Membrane-active Compounds Research Articles

Potent Synergism of the Combination of Fluconazole and Cyclosporine in Candida albicans

Several types of drugs currently used in clinical practice were screened in vitro for their potentiation of the antifungal effect of the fungistatic agent fluconazole (FLC) on Candida albicans. These drugs included inhibitors of multidrug efflux transporters, antimicrobial agents, antifungal agents, and membrane-active compounds with no antimicrobial activity, such as antiarrhythmic agents, proton pump inhibitors, and platelet aggregation inhibitors. Among the drugs tested in an agar disk diffusion assay, cyclosporine (Cy), which had no intrinsic antifungal activity, showed a potent antifungal effect in combination with FLC. In a checkerboard microtiter plate format, however, it was observed that the MIC of FLC, as classically defined by the NCCLS recommendations, was unchanged when FLC and Cy were combined. Nevertheless, if a different reading endpoint corresponding to the minimal fungicidal concentration needed to decrease viable counts by at least 3 logs in comparison to the growth control was chosen, the combination was synergistic (fractional inhibitory concentration index of <1). This endpoint fitted to the definition of MIC-0 (optically clear wells) and reflected the absence of the trailing effect, which is the result of a residual growth at FLC concentrations greater than the MIC. The MIC-0 values of FLC and Cy tested alone in C. albicans were >32 and >10 microg/ml, respectively, and decreased to 0.5 and 0.625 microg/ml when the two drugs were combined. The combination of 0.625 microg of Cy per ml with supra-MICs of FLC resulted in a potent antifungal effect in time-kill curve experiments. This effect was fungicidal or fungistatic, depending on the C. albicans strain used. Since the Cy concentration effective in vitro is achievable in vivo, the combination of this agent with FLC represents an attractive perspective for the development of new management strategies for candidiasis.

ELECTROPHYSIOLOGICAL ANALYSIS OF NG108-15 CELLS: AN IN VITRO MODEL FOR EVALUATING ANTAGONISTS OF MEMBRANE-ACTIVE COMPOUNDS

The utility of electrophysiological analysis of NG108-15 cells as an in vitro model system for evaluating antagonists of membrane-active compounds was investigated. This entailed the combined administration of the Ca ++ -selective ionophore A23187, which produces a concentration-dependent hyperpolarization of NG108-15 cells, with several antagonists shown to block the effects of A23187 in other model systems. Two series of experiments were conducted. In the first, the antagonists, that is, the Ca ++ -dependent K + (K Ca) channel blocker quinidine (QND), the calmodulin antagonist trifluoperazine (TFP), and the Ca ++ -channelblockerverapamil(VER),were administered simultaneously with A23187 (1 μM) and shown to block the development of the hyperpolarization in a concentration-dependent manner. In the second series of experiments, TFP (3 μM) or VER (3 μM) was administered only after the development of, and effectively reversed, anestablished A23187-induced hyperpolarization. These data demonstrate that this s...

Application of membrane-active peptides for nonviral gene delivery

A variety of membrane-modifying agents including pH-specific fusogenic or lytic peptides, bacterial proteins, lipids, glycerol, or inactivated virus particles have been evaluated for the enhancement of DNA–polycation complex-based gene transfer. The enhancement depends on the characteristics of both the cationic carrier for DNA and the membrane-modifying agent. Peptides derived from viral sequences such as the N-terminus of influenza virus haemagglutinin HA-2, the N-terminus of rhinovirus HRV2 VP-1 protein, and other synthetic or natural sequences such as the amphipathic peptides GALA, KALA, EGLA, JTS1, or gramicidin S have been tested. Ligand–polylysine-mediated gene transfer can be improved up to more than 1000-fold by membrane-active compounds. Other polycations like dendrimers or polyethylenimines as well as several cationic lipids provide a high transfection efficiency per se. Systems based on these polymers or lipids are only slightly enhanced by endosomolytic peptides or adenoviruses. Electroneutral cationic lipid–DNA complexes however can be strongly improved by the addition of membrane-active peptides.

