Low Divalent Cation Research Articles

Extractable latex yield from Taraxacum kok-saghyz roots is enhanced by increasing rubber particle buoyancy

Taraxacum kok-saghyz (TK) is a leading alternative rubber-producing plant that produces natural rubber (NR) latex in laticifers within its roots. Unlike the tropical rubber tree, Hevea brasiliensis, TK can be grown in temperate regions, such as the northern United States, as the basis of a domestic rubber production industry. To support such an industry, efficient, scalable rubber extraction techniques are needed. In this study, aqueous extraction was used to recover TK natural rubber latex (TNRL) from four harvests of greenhouse-produced TK roots with a new technique that improves rubber particle buoyancy in root homogenate by chelation. The new method includes harvest, cleaning, and homogenization of freshly harvested roots, followed by centrifugation, vacuum collection of latex, and purification. Treatment of homogenate with ethylenediaminetetraacetic acid (EDTA), a divalent cation chelator, allowed more than twice as much latex to be extracted from fresh homogenate. EDTA-treated rubber particles contained lower concentrations of divalent cations than untreated ones in fresh homogenate and homogenate stored for two weeks. Long term (three months) storage of root homogenate similarly yielded more latex than fresh homogenate and had low divalent cation rubber particle concentrations, indicating that heavy cations had slowly leached from the particles into the aqueous medium during storage. Rubber particle size was larger in this treatment suggestive of rubber particle agglomeration but this was prevented and reversed by EDTA. Removal of divalent cations via chelation increased particle packing density of particles in concentrated latex and appeared to increase the gel content of dried latex samples. Molecular characterization of dried samples of purified TNRL established that the rubber was of the high molecular weight needed for product manufacturing.

PH-tunable membrane-active polymers, NCMNP2a-x, and their potential membrane protein applications.

Accurate 3D structures of membrane proteins are essential for comprehending their mechanisms of action and designing specific ligands to modulate their activities. However, these structures are still uncommon due to the involvement of detergents in the sample preparation. Recently, membrane-active polymers have emerged as an alternative to detergents, but their incompatibility with low pH and divalent cations has hindered their efficacy. Herein, we describe the design, synthesis, characterization, and application of a new class of pH-tunable membrane-active polymers, NCMNP2a-x. The results demonstrated that NCMNP2a-x could be used for high-resolution single-particle cryo-EM structural analysis of AcrB in various pH conditions and can effectively solubilize BcTSPO with the function preserved. Molecular dynamic simulation is consistent with experimental data that shed great insights into the working mechanism of this class of polymers. These results demonstrated that NCMNP2a-x might have broad applications in membrane protein research.

Freshwater and Evaporite Brine Compositions on Hadean Earth: Priming the Origins of Life.

