Disruption Of Membrane Potential Research Articles

Pharmacotherapy Update: Daptomycin in the Management of Complicated Skin and Soft-tissue Infections

Because of the dramatic increase in severe infections caused methicillin-resistant Staphylococcus aureus (MRSA), including skin and skin structure infections (SSSI), and reports of vancomycin failures in the treatment of these infections, agents with better activity against MRSA are clearly needed. Daptomycin, the first cyclic lipopeptide, exerts its concentration dependent bactericidal activity against S. aureus through a calcium dependent formation of channels leading to disruption of the bacterial cell membrane potential. Daptomycin is 90% protein bound, has a half-life of 8–9 h, is eliminated through renal excretion, and has a good penetration into inflammatory skin blisters. This compound was shown to be non-inferior to the comparator in two double-blind randomized phase III studies including patients with complicated SSSI, using a dose of 4 mg/kg/day. In these studies, cure rates in the clinical and microbiology evaluable populations were 83.4% and 84.2% (95% CI, -4 to 5.6), and 84.7% and 85.9% (95% CI, -3.8 to 6.3) for daptomycin and comparator, respectively. Daptomycin also showed efficacy in prospective studies in patients with infective cellulitis or erysipelas, necrotizing soft tissue infection, and infected diabetic foot ulcers. Even though the standard dose of 4 mg/kg/day is considered appropriate for most SSSI, higher doses (e.g. 6 to 10 mg/kg/day) are currently suggested for patients with sepsis, necrotizing infection, prior glycopeptide failure, infections caused by vancomycin intermediate S. aureus (VISA) strains, renal insufficiency, burns, and in intravenous drug users. Overall, daptomycin is considered a well tolerated antibiotic. In clinical trials, drug discontinuation rate has been similar in patients receiving daptomycin compared with those treated with the comparators; however, muscular damage associated with increased serum creatine phopshokinase levels occurs in about 3% and 7% of the patients treated with 4 and 6 mg/kg/day, respectively. This reversible muscle toxicity appears to correlate with the daptomycin trough concentration and time of exposure.

Cytotoxic Activity of Isoliquiritigenin towards CCRF‐CEM Leukemia Cells and its Effect on DNA Damage

The cytotoxic activity of isoliquiritigenin (ISL) towards CCRF-CEM human T-cell leukemia cells and DNA damage induction were investigated in the present study. The anti-proliferative effect of ISL on CCRF-CEM cell line IN VITRO was analyzed. In order to further explore the underlying mechanism of cell growth inhibition of ISL towards CCRF-CEM cell line, the cell cycle distribution and mitochondrial membrane potential (DeltaPsim) disruption were measured. ISL exerted significant activity towards CCRF-CEM cells. The growth inhibitory ratio was concentration- and time-dependent with an IC(50) value of 18.38 microM. The cell cycle was arrested in the G2/M phase upon treatment with low doses of ISL. Mitochondrial membrane potential (DeltaPsim) disruption was observed at low ISL doses. The effect of ISL on DNA damage was analyzed by agarose gel electrophoresis and atomic force microscopy (AFM). Either ISL or Cu2+ failed to cause pBR322 DNA damage. However, plasmid DNA was damaged after treatment with ISL in the presence of Cu2+. Two forms of plasmid DNA closed circular and linear forms were visualized by AFM.

Telavancin: An Antimicrobial with a Multifunctional Mechanism of Action for the Treatment of Serious Gram‐Positive Infections

Telavancin is a once-daily lipoglycopeptide antibiotic structurally derived from vancomycin. It has broad-spectrum activity against gram-positive bacteria, including strains with reduced susceptibility to vancomycin. Telavancin's multifunctional mechanism of action, including inhibition of peptidoglycan synthesis and disruption of membrane potential, account for this enhanced activity as well as rapid bactericidal properties. In vitro activity has been demonstrated against a wide range of gram-positive pathogens such as multidrug-resistant Streptococcus pneumoniae, as well as methicillin-resistant, glycopeptide-intermediate, and vancomycin-resistant Staphylococcus aureus. The agent also displays activity against many gram-positive anaerobic organisms. Predictable linear pharmacokinetics have been demonstrated over a wide range of doses, with the most common adverse effects being taste disturbance and nausea. Clinical experience with telavancin in phase II and III studies for complicated skin and skin structure infections has shown it to have similar efficacy and tolerability compared with vancomycin and antistaphylococcal penicillins, and recently telavancin received an approvable letter from the United States Food and Drug Administration for this indication. Telavancin appears to be a promising agent for the treatment of serious infections caused by gram-positive pathogens, including drug-resistant pathogens. Further clinical experience will clarify its role in therapy.

