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Chagas Disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi, affecting 6-8 million people, mainly in Latin America. The medical treatment is based on two compounds, benznidazole and nifurtimox, with limited effectiveness and that produce severe side effects; consequently, there is an urgent need to develop new, safe, and effective drugs. Amphotericin B is the most potent antimycotic known to date. A21 is a derivative of this compound with the property of binding to ergosterol present in cell membranes of some organisms. In the search for a new therapeutic drug against T. cruzi, the objective of this work was to study the in vitro and in vivo effects of A21 derivative on T. cruzi. Our results show that the A21 increased the reactive oxygen species and reduced the mitochondrial membrane potential, affecting the morphology, metabolism, and cell membrane permeability of T. cruzi in vitro. Even more important was finding that in an in vivo murine model of infection, A21 in combination with benznidazole was able to reduce blood parasitemia, diminish the immune inflammatory infiltrate in skeletal muscle and rescue all the mice from death due to a virulent T. cruzi strain.
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This review examined a collection of studies regarding the molecular properties of some polyene antibiotic molecules as well as their properties in solution and in particular environmental conditions. We also looked into the proposed mechanism of action of polyenes, where membrane properties play a crucial role. Given the interest in polyene antibiotics as therapeutic agents, we looked into alternative ways of reducing their collateral toxicity, including semi-synthesis of derivatives and new formulations. We follow with studies on the role of membrane structure and, finally, recent developments regarding the most important clinical applications of these compounds.
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Safety studies are essential in drug development. This study evaluates the safety of Amphotericin A21 (AmB-A21), a derivative of amphotericin B with antifungal therapeutic potential. We performed a chronic toxicity study, a targeted organ study and a dermal irritation test. To evaluate chronic toxicity, 18 male adult rats were treated orally with AmB-21 (2 mg/kg) for 26 weeks. The effects on body-weight and animal health were measured, and haematological, clinical chemistry and histopathological tests were conducted on various organs. In the target organ toxicity study, male adult rats received a daily oral dose of AmB-21 (2 mg/kg) for 6 and 17 weeks; testicle histology and testosterone levels were then evaluated. For the dermal irritation study, AmB-21 (200 and 1000 mg/kg) was placed on the skin of adult male rabbits; macroscopic and microscopic studies, as well as haematological and clinical chemistry tests were then conducted. The chronic toxicity study revealed that AmB-21 caused testicle damage, and the testicle-targeted study showed structural alterations and changes in testosterone levels at 17 weeks. However, these alterations were no longer observed 8 weeks after discontinuation of treatment, and the testes showed very similar characteristics to those in the control group. The dermal irritation study showed skin thickening and reddening in rabbits treated with 2000 mg of AmB-A21 after 14 days of exposure. This same group also showed changes in liver enzymes, renal parameters and platelet levels. Based on our results, we consider AmB-21 to be a potential candidate for safe, long-term antifungal treatment given its reduced side effects.
Asunto(s)
Anfotericina B/toxicidad , Antifúngicos/toxicidad , Administración Oral , Anfotericina B/administración & dosificación , Anfotericina B/análogos & derivados , Animales , Antifúngicos/administración & dosificación , Masculino , Ratas , Pruebas de Toxicidad CrónicaRESUMEN
B lymphocytes are a leukocyte subset capable of developing several functions apart from differentiating into antibody-secreting cells. These processes are triggered by external activation signals that induce changes in the plasma membrane properties, regulated by the formation of different lipid-bilayer subdomains that are associated with the underlying cytoskeleton through different linker molecules, thus allowing the functional specialization of regions within the membrane. Among these, there are tetraspanin-enriched domains. Tetraspanins constitute a superfamily of transmembrane proteins that establish lateral associations with other molecules, determining its activity and localization. In this study, we identified TSPAN33 as an active player during B-lymphocyte cytoskeleton and plasma membrane-related phenomena, including protrusion formation, adhesion, phagocytosis, and cell motility. By using an overexpression model of TSPAN33 in human Raji cells, we detected a specific distribution of this protein that includes membrane microvilli, the Golgi apparatus, and extracellular vesicles. Additionally, we identified diminished phagocytic ability and altered cell adhesion properties due to the aberrant expression of integrins. Accordingly, these cells presented an enhanced migratory phenotype, as shown by its augmented chemotaxis and invasion rates. When we evaluated the mechanic response of cells during fibronectin-induced spreading, we found that TSPAN33 expression inhibited changes in roughness and membrane tension. Contrariwise, TSPAN33 knockdown cells displayed opposite phenotypes to those observed in the overexpression model. Altogether, our data indicate that TSPAN33 represents a regulatory element of the adhesion and migration of B lymphocytes, suggesting a novel implication of this tetraspanin in the control of the mechanical properties of their plasma membrane.
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Linfocitos B/metabolismo , Membrana Celular/metabolismo , Movimiento Celular/genética , Endocitosis/genética , Tetraspaninas/genética , Linfocitos B/ultraestructura , Sistemas CRISPR-Cas , Adhesión Celular/genética , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal , Microscopía Electrónica , Fagocitosis/genética , Estrés Mecánico , Tetraspaninas/metabolismoRESUMEN
We performed molecular dynamics simulations of a lipid bilayer consisting of POPC and cholesterol at temperatures from 283 to 308K and cholesterol concentrations from 0 to 50% mol/mol. The purpose of this study was to look for the existence of structural differences in the region delimited by these parameters and, in particular, in a region where coexistence of liquid disordered and liquid ordered phases has been proposed. Our interest in this range of concentration and temperature responds to the fact that polyene ionophore activity varies considerably along it. Two force fields, CHARMM36 and Slipids, were compared in order to determine the most suitable. Both force fields predict non-monotonic behaviors consistent with the existence of phase transitions. We found the presence of lateral structural heterogeneity, statistical in nature, in some of the bilayers occurring in this range of temperatures and sterol concentrations. This heterogeneity was produced by correlated ordering of the POPC tails and not due to cholesterol enrichment, and lasts for tens of nanoseconds. We relate these observations to the action of polyenes in these membranes.
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Colesterol/química , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , Microscopía de Fuerza Atómica , Transición de Fase , TemperaturaRESUMEN
The aim of this study was to investigate the factors that govern the activity and selectivity of two potent antimicrobial peptides (AMPs) using lipid membrane models of bacterial, erythrocyte and fungal cells. These models were used in calcein liposome leakage experiments to explore peptide efficiency. The AMPs (Pin2 and its variant Pin2[GVG]) showed highest affinity towards the bacterial models in the nanomolar range, followed by the erythrocyte and fungal systems. The presence of sterols modulated the variant's selectivity, while the wild type was unaffected. Liposome leakage experiments with Fluorescein Isothiocyanate-dextran (FITC)-dextran conjugates indicated that pore size depended on peptide concentration. Dynamic Light Scattering revealed peptide aggregation in aqueous solution, and that aggregate size was related to activity. The interacting peptides did not alter liposome size, suggesting pore forming activity rather than detergent activity. Atomic Force Microscopy showed differential membrane absorption, being greater in the bacterial model compared to the mammalian model, and pore-like defects were observed. Electrophysiological assays with the Tip-Dip Patch Clamp method provided evidence of changes in the electrical resistance of the membrane. Membrane potential experiments showed that liposomes were also depolarized in the presence of the peptides. Both peptides increased the Laurdan Generalized Polarization of the bacterial model indicating increased viscosity, on the contrary, no effect was observed with the erythrocyte and the fungal models. Peptide membrane insertion and pore formation was corroborated with Langmuir Pressure-Area isotherms and Brewster Angle Microscopy. Finally, molecular dynamics simulations were used to get an insight into the molecular mechanism of action.
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Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Liposomas Unilamelares/química , Animales , Péptidos Catiónicos Antimicrobianos/química , Bacterias , Membrana Celular/química , Membrana Eritrocítica/efectos de los fármacos , Hongos , Fluidez de la Membrana , Potenciales de la Membrana , Esteroles/química , ViscosidadRESUMEN
The effect of cholesterol and ergosterol on supported lipid bilayers composed of 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and egg sphingomyelin (eSM) in a 1/1â¯M ratio was studied using atomic force microscopy. The addition of ergosterol or cholesterol to these membranes considerably modifies both the structure and the dynamics of the domains present in them. The height of the eSM enriched domains increases with concentration of both sterols, but more markedly with ergosterol. The height of the POPC enriched domains increases with concentration in a similar manner for both sterols. This effect is larger for eSM than for POPC when ergosterol, not cholesterol, is present. Domain coverage increases with both sterols at 5â¯mol% but decreases at 20â¯mol% and almost disappears at 40â¯mol%. The size of the eSM enriched domains decreases with sterol concentration, more markedly with cholesterol. Bilayer rupture forces show that overall stiffness increases with the addition of 5â¯mol% cholesterol, but only for the eSM enriched domains with ergosterol at the same concentration. At larger sterol concentrations the stiffness of both regions becomes reduced. At 40â¯mol% sterol concentration, both membranes present the same rupture force value. To gain mechanistic insight into these observations we performed Quantum Mechanical calculations and Molecular Dynamics simulations of the sterol molecules. We found that conformational freedom for the sterol molecules is quite different. This difference might be behind the observed phenomena. Finally, the different action of sterols on membrane properties is related to the sterol-dependent ionophoretic activity of polyene antibiotics.