Proton Extrusion Research Articles

Genetic requirements for Staphylococcus aureus nitric oxide resistance and virulence.

Staphylococcus aureus exhibits many defenses against host innate immunity, including the ability to replicate in the presence of nitric oxide (NO·). S. aureus NO· resistance is a complex trait and hinges on the ability of this pathogen to metabolically adapt to the presence of NO·. Here, we employed deep sequencing of transposon junctions (Tn-Seq) in a library generated in USA300 LAC to define the complete set of genes required for S. aureus NO· resistance. We compared the list of NO·-resistance genes to the set of genes required for LAC to persist within murine skin infections (SSTIs). In total, we identified 168 genes that were essential for full NO· resistance, of which 49 were also required for S. aureus to persist within SSTIs. Many of these NO·-resistance genes were previously demonstrated to be required for growth in the presence of this immune radical. However, newly defined genes, including those encoding SodA, MntABC, RpoZ, proteins involved with Fe-S-cluster repair/homeostasis, UvrABC, thioredoxin-like proteins and the F1F0 ATPase, have not been previously reported to contribute to S. aureus NO· resistance. The most striking finding was that loss of any genes encoding components of the F1F0 ATPase resulted in mutants unable to grow in the presence of NO· or any other condition that inhibits cellular respiration. In addition, these mutants were highly attenuated in murine SSTIs. We show that in S. aureus, the F1F0 ATPase operates in the ATP-hydrolysis mode to extrude protons and contribute to proton-motive force. Loss of efficient proton extrusion in the ΔatpG mutant results in an acidified cytosol. While this acidity is tolerated by respiring cells, enzymes required for fermentation cannot operate efficiently at pH ≤ 7.0 and the ΔatpG mutant cannot thrive. Thus, S. aureus NO· resistance requires a mildly alkaline cytosol, a condition that cannot be achieved without an active F1F0 ATPase enzyme complex.

Modulations in extracellular calcium lead to H+-ATPase-dependent acid secretion: a clarification of PPI failure.

The H+,K+-ATPase was identified as the primary proton secretory pathway in the gastric parietal cell and is the pharmacological target of agents suppressing acid secretion. Recently, we identified a second acid secretory protein expressed in the parietal cell, the vacuolar H+-ATPase (V-type ATPase). The aim of the present study was to further characterize H+-ATPase activation by modulations in extracellular calcium via the calcium sensing receptor (CaSR). Isolated gastric glands were loaded with the pH indicator dye BCECF-AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester] to measure intracellular pH. Experiments were conducted in the absence of sodium and potassium to monitor H+-ATPase-specific transport activity. CaSR was activated with the calcimimetic R568 (400 nM) and/or by modulations in extracellular Ca2+. Elevation in calcium concentrations increased proton extrusion from the gastric parietal cell. Allosteric modification of the CaSR via R568 and calcium increased vacuolar H+-ATPase activity significantly (ΔpH/minlowCa2+(0.1mM) = 0.001 ± 0.001, ΔpH/minnormalCa2+(1.0mM) = 0.033 ± 0.004, ΔpH/minhighCa2+(5.0mM) = 0.051 ± 0.005). Carbachol significantly suppressed calcium-induced gastric acid secretion via the H+-ATPase under sodium- and potassium-free conditions. We conclude that the V-type H+-ATPase is tightly linked to CaSR activation. We observed that proton pump inhibitor (PPI) exposure does not modulate H+-ATPase activity. This elevated blood calcium activation of the H+-ATPase could provide an explanation for recurrent reflux symptoms while taking a PPI therapy. NEW & NOTEWORTHY This study emphasizes the role of the H+-ATPase in acid secretion. We further demonstrate the modification of this proton excretion pathway by extracellular calcium and the activation of the calcium sensing receptor CaSR. The novelty of this paper is based on the modulation of the H+-ATPase via both extracellular Ca (activation) and the classical secretagogues histamine and carbachol (inactivation). Both activation and inactivation of this proton pump are independent of PPI modulation.

