Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p)

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 μM) and thonzonium bromide (EC(50) = 69 μM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 μM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.     

Meta-analysis: Four-Drug, Three-Antibiotic, Non-bismuth-Containing "Concomitant Therapy" Versus Triple Therapy for Helicobacter pylori Eradication

Background: Low success rates with triple therapy for Helicobacter pylori infections have prompted search for alternatives. In one, a proton-pump inhibitor (PPI) and amoxicillin was followed by the PPI plus clarithromycin and a nitroimidazole (sequential therapy); in another, these four drugs were given concomitantly (concomitant therapy).
Aim: To compare concomitant therapy with standard triple therapy for H. pylori infection.
Methods: By searching PubMed, EMBASE, the Cochrane Central Register of Controlled Trials and abstracts of major gastrointestinal meeting, two independent reviewers systemically identified randomized controlled trials (RCT) comparing concomitant quadruple to standard triple therapies as well as studies reporting eradication rates of concomitant quadruple therapy in treatment of H. pylori . Pooled eradication rates and odds ratios (OR) with 95% confidence intervals (CI) were calculated, and univariable metaregression analysis for all extracted variables was conducted.
Results: We identified nine studies (10 treatment arms) including five qualifying RCTs (576 subjects) comparing concomitant (293 subjects, duration 3 to 5 days) and triple therapy (283 subjects, duration 5 to 10 days) and four other studies evaluating concomitant therapy (478 subjects, duration 3 to 7 days). Pooled estimates of the five RCTs showed superiority of concomitant therapy over triple therapy; with intention-to-treat) pooled OR of 2.86 (95% CI: 1.73–4.73) and per-protocol (PP) pooled OR of 3.52 (95% CI: 1.95–6.38). Considering all 10 treatment arms, the ITT eradication rate was 89.7% (95% CI: 86.8–92.1%) and PP was 92.9% (95% CI: 90.2–94.8%).
Conclusion: Concomitant therapy appears to be an effective alternative to triple therapy and is less complex than sequential therapy.     

Respiration-dependent proton translocation and the mechanism of protonmotive force generation in Nitrobacter winogradskyi

Abstract Proton translocation associated with electron flow to oxygen has been observed with cells of Nitrobacter winogradskyi in the presence of either potassium ferrocyanide or isoascorbate plus N , N , N ', N ' tetramethyl- p -phenylenediamine. The data are consistent with a proton pumping function for the terminal oxidase, cytochrome aa ₃, in this organism as the mechanism for generating a protonmotive force. The failure of previous work with Nitrobacter [4] to detect proton translocation linked to oxidation of nitrite, the physiological substrate, is discussed.     

Influence of sodium ion on heavy metal-induced inhibition of light-regulatd proton efflux and active carbon uptake in the cyanobacterium Anabaena flos-aquae

The light-induced proton efflux and active carbon uptake are inhibited by mercury and cadmium ions in Anabaena flos-aquae. The inhibitory effects of these heavy metal ions are reversed by 40 mM concentration of sodium. Here we report that light-induced proton efflux is sodium-dependent which leads to a characteristic enhancement in the rate of photosynthetic oxygen generation and carbon fixation. A low concentration (10 M) of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) significantly inhibited the rate of oxygen generation while 10 M carbonyl cyanide-m-chlorophenylhydrazone (CCCP) completely blocked the oxygen generation activity in the organism. The chlorophyll-a fluorescence yield indicates that little fluorescence quenching occurred in the absence of sodium ion. Increasing the extracellular sodium ion accelerated both the initial rate and the extent of fluorescence quenching. These results support the assumption that metal-induced inhibition of the photosynthetic machinery may be mediated by the movement of protons.     

Enhanced activity of deoxyuridine 5′-triphosphatase in regenerating rat liver

An enzyme, dUTPase, that catalyzes the conversion of dUTP to dUMP and PPi, was partially purified from regenerating rat livers. The molecular weight was estimated by gel filtration to be 60,000. The apparent Km for dUTP was 12 μM. No other deoxyribonucleoside triphosphates served as a substrate. This enzyme is active in the absence of added divalent cations or sulfhydryl reagents; the activity could be inhibited by EDTA and shows a broad pH optimum with no decrease in activity from pH 7 to 11. The specific activity of dUTPase in rat liver begins to rise 16 h after partial hepatectomy and reaches a maximum about 24 h after the operation, rising to at least 5 to 6 times the normal level.     

