Structural Insights into the Inhibition Site in the Phosphorylcholine Phosphatase Enzyme of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a highly pathogenic Gram-negative microorganism associated with high mortality levels in burned or immunosuppressed patients or individuals affected by cystic fibrosis. Studies support a colonization mechanism whereby P. aeruginosa can breakdown the host cell membrane phospholipids through the sequential action of two enzymes: (I) hemolytic phospholipase C acting upon phosphatidylcholine or sphingomyelin to produce phosphorylcholine (Pcho) and (II) phosphorylcholine phosphatase (PchP) that hydrolyzes Pcho to generate choline and inorganic phosphate. This coordinated action provides the bacteria with carbon, nitrogen, and inorganic phosphate to support growth. Furthermore, PchP exhibits a distinctive inhibition mechanism by high substrate concentration. Here, we combine kinetic assays and computational approaches such as molecular docking, molecular dynamics, and free-energy calculations to describe the inhibitory site of PchP, which shares specific residues with the enzyme's active site. Our study provides insights into a coupled inhibition mechanism by the substrate, allowing us to postulate that the integrity of the inhibition site is needed to the correct functioning of the active site. Our results allow us to gain a better understanding of PchP function and provide the basis for a rational drug design that might contribute to the treatment of infections caused by this important opportunistic pathogen.

Exploring the Properties of Unilamellar Vesicle Bilayers Formed by Ionic Liquid Surfactants for Future Applications in Nanomedicine.

Two ionic liquids (ILs) with amphiphilic properties composed of 1-butyl-3-methylimidazolium dioctylsulfosuccinate (bmim-AOT) and 1-hexyl-3-methylimidazolium dioctylsulfosuccinate (hmim-AOT) form unilamellar vesicles spontaneously simply by dissolving the IL-like surfactant in water. These novel vesicles were characterized using two different and highly sensitive fluorescent probes: 6-propionyl-2-(dimethylaminonaphthalene) (PRODAN) and trans-4-[4-(dimethylamino)-styryl]-1-methylpyridinium iodide (HC). These fluorescent probes provide information about the physicochemical properties of the bilayer, such as micropolarity, microviscosity, and electron-donor capacity. In addition, the biocompatibility of these vesicles with the blood medium was evaluated, and their toxicity was determined using Dictyostelium discoideum amoebas. First, using PRODAN and HC, it was found that the bilayer composition and the chemical structure of the ions at the interface produced differences between both amphiphiles, making the vesicles different. Thus, the bilayer of hmim-AOT vesicles is less polar, more rigid, and has a lower electron-donor capacity than those made by bmim-AOT. Finally, the results obtained from the hemolysis studies and the growth behavior of unicellular amoebas, particularly utilizing the D. discoideum assay, showed that both vesicular systems do not produce toxic effects up to a concentration of 0.02 mg/mL. This elegant assay, devoid of animal usage, highlights the potential of these newly organized systems for the delivery of drugs and bioactive molecules of different polarities.

Antioxidant and antimicrobial properties of tyrosol and derivative-compounds in the presence of vitamin B2. Assays of synergistic antioxidant effect with commercial food additives.

The main causes of food spoilage come from the process of oxidation and the contamination by microorganisms. For the purpose of increasing food shelf-life the industries employ different techniques, being the addition of preservatives, one of the most used. The aim of this contribution was to investigate the potential antioxidant properties of tyrosol (4-hydroxyphenethyl alcohol, 4-OH) and tyrosol derived isomers (2-hydroxyphenethyl alcohol, 2-OH and 3-hydroxyphenethyl alcohol, 3-OH) against reactive oxygen species (ROS) and the antimicrobial effect on Staphylococcus aureus and Escherichia coli. Furthermore, the type of antioxidant effect of substrates and commercial antioxidants mixtures was studied. Upon visible-light, the substrates interacted with the vitamin B2 and different ROS were generated. All the compounds deactivated O2(1Δg) and O2●-, whereas only 2-OH and 3-OH inhibited H2O2 and HO●. The substrates exhibited a synergistic antioxidant effect when combined with commercial antioxidants. 2-OH showed antimicrobial activity against strains tested.

Antioxidant ability of tyrosol and derivative-compounds in the presence of O2(1Δg)-species. Studies of synergistic antioxidant effect with commercial antioxidants.

The exposure of fatty products to environmental light can trigger lipid oxidation in food through a sensitized-photooxidation process, which involves the participation of the species singlet oxygen (O2(1Δg)). Therefore, preservation of food quality represents a subject of great economic interest to the food industry. In this sense, the phenolic compounds are natural antioxidants widely used in food industry. In this contribution we studied the interactions of phenolic derivatives (Phd), tyrosol and tyrosol derived isomers, with O2(1Δg) and their possible protective effect against the oxidative degradation of unsaturated fatty acids and amino acids. Besides, a potential synergistic interaction between Phd and antioxidants used in food industry were investigated. Phd substrates showed properties as antioxidant additives due to their high ability deactivating O2(1Δg) through a physical process and synergistic effect in the presence of commercial antioxidants. Phd presented an antioxidant protective effect toward O2(1Δg)-mediated degradation of methyl linoleate and tryptophan.

Reactive Oxygen Species-mediated Degradation of Antidiabetic Compounds: Cytotoxic Implications of Their Photodegradation Products.

