RESUMEN
Integrating graphene as an inorganic nanostructure within a hydrogel matrix enables the creation of a unique hybrid composite combining the peculiar chemical and physical properties of graphene with the high porosity and stability of hydrogels as for example agarose gel. As a consequence, the resulting material forms a double-network system providing advantages deriving from both the components. In this study, we present the synthesis of novel magnetic porous agarose-based graphene oxide microbeads for the adsorption and separation of positively charged aromatic molecules. The hydrogel-based graphene oxide beads revealed an ultrafast adsorption kinetics for positively charged aromatic dyes. We tested this material for the purification of fluorescent-tagged biomolecules. In addition, reduced graphene oxide microbeads were decorated with palladium nanoparticles, showing a high catalytic activity towards the reduction of dyes by sodium borohydride. Our results show that magnetic agarose based graphene microbeads with enhanced physical-chemical properties can be used for several biochemical applications.
RESUMEN
The biocompatibility of an implant depends upon the material it is composed of, in addition to the prosthetic device's morphology, mechanical and surface properties. Properties as porosity and pore size should allow, when required, cells penetration and proliferation. Stiffness and strength, that depend on the bulk characteristics of the material, should match the mechanical requirements of the prosthetic applications. Surface properties should allow integration in the surrounding tissues by activating proper communication pathways with the surrounding cells. Bulk and surface properties are not interconnected, and for instance a bone prosthesis could possess the necessary stiffness and strength for the application omitting out prerequisite surface properties essential for the osteointegration. In this case, surface treatment is mandatory and can be accomplished using various techniques such as applying coatings to the prosthesis, ion beams, chemical grafting or modification, low temperature plasma, or a combination of the aforementioned. Low temperature plasma-based techniques have gained increasing consensus for the surface modification of biomaterials for being effective and competitive compared to other ways to introduce surface functionalities. In this paper we review plasma processing techniques and describe potentialities and applications of plasma to tailor the interface of biomaterials.
Asunto(s)
Materiales Biocompatibles/química , Gases em Plasma , Materiales Biocompatibles/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Humanos , Microscopía de Fuerza Atómica , Microscopía Confocal , Espectroscopía de Fotoelectrones , Porosidad , Propiedades de Superficie , TemperaturaRESUMEN
Parkinson's disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons and by accumulation of alpha-synuclein (aS) aggregates in the surviving neurons. The dopamine catabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is a highly reactive and toxic molecule that leads to aS oligomerization by covalent modifications to lysine residues. Here we show that DOPAL-induced aS oligomer formation in neurons is associated with damage of synaptic vesicles, and with alterations in the synaptic vesicles pools. To investigate the molecular mechanism that leads to synaptic impairment, we first aimed to characterize the biochemical and biophysical properties of the aS-DOPAL oligomers; heterogeneous ensembles of macromolecules able to permeabilise cholesterol-containing lipid membranes. aS-DOPAL oligomers can induce dopamine leak in an in vitro model of synaptic vesicles and in cellular models. The dopamine released, after conversion to DOPAL in the cytoplasm, could trigger a noxious cycle that further fuels the formation of aS-DOPAL oligomers, inducing neurodegeneration.
Asunto(s)
Ácido 3,4-Dihidroxifenilacético/análogos & derivados , Multimerización de Proteína/efectos de los fármacos , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , alfa-Sinucleína/metabolismo , Ácido 3,4-Dihidroxifenilacético/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Membrana Celular/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/ultraestructura , Permeabilidad , Agregado de Proteínas , Agregación Patológica de Proteínas , Unión Proteica , Espectrometría de Masas en Tándem , alfa-Sinucleína/químicaRESUMEN
In this paper we test the ability of Raman micro-spectroscopy and Raman mapping to investigate the status of cells grown in adhesion on different substrates. The spectra of immortalized SH-SY5Y cells, grown on silicon and on metallic substrates are compared with those obtained for the same type of cells adhering on organic polyaniline (PANI), a memristive substrate chosen to achieve a living bio-hybrid system. Raman spectra give information on the status of the single cell, its local biochemical composition, and on the modifications induced by the substrate interaction. The good agreement between Raman spectra collected from cells adhering on different substrates confirms that the PANI, besides allowing the cell growth, doesn't strongly affect the general biochemical properties of the cell. The investigation of the cellular state in a label free condition is challenging and the obtained results confirm the Raman ability to achieve this information.
