Monitoring of the Proton Electrochemical Gradient in Reconstituted Vesicles: Quantitative Measurements of Both Transmembrane Potential and Intravesicular pH by Ratiometric Fluorescent Probes

Proteoliposomes carrying reconstituted yeast plasma membrane H⁺-ATPase in their lipid membrane or plasma membrane vesicles are model systems convenient for studying basic electrochemical processes involved in formation of the proton electrochemical gradient (Δμ_H ⁺) across the microbial or plant cell membrane. Δψ- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Δψ-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Δψ generated by the reconstituted H⁺-ATPase. Its Δψ response can be calibrated by the K⁺/valinomycin method and ratiometric mode of fluorescence measurements reduces undesirable artefacts. In situ pH-sensitive fluorescent probe pyranine was used for quantitative measurements of pH inside the proteoliposomes. Calibration of pH-sensitive fluorescence response of pyranine entrapped inside proteoliposomes was performed with several ionophores combined in order to deplete the gradients passively formed across the membrane. Presented model system offers a suitable tool for simultaneous monitoring of both components of the proton electrochemical gradient, Δψ and ΔpH. This approach should help in further understanding how their formation is interconnected on biomembranes and even how transport of other ions is combined to it.     

Asymmetric deformation of bubble shape: cause or effect of vortex-shedding?

Two perpendicular projections of rising bubbles were observed in counter-current downstream diverging flow. Evidently, the bubbles did not enter the boundary layer at the channel wall and a plug liquid flow assumption was acceptable in our experimental equipment. This confirmed that the experiment was appropriate for simulation of bubble rises in a quiescent liquid column. Recent data obtained by a high-speed camera permitted recording over a period of 60 s. Image analysis by a tailor-made program provided a time-series of quantities related to the position, size, and shape of bubbles. In addition to determination of the aspect ratio of the equivalent oblate ellipsoid, deviation from this shape was investigated in respect of the difference between the bubble’s centre of mass and the geometrical centre of bubble projection. Autocorrelation of the data indicated that the bubble inclination oscillated harmonically with a frequency of 5–10 Hz; cross correlation showed that the horizontal shift of the centre of mass, as well as the horizontal velocity, increased with increasing bubble inclination, and the vertical shift of the centre of mass increased with an increases in the absolute value of the bubble inclination. There is no significant phase shift in the oscillation of these quantities. The bulky bottom side of the bubbles is in accordance with the model of bubble oscillation induced by instability of the equilibrium of gravity and surface tension forces. The oscillation frequency dependence on surface forces (Eötvös number) is evident, while viscosity does not play a significant role in low-viscosity liquids. Therefore, vortex-shedding is more likely to be an effect of the oscillation and not its cause.     

Selective and Wash-Resistant Fluorescent Dihydrocodeinone Derivatives Allow Single-Molecule Imaging of μ-Opioid Receptor Dimerization

μ-Opioid receptors (μ-ORs) play a critical role in the modulation of pain and mediate the effects of the most powerful analgesic drugs. Despite extensive efforts, it remains insufficiently understood how μ-ORs produce specific effects in living cells. We developed new fluorescent ligands based on the μ-OR antagonist E-p-nitrocinnamoylamino-dihydrocodeinone (CACO), that display high affinity, long residence time and pronounced selectivity. Using these ligands, we achieved single-molecule imaging of μ-ORs on the surface of living cells at physiological expression levels. Our results reveal a high heterogeneity in the diffusion of μ-ORs, with a relevant immobile fraction. Using a pair of fluorescent ligands of different color, we provide evidence that μ-ORs interact with each other to form short-lived homodimers on the plasma membrane. This approach provides a new strategy to investigate μ-OR pharmacology at single-molecule level.     

Important Requirements for the Selection of Internal Standards during the Development of Desorption/Ionization Assays for Drug Quantification in Biological Matrices—A Practical Example

Desorption/ionization mass spectrometry (DI-MS) approaches allow for the rapid quantification of drugs in biological matrices using assays that can be validated according to regulatory guidelines. However, specific adaptations must be applied to create reliable quantification methods, depending on the approach and instrumentation used. In the present article, we demonstrate the importance of the molecular weight, the fragmentation pattern, and the purity of the internal standard for the development of matrix-assisted laser desorption/ionization (MALDI)-ion mobility (IM)-tandem MS and MS/MS methods. We present preliminary results of method development for the quantification of selinexor in microdialysis fluids with a stable isotopically labeled internal standard. In addition, we discuss the selection of internal standards for MALDI-MS assays using different instrumentations.     

Isolation,Characterization and In Silico Studies of Secondary Metabolites from the Whole Plant of Polygala inexpectata Peşmen & Erik

<em>Polygalaem> species are frequently used worldwide in the treatment of various diseases, such as inflammatory and autoimmune disorders as well as metabolic and neurodegenerative diseases, due to the large number of secondary metabolites they contain. The present study was performed on <em>Polygala inexpectataem>, which is a narrow endemic species for the flora of Turkey, and resulted in the isolation of nine known compounds, 6,3′-disinapoyl-sucrose (1), 6-<em>Oem>-sinapoyl,3′-<em>Oem>-trimethoxy-cinnamoyl-sucrose (tenuifoliside C) (2), 3′-<em>Oem>-(<em>Oem>-methyl-feruloyl)-sucrose (3), 3′-<em>Oem>-(sinapoyl)-sucrose (4), 3′-<em>Oem>-trimethoxy-cinnamoyl-sucrose (glomeratose) (5), 3′-<em>Oem>-feruloyl-sucrose (sibiricose A5) (6), sinapyl alcohol 4-<em>Oem>-glucoside (syringin or eleutheroside B) (7), liriodendrin (8), and 7,4′-di-<em>Oem>-methylquercetin-3-<em>Oem>-β-rutinoside (ombuin 3-<em>Oem>-rutinoside or ombuoside) (9). The structures of the compounds were determined by the spectroscopic methods including 1D-NMR (¹H NMR, ¹³C NMR, DEPT-135), 2D-NMR (COSY, NOESY, HSQC, HMBC), and HRMS. The isolated compounds were shown in an in silico setting to be accommodated well within the inhibitor-binding pockets of myeloperoxidase and inducible nitric oxide synthase and anchored mainly through hydrogen-bonding interactions and π-effects. It is therefore plausible to suggest that the previously established anti-inflammatory properties of some <em>Polygalaem>-derived phytochemicals may be due, in part, to the modulation of pro-inflammatory enzyme activities.     

