Rapid Membrane Depolarization Research Articles

Staphylococcus aureus α-toxin impairs early neutrophil localization via electrogenic disruption of store-operated calcium entry

The pore-forming S.aureus α-toxin (Hla) contributes to virulence and disease pathogenesis. While high concentrations of toxin induce cell death, neutrophils exhibit relative resistance to lysis, suggesting that the action of Hla may not be solely conferred by lytic susceptibility. Using intravital microscopy, we observed that Hla disrupts neutrophil localization and clustering early in infection. Hla forms a narrow, ion-selective pore, suggesting that Hla may dysregulate calcium or other ions to impair neutrophil function. We found that sub-lytic Hla did not permit calcium influx but caused rapid membrane depolarization. Depolarization decreases the electrogenic driving force for calcium, and concordantly, Hla suppressed calcium signaling invitro and invivo and calcium-dependent leukotriene B4 (LTB4) production, a key mediator of neutrophil clustering. Thus, Hla disrupts the early patterning of the neutrophil response to infection, in part through direct impairment of neutrophil calcium signaling. This early mis-localization of neutrophils may contribute to establishment of infection.

Antimicrobial Properties and Mode of Action of Cryptdin-4, a Mouse α-Defensin Regulated by Peptide Redox Structures and Bacterial Cultivation Conditions

Cryptdin-4 (crp4) is an enteric α-defensin derived from mice, and is a main mediator of immunity to oral infections and a determinant of the composition of the intestinal microbiota. Structurally, crp4 exists in two states: the oxidized form (crp4oxi), constrained by three invariant disulfide bonds, and the reduced form (crp4red) with six free thiol groups, both of which exist in the intestinal tract. In this study, the antibacterial mechanisms of crp4 in both forms under aerobic and anaerobic conditions were investigated using Escherichia coli (E. coli), an anaerobic facultative bacterium, as a model. Fluorescent dye studies revealed that both crp4oxi and crp4red exhibited antimicrobial activity against cells cultured under aerobic conditions via rapid membrane depolarization. Furthermore, the antioxidant treatment experiments suggested that only crp4oxi exhibited antimicrobial activity by the induction and accumulation of reactive oxygen species (ROS). However, under anaerobic culture conditions, the ability of both forms to disrupt the function of bacterial membranes decreased and activity was greatly reduced, but crp4red maintained some antimicrobial activity. This activity may be due to the inhibition of intracellular functions by DNA binding. Altogether, these data indicate that, according to its redox structure and the environmental redox conditions, crp4 could perform different antimicrobial activities via different mechanisms.

Relative Membrane Potential Measurements Using DISBAC2(3) Fluorescence in Arabidopsis thaliana Primary Roots.

In vivo microscopy of plants with high-frequency imaging allows observation and characterization of the dynamic responses of plants to stimuli. It provides access to responses that could not be observed by imaging at a given time point. Such methods are particularly suitable for the observation of fast cellular events such as membrane potential changes. Classical measurement of membrane potential by probe impaling gives quantitative and precise measurements. However, it is invasive, requires specialized equipment, and only allows measurement of one cell at a time. To circumvent some of these limitations, we developed a method to relatively quantify membrane potential variations in Arabidopsis thaliana roots using the fluorescence of the voltage reporter DISBAC2(3). In this protocol, we describe how to prepare experiments for agar media and microfluidics, and we detail the image analysis. We take an example of the rapid plasma membrane depolarization induced by the phytohormone auxin to illustrate the method. Relative membrane potential measurements using DISBAC2(3) fluorescence increase the spatio-temporal resolution of the measurements and are non-invasive and suitable for live imaging of growing roots. Studying membrane potential with a more flexible method allows to efficiently combine mature electrophysiology literature and new molecular knowledge to achieve a better understanding of plant behaviors. Key features Non-invasive method to relatively quantify membrane potential in plant roots. Method suitable for imaging seedlings root in agar or liquid medium. Straightforward quantification.