Mapping the Physiological Response of Oenococcus oeni to Ethanol Stress Using an Extended Genome-Scale Metabolic Model.

The effect of ethanol on the metabolism of Oenococcus oeni, the bacterium responsible for the malolactic fermentation (MLF) of wine, is still scarcely understood. Here, we characterized the global metabolic response in O. oeni PSU-1 to increasing ethanol contents, ranging from 0 to 12% (v/v). We first optimized a wine-like, defined culture medium, MaxOeno, to allow sufficient bacterial growth to be able to quantitate different metabolites in batch cultures of O. oeni. Then, taking advantage of the recently reconstructed genome-scale metabolic model iSM454 for O. oeni PSU-1 and the resulting experimental data, we determined the redistribution of intracellular metabolic fluxes, under the different ethanol conditions. Four growth phases were clearly identified during the batch cultivation of O. oeni PSU-1 strain, according to the temporal consumption of malic and citric acids, sugar and amino acids uptake, and biosynthesis rates of metabolic products – biomass, erythritol, mannitol and acetic acid, among others. We showed that, under increasing ethanol conditions, O. oeni favors anabolic reactions related with cell maintenance, as the requirements of NAD(P)+ and ATP increased with ethanol content. Specifically, cultures containing 9 and 12% ethanol required 10 and 17 times more NGAM (non-growth associated maintenance ATP) during phase I, respectively, than cultures without ethanol. MLF and citric acid consumption are vital at high ethanol concentrations, as they are the main source for proton extrusion, allowing higher ATP production by F0F1-ATPase, the main route of ATP synthesis under these conditions. Mannitol and erythritol synthesis are the main sources of NAD(P)+, countervailing for 51–57% of its usage, as predicted by the model. Finally, cysteine shows the fastest specific consumption rate among the amino acids, confirming its key role for bacterial survival under ethanol stress. As a whole, this study provides a global insight into how ethanol content exerts a differential physiological response in O. oeni PSU-1 strain. It will help to design better strategies of nutrient addition to achieve a successful MLF of wine.

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters.

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

Identification of Heterotrophic Zinc Mobilization Processes among Bacterial Strains Isolated from Wheat Rhizosphere (Triticum aestivum L.).

Soil and plant inoculation with heterotrophic zinc-solubilizing bacteria (ZSB) is considered a promising approach for increasing zinc (Zn) phytoavailability and enhancing crop growth and nutritional quality. Nevertheless, it is necessary to understand the underlying bacterial solubilization processes to predict their repeatability in inoculation strategies. Acidification via gluconic acid production remains the most reported process. In this study, wheat rhizosphere soil serial dilutions were plated on several solid microbiological media supplemented with scarcely soluble Zn oxide (ZnO), and 115 putative Zn-solubilizing isolates were directly detected based on the formation of solubilization halos around the colonies. Eight strains were selected based on their Zn solubilization efficiency and siderophore production capacity. These included one strain of Curtobacterium, two of Plantibacter, three strains of Pseudomonas, one of Stenotrophomonas, and one strain of Streptomyces In ZnO liquid solubilization assays, the presence of glucose clearly stimulated organic acid production, leading to medium acidification and ZnO solubilization. While solubilization by Streptomyces and Curtobacterium was attributed to the accumulated production of six and seven different organic acids, respectively, the other strains solubilized Zn via gluconic, malonic, and oxalic acids exclusively. In contrast, in the absence of glucose, ZnO dissolution resulted from proton extrusion (e.g., via ammonia consumption by Plantibacter strains) and complexation processes (i.e., complexation with glutamic acid in cultures of Curtobacterium). Therefore, while gluconic acid production was described as a major Zn solubilization mechanism in the literature, this study goes beyond and shows that solubilization mechanisms vary among ZSB and are strongly affected by growth conditions.IMPORTANCE Barriers toward a better understanding of the mechanisms underlying zinc (Zn) solubilization by bacteria include the lack of methodological tools for isolation, discrimination, and identification of such organisms. Our study proposes a direct bacterial isolation procedure, which prevents the need to screen numerous bacterial candidates (for which the ability to solubilize Zn is unknown) for recovering Zn-solubilizing bacteria (ZSB). Moreover, we confirm the potential of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a quick and accurate tool for the identification and discrimination of environmental bacterial isolates. This work also describes various Zn solubilization processes used by wheat rhizosphere bacteria, including proton extrusion and the production of different organic acids among bacterial strains. These processes were also clearly affected by growth conditions (i.e., solid versus liquid cultures and the presence and absence of glucose). Although highlighted mechanisms may have significant effects at the soil-plant interface, these should only be transposed cautiously to real ecological situations.

