Characterization of hydromedusan Ca2+-regulated photoproteins as a tool for measurement of Ca2+concentration

Calcium ion is a ubiquitous intracellular messenger, performing this function in many eukaryotic cells. To understand calcium regulation mechanisms and how disturbances of these mechanisms are associated with disease states, it is necessary to measure calcium inside cells. Ca²⁺-regulated photoproteins have been successfully used for this purpose for many years. Here we report the results of comparative studies on the properties of recombinant aequorin from Aequorea victoria, recombinant obelins from Obelia geniculata and Obelia longissima, recombinant mitrocomin from Mitrocoma cellularia, and recombinant clytin from Clytia gregaria as intracellular calcium indicators in a set of identical in vitro and in vivo experiments. Although photoproteins reveal a high degree of identity of amino acid sequences and spatial structures, and, apparently, have a common mechanism for the bioluminescence reaction, they were found to differ in the Ca²⁺ concentration detection limit, the sensitivity of bioluminescence to Mg²⁺, and the rates of the rise of the luminescence signal with a sudden change of Ca²⁺ concentration. In addition, the bioluminescence activities of Chinese hamster ovary cells expressing wild-type photoproteins also differed. The light signals of cells expressing mitrocomin, for example, slightly exceeded the background, suggesting that mitrocomin may be hardly used to detect intracellular Ca²⁺ without modifications improving its properties. On the basis of experiments on the activation of endogenous P2Y₂ receptor in Chinese hamster ovary cells by ATP, we suggest that wild-type aequorin and obelin from O. longissima are more suitable for calcium detection in cytoplasm, whereas clytin and obelin from O. geniculata can be used for calcium measurement in cell compartments with high Ca²⁺ concentration. Figure

Hydromedusan photoproteins differ in Ca²⁺ concentration detection limit, sensitivity of bioluminescence to Mg²⁺, and rates of rise of luminescence signal with a sudden change of [Ca²⁺] despite a high degree of identity of their amino acid sequences and spatial structures, and, apparently, a common mechanism for the bioluminescence reaction.     

xMAP-based analysis of three most prevalent staphylococcal toxins in Staphylococcus aureus cultures

Detection of staphylococcal toxins presents a great interest for medical diagnostics. Screening of clinical samples for the presence of several types of staphylococcal toxins using traditional methods—biological tests on animals or cell cultures as well as ELISA—is laborious. Multiplex detection methods would simplify testing. We have designed an xMAP-based assay to detect three staphylococcal toxins—enterotoxins A and B (SEA and SEB) and toxic shock syndrome toxin (TSST)—in cultural supernatants obtained from different strains of Staphylococcus aureus. The limits of detection of SEA, SEB, and TSST multiplex detection in S. aureus growth medium were 10, 1,000, and 5 pg/mL, respectively. Fifty-nine samples of S. aureus cultural supernatants were tested with the xMAP assay. The developed assay has proved highly effective detection of the natural toxins in the samples obtained due to bacterial cells cultivation. In prospect, the developed test system can be used in clinical diagnostics and in monitoring of foodstuffs and environmental objects.     

Two molybdenum pentacarbonyl complexes with electroneutral phosphane ligands: [bis(morpholin‐4‐yl)(pentafluoroethyl)phosphane‐κP]pentacarbonylmolybdenum(0) and pentacarbonyl[(pentafluoroethyl)bis(piperidin‐1‐yl)phosphane‐κP]molybdenum(0)

The crystal structures of the title compounds, [Mo{(C₄H₈NO)₂P(C₂F₅)}(CO)₅], (1a), and [Mo{(C₅H₁₀N)₂P(C₂F₅)}(CO)₅], (2a), were determined as part of a larger project that focuses on the synthesis and coordination chemistry of phosphane ligands possessing moderate (electroneutral, i.e. neither electron‐rich nor electron‐deficient) electronic characteristics. Both complexes feature a slightly distorted octahedral geometry at the metal center, due to the electronic and steric repulsions between two of the four equatorial CO groups and the pentafluoroethyl group attached to the phosphane ligand. Bond length and angle data for (1a) and (2a) support the conclusion that the free phosphane ligands are electroneutral. For complex (1a), the Mo—P, Mo—C_ax and Mo—C_eq(ave) bond lengths are 2.5063 (5), 2.018 (2) and 2.048 (2) Å, respectively, and for complex (2a) these values are 2.5274 (5), 2.009 (3) and 2.050 (3) Å, respectively. Geometric data for (1a) and (2a) are compared with similar data reported for analogous Mo(CO)₅ complexes.     

On a Mathematical Problem of Cusped Double-Layered Plates

The present paper is devoted to the system of degenerate partial differential equations arise from the investigation of elastic two layered prismatic shells. Harmonic vibration of cusped double-layered plates is consider. The weak setting of the BVPs in the case of the zero approximation of hierarchical models is considered. (© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim)     

Effect of Botanical Extracts on the Growth and Nutritional Quality of Field-Grown White Head Cabbage (Brassica oleracea var. capitata)