‘Random coil’ 1H chemical shifts obtained as a function of temperature and trifluoroethanol concentration for the peptide series GGXGG

Summary Proton chemical shifts of a series of disordered linear peptides (H-Gly-Gly-X-Gly-Gly-OH, with X being one of the 20 naturally occurring amino acids) have been obtained using 1D and 2D ¹H NMR at pH 5.0 as a function of temperature and solvent composition. The use of 2D methods has allowed some ambiguities in side-chain assignments in previous studies to be resolved. An additional benefit of the temperature data is that they can be used to obtain ‘random coil’ amide proton chemical shifts at any temperature between 278 and 318 K by interpolation. Changes of chemical shift as a function of trifluoroethanol concentration have also been determined at a variety of temperatures for a subset of peptides. Significant changes are found in backbone and side-chain amide proton chemical shifts in these ‘random coil’ peptides with increasing amounts of trifluoroethanol, suggesting that caution is required when interpreting chemical shift changes as a measure of helix formation in peptides in the presence of this solvent. Comparison of the proton chemical shifts obtained here for H-Gly-Gly-X-Gly-Gly-OH with those for H-Gly-Gly-X-Ala-OH [Bundi, A. and Wüthrich, K. (1979) Biopolymers, 18, 285–297] and for Ac-Gly-Gly-X-Ala-Gly-Gly-NH₂ [Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S. and Sykes, B.D. (1995) J. Biomol. NMR, 5, 67–81] generally shows good agreement for CH protons, but reveals significant variability for NH protons. Amide proton chemical shifts appear to be highly sensitive to local sequence variations and probably also to solution conditions. Caution must therefore be exercised in any structural interpretation based on amide proton chemical shifts.     

Characterisation of H+ and Cu2+ binding to humic and fulvic acids

Abstract

The nature of H⁺ and Cu²⁺ binding by soil-derived humic (HA) and fulvic (FA) acid was characterised using potentiometric titrations. The experimental data obtained showed that the derived proton balance equation was valid and capable of describing proton consumption by both polyelectrolytes. HA was found to be more acidic and more reactive as shown by its lower equivalent weight compared to FA. Acid consumption by HA during titrations was little affected at ionic strength (μ) up to 0.1 M although it was enhanced at higher μ. Displacement of protons by Cu²⁺ resulted in a nonlinear sigmoidal pattern suggesting the formation of different Cu-HA chelates, or existence of sites that differed in their affinities for Cu on the ligand. Different concentrations of added Cu appeared to favour one or both mechanisms, although the titration method could not differentiate which of the probable mechanisms was more dominant at a specific level of Cu added. Similar values were obtained for conditional stability constants using either the equation of Scatchard or Ruzic.     

Reversed-phase HPLC enantioseparation of pantoprazole using a teicoplanin aglycone stationary phase—Determination of the enantiomer elution order using HPLC-CD analyses

A direct HPLC method was developed for the enantioseparation of pantoprazole using macrocyclic glycopeptide-based chiral stationary phases, along with various methods to determine the elution order without isolation of the individual enantiomers. In the preliminary screening, four macrocyclic glycopeptide-based chiral stationary phases containing vancomycin (Chirobiotic V), ristocetin A (Chirobiotic R), teicoplanin (Chirobiotic T), and teicoplanin-aglycone (Chirobiotic TAG) were screened in polar organic and reversed-phase mode. Best results were achieved by using Chirobiotic TAG column and a methanol-water mixture as mobile phase. Further method optimization was performed using a face-centered central composite design to achieve the highest chiral resolution. Optimized parameters, offering baseline separation (resolution = 1.91 ± 0.03) were as follows: Chirobiotic TAG stationary phase, thermostated at 10°C, mobile phase consisting of methanol/20mM ammonium acetate 60:40 v/v, and 0.6 mL/min flow rate. Enantiomer elution order was determined using HPLC hyphenated with circular dichroism (CD) spectroscopy detection. The online CD signals of the separated pantoprazole enantiomers at selected wavelengths were compared with the structurally analogous esomeprazole enantiomer. For further verification, the inline rapid, multiscan CD signals were compared with the quantum chemically calculated CD spectra. Furthermore, docking calculations were used to investigate the enantiorecognition at molecular level. The molecular docking shows that the R-enantiomer binds stronger to the chiral selector than its antipode, which is in accordance with the determined elution order on the column—S- followed by the R-isomer. Thus, combined methods, HPLC-CD and theoretical calculations, are highly efficient in predicting the elution order of enantiomers.