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In this report, we investigated the hepatocytic uptake of rosuvastatin when administered with scutellarin (a Chinese herbal medicine) in rats and the role of organic anion transporting polypeptide 1B1 (OATP1B1) plays in the uptake. Forty-eight rats were randomly divided into two groups according to the medicine administered: rosuvastatin alone and rosuvastatin in combination with a series concentration of scutellarin. Rosuvastatin concentrations in blood and liver were measured using the liquid chromatography-tandem mass spectrometry (LC-MS) method. The uptake was also measured in rat primary hepatocytes and OATP1B1 transfected human embryonic kidney 293 T (HEK293T) cells. The uptake was investigated under the optimal intake conditions. The rosuvastatin Cmax and AUC 0-∞ in rat plasma increased 55% and 61%, respectively in the combination treatment group; and the liver scutellarin concentrations decreased 32%, 34%, and 33% at 1 h, 2 h, and 6 h, respectively. All scutellarin dosages (20, 50, and 100 μM) inhibited the uptake of rosuvastatin in rat primary hepatocytes (4.71%, 22.73%, and 45.89%). Scutellarin of 10 μM significantly inhibited the in vitro uptake of rosuvastatin in OATP1B1-HEK293T cells (P < 0.05), with an IC50 of 60.53 ± 5.74 μM. Scutellarin increases the plasma concentration of rosuvastatin and inhibits the uptake in rat primary hepatocytes and OATP1B1-HEK293T cells, suggesting a drug interaction between scutellarin and rosuvastatin and OATP1B1 as a potential mechanism.

The aim of this study was to develop a prodrug of ubiquinol-10 (UqH-10), the active form of ubiquinone-10 (Uq-10), for oral delivery. Bioavailability of UqH-10 is hampered by its high susceptibility to oxidation and water-insolubility. We prepared three novel N,N -dimethylglycine ester derivatives of UqH-10, including a 1-monoester (UqH-1-DMG), 4-monoester (UqH-4-DMG), and 1,4-bis-ester (UqH-DMG), and assessed their physicochemical properties in vitro and in vivo. UqH-DMG spontaneously formed an aqueous micelle solution comprising 20 nm particles at 36.5 °C. Cationic UqH-DMG formed nano-sized (5 nm) mixed-micelles with taurocholic acid. Reconversion of the derivatives to UqH-10 was accelerated in human liver microsomes. The oral bioavailability of UqH-10 after administration of UqH-derivatives or Uq-10 was determined in fasted and postprandial rats secreting normal and high levels of bile, respectively. In fasted rats, plasma UqH-10 after UqH-derivatives administration reached C max at 2-3 h and after Uq-10 administration, it remained low. The AUC 0-24h of UqH-10 after UqH-derivatives administration was 2-3-fold higher than that after Uq-10 administration. In postprandial rats, the T max of UqH-10 after UqH-derivatives administration was an hour earlier than after Uq-10 administration. In conclusion, cationic UqH-derivatives are convenient prodrugs that enhance UqH-10 bioavailability by forming nanosized mixed-micelles with intestinal bile acids.

Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO 2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O 2 , inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo═S) and oxo (Mo═O) bonds, were observed in the presence of azide (N 3 - ) or cyanate (OCN - ). Azide provided best protection against O 2 , resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO 2 - ) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN - ) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.

The free-radical theory of male infertility suggests that reactive oxygen species produced by the spermatozoa themselves are a leading cause of sperm dysfunction, including loss of sperm motility. However, the field is overshadowed on several fronts, primarily because: i) the probes used to measure reactive oxygen species (ROS) are imprecise; and ii) many reports suggesting that oxygen radicals are detrimental to sperm function add an exogenous source of ROS. Herein, a more reliable approach to measure superoxide anion production by human spermatozoa based on MS analysis is used. Furthermore, the formation of the lipid-peroxidation product 4-hydroxynonenal (4-HNE) during in vitro incubation using proteomics is also investigated. The data demonstrate that neither superoxide anion nor other free radicals that cause 4-HNE production are related to the loss of sperm motility during incubation. Interestingly, it appears that many of the 4-HNE adducted proteins, found within spermatozoa, originate from the prostate. A quantitative SWATH analysis demonstrate that these proteins transiently bind to sperm and are then shed during in vitro incubation. These proteomics-based findings propose a revised understanding of oxidative stress within the male reproductive tract., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Solute uptake and release by plant cells are frequently energized by coupling to H + influx supported by the proton motive force (pmf). The pmf results from a stable pH difference between the apoplast and the cytosol, with bulk values ranging from 4.9 to 5.8 and from 7.1 to 7.5, respectively, in combination with a negative electrical membrane potential. The P-type H + ATPases pumping H + from the cytosol into the apoplast at the expense of ATP hydrolysis are generally viewed as the only pmf source, exclusively linking membrane transport to energy metabolism. However, recent evidence suggests that pump activity may be insufficient to energize transport, particularly under stress conditions. Indeed, cytosolic H + scavenging and apoplastic H + generation by metabolism (denoted as 'active' buffering in contrast to the readily exhausted 'passive' matrix buffering) also stabilize the pH gradient. In the cytosol, H + scavenging is mainly associated with malate decarboxylation catalyzed by malic enzyme, and via the GABA shunt of the tricarboxylic acid (TCA) cycle involving glutamate decarboxylation. In the apoplast, formation of bicarbonate from CO 2 , the end-product of respiration, generates H + at pH ≥ 6. Membrane potential is stabilized by K + release and/or by anion uptake via ion channels. Finally, thermodynamic aspects of active buffering are discussed., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

L-lactic acidosis (L-LA) is the most common cause of metabolic acidosis in the critical care setting, which has been associated with a large increase in mortality. The purpose of this article is to provide clinicians with an overview of the biochemical and metabolic background required to understand the different pathophysiological mechanisms that may lead to the development of L-LA. We propose a classification based on whether the pathophysiology of L-LA is due predominantly to increased production or decreased removal of L-lactic acid. In this article, we provide an overview of the biochemical and metabolic aspects of glucose oxidation, the production and removal of L-lactic acid, and a discussion of the pathophysiology of the various causes of L-LA., (Copyright © 2019 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Volume-regulated anion channels (VRAC) are chloride channels activated in response to osmotic stress to regulate cellular volume and also participate in other cellular processes, including cell division and cell death. Recently, members of the LRRC8 family have been identified as the main contributors of VRAC conductance. LRRC8/VRAC is permeable to chloride ions but also exhibits significant permeability to various substrates that vary strongly in charge and size. In this study, we explored the intriguing ability of LRRC8/VRAC to transport glutathione (GSH), the major cellular reactive oxygen species (ROS) scavenger, and its involvement in epithelial-to-mesenchymal transition (EMT), a cellular process in which cellular oxidative status is a crucial step. First, in HEK293-WT cells, we showed that a hypotonic condition induced LRRC8/VRAC-dependent GSH conductance (P GSH /P Cl of ~0.1) and a marked decrease in intracellular GSH content. GSH currents and GSH intracellular decrease were both inhibited by DCPIB, an inhibitor of LRRC8/VRAC, and were not observed in HEK293-LRRC8A KO cells. Then, we induced EMT by exposing renal proximal tubule epithelial cells to the pleiotropic growth factor TGFβ1, and we measured the contribution of LRRC8/VRAC in this process by measuring (i) EMT marker expression (assessed both at the gene and protein levels), (ii) cell morphology and (iii) the increase in migration ability. Interestingly, pharmacologic targeting of LRRC8/VRAC (DCPIB) or RNA interference-mediated inhibition (LRRC8A siRNA) attenuated the TGFβ1-induced EMT response by controlling GSH and ROS levels. Interestingly, TGFβ1 exposure triggered DCPIB-sensitive chloride conductance. These results suggest that LRRC8/VRAC, due to its native permeability to GSH and thus its ability to modulate ROS levels, plays a critical role in EMT and might contribute to other physiological and pathophysiological processes associated with oxidative stress.