Asunto(s)
Compuestos de Anilina/química , Silicio/química , Adhesión Celular , Proliferación Celular , Supervivencia Celular , Humanos , Espectrometría Raman , Células Tumorales CultivadasRESUMEN
Plasma enhanced physical vapor depositions are extensively used to fabricate substrates for cell culture applications. One peculiarity of the plasma processes is the possibility to deposit thin films with reproducible chemical and physical properties. In the present work, a combinatorial plasma polymerization process was used to deposit thin carbon based films to promote cell adhesion, in the interest of testing cell proliferation as a function of the substrate chemical properties. Peculiarity of the combinatorial approach is the possibility to produce in just one deposition experiment, a set of surfaces of varying chemical moieties by changing the precursor composition. A full characterization of the chemical, physical and thermodynamic properties was performed for each set of the synthesized surfaces. X-ray photoelectron spectroscopy was used to measure the concentration of carboxyl, hydroxyl and amine functional groups on the substrate surfaces. A perfect linear trend between polar groups' density and precursors' concentration was found. Further analyses reveled that also contact angles and the correspondent surface energies of all deposited thin films are linearly dependent on the precursor concentration. To test the influence of the surface composition on the cell adhesion and proliferation, two cancer cell lines were utilized. The cell viability was assessed after 24 h and 48 h of cell culture. Experiments show that we are able to control the cell adhesion and proliferation by properly changing the thin film deposition conditions i.e. the concentration and the kind of chemical moiety on the substrate surface. The results also highlight that physical and chemical factors of biomaterial surface, including surface hydrophobicity and free energy, chemical composition, and topography, can altered cell attachment.
Asunto(s)
Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Polimerizacion , Propiedades de Superficie , HumectabilidadRESUMEN
Branched gold nanoparticles were grown on oxidized multiwalled carbon nanotubes by one-step reduction of gold chloride in water. The carbon nanotube/gold hybrids were used for the delivery of the anticancer drug doxorubicin hydrochloride into A549 lung cancer cell line. Doxorubicin (Dox) can be adsorbed in high quantity on both inner and outer surfaces of oxidized carbon nanotubes by π-π stacking interactions between doxorubicin aromatic groups and carbon nanotube (CNT) backbone. Carbon nanotube/gold hybrids display a broad absorption band in the red and near-infrared regions allowing their use for imaging applications. In vitro cellular tests showed that the nanostructures can efficiently transport and deliver doxorubicin inside the cells.
Asunto(s)
Antineoplásicos/farmacocinética , Doxorrubicina/farmacocinética , Sistemas de Liberación de Medicamentos , Oro/química , Neoplasias Pulmonares/tratamiento farmacológico , Nanopartículas del Metal/química , Nanotubos de Carbono/química , Humanos , Neoplasias Pulmonares/patología , Tamaño de la Partícula , Propiedades de Superficie , Células Tumorales CultivadasRESUMEN
The natural target of Staphylococcus aureus bicomponent gamma-hemolysins are leucocyte cell membranes. Because a proteinaceous receptor has not been found yet, we checked for the importance of the different membrane lipid compositions by measuring the activity of the toxin on several pure lipid model membranes. We investigated the effect of membrane thickness, fluidity, and presence of nonbilayer lipids and found that the toxin pore-forming ability increased in the presence of phosphocholines with short saturated acyl chains or with unsaturated chains even though not short. An increase of activity was also evident in the presence of cone-shaped lipids like phosphatidylethanolamine or diphytanoylphosphatidylcholine, whereas cylindrical lipids, like sphingomyelin, did not favor the activity. All these results suggest that gamma-hemolysins could bind to the bilayer only if the phosphatidylcholine (PC) head is freely accessible. This condition is satisfied by the concurrent presence of cholesterol and certain lipids, as highlighted by the so-called umbrella model (J. Huang and G. W. Feigenson, Biophys J 76:2142-2157, 1999). According to this model, cholesterol could help to a better exposition of PC head groups only if acyl chains are short or unsaturated. In fact, phosphatidylcholines with more than 13 carbon atoms acyl chains can cover cholesterol molecules; in this way, PC head groups pack tightly, rendering them inaccessible to the toxin, which thus shows a reduced pore-forming ability.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Permeabilidad de la Membrana Celular , Membrana Celular/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/metabolismo , Células Sanguíneas , Citometría de Flujo , Humanos , Membrana Dobles de Lípidos/metabolismo , Linfocitos/metabolismo , Fluidez de la Membrana , Microdominios de Membrana , Membranas Artificiales , Modelos Biológicos , PorosidadRESUMEN
gamma-Hemolysins are pore-forming toxins which develop from water-soluble monomers by combining two different 'albeit homologous' proteins. They form oligomeric pores in both cell and model membranes by undergoing a still poorly understood conformational rearrangement in the stem region. The stem is formed by three beta-strands, folded onto the core of the soluble protein and completely extended in the pore. We propose a new model to explain such a process. Seven double-cysteine mutants were developed by inserting one cysteine on the stretch that links the beta-hairpin to the core of the protein and another on different positions along the beta-strands. The membrane bound protein was blocked in a non-lytic state by S-S bond formation. Six mutants were oxidized as inactive intermediates, but became active after adding DTT. These results demonstrate that the stem extension can be temporarily frozen and that the beta-barrel formation occurs by beta-strand concerted step-by-step sliding.
