Proteomic characterization of donkey milk “caseome”

At present, compared with bovine milk, the characterization of donkey milk caseins is at a relatively early stage progress, and only limited data are related to its genetic polymorphism. In this work, the heterogeneity of donkey caseome was investigated using a proteomic approach, based on one- (PAGE, UTLIEF) and two-dimensional (PAGE → UTLIEF) electrophoresis, stained with either Coomassie Brilliant Blue or specific polyclonal antibodies, and structural MS analysis. These combined methodologies allowed the contemporary identification of donkey α_s1, α_s2, β and κ-CN with their related heterogeneity due to phosphorylation (α_s1, α_s2 and β-CN), glycosylation (κ-CN) and incorrect splicing of RNA in mRNA (deleted forms of α_s1-CN and β-CN). The results achieved showed 11 components for κ-CN, six phosphorylated components for β and α_s1-CN and three main phosphorylated components for α_s2-CN, each accounting for 10, 11 and 12 P/mole. At this regard, for the first time, the primary structure of the expressed protein corresponding to the only available donkey α_s2-CN cDNA sequence was determined. Furthermore β-CN was found in homozygous and heterozygous state for the occurrence of a genetic β-CN variant having a MW value 28 mass units higher than the common β-CN phenotype.     

Electronic and hydrogen bonding effects on the chain-breaking activity of sulfur-containing phenolic antioxidants

A kinetic and thermodynamic investigation of phenols para-substituted with thiyl (SR), sulfinyl (SOR), and sulfonyl (SO(2)R) groups and ortho-substituted with thiyl groups is reported. The effect of the sulfur substituents on the O-H bond dissociation enthalpy values, BDE(O-H), was measured by means of the EPR radical equilibration technique and the reactivity toward peroxyl radicals, k(inh), of these phenolic antioxidants was determined by inhibited autoxidation studies. An inverse correlation between these two parameters was found. A p-SMe substituent decreased the BDE(O-H) value to a lesser extent than a p-OMe group (-3.6 vs -4.4 kcal/mol), whereas the effect of the same groups in an ortho position showed an opposite trend (-0.85 vs -0.2 kcal/mol). The latter result is explained in terms of the different strength of the intramolecular hydrogen bond between the OH proton and the sulfur or oxygen substituents in ortho derivatives. ESI-MS analysis of the products formed by reacting the sulfides with peroxyl radicals from the azoinitiator AIBN revealed the formation of a complex mixture of products, which may play an important role in determining the overall antioxidant activity of the parent compounds.     

Enantioselective synthesis of homocarbocyclic-2′-oxo-3′-azanucleosides

The enantioselective synthesis of homocarbocyclic-2′oxo-3′-azanucleosides has been performed by cycloaddition reaction of the N-glycosyl nitrones with allyl nucleobases. The use of nitrones originated from two different carbohydrates, the N-ribosyl nitrone and the N-mannosyl nitrone, proceeded in a stereocontrolled and predictable manner with a good degree of enantioselectivity, so allowing an easy entry to both enantiomers.     

Temperature‐induced self‐assembly of degalactosylated xyloglucan at low concentration

Xyloglucan is a natural polysaccharide having a cellulose‐like backbone and hydroxyl groups‐rich side‐chains. In its native form the polymer is water‐soluble and forms gel only in presence of selected co‐solutes. When a given fraction of galactosyl residues are removed by enzymatic reaction, the polymer acquires the ability to form a gel in aqueous solution at physiological temperatures, a property of great interest for biomedical/pharmaceutical applications. This work presents data on the effect of a temperature increase on degalactosylated xyloglucan dispersed in water at concentration low enough not to run into macroscopic gelation. Results obtained over a wide interval of length scales show that, on increasing temperature, individual polymer chains and pre‐existing clusters self‐assemble into larger structures. The process implies a structural rearrangement over a few nanometers scale and an increase of dynamics homogeneity. The relation of these findings to coil‐globule transition and phase separation is discussed. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2015 , 53, 1727–1735     