A comparison of the effects of 1,4-naphthoquinone and 2-hydroxy-1,4-naphthoquinone (lawsone) on indole-3-acetic acid (IAA)-induced growth of maize coleoptile cells

The effects of 1,4-naphthoquinone (NQ) and 2-hydroxy-1,4-naphthoquinone (NQ-2-OH) on indole-3-acetic acid (IAA)-induced growth, medium pH changes and membrane potential (Em) in maize (Zea mays L.) coleoptile cells were determined. In addition, the redox cycling properties of both naphthoquinones were also compared. The dose-response curves constructed for the effects of NQ and NQ-2-OH on endogenous and IAA-induced growth differ in shape. It was found that NQ was by 10–50% more effective in inhibiting IAA-induced growth in maize coleoptile segments than NQ-2-OH. Simultaneous measurements of growth and external medium pH indicated that NQ and NQ-2-OH reduced or eliminated proton extrusion at all of the concentrations used, excluding NQ at 1 µM. It was found that both naphthoquinones at concentrations higher than 10 µM caused the depolarisation of the membrane potential (Em). Additionally, compared to the controls, NQ- and NQ-2-OH-exposure of coleoptile segments, at concentrations higher than 10 µM, caused an elevation of the hydrogen peroxide (H2O2) production and plasma membrane redox activity. The highest catalase activity was observed at 10 µM NQ and it was ca. 18-fold greater (at 4 h) than in the control medium. Moreover, it was also found that NQ and NQ-2-OH, at all concentrations studied, increased the malondialdehyde content of coleoptile segments at 4 h of the experiment. The data presented here are discussed taking into account the “acid growth hypothesis” of auxin action and the mechanisms by which naphthoquinones interact with biological systems.

Bicarbonate Recycling by HIF-1-Dependent Carbonic Anhydrase Isoforms 9 and 12 Is Critical in Maintaining Intracellular pH and Viability of Nucleus Pulposus Cells.

Intervertebral disc degeneration is a ubiquitous condition closely linked to chronic low-back pain. The health of the avascular nucleus pulposus (NP) plays a crucial role in the development of this pathology. We tested the hypothesis that a network comprising HIF-1α, carbonic anhydrase (CA) 9 and 12 isoforms, and sodium-coupled bicarbonate cotransporters (NBCs) buffer intracellular pH through coordinated bicarbonate recycling. Contrary to the current understanding of NP cell metabolism, analysis of metabolic-flux data from Seahorse XF analyzer showed that CO2 hydration contributes a significant source of extracellular proton production in NP cells, with a smaller input from glycolysis. Because enzymatic hydration of CO2 is catalyzed by plasma membrane-associated CAs we measured their expression and function in NP tissue. NP cells robustly expressed isoforms CA9/12, which were hypoxia-inducible. In addition to increased mRNA stability under hypoxia, we observed binding of HIF-1α to select hypoxia-responsive elements on CA9/12 promoters using genomic chromatin immunoprecipitation. Importantly, in vitro loss of function studies and analysis of discs from NP-specific HIF-1α null mice confirmed the dependency of CA9/12 expression on HIF-1α. As expected, inhibition of CA activity decreased extracellular acidification rate independent of changes in HIF activity or lactate/H+ efflux. Surprisingly, CA inhibition resulted in a concomitant decrease in intracellular pH that was mirrored by inhibition of sodium-bicarbonate importers. These results suggested that extracellular bicarbonate generated by CA9/12 is recycled to buffer cytosolic pH fluctuations. Importantly, long-term intracellular acidification from CA inhibition lead to compromised cell viability, suggesting that plasma-membrane proton extrusion pathways alone are not sufficient to maintain homeostatic pH in NP cells. Taken together, our studies show for the first time that bicarbonate buffering through the HIF-1α-CA axis is critical for NP cell survival in the hypoxic niche of the intervertebral disc. © 2017 American Society for Bone and Mineral Research.