Aims: Inflammasome activation plays a pivotal role in many inflammatory diseases. Given that connexin (Cx) channels regulate numerous cellular events leading to inflammasome activation, we determined whether and how connexin affected inflammasome activation and inflammatory cell injury. Results: Exposure of mouse peritoneal macrophages (PMs) to lipopolysaccharide (LPS) plus ATP caused NLRP3 inflammasome activation, together with an increased connexin43 (Cx43). Inhibition of Cx43 blunted inflammasome activation. Consistently, PMs from the Cx43 heterozygous mouse (Cx43 +/- ) exhibited weak inflammasome activation, in comparison with those from the Cx43 +/+ mouse. Further analysis revealed that inflammasome activation was preceded by an increased reactive oxygen species (ROS) production, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase 2 (NOX2), protein carbonylation, and mitogen-activated protein kinase (MAPK) activation. Suppression of ROS with antioxidant, downregulation of NOX2 with small interfering RNA (siRNA), or inhibition of NADPH oxidase or MAPKs with inhibitors blocked Cx43 elevation and inflammasome activation. Intriguingly, suppression of Cx43 also blunted NOX2 expression, protein carbonylation, p38 phosphorylation, and inflammasome activation. In a model of acute renal injury induced by LPS, the Cx43 +/- mouse exhibited a significantly lower level of blood interleukin-1β (IL-1β), blood urea nitrogen, and urinary protein, together with milder renal pathological changes and renal expression of NLRP3 and NOX4, as compared with the Cx43 +/+ mouse. Moreover, inhibition of gap junctions suppressed IL-1β- and tumor necrosis factor-α-induced expression of NOX4 in glomerular podocytes and tubular epithelial cells. Innovation and Conclusion: Our study indicates that Cx43 contributes to inflammasome activation and the progression of renal inflammatory cell injury through modulation of intracellular redox status. Cx43 could be a novel target for the treatment of certain inflammatory diseases.

The objective of this study was to evaluate expression of a cluster of genes encoding β-defensin antimicrobial peptides in neutrophils of postpartum cows in relation to prepartum dietary cation-anion difference (DCAD), vitamin D, and postpartum disease. Pregnant dry Holstein cows (28 nulliparous and 51 parous) at 255 d gestation were blocked by parity and randomly assigned to 4 prepartum diets of positive (+130 mEq/kg) or negative (-130 mEq/kg) DCAD and either 3 mg vitamin D 3 or 3 mg of 25-hydroxyvitamin D 3 per 11 kg of dry matter/d. Treatment diets were fed from 255 d of gestation until calving. Peripheral blood neutrophils of 35 parous cows were collected at 0 and 3 d after calving and stimulated with 0 or 100 ng/mL of lipopolysaccharide (LPS). Furthermore, serum Ca and incidences of postpartum diseases were recorded for all cows. The mRNA transcripts of β-defensin genes were quantified by real-time PCR, and data were analyzed with a general linear mixed model to test for fixed effects and interactions of day, level of DCAD, source of vitamin D, and incidence of disease. Effects of DCAD and vitamin D on neutrophil oxidative burst and phagocytosis were previously reported but were analyzed for effects of disease in the present study. Transcripts for DEFB1, DEFB3, DEFB4, DEFB5, DEFB7, DEFB10, and lingual antimicrobial peptide (LAP) in neutrophils were upregulated by LPS at 0 d but not at 3 d. Transcripts for DEFB4 and DEFB7 in LPS-stimulated neutrophils were greater in cows fed negative DCAD diets compared with positive DCAD. Source of vitamin D (vitamin D 3 vs. 25-hydroxyvitamin D 3 ) did not affect expression of β-defensins in neutrophils. Cows with postpartum subclinical hypocalcemia (serum Ca <2.0 mM) had decreased DEFB3, DEFB4, DEFB6, DEFB7, DEFB10, and LAP expression in LPS-stimulated neutrophils compared with cows that did not experience subclinical hypocalcemia. Likewise, DEFB4, DEFB6, DEFB7, DEFB10, and LAP in LPS-stimulated neutrophils at 3 d postpartum were positively associated with serum Ca at 0 d postpartum. Transcripts for DEFB7, DEFB10 and LAP also were less abundant in neutrophils from cows with metritis compared with healthy cows. In conclusion, feeding a prepartum negative DCAD to improve postpartum serum Ca resulted in greater neutrophil β-defensin expression, and greater neutrophil β-defensin expression was positively associated with postpartum health., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Protein translocation across the bacterial cytoplasmic membrane is an essential process catalyzed by the Sec translocase, which in its minimal form consists of the protein-conducting channel SecYEG, and the motor ATPase SecA. SecA binds via its positively charged N-terminus to membranes containing anionic phospholipids, leading to a lipid-bound intermediate. This interaction induces a conformational change in SecA, resulting in a high-affinity association with SecYEG, which initiates protein translocation. Here, we examined the effect of anionic lipids on the SecA-SecYEG interaction in more detail, and discovered a second, yet unknown, anionic lipid-dependent event that stimulates protein translocation. Based on molecular dynamics simulations we identified an anionic lipid-enriched region in vicinity of the lateral gate of SecY. Here, the anionic lipid headgroup accesses the lateral gate, thereby stabilizing the pre-open state of the channel. The simulations suggest flip-flop movement of phospholipid along the lateral gate. Electrostatic contribution of the anionic phospholipids at the lateral gate may directly stabilize positively charged residues of the signal sequence of an incoming preprotein. Such a mechanism allows for the correct positioning of the entrant peptide, thereby providing a long-sought explanation for the role of anionic lipids in signal sequence folding during protein translocation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Endogenous ions play important roles in the function and pharmacology of G-protein coupled receptors (GPCRs). Historically the evidence for ionic modulation of GPCR function dates to 1973 with studies of opioid receptors, where it was demonstrated that physiologic concentrations of sodium allosterically attenuated agonist binding. This Na + -selective effect was distinct from effects of other monovalent and divalent cations, with the latter usually counteracting sodium's negative allosteric modulation of binding. Since then, numerous studies documenting the effects of mono- and divalent ions on GPCR function have been published. While ions can act selectively and nonselectively at many sites in different receptors, the discovery of the conserved sodium ion site in class A GPCR structures in 2012 revealed the unique nature of Na + site, which has emerged as a near-universal site for allosteric modulation of class A GPCR structure and function. In this review, we synthesize and highlight recent advances in the functional, biophysical, and structural characterization of ions bound to GPCRs. Taken together, these findings provide a molecular understanding of the unique roles of Na + and other ions as GPCR allosteric modulators. We will also discuss how this knowledge can be applied to the redesign of receptors and ligand probes for desired functional and pharmacological profiles. SIGNIFICANCE STATEMENT: The function and pharmacology of GPCRs strongly depend on the presence of mono and divalent ions in experimental assays and in living organisms. Recent insights into the molecular mechanism of this ion-dependent allosterism from structural, biophysical, biochemical, and computational studies provide quantitative understandings of the pharmacological effects of drugs in vitro and in vivo and open new avenues for the rational design of chemical probes and drug candidates with improved properties., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Volume-regulated anion channels (VRACs) encoded by the leucine-rich repeat containing 8 ( LRRC8 ) gene family play critical roles in myriad cellular processes and might represent druggable targets. The dearth of pharmacological compounds available for studying VRAC physiology led us to perform a high-throughput screen of 1,184 of US Food and Drug Administration-approved drugs for novel VRAC modulators. We discovered the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, pranlukast, as a novel inhibitor of endogenous VRAC expressed in human embryonic kidney 293 (HEK293) cells. Pranlukast inhibits VRAC voltage-independently, reversibly, and dose-dependently with a maximal efficacy of only ~50%. The CysLT1R pathway has been implicated in activation of VRAC in other cell types, prompting us to test whether pranlukast requires the CysLT1R for inhibition of VRAC. Quantitative PCR analysis demonstrated that CYSLTR1 mRNA is virtually undetectable in HEK293 cells. Furthermore, the CysLT1R agonist leukotriene D4 had no effect on VRAC activity and failed to stimulate G q -coupled receptor signaling. Heterologous expression of the CysLT1R reconstituted LTD4-CysLT1R- G q -calcium signaling in HEK293 cells but had no effect on VRAC inhibition by pranlukast. Finally, we show the CysLT1R antagonist zafirlukast inhibits VRAC with an IC 50 of ~17 µM and does so with full efficacy. Our data suggest that both pranlukast and zafirlukast are likely direct channel inhibitors that work independently of the CysLT1R. This study provides clarifying insights into the putative role of leukotriene signaling in modulation of VRAC and identifies two new chemical scaffolds that can be used for development of more potent and specific VRAC inhibitors.

