Products of Cu(II)-catalyzed oxidation of alpha-synuclein fragments containing M1-D2 and H50 residues in the presence of hydrogen peroxide

Metal-catalyzed oxidation (MCO) of proteins is mainly a site-specific process in which one or a few amino acids at metal-binding sites on the protein are preferentially oxidized. The oxidation of proteins by MCO can lead to oxidation of amino acid residue side chains, cleavage of the peptide bonds and formation of covalent protein-protein cross-linked derivatives. In an attempt to elucidate the products of the copper(II)-catalyzed oxidation of the 29-56, M29-D30-56 and Ac-M29-D30-56 fragments of alpha-synuclein, high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) methods and Cu(II)/hydrogen peroxide as a model oxidizing system were employed. The peptide solution (0.50 mM) was incubated at 37 degrees C for 24 h with metal : peptide : hydrogen peroxide 1 : 1 : 4 molar ratio in phosphate buffer, pH 7.4. Oxidation targets for all studied peptides are the histidine residues coordinated to the metal ions. For the M29-D30-56 and Ac-M29-D30-56 peptides the oxidation of the methionine residue to methionine sulfoxide and sulfone is observed. The cleavage of the peptide bond M29-D30 for the M29-D30-56 peptide was detected as metal binding residues. The fragmentations of the M29-D30-56 peptide near the Lys residues were observed supporting the participation of this (Lys) residue in the coordination of the copper(II) ions.     

Structural characterization of the intra- and inter-repeat copper binding modes within the N-terminal region of "prion related protein" (PrP-rel-2) of zebrafish

The unique biology of prion proteins (PrPs) allied with the public-health risks posed by prion zoonoses, such as various animal neurodegenerations, has focused much attention on the molecular basis of the controls cross-species and on the similarities between PrPs from different species. Given the common feature of PrPs as Cu(2+) binding proteins, it appears relevant to compare the impact of Cu(2+) on the stability constants and structures of "physiological" complexes. After having comprehensively delineated the interaction of Cu(2+) with mammalian and avian PrPs, the stabilities and molecular structures of species generated by Cu(2+) interacting with the irregular repeated domain derived from Danio rerio zebrafish PrP-rel-2 were investigated. Copper complexes with different zebrafish PrP-rel-2 fragments were analyzed by potentiometric and spectroscopic techniques. The data were interpreted as to provide evidence of all investigated repeat units selectively binding Cu(2+) via the His imidazole(s). The structural models obtained from paramagnetic NMR showed an intra- or inter-copper binding according to the number of the His in the sequence. In comparison to the mammalian and avian cases, the enzymatic function referred to SOD-like activity was shown to be rather faint in the fish PrPs cases.     

Covalent binding of sensor phases--a recipe for stable potentials of solid-state ion-selective sensors

In this work, a new concept of the solid-state sensors free from EMF instabilities is proposed. In order to prevent the formation of an aqueous layer underneath the ion-selective membrane, instead of improving the hydrophobicity of the monolayer, the moieties terminated with acrylate groups were incorporated within the redox-active monolayer structure. It allowed to “sew” all phases of the sensor (i.e., the transducer, the intermediate layer and the ion-selective membrane) and to obtain a stable and durable ion-selective sensor. It is shown that newly designed monolayer containing both the ferrocene- and the acrylate-terminated molecules does not affect the working parameters of the electrode, such as selectivity or the slope of the calibration curve, although the EMF drift of the sensor is significantly reduced to 0.2 mV per day.     

Synthesis and structure of [2 x 2] molecular grid copper(II) and nickel(II) complexes with a new polydentate oxime-containing Schiff base ligand

A new polynucleating oxime-containing Schiff base ligand, 2-hydroxyimino- N'-[1-(2-pyridyl)ethylidene]propanohydrazone (H pop), has been synthesized and fully characterized. pH potentiometric, electrospray ionization mass spectrometric, and spectrophotometric studies of complex formation in H 2O/DMSO solution confirmed the preference for polynuclear complexes with 3d metal ions. Single-crystal X-ray diffraction analyses of [Ni 4( pop) 4(HCOO) 4].7H 2O ( 1), [Cu 4( pop-H) 4(HCOOH) 4].H 2O ( 2), and [Cu 4( pop-H) 4(H 2O) 4].9H 2O ( 3) indicated the presence of a [2 x 2] molecular grid structure in all three compounds but distinct configurations of the cores: a head-to-tail ligand arrangement with overall S 4 symmetry of the grid in the Cu (2+) complexes as opposed to a head-to-head ligand arrangement with (noncrystallographic) C 2 grid symmetry for the Ni (2+) complex. A cryomagnetic study of 3 revealed intramolecular ferromagnetic exchange between copper ions in the grid, while in 1, antiferromagnetic interactions between the metal ions were observed.     