Adenosine: Direct and Indirect Actions on Gastric Acid Secretion.

Composed by a molecule of adenine and a molecule of ribose, adenosine is a paradigm of recyclable nucleoside with a multiplicity of functions that occupies a privileged position in the metabolic and regulatory contexts. Adenosine is formed continuously in intracellular and extracellular locations of all tissues. Extracellular adenosine is a signaling molecule, able to modulate a vast range of physiologic responses in many cells and organs, including digestive organs. The adenosine A1, A2A, A2B, and A3 receptors are P1 purinergic receptors, G protein-coupled proteins implicated in tissue protection. This review is focused on gastric acid secretion, a process centered on the parietal cell of the stomach, which contains large amounts of H+/K+-ATPase, the proton pump responsible for proton extrusion during acid secretion. Gastric acid secretion is regulated by an extensive collection of neural stimuli and endocrine and paracrine agents, which act either directly at membrane receptors of the parietal cell or indirectly through other regulatory cells of the gastric mucosa, as well as mechanic and chemic stimuli. In this review, after briefly introducing these points, we condense the current body of knowledge about the modulating action of adenosine on the pathophysiology of gastric acid secretion and update its significance based on recent findings in gastric mucosa and parietal cells in humans and animal models.

Elucidation of antimicrobial activity and mechanism of action by N-substituted carbazole derivatives

Compounds belonging to a carbazole series have been identified as potent fungal plasma membrane proton adenosine triphophatase (H+-ATPase) inhibitors with a broad spectrum of antifungal activity. The carbazole compounds inhibit the adenosine triphosphate (ATP) hydrolysis activity of the essential fungal H+-ATPase, thereby functionally inhibiting the extrusion of protons and extracellular acidification, processes that are responsible for maintaining high plasma membrane potential. The compound class binds to and inhibits the H+-ATPase within minutes, leading to fungal death after 1–3h of compound exposure in vitro. The tested compounds are not selective for the fungal H+-ATPase, exhibiting an overlap of inhibitory activity with the mammalian protein family of P-type ATPases; the sarco(endo)plasmic reticulum calcium ATPase (Ca2+-ATPase) and the sodium potassium ATPase (Na+,K+-ATPase). The ion transport in the P-type ATPases is energized by the conversion of ATP to adenosine diphosphate (ADP) and phosphate and a general inhibitory mechanism mediated by the carbazole derivative could therefore be blocking of the active site. However, biochemical studies show that increased concentrations of ATP do not change the inhibitory activity of the carbazoles suggesting they act as allosteric inhibitors. Furthermore decreased levels of intracellular ATP would suggest that the compounds inhibit the H+-ATPase indirectly, but Candida albicans cells exposed to potent H+-ATPase-inhibitory carbazoles result in increased levels of intracellular ATP, indicating direct inhibition of H+-ATPase.