A new capillary electrophoresis method was developed to study the synergistic effect of superoxide dismutase and jujuboside A or B on scavenging superoxide anion radical in serum matrix respectively, in which superoxide anion radical was generated from pyrogallol autoxidation. The electrophoresis conditions, and the factors affecting the productive rate of purpurogallin, such as pyrogallol autoxidation product and the activity of superoxide dismutase, were optimized. Under optimal conditions, the content of superoxide dismutase in Gibco newborn calf serum was 7.06 mg/L, RSD was 2.01% and the average recovery was 98.4%. The values of IC 50 for jujuboside A and B in the serum matrix were 157.67 and 31.60 mg/L respectively, and they both had synergy on scavenging superoxide anion radical with superoxide dismutase, but there was no the dose-dependency on this synergy., (© 2019 John Wiley & Sons, Ltd.)

The role of arginines R64 and R89 at non-annular lipid binding sites of KcsA, on the modulation of channel activity by anionic lipids has been investigated. In wild-type (WT) KcsA reconstituted into asolectin lipid membranes, addition of phosphatidic acid (PA) drastically reduces inactivation in macroscopic current recordings. Consistent to this, PA increases current amplitude, mean open time and open probability at the single channel level. Moreover, kinetic analysis reveals that addition of PA causes longer open channel lifetimes and decreased closing rate constants. Effects akin to those of PA on WT-KcsA are observed when R64 and/or R89 are mutated to alanine, regardless of the added anionic lipids. We interpret these results as a consequence of interactions between the arginines and the anionic PA bound to the non-annular sites. NMR data shows indeed that at least R64 is involved in binding PA. Moreover, molecular dynamics (MD) simulations predict that R64, R89 and surrounding residues such as T61, mediate persistent binding of PA to the non-annular sites. Channel inactivation depends on interactions within the inactivation triad (E71-D80-W67) behind the selectivity filter. Therefore, it is expected that such interactions are affected when PA binds the arginines at the non-annular sites. In support of this, MD simulations reveal that PA binding prevents interaction between R89 and D80, which seems critical to the effectiveness of the inactivation triad. This mechanism depends on the stability of the bound lipid, favoring anionic headgroups such as that of PA, which thrive on the positive charge of the arginines., (Copyright © 2019. Published by Elsevier B.V.)

Background: Phosphorus is often present naturally in the soil as inorganic phosphate, Pi, which bio-availability is limited in many ecosystems due to low soil solubility and mobility. Plants respond to low Pi with a Pi Starvation Response, involving Pi sensing and long-distance signalling. There is extensive cross-talk between Pi homeostasis mechanisms and the homeostasis mechanism for other anions in response to Pi availability., Results: Recombinant Inbred Line (RIL) and Genome Wide Association (GWA) mapping populations, derived from or composed of natural accessions of Arabidopsis thaliana, were grown under sufficient and deficient Pi supply. Significant treatment effects were found for all traits and significant genotype x treatment interactions for the leaf Pi and sulphate concentrations. Using the RIL/QTL population, we identified 24 QTLs for leaf concentrations of Pi and other anions, including a major QTL for leaf sulphate concentration (SUL2) mapped to the bottom of chromosome (Chr) 1. GWA mapping found 188 SNPs to be associated with the measured traits, corresponding to 152 genes. One of these SNPs, associated with leaf Pi concentration, mapped to PP2A-1, a gene encoding an isoform of the catalytic subunit of a protein phosphatase 2A. Of two additional SNPs, associated with phosphate use efficiency (PUE), one mapped to AT5G49780, encoding a leucine-rich repeat protein kinase involved in signal transduction, and the other to SIZ1, a gene encoding a SUMO E3 ligase, and a known regulator of P starvation-dependent responses. One SNP associated with leaf sulphate concentration was found in SULTR2;1, encoding a sulphate transporter, known to enhance sulphate translocation from root to shoot under P deficiency. Finally, one SNP was mapped to FMO GS-OX4, a gene encoding glucosinolate S-oxygenase involved in glucosinolate biosynthesis, which located within the confidence interval of the SUL2 locus., Conclusion: We identified several candidate genes with known functions related to anion homeostasis in response to Pi availability. Further molecular studies are needed to confirm and validate these candidate genes and understand their roles in examined traits. Such knowledge will contribute to future breeding for improved crop PUE .

We constructed a two-photon fluorescence ratio probe (CST) for in situ quantitative real-time detection of mitochondrial O 2 ˙ - . Fluorescence imaging showed that O 2 ˙ - was over-generated from mitochondria and conveyed to the cytoplasm via voltage-dependent anion channels in hepatic ischemia-reperfusion mice, damaging the important functional protein aconitase in the cytoplasm.

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family. In this work, two members of this family, UCP5 (BMCP1, brain mitochondrial carrier protein 1 encoded by SLC25A14) and UCP6 (KMCP1, kidney mitochondrial carrier protein 1 encoded by SLC25A30) have been thoroughly characterized biochemically. They were overexpressed in bacteria, purified and reconstituted in phospholipid vesicles. Their transport properties and kinetic parameters demonstrate that UCP5 and UCP6 transport inorganic anions (sulfate, sulfite, thiosulfate and phosphate) and, to a lesser extent, a variety of dicarboxylates (e.g. malonate, malate and citramalate) and, even more so, aspartate and (only UCP5) glutamate and tricarboxylates. Both carriers catalyzed a fast counter-exchange transport and a very low uniport of substrates. Transport was saturable and inhibited by mercurials and other mitochondrial carrier inhibitors at various degrees. The transport affinities of UCP5 and UCP6 were higher for sulfate and thiosulfate than for any other substrate, whereas the specific activity of UCP5 was much higher than that of UCP6. It is proposed that a main physiological role of UCP5 and UCP6 is to catalyze the export of sulfite and thiosulfate (the H 2 S degradation products) from the mitochondria, thereby modulating the level of the important signal molecule H 2 S., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Endometrial epithelium exhibits a robust ion transport activity required for dynamical regulation of uterine fluid environment and thus embryo implantation. However, there still lacks a thorough understanding of the ion transport processes and regulatory mechanism in peri-implantation endometrial epithelium. As a gaseous signaling molecule or gasotransmitter, hydrogen sulfide (H 2 S) regulates a myriad of cellular and physiological processes in various tissues, including the modulation of ion transport proteins in epithelium. This study aimed to investigate the effects of H 2 S on ion transport across mouse endometrial epithelium and its possible role in embryo implantation. The existence of endogenous H 2 S in pregnant mouse uterus was tested by the detection of two key H 2 S-generating enzymes and measurement of H 2 S production rate in tissue homogenates. Transepithelial ion transport processes were electrophysiologically assessed in Ussing chambers on early pregnant mouse endometrial epithelial layers, demonstrating that H 2 S suppressed the anion secretion by blocking cystic fibrosis transmembrane conductance regulator (CFTR). H 2 S increased intracellular Cl - concentration ([Cl - ] i ) in mouse endometrial epithelial cells, which was abolished by pretreatment with the CFTR selective inhibitor CFTR inh -172. The cAMP level in mouse endometrial epithelial cells was not affected by H 2 S, indicating that H 2 S blocked CFTR in a cAMP-independent way. In vivo study showed that interference with H 2 S synthesis impaired embryo implantation. In conclusion, our study demonstrated that H 2 S inhibits the transepithelial anion secretion of early pregnant mouse endometrial epithelium via blockade of CFTR, contributing to the preparation for embryo implantation., (Copyright © 2019 Elsevier Inc. All rights reserved.)

S-type anion channels, which play important roles in plant anion (such as nitrate and chloride) transport, growth and development, abiotic stress responses and hormone signaling. However, there is far less information about this family in Rosaceae species. We performed a genome-wide analysis and identified SLAC/SLAH gene family members in pear (Pyrus bretschneideri) and four other species of Rosaceae. A total of 21 SLAC/SLAH genes were identified from the five Rosaceae species. Based on the structural characteristics and a phylogenetic analysis of these genes, the SLAC/SLAH gene family could be classified into three main groups. Transcriptome data demonstrated that PbrSLAC/SLAH genes were detected in all parts of the pear. PbrSLAC/SLAH genes were only located on the plasma membrane in transient expression experiments in Arabidopsis protoplasts cells. These results provide valuable information that increases our understanding of the evolution, expression and functions of the SLAC/SLAH gene family in higher plants., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The CLC family of proteins are involved in a variety of physiological processes to control cellular chloride concentration. Two distinct classes of CLC proteins, Cl - channels and Cl - /H + antiporters, have been functionally and structurally investigated over the last several decades. Previous studies have suggested that the conformational heterogeneity of the critical glutamate residue, Glu ex , could explain the transport cycle of CLC-type Cl - /H + antiporters. However, the presence of multiple conformations ( Up , Middle , and Down ) of the Glu ex has been suggested from combined structural snapshots of 2 different CLC antiporters: CLC-ec1 from Escherichia coli and cmCLC from a thermophilic red alga, Cyanidioschyzon merolae Thus, we aimed to investigate further the heterogeneity of Glu ex -conformations in CLC-ec1, the most deeply studied CLC antiporter, at both functional and structural levels. Here, we show that the crystal structures of the Glu ex mutant E148D and wild-type CLC-ec1 with varying anion concentrations suggest a structural intermediate, the " Midlow " conformation. We also found that an extra anion can be located above the external Cl - -binding site in the E148D mutant when the anion concentration is high. Moreover, we observed that a carboxylate in solution can occupy either the external or central Cl - -binding site in the ungated E148A mutant using an anomalously detectable short carboxylic acid, bromoacetate. These results lend credibility to the idea that the Glu ex can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter., Competing Interests: The authors declare no conflict of interest.