Insight into the antibacterial activity of lauric arginate against Escherichia coli O157:H7: Membrane disruption and oxidative stress

Lauric arginate (LAE), an amino acid-based cationic surfactant, has recently attracted great attentions for applications in food preservation due to its antimicrobial properties. This work aimed to unravel the possible antibacterial mechanisms of LAE against Escherichia coli O157:H7. After exposure to LAE at 50 μg/mL for 10 min, the microbial load decreased by 6.25-log units. According to the results of propidium iodide and N-phenyl-1-napthylamine fluorescent staining, LAE severely damaged the cytoplasmic and outer membranes of E. coli O157:H7, leading to a loss of membrane integrity. An increase in DiBAC4(3) fluorescent intensity was observed in LAE-treated cells, indicating a rapid membrane depolarization. Lipopolysaccharide and peptidoglycan could effectively block LAE-triggered inactivation of E. coli O157:H7 cells, suggesting the binding of LAE with cell membrane components. Moreover, LAE also resulted in an increase in intracellular reactive oxygen species levels and the coadministration of antioxidants (such as ascorbic acid and glutathione) could effectively quench LAE-mediated bacterial inactivation, indicating that oxidative stress was involved in the antibacterial action of LAE. In summary, the antibacterial activity of LAE against bacteria may be associated with a combined action of membrane damage and oxidative stress. This study provides a better understanding of the antibacterial mechanism of LAE.

Alkyltriphenylphosphonium turns naphthoquinoneimidazoles into potent membrane depolarizers against mycobacteria.

Due to its central role in energy generation and bacterial viability, mycobacterial bioenergetics is an attractive therapeutic target for anti-tuberculosis drug discovery. Building upon our work on antimycobacterial dioxonaphthoimidazoliums that were activated by a proximal positive charge and generated reactive oxygen species upon reduction by Type II NADH dehydrogenase, we herein studied the effect of a distal positive charge on the antimycobacterial activity of naphthoquinoneimidazoles by incorporating a trialkylphosphonium cation. The potency-enhancing properties of the linker length were affirmed by structure-activity relationship studies. The most active compound against M. tb H37Rv displayed good selectivity index (SI = 34) and strong bactericidal activity in the low micromolar range, which occurred through rapid bacterial membrane depolarization that resulted in depletion of intracellular ATP. Through this work, we demonstrated a switch of the scaffold's mode-of-action via relocation of positive charge while retaining its excellent antibacterial activity and selectivity.

Immobilizing Bactericides on Dental Resins via Electron Beam Irradiation

Polymerizable bactericides, such as quaternary ammonium compound–based monomers, have been intensively studied as candidates for immobilizing antibacterial components on dental resin. However, they predominantly exhibit a bacteriostatic behavior, rather than bactericidal, as the immobilized components are left with insufficient molecular movement to disrupt the bacterial surface structure through contact-mediated action. In this study, we developed a novel strategy to increase the density of the immobilized bactericide and enhance its antibacterial/antibiofilm properties by combining a surface-grafting technique with electron beam irradiation. A solution of the quaternary ammonium compound–based monomer, 12-methacryloyloxydodecylpyridinium bromide (MDPB), was coated on polymethyl methacrylate (PMMA) resin specimens at the concentrations of 30, 50, and 80 wt%. The coated resins were subsequently exposed to 10 MeV of electron beam irradiation at 50 and 100 kGy, followed by thermal stabilization at 60 °C. The antibacterial effect was evaluated by inoculating a Streptococcus mutans suspension on the coated PMMA resin samples, which exhibited bactericidal effects even after 28 d of aging (P < 0.05, Tukey’s honestly significant difference test). Transmission electron microscopy and bacteriolytic activity evaluation revealed that the S. mutans cells had sustained membrane depolarization. Furthermore, the antibiofilm effects against S. mutans and bacteria collected from human saliva were assessed. The thickness and the percentage of membrane-intact cells of the S. mutans and multispecies biofilms formed on the MDPB-immobilized surfaces were significantly lower than the uncoated PMMA specimens, even after 28-d aging (P < 0.05, Tukey’s honestly significant difference test). Thus, the immobilization of antibacterial MDPB via electron beam irradiation induced rapid membrane depolarization, increasing membrane permeability and eventually causing cell death. Our strategy substantially enhances the antibacterial properties of the resinous materials and inhibits biofilm formation, therefore demonstrating significant potential for preventing infectious diseases in the oral environment.

Identification of Escherichia coli Host Genes That Influence the Bacteriophage Lambda (λ) T4rII Exclusion (Rex) Phenotype.