The chemical composition of aqueous solutions during the Hadean era determined the availability of essential elements for prebiotic synthesis of the molecular building blocks of life. Here we conducted quantitative reaction path modeling of atmosphere-water-rock interactions over a range of environmental conditions to estimate freshwater and evaporite brine compositions. We then evaluated the solution chemistries for their potential to influence ribonucleotide synthesis and polymerization as well as protocell membrane stability. Specifically, solutions formed by komatiite and tonalite (primitive crustal rocks) weathering and evaporation-rehydration (drying-wetting) cycles were studied assuming neutral atmospheric composition over a wide range of values of atmospheric partial pressure of CO2 (PCO2) and temperatures (T). Solution pH decreased and total dissolved concentrations of inorganic P, Mg, Ca, Fe, and C (PT, MgT, CaT, FeT, and CT) increased with increasing PCO2. The PCO2 and T dictated how the solution evolved with regard to minerals precipitated and ions left in solution. At T = 75°C and PCO2 < 0.05 atm, the concentration ratio of magnesium to calcium ion concentrations (Mg2+/Ca2+) was < 1 and predominantly metal aluminosilicates (including clays), dolomite, gibbsite, and pyrite (FeS2) precipitated, whereas at PCO2 > 0.05 atm, Mg2+/Ca2+ was > 1 and mainly magnesite, dolomite, pyrite, chalcedony (SiO2), and kaolinite (Al2Si2O5) precipitated. At T = 75°C and PCO2 > 0.05 atm, hydroxyapatite (HAP) precipitated during weathering but not during evaporation, and so, PT increased with each evaporation-rehydration cycle, while MgT, CaT, and FeT decreased as other minerals precipitated. At T = 75°C and PCO2 ∼5 atm, reactions with komatiite provided end-of-weathering solutions with high enough Mg2+ concentrations to promote RNA-template directed and montmorillonite-promoted nonenzymatic RNA polymerization, but incompatible with protocell membranes; however, montmorillonite-promoted RNA polymerization could proceed with little or no Mg2+ present. Cyclically evaporating/rehydrating brines from komatiite weathering at T = 75°C and PCO2 ∼5 atm yielded the following: (1) high PT values that could promote ribonucleotide synthesis, and (2) low divalent cation concentrations compatible with amino acid-promoted, montmorillonite-catalyzed RNA polymerization and with protocell membranes, but too low for template-directed nonenzymatic RNA polymerization. For all PCO2 values, Mg2+ and PT concentrations decreased, whereas the HCO3- concentration increased within increasing temperature, due to the retrograde solubility of the minerals controlling these ions' concentrations; Fe2+ concentration increased because of prograde pyrite solubility. Tonalite weathering and cyclical wetting-drying reactions did not produce solution compositions favorable for promoting prebiotic RNA formation. Conversely, the ion concentrations compatible with protocell emergence, placed constraints on PCO2 of early Earth's atmosphere. In summary: (1) prebiotic RNA synthesis and membrane self-assembly could have been achieved even under neutral atmosphere conditions by atmosphere-water-komatiite rock interactions; and (2) constraints on element availability for the origins of life and early PCO2 were addressed by a single, globally operating mechanism of atmosphere-water-rock interactions without invoking special microenvironments. The present results support a facile origins-of-life hypothesis even under a neutral atmosphere as long as other favorable geophysical and planetary conditions are also met.

Functional diversity of isoprenoid lipids in Methylobacterium extorquens PA1.

Hopanoids and carotenoids are two of the major isoprenoid-derived lipid classes in prokaryotes that have been proposed to have similar membrane ordering properties as sterols. Methylobacterium extorquens contains hopanoids and carotenoids in their outer membrane, making them an ideal system to investigate the role of isoprenoid lipids in surface membrane function and cellular fitness. By genetically knocking out hpnE and crtB we disrupted the production of squalene and phytoene in M. extorquens PA1, which are the presumed precursors for hopanoids and carotenoids respectively. Deletion of hpnE revealed that carotenoid biosynthesis utilizes squalene as a precursor resulting in pigmentation with a C30 backbone, rather than the previously predicted canonical C40 phytoene-derived pathway. Phylogenetic analysis suggested that M. extorquens may have acquired the C30 pathway through lateral gene transfer from Planctomycetes. Surprisingly, disruption of carotenoid synthesis did not generate any major growth or membrane biophysical phenotypes, but slightly increased sensitivity to oxidative stress. We further demonstrated that hopanoids but not carotenoids are essential for growth at higher temperatures, membrane permeability and tolerance of low divalent cation concentrations. These observations show that hopanoids and carotenoids serve diverse roles in the outer membrane of M. extorquens PA1.

Biological insights from SMA-extracted proteins.

In the twelve years since styrene maleic acid (SMA) was first used to extract and purify a membrane protein within a native lipid bilayer, this technological breakthrough has provided insight into the structural and functional details of protein–lipid interactions. Most recently, advances in cryo-EM have demonstrated that SMA-extracted membrane proteins are a rich-source of structural data. For example, it has been possible to resolve the details of annular lipids and protein–protein interactions within complexes, the nature of lipids within central cavities and binding pockets, regions involved in stabilising multimers, details of terminal residues that would otherwise remain unresolved and the identification of physiologically relevant states. Functionally, SMA extraction has allowed the analysis of membrane proteins that are unstable in detergents, the characterization of an ultrafast component in the kinetics of electron transfer that was not possible in detergent-solubilised samples and quantitative, real-time measurement of binding assays with low concentrations of purified protein. While the use of SMA comes with limitations such as its sensitivity to low pH and divalent cations, its major advantage is maintenance of a protein's lipid bilayer. This has enabled researchers to view and assay proteins in an environment close to their native ones, leading to new structural and mechanistic insights.