Reactive oxygen species (ROS) are central bioenergetic markers linked to aortic stenosis (AS) development and severity. We sought to evaluate the time course and impact of ROS assessed by plasmatic superoxide anion (SA) among patients undergoing transcatheter aortic valve replacement (TAVR). Among 106 patients, SA significantly decreased after TAVR. Dropped values were measured 10 min after TAVR (0.590 ± 0.181 vs. 0.648 ± 0.193; p  < 0.001) and persistent at 3 days (0.611 ± 0.0.228 vs. 0.646 ± 0.199; p  = 0.033) and 30 days follow-up (0.572 ± 0.207 vs. 0.639 ± 0.199; p  = 0.005). Increased baseline SA (>75 percentile) was continuously associated with higher postprocedural SA values 10 min after valve expansion ( p  < 0.001), at 3 days ( p  < 0.001) and 30 days ( p  < 0.001). Higher baseline SA was linked to higher inflammatory response assessed by higher C-reactive protein values at day 1 and day 3. The composite endpoint of all-cause mortality and/or stroke and/or pacemaker implantation and/or significant paravalvular aortic regurgitation ≥mild at 30 days did not differ significantly according to SA baseline values ( p  = 0.055). This is the first report identifying a decrease in oxidative stress level after TAVR. Our observation leads to the hypothesis that oxidative stress biomarkers may survive the journey from bench to bedside in AS and TAVR and become new biomarkers with both diagnostic and prognostic values. Antioxid. Redox Signal. 31, 420-426.

Active sites of enzymes are highly optimized for interactions with specific substrates, thus binding of opportunistic ligands is usually observed only in the absence of native substrates or products. However, during growth of crystals required for structure determination enzymes are often exposed to conditions significantly divergent from the native ones, leading to binding of unexpected ligands to active sites even in the presence of substrates. Failing to recognize this possibility may lead to incorrect interpretation of experimental results and to faulty conclusions. Here, we present several examples of binding of a citrate anion to the active sites of E. coli L-asparaginases I and II, even in the presence of the native substrate, L-Asn. A part of this report focuses on a comprehensive re-interpretation of structural results published previously for complexes of type I L-asparaginase (EcAI) from E. coli. In two re-refined structures a citrate anion forms an acyl-enzyme reaction intermediate with the catalytic threonine. These results emphasize the importance of careful and critical analysis during interpretation of crystallographic data.

Channelrhodopsins (ChRs) are algal light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity. ChRs desensitize under continuous bright-light illumination, resulting in a significant decline of photocurrents. Here we describe a metagenomically identified family of phylogenetically distinct anion-conducting ChRs (designated MerMAIDs). MerMAIDs almost completely desensitize during continuous illumination due to accumulation of a late non-conducting photointermediate that disrupts the ion permeation pathway. MerMAID desensitization can be fully explained by a single photocycle in which a long-lived desensitized state follows the short-lived conducting state. A conserved cysteine is the critical factor in desensitization, as its mutation results in recovery of large stationary photocurrents. The rapid desensitization of MerMAIDs enables their use as optogenetic silencers for transient suppression of individual action potentials without affecting subsequent spiking during continuous illumination. Our results could facilitate the development of optogenetic tools from metagenomic databases and enhance general understanding of ChR function.

Enzyme-mediated catalysis is attributed to enzyme-substrate interactions, with models such as "induced fit" and "conformational selection" emphasizing the role of protein conformational transitions. The dynamic nature of the protein structure, thus, plays a crucial role in molecular recognition and substrate binding. As large-scale protein motions are coupled to water motions, hydration dynamics play a key role in protein dynamics, and hence, in enzyme catalysis. Here, microfluidic techniques and time-dependent fluorescence Stokes shift (TDFSS) measurements are employed to elucidate the role of nanoscopic water dynamics in the interaction of an enzyme, α-Chymotrypsin (CHT), with a substrate, Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC) in the cationic reverse micelles of benzylhexadecyldimethylammonium chloride (BHDC/benzene) and anionic reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT/benzene). The kinetic pathways unraveled from the microfluidic setup are consistent with the "conformational selection" fit for the interaction of CHT with AMC in the cationic reverse micelles, whereas an "induced fit" mechanism is indicated for the anionic reverse micelles. In the cationic reverse micelles of BHDC, faster hydration dynamics (≈550 ps) aid the pathway of "conformational selection", whereas in the anionic reverse micelles of AOT, the significantly slower dynamics of hydration (≈1600 ps) facilitate an "induced fit" mechanism for the formation of the final enzyme-substrate complex. The role of water dynamics in dictating the mechanism of enzyme-substrate interaction becomes further manifest in the neutral reverse micelles of Brij-30 and Triton X-100. In the former, the faster water dynamics aid the "conformational selection" pathway, whereas the significantly slower dynamics of water molecules in the latter are conducive to the "induced fit" mechanism in the enzyme-substrate interaction. Thus, nanoscopic water dynamics act as a switch in modulating the pathway of recognition of an enzyme (CHT) by the substrate (AMC) in reverse micelles., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

TMEM16F is activated by elevated intracellular Ca 2+ , and functions as a small-conductance ion channel and as a phospholipid scramblase. In contrast to its paralogs, the TMEM16A/B calcium-activated chloride channels, mouse TMEM16F has been reported as a cation-, anion-, or non-selective ion channel, without a definite conclusion. Starting with the Q559K mutant that shows no current rundown and less outward rectification in excised patch, we found that the channel shifted its ion selectivity in response to the change of intracellular Ca 2+ concentration, with an increased permeability ratio of Cl - to Na + (P Cl- /P Na+ ) at a higher Ca 2+ level. The gradual shift of relative ion permeability did not correlate with the channel activation state. Instead, it was indicative of an alteration of electrostatic field in the permeation pathway. The dynamic change of ion selectivity suggests a charge-screening mechanism for TMEM16F ion conduction, and it provides hints to further studies of TMEM16F physiological functions., Competing Interests: WY, TH, MH, YJ, LJ No competing interests declared, (© 2019, Ye et al.)

Acid-sensing ion channels have important functions in physiology and pathology, but the molecular composition of acid-activated chloride channels had remained unclear. We now used a genome-wide siRNA screen to molecularly identify the widely expressed acid-sensitive outwardly-rectifying anion channel PAORAC/ASOR. ASOR is formed by TMEM206 proteins which display two transmembrane domains (TMs) and are expressed at the plasma membrane. Ion permeation-changing mutations along the length of TM2 and at the end of TM1 suggest that these segments line ASOR's pore. While not belonging to a gene family, TMEM206 has orthologs in probably all vertebrates. Currents from evolutionarily distant orthologs share activation by protons, a feature essential for ASOR's role in acid-induced cell death. TMEM206 defines a novel class of ion channels. Its identification will help to understand its physiological roles and the diverse ways by which anion-selective pores can be formed., Competing Interests: FU, SB, KL, TD, Jv, TJ No competing interests declared, (© 2019, Ullrich et al.)

