Role of polyphosphate in regulation of the Streptomyces lividans KcsA channel

We examine the hypotheses that the Streptomyces lividans potassium channel KcsA is gated at neutral pH by the electrochemical potential, and that its selectivity and conductance are governed at the cytoplasmic face by interactions between the KcsA polypeptides and a core molecule of inorganic polyphosphate (polyP). The four polypeptides of KcsA are postulated to surround the end unit of the polyP molecule with a collar of eight arginines, thereby modulating the negative charge of the polyP end unit and increasing its preference for binding monovalent cations. Here we show that KcsA channels can be activated in planar lipid bilayers at pH 7.4 by the chemical potential alone. Moreover, one or both of the C-terminal arginines are replaced with residues of progressively lower basicity-lysine, histidine, valine, asparagine-and the effects of these mutations on conductance and selectivity for K⁺ over Mg²⁺ is tested in planar bilayers as a function of Mg²⁺ concentration and pH. As the basicity of the C-terminal residues decreases, Mg²⁺ block increases, and Mg²⁺ becomes permeant when medium pH is greater than the pI of the C-terminal residues. The results uphold the premise that polyP and the C-terminal arginines are decisive elements in KcsA channel regulation.     

Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method

Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) from the hyperthermophile Thermotoga maritima was biochemically characterized with the aim of establishing a colorimetric assay for inorganic pyrophosphate (PPi). When heterologously expressed in Escherichia coli, T. maritima PPDK (TmPPDK) was far more stable any other PPDK reported so far: it retained >90% of its activity after incubation for 1 h at 80 °C, and >80% of its activity after incubation for 20 min at pHs ranging from 6.5 to 10.5 (50 °C). In contrast to PPDKs from protozoa and plants, this TmPPDK showed very long-term stability at low temperature: full activity was retained even after storage for at least 2 years at 4 °C. TmPPDK was successfully applied to a novel colorimetric PPi assay, which employed (i) a PPi cycling reaction using TmPPDK and nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) from Saccharomyces cerevisiae and (ii) a NAD cycling reaction to accumulate reduced nitroblue tetrazolium (diformazan). This enabled detection of 0.2 μM PPi, making this method applicable for preliminary measurement of PPi levels in PCR products in an automatic clinical analyzer.     

Inorganic polyphosphate from the immunobiotic Lactobacillus rhamnosus CRL1505 prevents inflammatory response in the respiratory tract

Lactobacillus (L.) rhamnosus CRL1505 accumulates inorganic polyphosphate (polyP) in its cytoplasm in response to environmental stress. The aim of this study was to evaluate the potential effects of polyP from the immunobiotic CRL1505 on an acute respiratory inflammation murine animal model induced by lipopolysaccharide (LPS).First, the presence of polyP granules in the cytoplasm of CRL1505 strain was evidenced by specific staining. Then, it was demonstrated in the intracellular extracts (ICE) of CRL1505 that polyP chain length is greater than 45 phosphate residues. In addition, the functionality of the genes involved in the polyP metabolism (ppk, ppx1 and ppx2) was corroborated by RT-PCR. Finally, the possible effect of the ICE of CRL1505 strain containing polyP and a synthetic polyP was evaluated in vivo using a murine model of acute lung inflammation. It was observed that the level of cytokines pro-inflammatory (IL-17, IL-6, IL-2, IL-4, INF-γ) in serum was normalized in mice treated with ICE, which would indicate that polyP prevents the local inflammatory response in the respiratory tract. The potential application of ICE from L. rhamnosus CRL1505 as a novel bioproduct for the treatment of respiratory diseases is one of the projections of this work.     

Exploring emergent properties in cellular homeostasis using OnGuard to model K and other ion transport in guard cells

It is widely recognized that the nature and characteristics of transport across eukaryotic membranes are so complex as to defy intuitive understanding. In these circumstances, quantitative mathematical modeling is an essential tool, both to integrate detailed knowledge of individual transporters and to extract the properties emergent from their interactions. As the first, fully integrated and quantitative modeling environment for the study of ion transport dynamics in a plant cell, OnGuard offers a unique tool for exploring homeostatic properties emerging from the interactions of ion transport, both at the plasma membrane and tonoplast in the guard cell. OnGuard has already yielded detail sufficient to guide phenotypic and mutational studies, and it represents a key step toward ‘reverse engineering’ of stomatal guard cell physiology, based on rational design and testing in simulation, to improve water use efficiency and carbon assimilation. Its construction from the HoTSig libraries enables translation of the software to other cell types, including growing root hairs and pollen. The problems inherent to transport are nonetheless challenging, and are compounded for those unfamiliar with conceptual ‘mindset’ of the modeler. Here we set out guidelines for the use of OnGuard and outline a standardized approach that will enable users to advance quickly to its application both in the classroom and laboratory. We also highlight the uncanny and emergent property of OnGuard models to reproduce the ‘communication’ evident between the plasma membrane and tonoplast of the guard cell.     

