Anastassios N. Troganis

Phytochemicals in olive-leaf extracts and their antiproliferative activity against cancer and endothelial cells.

Olive oil compounds is a dynamic research area because Mediterranean diet has been shown to protect against cardiovascular disease and cancer. Olive leaves, an easily available natural material of low cost, share possibly a similar wealth of health benefiting bioactive phytochemicals. In this work, we investigated the antioxidant potency and antiproliferative activity against cancer and endothelial cells of water and methanol olive leaves extracts and analyzed their content in phytochemicals using LC-MS and LC-UV-SPE-NMR hyphenated techniques. Olive-leaf crude extracts were found to inhibit cell proliferation of human breast adenocarcinoma (MCF-7), human urinary bladder carcinoma (T-24) and bovine brain capillary endothelial (BBCE). The dominant compound of the extracts was oleuropein; phenols and flavonoids were also identified. These phytochemicals demonstrated strong antioxidant potency and inhibited cancer and endothelial cell proliferation at low micromolar concentrations, which is significant considering their high abundance in fruits and vegetables. The antiproliferative activity of crude extracts and phytochemicals against the cell lines used in this study is demonstrated for the first time.

Rapid and novel discrimination and quantification of oleanolic and ursolic acids in complex plant extracts using two-dimensional nuclear magnetic resonance spectroscopy-comparison with HPLC methods.

A novel strategy for NMR analysis of mixtures of oleanolic and ursolic acids that occur in natural products is described. These important phytochemicals have similar structure and their discrimination and quantification is rather difficult. We report herein the combined use of proton-carbon heteronuclear single-quantum coherence ((1)H-(13)C HSQC) and proton-carbon heteronuclear multiple-bond correlation ((1)H-(13)C HMBC) NMR spectroscopy, in the identification and quantitation of oleanolic acid (OA) and ursolic acid (UA)in plant extracts of the Lamiaceae and Oleaceae family. The combination of (1)H-(13)C HSQC and (1)H-(13)C HMBC techniques allows the connection of the proton and carbon-13 spins across the molecular backbone resulting in the identification and, thus, discrimination of oleanolic and ursolic acid without resorting to physicochemical separation of the components. The quantitative results provided by 2D (1)H-(13)C HSQC NMR data were obtained within a short period of time ( approximately 14min) and are in excellent agreement with those obtained by HPLC, which support the efficiency of the suggested methodology.

Structure and function of the myelin proteins : Current status and perspectives in relation to multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) characterized by demyelination and loss of neurological function, local macrophage infiltrate and neuroantigen-specific CD4(+)T cells. MS arises from complex interactions between genetic, immunological, infective and biochemical mechanisms. Although the circumstances of MS etiology remain hypothetical, one persistent theme involves immune system recognition of myelin-specific antigens derived from myelin basic protein, the most abundant extrinsic myelin membrane protein, and/or another equally suitable myelin protein or lipid. Knowledge of the biochemical and physico-chemical properties of myelin proteins and lipids, particularly their composition, organization, structure and accessibility with respect to the compacted myelin multilayers, becomes central to understanding how and why myelin-specific antigens become selected during the development of MS. This review focuses on the current understanding of the molecular basis of MS with emphasis: (i) on the physical-chemical properties, organization, morphology, and accessibility of the proteins and lipids within the myelin multilayers; (ii) on the structure-function relationships and characterization of the myelin proteins relevant to the manifestation and evolution of MS; (iii) on conformational relationships between myelin epitopes which might become selected during the development of MS; (iv) on the structure of MHC/HLA in complex with MBP peptides as well as with TCR, which is crucial to the understanding of the pathogenesis of MS with the ultimate goal of designed antigen-specific treatments.

1H NMR determination of hypericin and pseudohypericin in complex natural mixtures by the use of strongly deshielded OH groups.