Asunto(s)
Eritrocitos/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Modelos Biológicos , Animales , Cisteína/genética , Disulfuros/metabolismo , Electrofisiología , Membrana Eritrocítica/metabolismo , Hemólisis , Humanos , Cinética , Membranas Artificiales , Proteínas Mutantes/metabolismo , Mutación/genética , Oxidación-Reducción , Estructura Secundaria de Proteína , Conejos , Temperatura , Factores de TiempoRESUMEN
FP-A and FP-B are LDPs produced by the plant pathogen Pseudomonas fuscovaginae. As expected from their primary structure, they shared a similar mechanism of action with the better characterized SPs, synthesized by strains of Pseudomonas syringae pv. syringae. Indeed, they displayed hemolytic activity on human erythrocytes and were able to induce calcein release from LUVs: the effect was dependent on the concentration of the FPs and the lipid composition of the liposome and, in particular, it increased with the SM content of the membrane. The permeabilizing activity was further investigated on PLMs. FPs were able to open pores on pure POPC membranes. Pore opening was strongly voltage dependent: by switching the potential from negative to positive values, an increase in the absolute amplitude of transmembrane current was induced with simultaneous closure of pores. In 0.1 M KCl both FPs' pores had a conductance of 4 and 9 pS at - 140 mV and + 140 mV, respectively. Studies on ion selectivity indicated that FPs formed cation-selective channels.
Asunto(s)
Antiinfecciosos/química , Modelos Biológicos , Péptidos Cíclicos/química , Péptidos Cíclicos/fisiología , Pseudomonas/patogenicidad , Secuencia de Aminoácidos , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/química , Canales Iónicos/efectos de los fármacos , Membrana Dobles de Lípidos/química , Liposomas/química , Membranas Artificiales , Datos de Secuencia Molecular , Pseudomonas/química , Pseudomonas/genética , Relación Estructura-ActividadRESUMEN
The activity of the White Line Inducing Principle (WLIP) and tolaasin I, produced by virulent strains of Pseudomonas reactans and Pseudomonas tolaasii, respectively, was comparatively evaluated on lipid membranes. Both lipodepsipeptides were able to induce the release of calcein from large unilamellar vesicles. Their activity was dependent on the toxin concentration and liposome composition and in particular it increased with the sphingomyelin content of the membrane. Studies of dynamic light scattering suggested a detergent-like activity for WLIP at high concentration (> 27 microM). This effect was not detected for tolaasin I at the concentrations tested (< 28 microM). Differences were also observed in lipodepsipeptides secondary structure. In particular, the conformation of the smaller WLIP changed slightly when it passed from the buffer solution to the lipid environment. On the contrary, we observed a valuable increment in the helical content of tolaasin I which was inserted in the membrane core and oriented parallel to the lipid acyl chains.
