Resveratrol: from basic studies to bedside.

Plants produce a remarkable amount of low molecular mass natural products endowed with a large array of pivotal biological activities. Among these molecules, resveratrol (3,5,4'-trihydroxystilbene) has been identified as an important modulator of cell phenotype with a complex and pleiotropic mode of action. Extensive literature regarding its activity, mainly employing cellular models, suggests that this polyphenol controls cell proliferation, induces differentiation, and activates apoptosis and autophagy. The compound also modulates angiogenesis and inflammation. Similarly, studies on implanted cancers and chemical-induced tumors confirm the potential chemotherapeutical interest of the compound. Likewise, several reports clearly demonstrated, in animal models, that the compound might positively affect the development and evolution of chronic diseases including type 2 diabetes, obesity, coronary heart disease, metabolic syndrome, and neurogenerative pathologies. Finally, a number of investigations stated that the toxicity of the molecule is scarce. Despite these promising observations, few clinical trials have yet been performed to evaluate the effectiveness of the molecule both in prevention and treatment of human chronic disease. Preliminary findings therefore suggest the need for more extensive clinical investigations.

Histone deacetylase inhibitors upregulate p57Kip2 level by enhancing its expression through Sp1 transcription factor.

Histone deacetylase inhibitors (HDACIs) represent a new class of targeted anticancer agents. Here, we evaluate the effects of butyrate (BuA) and other HDACIs on p57(Kip2), a cyclin-dependent kinase inhibitor (cki). We observed that inhibitors of class I/II histone deacetylases (HDACs), but not of class III HDACs, induce a remarkable accumulation of p57(Kip2) in several cells. The cki upregulation is associated with an increased gene expression that was not prevented by cycloheximide, indicating that HDACIs affect directly p57(Kip2) transcription. The characterization of p57(Kip2) promoter indicates that the first 165 bp are mostly involved in the BuA effects. Chromatin immunoprecipitation studies demonstrated that the BuA treatment causes the recruitment of Sp1 transcription factor. The Sp1 importance was confirmed by the reduction of BuA effects by mithramycin A (an Sp1 antagonist) and, most stringently, by Sp1 downregulation due to Sp1 siRNA. Moreover, both the treatments reduce the p57(Kip2) transcription in untreated cells, suggesting that Sp1 is required for the constitutive cki expression. Studies employing plasmids containing parts of the 165 bp of p57(Kip2) promoter indicate that the promoter region between -87 and -113 bp, which includes two putative Sp1 consensus sequences, plays a critical role in the response to HDACIs. Since this p57(Kip2) promoter region also embraces the consensus sequence for the transcriptional repressor chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (CTIP2), we evaluated whether this factor is involved into the BuA effect. When CTIP2 was downregulated by a specific siRNA, we observed the enhancement of BuA activity on p57(Kip2) expression suggesting that CTIP2 might also be involved in HDACIs effects.

In vivo effect of the natural antioxidant hydroxytyrosol on cyclosporine nephrotoxicity in rats.

BACKGROUND: Cyclosporine A (CsA) is the first-line immunosuppressant used in transplant patients and in auto- immune diseases. Nephrotoxicity is the major limitation of CsA use. Although the mechanisms of nephrotoxicity have not been completely defined, some evidence suggests that reactive oxygen species (ROS) play a causal role. The present study was designed to investigate in vivo effects of hydroxytyrosol (DOPET), a natural olive oil antioxidant, on oxidative stress, renal histology and haemodynamic alterations induced in rats by CsA treatment. METHODS: Adult Sprague-Dawley rats were treated i.p. with CsA (15 mg/kg) alone or in combination with DOPET (20 mg/kg) for 3 weeks. At the end of the treatment, superoxide concentration within the cells of the abdominal aorta and renal artery was quantified from the oxidation of dihydroethidium (DHE) using fluorescence microscopic imaging analysis. In kidney tissues, lipid peroxidation was measured by thiobarbituric acid-reacting substances (TBARS) assay, glutathione level was assessed enzymatically and the expression of haem oxygenase-1 (HO-1) gene was evaluated by semiquantitative RT-PCR. Renal morphology was studied by classical histological techniques, while the glomerular filtration rate (GFR) was estimated by inulin clearance. Systemic blood pressure was monitored by the tail method and through the catheterization of the carotid artery. RESULTS: CsA administration increased superoxide concentration both in the aorta and in the renal artery, while DOPET completely prevented this effect. Higher levels of TBARS, a significant decrease in GSH and an upregulation of HO-1 mRNA were observed in the kidneys of CsA-treated rats. DOPET treatment reversed quantitatively these effects. However, CsA-dependent changes in renal histology were only partially reversed by DOPET. Finally, CsA induced a severe reduction in GFR and a significant increase in both systolic and diastolic blood pressure; the DOPET treatment had no significant effect on these haemodynamic alterations. CONCLUSION: The reported data indicate that effective DOPET protection from CsA-induced oxidative stress is associated with a mild effect on histological damages and does not affect the altered glomerular function and the hypertension, thus indicating that kidney injury by CsA is only in part dependent on oxidative stress.

