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Abstract: Sphingosine-1-phosphate (S1P) was found previously to inhibit [Na.sup.+]-[K.sup.+] ATPase in HepG2 cells. Whether fingolimod (FTY720), a S1P receptor (S1PR) agonist, similarly inhibits the ATPase is a question that needs [...]

Guttation is one of several visible physiological events of plant kingdom. The mechanism of this phenomenon is however, obscure and embedded deep into mysteries despite its vital significance for the plants and people. In this paper attempts have been made, in the light of recent discoveries, and new and novel findings, to review the past and present works reflecting on an integrated view of the mechanism of this phenomenon and its regulation. In this context, the gene transfer technology and other modern developments in instrumentation and experimentation have enabled to a large extent the understanding of the mechanism of plant secretion of liquid having solutes, both organic and inorganic, dissolved in it. In all probability, this process seems to originate primarily on account of root pressure that involves osmotic withdrawal and transport of water from growth medium and subsequently activities of living cells of xylem parenchyma at the expense of energy fuelled by ATP leading to energetically uphill co-transport of water and solutes in xylem vessels building hydrostatic pressure in the roots pushing the saps upward (Wegner in Journal of Experimental Botany 65:381-393, 2014; Singh 2016). This process though genetically governed, appears to be triggered and regulated by the interaction of several internal, external and edaphic factors finely-tuned by chemico-mechanosensors such as pH gradient, hormones, mechanical loading, turgor potential, membrane potential, temperature, light, touch, sound etc. influencing cellular contractile proteins and gating of aquaporins. The positive hydrostatic pressure so developed is, in turn, transmitted via stem to the leaves as release of water droplets of guttation through permanently open hydathodes located at the tips, margins, adaxial and abaxial surfaces of uninjured leaves. Keywords Abscisic acid * Aquaporins * Chemico-mechanosensory signal * Contractile proteins * Co-transport of water * [H.sup.+]-ATPases * Metabolic mechanism * Osmotic mechanism * Root pressure, CONTENTS Abstract I. Introduction II. Mode of Guttation III. Mechanism of Guttation: Chain of Events 1. Vital force theory of sap movement and exudation 2. Physical force theory of sap [...]

ATP is the dominant energy source in animals for mechanical and electrical work (for example, muscle contraction or neuronal firing). For chemical work, there is an equally important role for NADPH, which powers redox defence and reductive biosynthesis (1). The most direct route to produce NADPH from glucose is the oxidative pentose phosphate pathway, with malic enzyme sometimes also important (2,3). Although the relative contribution of glycolysis and oxidative phosphorylation to ATP production has been extensively analysed, similar analysis of NADPH metabolism has been lacking. Here we demonstrate the ability to directly track, by liquid chromatography-mass spectrometry, the passage of deuterium from labelled substrates into NADPH, and combine this approach with carbon labelling and mathematical modelling to measure NADPH fluxes. In proliferating cells, the largest contributor to cytosolic NADPH is the oxidative pentose phosphate pathway. Surprisingly, a nearly comparable contribution comes from serine-driven one-carbon metabolism, in which oxidation of methylene tetrahydrofolate to 10-formyl-tetrahydrofolate is coupled to reduction of [NADP.sup.+] to NADPH. Moreover, tracing of mitochondrial one-carbon metabolism revealed complete oxidation of 10-formyl-tetrahydrofolate to make NADPH. As folate metabolism has not previously been considered an NADPH producer, confirmation of its functional significance was undertaken through knockdown of methylenetetrahydrofolate dehydrogenase (MTHFD) genes. Depletion of either the cytosolic or mitochondrial MTHFD isozyme resulted in decreased cellular NADPH/[NADP.sup.+] and reduced/oxidized glutathione ratios (GSH/GSSG) and increased cell sensitivity to oxidative stress. Thus, although the importance of folate metabolism for proliferating cells has been long recognized and attributed to its function ofproducing one-carbon units for nucleic acid synthesis, another crucial function of this pathway is generating reducing power., Previous examination of NADPH production during cell growth has analysed metabolic fluxes in cells using [sup.13]C and [sup.14]C isotope tracers (4-7). For NADPH metabolism, however, carbon tracers alone are insufficient, [...]

