The molecular pathway of enrofloxacin, a fluoroquinolone antibiotic, through the outer membrane channel OmpF of Escherichia coli is investigated. High-resolution ion current fluctuation analysis reveals a strong affinity for enrofloxacin to OmpF, the highest value ever recorded for an antibiotic-channel interaction. A single point mutation in the constriction zone of OmpF, replacing aspartic acid at the 113 position with asparagine (D113N), lowers the affinity to a level comparable to other antibiotics. All-atom molecular dynamics simulations allow rationalizing the translocation pathways: wild-type OmpF has two symmetric binding sites for enrofloxacin located at each channel entry separated by a large energy barrier in the center, which inhibits antibiotic translocation. In this particular case, our simulations suggest that the ion current blockages are caused by molecules occupying either one of these peripheral binding sites. Removal of the negative charge on position 113 removes the central barrier and shifts the two peripheral binding sites to a unique central site, which facilitates translocation. Fluorescence steady-state measurements agree with the different location of binding sites for wild-type OmpF and the mutant. Our results demonstrate how a single-point mutation of the porin, and the resulting intrachannel shift of the affinity site, may substantially modify translocation.

In this work the critical micelle concentrations (cmc) of four bile salts, sodium cholate, sodium glycocholate, sodium deoxycholate, and sodium glycodeoxycholate, are determined and presented. Three independent noninvasive methodologies (potentiometry, derivative spectrophotometry, and light scattering) were used for cmc determination, at 25 degrees C with ionic strength adjusted to 0.10 M with NaCl. Spectrophotometric and potentiometric studies of some bile salts were also executed at various ionic strength values, thus allowing the influence of the ionic strength on the cmc value of the bile salt to be assessed. A critical comparison of the cmc values obtained with data collected from the literature is presented. Furthermore, this work makes an evaluation of the conceptual bases of different methodologies commonly used for cmc determination, since variations in the results obtained can be related mainly to different intrinsic features of the methods used (such as sensitivity or the need to include tracers or probes) or to the operational cmc definition applied. The undoubted definition of the experimental bile salt concentration that corresponds to cmc (operational cmc) is essential since in the case of these amphiphiles the formation of micelles is not as abrupt as in the case of ordinary association colloids. The biphasic nature of their aggregation leads to a "round-shaped" variation of the experimental parameters under analysis, which makes difficult the evaluation of the cmc values and can be responsible for the different results obtained.

A blueberry extract (A) and two anthocyanin-derived extracts (B and C) were prepared. The contents of polyphenols, flavonoids, anthocyanins, and anthocyanin-derived pigments of the extracts were determined for the first time. The pigment profile of blueberry extract A corresponded to 15 anthocyanins, whereas extract B was mainly composed of anthocyanin-pyruvic acid adducts of the blueberry original anthocyanins and extract C was mainly composed of the respective vinylpyranoanthocyanin-catechins (portisins). The extracts' abilities to inhibit lipid peroxidation, induced by 2,2'-azobis(2-methyl-propanimidamide) dihydrochloride in a liposomal membrane system were examined. The antioxidant capacities of the extracts were evaluated through monitoring oxygen consumption and by measuring the formation of conjugated dienes. All of the extracts provided protection of membranes against peroxyl radicals by increasing the induction time of oxidation. This effect increased with the polyphenol content and with the structural complexity of the anthocyanin-derived pigments of the extracts. The pigments present in extract C seemed to induce a higher protection of the liposome membranes toward oxidation. In addition, the antiradical properties and the reducing power of the extracts were determined by using DPPH and FRAP methods, respectively. The results from these assays were in agreement with those obtained with the liposome membranes.

The facilitated influx of moxifloxacin through the most abundant channel in the outer cell wall of gram-negative bacteria was investigated. Molecular modeling provided atomic details of the interaction with the channel surface, revealed the preferred orientation of the antibiotic along its pathway, and gave an estimated time necessary for translocation. High-resolution conductance measurements on single OmpF trimers allowed the passages of individual moxifloxacin molecules to be counted. The average mean residence time of 50 micros is in agreement with the predicted strong interaction from the modeling. In contrast, control measurements with nalidixic acid, a hydrophobic antibiotic that rather permeates across the lipid membrane, revealed a negligible interaction. The spectral overlap of tryptophan with moxifloxacin was suitable for a FRET study of the protein-antibiotic interaction. Combining molecular dynamics simulations with selective quenching identified an interaction of moxifloxacin with Trp61 inside the OmpF channel, whereas nalidixic acid showed preferential interaction with Trp214 on the channel exterior. An understanding of the detailed molecular interactions between the antibiotic and its preferred channel may be used to develop new antibiotics with improved uptake kinetics.

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