Structural and Functional Implications of the Interaction between Macrolide Antibiotics and Bile Acids

Macrolide antibiotics, such as azithromycin and erythromycin, are in widespread use for the treatment of bacterial infections. Macrolides are taken up and excreted mainly by bile. Additionally, they have been implicated in biliary system diseases and to modify the excretion of other drugs through bile. Despite mounting evidence for the interplay between macrolide antibiotics and bile acids, the molecular details of this interaction remain unknown. Herein, we show by NMR measurements that macrolides directly bind to bile acid micelles. The topology of this interaction has been determined by solvent paramagnetic relaxation enhancements (solvent PREs). The macrolides were found to be bound close to the surface of the micelle. Increasing hydrophobicity of both the macrolide and the bile acid strengthen this interaction. Both bile acid and macrolide molecules show similar solvent PREs across their whole structures, indicating that there are no preferred orientations of them in the bile micelle aggregates. The binding to bile aggregates does not impede macrolide antibiotics from targeting bacteria. In fact, the toxicity of azithromycin towards enterotoxic E. coli (ETEC) is even slightly increased in the presence of bile, as was shown by effective concentration (EC₅₀) values.     

Structural and dynamical characterization of poly-gamma-glutamic acid-based cross-linked nanoparticles

This work describes the formation of water-soluble hydrophilic nanoparticles from biosynthetic poly-γ-glutamic acid (PGA). Nanoparticles were formed by cross-linking using 2,2′-(ethylenedioxy) diethylamine in the presence of water-soluble carbodiimide. The structure was determined by nuclear magnetic resonance spectroscopy and the particle size by transmission electron microscopy (TEM), size exclusion chromatography (SEC), and dynamic light-scattering (DLS) measurements. The results from TEM, SEC, and DLS reveal that the particle size depends on the ratio of cross-linking. Particle size values measured by TEM were between 20 and 90 nm. Formation of cross-linked nanoparticles results in a dramatic viscosity drop compared to the viscosity of the corresponding solution of the parent PGA. The viscosity and DLS experiments disclose an intriguing interplay between intrachain and interchain cross-linking of the polymer chains, depending on the cross-linker density and polymer concentration. The SEC measurements show that the retention time of the major portion of particles increase because of the higher cross-linking ratio. At moderate cross-linker concentration, intramolecular cross-linking is the dominant process, whereas at higher cross-linker densities, the interpolymer cross-linking plays an important role. As a result, large clusters are also formed.     

THE ROLE OF DNA DAMAGE IN PM2 VIRAL INACTIVATION BY METHYLENE BLUE PHOTOSENSITIZATION

Abstract— This study investigates the importance of DNA damage in viral inactivation by phenothiazines and light. Phenothiazines, including methylene blue (MB), toluidine blue and azure B are of particular interest because of their ability to bind to nucleic acids in vitro. Initial studies employing phages T7, MS2 and PM2 indicated that both DNA and RNA phages as well as enveloped and nonenveloped phages can be inactivated by phenothiazine photosensiti-zation. PM2, which contains a lipid-protein bilayer and supercoiled DNA, was used for the mechanistic studies to model blood-borne viruses. Viral DNA damage was assessed following treatment of phage to known levels of viral inactivation by extracting the DNA and analyzing for both direct and piperidine-catalyzed strand cleavage by gel electrophoresis. DNA strand cleavage was found to be both sensitizer concentration and light dose dependent. Both viral inactivation and DNA damage were found to be oxygen-dependent events. In parallel experiments, strand cleavage of isolated PM2 DNA treated with MB and light was also found to be oxygen dependent, in contrast to some previous reports. Transfection studies, which measure the infectivity of the extracted viral DNA, indicated that DNA from MB-treated phage was just as capable of generating progeny virus as the untreated controls. It was therefore concluded that the observed DNA damage is not correlated with loss of phage infectivity.     

REASSESSMENT OF THE DIFFERENTIAL EFFECTS OF ULTRAVIOLET AND IONIZING RADIATION ON HIV PROMOTER: THE USE OF CELL SURVIVAL AS THE BASIS FOR COMPARISONS

