Crystallographic analysis of metal-ion binding to human ubiquitin

The metal-binding ability of human ubiquitin (hUb) towards a selection of biologically relevant metal ions and complexes has been probed. Different techniques have been used to obtain crystals suitable for crystallographic analysis. In the first type of experiments, crystals of hUb have been soaked in solutions containing copper(II) acetate and two metallodrugs, Zeise salt (K[PtCl(3)(η(2)-C(2)H(4))]·H(2)O) and cisplatin (cis-[PtCl(2)(NH(3))(2)]). The Zeise salt is used in a test for hepatitis, whereas cisplatin is one of the most powerful anticancer drugs in clinical use. The Zeise salt readily reacts with hUb crystals to afford an adduct with three platinum residues per protein molecule, Pt(3)-hUb. In contrast, copper(II) acetate and cisplatin were found to be unreactive for contact times up to one hour and to cause degradation of the hUb crystals for longer times. In the second type of experiments, hUb was cocrystallized with a solution of copper(II) or zinc(II) acetate or cisplatin. Zinc(II) acetate gives, at low metal-to-protein molar ratios (8:1), crystals containing one metal ion per three molecules of protein, Zn-hUb(3) (already reported in previous work), whereas at high metal-to-protein ratios (70:1) gives crystals containing three Zn(II) ions per protein molecule, Zn(3)-hUb. In contrast, once again, copper(II) acetate and cisplatin, even at low metal-to-protein ratios, do not give crystalline material. In the soaking experiment, the Zeise anion leads to simultaneous platination of His68, Met1, and Lys6. Present and previous results of cocrystallization experiments performed with Zn(II) and other Group 12 metal ions allow a comprehensive understanding of the metal-ion binding properties of hUb with His68 as the main anchoring site, followed by Met1 and carboxylic groups of Glu16, Glu18, Glu64, Asp21, and Asp32, to be reached. In the case of platinum, Lys6 can also be a binding site. The amount of bound metal ion, with respect to that of the protein, appears to be a relevant parameter influencing crystal packing.     

Soil properties, strontium isotopic signatures and multi-element profiles to authenticate the origin of vegetables from small-scale regions: illustration with early potatoes from southern Italy

We propose a method for the authentication of the origin of vegetables grown under similar weather conditions, in sites less than 10 km distance from the sea and distributed over a rather small scale area (58651 km(2)). We studied how the strontium (Sr) isotopic signature and selected elemental concentrations ([Mn], [Cu], [Zn], [Rb], [Sr] and [Cd]) in early potatoes from three neighbouring administrative regions in the south of Italy were related to the geological substrate (alluvial sediments, volcanic substrates and carbonate rocks) and to selected soil chemical properties influencing the bioavailability of elements in soils (pH, cation exchange capacity and total carbonate content). Through multiple-step multivariate statistics (PLS-DA) we could assign 26 potatoes (including two already commercialised samples) to their respective eight sites of production, corresponding to the first two types of geological substrates. The other 12 potatoes from four sites of production had similar characteristics in terms of the geological substrate (third type) and these soil properties could be grouped together. In this case, more discriminative parameters would be required to allow the differentiation between sites. The validation of our models included external prediction tests with data of potatoes harvested the year before and a study on the robustness of the uncertainties of the measurement results. Annual variations between multi-elemental and Sr isotopic fingerprints were observed in potatoes harvested from soils overlying carbonate rocks, stressing the importance of testing long term variations in authentication studies.     

Panchromatic ruthenium sensitizer based on electron-rich heteroarylvinylene π-conjugated quaterpyridine for dye-sensitized solar cells

The first example of a heteroarylvinylene π-conjugated quaterpyridine Ru(II) sensitizer (N1044) was synthesized and used in dye-sensitized solar cells; the dye has an effective panchromatic absorption band, covering the entire visible spectrum up to the NIR region, and superior electrochemical characteristics (HOMO/LUMO and bandgap energies) with respect to previous representative Ru(II) bi- and quaterpyridine sensitizers. A record IPCE curve ranging from 360 to 920 nm has been measured with a maximum of 65% at 646 nm and still 33% efficiency at 800 nm; this leads to substantially increased photocurrent (19.2 mA cm(-2)) when compared to the prototype N719 Ru(II) sensitizer.     

Development and validation of an HPLC-MS/MS method to quantify clopidogrel acyl glucuronide, clopidogrel acid metabolite, and clopidogrel in plasma samples avoiding analyte back-conversion

A new sensitive and fast quantitative analytical method for the simultaneous determination of clopidogrel, its main metabolite clopidogrel carboxylic acid, and the newly described acyl glucuronide metabolite, in human plasma samples, is presented. The analytical procedures (plasma storage, handling, and extract storage in the autosampler) were optimized in order to avoid back-conversion; a known drawback in measurements of clopidogrel. Clopidogrel acyl glucuronide was confirmed as a major source of back-conversion to the parent drug in the presence of methanol, and thorough stability experiments were carried out to find the most appropriate conditions for an accurate analysis of clopidogrel and the two metabolites. The method was validated by assessing selectivity, sensitivity, linearity, accuracy, and precision for all three analytes, in accordance to Food and Drug Administration guidelines. Spiked quality controls in plasma as well as incurred samples were used to verify back-conversion in the selected conditions, with results meeting European Medicines Agency acceptance criteria (concentrations within 80–120% of the first reading). The method was then applied to a pharmacokinetic study, and for the first time, a pharmacokinetic curve of clopidogrel acyl glucuronide in human plasma is presented. The concentrations ranged up to 1,048.684 ng/mL, with a mean of 470.268 ng/mL, while clopidogrel had a mean C _max of 1.348 ng/mL; these orders of magnitude show how much the back-conversion of this metabolite may influence clopidogrel quantification if it is not properly controlled.     