Asunto(s)
Proteínas Bacterianas/farmacología , Toxinas Bacterianas/farmacología , Membrana Celular/efectos de los fármacos , Depsipéptidos/farmacología , Lipoproteínas/farmacología , Permeabilidad/efectos de los fármacos , Pseudomonas/química , Secuencia de Aminoácidos , Antiinfecciosos/aislamiento & purificación , Antiinfecciosos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Toxinas Bacterianas/aislamiento & purificación , Membrana Celular/química , Membrana Celular/metabolismo , Depsipéptidos/química , Depsipéptidos/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Lipopolisacáridos/química , Lipopolisacáridos/metabolismo , Lipoproteínas/química , Lipoproteínas/aislamiento & purificación , Lípidos de la Membrana/química , Lípidos de la Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Modelos Biológicos , Datos de Secuencia MolecularRESUMEN
Equinatoxin II is a pore forming toxin produced by the sea anemone Actinia equina. It is able to kill very unspecifically most cell types by the membrane-perturbing action of an amphiphilic alpha-helix located at its N-terminal. A normally active N-terminal mutant, containing one single cys in the amphiphilic alpha-helix, becomes totally inactive when it is bound to avidin via a biotinylated linker. By choosing, as a linker, a peptide containing a tumor protease cleavage site, we were able to construct an enzymatically activable conjugate which should be selective for tumor cells. The introduced cleavage site was designed in order to be digested by both cathepsin B and matrix metalloproteases (MMPs). We confirmed that this conjugate could be activated in vitro by cathepsin B and MMPs. After having measured the enzymatic activity of fibrosarcoma and breast carcinoma cells, we analyzed the cytotoxic effect of the conjugate on the same lines and on human red blood cells (HRBC) as controls. We found that the conjugate was activated, at least in part, by the tumor cell lines used, whereas it was inactive on HRBC. That the activation process was dependent on the enzymatic action of cathepsin B and MMPs, was indicated by three lines of evidence: (1) binding occurred normally on all type of cells including HRBC which however were insensitive being devoid of enzymes; (2) the cytotoxic effect correlated with the amount of cathepsin B activity expressed by the cells; (3) conjugate activation was reduced by specific inhibitors of cathepsin B and MMPs. These results demonstrate the possibility of tumor cell killing by a pore-forming toxin conjugate specifically activated by tumor proteases.
Asunto(s)
Antineoplásicos/síntesis química , Venenos de Cnidarios/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Proteínas de Neoplasias/metabolismo , Péptidos/metabolismo , Animales , Antineoplásicos/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Catepsina B/metabolismo , Línea Celular Tumoral , Venenos de Cnidarios/química , Venenos de Cnidarios/genética , Reactivos de Enlaces Cruzados , Citotoxinas/administración & dosificación , Citotoxinas/química , Citotoxinas/genética , Femenino , Fibrosarcoma/tratamiento farmacológico , Fibrosarcoma/enzimología , Fibrosarcoma/patología , Humanos , Masculino , Metaloproteinasas de la Matriz/metabolismo , Mutación , Péptidos/uso terapéutico , Profármacos/síntesis química , Profármacos/metabolismo , Anémonas de Mar/químicaRESUMEN
Staphylococcus aureus strains causing human pathologies produce several toxins, including a pore-forming protein family formed by the single-component alpha-hemolysin and the bicomponent leukocidins and gamma-hemolysins. The last comprise two protein elements, S and F, that co-operatively form the active toxin. alpha-Hemolysin is always expressed by S. aureus strains, whereas bicomponent leukotoxins are more specifically involved in a few diseases. X-ray crystallography of the alpha-hemolysin pore has shown it is a mushroom-shaped, hollow heptamer, almost entirely consisting of beta-structure. Monomeric F subunits have a very similar core structure, except for the transmembrane stem domain which has to refold during pore formation. Large deletions in this domain abolished activity, whereas shorter deletions sometimes improved it, possibly by removing some of the interactions stabilizing the folded structure. Even before stem extension is completed, the formation of an oligomeric pre-pore can trigger Ca(2+)-mediated activation of some white cells, initiating an inflammatory response. Within the bicomponent toxins, gamma-hemolysins define three proteins (HlgA, HlgB, HlgC) that can generate two toxins: HlgA+HlgB and HlgC+HlgB. Like alpha-hemolysin they form pores in planar bilayers with similar conductance, but opposite selectivity (cation instead of anion) for the presence of negative charges in the ion pathway. gamma-Hemolysin pores seem to be organized as alpha-hemolysin, but should contain an even number of each component, alternating in a 1:1 stoichiometry.