Retinoic acid induces p27Kip1 nuclear accumulation by modulating its phosphorylation.

All-trans-retinoic acid (ATRA), the most biologically active metabolite of vitamin A, controls cell proliferation, apoptosis, and differentiation depending on the cellular context. These activities point to ATRA as a candidate for cancer therapy. A pivotal effect of the molecule is the modulation of p27Kip1, a cyclin-dependent kinase (CDK) inhibitor (CDKI). Here, we investigate the mechanisms by which ATRA regulates p27Kip1 level in LAN-5, a neuroblastoma cell line. When added to the cells, ATRA causes a rapid nuclear increase of p27Kip1, which clearly precedes growth arrest. The early buildup is not due to impairment of the CDKI degradation, in contrast to previous observations. Particularly, we did not detect the down-regulation of Skp2 and Cks1, two proteins involved in the nuclear ubiquitin-dependent p27Kip1 removal. Moreover, the morphogen does not impair the CDKI nuclear export and does not cause CDK2 relocalization. The characterization of CDKI isoforms by two-dimensional PAGE/immunoblotting showed that ATRA induces an early nuclear up-regulation of monophosphorylated p27Kip1. Immunologic studies established that this isoform corresponds to p27Kip1 phosphorylated on S10. The buildup of phospho(S10)p27Kip1 precedes the CDKI accumulation and increases its half-life. Finally, ATRA-treated nuclear LAN-5 extracts showed an enhanced capability of phosphorylating p27Kip1 on S10, thus explaining the nuclear up-regulation of the isoform. In conclusion, our data suggest a novel mechanism of ATRA antiproliferative activity, in which the morphogen rapidly up-regulates a nuclear kinase activity that phosphorylates p27Kip1 on S10. In turn, this event causes the stabilization of p27Kip1 and its accumulation in the nuclear compartment.

Homocysteine-induced endothelial cell adhesion is related to adenosine lowering and is not mediated by S-adenosylhomocysteine.

Hyperhomocysteinemia is a cardiovascular risk factor and may contribute to the pathogenesis of atherosclerosis by altering endothelial functions. The mechanism of homocysteine-induced cell adhesion has been here investigated using EA.hy 926 cells. Homocysteine induces a stereospecific, time- and dose-dependent cell adhesion which is prevented by adenosine. The dramatic increase of S-adenosylhomocysteine induced by adenosine-2',3'-dialdehyde does not cause cell adhesion, indicating that no apparent relationship exists between this process and intracellular S-adenosylhomocysteine content. Homocysteine-induced cell adhesion is abolished by pre-treatment with adenosine-2',3'-dialdehyde, demonstrating that the adenosine depletion caused by reversal of S-adenosylhomocysteine hydrolase reaction is responsible for homocysteine-induced cell damage.

Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the Archaeon Pyrococcus furiosus.