The F0F1 ATP synthase regulates human neutrophil migration through cytoplasmic proton extrusion coupled with ATP generation

Cytoplasmic alkalinization and extracellular adenosine triphosphate (ATP) signals are required for migration of chemokineactivated neutrophils, but the precise functions remain unclear. In this work, the effect of the plasma membrane-expressed F0F1-ATP synthase (FATPase) on human neutrophils was examined. We found F-ATPase to be involved in cytoplasm proton extrusion and extracellular ATP generation. Oligomycin A, an F-ATPase inhibitor that blocks proton transfer, inhibited cytoplasmic alkalinization, extracellular ATP generation, adhesion and chemotaxis in N-formyl-Met-Leu-Phe (fMLP)-stimulated neutrophils; however, adenosine diphosphate (ADP), a substrate and activator of F-ATPase, had the opposite effect. Further analysis revealed that cell surface F-ATPase can translocate to the leading edge of directional fMLP-stimulated neutrophils toward ADP hydrolyzed from pannexin 1 channel-released ATP, followed by F-ATPase-catalyzed ATP regeneration using ADP and protons transferred from the cytoplasm. Therefore, the membrane-expressed F-ATPase regulates human neutrophil migration via cytoplasm proton extrusion and extracellular ATP generation.

Reactivation of electron flow in chloroplasts of in vitro shootlets of apple through elimination of carbon source and evaluation of its activity by inhibitors of electron transport chain and chlorophyll fluorescence quenching

Electron transport chains (ETC) are drastically inactivated in chloroplasts of in vitro shootlets due to presence of carbon source in the growth media. This inactivation consequently creates limitations for in vitro studies on the role of chloroplasts in biotic and abiotic stresses. This research was carried out to evaluate reactivation of electron flow in chloroplasts ETC by elimination of carbon source and subsequent comparison of the effects of ETC inhibitors in the presence and absence of carbon source (i.e., sucrose) on the in vitro shootlets of apple rootstock, MM-111. All the tested ETC inhibitors, including uracil; glutaraldehyde, methyl viologen (MV) and DCMU triggered necrosis appearance on the in vitro apple shootlets exclusively on the carbon source-free media. Moreover, exposure of the in vitro shootlets to the various concentrations of ETC inhibitors demonstrated different severity of ETC inhibition by these inhibitors and their concentration-dependent effects on chloroplasts ETC. Uracil, the inhibitor of photosystem II, triggered the weakest shootlets necrosis, while DCMU exhibited no necrogenic effects at 50 mg/L and lower concentrations. MV, contrary to glutaraldehyde, did not have necrogenesis effects at 1 mg/L, but both of them caused sharp necrosis trigger on the shootlets as soon as 48–96 h after exposure to their effective concentrations. Evaluation of the shootlets H+-ATPase activity revealed that proton extrusion and pH decline in the growth media were mainly associated with the sucrose uptake by the shootlets, while no considerable pH decline observed in the presence of carbon source in the media. Finally, chloroplasts ETC activity of the shootlets was confirmed by higher percentage of O2 and less CO2 quantity in the atmospheres of the jars containing sucrose-free media, as well as by higher initial fluorescence (Fo) and maximum fluorescence (Fm) quenching parameters in the shootlets, in comparison with the sucrose-enriched condition.

Onion (Allium cepa L.) peel extract (OPE) regulates human sperm motility via protein kinase C-mediated activation of the human voltage-gated proton channel.