Reactive oxygen species (ROS) have been described in their double physiological function, helping in the maintenance of health as well as contributing to oxidative stress. Diabetes mellitus is a chronical disease nearly related to oxidative stress, whose treatment (in type II variant) consists in the administration of antidiabetic compounds (Andb) such as Gliclazide (Gli) and Glipizide (Glip). In this context, as Andb may be exposed to high ROS concentrations in diabetic patients, we have studied the potential ROS-mediated degradation of Gli and Glip through photosensitized processes, in the presence of Riboflavin (Rf) vitamin. We found that singlet oxygen (O2 (1 ∆g )) participated in the Rf-sensitized photodegradation of both Andb, and also superoxide radical anion in the case of Gli. Two principal products derived from O2 (1 ∆g )-mediated degradation of Gli were identified and their chemical structures characterized, through HPLC mass spectrometry. O2 (1 ∆g )-mediated degradation products and their toxicity was assayed on Vero cell line. These studies demonstrated that neither Gli nor its photoproducts caused cytotoxic effect under the experimental conditions assayed. Our results show strong evidences of ROS-mediated Andb degradation, which may involve the reduction or loss of their therapeutic action, as well as potential cytotoxicity derived from their oxidation products.

The activity of propolis in the scavenging of vitamin B2-photogenerated ROS.

OBJECTIVES: The study was focused on the activity of propolis from Amaicha del Valle, Argentina (ProAV) as a promoter and scavenger of Riboflavin (Rf)--photogenerated reactive oxygen species (ROS). METHODS: Through a kinetic and mechanistic study, employing stationary and time-resolved photochemical and electrochemical techniques, the protecting activity of ProAV was investigated. RESULTS: In the absence of light and Rf, ProAV exerted a relatively efficient inhibitory effect on 1,1-diphenyl-2-picrylhydrazyl radicals and acts as a protector of artificially promoted linoleic acid oxidation. Under aerobic visible-light-irradiation conditions, in the presence of Rf as the only light-absorber species, a complex picture of competitive processes takes place, starting with the quenching of singlet and triplet electronically excited states of Rf by ProAV. The species O2(1 g), O2(•-), H2O2, and OH(•) are generated and interact with ProAV. DISCUSSION: ProAV behaves as an efficient ROS scavenger. It is scarcely photo-oxidized by interaction with the mentioned ROS. Quantitative results indicate that ProAV is even more resistant to photo-oxidation than the recognized antioxidant trolox. Two dihydroxychalcones, mostly present in the ProAV composition, are responsible for the protecting activity of the propolis.

On the photooxidation of the multifunctional drug niclosamide. A kinetic study in the presence of vitamin B2 and visible light.

OBJECTIVES: The multifunctional drug niclosamide (NSD), extensively employed therapeutically, is a frequent pollutant of surface waters. Considering the environmental importance of photodegradative processes for this type of contaminant, the kinetic and mechanistic aspects of the possible visible-light-mediated photooxidation of NSD were studied under naturalistic conditions. METHODS: The visible-light absorber riboflavin (vitamin B2) was employed as a photosensitizer. The vitamin can usually be found in natural waters and is the most common endogenous photosensitizer in mammals. The interaction of NSD with electronically excited states of Rf and with photogenerated reactive oxygen species (ROS) was evaluated through conventional UV spectroscopy, laser flash photolysis, time-resolved phosphorescence detection of singlet molecular oxygen (O2((1)Δg)), and polarographic dosage of dissolved oxygen. RESULTS: Ground state NSD quenched the long-lived triplet excited state of Rf ((3)Rf*) and the photogenerated ROS (O2((1)Δg)) and superoxide radical anion (O2•−). As a result, NSD was photooxidized. The rate constants for the interaction NSD-O2((1)Δg) are particularly low, in the order of 10(6)/M/s, although the whole interaction is attributable to a pure reactive process. The O2((1)Δg) quenching was faster in alkaline medium, favored by the ionization of the NSD phenolic group. Under Rf-photosensitization, NSD was degraded very much more rapidly than phenol, the latter being considered a paradigmatic water-contaminant model compound. NSD may behave as an antioxidant in bio-environments, as demonstrated employing the photooxidizable amino acid tryptophan as a relevant biological target. DISCUSSION: The results indicate that a O2•−-mediated process is the main route for the Rf-sensitized photooxidation of NSD. Photodegradation of the biocide in the presence and absence of phenol and tryptophan was quantitatively evaluated, discussed, and interpreted in terms of competitive quenching processes of (3)Rf*, O2((1)Δg), and O2•− by the substrates.

On the natural fate of maleic hydrazide. Kinetic aspects of the photochemical and microbiological degradation of the herbicide.

Kinetic and mechanistic aspects of the photochemical and microbiological degradation of the herbicide Maleic Hydrazide (MH) have been studied. Riboflavin (Rf, vitamin B2) was employed as a main photosensitizer whereas Humic Acid (HA) was included as a second sensitizer in order to more closely simulate natural environmental conditions. MH quenches excited singlet and triplet states of Rf, with rate constants close to the diffusion limit. The herbicide and dissolved molecular oxygen competitively quench triplet excited Rf. As a consequence the reactive oxygen species (ROS), superoxide radical anion (O2(-·)), hydrogen peroxide (H2O2) and singlet molecular oxygen (O2((1)Δg)) are produced by electron- and energy-transfer processes, respectively, as demonstrated by auxiliary experiments employing selective auxiliary quenchers and the exclusive O2((1)Δg) generator Rose Bengal (RB). As a global result, the photodegradation of Rf is retarded, whereas MH is degraded by the generated ROS. The bacteria Pseudomonas aeruginosa (Ps) and Bacillus subtilis (Bs), recognized as contaminants surface-water and soil and microbial antagonists of phytopathogenic, were used in the microbiological experiments. Results of the individual incubation of both bacteria in in the presence of MH indicate a stimulation on the Ps growth, implying the biodegradation of the herbicide, whereas MH only exerted a bacteriostatic effect on Bs.