Claudia Bonechi

Effect of trans-resveratrol on induced oxidative stress in human sperm and in rat germinal cells

Resveratrol is a phytoalexin with antioxidant properties. We evaluated resveratrol toxicity in swim-up selected human sperm and in rat spermatocytes and spermatids separated by the STAPUT method. Resveratrol antioxidant activity was tested against lipid peroxidation (LPO) induced by tert-butyl hydroperoxide. LPO was evaluated using the C11-BODIPY(581/591) probe and transmission electron microscopy in samples incubated with and without resveratrol. LD50 for human sperm and rat spermatids was 50 μM; spermatocytes were more sensitive to resveratrol cytotoxicity. Sperm motility increased progressively at 30 μM, 15 μM and 6 μM. 15 μM resveratrol acts against LPO, preserving sperm chromatin and plasma membranes. LPO were more marked in spermatocytes than in spermatids and the effect of resveratrol was more evident in spermatocytes. In this study, the scavenger properties of resveratrol were demonstrated in vitro in human sperm and rat germ cells, thus resveratrol could be added to the media used in assisted reproduction techniques and cryopreservation when oxidative stress is exacerbated.

Using Liposomes as Carriers for Polyphenolic Compounds: The Case of Trans-Resveratrol

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenol found in various plants, especially in the skin of red grapes. The effect of resveratrol on human health is the topic of numerous studies. In fact this molecule has shown anti-cancer, anti-inflammatory, blood-sugar-lowering ability and beneficial cardiovascular effects. However, for many polyphenol compounds of natural origin bioavailability is limited by low solubility in biological fluids, as well as by rapid metabolization in vivo. Therefore, appropriate carriers are required to obtain efficient therapeutics along with low administration doses. Liposomes are excellent candidates for drug delivery purposes, due to their biocompatibility, wide choice of physico-chemical properties and easy preparation. In this paper liposome formulations made by a saturated phosphatidyl-choline (DPPC) and cholesterol (or its positively charged derivative DC-CHOL) were chosen to optimize the loading of a rigid hydrophobic molecule such as resveratrol. Plain and resveratrol loaded liposomes were characterized for size, surface charge and structural details by complementary techniques, i.e. Dynamic Light Scattering (DLS), Zeta potential and Small Angle X-ray Scattering (SAXS). Nuclear and Electron Spin magnetic resonances (NMR and ESR, respectively) were also used to gain information at the molecular scale. The obtained results allowed to give an account of loaded liposomes in which resveratrol interacted with the bilayer, being more deeply inserted in cationic liposomes than in zwitterionic liposomes. Relevant properties such as the mean size and the presence of oligolamellar structures were influenced by the loading of RESV guest molecules. The toxicity of all these systems was tested on stabilized cell lines (mouse fibroblast NIH-3T3 and human astrocytes U373-MG), showing that cell viability was not affected by the administration of liposomial resveratrol.

Effect of quercetin, rutin, naringenin and epicatechin on lipid peroxidation induced in human sperm.

Quercetin, rutin, naringenin, epicatechin are flavonoids with diverse properties, including antioxidant potential. We evaluated, in vitro, the cytotoxicity of these flavonoids (20, 30, 50, 100, 200, 400 μM) in swim-up selected human sperm. Antioxidant activity was tested against tert-butylhydroperoxide induced lipid peroxidation using a C11-BODIPY(581/591) probe and transmission electron microscopy. A significant concentration-dependent effect on sperm viability (P<0.001) and motility (P<0.001) was observed. Lipid peroxidation was decreased in samples treated with 30 μM quercetin (P<0.01) and 30 μM rutin (P<0.05) versus samples incubated with tert-butylhydroperoxide alone. Naringenin (50-100 μM) showed a low protective effect and epicatechin (200 μM) was not efficacious. Transmission electron microscopy analysis confirmed the protective action of rutin and in particular quercetin on damages induced by lipid peroxidation. These results underlined the antioxidant properties of quercetin and rutin. A possible role of these compounds in the supplementation of media used during semen handling warrants attention and further studies.

Solution structure of hyaluronic acid oligomers by experimental and theoretical NMR, and molecular dynamics simulation.

The conformational properties of hyaluronic acid (HA) oligomers in aqueous solution were investigated by combining high‐resolution NMR experimental results, theoretical simulation of NMR two‐dimensional (2D) spectra by Complete Relaxation Matrix Analysis (CORMA), and molecular dynamics calculations. New experimental findings recorded for the tetra‐ and hexasaccharides enabled the stiffness of the HA and its viscoelastic properties to be interpreted. In particular, rotating frame nuclear Overhauser effect spectroscopy spectra provided new information about the arrangement of the glycosidic linkage. From 13C NMR relaxation the rotational correlation time (τc) were determined. The τc were employed in the calculation of geometrical constraints, by using the MARDIGRAS algorithm. Restrained simulated annealing and 1 ns of unrestrained molecular dynamic simulations were performed on the hexasaccharide in a box of 1215 water molecules.