Necrotic pathway in human osteosarcoma Saos-2 cell death induced by chloroacetaldehyde

Chloroacetaldehyde, a metabolite of the anticancer drug ifosfamide, may be responsible for serious adverse effects like encephalopathy in ifosfamide chemotherapy. In this study, we demonstrate that chloroacetaldehyde, but not ifosfamide, induces cell death in human osteosarcoma Saos-2 cells and we investigated the mechanism by which this occurs. Chloroacetaldehyde above 30 micromol/l induced significant cell death in a time-dependent manner. Thiol compounds such as N-acetyl cysteine, glutathione and dithiothreitol protected the cells against chloroacetaldehyde-induced cell death, although other nonthiol compounds and the antioxidative enzymes superoxide dismutase and catalase did not, suggesting that reactive oxygen species might not mediate cell death. In cells exposed to chloroacetaldehyde, levels of both total thiols and glutathione were significantly reduced. Chloroacetaldehyde also collapsed the mitochondrial membrane potential of these cells, induced the release of cytochrome c from mitochondria to the cytosol and significantly reduced cellular ATP levels during the course of death. The mitochondrial potential collapse was also prevented by thiol compounds. Flow cytometric analyses by means of annexin-V and propidium iodide double staining and immunofluorescence staining of active caspase-3 revealed that cells subjected to a lethal dose of chloroacetaldehyde displayed features characteristic of necrosis and that caspase-3 was not activated in response to chloroacetaldehyde. Taken together, these findings suggest that Saos-2 cells exposed to chloroacetaldehyde die by necrosis resulting from a decrease in intracellular thiols, disruption of the mitochondrial membrane potential and the depletion of cellular ATP.

Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast

The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).

Yeast Model Uncovers Dual Roles of Mitochondria in the Action of Artemisinin

Artemisinins, derived from the wormwood herb Artemisia annua, are the most potent antimalarial drugs currently available. Despite extensive research, the exact mode of action of artemisinins has not been established. Here we use yeast, Saccharamyces cerevisiae, to probe the core working mechanism of this class of antimalarial agents. We demonstrate that artemisinin's inhibitory effect is mediated by disrupting the normal function of mitochondria through depolarizing their membrane potential. Moreover, in a genetic study, we identify the electron transport chain as an important player in artemisinin's action: Deletion of NDE1 or NDI1, which encode mitochondrial NADH dehydrogenases, confers resistance to artemisinin, whereas overexpression of NDE1 or NDI1 dramatically increases sensitivity to artemisinin. Mutations or environmental conditions that affect electron transport also alter host's sensitivity to artemisinin. Sensitivity is partially restored when the Plasmodium falciparum NDI1 ortholog is expressed in yeast ndi1 strain. Finally, we showed that artemisinin's inhibitory effect is mediated by reactive oxygen species. Our results demonstrate that artemisinin's effect is primarily mediated through disruption of membrane potential by its interaction with the electron transport chain, resulting in dysfunctional mitochondria. We propose a dual role of mitochondria played during the action of artemisinin: the electron transport chain stimulates artemisinin's effect, most likely by activating it, and the mitochondria are subsequently damaged by the locally generated free radicals.

Apoptotic effect of oridonin on NB4 cells and its mechanism

The anti-proliferation effects of oridonin on acute promyelocytic leukemia (APL) cells and its mechanisms were studied in vitro. NB4 cells as well as fresh leukemia cells obtained from APL patients in culture medium were treated with different concentrations of oridonin. Cell growth inhibition, apoptosis and related pathways were assessed by MTT assay as well as flow cytometry (FCM) and western blot analysis. The data revealed that oridonin (over 16 micromol/L) could inhibit the growth of NB4 cells by induction of apoptosis. Marked changes of cell apoptosis were observed very clearly by using electron microscopy and DNA fragmentation analysis after the cells exposed to oridonin for 48 h; Western blotting showed cleavage of the caspase-3 zymogen protein (32-kDa) with the appearance of its 20-kDa subunit as well as a cleaved 89-kDa fragment of 116-kDa PARP when apoptosis occurred. The expression of Bcl-2 was down-regulated remarkably accompanied by the disruption of the mitochondrial membrane potential (delta(psi)m). The anti-proliferative and apoptosis-inducing effects by oridonin in fresh APL cells were also found remarkably using Trypan Blue dye exclusion method and Wright's staining. We concluded that oridoning has significant anti-proliferative and apoptosis-inducing effects on NB4 cells by activation of caspase-3 and cleavage of PARP as well as by down regulation of Bcl-2 and disruption of the delta(psi)m. Furthermore, oridonin demonstrated apparent cell growth inhibition effects on fresh APL cells in vitro. The results indicated that oridonin may serve as a potential anti-leukemia reagent.

Mode of action of pyrazinamide: disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid.

Pyrazinamide is an important sterilizing drug that shortens tuberculosis (TB) therapy. However, the mechanism of action of pyrazinamide is poorly understood because of its unusual properties. Here we show that pyrazinoic acid, the active moiety of pyrazinamide, disrupted membrane energetics and inhibited membrane transport function in Mycobacterium tuberculosis. The preferential activity of pyrazinamide against old non-replicating bacilli correlated with their low membrane potential and the disruption of membrane potential by pyrazinoic acid and acid pH. Inhibitors of membrane energetics increased the antituberculous activity of pyrazinamide. These findings shed new light on the mode of action of pyrazinamide and may help in the design of new drugs that shorten therapy.

