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In this study, non-targeted 1 H NMR fingerprinting was used in combination with multivariate statistical analyses for the classification of Greek currants based on their geographical origins (Aeghion, Nemea, Kalamata, Zante, and Amaliada). As classification techniques, Principal Component Analysis (PCA) and Partial Least Squares Discriminant Analysis (PLS-DA) were carried out. To elucidate different components according to PDO (Protected Designation of Origin), products from Aeghion (Vostizza) were statistically compared with each one of the four other regions. PLS-DA plots ensure that currants from Kalamata, Nemea, Zante, and Amaliada are well classified with respect to the PDO currants, according to differences observed in metabolites. Results suggest that composition differences in carbohydrates, amino, and organic acids of currants are sufficient to discriminate them in correlation to their geographical origin. In conclusion, currants metabolites which mostly contribute to classification performance of such discriminant analysis model present a suitable alternative technique for currants traceability. The study results contribute information to the currants' metabolite fingerprinting by NMR spectroscopy and their geographical origin. PRACTICAL APPLICATION: This study presents an analytical approach for a high nutritional value Greek PDO product, Vostizza currant. A further research and implementation of this method in food industry, can be the key to food fraud incidents. Thus, application of this work opens up posibilities to "farm to table" mission.

Background and Purpose Fibrates are a class of drugs widely used to treat dyslipidaemias. They regulate lipid metabolism and act as PPARα agonists. Clinical trials demonstrate that besides changes in lipid profiles, fibrates decrease the incidence of cardiovascular events, with gemfibrozil exhibiting the most pronounced benefit. This study aims to characterize the effect of gemfibrozil on the activity and function of soluble guanylyl cyclase (sGC), the key mediator of NO signalling. Experimental Approach High-throughput screening of a drug library identified gemfibrozil as a direct sGC activator. Activation of sGC is unique to gemfibrozil and is not shared by other fibrates. Key Results Gemfibrozil activated purified sGC, induced endothelium-independent relaxation of aortic rings and inhibited platelet aggregation. Gemfibrozil-dependent activation was absent when the sGC haem domain was deleted, but was significantly enhanced when sGC haem was lacking or oxidized. Oxidation of sGC haem enhanced the vasoactive and anti-platelet effects of gemfibrozil. Gemfibrozil competed with the haem-independent sGC activators ataciguat and cinaciguat. Computational modelling predicted that gemfibrozil occupies the space of the haem group and interacts with residues crucial for haem stabilization. This is consistent with structure-activity data which revealed an absolute requirement for gemfibrozil's carboxyl group. Conclusions and Implications These data suggest that in addition to altered lipid and lipoprotein state, the cardiovascular preventive benefits of gemfibrozil may derive from direct activation and protection of sGC function. A sGC-directed action may explain the more pronounced cardiovascular benefit of gemfibrozil observed over other fibrates and some of the described side effects of gemfibrozil.

Arkadia (Rnf111), an E3 Ubiquitin (Ub) ligase, amplifies TGF-β signaling responses by targeting for degradation of the negative regulators Smad6/7 and the SnoN/Ski transcriptional repressors when they block the TGF-β effectors Smad2/3. The E3 ligase activity of Arkadia depends on its C-terminal RING-H2 domain that constitutes the docking site for the E2 Ub-conjugating enzyme carrying the activated Ub. We determined the nuclear magnetic resonance solution structure of Arkadia's RING-H2 domain and revealed a (β)ββα fold, fully consistent with the expected "cross-brace" mode of Zn(II)-ligation. In addition, the interaction of the Arkadia RING-H2 domain with its E2 partner enzyme (UbcH5b) was examined through chemical shift perturbation. Proteins 2012. © 2012 Wiley Periodicals, Inc.

Pleiotrophin (PTN) is a heparin-binding growth factor that plays a significant role in tumor growth and angiogenesis. We have previously shown that in order for PTN to induce migration of endothelial cells, binding to both ανβ3 integrin and its receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) is required. In the present study we show that a synthetic peptide corresponding to the last 25 amino acids of the C-terminal region of PTN (PTN112–136) inhibited angiogenesis in the in vivo chicken embryo chorioallantoic membrane (CAM) assay and PTN-induced migration and tube formation of human endothelial cells in vitro. PTN112–136 inhibited binding of PTN to ανβ3 integrin, and as shown by surface plasmon resonance (SPR) measurements, specifically interacted with the specificity loop of the extracellular domain of β3. Moreover, it abolished PTN-induced FAK Y397 phosphorylation, similarly to the effect of a neutralizing ανβ3-selective antibody. PTN112–136 did not affect binding of PTN to RPTPβ/ζ in endothelial cells and induced β3 Y773 phosphorylation and ERK1/2 activation to a similar extent with PTN. This effect was inhibited by down-regulation of RPTPβ/ζ by siRNA or by c-src inhibition, suggesting that PTN112–136 may interact with RPTPβ/ζ. NMR spectroscopy studies showed that PTN112–136 was characterized by conformational flexibility and absence of any element of secondary structure at room temperature, although the biologically active peptide segment 123–132 may adopt a defined structure at lower temperature. Collectively, our data suggest that although PTN112–136 induces some of the signaling pathways triggered by PTN, it inhibits PTN-induced angiogenic activities through inhibition of PTN binding to ανβ3 integrin. J. Cell. Biochem. 112: 1532–1543, 2011. © 2011 Wiley-Liss, Inc.