Onion (Allium cepa L.) and quercetin protect against oxidative damage and have positive effects on multiple functional parameters of spermatozoa, including viability and motility. However, the associated underlying mechanisms of action have not yet been identified. The aim of this study was to investigate the effect of onion peel extract (OPE) on voltage-gated proton (Hv1) channels, which play a critical role in rapid proton extrusion. This process underlies a wide range of physiological processes, particularly male fertility. The whole-cell patch-clamp technique was used to record the changes in Hv1 currents in HEK293 cells transiently transfected with human Hv1 (HVCN1). The effects of OPE on human sperm motility were also analyzed. OPE significantly activated the outward-rectifying proton currents in a concentration-dependent manner, with an EC50 value of 30μg/mL. This effect was largely reversible upon washout. Moreover, OPE induced an increase in the proton current amplitude and decreased the time constant of activation at 0 mV from 4.9±1.7 to 0.6±0.1sec (n=6). In the presence of OPE, the half-activation voltage (V1/2 ) shifted in the negative direction, from 20.1±5.8 to 5.2±8.7 mV (n=6), but the slope was not significantly altered. The OPE-induced current was profoundly inhibited by 10μm Zn2+ , the most potent Hv1 channel inhibitor, and was also inhibited by treatment with GF109203X, a specific protein kinase C (PKC) inhibitor. Furthermore, sperm motility was significantly increased in the OPE-treated groups. OPE exhibits protective effects on sperm motility, at least partially via regulation of the proton channel. Moreover, similar effects were exerted by quercetin, the major flavonoid in OPE. These results suggest OPE, which is rich in the potent Hv1 channel activator quercetin, as a possible new candidate treatment for human infertility.

Production of a potential liquid plant bio-stimulant by immobilized Piriformospora indica in repeated-batch fermentation process

Piriformospora indica, a mycorrhizal-like fungus able to establish associations with roots of a wide range of plants, supporting plant nutrition and increasing plant resistance and tolerance to stress, was shown to solubilise phosphate applied in the form of animal bone char (HABO) in fermentation systems. The process of P solubilisation was caused most likely by proton extrusion and medium pH lowering. The fungal mycelium was successfully immobilized/retained in a polyurethane foam carrier. Further employment of the immobilized mycelium in repeated-batch fermentation process resulted in at least 5 cycles of P solubilization. The concentration of soluble P increased during the experiment with 1.0 and 3.0 g HABO l−1 and at the end of the 5th batch cycle reached 40.8 and 120 mg l−1, respectively. The resulting final liquid product, without or with solubilized phosphate, was found to significantly increase plant growth and P plant uptake. It can be used as a biostimulant containing microbial plant growth-promoting substances and soluble P derived from renewable sources (HABO) thus supporting the development of sustainable agro-ecosystems.

Effect of quinoline based 1,2,3-triazole and its structural analogues on growth and virulence attributes of Candida albicans.

Candida albicans, along with some other non-albicans Candida species, is a group of yeast, which causes serious infections in humans that can be both systemic and superficial. Despite the fact that extensive efforts have been put into the discovery of novel antifungal agents, the frequency of these fungal infections has increased drastically worldwide. In our quest for the discovery of novel antifungal compounds, we had previously synthesized and screened quinoline containing 1,2,3-triazole (3a) as a potent Candida spp inhibitor. In the present study, two structural analogues of 3a (3b and 3c) have been synthesized to determine the role of quinoline and their anti-Candida activities have been evaluated. Preliminary results helped us to determine 3a and 3b as lead inhibitors. The IC50 values of compound 3a for C. albicans ATCC 90028 (standard) and C. albicans (fluconazole resistant) strains were 0.044 and 2.3 μg/ml, respectively while compound 3b gave 25.4 and 32.8 μg/ml values for the same strains. Disk diffusion, growth and time kill curve assays showed significant inhibition of C. albicans in the presence of compounds 3a and 3b. Moreover, 3a showed fungicidal nature while 3b was fungistatic. Both the test compounds significantly lower the secretion of proteinases and phospholipases. While, 3a inhibited proteinase secretion in C. albicans (resistant strain) by 45%, 3b reduced phospholipase secretion by 68% in C. albicans ATCC90028 at their respective MIC values. Proton extrusion and intracellular pH measurement studies suggested that both compounds potentially inhibit the activity of H+ ATPase, a membrane protein that is crucial for various cell functions. Similarly, 95–97% reduction in ergosterol content was measured in the presence of the test compounds at MIC and MIC/2. The study led to identification of two quinoline based potent inhibitors of C. albicans for further structural optimization and pharmacological investigation.