Asunto(s)
Toxinas Bacterianas/química , Canales Iónicos , Canales Iónicos/química , Staphylococcus aureus/metabolismo , Animales , Calcio/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Eliminación de Gen , Proteínas Hemolisinas/química , Humanos , Inflamación , Canales Iónicos/metabolismo , Iones , Modelos Moleculares , Mutación , Ósmosis , Porinas/química , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , ConejosRESUMEN
Syringopeptin 25A (SP(25)A) belongs to a family of cyclic lipodepsipeptides (LDPs) produced by the gram-negative bacterium Pseudomonas syringae, a phytopathogenic organism that affects several plants of agronomic interest. LDPs increase the permeability of plasma and, possibly, intracellular membranes in plant cells. Consistently, SP(25)A forms ion channels in planar lipid bilayers and other model membranes. Here we used sugar beet tonoplasts as a new biological model system to study toxin action. When applied to the vacuoles by a fast perfusion procedure, SP(25)A increases membrane permeability by forming discrete ion channels even at low applied potentials. The SP(25)A channel displays anion selectivity (with a Cl-/K+ permeability ratio of 6.7 +/- 1.3) and has intrinsic rectification properties that derive from a different channel conductance at negative and positive voltages, presumably owing to an asymmetric distribution of fixed charges on the pore. Substitution of chloride with different anions reveals the following selectivity sequence NO3- approximately Cl-> F- > gluconate-, suggesting that the permeation pore is filled with water. The properties of the SP(25)A channels in vacuolar membranes are similar to those observed in planar lipid membranes prepared with asolectin. This work provides a direct demonstration of toxin effects on a native plant membrane, extending to a biological system previous results obtained on artificial planar lipid membranes.
Asunto(s)
Beta vulgaris/fisiología , Canales Iónicos/biosíntesis , Péptidos Cíclicos/metabolismo , Pseudomonas/metabolismo , Vacuolas/metabolismo , Toxinas Bacterianas/administración & dosificación , Toxinas Bacterianas/metabolismo , Beta vulgaris/microbiología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/fisiología , Conductividad Eléctrica , Canales Iónicos/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Péptidos Cíclicos/administración & dosificación , Sensibilidad y Especificidad , Vacuolas/efectos de los fármacosRESUMEN
The high larvicidal effect of Bacillus sphaericus (Bs), a mosquito control agent, originates from the presence of a binary toxin (Bs Bin) composed of two proteins (BinA and BinB) that work together to lyse gut cells of susceptible larvae. We demonstrate for the first time that the binary toxin and its individual components permeabilize receptor-free large unilamellar phospholipid vesicles (LUVs) and planar lipid bilayers (PLBs) by a mechanism of pore formation. Calcein-release experiments showed that LUV permeabilization was optimally achieved at alkaline pH and in the presence of acidic lipids. BinA was more efficient than BinB, BinB facilitated the BinA effect, and their stoichiometric mixture was more effective than the full Bin toxin. In PLBs, BinA formed voltage-dependent channels of approximately 100-200 pS with long open times and a high open probability. Larger channels (> or =400 pS) were also observed. BinB, which inserted less easily, formed smaller channels (< or =100 pS) with shorter mean open times. Channels observed after sequential addition of the two components, or formed by their 1:1 mixture (w/w), displayed BinA-like activity. Bs Bin toxin was less efficient at forming channels than the BinA/BinB mixture, with channels displaying the BinA channel behavior. Our data support the concept of BinA being principally responsible for pore formation in lipid membranes with BinB, the binding component of the toxin, playing a role in promoting channel activity.
Asunto(s)
Bacillus/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/farmacología , Canales Iónicos/metabolismo , Proteínas Bacterianas/farmacología , Fluoresceínas/metabolismo , Concentración de Iones de Hidrógeno , Indicadores y Reactivos/metabolismo , Canales Iónicos/efectos de los fármacos , Membrana Dobles de Lípidos/metabolismo , Modelos Biológicos , Permeabilidad/efectos de los fármacosRESUMEN
The radius of the pore formed by sticholysin I and II (StI, StII) in erythrocytes and sticholysin I in lipid vesicles was investigated. The rate of colloid osmotic lysis of human erythrocytes, exposed to one of the toxins in the presence of sugars of different size, was measured. The relative permeability of each sugar was derived and the pore radius estimated with the Renkin equation. The radius was similar for sticholysin I and II and was independent of the reference sugar chosen and of the toxin concentration applied. It was also the same when erythrocytes were pretreated with different toxin doses in the presence of a polyethylene glycol (PEG) large enough to prevent lysis and thereafter transferred to solutions containing oligosaccharides of different size where they did lyse at different rates. The osmometric behavior of large unilamellar vesicles (LUV) was thereafter used to estimate the toxin lesion radius in a model system. LUV transferred to a hyperosmotic solution with a certain sugar immediately shrank and then re-swelled at a rate dependent on the bilayer permeability to water and sugar. When LUV were previously permeabilized with StI, only a fraction of them, namely those not carrying pores, continued to behave as osmometers. By increasing the size of the added sugar and approaching the pore radius, the fraction of osmometric LUV increased. Relative permeabilities were derived and used to estimate a channel radius around 1.2 nm, both for sugars and for PEGs. In conclusion the sticholysin pore has a constant size independent of toxin concentration and similar in natural and artificial membranes, suggesting it has a fixed predominant structure.
