Unambiguous NMR Structural Determination of (+)-Catechin—Laccase Dimeric Reaction Products as Potential Markers of Grape and Wine Oxidation

(+)-Catechin—laccase oxidation dimeric standards were hemi-synthesized using laccase from Trametes versicolor in a water-ethanol solution at pH 3.6. Eight fractions corresponding to eight potential oxidation dimeric products were detected. The fractions profiles were compared with profiles obtained with two other oxidoreductases: polyphenoloxidase extracted from grapes and laccase from Botrytis cinerea. The profiles were very similar, although some minor differences suggested possible dissimilarities in the reactivity of these enzymes. Five fractions were then isolated and analyzed by 1D and 2D NMR spectroscopy. The addition of traces of cadmium nitrate in the samples solubilized in acetone-d₆ led to fully resolved NMR signals of phenolic protons, allowing the unambiguous structural determination of six reaction products, one of the fractions containing two enantiomers. These products can further be used as oxidation markers to investigate their presence and evolution in wine during winemaking and wine ageing.     

A new approach to measuring the action spectrum for singlet oxygen production by human retinal lipofuscin

The human retinal pigment epithelial (RPE) layer contains a complex mixture of components called lipofuscin; this mixture forms with age and with various genetic disorders such as Stargardt's disease. Its presence may contribute to retinal deterioration via several mechanisms including photochemical processes. In the lipofuscin mixture, both type I and II mechanisms have been identified, with the latter consisting of the generation of singlet oxygen. Several components of that mixture have been identified, most notably a bis-retinoid pyridinium compound called A2E and its derivatives. Photo-oxidative studies on the compound A2E have revealed that its dominant photochemical mechanism is via free radical or type I processes. Because singlet oxygen is an important photooxidative intermediate in tissue, its generation in the RPE may contribute to retinal maculopathies. It is therefore necessary to determine which specific component(s) in the lipofuscin mixture produce singlet oxygen upon excitation with light. This was ascertained by evaluating the action spectrum for singlet oxygen production for the whole lipofuscin mixture using time-resolved spectroscopy. Singlet oxygen was generated by excitation of the sample at different wavelengths while maintaining a constant beam energy, and was directly detected by its phosphorescence decay at 1270 nm using a Ge photodiode. The action spectrum for singlet oxygen sensitization by the organic soluble portion of lipofuscin had an absorption maximum at ca 380 nm, which is to the blue of A2E (maximum at 430 nm). Compounds with a similar absorption maximum eluted in the HPLC earlier than A2E and were detected in human lipofuscin. The concentration of this component apparently increased in concentration in human RPE lipofuscin mixture as a function of age up to 90 years old.     

Structure-Lipophilicity Relationships of Neutral and Protonated β-Blockers,Part I,Intra- and Intermolecular Effects in Isotropic Solvent Systems

The objectives of this study were to validate new experimental techniques used to measure the log P of protonated drugs, and to investigate the inter- and intramolecular forces influencing the partitioning behavior of β-blockers in isotropic biphasic solvent systems. The lipophilicity parameters of a number of β-blockers were measured by two-phase titration, centrifugal partition chromatography (CPC), and cyclic voltammetry (CV) in one or more of the following solvent systems: octanol/water, 1,2-dichloroethane/water, and dibutyl ether/water. CV proved to be a promising technique for measuring the lipophilicity of protonated β-blockers. Derived parameters such as Δlog P (difference between log P in two different solvent systems, a parameter valid for a given solute in a given electrical form) and diff (difference between log P of two different electrical forms of a given solute, in the same system) yielded insights into inter- and intramolecular interactions characteristic of β-blockers. The relevance of these parameters in structure-permeation relationships is explored.     