Effects of elevated nutrients and CO2 emission scenarios on three coral reef macroalgae

Coral reef macroalgae are expected to thrive in the future under conditions that are deleterious to the health of reef-building corals. Here we examined how macroalgae would be affected by exposure to future CO2 emission scenarios (pCO2 and temperature), enriched nutrients and combinations of both. The species tested, Laurencia intricata (Rhodophyta), Turbinaria ornata and Chnoospora implexa (both Phaeophyceae), have active carbon-concentrating mechanisms but responded differently to the treatments. L. intricata showed high mortality under nutrient enriched RCP4.5 (“reduced” CO2 emission) and RCP8.5 (“business-as-usual” CO2 emission) and grew best under pre-industrial (PI) conditions, where it could take up carbon using external carbonic anhydrase combined, potentially, with proton extrusion. T. ornata’s growth rate showed a trend for reduction under RCP8.5 but was unaffected by nutrient enrichment. In C. implexa, highest growth was observed under PI conditions, but highest net photosynthesis occurred under RCP8.5, suggesting that under RCP8.5, carbon is stored and respired at greater rates while it is directed to growth under PI conditions. None of the species showed growth enhancement under future scenarios, nutrient enrichment or combinations of both. This leads to the conclusion that under such conditions these species are unlikely to pose an increasing threat to coral reefs.

Triple-edged therapy targeting intracellular alkalosis and extracellular acidosis in cancer.

Extracellular acidity and intracellular alkalinity are two of the characteristics hallmarks of malignant cells and their environment. This involves an inversion of the extracellular/intracellular pH gradient when compared with normal cells and it gives malignant cells proliferative and invasive advantages. Thus, the reversal of the pH gradient is a legitimate objective in the treatment of cancer and may be accomplished with drugs already used for other purposes and/or with specific new drugs that are currently being studied. The aim of this review is to describe a triple approach for reversing this gradient inversion using the concerted utilization of proton extrusion inhibitors, mitochondrial poisons and lysosomal poisons that should act synergistically through different mechanisms. The scheme presented here is compatible with almost all the chemotherapeutic protocols currently being used.

Na+/H+ exchanger-mediated hydrogen ion extrusion as a carcinogenic signal in triple-negative breast cancer etiopathogenesis and prospects for its inhibition in therapeutics.

Breast cancer is the leading cause of cancer-related death in women in Europe and North America, and metastasis is the primary cause of fatality in patients with breast cancer. While some breast cancers are quite treatable, the triple-negative breast cancers are more metastatic and resistant to chemotherapy. There is clearly an urgent need for better treatments for this form of the disease. Breast cancer is characterized by genetically complex intra-tumour heterogeneity, particularly within the triple-negative clinical subtype. This complicates treatment options, so the development of specifically targeted chemotherapy for less treatable forms is critical. Dysregulation of pH homeostasis is a common factor in breast tumour cells. This occurs in concert with a metabolic switch to aerobic glycolysis that occurs at the onset of oncogenic transformation. The Na+/H+ exchanger isoform 1 (NHE1) is the major pH regulatory protein involved in the increased proton extrusion of breast cancer cells. Its increased activity results in intracellular alkalinisation and extracellular acidification that drives cancer progression. The acidification of the extracellular tumour microenvironment also contributes to the development of chemotherapy resistance. In this review, we outline the role of H+ as a carcinogenic signal and the role and regulation of NHE1 as a trigger for metastasis. We review recent evidence supporting the use of pharmacological inhibitors of NHE1 as a viable treatment option for triple-negative breast cancer.