3D Raman imaging of systemic endothelial dysfunction in the murine model of metastatic breast cancer

It was recently reported in the murine model of metastatic breast cancer (4T1) that tumor progression and development of metastasis is associated with systemic endothelial dysfunction characterized by impaired nitric oxide (NO) production. Using Raman 3D confocal imaging with the analysis of the individual layers of the vascular wall combined with AFM endothelial surface imaging, we demonstrated that metastasis-induced systemic endothelial dysfunction resulted in distinct chemical changes in the endothelium of the aorta. These changes, manifested as a significant increase in the protein content (18 %) and a slight decrease in the lipid content (4 %), were limited to the endothelium and did not occur in the deeper layers of the vascular wall. The altered lipid to protein ratio in the endothelium, although more pronounced in the fixed vascular wall, was also observed in the freshly isolated unfixed vascular wall samples in the aqueous environment (12 and 7 % change of protein and lipid content, respectively). Our results support the finding that the metastasis induces systemic endothelial dysfunction that may contribute to cancer progression.

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Graphical Abstract Schematic illustration of methodology of sample preparation and measurement

     

Mixed Langmuir monolayers of gramicidin A and fluorinated alcohols

Mixed monolayers of gramicidin A (GA) and three alcohols, differing in the degree of fluorination, namely C18OH, F18OH, and F8H10OH have been investigated by means of: surface manometry (pi-A isotherms) and Brewster angle microscopy (BAM) aiming at finding appropriate molecules for incorporating gramicidin A for a biosensor design. Our results proved that only the semifluorinated alcohol is appropriate material for this purpose since it forms miscible and homogeneous monolayers with GA within the whole concentration range. The experimental results have been supported by the calculations of van der Waals energy profiles using the Insight II program. Both the hydrogenated and perfluorinated alcohols were found to aggregate at higher surface pressures, which exclude their application for gramicidin-based biosensor construction.     

7Li MR measures of blood lithium--correlation with chemical analysis data

Lithium is used in the treatment and prophylaxis of manic-depressive illness. A narrow therapeutic range of lithium (0.4-1.2 meq/l) requires constant monitoring of these levels to avoid neurotoxicity and overdose. It is general practice to measure plasma lithium levels as a guide to monitor the therapy. Efforts to predict the concentration of Li at its active sites in the brain have led to the use of red blood cells (RBCs) because they are similar to neuronal cells. Thus RBC lithium is a very relevant clinical parameter for monitoring therapy and to observe the changes at intracellular levels under varying treatment conditions. A measure of both plasma and RBC lithium may be of significant value to physicians and researchers as lithium profile for RBCs correlate more closely with the brain lithium than plasma lithium. Although methods to measure lithium in blood such as atomic absorption or flame photometry exist, a complete quantitation of both plasma and RBC lithium requires a tedious physical separation of the two components prior to chemical analysis. On the other hand, lithium MR technique, via the use of shift reagents, can provide both plasma and RBC lithium in a single study. Here we have performed a correlation study of lithium results obtained from MR with the vitros dry-slide method on blood samples from rats treated with lithium. The results show a high degree of correlation between the two methods. Additionally, the MR measurements made on dilute samples of blood indicate that small blood samples with lithium concentration in the neighborhood of 0.08 meq/l can be measured with high accuracy and reproducibility needed for clinical purposes.     

Time-resolved Laue diffraction of excited species at atomic resolution: 100 ps single-pulse diffraction of the excited state of the organometallic complex Rh2(μ-PNP)2(PNP)2·BPh4

The polychromatic Laue technique has been applied in 100 ps delay synchrotron pump-probe experiments of the triplet excited state of a Rh(I) dinuclear complex. The observed contraction of the Rh-Rh distance of 0.154 (13) ? is less than predicted by a series of theoretical calculations, a difference attributed to the constraining effect of the crystal lattice.     

Encapsulation of a guest sodium cation by iron(III) tris-(hydroxamate)s

An unprecedented encapsulation of an exogenous sodium ion by iron(III) tris(hydroxamate)s was observed upon crystallization of an iron(III) complex with isonicotinylhydroxamic acid. The sodium cation is bound by bridging coordination of the amide oxygen atoms from two mononuclear iron(III) fac-tris(hydroxamate)s.     

Activity-guided isolation and identification of free radical-scavenging components from various leaf extracts of Sorbus aria (L.) Crantz

Nine phenolics were obtained from the leaves of Sorbus aria (L.) Crantz by activity-directed isolation: isorhamnetin 3-O-β-glucopyranoside (1), astragalin (2), isoquercitrin (3), hyperoside (4), kaempferol 3-O-β-glucopyranoside-7-O-α-rhamnopyranoside (5), quercetin 3-O-β-glucopyranoside-7-O-α-rhamnopyranoside (6), rutin (7), chlorogenic acid (8) and neochlorogenic acid (9). The isolates were identified by spectral methods (UV, (1)H- and (13)C-NMR, COSY, HMQC and HMBC), and their free radical-scavenging activity was tested using the l,l-diphenyl-2-picrylhydrazyl (DPPH) method. The antioxidant potential of the different extracts obtained in the fractionation process was evaluated using the DPPH test in relation to the HPLC contents of the isolates 1-9, total phenolics and total proanthocyanidins. Among the analytes tested, superior activity was expressed by isoquercitrin (3, EC(50)?= 2.76 mg L(-1)) and the ethyl acetate extract (EC(50)?= 2.99 mg L(-1)). Five strongly active isolates 3, 6, 7, 8 and 9 were found to be major components and to be principally responsible for the radical-scavenging activity of S. aria extracts.