Effects of cations on biofilm formation and characteristics in integrated fixed film activated sludge process at different carbon and nitrogen loadings

Both divalent cations including calcium and magnesium play important roles for microbial aggregates in binding to negatively charged functional groups on bacterial surfaces, in extracellular polymeric substances (EPS), and on inorganic materials in flocs and biofilms. Monovalent cations such as sodium and potassium deteriorate the floc structure and physical properties. The Integrated Fixed Activated Sludge (IFAS) process employs fixed film media in the aerobic zone; therefore, both monovalent and divalent cations are involved in the process performances. In this study, the effects of cations indicated as the monovalent to divalent cations (M/D) ratio on the biofilm formation and characteristics, and on the IFAS performances for carbon and ammonium removals were evaluated. The experiments were conducted in three IFAS systems feeding with the same wastewater but different M/D ratios and two carbon and nitrogen loadings. The findings revealed that high monovalent with low divalent cations at the M/D ratios higher than 2.0 produced excessive polysaccharides in EPS resulting in high viscosity of activated sludge flocs causing viscous bulking with high SVI values, decreasing the biofilm formation, and increasing the biofilm sloughing. Increasing of both monovalent and carbon loading increased more polysaccharides in the EPS leading to the failures of IFAS system. Nitrification failed at higher M/D ratios because of less nitrifiers in flocs and biofilm. The M/D ratio less than 2.0 is suggested to minimize the excessive EPS production in the IFAS system, especially at high organic loading.

A comparison of SMA (styrene maleic acid) and DIBMA (di-isobutylene maleic acid) for membrane protein purification

The use of styrene maleic acid co-polymer (SMA) for membrane protein extraction and purification has grown in recent years. SMA inserts in the membrane and assembles into small discs of bilayer encircled by polymer, termed SMA lipid particles (SMALPs). This allows purification of membrane proteins whilst maintaining their lipid bilayer environment. SMALPs offer several improvements over conventional detergent approaches, however there are limitations, most notably a sensitivity to low pH and divalent cations. Recently it was shown that the aliphatic diisobutylene-maleic acid (DIBMA) copolymer, was also able to directly solubilise membranes forming DIBMALPs (DIBMA lipid particles), and that this polymer overcame some of the limitations of SMA. In this study the ability of DIBMA to solubilise and purify functional membrane proteins has been compared to SMA. It was found that DIBMA is able to solubilise several different membrane proteins from different expression systems, however for some proteins it gives a lower yield and lower degree of purity than SMA. DIBMA extracted G protein-coupled receptors retain ligand- and G protein-binding. DIBMALPS are larger than SMALPs and display a decreased sensitivity to magnesium. However the stability of DIBMALPs appears to be lower than SMALPs. The lower purity and lower stability are likely linked to the larger size of the DIBMALP particle. However, this also offers a potentially less rigid lipid environment which may be more amenable to protein dynamics. Therefore the optimal choice of polymer will depend on which features of a protein are to be investigated.

Insights on Structure and Threshold Detection Limits of Stichtite (Magnesium-Chromium Carbonate-Hydroxide) by Fourier Transform Infrared Analysis

Spectral features for natural stichtite at 1042, 1096, 1360, 1638, and 3482 cm−1 provide insights on mineral structure, with peaks consistent with OH− stretching modes bound to Mg or Cr, CO32− antisymmetric stretches and CO32− bound within the sample and molecular water. These Infrared (IR) data suggest natural stichtite forms at a pH of &gt;12 with increased water and decreased carbonate in the interlayer due to a smaller interlayer distance and unit cell. Higher pH favors lower divalent cation purity and may explain observed ranges of non-end member compositions in stichtite from localities around the world, and across geologic time. This constrains stichtite formation to a range of very high pH conditions and is consistent with active serpentinizing fluid vents and some mine wastes. IR has clear application for the detection and quantification of stichtite under field and laboratory conditions within the detection limits of 5% stichtite within a serpentine host. The size and grade of terrestrial stichtite deposits, and resolution of remote sensing instruments, suggest remote IR detection of stichtite is possible, and remote IR detection for Earth and Mars is discussed.

