Proton Pumping Activity Research Articles

Modulating Liposome Surface Charge for Maximized ATP Regeneration in Synthetic Nanovesicles.

In vitro reconstructed minimal respiratory chains are powerful tools to investigate molecular interactions between the different enzyme components and how they are influenced by their environment. One such system is the coreconstitution of the terminal cytochrome bo3 oxidase and the ATP synthase from Escherichia coli into liposomes, where the ATP synthase activity is driven through a proton motive force (pmf) created by the bo3 oxidase. The proton pumping activity of the bo3 oxidase is initiated using the artificial electron mediator short-chain ubiquinone and electron source DTT. Here, we extend this system and use either complex II or NDH-2 and succinate or NADH, respectively, as electron entry points employing the natural long-chain ubiquinone Q8 or Q10. By testing different lipid compositions, we identify that negatively charged lipids are a prerequisite to allow effective NDH-2 activity. Simultaneously, negatively charged lipids decrease the overall pmf formation and ATP synthesis rates. We find that orientation of the bo3 oxidase in liposomal membranes is governed by electrostatic interactions between enzyme and membrane surface, where positively charged lipids yield the desired bo3 oxidase orientation but hinder reduction of the quinone pool by NDH-2. To overcome this conundrum, we exploit ionizable lipids, which are either neutral or positively charged depending on the pH value. We first coreconstituted bo3 oxidase and ATP synthase into temporarily positively charged liposomes, followed by fusion with negatively charged empty liposomes at low pH. An increase of the pH to physiological values renders these proteoliposomes overall negatively charged, making them compatible with quinone reduction via NDH-2. Using this strategy, we not only succeeded in orienting the bo3 oxidase essentially unidirectionally into liposomes but also found up to 3-fold increased ATP synthesis rates through the usage of natural, long-chain quinones in combination with the substrate NADH compared to the synthetic electron donor/mediator pair.

Metformin and vitamin D combination therapy ameliorates type 2 diabetes mellitus-induced renal injury in male Wistar rats.

Diabetic kidney disease is a major microvascular diabetes mellitus (DM) complication clinically associated with a gradual renal function decline. Although metformin is a common drug for managing DM, however, monotherapy treatment with any antidiabetic drug will necessitate dosage increment since type 2 DM (T2DM) deteriorates over time due to the increasing pancreatic β-cell dysfunction and will eventually require a combination therapy approach with another antidiabetic medication. Vitamin D is a food supplement that has been proven to have antidiabetic and reno-protective activities. Hence, we explore the combination of vitamin D and metformin on T2DM-induced renal dysfunction. Thirty male Wistar rats were randomized into five (5) groups: control, diabetes untreated, diabetics treated with metformin, vitamin D, and vitamin D + metformin. Vitamin D and metformin significantly reversed DM-induced hyperglycemia, electrolyte imbalance, and dyslipidemia. Also, vitamin D and metformin reversed T2DM-induced increase in serum creatinine and urea and renal lactate, LDH, and oxido-inflammatory response. These observed alterations were accompanied by an increase in proton pump activities and modulation of Nrf2/Nf-κB and XO/UA signaling. This study revealed that vitamin D and/or metformin ameliorated T2DM-induced renal injury.

ZmCRK1 negatively regulates maize's response to drought stress by phosphorylating plasma membrane H+-ATPase ZmMHA2.

Drought severely affects crop growth and yields. Stomatal regulation plays an important role in plant response to drought stress. Light-activated plasma membrane-localized proton ATPase (PM H+-ATPase) mainly promoted the stomatal opening. Abscisic acid (ABA) plays a dominant role in the stomatal closure during drought stress. It is not clear how PM H+-ATPase is involved in the regulation of ABA-induced stomatal closure. We found that a CALCIUM-DEPENDENT PROTEIN KINASE RELATED KINASE 1 (ZmCRK1), and its mutant zmcrk1 exhibited slow water loss in detached leaves, high-survival rate after drought stress, and sensitivity to stomatal closure induced by ABA. The ZmCRK1 overexpression lines are opposite. ZmCRK1 interacted with the maize PM H+-ATPase ZmMHA2. ZmCRK1 phosphorylated ZmMHA2 at the Ser-901 and inhibited its proton pump activity. ZmCRK1 overexpression lines and zmmha2 mutants had low H+-ATPase activity, resulting in impaired ABA-induced H+ efflux. Taken together, our study indicates that ZmCRK1 negatively regulates maize drought stress response by inhibiting the activity of ZmMHA2. Reducing the expression level of ZmCRK1 has the potential to reduce yield losses under water deficiency.

