Lysosome pH-regulated H+ /K+ switching channel involved in maintaining lysosomal homeostasis.

Lysosomal pH setpoint and H+ homeostasis is key to the lysosome's functions. The Parkinson's disease-risk protein TMEM175, originally identified as lysosomal K+ channel, works as a H+ -activated H+ channel and discharges the lysosomal H+ store when it is hyper-acidified. Yang et al. indicate that TMEM175 is permeable for both K+ and H+ in the same pore and charges the lysosome with H+ under certain conditions. The charge and discharge functions are under regulation of the lysosomal matrix and glycocalyx layer. Their presented work indicates that TMEM175 performs as a multi-functional channel regulating lysosomal pH in response to physiological conditions.

Biosynthesis of silver nanoparticles using extracts of Stevia rebaudiana and evaluation of antibacterial activity.

The present study reveals a simple, non-toxic and eco-friendly method for the "green" synthesis of Ag-NPs using hydroponic and soil medicinal plant Stevia rebaudiana extracts, the characterization of biosynthesized nanoparticles, as well as the evaluation of their antibacterial activity. Transmission electronic microscopy (TEM) and Dynamic Light Scattering (DLS) analysis confirmed that biosynthesized Ag-NPs are in the nano-size range (50-100 nm) and have irregular morphology. Biogenic NPs demonstrate antibacterial activity against Escherichia coli BW 25,113, Enterococcus hirae ATCC 9790, and Staphylococcus aureus MDC 5233. The results showed a more pronounced antibacterial effect on E. coli growth rate, in comparison with Gram-positive bacteria, which is linked to the differences in the structure of bacterial cell wall. Moreover, the Ag-NPs not only suppressed the growth of bacteria but also changed the energy-dependent H+-fluxes across the bacterial membrane. The change of H+-fluxes in presence of H+-translocating systems inhibitor, N,N'-dicyclohexylcarbodiimide (DCCD), proves the effect of Ag-NPs on the structure and permeability of the bacterial membrane. Overall, our findings indicate that the Ag-NPs synthesized by medicinal plant Stevia extracts may be an excellent candidate as an alternative to antibiotics against the tested bacteria.

Transmembrane H+ fluxes and the regulation of neural induction in Xenopus laevis.

It has previously been reported that in ex vivo planar explants prepared from Xenopus laevis embryos, the intracellular pH (pHi) increases in cells of the dorsal ectoderm from stage 10.5 to 11.5 (i.e. 11-12.5 hpf). It was proposed that such increases (potentially due to H+ being extruded, sequestered, or buffered in some manner), play a role in regulating neural induction. Here, we used an extracellular ion-selective electrode to non-invasively measure H+ fluxes at eight locations around the equatorial circumference of intact X. laevis embryos between stages 9-12 (˜7-13.25 hpf). We showed that at stages 9-11, there was a small H+ efflux recorded from all the measuring positions. At stage 12 there was a small, but significant, increase in the efflux of H+ from most locations, but the efflux from the dorsal side of the embryo was significantly greater than from the other positions. Embryos were also treated from stages 9-12 with bafilomycin A1, to block the activity of the ATP-driven H+ pump. By stage 22 (24 hpf), these embryos displayed retarded development, arresting before the end of gastrulation and therefore did not display the usual anterior and neural structures, which were observed in the solvent-control embryos. In addition, expression of the early neural gene, Zic3, was absent in treated embryos compared with the solvent controls. Together, our new in vivo data corroborated and extended the earlier explant-derived report describing changes in pHi that were suggested to play a role during neural induction in X. laevis embryos.

Calcium/calmodulin-dependent protein kinase OsDMI3 positively regulates saline-alkaline tolerance in rice roots.