One of the main functions of the kidney is to excrete an acid load derived from both dietary and endogenous sources, thus maintaining the pH of other fluids in the body. Urine pH is also of particular interest in stone formers, since it determines the presence of either calcium phosphate or uric acid content in stones. Others have noted in epidemiological studies a rise in incidence of low pH-dependent uric acid stones with age, coinciding with a decrease in the incidence of high pH-dependent phosphate stones. Taken together, these trends are suggestive of a longitudinal decline in urine pH in stone-forming patients, and, if true, this could explain the observed trends in stone incidence. We studied 7,891 stone formers, all of whom collected a 24-h urine sample and matching serum. Multivariate modeling revealed that urine pH did indeed fall with age and particularly between the ages of 20 and 50 yr old in both men and women. We sought to explain this trend through the inclusion of traditionally understood determinants of urine pH such as urinary buffers, estimates of glomerular filtration, and dietary acid load, but these, taken together, accounted for but a small fraction of the pH fall. Gastrointestinal anion absorption was the strongest predictor of urine pH in all age groups, as we have previously reported in middle-aged normal men and women. However, we found that, despite a decreasing urine pH, gastrointestinal anion absorption increased monotonically with age. In fact, after adjustment for gastrointestinal anion absorption, urine pH declined more markedly, suggesting that bicarbonate-producing anion absorption is regulated in a manner that offsets the decline of urine pH.

Xenin-25 is a neurotensin-like peptide that is secreted by enteroendocrine cells in the small intestine. Xenin-8 is reported to augment duodenal anion secretion by activating afferent neural pathways. The intrinsic neuronal circuits mediating the xenin-25-induced anion secretion were characterized using the Ussing-chambered, mucosa-submucosa preparation from the rat ileum. Serosal application of xenin-25 increased the short-circuit current in a concentration-dependent manner. The responses were abolished by the combination of Cl - -free and HCO 3 - -free solutions. The responses were almost completely blocked by TTX (10 -6 M) but not by atropine (10 -5 M) or hexamethonium (10 -4 M). The selective antagonists for neurotensin receptor 1 (NTSR1), neurokinin 1 (NK1), vasoactive intestinal polypeptide (VIP) receptors 1 and 2 (VPAC1 and VPAC2, respectively), and capsaicin, but not 5-hydroxyltryptamine receptors 3 and 4 (5-HT 3 and 5-HT 4 ), NTSR2, and A803467, inhibited the responses to xenin-25. The expression of VIP receptors ( Vipr ) in rat ileum was examined using RT-PCR. The Vipr1 PCR products were detected in the submucosal plexus and mucosa. Immunohistochemical staining showed the colocalization of NTSR1 and NK1 with substance P (SP)- and calbindin-immunoreactive neurons in the submucosal plexus, respectively. In addition, NK1 was colocalized with noncholinergic VIP secretomotor neurons. Based on the results from the present study, xenin-25-induced Cl - / HCO 3 - secretion is involved in NTSR1 activation on intrinsic and extrinsic afferent neurons, followed by the release of SP and subsequent activation of NK1 expressed on noncholinergic VIP secretomotor neurons. Finally, the secreted VIP may activate VPAC1 on epithelial cells to induce Cl - / HCO 3 - secretion in the rat ileum. Activation of noncholinergic VIP secretomotor neurons by intrinsic primary afferent neurons and extrinsic afferent neurons by postprandially released xenin-25 may account for most of the neurogenic secretory response induced by xenin-25. NEW & NOTEWORTHY This study is the first to investigate the intrinsic neuronal circuit responsible for xenin-25-induced anion secretion in the rat small intestine. We have found that nutrient-stimulated xenin-25 release may activate noncholinergic vasoactive intestinal polypeptide (VIP) secretomotor neurons to promote Cl - / HCO 3 - secretion through the activation of VIP receptor 1 on epithelial cells. Moreover, the xenin-25-induced secretory responses are mainly linked with intrinsic primary afferent neurons, which are involved in the activation of neurotensin receptor 1 and neurokinin 1 receptor.

Avian hepatitis E virus (HEV) is the main causative agent of big liver and spleen disease in chickens. Due to the absence of a highly effective cell culture system, there are few reports about the interaction between avian HEV and host cells. In this study, organic anion-transporting polypeptide 1A2 (OATP1A2) from chicken liver cells was identified to interact with ap237, a truncated avian HEV capsid protein spanning amino acids 313 to 549, by a glutathione S -transferase (GST) pulldown assay. GST pulldown and indirect enzyme-linked immunosorbent assays (ELISAs) further confirmed that the extracellular domain of OATP1A2 directly binds with ap237. The expression levels of OATP1A2 in host cells are positively correlated with the amounts of ap237 attachment and virus infection. The distribution of OATP1A2 in different tissues is consistent with avian HEV infection in vivo Finally, when the functions of OATP1A2 in cells are inhibited by its substrates or an inhibitor or blocked by ap237 or anti-OATP1A2 sera, attachment to and infection of host cells by avian HEV are significantly reduced. Collectively, these results displayed for the first time that OATP1A2 interacts with the avian HEV capsid protein and can influence viral infection in host cells. The present study provides new insight to understand the process of avian HEV infection of host cells. IMPORTANCE The process of viral infection is centered around the interaction between the virus and host cells. Due to the lack of a highly effective cell culture system in vitro , there is little understanding about the interaction between avian HEV and its host cells. In this study, a total of seven host proteins were screened in chicken liver cells by a truncated avian HEV capsid protein (ap237) in which the host protein OATP1A2 interacted with ap237. Overexpression of OATP1A2 in the cells can promote ap237 adsorption as well as avian HEV adsorption and infection of the cells. When the function of OATP1A2 in cells was inhibited by substrates or inhibitors, attachment and infection by avian HEV significantly decreased. The distribution of OATP1A2 in different chicken tissues corresponded with that in tissues during avian HEV infection. This is the first finding that OATP1A2 is involved in viral infection of host cells., (Copyright © 2019 American Society for Microbiology.)

Aim: Anion/cation symport across cellular membranes may lead to cell apoptosis and be developed as a strategy for new anticancer drug discovery. Methodology: Four aza-crown ether-squaramide conjugates were synthesized and characterized. Their anion recognition, anion/cation symport, cytotoxicity and probable mechanism of action were investigated in details. Conclusion: These conjugates are able to form ion-pairing complexes with chloride anions and facilitate the transmembrane transport of anions via an anion/cation symport process. They can disrupt the cellular homeostasis of chloride anions and sodium cations and induce the basification of acidic organelles in live cells. These conjugates exhibit moderate cytotoxicity toward the tested cancer cells and trigger cell apoptosis by mediating the influx of chloride anions and sodium cations into live cells.

Root hairs, as lateral extensions of epidermal cells, provide large absorptive surfaces to the root and are major actors in plant hydromineral nutrition. In contact with the soil they also constitute a site of interactions between the plant and rhizospheric microorganisms. In legumes, initiation of symbiotic interactions with N 2 -fixing rhizobia is often triggered at the root hair cell membrane in response to nodulation factors secreted by rhizobia, and involves early signaling events with changes in H + , Ca 2+ , K + and Cl - fluxes inducing transient depolarization of the cell membrane. Here, we aimed to build a functional repertoire of the major root hair conductances to cations and anions in the sequenced legume model Medicago truncatula. Five root hair conductances were characterized through patch-clamp experiments on enzymatically recovered root hair protoplasts. These conductances displayed varying properties of voltage dependence, kinetics and ion selectivity. They consisted of hyperpolarization- and depolarization-activated conductances for K + , cations or Cl - . Among these, one weakly outwardly rectifying cationic conductance and one hyperpolarization-activated slowly inactivating anionic conductance were not known as active in root hairs. All five conductances were detected in apical regions of young growing root hairs using membrane spheroplasts obtained by laser-assisted cell-wall microdissection. Combined with recent root hair transcriptomes of M. truncatula, this functional repertoire of conductances is expected to help the identification of candidate genes for reverse genetics studies to investigate the possible role of each conductance in root hair growth and interaction with the biotic and abiotic environment., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

In this study, our fundamental research interest was to understand how negatively charged porphyrins could interact with a plant cell wall and further act inside cells. Thus, three anionic porphyrins differing in their anionic external groups (carboxylates, sulfonates, and phosphonates) were tested. First, the tobacco cell wall was isolated to monitor in vitro its interactions with the three different anionic porphyrins. Unexpectedly, these negatively charged molecules were able to bind to the negatively charged cell wall probably by weak bonds such as hydrogen bonds and/or electrostatic interactions when the tetrapyrrolic core was protonated. Moreover, we showed that at the pH of spent culture medium (4.5), the neutrality of the carboxylated porphyrin (TPPC) facilitated its cell wall crossing while the diffusion of the two other sulfonated (TPPS) or phosphonated (TPPP) porphyrins that remained anionic was delayed. Once inside Tobacco Bright Yellow-2 (TBY-2) cells, TPPC induced higher levels of production of both H 2 O 2 and malondialdehyde compared to TPPS after illumination. That result correlated well with strong cell death induction by photoactivated TPPC. Furthermore, reactive oxygen species-scavenging enzymes such as catalase, peroxidases, and superoxide dismutase were also strongly downmodulated in response to TPPC, while these enzymes were almost unchanged in response to photoactivated TPPS. To the best of our knowledge, this is the first study that took into account the whole story from interactions of porphyrins with a plant cell wall to their photodynamic activity inside the cells.