Nutraceuticals and functional foods are gaining more attention amongst consumers interested in nutritious food. The consumption of foodstuffs with a high content of phytochemicals has been proven to provide various health benefits. The application of biostimulants is a potential strategy to fortify cultivated plants with beneficial bioactive compounds. Nevertheless, it has not yet been established whether the proposed higher plants (St. John’s wort, giant goldenrod, common dandelion, red clover, nettle, and valerian) are appropriate for the production of potential bio-products enhancing the nutritional value of white cabbage. Therefore, this research examines the impact of botanical extracts on the growth and nutritional quality of cabbage grown under field conditions. Two extraction methods were used for the production of water-based bio-products, namely: ultrasound-assisted extraction and mechanical homogenisation. Bio-products were applied as foliar sprays to evaluate their impact on total yield, dry weight, photosynthetic pigments, polyphenols, antioxidant activity, vitamin C, nitrates, micro- and macroelements, volatile compounds, fatty acids, sterols, and sugars. Botanical extracts showed different effects on the examined parameters. The best results in terms of physiological and biochemical properties of cabbage were obtained for extracts from common dandelion, valerian, nettle, and giant goldenrod. When enriched with nutrients, vegetables can constitute a valuable component of functional food.     

Enhanced Physical and Mechanical Properties of Nitrile-Butadiene Rubber Composites with N-Cetylpyridinium Bromide-Carbon Black

The physical and mechanical properties of nitrile–butadiene rubber (NBR) composites with N-cetylpyridinium bromide-carbon black (CPB-CB) were investigated. Addition of 5 parts per hundred rubber (phr) of CPB-CB into NBR improved the tensile strength by 124%, vulcanization rate by 41%, shore hardness by 15%, and decreased the volumetric wear by 7% compared to those of the base rubber-CB composite.     

Study of diffusion in erythrocyte suspension using internal magnetic field inhomogeneity

Transport of water and ions through cell membranes plays an important role in cell metabolism. We demonstrate a novel technique to measure water transport dynamics using erythrocyte suspensions as an example. This technique takes advantage of inhomogeneous internal magnetic field created by the magnetic susceptibility contrast between the erythrocytes and plasma. The decay of longitudinal magnetization due to diffusion in this internal field reveals multi-exponential behavior, with one component corresponding to the diffusive exchange of water across erythrocyte membrane. The membrane permeability is obtained from the exchange time constant and is in good agreement with the literature values. As compared to the other methods, this technique does not require strong gradients of magnetic field or contrast agents and, potentially, can be applied in vivo.     

Online hyphenation of size-exclusion chromatography and gas-phase electrophoresis facilitates the characterization of protein aggregates

Gas-phase electrophoresis yields size distributions of polydisperse, aerosolized analytes based on electrophoretic principles. Nanometer-sized, surface-dry, single-charged particles are separated in a high laminar sheath flow of particle-free air and an orthogonal tunable electric field. Additionally, nano Electrospray Gas-Phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) data are particle-number based. Therefore, small particles can be detected next to larger ones without a bias, for example, native proteins next to their aggregates. Analyte transition from the liquid to the gas phase is a method inherent prerequisite. In this context, nonvolatile sample buffers influence results. In the worst case, the (bio-)nanoparticle signal is lost due to an increased baseline and unspecific clustering of nonvolatile components. We present a novel online hyphenation of liquid chromatography and gas-phase electrophoresis, coupling a size-exclusion chromatography (SEC) column to an advanced nES GEMMA. Via this novel approach, it is possible to (i) separate analyte multimers already present in liquid phase from aggregates formed during the nES process, (ii) differentiate liquid phase and spray-induced multimers, and (iii) to remove nonvolatile buffer components online before SEC–nES GEMMA analysis. Due to these findings, SEC–nES GEMMA has the high potential to help to understand aggregation processes in biological buffers adding the benefit of actual size determination for noncovalent assemblies formed in solution. As detection and characterization of protein aggregation in large-scale pharmaceutical production or sizing of noncovalently bound proteins are findings directly related to technologically and biologically relevant situations, we proposed the presented method to be a valuable addition to LC-MS approaches.     

Fundamental Characterization,Photophysics and Photocatalysis of a Base Metal Iron(II)-Cobalt(III) Dyad

A new base metal iron-cobalt dyad has been obtained by connection between a heteroleptic tetra-NHC iron(II) photosensitizer combining a 2,6-bis[3-(2,6-diisopropylphenyl)imidazol-2-ylidene]pyridine with 2,6-bis(3-methyl-imidazol-2-ylidene)-4,4′-bipyridine ligand, and a cobaloxime catalyst. This novel iron(II)-cobalt(III) assembly has been extensively characterized by ground- and excited-state methods like X-ray crystallography, X-ray absorption spectroscopy, (spectro-)electrochemistry, and steady-state and time-resolved optical absorption spectroscopy, with a particular focus on the stability of the molecular assembly in solution and determination of the excited-state landscape. NMR and UV/Vis spectroscopy reveal dissociation of the dyad in acetonitrile at concentrations below 1 mM and high photostability. Transient absorption spectroscopy after excitation into the metal-to-ligand charge transfer absorption band suggests a relaxation cascade originating from hot singlet and triplet MLCT states, leading to the population of the ³MLCT state that exhibits the longest lifetime. Finally, decay into the ground state involves a ³MC state. Attachment of cobaloxime to the iron photosensitizer increases the ³MLCT lifetime at the iron centre. Together with the directing effect of the linker, this potentially makes the dyad more active in photocatalytic proton reduction experiments than the analogous two-component system, consisting of the iron photosensitizer and Co(dmgH)₂(py)Cl. This work thus sheds new light on the functionality of base metal dyads, which are important for more efficient and sustainable future proton reduction systems.