Contribution of Proteorhodopsin to Light-Dependent Biological Responses in Hymenobacter nivis P3T Isolated from Red Snow in Antarctica.

Proteorhodopsin (PR) is a major family of microbial rhodopsins that function as light-driven outward proton pumps. PR is now widely recognized for its ecological importance as a molecule responsible for solar energy flow in various ecosystems on the earth. However, few concrete examples of the actual use of light by natural microorganisms via PR have been demonstrated experimentally. This study reveals one example of that in a cryophilic bacterium Hymenobacter nivis P3T isolated from red snow in Antarctica. The results demonstrate light-dependent biochemical and biological responses in H. nivis cells, such as the proton pump activity of H. nivis PR (HnPR), which leads to the production of proton motive force, cellular ATP production, and cell growth. In addition, the results of this study demonstrate the photochemical properties of a PR, namely, HnPR, in the membrane of a natural host bacterium. The photocycle of HnPR was much faster than other PRs even at 5 °C, indicating that the proton pump function of HnPR has adapted to the low-temperature environment of Antarctica. Although it is well-known that PR helps natural host microorganisms to use light energy, this study provides another concrete example for understanding the biological role of PR by demonstrating the link between the molecular functions of PR and the light-dependent biochemical and biological responses of a PR-bearing host.

Mechanistic insights into the plant biostimulant activity of a novel formulation based on rice husk nanobiosilica embedded in a seed coating alginate film.

Seed coating ensures the targeted delivery of various compounds from the early stages of development to increase crop quality and yield. Silicon and alginate are known to have plant biostimulant effects. Rice husk (RH) is a significant source of biosilica. In this study, we coated mung bean seeds with an alginate-glycerol-sorbitol (AGS) film with embedded biogenic nanosilica (SiNPs) from RH, with significant plant biostimulant activity. After dilute acid hydrolysis of ground RH in a temperature-controlled hermetic reactor, the resulting RH substrate was neutralized and calcined at 650°C. The structural and compositional characteristics of the native RH, the intermediate substrate, and SiNPs, as well as the release of soluble Si from SiNPs, were investigated. The film for seed coating was optimized using a mixture design with three factors. The physiological properties were assessed in the absence and the presence of 50 mM salt added from the beginning. The main parameters investigated were the growth, development, metabolic activity, reactive oxygen species (ROS) metabolism, and the Si content of seedlings. The results evidenced a homogeneous AGS film formation embedding 50-nm amorphous SiNPs having Si-O-Si and Si-OH bonds, 0.347 cm3/g CPV (cumulative pore volume), and 240 m2/g SSA (specific surface area). The coating film has remarkable properties of enhancing the metabolic, proton pump activities and ROS scavenging of mung seedlings under salt stress. The study shows that the RH biogenic SiNPs can be efficiently applied, together with the optimized, beneficial alginate-based film, as plant biostimulants that alleviate saline stress from the first stages of plant development.

Anti-inflammatory, anti-oxidant and anti-ulcer activities of aqueous lyophilizate of Markhamia lutea (Bignoniaceae).

