Journal: International Journal of Molecular Sciences

Article Title: The Antimicrobial Activity of Human Defensins at Physiological Non-Permeabilizing Concentrations Is Caused by the Inhibition of the Plasma Membrane H + -ATPases

doi: 10.3390/ijms25137335

Figure Lengend Snippet: Effect of microbicidal activity of human defensins on cellular K + -homeostasis. ( A ) Effect of extracellular K + on candidacidal activity. The candidacidal activity of defensins (NPIC) was tested using a cellular suspension of C. albicans in Tris buffer in the presence or absence of 50 mM KCl. ( B ) K + -efflux induced in C. albicans cells incubated with defensins. K + -release was measured after incubation (90 min, 37 °C) with defensins and compared with the total cellular K + -content (100%) of untreated cells. ( C ) Effect of inhibition of the K + -channel Tok1p on the candidacidal activity of defensins. Cells were preincubated for 15 min at 37 °C with 10 mM TEA (Tok1p inhibitor) before the addition of the peptides (NPIC). ( D ) Effect of extracellular K + on bactericidal activity of defensins. The bactericidal activity of defensins (NPIC) was tested using a cell suspension of P. aeruginosa in Tris buffer in the presence or absence of 50 mM KCl. A plate colony-counting method was used to determine the percentage of cell viability relative to the untreated control (100%). Human lactoferrin (hLf) and BM2 peptide or nystatin (Nys) and colistin (Cst) were used as positive or negative controls, respectively. Error bars represent the SD of the means determined from three independent experiments. Student’s t -test: * p < 0.05; ** p < 0.01; *** p < 0.001 relative to the control.

Article Snippet: BM2 peptide ( D -NH 2 -RRRFWWFRRR-CONH 2 ) was chemically synthesized and purified via high-performance liquid chromatography using GenScript (Piscataway, NJ, USA).

Techniques: Activity Assay, Suspension, Incubation, Inhibition, Control

Journal: International Journal of Molecular Sciences

Article Title: The Antimicrobial Activity of Human Defensins at Physiological Non-Permeabilizing Concentrations Is Caused by the Inhibition of the Plasma Membrane H + -ATPases

doi: 10.3390/ijms25137335

Figure Lengend Snippet: Influence of bioenergetic processes on the microbicidal activity of human defensins. ( A ) Viability of C. albicans cells treated with defensins (NPIC) in the presence of piericidin A (complex I inhibitor). ( B ) Viability of P. aeruginosa cells treated with the defensins HNP-1 or hBD-2 in the presence of piericidin A. In ( A , B ), cell suspensions (10 6 cells/mL) in Tris buffer were preincubated (for 15 min at 37 °C) with the inhibitor and then incubated with the defensins (NPIC). ( C ) Viability of fermenting L. lactis cells treated with a range of concentrations of HNP-1 (yellow bars) or hBD-2 (orange bars). The effect of defensins on membrane integrity was monitored by determining the percentage of propidium iodide (PI)-positive cells after treatment with HNP-1 (grey bars) or hBD-2 (dark bars). ( D ) Viability of P. aeruginosa cells treated with defensin HNP-1 or hBD-2 (NPIC) after preincubation (for 15 min at 37 °C) with 50 μM CCCP (protonophore). ( E ) Viability of P. aeruginosa cells treated with 50 μM CCCP alone at different extracellular pHs. ( F ) Viability of C. albicans cells treated with the defensins HNP-1 or hBD-2 in the presence of oligomycin A (mitochondrial ATP synthase inhibitor). Human lactoferrin (hLf) or BM2 peptide was used as a positive control. The percentage of cell viability relative to the untreated control (100%) was determined using a plate colony-counting method. Data are from at least three similar experiments, and bars indicate ± SD. Student’s t -test: * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: BM2 peptide ( D -NH 2 -RRRFWWFRRR-CONH 2 ) was chemically synthesized and purified via high-performance liquid chromatography using GenScript (Piscataway, NJ, USA).