Catabolite inactivation of the maltose transporter in nitrogen-starved yeast could be due to the stimulation of general protein turnover

Addition of glucose to Saccharomyces cerevisiae inactivates the maltose transporter. The general consensus is that this inactivation, called catabolite inactivation, is one of the control mechanisms developed by this organism to use glucose preferentially whenever it is available. Using nitrogen-starved cells (resting cells), it has been shown that glucose triggers endocytosis and degradation of the transporter in the vacuole. We now show that maltose itself triggers inactivation and degradation of its own transporter as efficiently as glucose. This fact, and the observation that glucose inactivates a variety of plasma membrane proteins including glucose transporters themselves, suggests that catabolite inactivation of the maltose transporter in nitrogen-starved cells is not a control mechanism specifically directed to ensure a preferential use of glucose. It is proposed that, in this metabolic condition, inactivation of the maltose transporter might be due to the stimulation of the general protein turnover that follows nitrogen starvation.     

Atomistic Simulation of New Materials: Pitfalls and Promises

Abstract

In this paper, we discuss some of the pitfalls that may be encountered using atomistic computer simulations of complex inorganic materials. The examples discussed are intended to illustrate the promising role that computer simulations can play in solid state chemistry, providing the pitfalls are avoided.     

Transport of cadmium across the apical membrane of epithelial cell lines

Cadmium (Cd) uptake and secretion across the apical membrane of epithelial cells was studied using LLC-PK1 cells cultured on Petri dishes and permeable membranes, respectively. Cd accumulation in cells from the apical medium was decreased by low temperature and metabolic inhibitors. A saturable tendency was observed between initial Cd accumulation and increased concentrations of Cd in the apical medium at 37 degrees C, but not at 4 degrees C. Co-incubation with ZnCl2 or CuCl2 competitively decreased Cd accumulation at 37 degrees C. A decrease in the pH of the apical medium markedly decreased Cd accumulation. Pretreatment of cells with an inorganic anion-exchange inhibitor significantly decreased Cd uptake at pH 7.4 in the presence of bicarbonate, but only marginally in its absence. A decrease in the pH of the apical medium increased the secretory (basolateral-to-apical) transport of Cd, with a concomitant decrease in the cellular accumulation of Cd. Co-incubation with Cd and tetraethylammonium, a typical substrate of the organic cation transporter, decreased Cd transport, with a concomitant increase in cellular Cd accumulation. The uptake and secretion of Cd across the apical membrane appear to be partly mediated via an inorganic anion exchanger and a H+ antiport of the organic cation transport system, respectively. Therefore, a decrease in pH of the apical medium markedly decreases Cd accumulation, possibly as a result of not only the decrease in Cd uptake via an inorganic anion exchanger, but also the increase in Cd secretion via the Cd2+/H+ antiport. Further evidence of the antiport was obtained from experiments using brush border membrane vesicles isolated from rat kidney and small intestine. In addition, passive diffusion of Cd appears to be decreased by low temperature and a decrease in pH.     

Alkaline inorganic pyrophosphatase from orchid leaves

An alkaline inorganic pyrophosphatase (IP) from leaves of an orchid, Aranda Christine 130 (Arachnis hookerana var. luteola × Vanda Hilo Blue) was purified by acetone precipitation and chromatography on Sephadex G-75 and DEAE-cellulose. The IP gave a single band on non-denaturing gel electrophoresis at pH 8.3 and its M, determined by gel filtration, was 28 000. The pH optimum was 9 and the IP required Mg²⁺ for its activity and stability. The IP exhibited high specificity for PPi and attained a maximum activity at a Mg²⁺: PPi ratio of 10:1. Other cations tested could not replace Mg²⁺ and they were also found to be inhibitory. The IP was also inhibited by EDTA and F^? but not by iodoacetamide.     

Nutrient content and ionic pattern in beech (Fagus sylvatica L.) from natural stands in Eastern Austria and ecological implications

Macro and Micro cation content, concentration of water soluble cations and anions, as well as nitrogen content in beech leaves from a variety of forest stands in the eastern part of Austria have been determined. Special attention has been focused on the highly air polluted Vienna Woods. A broad natural variability in total content of minerals, as well as in nitrogen content could be observed. In general, cation nourishment has been found to be adequate on all stands. The pattern of water soluble i.e. physiologically active cations, especially with regard to divalent cations, varies broadly among beeches from different provenances: Mg/Mn ratios differ within three orders of magnitude between dolomite and highly acidic, nutrient poor stands over silicate rock. The term mineral deficiency limit should, therefore, be used very carefully with respect to single nutrients. Organic anions (quinate, malate, citrate) clearly dominate over inorganic anions. The relative amount of the latter increased from NO₃, Cl, inorganic phosphate to SO₄. A concentration of SO₄ is markedly in leaves within the direct reach of SO₂ sources, its level, however, remaining well below the organic anions. Slight evidence of an antagonistic behavior between SO₄ and organic acids could be found. Total nitrogen as well as the ratio insoluble:soluble nitrogen were tendencially higher in Vienna Wood beeches, indicating an additional use of the airborne nitrogen. The high flexibility of beech with respect to mineral content and ionic balance is discussed with regard to the ecological distribution of beech and the susceptibility to air pollution.