A membrane-based recycling process for minimizing environmental effects inflicted by ion-exchange softening applications

A new process is presented for replacing the typical NaCl-based with a KCl-based solution, for regenerating water-softening ion-exchange resins. The incentive is three-fold: to minimize the release of detrimental brines to the environment, to augment K+ in humans’ diet at the expense of harmful Na+ and to improve the worth of the treated water with respect to irrigation and soil properties. Since KCl is 3–5 times more expensive than NaCl, the proposed process targets the recycling of the spent regeneration solution through a sequence of membrane processes (nanofiltration, diananofiltration, reverse osmosis) aimed at separating the divalent cations (hardness species) from K+, followed by concentration of a fraction of the K+ solution using RO to prepare it to serve as the next-cycle regeneration solution. Detailed results of all separation steps (apart from the relatively-simple RO step) are presented, along with a comprehensive cost analysis, that shows that the process becomes cost competitive (vs. the traditional NaCl-based regeneration) after merely five ion exchange/regeneration cycles. The three-step recycling technique resulted in the recovery of 92% of the K+ mass, along with 60% of the fresh water used in the process. After five adsorption/regeneration cycles the ion exchange breakthrough curve showed only slight deterioration due to a low divalent cations concentration that accumulated in the recycled regeneration solution ([Ca2+] + [Mg2+] ≈ 23 meq/L vs. ∼1000 meq/L of K+).

EDL Gated FET Biosensor Array for the Investigation of Ion Channels and Bioelectric Signals of Circulating Tumor Cells

This paper demonstrates a novel methodology to analyze the bioelectric signals of cells in different test media using highly sensitive Electrical Double Layer (EDL) gated AlGaN/GaN HEMT (High Electron Mobility Transistor) biosensor array. Circulating Tumor Cells (CTCs) derived from Colorectal Cancer (CRC) were the target of this experiment. Previously, we had demonstrated that our sensor can detect and count the number of captured cells with up to single cell resolution. Thereafter, we further discovered that our sensor can be used in detecting important fundamental bioelectric signals of cell. We have demonstrated that our FET (Field Effect Transistor) biosensor can dynamically monitor the changes in transmembrane potential of cells induced by high concentrations of extracellular KCl, low and high extracellular divalent cation concentrations. Additionally, cadmium ions were used to block the calcium channels present in the cell demonstrating an interesting application of bioelectric signal study. Thus, we demonstrate that our sensing methodology can replace the conventional methods to study fundamental cellular mechanisms such as ion channel gating.

Molecular Interactions of Lipopolysaccharide with an Outer Membrane Protein from Pseudomonas aeruginosa Probed by Solution NMR.

Pseudomonas aeruginosa is an opportunistic human pathogen causing pneumonias that are particularly severe in cystic fibrosis and immunocompromised patients. The outer membrane (OM) of P. aeruginosa is much less permeable to nutrients and other chemical compounds than that of Escherichia coli. The low permeability of the OM, which also contributes to Pseudomonas' significant antibiotic resistance, is augmented by the presence of the outer membrane protein H (OprH). OprH directly interacts with lipopolysaccharides (LPS) that constitute the outer leaflet of the OM and thus contributes to the structural stability of the OM. In this study, we used solution NMR spectroscopy to characterize the interactions between LPS and OprH in molecular detail. NMR chemical shift perturbations observed upon the addition of LPS to OprH in DHPC micelles indicate that this interaction is predominantly electrostatic and localized to the extracellular loops 2 and 3 and a number of highly conserved basic residues near the extracellular barrel rim of OprH. Single-site mutations of these residues were not enough to completely abolish binding, but OprH with cumulative mutations of Lys70, Arg72, and Lys103 no longer binds LPS. The dissociation constant (∼200 μM) measured by NMR is sufficient to efficiently bind LPS to OprH in the OM. This work highlights that solution NMR is suitable to study specific interactions of lipids with integral membrane proteins and provides a detailed molecular model for the interaction of LPS with OprH; i.e., an interaction that contributes to the integrity of the OM of P. aeruginosa under low divalent cation and antibiotic stress conditions. These methods should thus be useful for screening antibiotics that might disrupt OprH-LPS interactions and thereby increase the permeability of the OM of P. aeruginosa.

