Tea catechins, which are flavonoids and the main components of green tea extracts, are thought to have antibacterial and antioxidant activity. Several studies indicate that lipid membranes are one of the targets of the antibacterial activity of catechins. Studies using a suspension of large unilamellar vesicles (LUVs) indicate that catechin causes gradual leakage of internal contents from LUVs. However, the detailed characteristics of the interaction of catechins with lipid membranes remain unclear. In this study, we investigated the interaction of (-)-epigallocatechin gallate (EGCg), a major catechin in tea extract, with single giant unilamellar vesicles (GUVs) of egg phosphatidylcholine (egg PC) using phase-contrast fluorescence microscopy and the single GUV method. We prepared GUVs of lipid membranes of egg PC in a physiological ion concentration ( approximately 150 mM NaCl) using the polyethylene glycol-lipid method. Low concentrations of EGCg at and above 30 muM induced rapid leakage of a fluorescent probe, calcein, from the inside of single egg PC-GUVs; after the leakage, the GUVs changed into small lumps of lipid membranes. On the other hand, phase-contrast microscopic images revealed the detailed process of the EGCg-induced burst of GUVs, the decrease in their diameter, and their transformation into small lumps. The dependence of the fraction of burst GUVs on EGCg concentration was almost the same as that of the fraction of leaked GUV. This correlation strongly indicates that the leakage of calcein from the inside to the outside of the GUV occurred as a result of the burst of the GUV. The fraction of completely leaked GUV and the fraction of the burst GUV increased with time and also increased with increasing EGCg concentration. We compared the EGCg-induced leakage from single GUVs with EGCg-induced leakage from a LUV suspension. The analysis of the EGCg-induced shape changes shows that the binding of EGCg to the external monolayer of the GUV increases its membrane area, inducing an increase in its surface pressure. Small angle x-ray scattering experiments indicate that the intermembrane distance of multilamellar vesicles of PC membrane greatly decreased at EGCg concentrations above the threshold, suggesting that neighboring membranes came in close contact with each other. On the basis of these results, we discuss the mechanism of the EGCg-induced bursting of vesicles.

ESR (electron spin resonance) spectra of a fatty acid spin probe (16-doxylstearic acid, 16-DS) incorporated into an aqueous surfactant system composed of oleic acid and oleate molecules were measured between 10 and 50 °C up to a total oleic acid + oleate concentration of 50 mM. Depending on the total concentration and the pH, different types of oleic acid/oleate aggregates formed. At the two ends of the pH range investigated (above pH 10.4 and below pH 6.4), the ESR spectra of 16-DS were highly symmetric, enabling calculation of the microviscosities in the surfactant aggregates to be 4 cP and 6 cP, respectively. In the high pH range, the observed aggregates are micelles. On the other hand, in the low pH range the microviscosity was considerably lower than that of neat oleic acid (measured to be 11 cP), indicating that the obtained emulsion system was not composed of pure oleic acid droplets. We postulate that the surfactant molecules at low pH form condensed aggregates of lamellar bilayers. Asymmetric high-field ESR lines were obtained at intermediate pH between pH 6.4 and pH 10.4. This indicates that the probe molecules were present in two physically different aggregation states. We assigned the two aggregation states to be vesicles and nonlamellar aggregates (most likely nonspherical micelles), based on the observation made by microscopy and light scattering techniques. The analysis of the ESR lines by spectral simulation using a modified Bloch equation supports the coexistence of vesicles and nonlamellar aggregates through the entire intermediate pH range; the relative amount of the two aggregation forms depends critically on pH, temperature, and concentration. Furthermore, the spectral simulation indicated that particularly stable oleic acid/oleate vesicles are formed around pH 8.5, where the protonated and ionized species exist in a stoichiometric ratio.

Single mitochondria show the spontaneous fluctuations of DeltaPsim. In this study, to examine the mechanism of the fluctuations, we observed DeltaPsim in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate, or ascorbate plus TMPD to mitochondria induced polarization of the inner membrane followed by repeated cycles of rapid depolarizations and immediate repolarizations. ADP significantly decreased the frequency of the rapid depolarizations, but the ADP effect was counteracted by oligomycin. On the other hand, the rapid depolarizations did not occur when mitochondria were polarized by the efflux of K(+) from the matrix. The rapid depolarizations became frequent with the increase in the substrate concentration or pH of the buffer. These results suggest that the rapid depolarizations depend on the net translocation of protons from the matrix. The frequency of the rapid depolarizations was not affected by ROS scavengers, Ca(2+), CsA, or BA. In addition, the obvious increase in the permeability of the inner membrane to calcein (MW 623) that was entrapped in the matrix was not observed upon the transient depolarization. The mechanisms of the spontaneous oscillations of DeltaPsim are discussed in relation to the matrix pH and the permeability transitions.