The aim of this project was to investigate the interaction between the calcium-sensing receptor (CaSR) and proton extrusion by the V-ATPase and gastric-like isoform of the [H.sup.+]/[K.sup.+]-ATPase in the mouse nephron. Biochemical activity of [H.sup.+]-ATPases was analysed using a partially purified membrane fraction of mouse cortex and outer medullary region. The V-ATPase activity (sensitive to [10.sup.-7] mol x [L.sup.-1] bafilomycin) from the cortical and outer medullary region was significantly stimulated by increasing the [Ca.sup.2+.sub.o] (outside [Ca.sup.2+]), in a dose-dependent pattern. Gastric [H.sup.+]/[K.sup.+]-ATPase activity (sensitive to [10.sup.-5] mol x [L.sup.-1] Schering 28080) was also sensitive to changes in [Ca.sup.2+.sub.o] levels. A significant increase in V-ATPase activity was also observed when CaSR was stimulated with agonists such as 300 µmol x [L.sup.-1] [Gd.sup.3+] and 200 µmol x [L.sup.-1] neomycin, both in the cortex and outer medulla. The cortical and outer medullary gastric [H.sup.+]/[K.sup.+]-ATPase activity was also stimulated by [Gd.sup.3+] and neomycin. Finally, cortical V-ATPase activity was significantly stimulated by [10.sup.-9] mol x [L.sup.-1] angiotensin II, and the stimulation of CaSR in the presence of angiotensin significantly enhanced this effect, suggesting that an interaction in the intracellular signaling pathways is involved. In summary, CaSR stimulation enhances the biochemical activity of V-ATPase and gastric [H.sup.+]/[K.sup.+]-ATPase in both the cortical and outer medullary region of mouse kidney. Key words: mice, V-type proton ATPase, [H.sup.+]/[K.sup.+] ATPase, angiotensin II, CaSR (calcium-sensing receptor). Le but de ce projet etait d'examiner l'interaction du recepteur de detection de calcium CaSR (acronyme de Calcium-Sensing Receptor) avec l'extrusion de protons par la V-ATPase et l'isoforme de type gastrique de la [H.sup.+]/[K.sup.+]-ATPase dans le nephron de la souris. L'activite biochimique des [H.sup.+]-ATPases a ete mesuree sur une fraction membranaire partiellement purifiee du cortex et de la medulla externe du rein de souris. L'activite de la V-ATPase (activite sensible a [10.sup.-7] mol x [L.sup.-1] de bafilomycine) du cortex et de la medulla externe etait significativement stimulee par l'augmentation de [Ca.sup.2+.sub.e] ([Ca.sup.2+] externe), en fonction de la concentration. L'activite de la [H.sup.+]/[K.sup.+]-ATPase gastrique (activite sensible au compose Schering 28080 [10.sup.-5] mol x [L.sup.-1)] etait aussi sensible au changement de [Ca.sup.2+.sub.e]. Une augmentation significative de l'activite de la V-ATPase etait aussi observee lorsque le CaSR etait stimule par des agonistes comme 300 µmol x [L.sup.-1] de [Gd.sup.3+] et 200 µmol x [L.sup.-1] de neomycine, tant dans le cortex que dans la medulla externe. L'activite corticale et medullaire externe de la [H.sup.+]/[K.sup.+]-ATPase gastrique etait aussi stimulee par le [Gd.sup.3+] et la neomycine. Finalement, l'activite de la V-ATPase corticale etait significativement stimulee par [10.sup.-9] mol x [L.sup.-1] d'angiotensine II, et la stimulation du CaSR en presence d'angiotensine augmentait significativement cet effet, suggerant l'existence d'une interaction dans les sentiers de signalisation intracellulaire impliques. En resume, la stimulation du CaSR augmente l'activite biochimique de la V- ATPase et de la [H.sup.+]/[K.sup.+]-ATPase gastrique dans le cortex et la medulla externe du rein de la souris. [Traduit par la Redaction] Mots-cles: souris, ATPase a proton de type V, [H.sup.+]/[K.sup.+]-ATPase, angiotensine II, CaSR (Calcium-Sensing Receptor)., Introduction Systemic calcium homeostasis is critical to many cellular processes in multicellular organisms, including hormone secretion, muscle contraction, nerve conduction, exocytosis, and enzymatic reactions. In addition, calcium serves as an [...]