New chiral diphosphoramidite rhodium(I) complexes for asymmetric hydrogenation

Chiral 1,5‐cyclooctadiene rhodium(I) cationic complexes with C₂‐symmetric chelate diphosphoramidite ligands containing (R,R)‐1,2‐diaminocyclohexane as the backbone and two atropoisomeric biaryl units were easily synthesized and fully characterized by multinuclear one‐ and two‐dimensional NMR spectroscopy and elemental analysis. These complexes were used as catalysts in the asymmetric hydrogenation of dimethyl itaconate, methyl 2‐acetamidoacrylate and (Z)‐methyl‐2‐acetamido‐3‐phenylacrylate. The rhodium complexes derived from diphosphoramidite ligands that contain two (R) or (S) BINOL (2,2′‐dihydroxy‐1,1′‐binaphthyl) units proved to be efficient catalysts, giving complete conversion and very good enantioselectivity (up to 88% ee). An uncommon positive H₂ pressure effect on the enantioselectivity was observed in the hydrogenation of dimethyl itaconate catalyzed by Rh‐complex with diphosphoramidite ligand that contains two (S)‐binaphthol moieties. Copyright © 2014 John Wiley & Sons, Ltd.     

Host–Guest Chemistry of Aromatic‐Amide‐Linked Bis‐ and Tris‐Calix[4]pyrroles with Bis‐Carboxylates and Citrate Anion

A small library of polytopic receptors has been synthesized from meso‐p‐ and meso‐m‐aminophenylcalix[4]pyrroles and p‐ or m‐phthaloyl or trimesic chloride. Selected bis‐carboxylates and the citrate anion, which either exhibit altered distribution profiles in cancerous tissues in comparison with healthy tissues or are metabolites of carcinogenic substances (for example, trans,trans‐muconic acid from benzene exposure in humans) were tested as ligands. Varied affinities and binding modes were observed as a function of the number of calix[4]pyrroles and the topology of amide units present in each of the polytopic receptors. The structures of the 1:1 complexes derived by molecular modeling are in excellent agreement with the results of ¹H NMR complexation studies.     

Using high pressure to prepare polymorphs of the Ba2Co(1-x)Zn(x)S3 (0 ≤ x ≤ 1.0) compounds

In this work, high pressure was used as a tool to induce structural transition and prepare metastable polymorphs of ternary sulfides. Structural transformations under high pressure of compounds belonging to the Ba(2)Co(1-x)Zn(x)S(3) (0 ≤ x ≤ 1.0) series were studied using X-ray diffraction and electron microscopy. All members of the Ba(2)Co(1-x)Zn(x)S(3) series show the Ba(2)CoS(3)-type one-dimensional structure, but, after heating under pressure, the Ba(2)CoS(3) compound (x = 0) separates into BaS and the two-dimensional BaCoS(2-δ) (δ ≈ 0), while Ba(2)Co(1-x)Zn(x)S(3) compounds with x ≥ 0.25 maintain their one-dimensional features but rearrange into polymorphs showing the Ba(2)MnS(3)-type structure. All structural transformations can be linked to shortening in interchain metal-metal distances caused by the high pressure, and the role of the zinc in preventing loss of one-dimensionality is discussed.     

Eudragit as controlled release system for anti-inflammatory drugs: A comparison between DSC and dialysis experiments