Anatomical and Physiological Responses of Four Quinoa Cultivars to Salinity at Seedling Stage

Objectives: Salinity is one of the most important environmental stress factors that limits both seed germination and seedling develop. Seedling stage is the most susceptible stage of plants to salt stress. The aim of this work is to study the effect of salt stress on anatomical and physiological features of cotyledons and roots of the seedlings of four quinoa cultivars (Witulla, Wariponcho, Pasankalla and CICA). Methods/ Statistical Analysis: Seed germination and seedling growth was carried out under 0 and 200 mM NaCl during 10 days. Root length and diameter, and root proton extrusion were measured. Soluble sugars and proline concentrations were determined in both cotyledons and roots. Anatomical attributes of cotyledons (adaxial and abaxial stomatal area and density, palisade and spongy cell layers, palisade cell density) and roots (stele diameter and surface, metaxylem vessel diameter) were also analyzed. Findings: Salt stress caused physiological and anatomical changes in cotyledons and roots. Sucrose, glucose and fructose concentrations were more affected in cotyledons than in roots of salt-treated seedlings. Proline accumulation was more affected in roots. Saline stress affected differentially the anatomical attributes of quinoa cultivars. Application/Improvements: Quinoa seedlings when subjected to salt stress are able to develop a set of anatomical, physiological and biochemical adaptive mechanisms which seem to occur at different combinations among quinoa cultivars, but giving similar abilities to cope with the excess of salt and survive under extreme conditions.

Acute Effects of Vitamin C Exposure On Colonic Crypts: Direct Modulation of pH Regulation

Background/Aim: Colorectal cancer is still considered a leading cause of death in the United States and worldwide. One potential way to improve survival besides detection is to look to new therapeutic agents that can be taken prophylactically to reduce the risk of tumor formation. For cancer cells to grow and invade, a higher (more alkaline) intracellular pH must occur. We chose to examine a specific nutraceutical agent, which is Vitamin C. The acute effect of Vitamin C exposure on normal colonic crypts has been studied, providing some insight into how Vitamin C achieve its effect. Methods: Distal colon was excised from rats. Following enzymatic digestion single colonic crypts were isolated. Colonic crypts were loaded with pH sensitive dye to measure the intracellular pH changes. Crypts were exposed to solutions +/- Vitamin C. Results: 10 mM Vitamin C decreased Na⁺-dependent intracellular pH recovery. Vitamin C modulates SVCT leading to changes in proton extrusion. Vitamin C entry occurs via either SVCT2 on the basolateral membrane or by transcellular passive diffusion through tight junctions to the apical membrane and then active transport via SVCT1. Conclusion: Acute addition of Vitamin C to the basolateral membrane maintains low intracellular pH for a longer period which could halt and/or prevent tumor formation.

Biochemical and molecular mechanisms associated with Zn deficiency tolerance and signaling in rice (Oryza sativa L.)

ABSTRACTIn this study, zinc (Zn) deficiency caused a significant reduction in growth parameters and tissue Zn concentrations in BRRI 33 (sensitive) but not in Pokkali (tolerant). The increase of proton extrusion in both genotypes under high pH suggests that it gets triggered as a common consequence of reducing pH and solubilization of Zn. Real-time PCR showed pronounced upregulation of OsZIP4, OsDMAS1, OsNAS2 and OsPCS1 in Zn-deficient roots of Pokkali, and to a lesser extent in BRRI 33 only for OsZIP4 and OsPCS1. This suggests that OsDMAS1, OsNAS2 and OsPCS1 functions as secondary consequences leading to higher chelation and uptake of Zn under Zn deficiency in Pokkali. Further, a major increase in CAT, POD, SOD, GR and key metabolites suggests that high antioxidant defense plays a critical role in Zn deficiency tolerance in Pokkali. Further, Pokkali self-grafts and plants having Pokkali rootstock combined with BRRI 33 scion showed no significant decline in plant height, root dry matter and Zn concentration along with upregulation of Zn transporters (OsZIP4 and OsIRT1) under Zn deficiency, suggesting that signal driving mechanisms for Zn deficiency tolerance mechanisms are generated in the root and Zn-inefficient BRRI 33 is not capable of producing signals or sensing them.