This work was carried out with a view to determining the antioxidant, anti-inflammatory and anti-ulcer properties of the aqueous lyophilized extract of Markhamia lutea. In vitro proteinases inhibition, albumin denaturation, hemolysis of red blood cells by heat, inhibition ofthe proton pump H+/K+ATPase, FRAP (Ferric reducing antioxidant power) and DPPH (1,1-diphenyl-2-picrylhydrazyl) assays were performed. In vivo, cold water immersion-induced ulceration and methylene blue-induced ulceration was usedto determine the anti-ulcer properties of the lyophilizate (100, 200 and 300 mg/kg). In vitro, the lyophilizate (400 μg/mL) significantly inhibited protein denaturation (66.65 %), hemolysis of red blood cells (56.54 %), proteinase activity (69.22 %); then theIC50 was 26.31 μg/mL on proton pump activity. It has alsodeveloped a strong ferric reducing antioxidant power(EC50=52.96 mmol FeSO4/g) as well as free radicals scavenging activity (EC50=22.38 μg/mL). In vivo, the aqueous lyophilizate (200 and 300 mg/kg) protected the gastric mucosa (70.68 and 79.00 % protection respectively) and reduced (p<0.05) acetylcholine, calcium and corticosterone concentrations. A decrease in malondialdehyde level, an increased glutathione level and an increased in catalase andSOD activities were recorded. In the methylene blue test, it significantly increased gastric fluid pH, while reducing gastric volume and improving hematological parameters inulcer animals. In addition, the histological sections showthat the aqueous lyophilizate of M.lutea protected the gastric mucosa from the deleterious effects of stress. The aqueous lyophilizate of M.lutea has anti-ulcer properties thanks to its anti-inflammatory, antioxidant and anti-secretory properties.

Chloroplast envelope K+/H+ antiporters are involved in cytosol pH regulation.

Variations in light intensity induce cytosol pH changes in photosynthetic tissues, providing a possible signal to adjust a variety of biochemical, physiological and developmental processes to the energy status of the cells. It was shown that these pH changes are partially due to the transport of protons in or out of the thylakoid lumen. However, the ion transporters in the chloroplast that transmit these pH changes to the cytosol are not known. KEA1 and KEA2 are K+/H+ antiporters in the chloroplast inner envelope that adjust stromal pH in light-to-dark transitions. We previously determined that stromal pH is higher in kea1kea2 mutant cells. In this study, we now show that KEA1 and KEA2 are required to attenuate cytosol pH variations upon sudden light intensity changes in leaf mesophyll cells, showing they are important components of the light-modulated pH signalling module. The kea1kea2 mutant mesophyll cells also have a considerably less negative membrane potential. Membrane potential is dependent on the activity of the plasma membrane proton ATPase and is regulated by secondary ion transporters, mainly potassium channels in the plasma membrane. We did not find significant differences in the activity of the plasma membrane proton pump but found a strongly increased membrane permeability to protons, especially potassium, of the double mutant plasma membranes. Our results indicate that chloroplast envelope K+/H+ antiporters not only affect chloroplast pH but also have a strong impact on cellular ion homeostasis and energization of the plasma membrane.

The assembly factor Dmxl1 is required for H+(V)-ATPase assembly and function in mammals in vivo

The V-ATPase (proton pump) generates electrochemical gradients across cell membranes, driving essential biological processes. Critically, the two V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in serious pathologies such as cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting intercalated cells (IC) in the kidney. However, very little is known about the molecular regulation of V-ATPase in mammals. We have identified a novel interactor of the IC-enriched B1 subunit of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 protein (Dmxl1), aka Rabconnectin-3A (Rbcn3A). The yeast homologue of Dmxl1, Rav1p, is part of a protein complex that catalyzes the reversible assembly of the V1 and VO domains, thus regulating proton pumping activity. We, therefore, hypothesized that Dmxl1 is an assembly factor for the V-ATPase in mammalian cells. We generated kidney IC-specific Dmxl1 knockout (KO) mice and they had a high urine pH, similar to B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting of kidney lysates showed decreased B1 V-ATPase expression, but other V-ATPase subunits were normal. B1 (V1 domain) and a4 (VO domain) subunits were immunolocalized in ICs and apical localization quantified using FIJI. Both B1 and a4 were more intracellular in Dmxl1 KO ICs relative to control mice. A Pearson’s correlation analysis of B1 and a4 staining revealed a significant decrease in colocalization in Dmxl1 KO ICs, suggesting reduced B1/a4 assembly. In parallel, subcellular fractionation experiments revealed less V1 domain-associated B1 subunit in the membrane fraction relative to the cytosol. Furthermore, a Proximity Ligation Assay (PLA) was performed using probes against B1 and a4 to confirm decreased assembly of the two domains in KO ICs. When the two probes are within 40 μM of each other, like when the domains are assembled, this results in a fluorescent PLA punctum. As expected, we observed a significant decrease in the area of PLA puncta per cell in KO animals relative to control animals. These data demonstrate that the V-ATPase is less assembled in Dmxl1 KO ICs, relative to controls. We propose that loss of Dmxl1 reduces assembly of the V-ATPase holoenzyme, thereby inhibiting its proton pumping function. Dmxl1 may mediate recruitment of the V1 domain to the membrane and facilitate its assembly with the transmembrane VO domain. In the absence of Dmxl1, V1 is instead targeted for degradation, explaining the inability of these mice to properly acidify their urine. We conclude that Dmxl1 is a bona-fide mammalian V-ATPase assembly factor that functions similarly to its yeast homolog, Rav1p in affecting V-ATPase assembly. NIH/NIDDK R01DK121848 to DB. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Transceptor NRT1.1 and receptor-kinase QSK1 complex controls PM H+-ATPase activity under low nitrate