The (1)H NMR spectra of the commercially available compounds hypericin and its derivative pseudohypericin in CD(3)OH solutions indicate significantly deshielded signals in the region of 14-15 ppm. These resonances are attributed to the peri hydroxyl protons OH(6), OH(8) and OH(1), OH(13) of hypericins which participate in a strong six-membered ring intramolecular hydrogen bond with CO(7) and CO(14), respectively, and therefore, they are strongly deshielded. In the present work, we demonstrate that one-dimensional (1)H NMR spectra of hypericin and pseudohypericin, in Hypericum perforatum extracts show important differences in the chemical shifts of the hydroxyl groups with excellent resolution in the region of 14-15 ppm. The facile identification and quantification of hypericin and its derivative compound pseudohypericin was achieved, without prior HPLC separation, for two H. perforatum extracts from Greek cultivars and two commercial extracts: a dietary supplement, and an antidepressant medicine. The results were compared with those obtained from UV-vis and LC/MS measurements.

Prediction of cis/trans isomerization using feature selection and support vector machines

In protein structures the peptide bond is found to be in trans conformation in the majority of the cases. Only a small fraction of peptide bonds in proteins is reported to be in cis conformation. Most of these instances (>90%) occur when the peptide bond is an imide (X-Pro) rather than an amide bond (X-nonPro). Due to the implication of cis/trans isomerization in many biologically significant processes, the accurate prediction of the peptide bond conformation is of high interest. In this study, we evaluate the effect of a wide range of features, towards the reliable prediction of both proline and non-proline cis/trans isomerization. We use evolutionary profiles, secondary structure information, real-valued solvent accessibility predictions for each amino acid and the physicochemical properties of the surrounding residues. We also explore the predictive impact of a modified feature vector, which consists of condensed position-specific scoring matrices (PSSMX), secondary structure and solvent accessibility. The best discriminating ability is achieved using the first feature vector combined with a wrapper feature selection algorithm and a support vector machine (SVM). The proposed method results in 70% accuracy, 75% sensitivity and 71% positive predictive value (PPV) in the prediction of the peptide bond conformation between any two amino acids. The output of the feature selection stage is investigated in order to identify discriminatory features as well as the contribution of each neighboring residue in the formation of the peptide bond, thus, advancing our knowledge towards cis/trans isomerization.

On the Structural Basis of the Hypertensive Properties of Angiotensin II: A Solved Mystery or a Controversial Issue?

Angiotensin II (AII), Asp1-Arg2-Val3-Tyr4-Ile5-His6-Pro7-Phe8, the primary active hormone of the Renin-Angiotensin-System (RAS), is a major vasoconstrictor implicated in the cause of hypertension. To unravel the question of the biologically active conformation(s) of this flexible peptide hormone and to better understand the stereoelectronic requirements that lead to the molecular basis of hypertension, we will analyze research efforts in the identification of pharmacophoric groups of AII and three general approaches for structural characterisation: the free peptide-ligand approach, the receptor based approach, and approaches that target the peptide-receptor complex. The free peptide-ligand based approach can be further categorized to: (a) conformational analysis of AII and linear peptide analogues in aqueous solution; (b) the use of solvents of medium dielectric constants; (c) conformationally restricted analogues, with emphasis to cyclic analogues; (d) the use of receptor-simulating environments, and (e) non-peptide mimetics. The receptor and peptide-receptor based approaches can be categorised to: (a) The use of monoclonal antibodies and (b) the generic description of AII receptor sites through homology modelling and mutagenesis studies. These investigations, with particular emphasis to recent developments, have greatly assisted in the identification of pharmacophoric groups for receptor activation and the development of several models of AII-receptor complexes.

14N NMR relaxation times of several protein amino acids in aqueous solution—comparison with 17O NMR data and estimation of the relative hydration numbers in the cationic and zwitterionic forms

