Lutein, zeaxanthin and astaxanthin protect against DNA damage in SK-N-SH human neuroblastoma cells induced by reactive nitrogen species

The purpose of this study was to evaluate the ability of the predominant carotenoids (lutein and zeaxanthin) of the macular pigment of the human retina, to protect SK-N-SH human neuroblastoma cells against DNA damage induced by different RNOS donors. Although astaxanthin has never been isolated from the human eye, it was included in this study because its structure is very close to that of lutein and zeaxanthin and because it affords protection from UV-light. DNA damage was induced by GSNO-MEE, a nitric oxide donor, by Na(2)N(2)O(3), a nitroxyl anion donor and by SIN-1, a peroxynitrite-generating agent. DNA damage was assessed using the comet assay, a rapid and sensitive single cell gel electrophoresis technique able to detect primary DNA damage in individual cells. The tail moment parameter was used as an index of DNA damage. The values of tail moment increased in all the samples incubated with the RNOS donors, indicating DNA impairment. Data obtained show that the ability of zeaxanthin, lutein, and astaxanthin to reduce the DNA damage depends on the type of RNOS donor and the carotenoid concentration used. All the carotenoids studied were capable of protecting against DNA damage in neuroblastoma cells when the cells were exposed to GSNO-MEE. However, a different behaviour was present when the other two RNOS donors were used. The presence of a carotenoid alone (without an RNOS donor) did not cause DNA damage. Spectrophotometric studies showed that the order with which tested carotenoids reacted with RNOS was not always in agreement with the DNA protection results. The data from this study provides additional information on the activities of the macular pigment carotenoids of the human retina.     

Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries

Background

The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields.

Results

Here we present a novel esterase gene (lip3) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme. The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified from Escherichia coli, preferentially hydrolyzed short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 °C and a pH optimum of 8.0. Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, β-mercaptoethanol and high concentrations of salt. The enzyme was also highly activated by NaCl.

Conclusions

The biochemical characterization and homology model reveals a novel esterase originating from the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes.

     

Synthesis,characterization and magnetic properties of mixed-valence iron complexes with 2-pyridyl oximes

Abstract

Two new mixed-valence iron complexes with 2-pyridyl oximes, [Fe(mpko)₃Fe(H₂O)₂(NO₃)](NO₃)·2H₂O (1) (mpko^? = methyl(2-pyridyl)ketone oximate) and [{Fe(dpko)₃}₂Fe](ClO₄)·4H₂O (2) (dpko^? = bis(2-pyridyl)ketone oximate), have been prepared by reaction of Fe^III with mpkoH in methanol (1) and Fe^II with dpkoH in methanol/water (2). Dinuclear Fe^II(low-spin)Fe^III(high-spin) and trinuclear Fe^II(low-spin)Fe^III(high-spin)Fe^II(low-spin) cations are present in the crystal structure of 1 and 2, respectively. Intermolecular hydrogen bonds in 1 lead to weak antiferromagnetic interactions between pairs of neighboring Fe^III centers, which allows observation of single-ion zero-field splitting effects.     

A two-channel molecular dosimeter for the optical detection of copper(II)

A cyclam-like macrocycle with an integrated push-pull chromophore selectively detects Cu2+ inclusion through both orange-to-yellow colour change and quenching of the green fluorescence.     

Intramolecular carbohydrate-aromatic interactions and intermolecular van der Waals interactions enhance the molecular recognition ability of GM1 glycomimetics for cholera toxin

The design and synthesis of two GM1 glycomimetics, 6 and 7, and analysis of their conformation in the free state and when complexed to cholera toxin is described. These compounds, which include an (R)-cyclohexyllactic acid and an (R)-phenyllactic acid fragment, respectively, display significant affinity for cholera toxin. A detailed NMR spectroscopy study of the toxin/glycomimetic complexes, assisted by molecular modeling techniques, has allowed their interactions with the toxin to be explained at the atomic level. It is shown that intramolecular van der Waals and CH-pi carbohydrate-aromatic interactions define the conformational properties of 7, which adopts a three-dimensional structure significantly preorganized for proper interaction with the toxin. The exploitation of this kind of sugar-aromatic interaction, which is very well described in the context of carbohydrate/protein complexes, may open new avenues for the rational design of sugar mimics.     

Cyclic RGD Peptides Containing Azabicycloalkane Reverse‐Turn Mimics

The Fmoc‐protected lactams 3 and 4 were used to prepare cyclo(Arg‐Gly‐Asp‐lactam) 1 and cyclo(Arg‐Gly‐Asp‐Phe‐lactam) 2 , which contain the Arg‐Gly‐Asp (RGD) recognition motif. Their solid‐phase synthesis, conformational analysis, and binding to purified α_Vβ₃ and α_Vβ₅ integrins are reported. Compound 1 was found to act as an active and selective inhibitor of the α_Vβ₅ integrin.     

Conformational analysis of 1,2,3,4-tetrahydroisoquinolines

Ring and nitrogen inversion account for the conformational equilibria of 3-phenyl-1, 2,3, 4-tetrahydroiso-quinolines. In order to quantitate the relative contribution of each conformer to the equilibrium, we undertook a molecular mechanics study on several substituted 3-phenyl-1, 2, 3, 4-tetrahydroisoquinolines. Predictions from calculations were checked against cmr chemical shift data. No boat conformation contributed significantly to the equilibrium. A general result of our calculations is that in all cases the 3-phenyl group in the equatorial position is strongly favored (by at least 2.50 kcal/mole). For 3-phenyl-1, 2, 3, 4-tetrahydroisoquinolines without substitution at nitrogen, N-H in equatorial position is preferred over the axial conformer, although the energy difference between both is always small (0.30–1.10 kcal/mole). For the cis-1,3-disubstituted compounds the le'3e conformers are the only species present (at least 99.8%). The calculated energy differences between the la′3a conformer and the le′3e conformer are always large (3.80–6.10 kcal/mole for the NHe conformers and 3.60–3.80 kcal/mole for the NHa conformers). The lack of a γ_1a upfield shift at C3 also points to the preference for the pseudoequatorial-equatorial conformer. For N-methyl-3-phenyl-1,2,3,4-tetrahydroisoquinoline a preference for the NMe group in the equatorial position is predicted (0.60–2.00 kcal/mole). The small downfield shift at C4 (γ_Na = 0.5 ppm) is consistent with the equatorial NMe preference. For the cis-1,2,3-trisubstituted compounds no significant γ_1a effect at C3 (γ_1a = -0.2 and 1.0 ppm) or γ_Na effect at C4 (γ_Na = 0.1 and 0.4 ppm) is observed. For these compounds, deformations due to steric congestion are evidenced by the deviation from the values of the C4a-C8a-Cl-N and C4a-C4-C3-N torsional angles, as compared to less crowded 3-phenyl-1,2,3,4-tetrahydroisoquinolines. Here the heterocyclic ring adopts a distorted half-chair conformation.     

Multifunctional polymeric materials for biomedical applications

The work performed by our research group during the last few years in the area of bioerodible-biodegradable polymers as designed to the formulation of systems for the controlled delivery of drugs and as specific sorbents of uraemic toxins is broadly reviewed. In particular, attention has been focused on the strategies adopted in the preparation of functional polymers containing hydroxyl or carboxyl groups, suitable to establish specific bonding and non-bonding interactions with conventional and proteic drugs.