Brachyspira spp. cause diarrheal disease in multiple animal species by colonization of the colon, resulting in colitis, mucus induction, and disrupted ion transport. Unique to spirochete pathogenesis is the immense production of mucus, resulting in a niche mucin environment likely favoring spirochete colonization. Mucin rheological properties are heavily influenced by anionic secretion, and loss of secretory function has been implicated in diseases such as cystic fibrosis. Here, the effects on the agonist-induced electrogenic anionic secretory response by infectious colonic spirochete bacteria Brachyspira hyodysenteriae and Brachyspira hampsonii were assessed in the proximal, apex, and distal sections of colon in Ussing chambers. Activation of secretion via isoproterenol, carbachol, and forskolin/3-isobutyl-1-methylxanthine demonstrated a significantly decreased change in short-circuit current ( I sc ) in Brachyspira-infected pigs in all sections. Tissue resistances did not account for this difference, rather, it was attributed to a decrease in anionic secretion as indicated by a decrease in bumetanide inhibitable I sc . Quantitative RT-PCR and Western blot analyses determined that the major anionic channels of the epithelium were downregulated in diarrheic pigs paired with altered mucin gene expression. The investigated cytokines were not responsible for the downregulation of anion channel gene transcripts. Although IL-1α was upregulated in all segments, it did not alter cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in Caco-2 monolayers. However, a whole cell Brachyspira hampsonii lysate significantly reduced CFTR mRNA expression in Caco-2 monolayers. Together, these findings indicate that these two Brachyspira spp. may directly cause a decreased anionic secretory response in the porcine colon, supporting an altered mucin environment likely favoring spirochete colonization. NEW & NOTEWORTHY This research demonstrates for the first time that the niche mucin environment produced by two infectious spirochete spp. is supported by a decrease in the electrogenic anionic secretory response throughout the porcine colon. Our findings suggest that the host's cytokine response is not likely responsible for the decrease in anionic secretory function. Rather, it appears that Brachyspira spp. directly impede ion channel transcription and translation, potentially altering colonic mucin rheological properties, which may favor spirochete colonization.

Escherichia coli RecA (EcRecA) forms discrete foci that cluster at cell poles during normal growth, which are redistributed along the filamented cell axis upon induction of the SOS response. The plasma membrane is thought to act as a scaffold for EcRecA foci, thereby playing an important role in RecA-dependent homologous recombination. In addition, in vivo and in vitro studies demonstrate that EcRecA binds strongly to the anionic phospholipids. However, there have been almost no data on the association of mycobacterial RecA proteins with the plasma membrane and the effects of membrane components on their function. Here, we show that mycobacterial RecA proteins specifically interact with phosphatidylinositol and cardiolipin among other anionic phospholipids; however, they had no effect on the ability of RecA proteins to bind single-stranded DNA. Interestingly, phosphatidylinositol and cardiolipin impede the DNA-dependent ATPase activity of RecA proteins, although ATP binding is not affected. Furthermore, the ability of RecA proteins to promote DNA strand exchange is not affected by anionic phospholipids. Strikingly, anionic phospholipids suppress the RecA-stimulated autocatalytic cleavage of the LexA repressor. The Mycobacterium smegmatis RecA foci localize to the cell poles during normal growth, and these structures disassemble and reassemble into several foci along the cell after the induction of DNA damage. Taken together, these data support the notion that the interaction of RecA with cardiolipin and phosphatidylinositol, the major anionic phospholipids of the mycobacterial plasma membrane, may be physiologically relevant, as they provide a scaffold for RecA storage and may regulate recombinational DNA repair and the SOS response.

Microbial sulfate reduction (SR) by sulfate-reducing micro-organisms (SRM) is a primary environmental mechanism of anaerobic organic matter mineralization, and as such influences carbon and sulfur cycling in many natural and engineered environments. In industrial systems, SR results in the generation of hydrogen sulfide, a toxic, corrosive gas with adverse human health effects and significant economic and environmental consequences. Therefore, there has been considerable interest in developing strategies for mitigating hydrogen sulfide production, and several specific inhibitors of SRM have been identified and characterized. Specific inhibitors are compounds that disrupt the metabolism of one group of organisms, with little or no effect on the rest of the community. Putative specific inhibitors of SRM have been used to control sulfidogenesis in industrial and engineered systems. Despite the value of these inhibitors, mechanistic and quantitative studies into the molecular mechanisms of their inhibition have been sparse and unsystematic. The insight garnered by such studies is essential if we are to have a more complete understanding of SR, including the past and current selective pressures acting upon it. Furthermore, the ability to reliably control sulfidogenesis - and potentially assimilatory sulfate pathways - relies on a thorough molecular understanding of inhibition. The scope of this review is to summarize the current state of the field: how we measure and understand inhibition, the targets of specific SR inhibitors and how SRM acclimatize and/or adapt to these stressors.

Superoxide anion production by the phagocyte NADPH oxidase plays a crucial role in host defenses and inflammatory reaction. The phagocyte NADPH oxidase is composed of cytosolic components (p40phox, p47phox, p67phox, and Rac1/2) and the membrane flavocytochrome b558, which is composed of two proteins: p22phox and gp91phox/NOX2. p22phox plays a crucial role in the stabilization of gp91phox in phagocytes and is also a docking site for p47phox during activation. In the current study, we have used a yeast two-hybrid approach to identify unknown partners of p22phox. Using the cytosolic C-terminal region of p22phox as bait to screen a human spleen cDNA library, we identified the protein interacting with amyloid precursor protein tail 1 (PAT1) as a potential partner of p22phox. The interaction between p22phox and PAT1 was further confirmed by in vitro GST pulldown and overlay assays and in intact neutrophils and COSphox cells by coimmunoprecipitation. We demonstrated that PAT1 is expressed in human neutrophils and monocytes and colocalizes with p22phox, as shown by confocal microscopy. Overexpression of PAT1 in human monocytes and in COSphox cells increased superoxide anion production and depletion of PAT1 by specific small interfering RNA inhibited this process. These data clearly identify PAT1 as a novel regulator of NADPH oxidase activation and superoxide anion production, a key phagocyte function., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Ionic liquids (ILs) are widely used in various industrial applications. However, they are considered potential toxins in aquatic environments because of their physical stability and solubility. The growth inhibition and oxidative stress induced by four ionic liquids with different cations and anions on the green algae Scenedesmus obliquus was investigated in this study. The order of growth inhibition was 1‑hexyl‑3‑methylimidazolium nitrate ([HMIM]NO 3 ) > 1‑hexyl‑3‑methylimidazolium chloride ([HMIM]Cl) > N‑hexyl‑3‑metylpyridinium bromide ([HMPy]Br) > N‑hexyl‑3‑metylpyridinium chloride ([HMPy]Cl). Imidazolium IL had a higher growth inhibition effect than pyridinium IL, nitrate IL and bromide IL had a higher effect than chloride IL. Reactive oxygen species (ROS) level in S. obliquus increased with increasing IL concentrations. Green fluorescence in [HMIM]Cl treated algae showed increased brightness compared to the [HMPy]Cl treatment, and [HMIM]NO 3 treatment produced increased brightness compared to the [HMPy]Br treatment, suggesting that higher ROS levels were induced by [HMIM]Cl and [HMIM]NO 3 . Soluble protein, catalase (CAT), and superoxide dismutase (SOD) activities were stimulated at lower concentrations but were inhibited at higher concentrations. Regression analysis suggested that ROS level is the main index responsible for oxidative stress induced by the four ILs. The ILs induced oxidative damage on S. obliquus, and ROS in high concentration treatments could not be effectively removed by the antioxidant system, leading to oxidative damage and ultimately resulting in growth inhibition and cell death., (Copyright © 2018 Elsevier B.V. All rights reserved.)