NRT1.1, a nitrate transceptor, plays an important role in nitrate binding, sensing, and nitrate-dependent lateral root (LR) morphology. However, little is known about NRT1.1-mediated nitrate signaling transduction through plasma membrane (PM)-localized proteins. Through in-depth phosphoproteome profiling using membranes of Arabidopsis roots, we identified receptor kinase QSK1 and plasma membrane H+-ATPase AHA2 as potential downstream components of NRT1.1 signaling in a mild low-nitrate (LN)-dependent manner. QSK1, as a functional kinase and molecular link, physically interacts with NRT1.1 and AHA2 at LN and specifically phosphorylates AHA2 at S899. Importantly, we found that LN, not high nitrate (HN), induces formation of the NRT1.1-QSK1-AHA2 complex in order to repress the proton efflux into the apoplast by increased phosphorylation of AHA2 at S899. Loss of either NRT1.1 or QSK1 thus results in a higher T947/S899 phosphorylation ratio on AHA2, leading to enhanced pump activity and longer LRs under LN. Our results uncover a regulatory mechanism in which NRT1.1, under LN conditions, promotes coreceptor QSK1 phosphorylation and enhances the NRT1.1-QSK1 complex formation to transduce LN sensing to the PM H+-ATPase AHA2, controlling the phosphorylation ratio of activating and inhibitory phosphorylation sites on AHA2. This then results in altered proton pump activity, apoplast acidification, and regulation of NRT1.1-mediated LR growth.

The peculiarities of cell elongation growth of cereal coleoptiles under normal and flooding conditions

The review examines modern knowledge on the mechanisms of the early stages of plant cell elongation growth. Coleoptiles are used as a model object representing juvenile organs of cereal seedlings. Elongation growth is considered to be a protective morphophysiological stage of seedling development during hypogeal germination. The molecular mechanisms of elongation growth include: changes in the properties of the cell wall, activation of proton pumps, as well as aquaporins of plasma membrane and tonoplast. Particular attention is paid to the hormonal system of regulation, including auxin and ethylene. Coleoptiles of rice, a semi-aquatic plant tolerant to oxygen deficiency, demonstrate that the mechanisms of elongation growth are changing intensively under submergence, but they completely ensure cell growth. There is also a redistribution of importance and abundance between phytohormones. The data presented in the review indicate the necessity to continue investigations on the mechanisms of elongation growth under normal and stress conditions.

Engineering yeast with a light-driven proton pump system in the vacuolar membrane

BackgroundThe supply of ATP is a limiting factor for cellular metabolism. Therefore, cell factories require a sufficient ATP supply to drive metabolism for efficient bioproduction. In the current study, a light-driven proton pump in the vacuolar membrane was constructed in yeast to reduce the ATP consumption required by V-ATPase to maintain the acidification of the vacuoles and increase the intracellular ATP supply for bioproduction.ResultsDelta rhodopsin (dR), a microbial light-driven proton-pumping rhodopsin from Haloterrigena turkmenica, was expressed and localized in the vacuolar membrane of Saccharomyces cerevisiae by conjugation with a vacuolar membrane-localized protein. Vacuoles with dR were isolated from S. cerevisiae, and the light-driven proton pumping activity was evaluated based on the pH change outside the vacuoles. A light-induced increase in the intracellular ATP content was observed in yeast harboring vacuoles with dR.ConclusionsYeast harboring the light-driven proton pump in the vacuolar membrane developed in this study are a potential optoenergetic cell factory suitable for various bioproduction applications.