Eukaryotic ribosomes are assembled by a complex pathway that extends from the nucleolus to the cytoplasm and is powered by many energy-consuming enzymes (1-3). Nuclear export is a key, irreversible step in pre-ribosome maturation (4-8), but mechanisms underlying the timely acquisition of export competence remain poorly understood. Here we show that a conserved Saccharomyces cerevisiae GTPase Nug2 (also known as Nog2, and as NGP-1, GNL2 or nucleostemin 2 in human (9)) has a key role in the timing of export competence. Nug2 binds the inter-subunit face of maturing, nucleoplasmic pre-60S particles, and the location clashes with the position of Nmd3, a key pre-60S export adaptor (10). Nug2 and Nmd3 are not present on the same pre-60S particles, with Nug2 binding before Nmd3. Depletion of Nug2 causes premature Nmd3 binding to the pre-60S particles, whereas mutations in the G-domain of Nug2 block Nmd3 recruitment, resulting in severe 60S export defects. Two pre60S remodelling factors, the Real ATPase and its co-substrate Rsa4, are present on Nug2-associated particles, and both show synthetic lethal interactions with nug2 mutants. Release of Nug2 from pre60S particles requires both its [K.sup.+]-dependent GTPase activity and the remodelling ATPase activity of Real. We conclude that Nug2 is a regulatory GTPase that monitors pre-60S maturation, with release from its placeholder site linked to recruitment of the nuclear export machinery. CEN, LEU2, [P.sub. Ref. (21) & This Study GAL1-10]-GFP-realDAA This Study, The conserved GTPase Nug2 (Extended Data Fig. 1) is associated with several pre-60S particles located in the nucleoplasm, but was not detected on particles with a known cytoplasmic location (see [...]

The conversion of chemical energy into mechanical force by AAA+ (ATPases associated with diverse cellular activities) ATPases is integral to cellular processes, including DNA replication, protein unfolding, cargo transport and [...]

Abstract Gastroesophageal reflux disorder (GERD), a common disorder in the Western world, can lead to complications that include esophageal stricture and esophageal adenocarcinoma. Multiple challenges are associated with GERD treatment. [...]

The sarcoplasmic reticulum [Ca.sup.2+]-ATPase transports two [Ca.sup.2+] per ATP hydrolyzed from the cytoplasm to the lumen against a large concentration gradient. During transport, the pump alters the affinity and accessibility for [Ca.sup.2+] by rearrangements of transmembrane helices. In this study, all-atom molecular dynamics simulations were performed for wild-type [Ca.sup.2+]-ATPase in the [Ca.sup.2+]-bound form and the Gin mutants of Glu771 and Glu908. Both of them contribute only one carboxyl oxygen to site I [Ca.sup.2+], but only Glu771GIn completely looses the [Ca.sup.2+]-binding ability. The simulations show that: (i) For Glu771GIn, but not Glu908GIn, coordination of [Ca.sup.2+] was critically disrupted. (ii) Coordination broke at site II first, although Glu771 and Glu908 only contribute to site I. (iii) A water molecule bound to site I [Ca.sup.2+] and hydrogen bonded to Glu771 in wild-type, drastically changed the coordination of [Ca.sup.2+] in the mutant. (iv) Water molecules flooded the binding sites from the lumenal side. (v) The side chain conformation of Ile775, located at the head of a hydrophobic cluster near the lumenal surface, appears critical for keeping out bulk water. Thus the simulations highlight the importance of the water molecule bound to site I [Ca.sup.2+] and point to a strong relationship between [Ca.sup.2+]-coordination and shielding of bulk water, providing insights into the mechanism of gating of ion pathways in cation pumps. sarco(endo)plasmic reticulum [Ca.sup.2+]-ATPase | ion pump | mutation doi/ 10.1073/pnas.1015819107