Two‐Step Oxidation Synthesis of Sulfur with a Red Aggregation‐Induced Emission

Sulfur is not normally considered a light‐emitting material, even though there have been reports of a dim luminescence of this compound in the blue‐to‐green spectral region. Now, it is shown how to make red‐emissive sulfur by a two‐step oxidation approach using elemental sulfur and Na₂S as starting materials, with a high photoluminescence quantum yield of 7.2 %. Polysulfide is formed first and is partially transformed into Na₂S₂O₃ in the first step, and then turns back to elemental S in the second step. The elevated temperature and relatively oxygen‐deficient environment during the second step transforms Na₂S₂O₃ into Na₂SO₃ incorporated with oxygen vacancies, thus resulting in the formation of a solid‐state powder consisting of elemental S embedded in Na₂SO₃. It shows aggregation‐induced emission properties, attributed to the influence of oxygen vacancies on the emission dynamics of sulfur by providing additional lower energy states that facilitate the radiative relaxation of excitons.     

Stability and Removal of Benzophenone-Type UV Filters from Water Matrices by Advanced Oxidation Processes

Benzophenone (BP) type UV filters are common environmental contaminants that are posing a growing health concern due to their increasing presence in water. Different studies have evidenced the presence of benzophenones (BP, BP-1, BP-2, BP-3, BP-4, BP-9, HPB) in several environmental matrices, indicating that conventional technologies of water treatment are not able to remove them. It has also been reported that these compounds could be associated with endocrine-disrupting activities, genotoxicity, and reproductive toxicity. This review focuses on the degradation kinetics and mechanisms of benzophenone-type UV filters and their degradation products (DPs) under UV and solar irradiation and in UV-based advanced oxidation processes (AOPs) such as UV/H₂O₂, UV/persulfate, and the Fenton process. The effects of various operating parameters, such as UV irradiation including initial concentrations of H₂O₂, persulfate, and Fe²⁺, on the degradation of tested benzophenones from aqueous matrices, and conditions that allow higher degradation rates to be achieved are presented. Application of nanoparticles such as TiO₂, PbO/TiO₂, and Sb₂O₃/TiO₂ for the photocatalytic degradation of benzophenone-type UV filters was included in this review.     

Sound propagation above a porous road surface with extended reaction by boundary element method

Acoustic impedance of an absorbing interface is easily introduced in boundary element codes provided that a local reaction is assumed. But this assumption is not valid in the case of porous road surface. A two-domain approach was developed for the prediction of sound propagation above a porous layer that takes into account the sound propagation inside the porous material. The porous material is modeled by a homogeneous dissipative fluid medium. An alternative to this time consuming two-domain approach is proposed by using the grazing incidence approximate impedance in the traditional single-domain boundary element method (BEM). It can be checked that this value is numerically consistent with the surface impedance calculated at the interface from the pressure and surface velocity solutions of the two-domain approach. The single-domain BEM introducing this grazing incidence impedance is compared in terms of sound attenuation with analytical solutions and two-domain BEM. The comparison is also performed with the single-domain BEM using the normal incidence impedance, and reveals a much better accuracy for the prediction of sound propagation above a porous interface.     

Some physical properties of KCl+Pb2+ crystals

This paper presents the results of measurements of the mechanical and electrical properties of as-received pure and lead-doped crystals of potassium chloride. The critical resolved shear stress of the crystals obeys the Franks relationship ent/h124431p2u3h5220/10582_2005_Article_BF01587448_TeX2GIFIE1.gif" alt=" $$\tau _A^* \propto \surd c_{Pb^{2 + } } $$ " align="middle" border="0"> in the whole concentration range. In addition, the correlation betweenent/h124431p2u3h5220/xxlarge964.gif" alt="tau" align="BASELINE" BORDER="0"> ₀ and the Vickers hardness numberH was found and the equation is of the forment/h124431p2u3h5220/xxlarge964.gif" alt="tau" align="BASELINE" BORDER="0"> ₀=k (H–H ₀). The solubility of Pb²⁺ in as-received KCl crystals was observed, from measurements of the electrical conductivity, to be low—the successive saturation of the solid solution started already in an environment of 5 mole ppm in agreement with data from the mechanical measurements. The density of cation vacancies and their mobility were represented by the following euqationsent/h124431p2u3h5220/xxlarge956.gif" alt="mgr" align="MIDDLE" BORDER="0"> ₁ T=6·25&times;10⁴ exp(–·75/kT) cm² K/volt. sec,n ₁=6·95&times;10²³ exp(–2·12/kT) vacancies/cm³ The value of 0sd46±0·02 eV was found as a rough estimate of the association energy.The authors wish to express their thanks to Professor Dr. J. Z.Damm and Ing. E.Mariani for their stimulating interest in the present work.