Omega 3 fatty acids preserve testicular function by ameliorating BPF-induced dysthyroidism: role of p53/Bcl-2 signaling and proton pump activities.

Bisphenol F (BPF) is an endocrinedisrupting chemical, but information about its effect on thyroid hormones has not been fully explored. Omega 3 fatty acids (O3FA), on the other hand, are antioxidant and antiapoptotic agents. Therefore, this study explored the role and associated molecular mechanism of O3FA in BPF-induced hypothyroidism-mediated testicular dysfunction in male Wistar rats. Twenty (20) male Wistar rats were randomized into four groups (n=5/group), namely: the control group; the BPF treated group (30 mg/kg of BPF); and the intervention groups (30mg/kg BPF + 100mg/kg O3FA (BPF+O3FA-L) and 30mg/kg BPF + 300mg/kg of O3FA for 28 days). Low and high doses of O3FA ameliorated BPF-induced hypothyroidism-mediated reduction in sperm quality, testosterone, luteinizing hormone, follicle-stimulating hormone, catalase, superoxide dismutase, total antioxidant capacity, and nuclear factor erythroid 2-related factor 2 and increases in estrogen, malondialdehyde, c-reactive protein, interleukin 1 beta, caspase 3. Furthermore, O3FA prevented BPF-induced Na+/K+-ATPase and Ca2+-ATPase dysfunction, estrogen receptor beta overexpression, and tumor protein P53 (p53)/ b-cell lymphoma 2 (Bcl-2) imbalance. This study showed that O3FA ameliorated BPF-induced dysthyroidism-mediated testicular dysfunction by preventing proton pump dysfunction and p53/BCl-2 imbalance.

Plasmodium falciparum utilizes pyrophosphate to fuel an essential proton pump in the ring stage and the transition to trophozoite stage.

During asexual growth and replication cycles inside red blood cells, the malaria parasite Plasmodium falciparum primarily relies on glycolysis for energy supply, as its single mitochondrion performs little or no oxidative phosphorylation. Post merozoite invasion of a host red blood cell, the ring stage lasts approximately 20 hours and was traditionally thought to be metabolically quiescent. However, recent studies have shown that the ring stage is active in several energy-costly processes, including gene transcription, protein translation, protein export, and movement inside the host cell. It has remained unclear whether a low glycolytic flux alone can meet the energy demand of the ring stage over a long period post invasion. Here, we demonstrate that the metabolic by-product pyrophosphate (PPi) is a critical energy source for the development of the ring stage and its transition to the trophozoite stage. During early phases of the asexual development, the parasite utilizes Plasmodium falciparum vacuolar pyrophosphatase 1 (PfVP1), an ancient pyrophosphate-driven proton pump, to export protons across the parasite plasma membrane. Conditional deletion of PfVP1 leads to a delayed ring stage that lasts nearly 48 hours and a complete blockage of the ring-to-trophozoite transition before the onset of parasite death. This developmental arrest can be partially rescued by an orthologous vacuolar pyrophosphatase from Arabidopsis thaliana, but not by the soluble pyrophosphatase from Saccharomyces cerevisiae, which lacks proton pumping activities. Since proton-pumping pyrophosphatases have been evolutionarily lost in human hosts, the essentiality of PfVP1 suggests its potential as an antimalarial drug target. A drug target of the ring stage is highly desired, as current antimalarials have limited efficacy against this stage.

Mitochondrion of the Trypanosoma brucei long slender bloodstream form is capable of ATP production by substrate-level phosphorylation.

The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the FoF1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the FoF1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨm. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨm. In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.