The Na/K pump is a P-type ATPase that exchanges three intracellular [Na.sup.+] ions for two extracellular [K.sup.+] ions through the plasma-lemma of nearly all animal cells. The mechanisms involved in cation selection by the pump's ion-binding sites (site I and site II bind either [Na.sup.+] or [K.sup.+]; site III binds only [Na.sup.+]) are poorly understood. We studied cation selectivity by outward-facing sites (high [K.sup.+] affinity) of Na/K pumps expressed in Xenopus oocytes, under voltage clamp. [Guanidinium.sup.+] , [methylguanidinium.sup.+], and [aminoguanidinium.sup.+] produced two phenomena possibly reflecting actions at site III: (i) voltage-dependent inhibition (VDI) of outwardly directed pump current at saturating [K.sup.+], and (ii) induction of pump-mediated, guanidinium-derivative-carried inward current at negative potentials without [Na.sup.+] and [K.sup.+]. In contrast, [formamidinium.sup.+] and [acetamidinium.sup.+] induced [K.sup.+]-like outward currents. Measurement of ouabain-sensitive ATPase activity and radiolabeled cation uptake confirmed that these cations are external [K.sup.+] congeners. Molecular dynamics simulations indicate that bound organic cations induce minor distortion of the binding sites. Among tested metals, only [Li.sup.+] induced [Na.sup.+]-like VDI, whereas all metals tested except [Na.sup.+] induced [K.sup.+]-like outward currents. Pump-mediated [K.sup.+]like organic cation transport challenges the concept of rigid structural models in which ion specificity at site I and site II arises from a precise and unique arrangement of coordinating ligands. Furthermore, actions by [guanidinium.sup.+] derivatives suggest that [Na.sup.+] binds to site III in a hydrated form and that the inward current observed without external [Na.sup.+] and [K.sup.+] represents cation transport when normal occlusion at sites I and II is impaired. These results provide insights on external ion selectivity at the three binding sites. ion selectivity | Na,K-ATPase | pump current | voltage-dependent | guanidinium doi/ 10.1073/pnas.1004214107

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.05.490 Byline: Limor Daniel, Nir Etkovitz, Shirel Rosenwasser Weiss, Sara Rubinstein, Debby Ickowicz, Haim Breitbart Keywords: Acrosome reaction; Calcium; Kinases; Signal transduction; Sperm capacitation; Ouabain Abstract: The sperm acrosome reaction occurs after the binding of the capacitated sperm to the egg zona pellucida. This study describes a novel mode of regulation of the sperm epidermal growth factor receptor (EGFR) under physiological conditions and its relevance to the acrosome reaction. Ouabain, a known Na/K ATPase blocker is present in the blood and in the female reproductive tract. We show here that physiological concentrations (nM) of ouabain enhance phosphorylation of EGFR on tyr-845, stimulate Ca.sup.2+ influx and induce the acrosome reaction in sperm. These effects could be seen only in the presence of very low concentrations of EGF (0.1ng/ml or 0.016 nM) added together with nano-molar ouabain. Phosphorylation, Ca.sup.2+ influx, and the acrosome reaction are inhibited by an EGFR blocker, suggesting that trans-activation of the EGFR is involved. Moreover, our data revealed that protein kinase A and the family of tyrosine kinase, SRC, shown before to be involved in EGFR activation in sperm, mediate the acrosome reaction induced by ouabain. Ouabain alone (without EGF) at relatively high concentration (10A[micro]M) could enhance EGFR phosphorylation, Ca.sup.2+ influx and acrosome reaction, and these processes were inhibited by EGFR blockers. Moreover, we show here that PKA and SRC family are involved in the activation of EGFR by 10A[micro]M ouabain, further demonstrating that ouabain induces the acrosome reaction by a mechanism mediated by the trans-activation of EGFR. In conclusion, this study describes an interesting regulatory path of EGFR by physiological concentrations of ouabain and EGF found in the female reproductive tract. Neither of these compounds can activate the EGFR alone at such low physiological levels; however, when both are present, the interaction of ouabain with the Na/K ATPase leads to the priming of the EGFR, which undergoes its full activation by EGF. Author Affiliation: The Mina and Everard Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel Article History: Received 7 January 2010; Revised 25 March 2010; Accepted 15 May 2010 Article Note: (footnote) [star] Funding: This work was supported by the IHEL Foundation to H.B.