The 14N nuclear magnetic resonance (NMR) linewidths of the alpha-amino groups of several protein amino acids were measured in aqueous solution, with and without composite proton decoupling, to estimate the effect of proton exchange and molecular weight on the linewidths. It is shown that, contrary to earlier claims, the increase in the linewidth at low pH is not exclusively due to the effect of proton exchange broadening. The 14N linewidths, under composite proton decoupling, increase with the bulk of the amino acid, and increase at low pH. Statistical treatment of the experimental 14N and literature 17O NMR data was performed assuming two models: (i) an isotropic molecular reorientation of a rigid sphere in a medium of viscosity eta, (ii) a stochastic diffusion of the amino and carboxyl groups comprising contributions from internal (tauint) and overall (taumol) motions. Assuming a single correlation time from overall molecular reorientation (taumol), then, a linear correlation was found between the linewidths and the molecular weights of the protein amino acids at the pH values 0.5 and 6.0, which are characteristic of the cationic and zwitterionic forms, respectively. The slopes of the straight-lines were found to be dependent of pH for 14N, contrary to the 17O linear correlations whose slopes were found to be independent of pH. Assuming effective correlation times of the amino and carboxyl groups, which comprise contributions from the internal (tauint) and overall (taumol) motions, then, a significant improvement of the statistics of the regression analysis was observed. The 14N relaxation data, in conjunction with 17O NMR linewidths, can be interpreted by assuming that the 14N quadrupole coupling constants (NQCCs) are influenced by the protonation state of the carboxyl group, the 17O NQCCs remain constant, and the cationic form of the amino acids is hydrated by an excess of 1-3 molecules of water relative to the zwitterionic state.

Structural elucidation of Leuprolide and its analogues in solution: insight into their bioactive conformation

Leuprolide [dLeu6, NHEt10]GnRH, a potent gonadotropin-releasing hormone (GnRH) agonist, is used in a wide variety of hormone-related diseases like cancer and endometriosis. In this report, the conformational behaviour of Leuprolide and its linear synthetic analogues, namely [Tyr5(OMe), dLeu6, Aze9, NHEt10]GnRH (1) and [Tyr5(OMe), dLeu6, NHEt10]GnRH (2) have been studied in DMSO and H2O solutions by means of 2D nuclear magnetic resonance (NMR) experiments and detailed molecular dynamics (MD) simulations. The aim was to identify the conformational requirements of GnRH analogues for agonistic activity. This approach is of value as no crystallographic data are available for the GnRH receptor (G protein-coupled receptor, GPCR). The NOE data indicate the existence of a β-turn type I in the 2–5 segments of Leuprolide and its linear analogues in the case of using DMSO-d6 as solvent, whereas a β-turn type II in the 3–6 segments is indicated using D2O as solvent. The final structures fulfil the conformational requirements that are known, in the literature, to play a significant role in receptor recognition and activation. Finally, the linear analogues (1) and (2) are biologically active when tested against the human breast cancer cell line, MCF-7.

NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY: BASIC PRINCIPLES AND PHENOMENA, AND THEIR APPLICATIONS TO CHEMISTRY, BIOLOGY AND MEDICINE

Nuclear Magnetic Resonance (NMR) spectroscopy has made a tremendous impact in many areas of chemistry, biology and medicine. In this report a student-oriented approach is presented, which enhances the ability of students to comprehend the basic concepts of NMR spectroscopy and the NMR spectra of various nuclei. The origin of chemical shifts, coupling constants, spin relaxation and the Nuclear Overhauser Effect (NOE) will be discussed and their relation to molecular structure will be provided. A wide range of applications of NMR spectroscopy is presented, including exchange phenomena, the identification and structural studies of complex biomolecules, such as proteins, applications to food analysis, clinical studies, NMR as a microscope and magnetic tomography. [Chem. Educ. Res. Pract. Eur., 2002, 3, 229-252]

Modes of association of concanavalin A with α-d-glycosides

Abstract Complexes of Con A with α- d -glycosides were studied using 11 H-NMR, ESR and fluorescence methods. Correlation times,τ c , for the interaction of the aglycon protons with the manganese ion, present at the S1 site of the protein, were calculated from T 1 measurements at two frequencies. The protons of aromatic aglycons have τ c values comparable to the rotational correlation time of the protein molecule, whereas those of non-aromatic aglycons have τ c S 10 to 100 times lower. The correlation times were combined with the experimentally acquired paramagnetic contributions to proton relaxation due to the presence of the manganese ion to yield manganese-proton distances. These distances show that aromatic aglycons bave additional favorable contacts with the protein which stabilize the lectin-saccharide interaction. The results are compared to the crystal structure of the methyl α- d -glycopyranoside complex with Con A and to models earlier proposed for the binding of monosaccharides to Con A.