Abstract: Effects of different radiation treatments on the human immunodeficiency virus-1 (HIV) promoter were reassessed for exposures comparable to those encountered in clinical or cosmetic practice, using survival of the host cell as a basis for comparisons. The exposures were performed with two ultraviolet radiation sources commonly used as medical or cosmetic devices (UVASUN 2000 and FS20 lamps), a germicidal (G15T8) lamp and an X-ray machine. The UVC component of the FS20 lamp was filtered out. The emission spectra of the lamps were determined. The characteristics of these sources allowed us to discriminate among effects of UVA1 (340–400 nm), UVB + UVA2 (280–340 nm) and UVC (254 nm) radiations. Effects of irradiation were ascertained using cultures of HeLa cells stably transfected with the HIV promoter linked to a reporter—chloramphenicol acetyl transferase—gene. The exposures used caused at least two logs of cell killing. In this cytotoxicity range, UVA1 or X radiations had no effect on the HIV promoter, whereas UVB + UVA2 or UVC radiations activated the HIV promoter in a fluence-dependent manner. Survivals following exposure to UVB + UVA2 or UVC radiation were (1) at the lowest measurable HIV promoter activation, 30 and 20%, respectively, (2) at one-half maximal activation, 6 and 3%, respectively and (3) at the maximal activation, 0.5 and 0.2%, respectively. The results suggest that, among the radiations studied, UVB is the most important modality from the viewpoint of its potential effects on HIV-infected individuals, since (1) UVA1 or X radiations have no effects on the HIV promoter, (2) human exposure to UVC radiation is infrequent and (3) human UVB exposure is very common.     

Inhibition of Acinetobacter baumannii biofilm formation on a methacrylate polymer containing a 2-aminoimidazole subunit

A polymeric composite containing a 2-aminoimidazole derivative was synthesized. It was found that this polymer was resistant to biofilm colonization by Acinetobacter baumannii, no leaching of the 2-aminoimidazole derivative was observed after 2 weeks of treatment with deionized water, and the resulting polymer was not hemolytic.     

pH-dependent spectral properties of para-aminobenzoic acid and its derivatives

The local environment dictates the structural and functional properties of many important chemical and biological systems. The impact of pH on the photophysical properties of a series of para-aminobenzoic acids is examined using a combination of experimental spectroscopy and quantum chemical calculations. Following photoexcitation, PABA derivatives may undergo an intramolecular charge transfer (ICT) resulting in the formation of a zwitterionic species. The thermodynamics of the excited state reaction and temperature-dependence of the radiative emission processes are evaluated through variable temperature fluorescence spectroscopy carried out in a range of aqueous buffers. Quantum chemical calculations are used to analyze structural changes with modifications at the amine position and different protonation states. The ICT is only observed in the tertiary amine, which calculations show has more sp(2) character than the primary or secondary amines. Thermodynamic analysis indicates the ICT reaction is driven by entropy.     

Variability of essential oils of Betonica officinalis (Lamiaceae) from different wild populations in Kosovo

The aerial parts and roots of Betonica officinalis were collected from three localities characterized by different ecological conditions to study the natural variability of the chemical composition of the essential oils in this plant. The leaves and inflorescences were collected during the flowering time, whereas the roots were collected at the end of the vegetative period. The plant material was dried at room temperature. The essential oils were obtained by micro-steam hydrodistillation and analyzed using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Overall, 69 constituents were identified in all localities and plant parts. The main compositions of the leaves in all localities were alpha-pinene, 1-octen-3-ol, beta-bourbonene, (E)-caryophyllene and germacrene D. The essential oil of the inflorescences was characterized by these main constituents: alpha-pinene, (E)-caryophyllene and trans-beta-farnesene. In all localities, the percentages of alpha-pinene and (E)-caryophyllene were higher in the inflorescences than in the leaves, whereas nonane was the main constituent in the roots.     

NON-NUCLEAR DAMAGE AND CELL LYSIS ARE INDUCED BY UVA, BUT NOT UVB OR UVC, RADIATION IN THREE STRAINS OF L5178Y CELLS

The potential to induce non-nuclear changes in mammalian cells has been examined for (1) UVA1 radiation (340–400 nm, UVASUN 2000 lamp), (2) UVA + UVB (peak at 313 nm) radiation (FS20 lamp), and (3) UVC (254 nm) radiation (GI5T8 lamp). The effects of irradiation were monitored in vitro using three strains of L5178Y (LY) mouse lymphoma cells that markedly differ in sensitivity to UV radiation. Comparisons were made for the effects of approximately equitoxic fluences that reduced cell survival to 1–15%. Depending on the cell strain, the fluences ranged from 830 to 1600 kJ/m² for the UVASUN lamp, 75 to 390 J/m² for the FS20 lamp and 3.8 to 17.2 J/m² for the G15T8 lamp. At the exposure level used in this study, irradiation with the UVASUN, but not the FS20 or G15T8, lamp induced a variety of non-nuclear changes including damage to cytoplasmic organelles and increased plasma membrane permeability and cell lysis. Cell lysis and membrane permeabilization were induced by the UVA1 emission of the UVASUN lamp, but not by its visible + IR components (>400 nm). The results show that the plasma membrane and other organelles of LY cells are highly sensitive to UVA1 but not to UVB or UVC radiation. Also UVA1, but not UVB or UVC radiation, causes rapid and extensive lysis of LY cells. In conclusion, non-nuclear damage contributes substantially to UVA cytotoxicity in all three strains of LY cells.