TRPM7 is a molecular substrate of ATP-evoked P2X7-like currents in tumor cells.

Within the ion channel-coupled purine receptor (P2X) family, P2X7 has gained particular interest because of its role in immune responses and in the growth control of several malignancies. Typical hallmarks of P2X7 are nonselective and noninactivating cation currents that are elicited by high concentrations (0.1-10 mM) of extracellular ATP. Here, we observe spurious ATP-induced currents in HEK293 cells that neither express P2X7 nor display ATP-induced Ca(2+) influx or Yo-Pro-1 uptake. Although the biophysical properties of these ionic currents resemble those of P2X7 in terms of their reversal potential close to 0 mV, nonrectifying current-voltage relationship, current run-up during repeated ATP application, and augmentation in bath solutions containing low divalent cation (DIC) concentrations, they are poorly inhibited by established P2X7 antagonists. Because high ATP concentrations reduce the availability of DICs, these findings prompted us to ask whether other channel entities may become activated by our experimental regimen. Indeed, a bath solution with no added DICs yields similar currents and also a rapidly inactivating Na(+)-selective conductance. We provide evidence that TRPM7 and ASIC1a (acid-sensing ion channel type Ia)-like channels account for these noninactivating and phasic current components, respectively. Furthermore, we find ATP-induced currents in rat C6 glioma cells, which lack functional P2X receptors but express TRPM7. Thus, the observation of an atypical P2X7-like conductance may be caused by the activation of TRPM7 by ATP, which scavenges free DICs and thereby releases TRPM7 from permeation block. Because TRPM7 has a critical role in controlling the intracellular Mg(2+) homeostasis and regulating tumor growth, these data imply that the proposed role of P2X7 in C6 glioma cell proliferation deserves reevaluation.

Local electrostatic interactions determine the diameter of fusion pores

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition.

Pannexin 1 channels play essential roles in urothelial mechanotransduction and intercellular signaling.

Urothelial cells respond to bladder distension with ATP release, and ATP signaling within the bladder and from the bladder to the CNS is essential for proper bladder function. In other cell types, pannexin 1 (Panx1) channels provide a pathway for mechanically-induced ATP efflux and for ATP-induced ATP release through interaction with P2X7 receptors (P2X7Rs). We report that Panx1 and P2X7R are functionally expressed in the bladder mucosa and in immortalized human urothelial cells (TRT-HU1), and participate in urothelial ATP release and signaling. ATP release from isolated rat bladders induced by distention was reduced by the Panx1 channel blocker mefloquine (MFQ) and was blunted in mice lacking Panx1 or P2X7R expression. Hypoosmotic shock induced YoPro dye uptake was inhibited by MFQ and the P2X7R blocker A438079 in TRT-HU1 cells, and was also blunted in primary urothelial cells derived from mice lacking Panx1 or P2X7R expression. Rinsing-induced mechanical stimulation of TRT-HU1 cells triggered ATP release, which was reduced by MFQ and potentiated in low divalent cation solution (LDPBS), a condition known to enhance P2X7R activation. ATP signaling evaluated as intercellular Ca2+ wave radius was significantly larger in LDPBS, reduced by MFQ and by apyrase (ATP scavenger). These findings indicate that Panx1 participates in urothelial mechanotransduction and signaling by providing a direct pathway for mechanically-induced ATP release and by functionally interacting with P2X7Rs.