Techniques: Activity Assay, Incubation, Membrane, Positive Control, Control

Journal: International Journal of Molecular Sciences

Article Title: The Antimicrobial Activity of Human Defensins at Physiological Non-Permeabilizing Concentrations Is Caused by the Inhibition of the Plasma Membrane H + -ATPases

doi: 10.3390/ijms25137335

Figure Lengend Snippet: Effect of defensins on the total ATP level, the electrical potential of the plasma membrane, and the glucose-induced external acidification in C. albicans . Cells were preincubated (for 15 min at 37 °C) with defensins (NPIC) or with other peptides [5 μM human lactoferrin (hLf), 0.25 μM BM2 peptide] used as positive controls. ( A ) Increment in ATP levels in C. albicans cells exposed to defensins. The percentage change in total cellular ATP after 30 min treatment with the peptides (NPIC) compared to the untreated control is shown. ( B ) Relative level of membrane electrical potential of C. albicans treated with defensins. Percentage of DiBAC 4 (3)-positive cells (depolarized cells) exposed to defensins relative to untreated control (100%). ( C ) Effect of defensins on glucose-dependent external acidification. Glucose was added to induce the proton pumping activity of Pma1p H + -ATPase as indicated with external acidification in control assays. HNP-1 (grey line), HNP-4 (orange line), hBD-2 (blue line), hBD-3 (green line), BM2 peptide (violet line), human lactoferrin (red line), and control (black line). Data are from at least three similar experiments, and bars indicate ± SD. In ( C ), only the mean data ( n = 3) are shown, and the bars representing the ±SD (coefficient of variation of ≤10%) are omitted for clarity.

Article Snippet: BM2 peptide ( D -NH 2 -RRRFWWFRRR-CONH 2 ) was chemically synthesized and purified via high-performance liquid chromatography using GenScript (Piscataway, NJ, USA).

Techniques: Clinical Proteomics, Membrane, Control, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H + -ATPases in Target Pathogens

doi: 10.3390/ijms25179672

Figure Lengend Snippet: Potassium efflux induced by hLf γ-core-containing peptides. ( A , D ) Kinetics of K + efflux. C. albicans or P. aeruginosa (10 7 cells/mL) were incubated with kaliocin-1, Kdp15, human lactoferrin (hLf), and Lfpep. hLf and Lfpep were used as negative and positive controls of the membrane permeabilization, respectively. The effect of antifungal BM2 peptide was evaluated. The extracellular K + concentration was quantified at different times. ( B , C , E ) Effect of extracellular K + and TEA + on microbicidal activity. ( B , E ) microbicidal activity was assessed in Tris buffer in the absence or in the presence of 50 mM KCl or ( C ) 10 mM TEA + . Cell viability was determined by plating aliquots of the cell suspensions. The percentage of viable cells was determined relative to that for cells incubated without peptides (control). Results are the mean ± SD from duplicates of at least three independent determinations. * p < 0.05, ** p < 0.01, and *** p < 0.001 were used.

Article Snippet: The antifungal peptide BM2 ( D -NH 2 -RRRFWWFRRR-CONH 2 , D-form amino acids) described by Monk et al. [ ] was synthesized according to the conventional Fmoc chemistry and purchased from Genosphere Biotechnologies (Clamart, France).

Techniques: Incubation, Membrane, Concentration Assay, Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H + -ATPases in Target Pathogens

doi: 10.3390/ijms25179672

Figure Lengend Snippet: Effect of cellular respiration on the microbicidal activity of hLf γ-core-containing peptides. ( A , C ) Oxygen consumption was measured in C. albicans and P. aeruginosa cellular suspensions in the absence (control) or the presence of kaliocin-1 (Kal-1), Kdp15, or human lactoferrin (hLf), and piericidin A (positive control). ( B , D ) Effect of cellular respiration on the microbicidal activity of hLf γ-core-containing peptides. Cells (10 6 cells/mL) were pre-incubated (15 min, 37 °C) with 32 μM piericidin A and incubated for 90 min at 37 °C with the peptides hLf or BM2. The cell viability was determined by means of the plate counting method. The results are the mean ± SD from duplicates of at least three independent experiments. * p < 0.05, ** p < 0.01, and *** p < 0.001 were used.