MEMBRANE POTENTIAL AND RESISTANCE CHANGES IN NG108-15 CELLS: AN IN VITRO MODEL TO STUDY MEMBRANE-ACTIVE COMPOUNDS

The objective of this work was to develop an in vitro model to screen possible antidotal treatments against membrane-active compounds. Toward this end, studies were conducted to examine changes in membrane potential and resistance induced by several test compounds, i.e., gramicidin D, ouabain, and minoxidil, in differentiated NG108-15 hybrid (mouse neuroblastoma X rat glioma) cells using intracellular electrodes and standard current clamp techniques. In general, changes observed were consistent with known actions of these compounds and their effects demonstrated in a number ofother systems.For example, the channel-forming ionophore gramicidin D produced severe membrane depolarization with markedly reduced resistance. Also, the Na+-K+ pump blocker ouabain produced membrane depolarization, and the potassium channel activator minoxidil produced membrane hyperpolarization. Neither ouabain nor minoxidil significantly altered input resistanceof theNG108-15 cells.Thenature of theobserved alterations combined wit...

High throughput assay to detect compounds that enhance the proton permeability of Candida albicans membranes.

We describe a 96-well microtiter plate format assay to detect changes in proton permeability in membranes of the pathogenic yeast, Candida albicans. Candida albicans cells were incubated with the lipophilic ester of 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), a pH-sensitive fluorescein derivative. Inside the cells, BCECF was released and trapped in the vacuole. Compounds that destroyed membrane integrity increased the pH value of the vacuole due to proton leakage into the cytoplasm. This was paralleled by an increase in BCECF fluorescence intensity, which could be quantified. The test assay was validated with amphotericin B, as well as with other membrane-active compounds known to increase membrane permeability. Possible applications and limitations of this assay in the field of antifungal drug discovery are discussed.

35] Use of planar lipid bilayer membranes for rapid screening of membrane active compounds

Planar lipid bilayer membranes (BLMs) have been in use for more than three decades for the study of membrane active compounds) BLMs provide a unique environment that allows the assay of the functional activity of carriers and channels translocating ions across membrane. Although BLMs are in vitro systems that have been used extensively for the measurement of biophysical properties of carriers and channels, their relevance to in vivo phenomena is discussed repeatedly through the more recent technology of patch clamping that has shown that channel properties measured in BLMs correspond qualitatively and often quantitatively to those observed in whole cells. This chapter describes a new BLM perfusion chamber that allows rapid, reversible, and quantitative assay of compounds on the bilayers.

The potential management of resistant infections with non-antibiotics

The antimicrobial activity of synthetic, non-chemotherapeutic compounds, such as the phenothiazine, methylene blue, has been known since the time of Ehrlich (1854-1915). In this context the term 'non-antibiotics' is taken to include a variety of compounds which are employed in the management of pathological conditions of a non-infectious aetiology, but which modify cell permeability and have been shown to exhibit broad-spectrum antimicrobial activity. The antimicrobial properties of compounds such as phenothiazines, as well as those of other neurotropic compounds, have only been investigated sporadically, and their application to management of microbial infections has not been evaluated. A review of the literature, coupled with a number of more recent investigations, suggests that some of these and other membrane-active compounds enhance the activity of conventional antibiotics, eliminate natural resistance to specific antibiotics (reversal of resistance) and exhibit strong activity against multi-drug resistant forms of Mycobacterium tuberculosis. Thus non-antibiotics may have a significant role in the management of certain bacterial infections.

Resistance and vulnerability of crustaceans to cytolytic sea anemone toxins

Crustaceans ( Mithraculus, Neopetrolisthes, Periclimenes, Stenorhynchus sp.) living in association with sea anemones, shore crabs ( Metopograpsus oceanicus) and brine shrimps ( Artemia salina) were found to be resistent to the exposure of cytolytic sea anemone toxins (up to 100 μg/ml) and to other membraneactive compounds such as gramicidin A and saponin. The gill filaments of the crustaceans were not affected, indicating that the chitin layer protects the epithelium from the action of the cytolytic toxins. However, crustaceans are highly susceptible to sea anemone toxins when injected parenterally.

Membrane stiffness and channel function.

Alterations in the stiffness of lipid bilayers are likely to constitute a general mechanism for modulation of membrane protein function. Gramicidin channels can be used as molecular force transducers to measure such changes in bilayer stiffness. As an application, we show that N-type calcium channel inactivation is shifted reversibly toward negative potentials by synthetic detergents that decrease bilayer stiffness. Cholesterol, which increases bilayer stiffness, shifts channel inactivation toward positive potentials. The voltage activation of the calcium channels is unaffected by the changes in stiffness. Changes in bilayer stiffness can be predicted from the molecular shapes of membrane-active compounds, which suggests a basis for the pharmacological effects of such compounds.