Human ACE is a central component of the renin-angiotensin system and a major therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE.

Angiotensin-converting enzyme (ACE) is a key molecule of the renin-angiotensin-aldosterone system which is responsible for the control of blood pressure. For over 30 years it has become the target for fighting off hypertension. Many inhibitors of the enzyme have been synthesized and used widely in medicine despite the lack of ACE structure. The last 5 years the crystal structure of ACE separate domains has been revealed, but in order to understand how the enzyme works it is necessary to study its structure in solution. We present here the cloning, overexpression in Escherichia coli, purification and structural study of the Ala(959) to Ser(1066) region (ACE_C) that corresponds to the C-catalytic domain of human somatic angiotensin-I-converting enzyme. ACE_C was purified under denatured conditions and the yield was 6 mg/l of culture. Circular dichroism (CD) spectroscopy indicated that 1,1,1-trifluoroethanol (TFE) is necessary for the correct folding of the protein fragment. The described procedure can be used for the production of an isotopically labelled ACE(959-1066) protein fragment in order to study its structure in solution by NMR spectroscopy.

The anthrax toxin of the bacterium Bacillus anthracis consists of three distinct proteins, one of which is the anthrax lethal factor (LF). LF is a gluzincin Zn-dependent, highly specific metalloprotease with a molecular mass of ∼90 kDa that cleaves most isoforms of the family of mitogen-activated protein kinase kinases (MEKs/MKKs) close to their amino termini, resulting in the inhibition of one or more signaling pathways. Previous studies on the crystal structures of uncomplexed LF and LF complexed with the substrate MEK2 or a MKK-based synthetic peptide provided structure-activity correlations and the basis for the rational design of efficient inhibitors. However, in the crystallographic structures, the substrate peptide was not properly oriented in the active site because of the absence of the catalytic zinc atom. In the current study, docking and molecular dynamics calculations were employed to examine the LF-MEK/MKK interaction along the catalytic channel up to a distance of 20 A from the zinc atom. This residue-specific view of the enzyme-substrate interaction provides valuable information about: (i) the substrate selectivity of LF and its inactivation of MEKs/MKKs (an issue highly important not only to anthrax infection but also to the pathogenesis of cancer), and (ii) the discovery of new, previously unexploited, hot-spots of the LF catalytic channel that are important in the enzyme/substrate binding and interaction.

Conformational analysis of angiotensin I (AI) and II (AII) peptides has been performed through 2D (1) H-NMR spectroscopy in dimethylsulfoxide and 2,2,2-trifluoroethanol/H-2 O. The solution structural models of AI and AII have been determined in dimethylsulfoxide using NOE distance and (3) J (HNHalpha) coupling constants. Finally, the AI family of models resulting from restrained energy minimization (REM) refinement, exhibits pairwise rmsd values for the family ensemble 0.26 +/- 0.13 Angstrom, 1.05 +/- 0.23 Angstrom, for backbone and heavy atoms, respectively, and the distance penalty function is calculated at 0.075 +/- 0.006 Angstrom(2) . Comparable results have been afforded for AII ensemble (rmsd values 0.30 +/- 0.22 Angstrom, 1.38 +/- 0.48 Angstrom for backbone and heavy atoms, respectively; distance penalty function is 0.029 +/- 0.003 Angstrom(2) ). The two peptides demonstrate similar N-terminal and different C-terminal conformation as a consequence of the presence/absence of the His9-Leu10 dipeptide, which plays an important role in the different biological function of the two peptides. Other conformational variations focused on the side-chain orientation of aromatic residues, which constitute a biologically relevant hydrophobic core and whose inter-residue contacts are strong in dimethylsulfoxide and are retained even in mixed organic-aqueous media. Detailed analysis of the peptide structural features attempts to elucidate the conformational role of the C-terminal dipeptide to the different binding affinity of AI and AII towards the AT(1) receptor and sets the basis for understanding the factors that might govern free- or bound-depended AII structural differentiation. European Journal of Biochemistry

Temperature dependencies of the 1H NMR spectra of asymmetric double-decker complexes of DyH(oep)(tpp) (I) and LuH(oep)(tpp) (II), where tpp is tetraphenylporphyrin and oep is octaethylporphyrin, have been analyzed. The kinetic and thermodynamic parameters for intramolecular phenyl ring rotations have been determined. The rotation in (I) is characterized by k(298 K) = 11.4 s−1, ΔH‡ = 48.0±6.0 kJ mol−1, while in (II) the derived parameters are found to be k(298 K) = 14.0 s−1, ΔH‡ = 65.5±7.1 kJ mol−1. The possible reasons for the different characteristics of these complexes are discussed. The structural parameters calculated for I and YbH(oep)(tpp) (III), indicate close similarities between the complexes. Nevertheless, it appears that the principal values of the molar paramagnetic susceptibility tensor (χi) differ significantly in these complexes.

We report the design and synthesis through solid phase 9-flourenylmethoxycarbonyl (Fmoc) chemistry of the two angiotensin-I converting enzyme active sites possessing the general sequence HEMGHX23EAIGDX3. Their zinc-binding properties were monitored in solution through high-resolution 1H-NMR. The obtained data were analyzed in terms of chemical shift differences. The results indicate that zinc binds to the HEMGH and the EAIGD characteristic motifs, and suggest possible coordination modes of zinc in the native enzyme. © 2003 Wiley Periodicals, Inc. Biopolymers 69: 244–252, 2003