We investigated whether human serum albumin (HSA) in suspended human hepatocytes would affect the uptake clearance of anionic drugs with high binding to HSA and improve the extrapolation of in vivo hepatic clearance from in vitro uptake clearance by the hepatocytes via the "albumin-mediated" hepatic uptake mechanism. The uptake clearances for total forms ( PS inf ) and for unbound forms ( PS u,inf ) of 11 anionic drugs [all of which were organic anion-transporting polypeptide (OATP) substrates] were determined with suspended human hepatocytes in varying concentrations of HSA. The fraction of unbound drugs ( f u ) was determined using an equilibrium dialysis at the various HSA concentrations. The PS inf values decreased with increasing concentrations of HSA, whereas the unbound uptake clearances ( PS u,inf (+) = PS inf / f u ) in the presence of HSA increased substantially, thus demonstrating the "albumin-mediated" hepatic uptake mechanism. The relationships between PS inf and HSA concentration were well described by the previously proposed facilitated-dissociation model, in which the drug-albumin complex interacts with the cell surface, enhancing the dissociation of the complex and providing unbound drug for hepatic uptake. Furthermore, the PS u,inf (+) values in in vivo conditions (at 5% HSA) were predicted from those obtained in isolated hepatocytes on the basis of the facilitated-dissociation model, revealing compatibility with the overall hepatic intrinsic clearance in vivo. We conclude that the "facilitated-dissociation" model is useful for describing the "albumin-mediated" hepatic uptake phenomenon of OATP drugs and to predict hepatic uptake clearance in vivo., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Integral membrane proteins of the divalent anion/Na⁺ symporter (DASS) family are conserved from bacteria to humans. DASS proteins typically mediate the coupled uptake of Na⁺ ions and dicarboxylate, tricarboxylate, or sulfate. Since the substrates for DASS include key intermediates and regulators of energy metabolism, alterations of DASS function profoundly affect fat storage, energy expenditure and life span. Furthermore, loss-of-function mutations in a human DASS have been associated with neonatal epileptic encephalopathy. More recently, human DASS has also been implicated in the development of liver cancers. Therefore, human DASS proteins are potentially promising pharmacological targets for battling obesity, diabetes, kidney stone, fatty liver, as well as other metabolic and neurological disorders. Despite its clinical relevance, the mechanism by which DASS proteins recognize and transport anionic substrates remains unclear. Recently, the crystal structures of a bacterial DASS and its humanized variant have been published. This article reviews the mechanistic implications of these structures and suggests future work to better understand how the function of DASS can be modulated for potential therapeutic benefit.

A water-soluble curcumin lysinate incorporated into hydroxypropyl-β-cyclodextrin (NDS27) has been developed and shown anti-inflammatory properties but no comparative study has been made in parallel with its parent molecule, curcumin on polymorphonuclear neutrophils (PMNs) and myeloperoxidase (MPO) involved in inflammation. The effect of NDS27, its excipients (hydroxypropyl-β-cyclodextrin and lysine), curcumin lysinate and curcumin were compared on the release of superoxide anion by PMNs using a chemiluminescence assay and on the enzymatic activity of MPO. It was shown that curcumin and NDS27 exhibit similar inhibition activities on superoxide anion release by stimulated PMNs but also on MPO peroxidase and halogenation activities. The action mechanism of curcumin and NDS27 on the MPO activity was refined by stopped-flow and docking analyses. We demonstrate that both curcumin and NDS27 are reversible inhibitors of MPO by acting as excellent electron donors for redox intermediate Compound I (∼10 7  M -1  s -1 ) but not for Compound II (∼10 3  M -1  s -1 ) in the peroxidase cycle of the enzyme, thereby trapping the enzyme in the Compound II state. Docking calculations show that curcumin is able to enter the enzymatic pocket of MPO and bind to the heme cavity by π-stacking and formation of hydrogen bonds involving substituents from both aromatic rings. Hydroxypropyl-β-cyclodextrin is too bulky to enter MPO channel leading to the binding site suggesting a full release of curcumin from the cyclodextrin thereby allowing its full access to the active site of MPO. In conclusion, the hydroxypropyl-β-cyclodextrin of NDS27 enhances curcumin solubilization without affecting its antioxidant capacity and inhibitory activity on MPO., (Copyright © 2018 Elsevier B.V. All rights reserved.)

There are a number of mammalian anion channel types associated with cell volume changes. These channel types are classified into two groups: volume-activated anion channels (VAACs) and volume-correlated anion channels (VCACs). VAACs can be directly activated by cell swelling and include the volume-sensitive outwardly rectifying anion channel (VSOR), which is also called the volume-regulated anion channel; the maxi-anion channel (MAC or Maxi-Cl); and the voltage-gated anion channel, chloride channel (ClC)-2. VCACs can be facultatively implicated in, although not directly activated by, cell volume changes and include the cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, the Ca 2+ -activated Cl - channel (CaCC), and the acid-sensitive (or acid-stimulated) outwardly rectifying anion channel. This article describes the phenotypical properties and activation mechanisms of both groups of anion channels, including accumulating pieces of information on the basis of recent molecular understanding. To that end, this review also highlights the molecular identities of both anion channel groups; in addition to the molecular identities of ClC-2 and CFTR, those of CaCC, VSOR, and Maxi-Cl were recently identified by applying genome-wide approaches. In the last section of this review, the most up-to-date information on the pharmacological properties of both anion channel groups, especially their half-maximal inhibitory concentrations (IC 50 values) and voltage-dependent blocking, is summarized particularly from the standpoint of pharmacological distinctions among them. Future physiologic and pharmacological studies are definitely warranted for therapeutic targeting of dysfunction of VAACs and VCACs., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

NADPH oxidases (NOX) are transmembrane enzymes, which catalyze the formation of reactive oxygen species (ROS). In humans and most mammals, the NOX family comprises seven members, namely, NOX1-5 and the dual oxidases DUOX1 and 2. The primary product of most NOX isoforms is the superoxide radical anion O 2 ċ- , which is rapidly dismutated in hydrogen peroxide (H 2 O 2 ), while NOX4 and DUOX mostly generate H 2 O 2. ROS are multifunctional molecules in tissues, and NOX-derived ROS cellular functions are as diverse as microbial killing (NOX2), thyroid hormone synthesis (DUOX2), or otoconia formation in the inner ear (NOX3). NOX are potential pharmacological targets in numerous diseases such as diabetes, fibrosis, and brain ischemia, and NOX inhibitors are currently under development. Here we describe two cellular assays to detect extracellular O 2 ċ- and H 2 O 2 in cells overexpressing specific NOX isoforms and their subunits.

The cystic fibrosis transmembrane conductance regulator (CFTR) belongs to the ATP binding cassette (ABC) transporter superfamily but functions as an anion channel crucial for salt and water transport across epithelial cells. CFTR dysfunction, because of mutations, causes cystic fibrosis (CF). The anion-selective pore of the CFTR protein is formed by its two transmembrane domains (TMDs) and regulated by its cytosolic domains: two nucleotide binding domains (NBDs) and a regulatory (R) domain. Channel activation requires phosphorylation of the R domain by cAMP-dependent protein kinase (PKA), and pore opening and closing (gating) of phosphorylated channels is driven by ATP binding and hydrolysis at the NBDs. This review summarizes available information on structure and mechanism of the CFTR protein, with a particular focus on atomic-level insight gained from recent cryo-electron microscopic structures and on the molecular mechanisms of channel gating and its regulation. The pharmacological mechanisms of small molecules targeting CFTR's ion channel function, aimed at treating patients suffering from CF and other diseases, are briefly discussed.

Hydroethidine is a fluorogenic probe that in the presence of the superoxide radical anion is oxidized to a red fluorescent product, 2-hydroxyethidium. In cells, hydroethidine is also oxidized to other products, including red fluorescent ethidium. Thus, selective monitoring of 2-hydroxyethidium is required for specific detection of the superoxide radical anion. Here, we provide protocols for HPLC- and LC-MS-based quantitation of 2-hydroxyethidium, among other oxidation products. Also, a protocol for continuous sampling for real-time monitoring of superoxide production using rapid HPLC measurements of 2-hydroxyethidium is described.