The redox state of endogenous pyridine nucleotides can determine both the degree of mitochondrial oxidative stress and the solute selectivity of the permeability transition pore

Acetoacetate, an NADH oxidant, stimulated the ruthenium red-insensitive rat liver mitochondrial Ca 2+ efflux without significant release of state-4 respiration, disruption of membrane potential (Δ ψ) or mitochondrial swelling. This process is compatible with the opening of the currently designated low conductance state of the permeability transition pore (PTP) and, under our experimental conditions, was associated with a partial oxidation of the mitochondrial pyridine nucleotides. In contrast, diamide, a thiol oxidant, induced a fast mitochondrial Ca 2+ efflux associated with a release of state-4 respiration, a disruption of Δ ψ and a large amplitude mitochondrial swelling. This is compatible with the opening of the high conductance state of the PTP and was associated with extensive oxidation of pyridine nucleotides. Interestingly, the addition of carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone to the acetoacetate experiment promoted a fast shift from the low to the high conductance state of the PTP. Both acetoacetate and diamide-induced mitochondrial permeabilization were inhibited by exogenous catalase. We propose that the shift from a low to a high conductance state of the PTP can be promoted by the oxidation of NADPH. This impairs the antioxidant function of the glutathione reductase/peroxidase system, strongly strengthening the state of mitochondrial oxidative stress.

Ca 2+-dependent permeabilization of the inner mitochondrial membrane by 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS)

We have recently shown that the permeabilization of the inner mitochondrial membrane by Ca 2+ plus prooxidants is associated with oxidation of protein thiols forming cross-linked protein aggregates (Fagian, M.M., Pereira-da-Silva, L., Martins, I.S. and Vercesi, A.E. (1990) J. Biol. Chem. 265, 19955–19960). In this study we show that the incubation of rat liver mitochondria in the presence of the thiol reagent 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and Ca 2+ caused production of membrane protein aggregates, mitochondrial swelling, disruption of membrane potential and Ca 2+ release. The presence of DTT prevented but did not reverse the elimination of Δψ induced by DIDS. EGTA prevented Δψ elimination and decreased the amount of protein aggregates, suggesting that the binding of CA 2+ to some membrane protein may expose buried thiols to react with DIDS. Reversal of collapsed Δψ by EGTA indicates that DIDS-induced protein aggregates require the presence of Ca 2+ for significant membrane permeabilization. Cyclosporin A prevented mitochondrial swelling, suggesting that DIDS-induced membrane protein polymerization mimics the condition designated as Ca 2+-induced permeabilization transition of mitochondria. The lack of oxidation of pyridine nucleotides or significant lipid peroxidation by DIDS supports the notion that membrane permeabilization by this compound is mediated by its interaction with membrane proteins.

The mode of action of SA-FF22, a lantibiotic isolated from Streptococcus pyogenes strain FF22.

SA-FF22 is a lanthionine-containing bacteriocidal peptide isolated from Streptococcus pyogenes strain FF22. The peptide interacts closely with non-energised artificial phospholipid vesicles, as evidenced by a 'blue shift' in the fluorescent emissions associated with a tryptophan residue within the peptide sequence. Furthermore, SA-FF22 induced efflux of radiolabelled amino acids from artificially energised cytoplasmic membrane vesicles and arrested uptake of amino acids by intact cells. By measuring the decrease in membrane potential of both starved and energised SA-FF22-treated cells, and through the use of artificial planar membranes, a potential of approximately 100 mV was deduced as the minimum required to induce pore formation by SA-FF22. This threshold potential is independent of the orientation of the applied voltage (i.e. trans or cis orientations are equally effective). Single channel conductance measurements suggested that the pores formed by SA-FF22 are relatively unstable, short-lived and approximately 0.5-0.6 nm in diameter. This is somewhat smaller than those of the previously described, pore-forming lantibiotics and should not allow significant efflux of large molecules such as ATP. Thus, death of affected cells seems to result from membrane-potential disruption and subsequent exhaustion of the cells.

Protective effect of trifluoperazine on the mitochondrial damage induced by Ca 2+ plus prooxidants

Isolated rat liver mitochondria undergo extensive swelling and disruption of membrane potential when they accumulate Ca 2+ in the presence of a prooxidant such as diamide or t-butylhydroperoxide. The phenothiazinic drug trifluoperazine, at concentrations (15–35 μM) which do not inhibit respiration or the influx of Ca 2+ into mitochondria, significantly protected mitochondria against the deleterious effects of Ca 2+ plus a prooxidant. In contrast, at concentrations higher than 100 μM the drug potentiated these deleterious effects of Ca 2+ and prooxidants and had a damaging effect per se on the inner mitochondrial membrane. It is proposed that the protection conferred by the drug is mediated by changes in membrane protein structure that decrease the production of protein thiol cross-linkings which occur when mitochondria accumulate calcium under oxidant stress conditions.