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Different mechanisms of action of antimicrobial peptides: insights from fluorescence spectroscopy
Full text linkI peptidi antimicrobici (AMPs) sono oligopeptidi naturali dotati di una forte attività battericida. Molti di essi distruggono i batteri interagendo con le loro membrane e perturbandone la permeabilità. A causa di questo meccanismo di azione, che evidentemente non induce resistenza, essi sono studiati come candidati promettenti per lo sviluppo di nuovi farmaci antibiotici per fronteggiare l’insorgenza di batteri resistenti a più farmaci. Sono stati proposti diversi modelli per il loro meccanismo di perturbazione della membrana, ma i dettagli molecolari di questo processo sono ancora dibattuti. Perciò, è importante conoscere le informazioni strutturali sull’interazione con i doppi strati lipidici per comprendere in dettaglio il loro meccanismo di destabilizzare la membrana e per progettare razionalmente nuovi e potenti analoghi peptidici.
Nel presente lavoro, è stata utilizzata la spettroscopia di fluorescenza insieme ad altre tecniche chimico-fisiche, come la riflettività di neutroni, la bilancia Langmuir-Blodgett e simulazioni di dinamica molecolare, per caratterizzare il meccanismo di azione del PMAP-23, scelto come esempio tipico tra i peptidi antimicrobici cationici ed elicoidali. Allo scopo di discutere i diversi meccanismi di azione dei peptidi antimicrobici, e per evidenziare la potenzialità dei metodi spettroscopici nel discriminare i diversi modelli, i risultati ottenuti con il PMAP-23 sono stati confrontati sistematicamente con quelli ottenuti precedentemente per la tricogina GA IV, peptide neutro della famiglia dei peptaiboli.
Sia i dati sperimentali che quelli teorici indicano che il PMAP-23 si colloca appena al di sotto delle teste polari della membrana, con un’orientazione prevalentemente parallela al piano del doppio strato. Questi risultati, insieme ai dati sperimentali sul rilascio indotto dal peptide da liposomi grandi e giganti, sulla traslocazione lipidica e sullo scambio del peptide tra vescicole, supportano un meccanismo di azione conforme con il modello “a tappeto”. Secondo questo modello, l’accumulo di peptide sulla superficie esterna del doppio strato causa una differenza di tensione superficiale tra i due strati della membrana, portando alla formazione dei difetti che inducono il rilascio. Ulteriori esperimenti hanno escluso il ruolo specifico di alcuni residui del PMAP-23 nella sua attività di perturbare la membrana.
Infine, è stato proposto e discusso un modello per descrivere la cinetica di rilascio indotta dai peptidi antimicrobici. Le cinetiche di rilascio sono solitamente molto lente (richiedono dai minuti alle ore per lo svuotamento completo delle vescicole) e mostrano un comportamento bifasico. In questa tesi, sono state affrontate sistematicamente tutte le possibili opzioni per individuare la causa della fase lenta del rilascio. Gli esperimenti di fluorescenza hanno dimostrato che tutti i processi coinvolti nell’interazione peptide-membrana (associazione peptide-membrana, aggregazione e traslocazione del peptide) avvengono in una scala di tempi molto più corta rispetto al rilascio. Inoltre, i peptidi scambiano continuamente tra le vescicole e questo causa necessariamente delle fluttuazioni nel tempo nel numero di molecole di peptide legate ad ogni liposoma, e nella formazione di pori. Data la dimensione, relativamente piccola, delle vescicole, il numero di peptidi legati può essere piuttosto piccolo e le fluttuazioni significative. Perciò, secondo il nostro modello, la fase rapida iniziale del rilascio è causata da quelle vescicole che già contengono almeno un poro, in seguito alla distribuzione casuale di peptidi tra liposomi, mentre la fase lenta è associata al tempo necessario a raggiungere di volta in volta il numero di peptidi legati necessario per la formazione del poro nella vescicola ancora intatta. Le fluttuazioni dovute allo scambio del peptide tra i liposomi sono perciò il passo limitante del meccanismo lento, che alla fine porta al rilascio di tutte le vescicole.Antimicrobial peptides (AMPs) are natural oligopeptides endowed with a strong bactericidal activity. Most of them kill bacteria by interacting with their membranes and perturbing their permeability. Due to this mechanism of action, which apparently does not induce bacterial resistance, they are investigated as promising candidates for the development of new antibiotic drugs to face the insurgence of multidrug resistant bacteria. Several different models have been proposed for their mechanism of membrane perturbation, but the molecular details of this process are still debated. Therefore, structural data on their interaction with lipid bilayers are essential for a detailed understanding of their mechanism of membrane destabilization, and for the rational design of new, potent and selective peptidomimetic analogues.