Interactions of the three transported Na ions with the Na/K pump remain incompletely understood. Na/K pump crystal structures show that the extended C terminus of the Na,K--adenosine triphosphatase (ATPase) [alpha] subunit directly contacts transmembrane helices. Deletion of the last five residues (KETYY in almost all Na/K pumps) markedly lowered the apparent affinity for Na activation of pump phosphorylation from ATP, a reflection of cytoplasmic Na affinity for forming the occluded E1P(Na3) conformation. ATPase assays further suggested that C-terminal truncations also interfere with low affinity Na interactions, which are attributable to extracellular effects. Because extracellular Na ions traverse part of the membrane's electric field to reach their binding sites in the Na/K pump, their movements generate currents that can be monitored with high resolution. We report here electrical measurements to examine how Na/K pump interactions with extracellular Na ions are influenced by C-terminal truncations. We deleted the last two (YY) or five (KESYY) residues in Xenopus laevis [alpha]1 Na/K pumps made ouabain resistant by either of two kinds of point mutations and measured their currents as 10-mM ouabain--sensitive currents in Xenopus oocytes after silencing endogenous Xenopus Na/K pumps with 1 [micro]M ouabain. We found the low affinity inhibitory influence of extracellular Na on outward Na/K pump current at negative voltages to be impaired in all of the C-terminally truncated pumps. Correspondingly, voltage jump--induced transient charge movements that reflect pump interactions with extracellular Na ions were strongly shifted to more negative potentials; this signals a several-fold reduction of the apparent affinity for extracellular Na in the truncated pumps. Parallel lowering of Na affinity on both sides of the membrane argues that the C-terminal contacts provide important stabilization of the occluded E1P(Na3) conformation, regardless of the route of Na ion entry into the binding pocket. Gating measurements of palytoxin-opened Na/K pump channels additionally imply that the C-terminal contacts also help stabilize pump conformations with occluded K ions. doi/10.1085/jgp.201010407

Solar disinfection (SODIS) is used as an effective and inexpensive tool to improve the microbiological quality of drinking water in developing countries where no other means are available. Solar UVA light is the agent that inactivates bacteria during the treatment. Damage to bacterial membranes plays a crucial role in the inactivation process. This study showed that even slightly irradiated cells (after less than 1 h of simulated sunlight) were strongly affected in their ability to maintain essential parts of their energy metabolism, in particular of the respiratory chain (activities of NADH oxidase, succinate oxidase and lactate oxidase were measured). The cells' potential to generate ATP was also strongly inhibited. Many essential enzymes of carbon metabolism (glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase and malate dehydrogenase) and defence against oxidative stress (catalases and glutathione-disulfide reductase) were reduced in their activity during SODIS. The work suggests that damage to membrane enzymes is a likely cause of membrane dysfunction (loss of membrane potential and increased membrane permeability) during UVA irradiation. In this study, the first targets on the way to cell death were found to be the respiratory chain and [F.sub.1][F.sub.0] ATPase. DOI 10.1099/mic.0.038471-0

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important pathogen and a causative agent of gastroenteritis. During infection, S. Typhimurium assembles molecular-needle complexes termed type III secretion (T3S) systems to translocate effector proteins from the bacterial cytoplasm directly into the host cell. The T3S signals that direct the secretion of effectors still remain enigmatic. SopD is a key T3S effector contributing to the systemic virulence of S. Typhimurium and the development of gastroenteritis. We have scrutinized the distribution of the SopD T3S signals using in silico analysis and a targeted deletion approach. We show that amino acid residues 6-10 act as the N-terminal secretion signal for Salmonella pathogenicity island 1 (SPI-1) T3S. Furthermore, we show that two putative C-terminal helical regions of SopD are essential for its secretion and also help prevent erroneous secretion through the flagellar T3S machinery. In addition, using protein-protein interaction assays, we have identified an association between SopD and the SPI-1 T3S system ATPase, InvC. These findings demonstrate that T3S of SopD involves multiple signals and protein interactions, providing important mechanistic insights into effector protein secretion. DOI 10.1099/mic.0.038117-0