Improved production of β-carotene in light-powered Escherichia coli by co-expression of Gloeobacter rhodopsin expression

BackgroundProviding sufficient and usable energy for the cell factory has long been a heated issue in biosynthesis as solar energy has never been rooted out from the strategy for improvement, and turning Escherichia coli (E. coli) into a phototrophic host has multiple captivating qualities for biosynthesis. In this study, β-carotene was a stable compound for production in E. coli with the expression of four enzymes (CrtE, CrtB, CrtI, CrtY) for production due to its light-harvesting feature as an antenna pigment and as an antioxidant and important precursor for human health. The expression of Gloeobacter rhodopsin (GR) in microbial organisms was proved to have potential for application.ResultsThe expression of fusion protein, GR-GFP, in E. coli showed visible GFP signal under fluorescent microscopy, and its in vivo proton pumping activity signal can be detected in induced photocurrent by electrodes on the chip under intervals of illumination. To assess the phototrophic synthesis ability of the host strain compared to wild-type and vector control strain in chemostat batch with illumination, the expression of red fluorescent protein (RFP) as a target protein showed its yield improvement in protein assay and also reflected its early dominance in RFP fluorescence signal during the incubation, whereas the synthesis of β-carotene also showed yield increase by 1.36-fold and 2.32-fold compared with its wildtype and vector control strain. To investigate the effect of GR-GFP on E. coli, the growth of the host showed early rise into the exponential phase compared to the vector control strain and glucose turnover rate was elevated in increased glucose intake rate and upregulation of ATP-related genes in glycolysis (PtsG, Pgk, Pyk).ConclusionWe reported the first-time potential application of GR in the form of fusion protein GR-GFP. Expression of GR-GFP in E. coli improved the production of β-carotene and RFP. Our work provides a strain of E. coli harboring phototrophic metabolism, thus paving path to a more sustainable and scalable production of biosynthesis.

Anionic Phospholipids Stimulate the Proton Pumping Activity of the Plant Plasma Membrane P-Type H+-ATPase.

The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H+-ATPase Arabidopsis thaliana H+-ATPase isoform 2 (AHA2) upon purification and reconstitution into liposomes of defined lipid compositions. We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. This activation was independent of the cytoplasmic C-terminal regulatory domain of the pump. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partially conserved in functionally different P-type ATPases from different organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity.

Mutations in type II Golgi-localized proton pyrophosphatase AVP2;1/VHP2;1 affect pectic polysaccharide rhamnogalacturonan-II and alter root growth under low boron condition in Arabidopsis thaliana.

The essential plant nutrient boron is required for the crosslinking of the pectin polysaccharide, rhamnogalacturonan II (RG-II). The synthesis of the pectic polysaccharides takes place in the Golgi apparatus, acidified by proton pumps. AVP2;1/VHP2;1 is a type II proton pyrophosphatase localized in the Golgi apparatus, which possesses proton pumping activity coupled with pyrophosphate hydrolysis. Its activity and expression patterns have been previously revealed but its role in plants remains unknown. The aim of the present work therefore was to explore the physiological role of AVP2;1 in Arabidopsis thaliana. In the screening of mutants under low boron, a mutant carrying a missense mutation in AVP2;1 was isolated. This mutant showed increased primary root growth under low boron conditions but no significant difference under normal boron condition compared to wild type plants. T-DNA insertion caused similar growth, suggesting that reduced function of AVP2;1 was responsible. Root cell observation revealed an increase in meristematic zone length, cell number in meristem and length of matured cell in avp2;1 mutants compared to wild type under low boron. Calcium concentration was reduced in mutant root cell wall under low boron. RG-II specific sugars also tended to be decreased in mutant root cell wall under low and normal boron conditions. These results suggest that changes in cell wall component by mutations in AVP2;1 may possibly explain the increased root length of mutants under low boron. This supports the idea that AVP2;1 plays a role in pH homoeostasis in Golgi apparatus for pectin synthesis.

14-3-3 Proteins and the Plasma Membrane H+-ATPase Are Involved in Maize (Zea mays) Magnetic Induction.