The T4rII exclusion (Rex) phenotype is the inability of T4rII mutant bacteriophage to propagate in hosts (Escherichia coli) lysogenized by bacteriophage lambda (λ). The Rex phenotype, triggered by T4rII infection of a rex+ λ lysogen, results in rapid membrane depolarization imposing a harsh cellular environment that resembles stationary phase. Rex "activation" has been proposed as an altruistic cell death system to protect the λ prophage and its host from T4rII superinfection. Although well studied for over 60 years, the mechanism behind Rex still remains unclear. We have identified key nonessential genes involved in this enigmatic exclusion system by examining T4rII infection across a collection of rex+ single-gene knockouts. We further developed a system for rapid, one-step isolation of host mutations that could attenuate/abrogate the Rex phenotype. For the first time, we identified host mutations that influence Rex activity and rex+ host sensitivity to T4rII infection. Among others, notable genes include tolA, ompA, ompF, ompW, ompX, ompT, lpp, mglC, and rpoS They are critical players in cellular osmotic balance and are part of the stationary phase and/or membrane distress regulons. Based on these findings, we propose a new model that connects Rex to the σS, σE regulons and key membrane proteins.

Caspase-3 Activation Correlates With the Initial Mitochondrial Membrane Depolarization in Neonatal Cerebellar Granule Neurons.

In this study we evaluated the effect of the reduction in the endoplasmic reticulum calcium concentration ([Ca2+]ER), changes in the cytoplasmic calcium concentration ([Ca2+]i), alteration of the mitochondrial membrane potential, and the ER stress in the activation of caspase-3 in neonatal cerebellar granule cells (CGN). The cells were loaded with Fura-2 to detect changes in the [Ca2+]i and with Mag-fluo-4 to measure variations in the [Ca2+]ER or with TMRE to follow modifications in the mitochondrial membrane potential in response to five different inducers of CGN cell death. These inducers were staurosporine, thapsigargin, tunicamycin, nifedipine and plasma membrane repolarization by switching culture medium from 25 mM KCl (K25) to 5 mM KCl (K5). Additionally, different markers of ER stress were determined and all these parameters were correlated with the activation of caspase-3. The different inducers of cell death in CGN resulted in three different levels of activation of caspase-3. The highest caspase-3 activity occurred in response to K5. At the same time, staurosporine, nifedipine, and tunicamycin elicited an intermediate activation of caspase-3. Importantly, thapsigargin did not activate caspase-3 at any time. Both K5 and nifedipine rapidly decreased the [Ca2+]i, but only K5 immediately reduced the [Ca2+]ER and the mitochondrial membrane potential. Staurosporine and tunicamycin increased the [Ca2+]i and they decreased both the [Ca2+]ER and mitochondrial membrane potential, but at a much lower rate than K5. Thapsigargin strongly increased the [Ca2+]i, but it took 10 min to observe any decrease in the mitochondrial membrane potential. Three cell death inducers -K5, staurosporine, and thapsigargin- elicited ER stress, but they took 30 min to have any effect. Thapsigargin, as expected, displayed the highest efficacy activating PERK. Moreover, a specific PERK inhibitor did not have any impact on cell death triggered by these cell death inducers. Our data suggest that voltage-gated Ca2+ channels, that are not dihydropyridine-sensitive, load the ER with Ca2+ and this Ca2+ flux plays a critical role in keeping the mitochondrial membrane potential polarized. A rapid decrease in the [Ca2+]ER resulted in rapid mitochondrial membrane depolarization and strong activation of caspase-3 without the intervention of the ER stress in CGN.

Cellular mechanisms underlying the rapid depolarization caused by oxygen and glucose deprivation in layer III pyramidal cells of the somatosensory cortex

Cortical pyramidal neurons show rapid and irreversible membrane depolarization in response to oxygen-glucose depolarization (OGD). In this study, we investigated cellular mechanisms responsible for rapid depolarization caused by OGD in layer III pyramidal neurons of the mouse somatosensory cortex. When OGD solution was perfused in the presence of Ca2+ chelator and inhibitors of ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs) in the pipette solution or in the presence of inhibitors of NMDA receptors (NMDARs), voltage-gated Ca2+ channels (VGCCs), and canonical transient receptor potential (TRPC) channels in the perfusion solution, the latency of the rapid depolarization was significantly prolonged compared to the control. In addition, when OGD solution was perfused in the presence of scavengers of nitric oxide and reactive oxygen species in the perfusion solution or in the presence of calcineurin inhibitors in the pipette solution, the latency of the rapid depolarization was significantly prolonged compared to the control. These data indicate that OGD-induced intracellular Ca2+ increases mediated by Ca2+ influx through NMDARs, VGCCs and TRPC channels as well as by Ca2+ release from RyRs and IP3Rs lead to mitochondrial impairment, which may facilitate the generation of the rapid depolarization via dysfunction of Na+-K+-ATPase due to decreased ATP production.