Asunto(s)
Venenos de Cnidarios/farmacología , Membrana Eritrocítica/efectos de los fármacos , Proteínas Hemolisinas/farmacología , Canales Iónicos/efectos de los fármacos , Animales , Coloides , Fluorescencia , Membranas Intracelulares/fisiología , Cinética , Liposomas , Matemática , Lípidos de la Membrana/metabolismo , Oligosacáridos/fisiología , Compuestos Orgánicos , Presión Osmótica , Polietilenglicoles/farmacología , Anémonas de Mar , Factores de TiempoRESUMEN
Sticholysin I and II (St I and St II), two basic cytolysins purified from the Caribbean sea anemone Stichodactyla helianthus, efficiently permeabilize lipid vesicles by forming pores in their membranes. A general characteristic of these toxins is their preference for membranes containing sphingomyelin (SM). As a consequence, vesicles formed by equimolar mixtures of SM with phosphatidylcholine (PC) are very good targets for St I and II. To better characterize the lipid dependence of the cytolysin-membrane interaction, we have now evaluated the effect of including different lipids in the composition of the vesicles. We observed that at low doses of either St I or St II vesicles composed of SM and phosphatidic acid (PA) were permeabilized faster and to a higher extent than vesicles of PC and SM. As in the case of PC/SM mixtures, permeabilization was optimal when the molar ratio of PA/SM was ~1. The preference for membranes containing PA was confirmed by inhibition experiments in which the hemolytic activity of St I was diminished by pre-incubation with vesicles of different composition. The inclusion of even small proportions of PA into PC/SM LUVs led to a marked increase in calcein release caused by both St I and St II, reaching maximal effect at ~5 mol % of PA. Inclusion of other negatively charged lipids (phosphatidylserine (PS), phosphatidylglycerol (PG), phosphatidylinositol (PI), or cardiolipin (CL)), all at 5 mol %, also elicited an increase in calcein release, the potency being in the order CL approximately PA >> PG approximately PI approximately PS. However, some boosting effect was also obtained, including the zwitterionic lipid phosphatidylethanolamine (PE) or even, albeit to a lesser extent, the positively charged lipid stearylamine (SA). This indicated that the effect was not mediated by electrostatic interactions between the cytolysin and the negative surface of the vesicles. In fact, increasing the ionic strength of the medium had only a small inhibitory effect on the interaction, but this was actually larger with uncharged vesicles than with negatively charged vesicles. A study of the fluidity of the different vesicles, probed by the environment-sensitive fluorescent dye diphenylhexatriene (DPH), showed that toxin activity was also not correlated to the average membrane fluidity. It is suggested that the insertion of the toxin channel could imply the formation in the bilayer of a nonlamellar structure, a toroidal lipid pore. In this case, the presence of lipids favoring a nonlamellar phase, in particular PA and CL, strong inducers of negative curvature in the bilayer, could help in the formation of the pore. This possibility is confirmed by the fact that the formation of toxin pores strongly promotes the rate of transbilayer movement of lipid molecules, which indicates local disruption of the lamellar structure.
Asunto(s)
Permeabilidad de la Membrana Celular , Venenos de Cnidarios/metabolismo , Proteínas Hemolisinas/metabolismo , Liposomas/química , Liposomas/metabolismo , Lípidos de la Membrana/metabolismo , Anémonas de Mar , Animales , Cardiolipinas/metabolismo , Fluoresceínas/metabolismo , Hemólisis , Modelos Biológicos , Compuestos Orgánicos , Concentración Osmolar , Ácidos Fosfatidicos/metabolismo , Fosfatidilgliceroles/metabolismo , Fosfatidilinositoles/metabolismo , Fosfatidilserinas/metabolismo , Espectrometría de Fluorescencia , Electricidad EstáticaRESUMEN
Through the analysis of a series of 25 peptides composed of various portions of the histatin 5 sequence, we have identified P-113, a 12-amino-acid fragment of histatin 5, as the smallest fragment that retains anticandidal activity comparable to that of the parent compound. Amidation of the P-113 C terminus increased the anticandidal activity of P-113 approximately twofold. The three histidine residues could be exchanged for three hydrophobic residues, with the fragment retaining anticandidal activity. However, the change of two or more of the five basic (lysine and arginine) residues to uncharged residues resulted in a substantial loss of anticandidal activity. A synthetic D-amino-acid analogue, P-113D, was as active against Candida albicans as the L-amino-acid form. In vitro MIC tests in low-ionic-strength medium showed that P-113 has potent activity against Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis. These results identify P-113 as a potential antimicrobial agent in the treatment of oral candidiasis.