We report here the characterization of the first mammalian-like purine nucleoside phosphorylase from the hyperthermophilic archaeon Pyrococcus furiosus (PfPNP). The gene PF0853 encoding PfPNP was cloned and expressed in Escherichia coli and the recombinant protein was purified to homogeneity. PfPNP is a homohexamer of 180 kDa which shows a much higher similarity with 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) than with purine nucleoside phosphorylase (PNP) family members. Like human PNP, PfPNP shows an absolute specificity for inosine and guanosine. PfPNP shares 50% identity with MTAP from P. furiosus (PfMTAP). The alignment of the protein sequences of PfPNP and PfMTAP indicates that only four residue changes are able to switch the specificity of PfPNP from a 6-oxo to a 6-amino purine nucleoside phosphorylase still maintaining the same overall active site organization. PfPNP is highly thermophilic with an optimum temperature of 120 degrees C and is characterized by extreme thermodynamic stability (T(m), 110 degrees C that increases to 120 degrees C in the presence of 100 mm phosphate), kinetic stability (100% residual activity after 4 h incubation at 100 degrees C), and remarkable SDS-resistance. Limited proteolysis indicated that the only proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is not necessary for the integrity of the active site. By integrating biochemical methodologies with mass spectrometry we assigned three pairs of intrasubunit disulfide bridges that play a role in the stability of the enzyme against thermal inactivation. The characterization of the thermal properties of the C254S/C256S mutant suggests that the CXC motif in the C-terminal region may also account for the extreme enzyme thermostability.

Accumulation of altered aspartyl residues in erythrocyte proteins from patients with Down's syndrome.

Spontaneous protein deamidation of labile Asn residues, generating L-isoaspartates and D-aspartates, is associated with cell aging and is enhanced by an oxidative microenvironment; to minimize the damage, the isoaspartate residues can be 'repaired' by a specific L-isoaspartate (D-aspartate) protein O-methyltransferase (PIMT). As both premature aging and chronic oxidative stress are typical features of Down's syndrome (DS), we tested the hypothesis that deamidated proteins may build up in trisomic patients. Blood samples were obtained from children with karyotypically confirmed full trisomy 21 and from age-matched healthy controls. Using recombinant PIMT as a probe, we demonstrated a dramatic rise of L-isoaspartates in erythrocyte membrane proteins from DS patients. The content of D-aspartate was also significantly increased. The integrity of the repair system was checked by evaluating methionine transport, PIMT specific activity, and intracellular concentrations of adenosylmethionine and adenosylhomocysteine. The overall methylation pathway was directly monitored by incubating fresh red blood cells with methyl-labeled methionine; a three-fold increase of protein methyl esters was detected in trisomic children. Deamidated species include ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3; ankyrin and band 4.1 were significantly hypermethylated in DS. When DS red blood cells were subjected to oxidative treatment in vitro, the increase of protein deamidation paralleled lipid peroxidation and free radical generation. We observed a similar pattern in Epstein-Barr virus B-lymphocytes from trisomic patients. In conclusion, our findings support the hypothesis that protein instability at asparagine sites is a biochemical feature of DS, presumably depending upon the oxidative microenvironment. The possible pathophysiological implications are discussed.

Effect of reddening-ripening on the antioxidant activity of polyphenol extracts from cv. 'Annurca' apple fruits.

Apple is among the most consumed fruits worldwide, and several studies suggest that apple polyphenols could play a role in the prevention of degenerative diseases. 'Annurca' apple fruit undergoes, after harvest, a typical reddening treatment to turn the apples' skin red, and it is noted for its high firmness. This paper reports the effect of reddening-ripening treatment on polyphenol concentration and antioxidant activity of both peel and flesh extracts. The in vitro antioxidant properties have been compared with the protective effect against the cytotoxic effects of reactive oxygen species using Caco-2 cells as model system. Pretreatment of cells with different polyphenolic apple extracts provides a remarkable protection against oxidative damage. This effect seems to be associated with the antioxidant activity of 'Annurca' apple polyphenolic compounds. The flesh has antioxidant properties comparable to those possessed by the peel. Neither the reddening nor the fruit conservation causes changes in the antioxidant properties possessed by this apple variety. The data indicate that polyphenolic compounds in 'Annurca' apples are relatively stable in the peel and also in the flesh; therefore, the health benefits of polyphenols should be maintained during long-term storage. Finally, a diet rich in apple antioxidants could exert a beneficial effect in the prevention of intestinal pathologies related to the production of reactive oxygen species.

A novel hyperthermostable 5'-deoxy-5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus.