Soil saline-alkalization is a major environmental stress that impairs plant growth and crop productivity. Plant roots are the primary site for the perception of soil stresses; however, the regulation mechanism engaged in the saline-alkaline stress response in plant roots is not well understood. In this study, we identified how a rice Ca2+/calmodulin-dependent protein kinase, OsDMI3, confers saline-alkaline tolerance in rice root growth. We measured the OsDMI3 activity by an in-gel kinase assay, Na+ content by NaHCO3 treatment, and Na+ and H+ fluxes by noninvasive micro-test technology (NMT). Furthermore, a real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis was performed to identify the genes upregulated in response to NaHCO3 treatment in rice roots. The results showed that NaHCO3 significantly increased OsDMI3 expression and activity in rice roots. This was consistent with the results of Na+ content and NMT that indicated OsDMI3 promoted root elongation under saline-alkaline stress by reducing root Na+ and H+ influx. Moreover, real-time RT-PCR analysis revealed that OsDMI3 up-regulated the transcript levels of OsSOS1 and PM-H+-ATPase genes OsA3 and OsA8 in saline-alkaline stressed rice plants. Collectively, our results suggest that OsDMI3 could promote saline-alkaline tolerance in rice roots by modulating the Na+ and H+ influx. These findings provide an important genetic target for protection of growth in rice exposed to saline-alkaline stress.

Function of NHX-type transporters in improving rice tolerance to aluminum stress and soil acidity.

MAIN CONCLUSION: In this study, we show that ectopic expression of either HtNHX1 or HtNHX2, from Helianthus tuberosus plant (located at vacuolar and endosome membranes, respectively), in rice plants could enhance its tolerance to aluminum (Al3+) stress and soil acidity. Plant sodium (potassium)/proton (Na+(K+)/H+ antiporters of the NHX family have been extensively characterized as they are related to the enhancement of salt tolerance. However, no previous study has reported NHX transporter functions in plant tolerance to Al3+ toxicity. In this study, we demonstrate their role as a component of the Al3+ stress tolerance mechanism. We show that the ectopic expression of either HtNHX1 or HtNHX2 , from Helianthus tuberosus plant, in rice (located at vacuole and endosome, respectively) could also enhance rice tolerance to Al3+ stress and soil acidity. Expression of either HtNHX1 or HtNHX2 reduced the inhibitory effect of Al3+ on the rice root elongation rate; both genes were reported to be equally effective in improvement of stress conditions. Expression of HtNHX1 enhanced Al3+-trigged-secretion of citrate acids, rhizosphere acidification, and also reduced K+ efflux from root tissues. In contrast, expression of HtNHX2 prevented Al3+-trigged-decrease of H+ influx into root tissues. Al3+-induced damage of the cell wall extensibility at the root tips was impaired by either HtNHX1 or HtNHX2. Co-expression of HtNHX1 and HtNHX2 further improved rice growth, particularly under the Al3+ stress conditions. The results demonstrate that HtNHX1 and HtNHX2 improved rice tolerance to Al3+ via different mechanisms by altering the K+ and H+ fluxes and the cell wall structure.

Antibacterial activities of transient metals nanoparticles and membranous mechanisms of action.

Various transient metal and metal oxide nanoparticles (NPs) have shown pronounced biological activity, including antibacterial action against different Gram-negative and Gram-positive bacteria including pathogens and drug-resistant ones. Thus, NPs can be applied in nanotechnology for controlling bacterial growth as well as in biomedicine for the treatment of various diseases. However, the mechanisms of these effects are not clear yet. This review is focused on the antibacterial effects of transient metal NPs, especially iron oxide (Fe3O4) and Ag NPs on Escherichia coli wild type and antibiotic-resistant strains. Ag NPs show more pronounced bactericidal effect than Fe3O4 NPs. Moreover, Ag NPs display more expressed antibacterial effect at low concentrations. Interestingly, kanamycin-resistant strain is more susceptible to Fe3O4 NPs than wild type strain. In order to explain the possible mechanisms of NP effects, in addition to the production of reactive oxygen species causing damage in cells, particularly, their membranes, the changes in the membrane-associated H+-translocating FOF1-ATPase activity, H+-fluxes through the bacterial membrane, redox potential and hydrogen yield by membrane-associated enzymes-hydrogenases, are discussed. We observed from the results that FOF1-ATPase could be a main target for NPs. A scheme of possible action mechanism is proposed.