A comparative study between the release of Ibuprofen (IBU) from Eudragit RS100^® (RS) and RL100^® (RL) nanosuspensions as well as the free drug to a biological model membrane, consisting of dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles (MLV), was carried out by DSC technique. The aim was to assess the suitability of such calorimetric technique to determine the kinetics of drug release from a polymer system, compared with a classical release test by dialysis method. Nanosuspensions were prepared by a modification of the quasi-emulsion solvent diffusion technique (QESD), a particular approach to the general solvent-change method. This kind of system was planned for the ophthalmic release of non-steroidal anti-inflammatory drugs in ocular diseases associated with inflammatory processes (i.e. post-cataract surgery or uveitis). The drug release was monitored by differential scanning calorimetry (DSC), following the effects exerted by IBU on the thermotropic behaviour of DMPC multilamellar vesicles. IBU affects the main transition temperature (T_m) of phospholipid vesicles, causing a shift towards lower values, driven by the drug fraction entering the lipid bilayer. The obtained values have been used as a calibration curve. DSC was performed on suspensions of blank liposomes added to fixed amounts of unloaded and IBU-loaded Eudragit RS100^® and RL100^® nanosuspensions as well as to powdered free drug. The T_m shifts caused by the drug released from the polymer system or by the free drug, during incubation cycles at 37 °C, were compared to the calibration curve in order to obtain the fraction of drug released. The results were also compared with in vitro dialysis release experiments. The suitability of the two different techniques to follow the drug release as well as the differences between the RL and RS polymer systems was compared, confirming the efficacy of DSC for studying the release from polymer nanoparticulate systems. Explanation of the different rate of kinetic release could be due to void liposomes, which represent a better up-taking system than the aqueous solution phase in the dialysis experiments.     

Microcalorimetric study of thermal unfolding of lysozyme in water/glycerol mixtures: an analysis by solvent exchange model

Folded protein stabilization or destabilization induced by cosolvent in mixed aqueous solutions has been studied by differential scanning microcalorimetry and related to difference in preferential solvation of native and denatured states. In particular, the thermal denaturation of a model system formed by lysozyme dissolved in water in the presence of the stabilizing cosolvent glycerol has been considered. Transition temperatures and enthalpies, heat capacity, and standard free energy changes have been determined when applying a two-state denaturation model to microcalorimetric data. Thermodynamic parameters show an unexpected, not linear, trend as a function of solvent composition; in particular, the lysozyme thermodynamic stability shows a maximum centered at water molar fraction of about 0.6. Using a thermodynamic hydration model based on the exchange equilibrium between glycerol and water molecules from the protein solvation layer to the bulk, the contribution of protein-solvent interactions to the unfolding free energy and the changes of this contribution with solvent composition have been derived. The preferential solvation data indicate that lysozyme unfolding involves an increase in the solvation surface, with a small reduction of the protein-preferential hydration. Moreover, the derived changes in the excess solvation numbers at denaturation show that only few solvent molecules are responsible for the variation of lysozyme stability in relation to the solvent composition.     

Copper(I) and copper(II) inhibit Aβ peptides proteolysis by insulin-degrading enzyme differently: implications for metallostasis alteration in Alzheimer's disease

Accumulation of neurotoxic amyloid-β peptide (Aβ) and alteration of metal homeostasis (metallostasis) in the brain are two main factors that have been very often associated with neurodegenerative diseases, such as Alzheimer's disease (AD). Aβ is constantly produced from the amyloidprecursor-protein APP precursor and immediately catabolized under normal conditions, whereas dysmetabolism of Aβ and/or metal ions seems to lead to a pathological deposition. Although insulin-degrading enzyme (IDE) is the main metalloprotease involved in Aβ degradation in the brain being up-regulated in some areas of AD brains, the role of IDE for the onset and development of AD is far from being understood. Moreover, the biomolecular mechanisms involved in the recognition and interaction between IDE and its substrates are still obscure. In spite of the important role of metals (such as copper, aluminum, and zinc), which has brought us to propose a "metal hypothesis of AD", a targeted study of the effect of metallostasis on IDE activity has never been carried out. In this work, we have investigated the role that various metal ions (i.e., Cu(2+), Cu(+), Zn(2+), Ag(+), and Al(3+)) play in modulating the interaction between IDE and two Aβ peptide fragments, namely Aβ(1-16) and Aβ(16-28). It was therefore possible to identify the direct effect that such metal ions have on IDE structure and enzymatic activity without interferences caused by metal-induced substrate modifications. Mass spectrometry and kinetic studies revealed that, among all the metal ions tested, only Cu(2+), Cu(+), and Ag(+) have an inhibitory effect on IDE activity. Moreover, the inhibition of copper(II) is reversed by adding zinc(II), whereas the monovalent cations affect the enzyme activity irreversibly. The molecular basis of their action on the enzyme is also discussed on the basis of computational investigations.