The geomagnetic field (GMF) is a natural component of the biosphere, and, during evolution, all organisms experienced its presence while some evolved the ability to perceive magnetic fields (MF). We studied the response of 14-3-3 proteins and the plasma membrane (PM) proton pump H+-ATPase to reduced GMF values by lowering the GMF intensity to a near-null magnetic field (NNMF). Seedling morphology, H+-ATPase activity and content, 14-3-3 protein content, binding to PM and phosphorylation, gene expression, and ROS quantification were assessed in maize (Zea mays) dark-grown seedlings. Phytohormone and melatonin quantification were also assessed by LG-MS/MS. Our results suggest that the GMF regulates the PM H+-ATPase, and that NNMF conditions alter the proton pump activity by reducing the binding of 14-3-3 proteins. This effect was associated with both a reduction in H2O2 and downregulation of genes coding for enzymes involved in ROS production and scavenging, as well as calcium homeostasis. These early events were followed by the downregulation of IAA synthesis and gene expression and the increase in both cytokinin and ABA, which were associated with a reduction in root growth. The expression of the homolog of the MagR gene, ZmISCA2, paralleled that of CRY1, suggesting a possible role of ISCA in maize magnetic induction. Interestingly, melatonin, a widespread molecule present in many kingdoms, was increased by the GMF reduction, suggesting a still unknown role of this molecule in magnetoreception.

Prediction of gastroesophageal reflux episodes by smooth muscle electromyography: A translational study in rats and adolescents

AimsOur aim was to measure the myoelectric modifications during gastric acid secretion along with the gastric pH in a rat model and to detect the gastrointestinal (GI) myoelectric changes in adolescents suffering from gastroesophageal reflux disease (GERD) along with the esophageal pH measurement. Main methodsIn anesthetized rats, gastric acid secretion was initiated with intragastric histamine (50 mg/kg), and gastric pH, GI myoelectric activity and mechanical GI contractions were measured with intragastric pH electrode, subcutaneously implanted smooth muscle electromyography (SMEMG) electrodes and organ implanted strain gauges, respectively. In the clinical study, esophageal pH and GI myoelectric activity were measured in adolescents suffering from GERD with intraesophageal pH electrode and SMEMG electrodes placed on the abdominal surface, respectively. The SMEMG records were analyzed by fast Fourier transformation (FFT) and power spectrum density maximum (PsDmax) values were calculated for the GI segments. Key findingsIn rats, histamine initiated an immediate increase in gastric PsDmax, which preceded the significant reduction in gastric pH by 75 min. The myoelectric change was independent of mechanical GI contractions. In adolescents, the GERD episodes were preceded by a significant increase in gastric PsDmax 45 min earlier. These changes were independent of motion or meals. SignificanceIncreased gastric myoelectric activity during histamine stimulation or GERD might be linked to the enhanced activity of the gastric proton pump, indicating a link between gastric acid secretion and GERD episodes. It is supposed that SMEMG might be a tool for predicting forthcoming reflux episodes in GERD.

Regulation of proteolysis in bovine cumulus cells with possible inclusion of proton pump activators.

The aim of this study was to reveal the effects of V-ATPase proton pump activation on lysosomal acidity and protein degradation in cultured cumulus cells. Cumulus cells from bovine ovaries were cultured in the presence of 10 and 50 μM doses of V-ATPase proton pump activators PIP2, PMA and DOG for 12 and 24 h. At the end of the culture period, the level of protein degradation was evaluated through DQ-Red-BSA analysis and the lysosomes were detected through a fluorescent probe. In addition, total and phosphorylated MAPK1/3 and AKT protein levels of cumulus cells were determined through Western blotting. PIP2 and PMA were shown to increase protein degradation and lysosomal acidity in cultured bovine cumulus cells, whereas DOG did not have any significant effects on these cells. Total and phosphorylated MAPK and AKT protein levels were higher in PIP2 and PMA groups compared with the control and DOG. It was concluded that particular proton pump activators can enhance protein degradation and lysosomal acidification in cultured bovine cumulus cells without having detrimental effects on cell signalling members required for cell viability and proper functioning. Due to the cellular interactions, increasing the lysosomal activity in cumulus cells in the culture environment could also affect the removal of protein aggregates in the oocytes. This strategy could be effective for improving in vitro maturation of the oocytes by providing proteostasis.