Aspirin modulates LPS-induced nitric oxide release in rat glial cells

Nitric oxide and prostaglandins are among the numerous substances released by activated glial cells. The aim of this study was to evaluate the effect of high-level aspirin on iNOS expression in cultured rat glial cells treated with lipopolysaccharide (LPS) as pathological stimulator. Using Western Blotting, we verified that aspirin enhanced LPS-induced iNOS expression and the presence of 15-deoxy-Delta(12,14)-prostaglandin (15d-PGJ(2)) suppressed this aspirin effect. However, the exposure of LPS-treated glial cells to aspirin resulted in a decrease of NO production. These results suggest that aspirin interferes with the cross-talk of prostaglandins and NO, blocking the endogenous negative control exerted by COX products on iNOS expression. On the other side, aspirin seems to act directly on iNOS reducing its activity, even if it does not completely block NO release by LPS-stimulated glial cells. Then aspirin could maintain homeostatic functions of NO, while it prevents toxic effects, corresponding to high NO concentrations. (c) 2005 Elsevier Ireland Ltd. All rights reserved

Effect of polyphenolic compounds on the proteolytic activities of constitutive and immuno-proteasomes

The effect of several polyphenols on the 20S proteasomes, both the constitutive and the LMP proteasomes, isolated from bovine tissues, has been investigated. Polyphenolic compounds show many biological activities such as antiviral, antibacterial, antifungal, anti-inflammatory, antimutagenic, and antiallergic activities. However, the molecular mechanism underlying these effects has not been identified. It is well established that polyphenols possess inhibitory activities on several enzymes and among them the 20S proteasome. In the present work, the ChT-L, BrAAP, PGPH, and T-L activities of the isolated constitutive and immuno-proteasomes were assayed in order to get an overall information on the polyphenols binding to the complexes. The effects of the polyphenols on the proteasomal activities were analyzed, taking into account the different subunits composition of the two complexes. Furthermore the same activities were measured on whole extracts from cancer cells exposed to EGCG and gallic acid, evaluating, also, their antioxidant action under oxidative stress. EGCG and gallic acid are able to affect the 20S proteasomes functionality, depending on the complex subunit composition and, in cell extracts, they behave both as antioxidants and proteasome effectors

Structure-function relationships in bovine thymus 20S proteasome: a fluorimetric study

The structure-function relationships occurring on the bovine thymus 20S proteasome, which exhibits the features of an immunoproteasome, have been studied. The investigation has been performed, essentially, using a fluorimetric approach, taking advantage either of the sensitivity of the complex to sodium dodecil sulfate and chaotropic agents (urea and guanidine hydrochloride) or of the presence, on the molecule, of a high number of tryptophan residues. The results obtained indicate that the perturbation or the oxidation of these residues affect the catalytic events taking place on the thymus proteasome and that the functional effects determined by SDS and chaotropic agents most likely occur through a series of progressive structural modifications leading to an inactive molecule. The presence of structural intermediates in the proteasome inactivation process suggests that thymus proteasome is a molecule characterized, at the same time, by structural flexibility (modulation of active sites) and structural stability (maintaining of the quaternary structure) in agreement with its crucial role in the cell life cycle. (C) 2001 Elsevier Science B.V. All rights reserved

Peroxynitrite-mediated oxidation of the C85S/C152E mutant of dihydrofolate reductase from Escherichia coli: functional and structural effects

Peroxynitrite is a potent reactive oxygen species that is believed to mediate deleterious protein modifications in a wide variety of neurodegenerative disorders. In this study, we have analysed the effects of oxidative damage induced by peroxynitrite on a cysteine-free mutant of dihydrofolate reductase (SE-DHFR), from a functional and a structural point of view. The peroxynitrite-mediated oxidation results in the inhibition, concentration-dependent, of the catalytic activity. This effect is strongly influenced by the HCO(3)(-)/CO(2) buffering system, that we observed to significantly affect the yield of protein oxidation by modulating the peroxynitrite-induced modification of aromatic residues. Because of this effect, in presence of bicarbonate system, we have observed a protection of enzymatic activity of SE-DHFR with regard to peroxynitrite. The thermodynamic stability of the oxidized protein has been studied in comparison with the non-oxidized protein by differential scanning calorimetry. The thermodynamic parameters obtained showed a decrease of stability of SE-DHFR upon oxidation, evaluated in terms of Gibbs free energy of about 1.25 kcal/mol at 25 degrees C, with respect to the non-oxidized protein. Together, these data indicate that structural and functional alterations induced by peroxynitrite may play a direct role in compromising DHFR function in multiple pathological conditions

Mechanism of inhibition of wt-dihydrofolate reductase from E. coli by tea epigallocatechin-gallate.

Hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) is the rate-controlling enzyme of cholesterol synthesis, and owing to its biological and pharmacological relevance, researchers have investigated several compounds capable of modulating its activity with the hope of developing new hypocholesterolemic drugs. In particular, polyphenol-rich extracts were extensively tested for their cholesterol-lowering effect as alternatives, or adjuvants, to the conventional statin therapies, but a full understanding of the mechanism of their action has yet to be reached. Our work reports on a detailed kinetic and equilibrium study on the modulation of HMGR by the most-abundant catechin in green tea, epigallocatechin-3-gallate (EGCG). Using a concerted approach involving spectrophotometric, optical biosensor, and chromatographic analyses, molecular docking, and site-directed mutagenesis on the cofactor site of HMGR, we have demonstrated that EGCG potently inhibits the in vitro activity of HMGR (K(i) in the nanomolar range) by competitively binding to the cofactor site of the reductase. Finally, we evaluated the effect of combined EGCG-statin administration

Interplay between 20S proteasomes and prion proteins in scrapie disease.

Scrapie is a transmissible spongiform encephalopathy affecting the central nervous system in sheep. The key event in such neurodegeneration is the conversion of the normal prion protein (PrPC) into the pathological isoform (PrPSc). Misfolded prion proteins are normally degraded by the proteasome. This work, analyzing models of scrapie disease, describes the in vivo relationship between the proteasome and prions. We report that the disease is associated with an increase of proteasome functionality, most likely as a means of counteracting the increased levels of oxidative stress. Here, we show that prions coprecipitate with the 20S proteasome and that they colocalize within the same neuron, thus raising the possibility that PrP interacts with the proteasome in both normal and diseased brain, affecting substrate trafficking and proteasome functionality. This interaction, inducing proteasome activation, leads to different neuronal alterations and triggers apoptosis. Furthermore, testing the effects of isolated PrPC on purified 20S proteasomes, we obtain a concentration- and proteasome composition-dependent decrease in the complex activity. (C) 2009 Wiley-Liss, Inc

Wheat sprout extract-induced apoptosis in human cancer cells by proteasomes modulation.

Natural occurring modulators of proteasome functionality are extensively investigated for their implication in cancer therapy. On the basis of our previous evidences both on proteasomal inhibition by monomeric polyphenols, and on the characterization of wheat sprout hydroalcoholic extract, herein we thoroughly report on a comparative study of the effect of wheat sprout extract on both normal and tumour cells. Treatment of isolated 20S proteasomes with wheat sprout extracts induced a gradual inhibition of all proteasome activities. Next, two wheat sprout extract components were separated: a polyphenol and a protein fraction. Both components exerted an in vitro inhibitory effect on proteasome activity. HeLa tumour cells and FHs 74 Int normal cells were exposed to both fractions, resulting in different rates of proteasome inhibition, with tumour cells showing a significantly higher degree of proteasome impairment and apoptosis induction. Furthermore, a decrease in proteasome activities and in cell survival of the human plasmacytoma RPMI 8226 cell line, upon the same treatments, was observed. Collectively, our results provide additional evidences supporting the possible use of natural extracts as coadjuvants in cancer treatments

Wheat sprout extract induces changes on 20S proteasomes functionality.

Wheat sprouts contain a very high level of organic phosphates and a powerful cocktail of different molecules such as enzymes, reducing glycosides and polyphenols. The antioxidant properties of wheat sprouts have been widely documented and it has been shown that they are able to protect DNA against free-radicals mediated oxidative damage. Furthermore, we have recently reported on the effects of several polyphenols on 20S proteasomes, underlying the dual role of epigallocatechin-3-gallate as an antioxidant and a proteasome effector in cancer cells. The aim of this study was to investigate the effects of wheat sprout extracts on 20S proteasome functionality. Wheat sprout extracts have been analysed and characterized for their polyphenolic content using the Folin-Ciocalteau reagent and RP-HPLC technique. Comparing our data with a polyphenol standard mixture we identified five different polyphenols: gallic acid, epigallocatechin-3-gallate, epigallocatechin, epicatechin and catechin. The treatment of isolated 20S proteasomes with the extract induced a gradual inhibition of all the tested components, ChT-L, T-L, PGPH and BrAAP, in both the complexes. At low extract concentration a slight activation of the enzyme was evident only for the BrAAP component of the constitutive enzyme and the ChT-L activity of the immunoproteasome. b-casein degradation rate decreased, particularly with the immunoproteasome. Human Colon adenocarcinoma (Caco) cells, stimulated with 12-O-tetradecanoylphorbol-13-acetate, showed activation of the 20S proteasome activities at short incubation times and an increase in intracellular oxidative proteins. Cells treatment with wheat sprout extract led to proteasome inhibition in unstimulated cells and attenuated the effects mediated by TPA. Finally, exposure to the extract affected the expression levels of pro-apoptotic proteins