Calcium ions tune the zinc-sequestering properties and antimicrobial activity of human S100A12

Human S100A12 is a host-defense protein expressed and released by neutrophils that contributes to innate immunity. Apo S100A12 is a 21 kDa antiparallel homodimer that harbors two Ca(ii)-binding EF-hand domains per subunit and exhibits two His₃Asp motifs for chelating transition metal ions at the homodimer interface. In this work, we present results from metal-binding studies and microbiology assays designed to ascertain whether Ca(ii) ions modulate the Zn(ii)-binding properties of S100A12 and further evaluate the antimicrobial properties of this protein. Our metal-depletion studies reveal that Ca(ii) ions enhance the ability of S100A12 to sequester Zn(ii) from microbial growth media. We report that human S100A12 has antifungal activity against Candida albicans, C. krusei, C. glabrata and C. tropicalis, all of which cause human disease. This antifungal activity is Ca(ii)-dependent and requires the His₃Asp metal-binding sites. We expand upon prior studies of the antibacterial activity of S100A12 and report Ca(ii)-dependent and strain-selective behavior. S100A12 exhibits in vitro growth inhibitory activity against Listeria monocytogenes. In contrast, S100A12 has negligible effect on the growth of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1. Loss of functional ZnuABC, a high-affinity Zn(ii) import system, increases the susceptibility of E. coli and P. aeruginosa to S100A12, indicating that S100A12 deprives these mutant strains of Zn(ii). To evaluate the Zn(ii)-binding sites of S100A12 in solution, we present studies using Co(ii) as a spectroscopic probe and chromophoric small-molecule chelators in Zn(ii) competition titrations. We confirm that S100A12 binds Zn(ii) with a 2 : 1 stoichiometry, and our data indicate sub-nanomolar affinity binding. Taken together, these data support a model whereby S100A12 uses Ca(ii) ions to tune its Zn(ii)-chelating properties and antimicrobial activity.     

Hydrogenolysis of hexane on supported rhodium catalysts

Hexane was reacted in mixtures with excess hydrogen on 5% Rh on different supports: Al₂O₃ and SiO₂, the latter catalyst in two states: after reduction at 603 K (LT) and after a prereduction at 1253 K (HT). The main reaction was hydrogenolysis. The catalysts were characterized with the fragment composition at different temperatures and hydrogen pressures. Two surface states could be distinguished: one with more hydrogen favored single rupture, the other with less hydrogen preferred multiple fragmentation. The transition between these states could be rather abrupt, as the surface hydrogen availability changed. The tendency to produce multiple fragments increased in the order Rh/Al₂O₃< Rh/SiO₂-LT < Rh/SiO₂-HT.     

Near-Surface Structure of Plasma Polymer Films Affects Surface Behavior in Water and its Interaction with Proteins

Using low pressure plasma polymerization, nano-scaled oxygen-rich plasma polymer films (CO) were deposited onto pristine silicon wafers as well as on nitrogen-containing plasma polymer (CN) model surfaces. We investigate the influence of the nature of the substrate as well as a potential sub-surface effect emerging from the buried CO/CN interface, just nanometers below the surface. X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry revealed two important phenomena that occurred during the deposition of the terminal CO layer: (1) a strong degree of oxidation, already for 1 nm nominal thickness, and (2) a gradual transition in chemical composition between the two layers, clearly indicating that effectively a vertical chemical gradient results, even when a two-step coating process was applied. Such terminal gradient film structures were used to study film stability in aqueous environments. Molecular rearrangements were scrutinized in the top-surface in contact with water and we found that the top-surface chemistry and wetting properties of the oxygen-rich termination layer matched those of thick CO reference coatings. Nevertheless, the adsorption of green fluorescent protein (GFP) was observed to be sensitive to the CO terminal layer thickness. Namely, an enhanced protein adsorption was observed for 1–2 nm thick CO layers on CN, whereas a significantly reduced protein adsorption was seen on ≥?3 nm thick CO terminal layers. We conclude that both, surface and sub-surface conditions significantly affect protein adsorption as opposed to the traditional consideration of surface properties alone.     

Optimization of a nano‐enzymatic reactor for on‐line tryptic digestion of polypeptide conjugates by capillary electrophoresis

This work aims at studying the optimization of an on‐line capillary electrophoresis (CE)‐based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on‐line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.     

Search for the decay B+ --> K+nunu

We search for the rare flavor-changing neutral-current decay B(+)--> K(+)nunu in a data sample of 82 fb(-1) collected with the BABAR detector at the PEP-II B-factory. Signal events are selected by examining the properties of the system recoiling against either a reconstructed hadronic or semileptonic charged-B decay. Using these two independent samples we obtain a combined limit of B(B(+)-->K(+)nunu ) < 5.2 x 10(-5) at the 90% confidence level. In addition, by selecting for pions rather than kaons, we obtain a limit of B(B+-->pi(+)nunu) < 1.0 x 10(-4) using only the hadronic B reconstruction method.