In this work, fluorescence spectroscopy was employed, together with other physico-chemical techniques, such as neutron reflectivity, Langmuir-Blodgett film balance and molecular dynamics simulations, to characterize the mechanism of action of PMAP-23, chosen as a typical example of cationic, helical AMPs. In order to discuss the different mechanisms of action of AMPs, and to evidence the potential of spectroscopic methods to discriminate between the different models, the results obtained with PMAP-23 were systematically compared with those previously reported for the peptaibol trichogin GA IV.
Both experimental and theoretical data indicate a PMAP-23 location just below the polar headgroups of the membrane, with an orientation essentially parallel to the bilayer plane. These findings, together with experimental results on peptide-induced leakage from large and giant vesicles, lipid flip-flop and peptide exchange between vesicles, support a mechanism of action consistent with the “carpet” model. According to this model, peptide accumulation in the outer leaflet of the bilayer causes a difference in surface tension between the two layers of the membrane, which is then released though the formation of defects, thus inducing membrane leakage. Further experiments excluded the specific role of some PMAP-23 residues in its membrane-perturbing activity .
Finally, a kinetic model describing the kinetics of vesicle leakage induced by antimicrobial peptides was proposed and discussed. The leakage kinetics are usually very slow, requiring minutes to hours for complete release of vesicle contents, and exhibit a biphasic behavior. In this thesis, all the possible options for the rate-limiting step of the slow leakage process were systematically studied. Fluorescence experiments demonstrated that all processes involved in peptide-membrane interaction (peptide-membrane association, peptide aggregation and peptide translocation) take place in a time-scale much shorter than the leakage kinetics. However, peptides are continually exchanging among vesicles, and this necessarily causes fluctuations over time in the number of peptide molecules bound to each liposome, and in the formation of pores. Due to the relatively small size of the vesicles, the number of bound peptides can be rather small, and its fluctuations significant. Therefore, according to our model, the fast initial leakage is caused by those vesicles which, after the initial random distribution of peptides among liposomes, already contain at least one pore, while the slower release is associated to the time needed to occasionally reach the critical number of bound peptides necessary for pore formation in an intact vesicle. Fluctuations due to peptide exchange among vesicles are therefore the rate-limiting step of such a slow mechanism, that ultimately leads to the leakage of all the loaded vesicles
Orthonormal Gas Chromatography Sets Of Extra Virgin Olive Oil,
No full textStarting from gas-chromatograms of samples of extra virgin olive oil, choosing most significant peaks, authors try to
define a standard ortho-normal base be deposited for quality valuatio
Polymorphic Behavior in Protein-Surfactant Mixtures: The Water-Bovine SerumAlbumin-Sodium Taurodeoxycholate System
No full textMixtures containing water, bovine serum albumin (BSA), and sodium taurodeoxycholate (NaTDChave been investigated. Depending on the concentration of both solutes and the pH, solutions, precipitates, and gels are formed. At high concentrations, a gel, extending on both sides of the charge neutralization line, and two-phase regions are observed. The thermal gelation threshold, the temperature above which G> G, depends on BSA and NaTDC content and is concomitant to moderate heat effects, inferred by differential scanning calorimetry (DSC). Water self-diffusion in the gels is slightly slower than that in the bulk and poorly sensitive to composition: it is about 65% the value of neat H2O in a wide concentration range, irrespective of the BSA, or NaTDC, concentration. The 23Na T1 and T2 values, measured at 105.75 MHz on BSA-NaTDC gels, indicate that the motions determining the NMR relaxation of the sodium ions in the hydration layer of the protein-surfactant aggregates are not slow
Flavours Correlation Determination by Non Supervised Neural Network in Virgin Olive Oil
No full textOlive Oil is the first food product for which the assessment of the organoleptic quality by means of the Panel Test (PT) was introduced at the level of European Union legislation as a criterion for the sensory evaluation in the Reg. EU 1348/2013 annex XII (revision of Reg. EEC 2568/91). The standard establishes that olive oil must be tasted according to the rigid standards and regulations of the Panel Test (PT) that is carried out by a group of at least 8 professional tasters with a chief, that, through the tasting, give a numerical subjective evaluation (from 0 to 5) to many questions about flavours, in a questionnaire. The ensemble of the scores, reached by each components, defines the organoleptic quality. The amount of these numbers suggests, for better readability, the use of an unsupervised Artificial Neural Network (ANN), which identifies the best two-dimensional representation of the samples that have stimulated the PT. Analysing the coefficients relating a flavour to output neurons of the Kohonen Map, it is possible to individuate the heaviest. If it happens that more heavier coefficients inherent to different flavours, flights the same neuron, an internal correlation will be searched
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
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