Red cell echinocytogenesis is correlated to the recruitment of external band-3 conformations

For human erythrocytes the bilayer-couple model predicts a reversible crenation for increasing ratios of external to internal membrane monolayer area. This can be proven experimentally by changing the ratio of lipids in the two monolayers. Under physiological conditions with no addition of membrane-active compounds, active lipid translocation and passive flip-flop are comparatively slow. Nevertheless, individual erythrocytes underwent dramatic shape changes within seconds when the suspension medium was changed. After cell suspension in low ionic strength solutions a rapid formation of echinocytes was observed. This suspension effect was also found at high ionic concentrations when Cl − was replaced by SO 4 2−, an ion transported very slowly by band 3. The band-3 anion-exchange protein occupies about 10% of the total membrane area. Band-3 conformers are asymmetrically distributed with about 90% of the transport sites facing the inside. Specific band-3 inhibitors can induce echinocytes. These inhibitors are known to recruit the band-3 conformation with the transport site exposed to the external medium, e.g. 4,4′-diisothio-cyanatostilbene-2,2′-disulfonic acid, 4-acet-amido-4′-isothiocyanatostilbene-2,2′-disulfonic acid, flufenamic acid, furosemide and salicylic acid. Other inhibitors, e.g. phenylglyoxal and niflumic acid, which do not recruit a certain conformation, are not echinocytogenic. Physiologically, band 3 ensures the equilibration of internal and external anions like Cl − and HCO 3 − which are distributed according to the transmembrane potential (TMP). In the literature a correlation of the TMP and the band-3 conformation, as well as a correlation of the TMP and the erythrocyte shape, is described. In the proposed model, conformational changes may significantly alter the monolayer area ratio. Low external Cl − concentrations, negative TMPs or inhibitors may recruit the band-3 transport site towards the outside solution. It is proposed that this increases the bulk of the protein in the external monolayer and subsequently induces an echinocytogenic shape transformation according to the bilayer couple model.

Do band 3 protein conformational changes mediate shape changes of human erythrocytes?

The bilayer-couple model predicts a reversible membrane crenation for an increasing ratio of external to internal monolayer area. This was comprehensively proven. However, individual erythrocytes may undergo dramatic shape changes within seconds when the suspension medium is changed. In contrast, under physiological conditions with no addition of membrane active compounds, active phospholipid translocation and passive flip-flops are comparatively slow. We propose that conformational changes of the anion-exchange protein, band 3, may rapidly alter the monolayer area ratio. Band 3 occupies about 10% of the total membrane area of human erythrocytes. Under physiological conditions, its conformers are asymmetrically distributed with about 90% of the transport sites facing the cytoplasm. This distribution is altered when external conformations are recruited by changing the transmembranous Cl- gradient, the external pH, or by the application of inhibitors. In experiments, recruitment by low ionic strength caused a rapid, temporary formation of echinocytes. This suspension effect could also be found at high ionic concentrations, when Cl- was replaced by SO4(2-). Inhibitors known to recruit the external band 3 conformation, like DIDS, SITS and flufenamic acid, are echinocytogenic. For inhibitors not recruiting a certain conformation, e.g. phenylglyoxal and niflumic acid, no shape effect was found. Since band 3 ensures a fast equilibrium of internal and external anions these ions are usually distributed according to the transmembrane potential (TMP). In the literature, a correlation of TMP and band 3 conformation, as well as a correlation of TMP and red cell shape, is described. In the proposed model, low external Cl- concentrations, inhibitors, or a negative TMP may recruit the transport sit outwards.(ABSTRACT TRUNCATED AT 250 WORDS)

The Evaluation of a Multi-endpoint Cytotoxicity Assay System

The aim of this study was to evaluate a multi-endpoint cytotoxicity screening method using V79 cells based on four different endpoints of cytotoxicity: trypan blue exclusion, reduction of XTT, neutral red uptake and total protein content. In addition, cell morphology was routinely observed after each treatment. Seven compounds were studied, which can be divided into five classes: protein synthesis inhibitors (cycloheximide, actinomycin D); inhibitors of cell division (bleomycin, vincristine); membrane-active compounds (Triton X-100); lysosomotropic agents (ammonium chloride); and general toxicants (sodium chloride). We obtained a variety of different toxicity profiles, which may be useful in defining the mechanisms of toxic action of these compounds. The multi-endpoint screening system proved to be readily applicable, robust and rapid, and gave reliable toxicity results over a wide range of chemical concentrations.