Cloning and Characterization of a Wheat Homologue of Apurinic/Apyrimidinic Endonuclease Ape1L

BackgroundApurinic/apyrimidinic (AP) endonucleases are key DNA repair enzymes involved in the base excision repair (BER) pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which includes E. coli Xth, human APE1 and Arabidopsis thaliana AtApe1L has been isolated and its protein product purified and characterized.Methodology/Principal FindingsWe report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 3′-repair phosphodiesterase, 3′-phosphatase and 3′→5′ exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg2+ and Ca2+ (5–10 mM) to the reaction mixture inhibited TaApe1L whereas the presence of Mn2+, Co2+ and Fe2+ cations (0.1–1.0 mM) strongly stimulated all its DNA repair activities. Optimization of the reaction conditions revealed that the wheat enzyme requires low divalent cation concentration (0.1 mM), mildly acidic pH (6–7), low ionic strength (20 mM KCl) and has a temperature optimum at around 20°C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 3′-blocking sugar-phosphate and 3′-phosphate groups with good efficiency (k cat/K M = 630 and 485 μM−1·min−1, respectively) but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1.Conclusions/SignificanceTaken together, these data establish the DNA substrate specificity of the wheat AP endonuclease and suggest its possible role in the repair of DNA damage generated by endogenous and environmental factors.

Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis

Our understanding of focal and segmental glomerulosclerosis (FSGS) has advanced significantly from the studies of rare, monogenic forms of the disease. These studies have demonstrated the critical roles of multiple aspects of podocyte function in maintaining glomerular function. A substantial body of research has suggested that the integral membrane protein podocalyxin (PODXL) is required for proper function of podocytes, possibly by preserving the patency of the slit diaphragm by negative charge-based repulsion. Exome sequencing of affected cousins from an autosomal dominant pedigree with FSGS identified a co-segregating private variant, PODXL p.L442R, affecting the transmembrane region of the protein. Of the remaining 11 shared gene variants, two segregated with disease but their gene products were not detected in the glomerulus. In comparison to wild type, this disease-segregating PODXL variant facilitated dimerization. By contrast, this change does not alter protein stability, extracellular domain glycosylation, cell surface expression, global subcellular localization, or interaction with its intracellular binding partner ezrin. Thus, a variant form of PODXL remains the most likely candidate causing FSGS in one family with autosomal dominant inheritance, but its full effect on protein function remains unknown. Our work highlights the challenge faced in the clinical interpretation of whole exome data for small pedigrees with autosomal dominant diseases.

The phenothiazine-class antipsychotic drugs prochlorperazine and trifluoperazine are potent allosteric modulators of the human P2X7 receptor

P2X7, an ATP-gated cation channel, is involved in immune cell activation, hyperalgesia and neuropathic pain. By regulating cytokine release in the brain, P2X7 has been linked to the pathophysiology of mood disorders and schizophrenia. We here assess the impact of 123 drugs that act in the central nervous system on human P2X7. Most prominently, the tricyclic antipsychotics prochlorperazine (PCP) and trifluoperazine (TFP) potently inhibited P2X7-mediated Ca2+ entry, dye permeation and ionic currents. In divalent cation-containing bath solutions or after prolonged incubation, ATP-evoked P2X7 currents were inhibited by 10 μM PCP. This effect was not related to dopamine receptor antagonism. Surprisingly, PCP co-applied with ATP enhanced inward currents in bath solutions with low divalent cation concentrations. Intracellular perfusion with PCP did not substitute for the extracellularly applied drug, indicating that its binding sites are accessible from the extracellular space. Since P2X7 current potentiation by PCP was voltage-dependent, at least one site may be located within the electrical field of the membrane. While the channel opening and closure kinetic was altered by PCP, the apparent affinity of ATP remained unchanged (potentiation) or changed slightly (inhibition). Measurements in human monocyte-derived macrophages confirmed the PCP-induced inhibition of ATP-evoked Ca2+ influx, Yo-Pro-1 permeability, and whole cell currents. Interestingly, neither heterologously expressed rat or mouse P2X7 nor native P2X7 in rat astrocyte cultures or in mouse bone marrow-derived macrophages were inhibited by perazines with a similar potency. We conclude that perazine-type neuroleptics are potent, but species-selective allosteric modulators of human but not murine P2X7 receptors.