Effects of iron oxide (Fe3 O4 ) nanoparticles on Escherichia coli antibiotic-resistant strains.

AIMS: Antibiotic resistance of different bacteria requires the development of alternative approaches for overcoming this phenomenon. The antibacterial effects of iron oxide (Fe3 O4 ) nanoparticles (NPs) (from 50 to 250 µg ml-1 ) on Escherichia coli antibiotic-resistant strains have been aimed. METHODS AND RESULTS: The study was performed with ampicillin-resistant E. coli DH5α-pUC18 and kanamycin-resistant E. coli pARG-25 stains. Specific growth rate of bacteria (µ), lag phase duration and colony-forming units (CFU) were determined to evaluate growth properties. Fe3 O4 NPs (average size of 10·64 ± 4·73 nm) coated with oleic acid and synthesized by modified co-precipitation method were used. The medium pH, H+ efflux, membrane H+ conductance, redox potential determinations and H2 yield assay were done using potentiometer methods. Growth properties were changed by NPs in concentration-dependent manner. NPs decreased (up to twofold) H+ -fluxes through bacterial membrane more in E. coli in the presence of the N,N'-dicyclohexylcarbodiimide, inhibitor of ATPase, indicating that antibacterial activity of these NPs was connected with ATP-associated metabolism. Membrane-associated H2 production was lowered up to twofold. Moreover, the synergetic interactions of NPs and antibiotics were found: combination of NPs and antibiotics provided the higher H+ conductance, lower H+ -fluxes and H2 yield. CONCLUSIONS: Fe3 O4 NPs can be suggested as alternative antibacterial agents, which can substitute antibiotics in different applications. SIGNIFICANCE AND IMPACT OF THE STUDY: The antibacterial effects of Fe3 O4 NPs on the growth properties and membrane activity of E. coli antibiotic-resistant strains have been demonstrated. These NPs have potential as antibacterial agents, which can substitute for antibiotics in bacterial disease treatment in biomedicine, pharmaceutical and environmental applications.

NaCl-induced expression of AtVHA-c5 gene in the roots plays a role in response of Arabidopsis to salt stress.

KEY MESSAGE: Suppression of AtVHA-c5 expression results in changes in H+ and Na+ fluxes of roots, and increase sensitivity to salt in Arabidopsis. Vacuolar-type H+-ATPase (V-ATPase), a multisubunit endomembrane proton pump, is essential in plant growth and response to environmental stresses. In the present study, the function of Arabidopsis V-ATPase subunit c5 (AtVHA-c5) gene in response to salt stress was investigated. Subcellular localization showed that AtVHA-c5 was mainly localized to endosomes and the vacuolar membrane in Arabidopsis. The analysis of quantitative real-time PCR showed that expression of AtVHA-c5 gene was induced by NaCl stress. Histochemical analysis revealed that AtVHA-c5 was expressed in the root epidermis of untreated Arabidopsis and in the whole root elongation zone after NaCl treatment. Phenotypic analysis showed that the atvha-c5 mutant is sensitive to high NaCl as compared to the wild type. The non-invasive micro-test technology measurement demonstrated that the net H+ and Na+ efflux in the root elongation zone of the atvha-c5 mutant was weaker than that of the wild type under NaCl treatment, suggesting that H+ and Na+ fluxes in atvha-c5 roots are impaired under NaCl stress. Moreover, compared to the wild type, the expression of AtSOS1 (salt overly sensitive 1) and AtAHA1 (plasma membrane H+-ATPase 1) were down-regulated in atvha-c5 roots under NaCl stress. Overall, our results indicate that AtVHA-c5 plays a role in Arabidopsis root response to NaCl stress by influencing H+ and Na+ fluxes.