In vitro activation of lipophilic tributyltins by superoxide produces tributylstannyl superoxo radicals, proposed initiators of lipid peroxidation: an EPR model study.

Tri-n-butyltin(TBT) compounds having the chemical formula (C4H9)3SnX are broad-spectrum biocidal agents whose toxic effect is primarily at the membrane level. Red blood cells (RBC) exposed to micromolar concentrations of tributyltin compounds (TBTX) undergo morphological changes and hemolysis. Determination of the mechanism of action whereby TBT elicits membrane damage continues to be a challenging endeavor. Because xenobiotic TBT+ and endogenous O2.- have been found to penetrate and alter RBC membrane function, it is hypothesized that they may combine chemically within the RBC hydrophobic lipid bilayer during TBT insult to initiate lipid peroxidative processes. The present study has been designed (1) to determine if TBT+ and O2.- combine chemically in aprotic media and, if so, (2) to characterize any free-radical complex(es) generated. The reactions of the membrane-active TBTX compounds (X = OCH3, Cl, Br, or I) with O2.- have been investigated in the aprotic solvent system cis-dicyclohexano-18-crown-6 ether/DMSO using EPR techniques. When incremental amounts of each TBT halide were added to O2.- solutions at room temperature, the EPR signal characteristic of O2.- diminished in intensity and disappeared when a 1:1 O2.-/TBT+ mole ratio was attained. Although the same phenomenon was observed for all TBT halides used, only the KO2/TBTI reaction produced detectable amounts of a new oxygen-centered free-radical complex. The EPR spectral parameters calculated from the product anisotropic frozen glass spectra were gx = 2.054, a(x) = 31.7 G, gy = 2.021, gz = 2.002, gav = 2.026.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of magainins on ameba and cyst stages of Acanthamoeba polyphaga.

Amebic keratitis produced by Acanthamoeba spp. is an increasingly important ocular infection in extended-use contact lens wearers. Problems associated with the infection are compounded by the lack of effective and well-tolerated chemotherapeutic agents. The magainins, a group of naturally occurring and synthetic membrane-active peptide compounds, have been shown to be active in vitro against a clinical isolate of Acanthamoeba polyphaga. Two magainins tested extensively had minimal inhibitory and minimal amebicidal values of 20 and 25 micrograms/ml for magainin MSI-103 and 25 and 40 micrograms/ml for magainin MSI-94, respectively. Both amebastatic and amebicidal activities are enhanced by combining the magainins with silver nitrate (200 micrograms/ml) and/or other marginally effective antimicrobial agents. These combinations have activity against both trophic and cystic stages in the Acanthamoeba life cycle and have promise as antimicrobial agents in the treatment of amebic keratitis.

Effects of hyperthermia and membrane-active compounds or low pH on the membrane fluidity of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells heated in the presence of the membrane-active agents procaine, ethanol, butylated hydroxytoluene (BHT) and glycerol were analysed for changes in fluorescence polarization of the lipid probe (1-[4(trimethylamino)phenyl]-6-phenyl-1,3,5-hexatriene, an indicator of plasma membrane fluidity). Cells were heated in normal and acid (pH 6.6) medium. Procaine, ethanol, BHT and low pH sensitized cells to heat-killing. Procaine, ethanol and BHT decreased fluorescence polarization (increased plasma membrane fluidity) significantly. Polarization distributions for cells heated with these sensitizers were broadened substantially and were skewed toward lower polarization values. Glycerol, a heat-protector, inhibited changes in fluorescence polarization due to heating at temperatures up to 45.0 degrees C. Heating cells at either 42 or 45 degrees C in pH 6.6 medium had no significant effect on the fluorescence polarization compared with controls, while survival was reduced substantially. Thus, heat-sensitization of low pH cannot be ascribed to changes in membrane fluidity. The changes in membrane fluidity caused by other sensitizers and protector indicate that membrane fluidity changes may be a contributing cause of cell killing by hyperthermia.