Superposition of anion and cation exchange for removal of natural water ions

Ion exchange in a fluidized bed has been shown to be an effective design for anion and cation exchange. However, little is known about combining anion and cation exchange resins in the same vessel. More specifically, can combined ion exchange be predicted by adding the removal of anions during anion exchange to the removal of cations during cation exchange? Results indicated that combined ion exchange can reduce precipitation of sparingly soluble minerals, such as CaCO3, in the bulk solution by simultaneously removing calcium and bicarbonate. Inorganic cations dominated the ion exchange behavior during these processes, and organic anions such as dissolved organic matter (DOM) behaved the same in combined ion exchange as in anion exchange. Results showed that it is possible to estimate sulfate and magnesium removal during combined ion exchange by rules of superposition, as well as calcium when initial concentration of calcium is low. Synthetic waters with varying divalent cations were compared to a natural groundwater, both of which had low organics and high divalent cations. Results showed similar behavior between natural and synthetic waters with varying types of DOM. The effect of combined ion exchange on separation factors is also calculated and discussed.

Rheological Study on ATBS‐AM Copolymer‐Surfactant System in High‐Temperature and High‐Salinity Environment

Experimental studies were conducted to evaluate the rheological properties of surfactant‐polymer (SP) system. This SP system consists of a copolymer of acrylamide (AM) and acrylamido tertiary butyl sulfonate (ATBS) and sodium dodecyl sulphate (SDS) surfactant. Effects of surfactant concentration, temperature, polymer concentration, and salinity on rheological properties of SP system were investigated by means of oscillation and shear measurements. Comparison with classical partially hydrolyzed polyacrylamide (HPAM) was made. For the same temperature range, the viscosity drop for HPAM was about four times higher than the viscosity drop for ATBS‐AM copolymer. In deionized water, viscosity of both polymers and SP systems was very high as compared to viscosity in saline water. Viscosity reduction of ATBS‐AM copolymer was higher for salts having divalent cations. The SP system showed precipitation in presence of divalent cations. It worked well with monovalent cations even at relatively high salinities. The addition of 0.1% surfactant to the polymer resulted in a 60% decrease in the viscosity. Some interfacial rheological experiments were also carried out to investigate the behaviors on the interface between SP solutions and oil. Addition of 0.1% surfactant showed a 65% decrease in G′ at SP solution‐oil interface. SP system consisting of ATBS‐AM and SDS showed better performance at high temperature compared to HPAM‐SDS system. Due to precipitation, the SP system should be restricted to environment having low divalent cations.

Phosphorylation of Podocalyxin (Ser415) Prevents RhoA and Ezrin Activation and Disrupts Its Interaction with the Actin Cytoskeleton

Podocalyxin (PC) is a polysialylated, anti-adhesin that is essential for maintaining foot process architecture and the integrity of the glomerular filtration barrier. We showed previously that PC is firmly attached to the actin cytoskeleton through ezrin, that in puromycin aminonucleoside (PAN)-mediated nephrosis the PC-ezrin-actin complex is disrupted, and that PC is uncoupled from actin. However, the precise mechanisms involved remained unknown. Here we show that detachment of PC from actin is regulated by phosphorylation of PC. PC is hyperphosphorylated at serines in PAN- and protamine sulfate (PS)-treated rat glomeruli. We determined that PC is a substrate of PKC and that the site of phosphorylation is Ser415, located within the juxtamembrane, ezrin-binding domain of the cytoplasmic tail of PC. Mutation of Ser415 to the phosphomimetic residues Glu (S415E) or Asp (S415D) interfered with direct binding of the PC cytoplasmic tail to ezrin in vitro. Moreover, stable expression of a phosphomimetic (S415E) PC mutant but not the WT or the phosphorylation-deficient (S415A) PC mutant, disrupted PC-ezrin-actin interaction, failed to activate RhoA, and the cytoskeletal linker, ezrin, remained inactive. Our data indicate that phosphorylation of PC at Ser415 prevents attachment of PC and ezrin to actin and highlights the strategic position of Ser415 and direct binding of PC to ezrin in regulating podocyte foot process architecture.