In Vitro and Ex Vivo Efficacy of Novel Trp-Arg Rich Analogue of α-MSH against Staphylococcus aureus.

Antimicrobial peptides (AMPs), an essential component of innate immunity, are very important resources for human therapeutics to counter the current threat of drug resistance. We have previously established that one such AMP, α-melanocyte stimulating hormone (α-MSH), an endogenous neuropeptide, and its derivatives have potent antimicrobial activity against Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). However, the immense potential of α-MSH for therapeutic development against staphylococcal infections is marred by its reduced efficacy in the presence of standard microbiological culture medium. To overcome this issue, in this study, we designed a series of five novel analogues of the C-terminal fragment of α-MSH, i.e., α-MSH(6–13), by replacing uncharged and less hydrophobic residues with tryptophan and arginine to increase the hydrophobicity and cationic charge of the peptide, respectively. While all of the peptides showed a preferential interaction with negatively charged phospholipid vesicles, the most hydrophobic and cationic peptide, i.e., Ana-5, exhibited the highest activity against S. aureus cells while maintaining cell selectivity. Moreover, Ana-5 could retain its activity even in complex media like the Mueller Hinton broth and displayed rapid bactericidal activity in the presence of serum. Ana-5 also caused rapid bacterial membrane depolarization, permeabilization, and cell lysis and was able to bind to polyanionic plasmid DNA suggesting a possible dual mode of action of the peptide. Importantly, Ana-5 was able to eradicate intracellular S. aureus in fibroblast cells similar to conventional antibiotics. Collectively, in the present study, we obtained a potent α-MSH-based analogue with excellent staphylocidal potency in microbial growth medium and ex vivo efficacy, which may translate into therapeutic application.

6-Bromoindolglyoxylamido derivatives as antimicrobial agents and antibiotic enhancers.

The combination of increased incidence of drug-resistant strains of bacteria and a lack of novel drugs in development creates an urgency for the search for new antimicrobials. Initial screening of compounds from an in-house library identified two 6-bromoindolglyoxylamide polyamine derivatives (3 and 4) that exhibited intrinsic antimicrobial activity towards Gram-positive bacteria, Staphylococcus aureus and S. intermedius with polyamine 3 also displaying in vitro antibiotic enhancing properties against the resistant Gram-negative bacterium Pseudomonas aeruginosa. A series of 6-bromo derivatives (5-15) were prepared and biologically evaluated, identifying analogues with enhanced antibacterial activity towards Escherichia coli and with moderate to excellent antifungal properties. Polyamine 3, which includes a spermine chain, was the most potent of the series - its mechanism of action was attributed to rapid membrane permeabilization and depolarization in both Gram-positive and Gram-negative bacteria.

Cysteine: A multifaceted amino acid involved in signaling, plant resistance and antifungal development

Early effects induced by cysteine were monitored using the model of Mimosa pudica pulvinar cells. Rapid dose-dependent membrane depolarization (within seconds) and modification of proton secretion (within minutes) were triggered at cysteine concentrations higher than 0.1 mM. These effects did not result from a modification of the plasma membrane H+-ATPase activity nor from a protonophore effect as shown by assays on plasma membrane vesicles isolated from pulvinar tissues. In a 0.5–10 mM range, cysteine inhibited the ion-driven turgor-mediated seismonastic reaction of Mimosa pudica primary pulvini and the dark-induced movement of Cassia fasciculata leaflets. At concentrations higher than 1 mM, it induced a long-lasting leaflet necrosis dependent on the concentration and treatment duration. Electron microscopy showed that cysteine induced important damage in the nucleus, mitochondria, endoplasmic reticulum and Golgi of the M. pudica motor cell. Cysteine inhibited in a concentration-dependent manner, from 0.5 to 20 mM, both the mycelial growth and the spore germination of the fungal pathogens Phaeomoniella chlamydospora and Phaeoacremonium minimum implicated in esca disease of grapevines. Using [35S] cysteine, we showed that the amino acid was absorbed following leaf spraying, translocated from leaves to other parts of grapevine cuttings and accumulated within trunks and roots. Therefore, cysteine showed relevant properties to be a candidate able to control fungal diseases either by acting as an early signal directing plant host reaction or/and by acting directly on fungal development.

Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis.

Antimicrobial peptides (AMPs) are a diverse group of molecules which play an important role in the innate immune response. Bovine NK-lysins, a type of AMP, have been predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Bovine NK-lysin-derived peptides demonstrate antimicrobial activity against various bacterial pathogens, including several involved in bovine respiratory disease complex (BRDC) in cattle; however, such studies are yet to be performed with one important contributor to the BRDC, Mycoplasma bovis. Therefore, the goal of this study was to assess the antimicrobial activity of bovine NK-lysin-derived peptides on M. bovis. Thirty-mer synthetic peptides corresponding to the functional region helices 2 and 3 of bovine NK-lysins NK1, NK2A, NK2B, and NK2C were evaluated for killing activity on M. bovis isolates. Among four peptides, NK2A and NK2C showed the highest antimicrobial activity against the M. bovis isolates tested. All four NK-lysin peptides induced rapid plasma membrane depolarization in M. bovis at two concentrations tested. However, based on propidium iodide uptake, only NK2A and NK2C appeared capable of causing structural damage to M. bovis plasma membrane. Confocal microscopy, flow cytometry, and transmission electron microscopy further suggested NK-lysin-induced damage to the plasma membrane. Taken together, the findings in this study suggest that plasma membrane depolarization alone was insufficient to induce lethality, but disruption/permeabilization of the M. bovis plasma membrane was the cause of lethality.

Non-linear amplification of graded voltage signals in the first-order visual interneurons of the butterfly Papilio xuthus.

Lamina monopolar cells (LMCs) are the first-order visual interneurons of insects and crustacea, primarily involved in achromatic vision. Here, we investigated morphological and electrophysiological properties of LMCs in the butterfly Papilio xuthus Using intracellular recording coupled with dye injection, we found two types of LMCs. Cells with roundish terminals near the distal surface of the medulla demonstrating no or small depolarizing spikes were classified as L1/2. Cells with elongated terminals deep in the medulla that showed prominent spiking were classified as L3/4. The majority of LMCs of both types had broad spectral sensitivities, peaking between 480 and 570 nm. Depending on the experimental conditions, spikes varied from small to action potential-like events, with their amplitudes and rates decreasing as stimulus brightness increased. When the eye was stimulated with naturalistic contrast-modulated time series, spikes were reliably triggered by high-contrast components of the stimulus. Spike-triggered average functions showed that spikes emphasize rapid membrane depolarizations. Our results suggest that spikes are mediated by voltage-activated Na+ channels, which are mainly inactivated at rest. Strong local minima in the coherence functions of spiking LMCs indicate that the depolarizing conductance contributes to the amplification of graded responses even when detectable spikes are not evoked. We propose that the information transfer strategies of spiking LMCs change with light intensity. In dim light, both graded voltage signals and large spikes are used together without mutual interference, as a result of separate transmission bandwidths. In bright light, signals are non-linearly amplified by the depolarizing conductance in the absence of detectable spikes.

A quinolinol-based small molecule with anti-MRSA activity that targets bacterial membrane and promotes fermentative metabolism.

In a loss-of-viability screen of small molecules against methicillin resistant S. aureus (MRSA) USA300, we found a small molecule, designated DNAC-2, which has an MIC of 8 μg/ml. DNAC-2 is a quinolinol derivative that is bactericidal at 2X MIC. Macromolecular synthesis assays at 2X MIC of DNAC-2 inhibited DNA, cell wall, RNA and protein synthesis within fifteen to thirty minutes of treatment when compared to the untreated control. Transmission electron microscopy of DNAC-2 treated cells revealed a significantly thicker cell wall and impaired daughter cell separation. Exposure of USA300 cells to 1X MIC of DNAC-2 resulted in mislocalization of PBP2 away from the septum in an FtsZ independent manner. In addition, membrane localization with FM4-64, as well as depolarization study with DiOC2 and lipophilic cation TPP+ displayed membrane irregularities and rapid membrane depolarization in DNAC-2 treated cells vs. untreated control. However, DNAC-2 exhibited almost no toxicity towards eukaryotic membranes. Notably, DNAC-2 drives energy generation towards substrate level phosphorylation and the bacteria become more sensitive to DNAC-2 under anaerobic conditions. We propose that DNAC-2 affects USA300 by targeting the membrane, leading to partial membrane depolarization and subsequently affecting aerobic respiration and energy-dependent functional organization of macromolecular biosynthetic pathways. The multiple effects may have the desirable consequence of limiting the emergence of resistance to DNAC-2.