A semi-Markov process with an inverse Gaussian distribution as sojourn time

Let X_n denote the state of a device after n repairs. We assume that the time between two repairs is the time τ taken by a Wiener process {W(t), t ? 0}, starting from w₀ and with drift μ < 0, to reach c ∈ [0, w₀). After the nth repair, the process takes on either the value X_n?1 + 1 or X_n?1 + 2. The probability that X_n = X_n?1 + j, for j = 1, 2, depends on whether τ ? t₀ (a fixed constant) or τ > t₀. The device is considered to be worn out when X_n ? k, where k ∈ {1, 2, …}. This model is based on the ones proposed by Rishel (1991) [1] and Tseng and Peng (2007) [2]. We obtain an explicit expression for the mean lifetime of the device. Numerical methods are used to illustrate the analytical findings.     

Nonlinear interaction between self- and parametrically excited wind-induced vibrations

Nonlinear Dynamics - The aeroelastic behavior of a planar prismatic visco-elastic structure, subject to a turbulent wind, flowing orthogonally to its plane, is studied in the nonlinear field. The...     

Enantioselective Alternating Olefin-Carbon Monoxide Copolymerization: A new concept for activity and stereoselectivity

Cationic Pd-complexes modified by dicyclohexyl{(R)-1-[(S)-2-(diphenylphosphino)ferrocenyl]ethyl}phosphine ( 1a ) give very active catalytic systems for the regioregular isotactic specific copolymerization of propene with CO. Other alk-1-enes also give stereoregular and regioregular copolymers, even if with lower productivity. The copolymers are isolated as poly(4-alkyl-tetrahydrofuran-2,2,5,5-tetrayl-2-oxy-2-methylenes) B in the solid state and give the isomeric poly(2-alkyl-1-oxopropane-1,3-diyls) A by dissolution in (CF₃)₂CHOH. Solid polymer A (R = Et) is formed back at least partially when the dissolved material is reprecipitated from MeOH. The use of the related (ferrocenyl)diphosphine ligands 1b ? e and 2 as the catalyst modifier shows that the presence of both elements of chirality and of large substituents on the P-atoms of the ligand is necessary to achieve good stereocontrol, and that the large difference in basicity between the two P-atoms is probably the reason for the good catalytic activity.     

Synthesis,structural analysis,and self-assembly of phenylene ethynylene oligomers and their ?F, ?CF3, and ?CH3 substituted derivatives

Oligomers consisting of aromatic building blocks separated by alkynyl units were synthesized by Sonogashira cross-coupling of aryl halides with terminal acetylenes. Strong electron acceptors such as  F and  CF₃ and weak electron donors like  CH₃ were placed as substituents on one of the benzene rings. Acetyl-protected sulfhydryl groups were attached to one end of these molecules to promote their self-organization on gold surfaces. The electron-transport properties of such self-assembled monolayers (SAMs) are highly sensitive to the local order of the molecules in the solid state. Single crystals were analyzed by X-ray diffraction experiments that revealed structural details that could lead to a better understanding of the electron-transport properties. The unsymmetrical substitution of the aromatic rings by electron-active groups in the ortho-, meta-, or para positions resulted in changes of such molecular parameters as bonding and torsion angles and planarity. These parameters, in turn, can affect the angle of the molecular attachment to a gold substrate and the density of the resulting SAMs. Patterned SAMs of some of these molecules and comparison alkane thiols were obtained on gold by microcontact printing or flooding. The SAM thickness was determined by spectroscopic ellipsometry. Surface potential differences between adjacent SAMs or between SAMs and the gold substrate were measured by scanning surface potential microscopy under ambient conditions. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 541–550, 2004     

Solid‐state 13C and 129Xe NMR study of poly(vinyl alcohol) and poly(vinyl alcohol)/lactosilated chitosan gels

Dry and hydrated poly(vinyl alcohol) (PVA) gels with 55% (a‐PVA) and 61% (s‐PVA) syndiotacticity and related PVA/lactyl chitosan (LC) blends have been investigated with ¹²⁹Xe and cross‐polarization/magic‐angle‐spinning ¹³C NMR techniques. Although the dry gels exhibit two broad ¹²⁹Xe resonances in the slow‐to‐intermediate exchange limit, both hydrated gels show three resonances. The corresponding dry blends exhibit two signals, the chemical shifts and line widths of which change with respect to those of pure PVA, whereas one (a‐PVA/LC) or two (s‐PVA/LC) signals appear in the spectra of the hydrated blends. A comparative analysis of the data demonstrates that LC rearranges the domains of the polymeric matrix in both the dry and hydrated blends according to the syndiotacticity of the PVA chains. Information on the molecular motions of the amorphous and swollen polymeric domains in the kilohertz range has been obtained from an analysis of the spin‐lattice relaxation times. These data indicate that the dynamics and arrangement of the PVA chains in the gels are strongly affected by their tacticity and the addition of the copolymer LC. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 3123–3131, 2003     

Os(bpy)2(CO)(enIA)][OTf]2: a novel sulfhydryl-specific metal-ligand complex

The synthesis and physical-chemical characterization of the metal-ligand complex [Os(bpy)2(CO)(enIA)][OTf]2 (where enIA = ethylenediamine iodoacetamide) with a sulfhydryl-specific functional group is described. The UV and visible absorption and luminescence emission, including lifetime and steady-state anisotropy, are reported for the free probe and the probe covalently linked to two test proteins. The spectroscopic properties of the probe are unaffected by chemical modification and subsequent covalent linkage to the proteins. The luminescence lifetime in aqueous buffer is approximately 200 ns and the limiting anisotropy is greater than 0.125, suggesting a potentially useful probe for biophysical investigations.