Chiral recognition by the copper(II) complex of 6-deoxy-6-N-(2-methylaminopyridine)-β-cyclodextrin

A modified β-cyclodextrin bearing a 2-aminomethylpyridine binding site for copper(II) (6-deoxy-6-[N-(2-methylamino)pyridine)]-β-cyclodextrin, CDampy was synthesized by C₆-monofunctionalization. The acid-base properties of the new ligand in aqueous solution were investigated by potentiometry and calorimetry, and its conformations as a function of pH were studied by NMR and circular dichroism (c.d.). The formation of binary copper(II) complexes was studied by potentiometry, EPR, and c.d. The copper(II) complex was used as chiral selector for the HPLC enantiomeric separation of underivatized aromatic amino acids. Enantioselectivity in the overall stability constants of the ternary complexes with D- or L-Trp was detected by potentiometry, whereas the complexes of the Ala enantiomers did not show any difference in stability. These results were consistent with a preferred cis coordination of the amino group of the ligand and of the amino acid in the ternary complexes (“cis effect”), which leads to the inclusion of the aromatic side chain of D-Trp, but not of that of L-Trp. In Trp-containing ternary complexes, the two enantiomers showed differences in the fluorescence lifetime distribution, consistent with only one conformer of D-Trp and two conformers of L-Trp, and the latter were found to be more accessible to fluorescence quenching by acrylamide and KI. Chirality 9:341–349, 1997. © 1997 Wiley-Liss, Inc.     

Structural and mechanical properties of UV-photo-cross-linked poly(N-vinyl-2-pyrrolidone) hydrogels

Biocompatible poly( N-vinyl-2-pyrrolidone) (PVP) hydrogels have been produced by UV irradiation of aqueous polymer mixtures, using a high-pressure mercury lamp. The resulting materials have been characterized by a combination of experimental techniques, including rheology, small-angle neutron scattering (SANS), electron paramagnetic resonance (EPR), and pulsed gradient spin-echo nuclear magnetic resonance (PGSE-NMR), to put in evidence the relationship between the microstructural properties and the macrofunctional behavior of the gels. Viscoelastic measurements showed that UV photo-cross-linked PVP hydrogels present a strong gel mechanical behavior and viscoelastic moduli values similar to those of biological gels. The average distance between the cross-linking points of the polymer network was estimated from the hydrogels elastic modulus. However, SANS measurements showed that the network microstructure is highly inhomogeneous, presenting polymer-rich regions more densely cross-linked, surrounded by a water-rich environment. EPR and PGSE-NMR data further support the existence of these water-rich domains. Inclusion of a third component, such as glycerol, in the PVP aqueous mixture to be irradiated has been also investigated. A small amount of glycerol (<3% w/w) can be added keeping satisfactory properties of the hydrogel, while higher amounts significantly affect the cross-linking process.     

Protective effect and relation structure-activity of nonivamide and iododerivatives in several models of lipid oxidation

The introduction of an iodine atom on the vanillyl moiety of nonivamide causes a switch in the vanilloid activity (TRPV1 antagonism versus TRPV1 desensitization) and nullifies the aversive properties of capsaicinoids. In the present study we investigated the effect of iodination in the vanillyl moiety on the antioxidant activity of capsaicinoids. To this aim, we have compared the protective effects of nonivamide and three iododerivatives, 2-iodononivamide (2IN), 5-iodononivamide (5IN), and 6-iodononivamide (6IN) in a series of in vitro models of lipid oxidation, namely the autoxidation and the FeCl₃-mediated oxidation of linoleic acid at 37 °C and the thermal (140 °C), solvent-free oxidation of cholesterol. All tested compounds could protect linoleic acid and cholesterol against oxidative degradation, the order of potency being: nonivamide > 5IN > 6IN ≈ 2IN. Our results show that, in general, the introduction of an iodine atom on the vanillyl moiety of nonivamide causes a decrease in the antioxidant activity, and this effect is sensitive to the position of iodine on the aromatic ring, with 5IN substantially retaining the efficacy of nonivamide to protect linoleic and cholesterol against free radical attack. Moreover, the pre-treatment with 5IN, at noncytotoxic concentrations, significantly preserved LDL from Cu²⁺-induced oxidative damage at 37 °C for 2 h, inhibiting the reduction of polyunsaturated fatty acids and cholesterol and the increase of their oxidative products. The results of the present work suggest that 5IN exerts useful antioxidant properties in different in vitro systems of lipid peroxidation. This, coupled to its lacks of pungency and TRPV1 inhibiting properties, qualifies this phenolic compound as an attractive candidate for further investigations in vivo.     

Potential allelopathic effect of neo-clerodane diterpenes from Teucrium chamaedrys (L.) on stenomediterranean and weed cosmopolitan species

The allelopathic effects of neo-clerodane diterpenes, isolated from Teucrium chamaedrys (L.), have been evaluated on the seed germination and seedling growth of four coexisting Mediterranean species (Dactylis hispanica, Petrorhagia velutina, Phleum subulatum and Petrorhagia saxifraga) and two cosmopolitan weeds (Amaranthus retroflexus and Avena fatua). All of the structures have been elucidated on the basis of their spectroscopic features. The bioassays data, analyzed by principal component analysis, showed more negative effects on weeds respect to coexisting species. Moreover D. hispanica, P. velutina, P. subulatum showed both stimulating or inhibiting effects depending on the type of metabolite and the concentration used in the test.     

Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs

With no known exceptions, every published microarray study to determine differential mRNA levels in eukaryotes used RNA extracted from whole cells. It is assumed that the use of whole cell RNA in microarray gene expression analysis provides a legitimate profile of steady-state mRNA. Standard labeling methods and the prevailing dogma that mRNA resides almost exclusively in the cytoplasm has led to the long-standing belief that the nuclear RNA contribution is negligible. We report that unadulterated cytoplasmic RNA uncovers differentially expressed mRNAs that otherwise would not have been detected when using whole cell RNA and that the inclusion of nuclear RNA has a large impact on whole cell gene expression microarray results by distorting the mRNA profile to the extent that a substantial number of false positives are generated. We conclude that to produce a valid profile of the steady-state mRNA population, the nuclear component must be excluded, and to arrive at a more realistic view of a cell''s gene expression profile, the nuclear and cytoplasmic RNA fractions should be analyzed separately.     

Biochemical characterization of a recombinant short-chain NAD(H)-dependent dehydrogenase/reductase from Sulfolobus acidocaldarius

The gene encoding a novel alcohol dehydrogenase that belongs to the short-chain dehydrogenases/reductases (SDRs) superfamily was identified in the aerobic thermoacidophilic crenarchaeon Sulfolobus acidocaldarius strain DSM 639. The saadh gene was heterologously overexpressed in Escherichia coli, and the protein (SaADH) was purified to homogeneity and characterized. SaADH is a tetrameric enzyme consisting of identical 28,978-Da subunits, each composed of 264 amino acids. The enzyme has remarkable thermophilicity and thermal stability, displaying activity at temperatures up to 75°C and a 30-min half-inactivation temperature of ~90°C, and shows good tolerance to common organic solvents. SaADH has a strict requirement for NAD(H) as the coenzyme, and displays a preference for the reduction of alicyclic, bicyclic and aromatic ketones and α-keto esters, but is poorly active on aliphatic, cyclic and aromatic alcohols, and shows no activity on aldehydes. The enzyme catalyses the reduction of α-methyl and α-ethyl benzoylformate, and methyl o-chlorobenzoylformate with 100% conversion to methyl (S)-mandelate [17% enantiomeric excess (ee)], ethyl (R)-mandelate (50% ee), and methyl (R)-o-chloromandelate (72% ee), respectively, with an efficient in situ NADH-recycling system which involves glucose and a thermophilic glucose dehydrogenase. This study provides further evidence supporting the critical role of the D37 residue in discriminating NAD(H) from NAD(P)H in members of the SDR superfamily.     

The distribution of mannose-6-phosphate receptors changes from newborns to adults in rat liver

The co-existence of two types of mannose-6-phosphate receptors (CD-MPR and CI-MPR) in most cell types is still not well explained. Some evidence suggests that the CI-MPR could be actively involved in the regulation of growth factors in the early stages of mammalian organ development. In this study, it was demonstrated that both receptors are distributed in a non-overlapping fashion in rat liver, and that the distribution of CI-MPR changes over a percoll gradient between newborn and adult animals. By using marker proteins it was observed that in newborns the CI-MPR is located both in intracellular fractions and in fractions that coincide with a plasma membrane marker, whereas in adults it is only detected in intracellular fractions. It was also noted that N-acetyl-β-d-glucosaminidase distribution is closer to CI-MPR than to CD-MPR and that acid phosphatase did not match with any receptor. This evidence may also suggest that both receptors have different functions, mainly at early stages in the development of organs.     

Hydroxytyrosol glucuronides protect renal tubular epithelial cells against H(2)O(2) induced oxidative damage

Hydroxytyrosol (2-(3′,4′-dihydroxyphenyl)ethanol; HT), the most active ortho-diphenolic compound, present either in free or esterified form in extravirgin olive oil, is extensively metabolized in vivo mainly to O-methylated, O-sulfated and glucuronide metabolites. We investigated the capacity of three glucuronide metabolites of HT, 3′-O-β-d-glucuronide and 4′-O-β-d-glucuronide derivatives and 2-(3′,4′-dihydroxyphenyl)ethanol-1-O-β-d-glucuronide, in comparison with the parent compound, to inhibit H₂O₂ induced oxidative damage and cell death in LLC-PK1 cells, a porcine kidney epithelial cell line. H₂O₂ treatment exerted a toxic effect inducing cell death, interacting selectively within the pro-death extracellular-signal relate kinase (ERK 1/2) and the pro-survival Akt/PKB signaling pathways. It also produced direct oxidative damage initiating the membrane lipid peroxidation process. None of the tested glucuronides exhibited any protection against the loss in renal cell viability. They also failed to prevent the changes in the phosphorylation states of ERK and Akt, probably reflecting their inability to enter the cells, while HT was highly effective. Notably, pretreatment with glucuronides exerted a protective effect at the highest concentration tested against membrane oxidative damage, comparable to that of HT: the formation of malondialdehyde, fatty acid hydroperoxides and 7-ketocholesterol was significantly inhibited.