Asunto(s)
Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Proteínas y Péptidos Salivales/farmacología , Saralasina/farmacología , Secuencia de Aminoácidos , Farmacorresistencia Microbiana , Histatinas , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fragmentos de Péptidos/farmacología , Saralasina/química , Homología de Secuencia de AminoácidoAsunto(s)
Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Membrana Dobles de Lípidos/química , Proteínas Bacterianas/química , Toxinas Bacterianas/química , Electroquímica/instrumentación , Electroquímica/métodos , Electrofisiología/instrumentación , Electrofisiología/métodos , Canales Iónicos/fisiología , Membranas ArtificialesRESUMEN
In this study, the charge selectivity of staphylococcal alpha-hemolysin (alphaHL), a bacterial pore-forming toxin, is manipulated by using cyclodextrins as noncovalent molecular adapters. Anion-selective versions of alphaHL, including the wild-type pore and various mutants, become more anion selective when beta-cyclodextrin (betaCD) is lodged within the channel lumen. By contrast, the negatively charged adapter, hepta-6-sulfato-beta-cyclodextrin (s(7)betaCD), produces cation selectivity. The cyclodextrin adapters have similar effects when placed in cation-selective mutant alphaHL pores. Most probably, hydrated Cl(-) ions partition into the central cavity of betaCD more readily than K(+) ions, whereas s(7)betaCD introduces a charged ring near the midpoint of the channel lumen and confers cation selectivity through electrostatic interactions. The molecular adapters generate permeability ratios (P(K+)/P(Cl-)) over a 200-fold range and should be useful in the de novo design of membrane channels both for basic studies of ion permeation and for applications in biotechnology.
Asunto(s)
Toxinas Bacterianas/química , Ciclodextrinas/química , Exotoxinas/química , Proteínas Hemolisinas/química , Aniones , Toxinas Bacterianas/genética , Cationes , Exotoxinas/genética , Proteínas Hemolisinas/genética , MutagénesisRESUMEN
Pseudomonas syringae pv. syringae produces two groups of cyclic lipodepsipeptides (LDPs): the nona-peptides syringomycins, syringostatins, and syringotoxin (ST), and the more complex syringopeptins composed of either 22 or 25 amino acid residues (SP22 and SP25). Both classes of peptides significantly contribute to bacterial pathogenesis and their primary target of action seems to be the plasma membrane. We studied and compared the activity of some members of these two classes of LDPs on red blood cells and on model membranes (monolayers and unilamellar vesicles). All peptides induced red blood cell hemolysis. The mechanism was apparently that of a colloid-osmotic shock caused by the formation of pores, as it could be prevented by osmoticants of adequate size. Application of the Renkin equation indicated a radius of approximately 1 nm for the lesions formed by syringopeptins SP22A and SP25A, whereas those formed by syringomycin E (SRE) had a variable, dose-dependent size ranging from 0.7 up to 1.7 nm. All tested LDPs displayed surface activity, forming peptide monolayers with average molecular areas of 1.2 nm2 (SRE), 1.5 nm2 (SP22A), and 1.3 nm2 (SP25A). They also partitioned into preformed lipid monolayers occupying molecular areas that ranged from 0.6 to 1.7 nm2 depending on the peptide and the lipid composition of the film. These LDPs formed channels in lipid vesicles as indicated by the release of an entrapped fluorescent dye (calcein). The extent of permeabilization was dependent on the concentration of the peptide and the composition of the lipid vesicles, with a preference for those containing a sterol. From the dose dependence of the permeabilization it was inferred that LDPs increased membrane permeability by forming oligomeric channels containing from four to seven monomers. On average, syringopeptin oligomers were smaller than SRE and ST oligomers.