Article Snippet: The antifungal peptide BM2 ( D -NH 2 -RRRFWWFRRR-CONH 2 , D-form amino acids) described by Monk et al. [ ] was synthesized according to the conventional Fmoc chemistry and purchased from Genosphere Biotechnologies (Clamart, France).

Techniques: Activity Assay, Control, Positive Control, Incubation

Journal: International Journal of Molecular Sciences

Article Title: The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H + -ATPases in Target Pathogens

doi: 10.3390/ijms25179672

Figure Lengend Snippet: Effect of inhibition of mitochondrial ATP synthase on the candidacidal activity of hLf γ-core-containing peptides. Candidacidal effect of IC 50 of kaliocin-1 (Kal-1) and Kdp15 and 0.25 μM BM2 on cells (10 6 cells/mL) pre-incubated without or with 16 μg/mL oligomycin A. Cell viability was determined by plating aliquots of the cell suspensions and the percentage of viable cells was determined relative to that for cells incubated without peptides. Human lactoferrin (hLf) and Lfpep were used as positive and negative controls, respectively. Data are the mean ± SD from at least three experiments. ** p < 0.01, and *** p < 0.001 were used.

Article Snippet: The antifungal peptide BM2 ( D -NH 2 -RRRFWWFRRR-CONH 2 , D-form amino acids) described by Monk et al. [ ] was synthesized according to the conventional Fmoc chemistry and purchased from Genosphere Biotechnologies (Clamart, France).

Techniques: Inhibition, Activity Assay, Incubation

Journal: International Journal of Molecular Sciences

Article Title: The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H + -ATPases in Target Pathogens

doi: 10.3390/ijms25179672

Figure Lengend Snippet: Effect of hLf γ-core-containing peptides on PM H + -ATPases. ( A ) Effect of hLf γ-core-containing peptides on glucose-dependent external acidification. C. albicans cells (10 7 cells/mL) in 50 mM KCl were preincubated with the peptides (2 × IC 50 ) for 20 min. Glucose (final concentration of 2.5 mM) was then added to induce H + efflux via Pma1p H + -ATPase, as indicated by the subsequent external acidification. For clarity, only one set of three independent experiments and values measured every 5 min are represented. ( B ) Effect of hLf γ-core-containing peptides on intracellular ATP (iATP) levels. Measurement of iATP of C. albicans cells ( B ) cells in the presence of kaliocin-1 (Kal-1) or Kdp15. The PM H + -ATPases inhibitors, human lactoferrin (hLf), and the peptide BM2 were used as positive controls. The total cellular iATP concentrations were determined after 90 min of treatment. The mean data ± SD are indicated as the percent change compared to that for the untreated control sample.

Article Snippet: The antifungal peptide BM2 ( D -NH 2 -RRRFWWFRRR-CONH 2 , D-form amino acids) described by Monk et al. [ ] was synthesized according to the conventional Fmoc chemistry and purchased from Genosphere Biotechnologies (Clamart, France).

Techniques: Concentration Assay, Control

Journal: PLoS Computational Biology

Article Title: Scalable Parameter Estimation for Genome-Scale Biochemical Reaction Networks

doi: 10.1371/journal.pcbi.1005331

Figure Lengend Snippet: List of investigated models and their properties.

Article Snippet: From the biomodels database we considered models for the regulation of insulin signaling by oxidative stress (BM1) [ ], the sea urchin endomesoderm network (BM2) [ ], and the ErbB sigaling pathway (BM3) [ ].

Techniques: