RESUMEN
The essential oil (EO) of Schinus areira L. (Anacardiaceae) leaves has shown antibacterial activity against Staphylococcus aureus. In this study, we aimed to unravel the mechanisms of its antibacterial action by using bacterial cells and model membranes. First, the integrity of the S. aureus membrane was evaluated by fluorescence microscopy. It was observed that there was an increase in the permeability of cells that was dependent on the EO concentration as well as the incubation time. For a deep comprension of the action of the EO on the lipids, its effect on the membrane fluidity was evaluated on DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine): DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-1'-rac-glycerol) (5:1) liposomes by dynamic light scattering and by using Laurdan doped liposomes. The results indicate that EO produces changes in lipid membrane packing, increasing the fluidity, reducing the cooperative cohesive interaction between phospholipids and increasing access of water or the insertion of some components of the EO to the interior of the membrane. In addition, the potential effect of EO on intracellular targets, such as the increase of cytosolic reactive oxygen species (ROS) and DNA damage, were analyzed. The EO was capable of increasing the production of ROS as well as inducing a partial DNA degradation. Finally, the effect of EO on S. aureus biofilm was tested. These assays showed that EO was able to inhibit the biofilm formation, and also eradicate preformed biofilms. The results show, that the EO seems to have several bacterial targets involved in its antibacterial activity, from the bacterial membrane to DNA. Furthermore, the antibacterial action affects not only planktonic cells but also biofilms; reinforcing the potential application of this EO.
Asunto(s)
Antiinfecciosos , Staphylococcus aureus Resistente a Meticilina , Aceites Volátiles , Staphylococcus aureus , Aceites Volátiles/farmacología , Schinus , Liposomas , Plancton , Especies Reactivas de Oxígeno , Antiinfecciosos/farmacología , Antibacterianos/farmacología , Biopelículas , Pruebas de Sensibilidad MicrobianaRESUMEN
During the storage of Prosopis alba pods, substantial quantitative and qualitative losses were observed. One of the main factors is the seed beetle Rhipibruchus picturatus. A key strategy to develop new pest control management is the use of essential oils (EOs) due they are efficient, less toxic, and less persistent in the environment compared to synthetic pesticides. In this context, seeds and leaves of Schinus areira L. (Anacardiaceae) EOs and Citrus spp. EO were studied in the present work. In the leaves of S. areira EO, 1-epi-cadinol, sesquiterpenoid alcohol, was the major compound. On the other hand, the main compounds of the EO extracted from S. areira seeds are the monoterpenes sabinene, and α-pinene. Finally, in the Citrus EO, limonene is the principal component. The three EOs obtained exhibited insecticidal activity against R. picturatus, being the first report of the use of EOs against this insect pest. The best insecticidal results were obtained with the leaves of S. areira EO. Moreover, this EO inhibits the acetylcholinesterase enzyme in vitro assays. Molecular docking studies on acetylcholinesterase (AChE) suggest that the main components of the leaves of S, areira EOs, bind to the active site of the enzyme, in good agreement with in vitro competitive inhibition against AChE observed for this EO. The data obtained demonstrate the potential use of Schinus areira EOs in the development of new storage pest control strategies.
Asunto(s)
Anacardiaceae , Escarabajos , Insecticidas , Aceites Volátiles , Acetilcolinesterasa , Anacardiaceae/química , Animales , Insecticidas/química , Insecticidas/farmacología , Simulación del Acoplamiento Molecular , Aceites Volátiles/química , Aceites Volátiles/farmacologíaRESUMEN
Cholesterol is responsible for the plasticity of plasma membranes and is involved in physiological and pathophysiological responses. Cholesterol homeostasis is regulated by oxysterols, such as 25-hydroxycholesterol. The presence of 25-hydroxycholesterol at the membrane level has been shown to interfere with several viruses' entry into their target cells. We used atomic force microscopy to assess the effect of 25-hydroxycholesterol on different properties of supported lipid bilayers with controlled lipid compositions. In particular, we showed that 25-hydroxycholesterol inhibits the lipid-condensing effects of cholesterol, rendering the bilayers less rigid. This study indicates that the inclusion of 25-hydroxycholesterol in plasma membranes or the conversion of part of their cholesterol content into 25-hydroxycholesterol leads to morphological alterations of the sphingomyelin (SM)-enriched domains and promotes lipid packing inhomogeneities. These changes culminate in membrane stiffness variations.
Asunto(s)
Membrana Celular/química , Hidroxicolesteroles/química , Colesterol/química , Membrana Dobles de Lípidos/química , Lípidos/química , Microscopía de Fuerza Atómica/métodos , Esfingomielinas/químicaRESUMEN
In the last years, the decreasing effectiveness of conventional antimicrobial-drugs has caused serious problems due to the rapid emergence of multidrug-resistant pathogens. This situation has brought attention to other antimicrobial agents like antimicrobial peptides (AMPs), for being considered an alternative to conventional drugs. These compounds target bacterial membranes for their activity, which gives them a broad spectrum of action and less probable resistance development. That is why the peptide-membrane interaction is a crucial aspect to consider in the study of AMPs. The aim of this work was the characterization of the "de novo" designed peptide P1, studying its interactions with model membranes (i.e. liposomes of DMPC:DMPG 5:1) in order to evaluate the final position of the peptide upon interacting with the membrane. Also, we tested the effects of the peptide in gram-positive and gram-negative bacteria. Later, by spectroscopic methods, the ability of the peptide to permeabilize the inner and outer membrane of E. coli and plasmatic membrane of S. aureus was assessed. The results obtained confirmed that P1 can disrupt both membranes, showing some difference in its activity as a function of the nature of each bacterial cell wall, confirming higher effects on gram-positive S. aureus. Finally, we also showed the ability of P1 to inhibit biofilms of that gram-positive bacterium. All data obtained in this work allowed us to propose a model, where the first interactions of the peptide with the bacterial envelope, seem to depend on the gram-negative and gram-positive cell wall structure. After that first interaction, the peptide is stabilized by Trp residues depth inserted into the hydrocarbon region, promoting several changes in the organization of the lipid bilayer, following a carpet-like mechanism, which results in permeabilization of the membrane, triggering the antimicrobial activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/metabolismo , Membranas Artificiales , Antibacterianos/farmacología , Biopelículas , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Cinética , Pruebas de Sensibilidad Microbiana , PermeabilidadRESUMEN
Colistin is a polymyxin antibiotic (polymyxin E) that has in recent years re-emerged as an option for treatment of multidrug-resistant bacteria. Recently, the re-introduction of colistin resulted in the appearance of colistin-resistant bacteria, which is usually caused by LPS modifications. The fact that this modification is mediated by a plasmid carrying the mcr-1 gene, implies a horizontal transfer of colistin resistance. In Argentina, the National Reference Laboratory in Antimicrobial Resistance (NRLAR), has recently screened several bacteria for the MCR-1 plasmid, detecting nine Escherichia coli isolates carrying the plasmid with the mcr-1 gene, among others. In this context, we proposed to assess the effect of surface charge modifications induced by the plasmid MCR-1 and its impact on the resulting colistin resistance in two clinical isolates of colistin-resistant E. coli. Using zeta potential assays, we confirmed the reduction of negative charge exposure on clinical isolates compared to the reference strain of E. coli. In addition, through permeabilization assays, we were able to correlate this reduction in charge exposure with the extent of damage to the bacterial membrane. The fact that this surface charge modification through substitution of lipid A is plasmid encoded, represents an important concern for future antimicrobial peptide drug development.
Asunto(s)
Colistina/farmacología , Farmacorresistencia Bacteriana , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Argentina , Permeabilidad de la Membrana Celular , Escherichia coli/citología , HumanosRESUMEN
Recently, it was demonstrated that 25-hydroxycholesterol (25HC), an oxidized cholesterol derivative, inhibits human immunodeficiency virus type 1 (HIV) entry into its target cells. However, the mechanisms involved in this action have not yet been established. The aim of this work was to study the effects of 25HC in biomembrane model systems and at the level of HIV fusion peptide (HIV-FP). Integration of different biophysical approaches was made in the context of HIV fusion process, to clarify the changes at membrane level due to the presence of 25HC that result in the suppressing of viral infection. Lipid vesicles mimicking mammalian and HIV membranes were used on spectroscopy assays and lipid monolayers in surface pressure studies. Peptide-induced lipid mixing assays were performed by Förster resonance energy transfer to calculate fusion efficiency. Liposome fusion is reduced by 50% in the presence of 25HC, comparatively to cholesterol. HIV-FP conformation was assessed by infrared assays and it relies on sterol nature. Anisotropy, surface pressure and dipole potential assays indicate that the conversion of cholesterol in 25HC leads to a loss of the cholesterol modulating effect on the membrane. With different biophysical techniques, we show that 25HC affects the membrane fusion process through the modification of lipid membrane properties, and by direct alterations on HIV-FP structure. The present data support a broad antiviral activity for 25HC.
Asunto(s)
Fármacos Anti-VIH/farmacología , Proteína gp41 de Envoltorio del VIH/química , Hidroxicolesteroles/farmacología , Fusión de Membrana/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Antivirales/farmacología , Colesterol/química , Colesterol/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Humanos , Hidroxicolesteroles/química , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Fluidez de la Membrana/efectos de los fármacos , Fluidez de la Membrana/fisiología , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
The aim of the present study was to evaluate the effects of freeze-drying in the presence of trehalose as a cryoprotectant, followed by incubation in synthetic wine, on surface damage, viability and l-malic acid consumption of the oenological strain Oenococcus oeni UNQOe 73.2. After freeze-drying, no significant differences were observed in the number of viable cells (for both acclimated and non-acclimated cultures) respect to the fresh culture. In contrast, loss of viability was observed after wine incubation for 24â¯h, being acclimated freeze-dried cells the best conditions for this. After the preservation process, small changes in cell morphology were observed by Atomic Force Microscopy (AFM). The Zeta potential and AFM showed that 24â¯h of wine incubation was enough to induce several cell surface modifications. Plate count data allowed us to establish that surface damage is an important factor for loss of viability, regardless of the acclimation treatment. Although the number of surviving O. oeni cells decreased dramatically after incubation in synthetic wine for 15 days, the consumption of l-malic acid was higher than 70%, with freeze-dried cells showing a better performance than fresh cultures. These results demonstrate that O. oeni freeze-dried cultures could be applied to direct wine inoculation, to conduct malolactic fermentation, maintaining its technological properties and reducing the time and costs of the winemaking process.
Asunto(s)
Membrana Celular/patología , Crioprotectores/farmacología , Liofilización/métodos , Malatos/metabolismo , Oenococcus/citología , Trehalosa/farmacología , Vino/microbiología , Aclimatación , Fermentación , Microscopía de Fuerza AtómicaRESUMEN
In previous works, it was shown that S-layer proteins from Lactobacillus kefir were able to recrystallize and stabilize liposomes, this feature reveling a great potential for developing liposomal-based carriers. Despite previous studies on this subject are important milestones, a number of questions remain unanswered. In this context, the feasibility of S-layer proteins as a biomaterial for drug delivery was evaluated in this work. First, S-layer proteins were fully characterized by electron microscopy, 2D-electrophoresis, and anionic exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). Afterward, interactions of S-layer proteins with model lipid membranes were evaluated, showing that proteins adsorb to the lipid surface following a non-fickean or anomalous diffusion, when positively charged lipid were employed, suggesting that electrostatic interaction is a key factor in the recrystallization process on these proteins. Finally, the interaction of S-layer coated liposomes with Caco-2 cell line was assessed: First, cytotoxicity of formulations was tested showing no cytotoxic effects in S-layer coated vesicles. Second, by flow cytometry, it was observed an increased ability to transfer cargo molecules into Caco-2 cells from S-layer coated liposomes in comparison to control ones. All data put together, supports the idea that a combination of adhesive properties of S-layer proteins concomitant with higher stability of S-layer coated liposomes represents an exciting starting point in the development of new drug carriers.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Lactobacillus/química , Liposomas/química , Glicoproteínas de Membrana/metabolismo , Proteínas Bacterianas/química , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Difusión , Liberación de Fármacos , Humanos , Punto Isoeléctrico , Kéfir , Liposomas/farmacología , Glicoproteínas de Membrana/química , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
A set of lipopeptides was recently reported for their broad-spectrum antiviral activity against viruses belonging to the Paramyxoviridae family, including human parainfluenza virus type 3 and Nipah virus. Among them, the peptide with a 24-unit PEG linker connecting it to a cholesterol moiety (VG-PEG24-Chol) was found to be the best membrane fusion inhibitory peptide. Here, we evaluated the interaction of the same set of peptides with biomembrane model systems and isolated human peripheral blood mononuclear cells (PBMC). VG-PEG24-Chol showed the highest insertion rate and it was among the peptides that induced a larger change on the surface pressure of cholesterol rich membranes. This peptide also displayed a high affinity towards PBMC membranes. These data provide new information about the dynamics of peptide-membrane interactions of a specific group of antiviral peptides, known for their potential as multipotent paramyxovirus antivirals.
Asunto(s)
Antivirales/química , Membrana Celular/química , Lipopéptidos/química , Polietilenglicoles/química , Antivirales/metabolismo , Antivirales/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colesterol/química , Humanos , Leucocitos Mononucleares/química , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Lipopéptidos/metabolismo , Lipopéptidos/farmacología , Liposomas/química , Paramyxovirinae/química , Relación Estructura-ActividadRESUMEN
In order to give a physical meaning to each region of the membrane we define the interphase as the region in a lipid membrane corresponding to the polar head groups imbibed in water with net different properties than the hydrocarbon region and the water phase. The interphase region is analyzed under the scope of thermodynamics of surface and solutions based on the definition of Defay-Prigogine of an interphase and the derivation that it has in the understanding of membrane processeses in the context of biological response. In the view of this approach, the complete monolayer is considered as the lipid layer one molecule thick plus the bidimensional solution of the polar head groups inherent to it (the interphase region). Surface water activity appears as a common factor for the interaction of several aqueous soluble and surface active proteins with lipid membranes of different composition. Protein perturbation can be measured by changes in the surface pressure of lipid monolayers at different initial water surface activities. As predicted by solution chemistry, the increase of surface pressure is independent of the particle nature that dissolves. Therefore, membranes give a similar response in terms of the determined surface states given by water activity independent of the protein or peptide.
Asunto(s)
Lípidos/química , Agua/química , Modelos Teóricos , Presión , Propiedades de SuperficieRESUMEN
Rigid amphipathic fusion inhibitors (RAFIs) are lipophilic inverted-cone-shaped molecules thought to antagonize the membrane curvature transitions that occur during virus-cell fusion and are broad-spectrum antivirals against enveloped viruses (Broad-SAVE). Here, we show that RAFIs act like membrane-binding photosensitizers: their antiviral effect is dependent on light and the generation of singlet oxygen ((1)O(2)), similar to the mechanistic paradigm established for LJ001, a chemically unrelated class of Broad-SAVE. Photosensitization of viral membranes is a common mechanism that underlies these Broad-SAVE.
Asunto(s)
Antivirales/farmacología , Membrana Celular/efectos de los fármacos , Herpes Simple/virología , Herpesvirus Humano 1/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Antivirales/química , Membrana Celular/efectos de la radiación , Membrana Celular/virología , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Herpesvirus Humano 1/efectos de la radiación , Humanos , LuzRESUMEN
LJ001 is a lipophilic thiazolidine derivative that inhibits the entry of numerous enveloped viruses at non-cytotoxic concentrations (IC50 ≤ 0.5 µM), and was posited to exploit the physiological difference between static viral membranes and biogenic cellular membranes. We now report on the molecular mechanism that results in LJ001's specific inhibition of virus-cell fusion. The antiviral activity of LJ001 was light-dependent, required the presence of molecular oxygen, and was reversed by singlet oxygen ((1)O2) quenchers, qualifying LJ001 as a type II photosensitizer. Unsaturated phospholipids were the main target modified by LJ001-generated (1)O2. Hydroxylated fatty acid species were detected in model and viral membranes treated with LJ001, but not its inactive molecular analog, LJ025. (1)O2-mediated allylic hydroxylation of unsaturated phospholipids leads to a trans-isomerization of the double bond and concurrent formation of a hydroxyl group in the middle of the hydrophobic lipid bilayer. LJ001-induced (1)O2-mediated lipid oxidation negatively impacts on the biophysical properties of viral membranes (membrane curvature and fluidity) critical for productive virus-cell membrane fusion. LJ001 did not mediate any apparent damage on biogenic cellular membranes, likely due to multiple endogenous cytoprotection mechanisms against phospholipid hydroperoxides. Based on our understanding of LJ001's mechanism of action, we designed a new class of membrane-intercalating photosensitizers to overcome LJ001's limitations for use as an in vivo antiviral agent. Structure activity relationship (SAR) studies led to a novel class of compounds (oxazolidine-2,4-dithiones) with (1) 100-fold improved in vitro potency (IC50<10 nM), (2) red-shifted absorption spectra (for better tissue penetration), (3) increased quantum yield (efficiency of (1)O2 generation), and (4) 10-100-fold improved bioavailability. Candidate compounds in our new series moderately but significantly (p≤0.01) delayed the time to death in a murine lethal challenge model of Rift Valley Fever Virus (RVFV). The viral membrane may be a viable target for broad-spectrum antivirals that target virus-cell fusion.
Asunto(s)
Antivirales/farmacología , Oxazoles/farmacología , Rodanina/análogos & derivados , Virus de la Fiebre del Valle del Rift/efectos de los fármacos , Tionas/farmacología , Acoplamiento Viral/efectos de los fármacos , Internalización del Virus/efectos de los fármacos , Animales , Antivirales/química , Disponibilidad Biológica , Membrana Celular/efectos de los fármacos , Membrana Celular/virología , Ratones , Ratones Endogámicos BALB C , Fosfolípidos/química , Fosfolípidos/metabolismo , Fármacos Fotosensibilizantes/farmacología , Rodanina/farmacología , Fiebre del Valle del Rift/tratamiento farmacológico , Oxígeno Singlete/química , Relación Estructura-ActividadRESUMEN
It was reported recently that a new aryl methyldiene rhodanine derivative, LJ001, and oxazolidine-2,4-dithione, JL103, act on the viral membrane, inhibiting its fusion with a target cell membrane. The aim of the present study was to investigate the interactions of these two active compounds and an inactive analogue used as a negative control, LJ025, with biological membrane models, in order to clarify the mechanism of action at the molecular level of these new broad-spectrum enveloped virus entry inhibitors. Fluorescence spectroscopy was used to quantify the partition and determine the location of the molecules on membranes. The ability of the compounds to produce reactive oxygen molecules in the membrane was tested using 9,10-dimethylanthracene, which reacts selectively with singlet oxygen (1O2). Changes in the lipid packing and fluidity of membranes were assessed by fluorescence anisotropy and generalized polarization measurements. Finally, the ability to inhibit membrane fusion was evaluated using FRET. Our results indicate that 1O2 production by LJ001 and JL103 is able to induce several changes on membrane properties, specially related to a decrease in its fluidity, concomitant with an increase in the order of the polar headgroup region, resulting in an inhibition of the membrane fusion necessary for cell infection.
Asunto(s)
Antivirales/farmacología , Proteína gp41 de Envoltorio del VIH/antagonistas & inhibidores , Lípidos de la Membrana/metabolismo , Oxígeno Singlete/fisiología , Proteínas Virales de Fusión/antagonistas & inhibidores , Secuencia de Aminoácidos , Antivirales/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Proteína gp41 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/metabolismo , Datos de Secuencia Molecular , Rodanina/análogos & derivados , Rodanina/química , Rodanina/farmacología , Proteínas Virales de Fusión/genética , Proteínas Virales de Fusión/metabolismoRESUMEN
Targeting membranes of enveloped viruses represents an exciting new paradigm to explore on the development of broad-spectrum antivirals. Recently, broad-spectrum small-molecule antiviral drugs were described, preventing enveloped virus entry at an intermediate step, after virus binding but before virus-cell fusion. Those compounds, including an oxazolidine-2,4-dithione named JL103 that presented the most promissing results, act deleteriously on the virus envelope but not at the cell membrane level. In this work, by using atomic force microscopy (AFM), we aimed at unraveling the effects that JL103 is able to induce in the lipid membrane architecture at the nanoscale. Our results indicate that singlet oxygen produced by JL103 decreases membrane thickness, with an expansion of the area per phospholipid, by attacking the double bonds of unsaturated phospholipids. This membrane reorganization prevents the fusion between enveloped virus and target cell membranes, resulting in viral entry inhibition. FROM THE CLINICAL EDITOR: The recent development of a family of innovative broad-spectrum small-molecule antiviral drugs that block virus cell entry has provided exciting armors against viruses. In this research paper, the authors utilize atomic force microscopy to investigate the mechanism of action of viral blockade. The findings have resulted in new understanding of cell membrane behavior, which may help in further drug design.
Asunto(s)
Antivirales/farmacología , Membrana Celular/efectos de los fármacos , Lípidos de la Membrana/metabolismo , Oxazoles/farmacología , Oxígeno Singlete/metabolismo , Internalización del Virus/efectos de los fármacos , Antivirales/química , Membrana Celular/química , Membrana Celular/metabolismo , Lípidos de la Membrana/química , Microscopía de Fuerza Atómica , Modelos Moleculares , Oxazoles/química , Fosfolípidos/química , Fosfolípidos/metabolismo , Tionas/química , Tionas/farmacologíaRESUMEN
Surface water activity appears as a common factor when the interaction of several aqueous soluble and surface active proteins with lipid membranes of different compositions is measured by the changes in surface pressure of a lipid monolayer. The perturbation of the lipid surface caused by aqueous soluble proteins depends on the composition of the hydrocarbon phases, either modified by unsaturated bonds in the acyl chains or by inclusion of cholesterol. The cut-off (critical) surface pressure in monolayers, at which no effect of the proteins is found, is related to the composition of the head group region. The perturbation of surface pressure is produced by proteins when the area per lipid is above just 4% larger than that corresponding to the hydration shell of the phospholipid head groups found in the cut-off. This area excess gives place to regions in which the chemical potential of water changes with respect to bulk water. According to the Defay-Prigogine relation this interfacial water activity is the reason of the surface pressure increase induced by aqueous soluble proteins injected in the subphase. As predicted by solution chemistry, the increase of surface pressure is independent of the protein nature but depends on the water surface state determined by the lipid composition.
Asunto(s)
Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Membranas/química , Modelos Teóricos , Proteínas/química , Agua/química , Lípidos de la Membrana/metabolismo , Membranas/metabolismo , Proteínas/metabolismo , Propiedades de Superficie , Tensión Superficial , Termodinámica , Agua/metabolismoRESUMEN
OBJECTIVES: The aim of the present work was to evaluate the interaction of two new HIV fusion inhibitors {HIVP3 [C34-polyethylene glycol (PEG)4-cholesterol] and HIVP4 [(C34-PEG4)2-cholesterol]} with membrane model systems and human blood cells in order to clarify where and how the fusion inhibitors locate, allowing us to understand their mechanism of action at the molecular level, and which strategies may be followed to increase efficacy. METHODS: Lipid vesicles with defined compositions were used for peptide partition and localization studies, based on the intrinsic fluorescence of HIVP3 and HIVP4. Lipid monolayers were employed in surface pressure studies. Finally, human erythrocytes and peripheral blood mononuclear cells (PBMCs) isolated from blood samples were used in dipole potential assays. RESULTS: Membrane partition, dipole potential and surface pressure assays indicate that the new fusion inhibitors interact preferentially with cholesterol-rich liquid-ordered membranes, mimicking biological membrane microdomains known as lipid rafts. HIVP3 and HIVP4 are able to interact with human erythrocytes and PBMCs to a similar degree as a previously described simpler drug with monomeric C34 and lacking the PEG spacer, C34-cholesterol. However, the pocket-binding domain (PBD) of both HIVP3 and HIVP4 is more exposed to the aqueous environment than in C34-cholesterol. CONCLUSIONS: The present data allow us to conclude that more efficient blocking of HIV entry results from the synergism between the membranotropic behaviour and the enhanced exposure of the PBD.
Asunto(s)
Proteína gp41 de Envoltorio del VIH/farmacología , Inhibidores de Fusión de VIH/farmacología , Fragmentos de Péptidos/farmacología , Membrana Celular/metabolismo , Eritrocitos/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Unión ProteicaRESUMEN
The increased emergence of antibiotic-resistant bacteria is a serious health problem worldwide. In this sense, silver nanoparticles (AgNPs) have received increasing attention for their antimicrobial activity. In this context, the goal of this study was to produce AgNPs by a green synthesis protocol using an aqueous leaf extract of Schinus areira as biocomposite to later characterize their antimicrobial action. The nanomaterials obtained were characterized by UVâvis spectroscopy, DLS, TEM, and Raman, confirming the presence of quasi-spherical AgNPs with a negative surface charge and diameter around 11 nm. Afterward, the minimum inhibitory and bactericidal concentration of the AgNPs against Staphylococcus aureus and Escherichia coli were obtained, showing high antibacterial activity. In both of the examined bacteria, the AgNPs were able to raise intracellular ROS levels. In E. coli, the AgNPs can harm the bacterial membrane as well. Overall, it can be concluded that it was possible to obtain AgNPs with colloidal stability and antibacterial activity against Gram-positive and Gram-negative bacteria. Our findings point to at least two separate mechanisms that can cause cell death, one of which involves bacterial membrane damage and the other of which involves intracellular ROS induction.
Asunto(s)
Antibacterianos , Nanopartículas del Metal , Antibacterianos/química , Plata/farmacología , Plata/química , Schinus , Nanopartículas del Metal/química , Escherichia coli , Especies Reactivas de Oxígeno , Bacterias Gramnegativas , Bacterias Grampositivas , Bacterias , Extractos Vegetales/farmacología , Extractos Vegetales/química , Pruebas de Sensibilidad MicrobianaRESUMEN
The aim of this study was to evaluate fifty-three Lactobacillus plantarum isolates obtained from a Patagonian red wine, molecularly identified and typified using RAPD analysis, in order to select starter cultures for malolactic fermentation (MLF). The results obtained suggest a considerable genetic diversity, taking into account that all L. plantarum isolates were obtained from one cellar and one vintage. Based on the capacity to tolerate a concentration of 14 % ethanol in MRS broth for 2 days, eight isolates were selected for the subsequent analysis. The incidence of various wine stress factors (ethanol, acid pH, lysozyme and sulfur dioxide) on isolates growth was studied. Besides, glucosidase and tannase activities were evaluated, and the presence of genes involved in the synthesis of biogenic amines was examined by PCR. A previously characterized indigenous Oenococcus oeni strain was included with comparative purposes. Differences in technologically relevant characteristics were observed among the eight L. plantarum selected isolates, revealing an isolate-dependent behavior. Detectable glucosidase and tannase activities were found in all isolates. The presence of genes encoding histidine and tyrosine descarboxylases and putrescine carbamoyltransferase was not detected. The ability of L. plantarum isolates to grow and consume L-malic acid in simulated laboratory-scale vinifications revealed that two of them could be considered as possible MLF starter cultures for Patagonian red wines. These isolates will be subjected to further analysis, for a final winery technological characterization.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Glucosidasas/metabolismo , Ácido Láctico/metabolismo , Lactobacillus plantarum/aislamiento & purificación , Lactobacillus plantarum/metabolismo , Vino/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Fermentación/fisiología , Concentración de Iones de Hidrógeno , Lactobacillus plantarum/genética , Lactobacillus plantarum/crecimiento & desarrollo , Malatos/metabolismo , Técnica del ADN Polimorfo Amplificado AleatorioRESUMEN
Prokaryotes commonly present outer cell wall structures composed of a crystalline array of proteinaceous subunits, known as surface layers (S-layers). The ORF encoding the S-layer protein (SlpA) of Lactobacillus brevis was cloned into Lactococcus lactis under the transcriptional control of the xylose-inducible expression system (XIES). SlpA was secreted into the extracellular medium, as determined by immunoblotting, and assays on the kinetics of SlpA production revealed that repression of the system with glucose did not require the depletion of xylose from the medium that allows transitory ORF expression. The successful use of XIES to express S-layer proteins in the versatile and generally recognized as safe species L. lactis opens new possibilities for an efficient production and isolation of SlpA S-layer protein for its various applications in biotechnology and importantly as an antigen-carrying vehicle.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Levilactobacillus brevis/genética , Proteínas Bacterianas/genética , Western Blotting , Clonación Molecular , Medios de Cultivo/química , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Glucosa/metabolismo , Xilosa/metabolismoRESUMEN
Viruses remain one of the leading causes of animal and human disease. Some animal viral infections spread sporadically to human populations, posing a serious health risk. Particularly the emerging viral zoonotic diseases such as the novel, zoonotic coronavirus represent an actual challenge for the scientific and medical community. Besides human health risks, some animal viral infections, although still not zoonotic, represent important economic loses to the livestock industry. Viral infections pose a genuine concern for which there has been an increasing interest for new antiviral molecules. Among these novel compounds, antiviral peptides have been proposed as promising therapeutic options, not only for the growing body of evidence showing hopeful results but also due to the many adverse effects of chemical-based drugs. Here we review the current progress, key targets and considerations for the development of antiviral peptides (AVPs). The review summarizes the state of the art of the AVPs tested in zoonotic (coronaviruses, Rift Valley fever viruses, Eastern Equine Encephalitis Virus, Dengue and Junín virus) and also non-zoonotic farm animal viruses (avian and cattle viruses). Their molecular target, amino acid sequence and mechanism of action are summarized and reviewed. Antiviral peptides are currently on the cutting edge since they have been reported to display anti-coronavirus activity. Particularly, the review will discuss the specific mode of action of AVPs that specifically inhibit the fusion of viral and host-cell membranes for SARS-CoV-2, showing in detail some important features of the fusion inhibiting peptides that target the spike protein of these risky viruses.