A broad-spectrum bactericidal lipopeptide with anti-biofilm properties

Previous studies of the oligoacyllysyl (OAK) series acyl-lysyl-lysyl-aminoacyl-lysine-amide, suggested their utility towards generating robust linear lipopeptide-like alternatives to antibiotics, although to date, none exhibited potent broad-spectrum bactericidal activity. To follow up on this premise, we produced a new analog (C14KKc12K) and investigated its properties in various media. Mechanistic studies suggest that C14KKc12K uses a non-specific membrane-disruptive mode of action for rapidly reducing viability of Gram-negative bacteria (GNB) similarly to polymyxin B (PMB), a cyclic lipopeptide used as last resort antibiotic. Indeed, C14KKc12K displayed similar affinity for lipopolysaccharides and induced cell permeabilization associated with rapid massive membrane depolarization. Unlike PMB however, C14KKc12K was also bactericidal to Gram-positive bacteria (GPB) at or near the minimal inhibitory concentration (MIC), as assessed against a multispecies panel of >50 strains, displaying MIC50 at 3 and 6 µM, respectively for GPB and GNB. C14KKc12K retained activity in human saliva, reducing the viability of cultivable oral microflora by >99% within two minutes of exposure, albeit at higher concentrations, which, nonetheless, were similar to the commercial gold standard, chlorhexidine. This equipotent bactericidal activity was also observed in pre-formed biofilms of Streptococcus mutans, a major periodontal pathogen. Such compounds therefore, may be useful for eradication of challenging poly-microbial infections.

Insight into the mechanism of action of temporin-SHa, a new broad-spectrum antiparasitic and antibacterial agent

Antimicrobial peptides (AMPs) are promising drugs to kill resistant pathogens. In contrast to bacteria, protozoan parasites, such as Leishmania, were little studied. Therefore, the antiparasitic mechanism of AMPs is still unclear. In this study, we sought to get further insight into this mechanism by focusing our attention on temporin-SHa (SHa), a small broad-spectrum AMP previously shown to be active against Leishmania infantum. To improve activity, we designed analogs of SHa and compared the antibacterial and antiparasitic mechanisms. [K3]SHa emerged as a highly potent compound active against a wide range of bacteria, yeasts/fungi, and trypanosomatids (Leishmania and Trypanosoma), with leishmanicidal intramacrophagic activity and efficiency toward antibiotic-resistant strains of S. aureus and antimony-resistant L. infantum. Multipassage resistance selection demonstrated that temporins-SH, particularly [K3]SHa, are not prone to induce resistance in Escherichia coli. Analysis of the mode of action revealed that bacterial and parasite killing occur through a similar membranolytic mechanism involving rapid membrane permeabilization and depolarization. This was confirmed by high-resolution imaging (atomic force microscopy and field emission gun-scanning electron microscopy). Multiple combined techniques (nuclear magnetic resonance, surface plasmon resonance, differential scanning calorimetry) allowed us to detail peptide-membrane interactions. [K3]SHa was shown to interact selectively with anionic model membranes with a 4-fold higher affinity (KD = 3 x 10−8 M) than SHa. The amphipathic α-helical peptide inserts in-plane in the hydrophobic lipid bilayer and disrupts the acyl chain packing via a detergent-like effect. Interestingly, cellular events, such as mitochondrial membrane depolarization or DNA fragmentation, were observed in L. infantum promastigotes after exposure to SHa and [K3]SHa at concentrations above IC50. Our results indicate that these temporins exert leishmanicidal activity via a primary membranolytic mechanism but can also trigger apoptotis-like death. The many assets demonstrated for [K3]SHa make this small analog an attractive template to develop new antibacterial/antiparasitic drugs.

Calcium-Induced Mitochondrial Permeability Transitions: Parameters of Ca2+ Ion Interactions with Mitochondria and Effects of Oxidative Agents.