A PVA/PVP hydrogel for human lens substitution: Synthesis, rheological characterization, and in vitro biocompatibility

To overcome opacification and absence of accommodation of human lens substitutes a new poly(vinyl alcohol) (PVA)/poly(N-vinyl-2- pyrrolidinone) (PVP) based hydrogel (PPS31075) was realised. The Infrared Spectroscopy and the mechanical spectra confirmed the successful occurrence of crosslinking reaction. The rheological analysis pointed out a behavior comparable with that of young human lens in terms of complex shear modulus and accommodation capability. Further analysis in terms of optical properties, water content measurements, diffusion coefficient, cytotoxicity, and human capsular cell adhesion confirmed the applicability of such a hydrogel as potential human lens substitute.

Interaction of Quercetin and Its Conjugate Quercetin 3-O-β-D-Glucopyranoside with Albumin as Determined by NMR Relaxation Data

NMR methodology has been developed in order to study phytochemical-macromolecular receptor interactions. This approach is based on the analysis of proton selective spin-lattice relaxation rate enhancements of the ligand in the presence of the macromolecule, to calculate an affinity index, [ A] L (T) , related to the strength of the interaction process. This index has been modified by normalization to the relaxation rate of the free ligand, in order to take into account the effects of motional anisotropies and different proton densities. The normalized affinity index, [ A N ] L (T) , isolates the contribution due to a decrease in the ligand dynamics caused by the binding with the protein. This methodology has been applied to the interaction between two flavonoids (quercetin, 1, and quercetin 3-O-beta-D-glucopyranoside, 2) and bovine serum albumin (BSA). The calculated values of the affinity indexes and thermodynamic equilibrium constants suggested a much stronger capacity of 1 to interact with BSA when compared with its glucosylated derivative, 2.

Modelling energy fluxes in remote wetland ecosystems with the help of remote sensing

The study of the material and energy fluxes that characterise an ecosystem is fundamental to understanding the behaviour of that system and its resiliency in the face of changing local and global conditions. In the present research, an extended energy balance model was used to examine the fluxes that characterise the energy exchange between a large wetland area and its environment. For each flux term, a number of modelling approaches was compared. Modelling was complemented through the use of irradiance measurements from satellite data. The model output shows the daily and season variations in each energy term, as well as a comparison of the modelling approaches. The final output of the model is validated with the measured hourly variation in the enthalpy (water temperature) of the lagoon. A comparison is made of the relative importance of each energy term in the overall balance of the system. This physical based model is being used to examine possible modifications of local and global environments to the wetland energy balance. The model is also being combined with fauna models to examine the relation between climate and population characteristics (habitat). The present work is directed towards the study of a subtropical lagoon in the internationally important wetland, the Esteros del Ibera in Argentina.

In vivo 13C-NMR and modelling study of metabolic yield response to ethanol stress in a wild-type strain of Saccharomyces cerevisiae

In this paper the combined use of in vivo 13C‐nuclear magnetic resonance spectroscopy and mathematical modelling allowed the analysis of the response to ethanol stress in a wild‐type strain of Saccharomyces cerevisiae, in terms of a reduced metabolic activity. The model developed succeeded in describing and interpreting the effects of increasing concentrations of exogenous ethanol. In particular, the ratio between the kinetic constants associated with ethanol production and glucose consumption gave the estimation of the metabolic yield of the processes in perfect agreement with experimental results.

Nuclear relaxation studies in ligand-macromolecule affinity index determinations

Abstract An ‘affinity index’, representing the global affinity between a ligand and a biomacromolecular receptor, is proposed. It is determined as the slope of the linear relations between ΔR1SE and the receptor concentration and has the advantage of providing a measure of the ligand-biomacromolecule global affinity which is independent of the number of interaction sites. The method was applied to the calculation of the lamotrigine-albumin affinity index.

Study of bradykinin conformation in the presence of model membrane by Nuclear Magnetic Resonance and molecular modelling

The conformation of bradykinin (BK), Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9, was investigated by Nuclear Magnetic Resonance (NMR) spectroscopy and Monte Carlo simulation in two different media, i.e. in pure aqueous solution and in the presence of phospholipid vesicles. Monolamellar liposomes are a good model for biological membranes and mimic the environment experienced by bradykinin when interacting with G-protein coupled receptors (GPCRs). The NMR spectra showed that lipid bilayers induced a secondary structure in the otherwise inherently flexible peptide. The results of ensemble calculations revealed conformational changes occurring rapidly on the NMR time scale and allowed for the identification of different families of conformations that were averaged to reproduce the NMR observables. These structural results supported the hypothesis of the central role played by the peptide C-terminal domain in biological environments, and provided an explanation for the different biological behaviours observed for bradykinin