Salivary Metabolome and Soccer Match: Challenges for Understanding Exercise induced Changes.

Saliva samples of seventeen soccer players were analyzed by nuclear magnetic resonance before and after an official match. Two different ways of normalizing data are discussed, using total proteins and total metabolite concentrations. Changes in markers related to energy, hydration status, amino acids and other compounds were found. The limits and advantages of using saliva to define the systemic responses to exercise are examined, both in terms of data normalization and interpretation, and the time that the effect lasts in this biofluid, which is shorter to that commonly observed in blood. The heterogeneous nature and different timing of the exercise developed by players also plays an important role in the metabolic changes that can be measured. Our work focuses mainly on three different aspects: The effect that time sampling has on the observed effect, the type of normalization that is necessary to perform in order to cope with changes in water content, and the metabolic response that can be observed using saliva.

Early metabolic response to acute myocardial ischaemia in patients undergoing elective coronary angioplasty.

Objective: Balloon-induced transient coronary ischaemia represents a model of myocardial ischaemia and reperfusion. We are interested in the very early systemic metabolic response to this event. Methods: Blood samples of patients with stable angina (SA) were collected before and after coronary angioplasty. Serum metabolic profiles were obtained using nuclear magnetic resonance spectroscopy. Univariate and multivariate analyses were used to investigate changes in metabolite concentrations. Results: Thirty-four consecutive patients with SA, undergoing elective coronary angioplasty at Policlinico Umberto I of Rome, were included in this study. Changes in metabolites concentration induced by balloon occlusion in venous and arterial sera were detected. In both serum types, a significant increase in ketone bodies, 2-hydroxybutyrate, glutamine and O-acetylcarnitine concentration is observed, while alanine, lactate, phenylalanine and tyrosine decreased after intervention. Most significant metabolic changes were detected in arterial serum. Conclusions: Our study points out two main global metabolic changes in peripheral blood after balloon-induced coronary ischaemia: ketone bodies increase and lactate decrease. Both could be related to compensation mechanisms finalised to fulfil heart's needs after short period of myocardial ischaemia and probably after reperfusion.

Structural evidence of N6-isopentenyladenosine as a new ligand of farnesyl pyrophosphate synthase.

N6-isopentenyladenosine (i6A), a modified nucleoside belonging to the cytokinin family, has shown in humans many biological actions, including antitumoral effects through the modulation of the farnesyl pyrophosphate synthase (FPPS) activity. To investigate the relationship between i6A and FPPS, we undertook an inverse virtual screening computational target searching, testing i6A on a large panel of 3D protein structures involved in cancer processes. Experimentally, we performed an NMR investigation of i6A in the presence of FPPS protein. Both inverse virtual screening and saturation transfer difference (STD) NMR outcomes provided evidence of the structural interaction between i6A and FPPS, pointing to i6A as a valuable lead compound in the search of new ligands endowed with antitumoral potential and targeting FPPS protein.

Structural features of the C8 antiviral peptide in a membrane-mimicking environment.

C8, a short peptide characterized by three regularly spaced Trp residues, belongs to the membrane-proximal external functional domains of the feline immunodeficiency virus coat protein gp36. It elicits antiviral activity as a result of blocking cell entry and exhibits membranotropic and fusogenic activities. Membrane-proximal external functional domains of virus coat proteins are potential targets in the development of new anti-HIV drugs that overcome the limitations of the current anti-retroviral therapy. In the present work, we studied the conformation of C8 and its interaction with micellar surfaces using circular dichroism, nuclear magnetic resonance and fluorescence spectroscopy. The experimental data were integrated by molecular dynamics simulations in a micelle-water system. Our data provide insight into the environmental conditions related to the presence of the fusogenic peptide C8 on zwitterionic or negatively charged membranes. The membrane charge modulates the conformational features of C8. A zwitterionic membrane surface induces C8 to assume canonical secondary structures, with hydrophobic interactions between the Trp residues and the phospholipid chains of the micelles. A negatively charged membrane surface favors disordered C8 conformations and unspecific superficial interactions, resulting in membrane destabilization.

Omega-3 fatty acids regulate the interaction of the Alzheimer's aß(25-35) peptide with lipid membranes.

Polyunsaturated omega-3 fatty acids are increasingly proposed as dietary supplements able to reduce the risk of development or progression of the Alzheimer's disease (AD). To date, the molecular mechanism through which these lipids act has not been yet univocally identified. In this work, we investigate whether omega-3 fatty acids could interfere with the fate of the Alzheimer-related amyloid peptide by tuning the microstructural and dynamical properties of the neuronal membrane. To this aim, the influence of the omega-3 lipid, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine [22:6(cis)PC] on the biophysical properties of lipid bilayers, and on their interaction with the amyloid peptide fragment Aß(25-35) has been investigated by Electron Spin Resonance (ESR), using spin-labeled phospholipids. The results show that the peptide selectively interacts with bilayers enriched in cholesterol (Chol) and sphingomyelin (SM). [22:6(cis)PC] enhances the Aß(25-35)/membrane interaction, favoring a deeper internalization of the peptide among the lipid acyl chains and, consequently, hindering its pathogenic self-aggregation.

Solvent independent conformational propensities of [1,2,3]triazolyl-bridged parathyroid hormone-related peptide-derived cyclo-nonapeptide analogues.

The recently introduced Cu(I)-catalyzed azide-alkyne 1,3-dipolar Huisgen cycloaddition as a prototypic "click chemistry reaction" presented an opportunity for introducing the 1,4-disubstituted-[1,2,3]triazolyl moiety as a new isostere for peptide bonds in the backbone. Previous study in our lab focused on the synthesis of a model i-to-i+4 side chain-to-side chain 1,4- and 4,1-disubstituted-[1,2,3]triazolyl-bridged cyclo-nonapeptide I (Scheme 1) as analogues of its structurally related helical i-to-i+4 lactam-bridged cyclo-nonapeptide [Lys¹³ (&¹), Asp¹7 (&²)]parathyroid hormone related peptide (PTHrP)(11-19)NH2 (1) a truncated version of the α-helical and potent parathyroid hormone receptor 1 agonist [Lys¹³ (&¹), Asp¹7 (&²)]PTHrP(1-34)NH2, (2,3) N(α) -Ac-Lys-Gly-Lys(&¹)-Ser-Ile-Gln-Asp(&²)-Leu-Arg-NH2]. Systematic [1,2,3]triazolyl-containing bridge structure-conformation relationship studies in hexafluoroacetone/water mixture included incorporation of bridges varied in size and position and orientation of the triazolyl ring within the bridge. These studies revealed that the size of methylene bridge flanking triazolyl moiety is critical to reproduce in the heterodetic cyclo-nonapeptides. The conformational features of the analogues cyclo-nonapeptide in which Lys¹³ and Asp¹7 are bridged by the isosteric lactam. Here, we extend our conformational analysis to dimethyl sulfoxide/water mixture in an effort to characterize inherent conformational properties of the heterodetic cyclopeptides that are solvent independent. Our present study shows that the physicochemical properties of the structure-supporting solvent cannot override the effect of the size of methylene bridge to form helical mimetic structures. Moreover, we prove that [1,2,3]triazolyl ring is not a simple bioisostere of lactam bond, but it affects the secondary structure of the peptide, in relation to its positioning orientation.

Designed glucopeptides mimetics of myelin protein epitopes as synthetic probes for the detection of autoantibodies, biomarkers of multiple sclerosis.

We previously reported that CSF114(Glc) detects diagnostic autoantibodies in multiple sclerosis sera. We report herein a bioinformatic analysis of myelin proteins and CSF114(Glc), which led to the identification of five sequences. These glucopeptides were synthesized and tested in enzymatic assays, showing a common minimal epitope. Starting from that, we designed an optimized sequence, SP077, showing a higher homology with both CSF114(Glc) and the five sequences selected using the bioinformatic approach. SP077 was synthesized and tested on 50 multiple sclerosis patients' sera, and was able to detect higher antibody titers as compared to CSF114(Glc). Finally, the conformational properties of SP077 were studied by NMR spectroscopy and structure calculations. Thus, the immunological activity of SP077 in the recognition of specific autoantibodies in multiple sclerosis patients' sera may be ascribed to both the optimized design of its epitopic region and the superior surface interacting properties of its C-terminal region.

Antifungal peptides at membrane interaction.

Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Furthermore, resistance development and fungistatic rather than fungicidal activities represent limitations of current antifungal therapy. Therefore an urgent need for a new generation of antifungal agents remains. We recently synthesised a set of linear and cyclic peptides characterized by sequences typical of membrane-active antimicrobial peptides (AMP). AMT2, cyclo-AMT2, AMT3 and cyclo-AMT3 (Scheme 1) were tested against different yeast species and exhibited general antifungal activity, with a specificity against Cryptococcus neoformans. To evaluate the role of the membrane cell in the mechanism of antifungal activity, we investigated the conformational behaviour of AMT2, cyclo-AMT2, AMT3 and cyclo-AMT3 in different bio-membrane mimicking systems using a combined approach based on spectroscopy and microscopy techniques. Our data highlight the behaviour of the peptides to interact with the bilayer surface, excluding their ability to destabilize or permeabilize the fungal cell wall. Microbial membrane, indeed, may be an important platform for specific interactions of peptides with specific targets involved in the cell wall synthesis.

Binding of the hemopressin peptide to the cannabinoid CB1 receptor: structural insights.

Hemopressin, a bioactive nonapeptide derived from the α1 chain of hemoglobin, was recently shown to possess selective antagonist activity at the cannabinoid CB(1) receptor [Heimann, A. S., et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 20588-20593]. CB(1) receptor antagonists have been extensively studied for their possible therapeutic use in the treatment of obesity, drug abuse, and heroin addiction. In particular, many compounds acting as CB(1) receptor antagonists have been synthesized and subjected to experiments as possible anti-obesity drugs, but their therapeutic application is still complicated by important side effects. Using circular dichroism and nuclear magnetic resonance spectroscopy, this work reports the conformational analysis of hemopressin and its truncated, biologically active fragment hemopressin(1-6). The binding modes of both hemopressin and hemopressin(1-6) are investigated by molecular docking calculations. Our conformational data indicate that regular turn structures in the central portion of hemopressin and hemopressin(1-6) are critical for an effective interaction with the receptor. The results of molecular docking calculations, indicating similarities and differences in comparison to the most accepted CB(1) pharmacophore model, suggest the possibility of new chemical scaffolds for the design of new CB(1) antagonist lead compounds.

Synthesis of new antifungal peptides selective against Cryptococcus neoformans.

Many drugs are available for the treatment of systemic or superficial mycoses, but only a limited number of them are effective antifungal drugs, devoid of toxic and undesirable side effects. Furthermore, resistance development and fungistatic rather than fungicidal activities represent limitations of current antifungal therapy. Therefore there remains an urgent need for a new generation of antifungal agents. According to a polypharmacological approach, the present work concerns the synthesis and antifungal activity of a set of peptides designed to simultaneously target the fungal cell surface and lanosterol demethylase, a key enzyme involved in ergosterol synthesis. Our peptides include amino acid sequences characteristic of membrane-active antimicrobial peptides (AMP), and due to the presence of His residues, they carry the imidazole ring characteristic of azole compounds. The peptides synthesized by us, were tested against different yeast species, and displayed general antifungal activity, with a therapeutically promising antifungal specificity against Cryptococcus neoformans.

Effect of flavonoids on the Abeta(25-35)-phospholipid bilayers interaction.

Amyloid-beta peptide (Abeta) is the major component of amyloid deposits found in the brain tissue of Alzheimer patients. The tendency of amyloid peptide to form amyloid plaques is known to be related to the features of the plasma membrane. Flavonoids, a group of naturally occurring molecules, exert beneficial properties to human health thanks to their antioxidant property; this property depends on their capacity to interact and permeate the cell membrane lipid bilayer. In the present research we report an Electron Paramagnetic Resonance (EPR) investigation of 2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes interacting with the beta-amyloid fragment Abeta(25-35), in the presence of flavonoids rutin, quercetin, naringin and naringenin. Our results, evidencing a flavonoid-dependent rigidifying effect of the bilayer, may provide the molecular basis to explain the known neuroprotective effect of flavonoid compounds.