Ab Initio Investigation of Interactions between Models of Membrane-Active Compounds and Polar Groups of Membranes: Complexes Involving Amine, Ether, Amide, Phosphate, and Carboxylate

Ab initio (MINI-1) molecular orbital calculations were performed on model systems to investigate the hydrogen bonds and proton transfer between antiarrhythmics and polar groups of the cell membrane, Methylamine cation, dimethyl ether, and N-methylacetamide served as models of associative sites for the antiarrhythmics mexiletine and tocainide. Formate and phosphate anions, the methylamine cation, and formamide were chosen as models for the membrane polar groups. Protonated methylamine forms a very strong complex with the formate and phosphate anions. However, the formate COO- group is a better proton acceptor than the phosphate PO-4 group. The effect of specific hydration on the proton potential functions was investigated in the HCOO-… +HNH2CH3 and H2PO4 … +HNH2CH3 systems. The proton potential functions, calculated at the equilibrium distances RO … N, with a single minimum were found. The ab initio calculations at the longer RO … N = 0.275nm distance indicate double-minimum potentials. The increasing hydration stabilizes a second minimum corresponding to the charged O- … +HN structures. The complexes involving the amide and ether groups of tocainide and mexiletine and the protonated primary amine group of the membrane are considerably weaker. The weakest hydrogen bonds are formed by the amine group of the drug (in its neutral and ionized state) with the peptide group.

A Therapeutic Panorama of the Spongiform Encephalopathies

The spongiform encephalopathies are a group of uniformly fatal, transmissible amyloidoses of humans and animals, for which the causative agents have not yet been precisely defined; in some respects they resemble viruses, but in other respects appear to be replicating host polypeptides. A vast array of anti-infective and other drugs has been studied in animal models, among which a few membrane-active compounds (heteropolyanions and amphotericin B) consistently prolong the course of infection, and occasionally even prevent the illness. However, because no form of therapy has any effect when given after the disease becomes clinically manifest, and because there is no laboratory test to detect preclinical infection, therapeutic efforts in humans have been predictably unsuccessful. If infection and disease turn out to depend upon the pathological accumulation of an amyloidogenic host protein, the prospects for future therapy may include genetic engineering and perhaps even the ‘poisoning’ of protein crystal growth.

Components of Essential Oils as Membrane-Active Compounds

Components of Essential Oils as Membrane-Active Compounds

Scope and limitations of in vivo brain dialysis: A comparison of its application to various neurotransmitter systems

Brain dialysis is rapidly becoming a routine research method with a wide range of applications. Since 1982 this sampling technique is frequently used as a method to study the in vivo release of endogenous neurotransmitters such as dopamine, noradrenaline, serotonin, acetylcholine and certain amino acids. In this review most of the studies that have appeared in this field, are evaluated. Special attention was given to the question whether the neurotransmitter content in the dialysate is related to neurotransmission. Criteria such as the presence of a high tissue/dialysate concentration ratio, the sensitivity of the transmitters to membrane active compounds and the occurence of receptor-mediated effects, are discussed. It is concluded that dopamine, noradrenaline and acetylcholine found in the dialysate are directly derived from neurotransmission, whereas the overflow of excitatory amino acid neurotransmitters is related to neurogenic as well as to metabolic events.

The antibacterial activity of the psychopharmacological agent clopenthixol and its two main metabolites.

The antibacterial effect of the stereo-isomeric compounds cis(Z)- and trans(E)-clopenthixol and the two main metabolites of clopenthixol in man, N-dealkyl-clopenthixol and clopenthixol sulfoxide, was examined in 24 Gram-positive and 37 Gram-negative bacterial strains in vitro. The antibacterial potency of the drugs towards the Gram-positive strains, measured as IC50, was: N-dealkyl-clopenthixol, 6.2 microM (3.7 micrograms/ml), trans(E)-clopenthixol, 16 microM (7.6 micrograms/ml) and cis(Z)-clopenthixol, 37 microM (17.5 micrograms/ml), and clopenthixol sulfoxide was inactive in the investigated area. Against the Gram-negative strains the drugs are less potent. A therapeutic application of these results, especially in the case of trans(E)-clopenthixol, which possesses no neuroleptic activity, requires in vivo testing in an animal model. However, the in vitro model employed might also be useful in the study of such agents and other membrane-active compounds with regard to their interaction with biological membranes.