Modifications of the chemical structure of phenolics differentially affect physiological activities in pulvinar cells of Mimosa pudica L. II. Influence of various molecular properties in relation to membrane transport.

Early prediction of compound absorption by cells is of considerable importance in the building of an integrated scheme describing the impact of a compound on intracellular biological processes. In this scope, we study the structure-activity relationships of several benzoic acid-related phenolics which are involved in many plant biological phenomena (growth, flowering, allelopathy, defense processes). Using the partial least squares (PLS) regression method, the impact of molecular descriptors that have been shown to play an important role concerning the uptake of pharmacologically active compounds by animal cells was analyzed in terms of the modification of membrane potential, variations in proton flux, and inhibition of the osmocontractile reaction of pulvinar cells of Mimosa pudica leaves. The hydrogen bond donors (HBD) and hydrogen bond acceptors (HBA), polar surface area (PSA), halogen ratio (Hal ratio), number of rotatable bonds (FRB), molar volume (MV), molecular weight (MW), and molar refractivity (MR) were considered in addition to two physicochemical properties (logD and the amount of non-dissociated form in relation to pKa). HBD + HBA and PSA predominantly impacted the three biological processes compared to the other descriptors. The coefficient of determination in the quantitative structure-activity relationship (QSAR) models indicated that a major part of the observed seismonasty inhibition and proton flux modification can be explained by the impact of these descriptors, whereas this was not the case for membrane potential variations. These results indicate that the transmembrane transport of the compounds is a predominant component. An increasing number of implicated descriptors as the biological processes become more complex may reflect their impacts on an increasing number of sites in the cell. The determination of the most efficient effectors may lead to a practical use to improve drugs in the control of microbial attacks on plants.

Rapid regulation of the plasma membrane H⁺-ATPase activity is essential to salinity tolerance in two halophyte species, Atriplex lentiformis and Chenopodium quinoa.

BACKGROUND AND AIMS: The activity of H(+)-ATPase is essential for energizing the plasma membrane. It provides the driving force for potassium retention and uptake through voltage-gated channels and for Na(+) exclusion via Na(+)/H(+) exchangers. Both of these traits are central to plant salinity tolerance; however, whether the increased activity of H(+)-ATPase is a constitutive trait in halophyte species and whether this activity is upregulated at either the transcriptional or post-translation level remain disputed. METHODS: The kinetics of salt-induced net H(+), Na(+) and K(+) fluxes, membrane potential and AHA1/2/3 expression changes in the roots of two halophyte species, Atriplex lentiformis (saltbush) and Chenopodium quinoa (quinoa), were compared with data obtained from Arabidopsis thaliana roots. KEY RESULTS: Intrinsic (steady-state) membrane potential values were more negative in A. lentiformis and C. quinoa compared with arabidopsis (-144 ± 3·3, -138 ± 5·4 and -128 ± 3·3 mV, respectively). Treatment with 100 mm NaCl depolarized the root plasma membrane, an effect that was much stronger in arabidopsis. The extent of plasma membrane depolarization positively correlated with NaCl-induced stimulation of vanadate-sensitive H(+) efflux, Na(+) efflux and K(+) retention in roots (quinoa > saltbush > arabidopsis). NaCl-induced stimulation of H(+) efflux was most pronounced in the root elongation zone. In contrast, H(+)-ATPase AHA transcript levels were much higher in arabidopsis compared with quinoa plants, and 100 mm NaCl treatment led to a further 3-fold increase in AHA1 and AHA2 transcripts in arabidopsis but not in quinoa. CONCLUSIONS: Enhanced salinity tolerance in the halophyte species studied here is not related to the constitutively higher AHA transcript levels in the root epidermis, but to the plant's ability to rapidly upregulate plasma membrane H(+)-ATPase upon salinity treatment. This is necessary for assisting plants to maintain highly negative membrane potential values and to exclude Na(+), or enable better K(+) retention in the cytosol under saline conditions.