We evaluated the parameters of Ca2+-induced mitochondrial permeability transition (MPT) pore formations, Ca2+ binding constants, stoichiometry, energy of activation, and the effect of oxidative agents, tert-butyl hydroperoxide (tBHP), and hypochlorous acid (HOCl), on Ca2+ -mediated process in rat liver mitochondria. From the Hill plot of the dependence of MPT rate on Ca2+ concentration, we determined the order of interaction of Ca2+ ions with the mitochondrial sites, n = 3, and the apparent Kd = 60 ± 12µM. We also found the apparent Michaelis-Menten constant, Km, for Ca2+ interactions with mitochondria to be equal to 75 ± 20µM, whereas that in the presence of 300µM tBHP was 120 ± 20µM. Using the Arrhenius plots of the temperature dependences of apparent mitochondrial swelling rate at various Ca2+ concentrations, we calculated the activation energy of the MPT process. ΔEa was 130 ± 20kJ/mol at temperatures below the break point of the Arrhenius plot (30-34 °C) and 50 ± 9kJ/mol at higher temperatures. Ca2+ ions induced rapid mitochondrial NADH depletion and membrane depolarization. Prevention of the pore formation by cyclosporin A inhibited Ca2+-dependent mitochondrial depolarization and Mg2+ ions attenuated the potential dissipation. tBHP (10-150µM) dose-dependently enhanced the rate of MPT opening, whereas the effect of HOCl on MPT depended on the ratio of HOCl/Ca2+. The apparent Km of tBHP interaction with mitochondria in the swelling reaction was found to be Km = 11 ± 3µM. The present study provides evidence that three calcium ions interact with mitochondrial site with high affinity during MPT. Ca2+-induced MPT pore formations due to mitochondrial membrane protein denaturation resulted in membrane potential dissipation. Oxidants with different mechanisms, tBHP and HOCl, reduced mitochondrial membrane potential and oxidized mitochondrial NADH in EDTA-free medium and had an effect on Ca2+-induced MPT onset.

High Antibacterial Activity of Functionalized Chemically Exfoliated MoS2.

In view of the implications of inherent resistance of pathogenic bacteria, especially ESKAPE pathogens toward most of the commercially available antibiotics and the importance of these bacteria-induced biofilm formation leading to chronic infection, it is important to develop new-generation synthetic materials with greater efficacy toward antibacterial property. In addressing this issue, this paper reports a proof-of-principle study to evaluate the potential of functionalized two-dimensional chemically exfoliated MoS2 (ce-MoS2) toward inhibitory and bactericidal property against two representative ESKAPE pathogenic strain-a Gram-positive Staphylococcus aureus (MRSA) and a Gram-negative Pseudomonas aeruginosa. More significantly, the mechanistic study establishes a different extent of oxidative stress together with rapid membrane depolarization in contact with ce-MoS2 having ligands of varied charge and hydrophobicity. The implication of our results is discussed in the light of the lack of survivability of planktonic bacteria and biofilm destruction in vitro. A comparison with widely used small molecules and other nanomaterial-based therapeutics conclusively establishes a better efficacy of 2D ce-MoS2 as a new class of antibiotics.

The defence elicitor AsES causes a rapid and transient membrane depolarization, a triphasic oxidative burst and the accumulation of nitric oxide

The newly characterized elicitor AsES obtained from Acremonium strictum induces a strong defence response in strawberry plants and confers plants resistance against the fungal pathogen Colletotricum acutatum the casual agent of anthracnose disease. Previous studies showed that AsES causes the accumulation of reactive oxygen species (ROS) that peaked 4 h post treatment (hpt), but due to the experimental approach used it was not clear whether the accumulation of ROS observed was intracellular or extracellular or took place as a single peak. By using a different experimental setup, a more complex early events associated to the activation of the innate immunity were observed. In this paper we report that strawberry plant cells treated with AsES exhibits a triphasic production of H2O2 and a rapid intracellular accumulation of NO. The first phase consists in a progressive extracellular accumulation of H2O2 that starts immediately after the treatment with AsES and is preceded by a rapid and transient cell membrane depolarization. During this phase takes place also a rapid intracellular accumulation of NO. Microscopic observations of mesophyll cells treated with AsES reveals that NO accumulates at the chloroplast. After the first extracellular H2O2 production phase, two intracellular H2O2 accumulation events occur, the first 2 hpt, and the second 7 hpt. Cells treated with AsES also show a transient increase of ion leakage, and a progressive alkalinization of the extracellular medium.