The main objective of this study was to determine how feeding different dietary calcium (Ca) concentrations in combination with a negative dietary cation-anion difference (DCAD) would affect the cow's response to induced hypocalcemia. We conducted an experiment with multiparous, nonlactating, nonpregnant Holstein cows fed a negative DCAD (average -18.2 across all diets) for 21 d with low (LC; 0.45% Ca; n = 5), medium (MC; 1.13% Ca; n = 6), or high (HC; 2.02% Ca; n = 6) concentrations of dietary Ca. Urine and blood samples were collected and urine pH measured daily during the 21-d feeding period prior to hypocalcemia challenge. Cows were then subjected to a controlled induction of hypocalcemia to determine how dietary Ca intake affected the response to a hypocalcemia challenge. On days 22, 23, and 24, hypocalcemia was induced with an intravenous infusion of 5% EGTA in 2 different cows from each treatment daily. During infusion, blood samples were collected every 15 min until 60% of prechallenge ionized calcium (iCa) concentrations were achieved. Samples were collected postinfusion at 0, 2.5, 5, 10, 15, 30, and every 30 min thereafter until 90% of prechallenge iCa was reached. Blood pH, hematocrit, and serum total Ca (tCa), sodium (Na), potassium (K), phosphorous (P), magnesium (Mg), and serotonin did not differ (P > 0.05) among treatments during the feeding period. Blood iCa (P = 0.04) and glucose (P = 0.03) were significantly elevated in HC compared with LC and MC cows during the feeding period. Urine pH was less than 6.0 in all cows, but was lowest in LC (P = 0.02) compared with MC and HC cows during the feeding period. Urine Ca, P, Mg, and deoxypyridinoline did not differ among treatments (P > 0.05). Cows fed HC maintained higher concentrations of iCa (P = 0.03) during the challenge period than MC (P = 0.04), and LC (P = 0.004), and required a longer time to reach 60% of whole blood iCa, and required more EGTA to reach 60% iCa than MC or LC cows (P = 0.01). Serum tCa decreased in all cows during infusion (P < 0.0001) but did not differ among treatments. Serotonin concentrations were elevated in MC cows compared with HC and LC cows during EGTA infusion (P = 0.05), suggesting an interdependent relationship between iCa and serotonin. Cows fed HC had a slower rate of decrease in iCa, but not tCa, when induced with hypocalcemia, indicating potential metabolic benefits of feeding higher dietary Ca in combination with a negative DCAD.

The barrier function of the human epidermis is constantly challenged by environmental osmotic fluctuations. Hypotonic stress triggers cell swelling, which is counteracted by a compensatory mechanism called regulatory volume decrease (RVD) involving volume-regulated anion channels (VRACs). Recently, it was discovered that VRACs are composed of LRRC8 heteromers and that LRRC8A functions as the essential VRAC subunit in various mammalian cell types; however, the molecular identity of VRACs in the human epidermis remains to be determined. Here, we investigated the expression of LRRC8A and its role in hypotonic stress response of human keratinocytes. Immunohistological staining showed that LRRC8A is preferentially localized in basal and suprabasal epidermal layers. RNA sequencing revealed that LRRC8A is the most abundant subunit within the LRRC8 gene family in HaCaT cells as well as in primary normal human epidermal keratinocytes (NHEKs). To determine the contribution of LRRC8A to hypotonic stress response, we generated HaCaT- and NHEK-LRRC8A knockout cells by using CRISPR-Cas9. I - influx assays using halide-sensitive YFP showed that LRRC8A is crucially important for mediating VRAC activity in HaCaTs and NHEKs. Moreover, cell volume measurements using calcein-AM dye further revealed that LRRC8A also substantially contributes to RVD. In summary, our study provides new insights into hypotonic stress response and suggests an important role of LRRC8A as VRAC component in human keratinocytes., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Optogenetic silencing allows time-resolved functional interrogation of defined neuronal populations. However, the limitations of inhibitory optogenetic tools impose stringent constraints on experimental paradigms. The high light power requirement of light-driven ion pumps and their effects on intracellular ion homeostasis pose unique challenges, particularly in experiments that demand inhibition of a widespread neuronal population in vivo. Guillardia theta anion-conducting channelrhodopsins (GtACRs) are promising in this regard, due to their high single-channel conductance and favorable photon-ion stoichiometry. However, GtACRs show poor membrane targeting in mammalian cells, and the activity of such channels can cause transient excitation in the axon due to an excitatory chloride reversal potential in this compartment. Here, we address these problems by enhancing membrane targeting and subcellular compartmentalization of GtACRs. The resulting soma-targeted GtACRs show improved photocurrents, reduced axonal excitation and high light sensitivity, allowing highly efficient inhibition of neuronal activity in the mammalian brain.

The passive membrane permeability of drugs may be potentially predicted by phospholipid-water sorption coefficient (K PLIPW ) through immobilized artificial membrane (IAM) chromatography. However, for predominantly ionized compounds, unexpected confounding electrostatic interaction would influence the accurate determination of K PLIPW , accompanied by the mobile phase with varied pH and ionic strength. In order to measure the intrinsic phospholipid-water sorption coefficient (K IAM,intr ) for diverse analytes, a mathematic model by characterizing the confounding electrostatic effects was developed in this study. Based on the developed electrostatic model, additional electrostatic repulsion/attraction interactions with the charged IAM surface were characterized for organic cations/anions. Considering that most of the IAM chromatography data was determined under physiological conditions, the K PLIPW values of all compounds were calculated with the ionic strength of 0.1 M and the pH value of 7. Generally, K PLIPW was hardly affected by the pH and ionic strength of mobile phase for those electroneutral compounds. K IAM,intr was 0.28 log units smaller than apparent phospholipid-water sorption coefficient (K IAM,app ) for cations. In contrast, K IAM,intr was larger than K IAM,app by 0.28 log units for anions. Herein, K IAM,intr of diverse analytes can be easily calculated by the developed electrostatic model. For application, K IAM,intr was further compared with intrinsic Caco-2 permeability, and good sigmoidal correlations (R 2  = 0.76) were established between them for organic cations and neutral compounds but not for organic anions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The basolateral membrane anion conductance of the retinal pigment epithelium (RPE) is a key component of the transepithelial Cl - transport pathway. Although multiple Cl - channels have been found to be expressed in the RPE, the components of the resting Cl - conductance have not been identified. In this study, we used the patch-clamp method to characterize the ion selectivity of the anion conductance in isolated mouse RPE cells and in excised patches of RPE basolateral and apical membranes. Relative permeabilities ( P A / P Cl ) calculated from reversal potentials measured in intact cells under bi-ionic conditions were as follows: SCN - >> ClO 4 - > [Formula: see text] > I - > Br - > Cl - >> gluconate. Relative conductances ( G A / G Cl ) followed a similar trend of SCN - >> ClO 4 - > [Formula: see text] > I - > Br - ≈Cl - >> gluconate. Whole cell currents were highly time-dependent in 10 mM external SCN - , reflecting collapse of the electrochemical potential gradient due to SCN - accumulation or depletion intracellularly. When the membrane potential was held at -120 mV to minimize SCN - accumulation in cells exposed to 10 mM SCN - , the instantaneous current reversed at -90 mV, revealing that P SCN / P Cl is approximately 500. Macroscopic current recordings from outside-out patches demonstrated that both the basolateral and apical membranes exhibit SCN - conductances, with the basolateral membrane having a larger SCN - current density and higher relative permeability for SCN - . Our results suggest that the RPE basolateral and apical membranes contain previously unappreciated anion channels or electrogenic transporters that may mediate the transmembrane fluxes of SCN - and Cl - .

Natural flavonoids are ubiquitous in dietary plants and vegetables and have been proposed to have antiviral, antioxidant, cardiovascular protective, and anticancer effects. Volume-regulated anion channels (VRACs), which are essential for cell volume regulation, have been proposed to play a key role in cell proliferation and migration, apoptosis, transepithelial transport, and cancer development. In this study, we screened a group of 53 structurally related natural flavonoids and three synthetic flavonoids for their inhibitory activities on VRAC currents. A whole-cell patch technique was used to record VRAC currents in the human embryonic kidney (HEK) 293 and human umbilical vein endothelial (HUVEC) cells. The 5'-bromo-2-deoxyuridine (BrdU) assay technique was used to investigate cell proliferation. At 100 μM, 34 of 53 compounds significantly inhibited hypotonic extrasolution-induced VRAC currents by > 50% in HEK293 cells. Among these compounds, luteolin, baicalein, eupatorin, galangin, quercetin, fisetin, karanjin, Dh-morin, genistein, irisolidone, and prunetin exhibited the highest efficacy for VRAC blockade (the mean inhibition > 80%) with IC 50 s of 5-13 μM and E max s of about 87-99%. We also studied the effects of three synthetic flavonoids on VRAC currents in HEK293 cells. Flavoxate showed high inhibition efficacy toward VRAC currents (IC 50  = 2.3 ± 0.3 μM; E max  = 91.8% ± 2.7%). Finally, these flavonoids inhibited endogenous VRAC currents and cell proliferation in endothelial cells. This study demonstrates that natural and synthetic flavonoids are potent VRAC current inhibitors, and VRAC inhibition by flavonoids might be responsible for their anti-angiogenic effects.