.3 +/- 0.6 mV (SEM). HgCl2 (10 microM) added to the medium inhibited AIB flux and AIB-stimulated Na+ flux by 45-50% but did not change the coupling ratio. HgCl2 (10 microM) does not inhibit the basal Na+ flux nor does it affect cellular Na+ or K+ content. In physiological medium cotransport is electrogenic. The membrane potential of Ehrlich cells in physiological medium is -22.3 +/- 0.8 mV (SEM) and depolarizes to -16.7 +/- 0.7 mV (SEM) upon addition of AIB. Under these conditions the coupling ratio was highly variable but the ratio of codepression is 0.90 +/- 0.02 (SEM) in the presence of HgCl2 (10 microM). These results are consistent with a model (Smith and Robinson, 1981) in which the stoichiometry is one cosubstrate molecule per molecule of alpha-AIB. We suggest that H+ provides the alternative cosubstrate in this low Na+ environment and that in high Na+ medium the Na+:AIB stoichiometry approaches 1:1.