We report herein the first molecular characterization of 5'-deoxy-5'-methylthio-adenosine phosphorylase II from Sulfolobus solfataricus (SsMTAPII). The isolated gene of SsMTAPII was overexpressed in Escherichia coli BL21. Purified recombinant SsMTAPII is a homohexamer of 180 kDa with an extremely low Km (0.7 microm) for 5'-deoxy-5'-methylthioadenosine. The enzyme is highly thermophilic with an optimum temperature of 120 degrees C and extremely thermostable with an apparent Tm of 112 degrees C that increases in the presence of substrates. The enzyme is characterized by high kinetic stability and remarkable SDS resistance and is also resistant to guanidinium chloride-induced unfolding with a transition midpoint of 3.3 m after 22-h incubation. Limited proteolysis experiments indicated that the only one proteolytic cleavage site is localized in the C-terminal region and that the C-terminal peptide is necessary for the integrity of the active site. Moreover, the binding of 5'-deoxy-5'-methylthioadenosine induces a conformational transition that protected the enzyme against protease inactivation. By site-directed mutagenesis we demonstrated that Cys259, Cys261 and Cys262 play an important role in the enzyme stability since the mutants C259S/C261S and C262S show thermophilicity and thermostability features significantly lower than those of the wild-type enzyme. In order to get insight into the physiological role of SsMTAPII a comparative kinetic analysis with the homologous 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) was carried out. Finally, the alignment of the protein sequence of SsMTAPII with those of SsMTAP and human 5'-deoxy-5'-methylthioadenosine phosphorylase (hMTAP) shows several key residue changes that may account why SsMTAPII, unlike hMTAP, is able to recognize adenosine as substrate.

Antioxidants induce different phenotypes by a distinct modulation of signal transduction.

Antioxidants are known to exert a preventive activity against degenerative diseases. Here, we investigated the mechanism of action of three antioxidants: resveratrol, which causes differentiation of HL-60 cells, and hydroxytyrosol and pyrrolidine dithiocarbamate which, in the same model system, activate apoptosis. The expression profile of hydroxytyrosol-treated cells showed the up-regulation of several genes, including c-jun and egr1. Pyrrolidine dithiocarbamate activates both genes, while resveratrol increases uniquely egr1. A selective modulation of signalling pathway explained this finding. All antioxidants up-regulate Erk1/2, while only hydroxytyrosol and pyrrolidine dithiocarbamate activate c-Jun N-terminal kinase (JNK). Since JNK induces apoptosis by Bcl-2 phosphorylation, we investigated this event. Bcl-2 phosphorylation was increased by hydroxytyrosol and pyrrolidine dithiocarbamate and not by resveratrol. Our results indicate that the different phenotypical effects of antioxidants correlate with modulation of selective transduction pathways.

Oleuropein prevents oxidative myocardial injury induced by ischemia and reperfusion.

The potential protective effects of oleuropein, a dietary antioxidant of olive oil, has been investigated in the isolated rat heart. The organs were subjected to 30 minutes of no-flow global ischemia and then reperfused. At different time intervals, the coronary effluent was collected and assayed for creatine kinase activity as well as for reduced and oxidized glutathione. In addition, the extent of lipid peroxidation was evaluated by measuring thiobarbituric acid reactive substance concentration in cardiac muscle. Pretreatment with 20 microg/g oleuropein before ischemia resulted in a significant decrease in creatine kinase and reduced glutathione release in the perfusate. The protective effect of oleuropein against the post-ischemic oxidative burst was investigated by measuring the release, in the coronary effluent, of oxidized glutathione, a sensitive marker of heart's exposure to oxidative stress. Reflow in ischemic hearts was accompanied by a prompt release of oxidized glutathione; in ischemic hearts pretreated with oleuropein, this release was significantly reduced. Membrane lipid peroxidation was also prevented by oleuropein. The reported data provide the first experimental evidence of a direct cardioprotective effect of oleuropein in the acute events that follow coronary occlusion, likely because of its antioxidant properties. This finding strengthens the hypothesis that the nutritional benefit of olive oil in the prevention of coronary heart disease can be also related to the high content of oleuropein and its derivatives. Moreover, our data, together with the well documented antithrombotic and antiatherogenic activity of olive oil polyphenols, indicate these antioxidants as possible therapeutic tools for the pharmacological treatment of coronary heart disease as well as in the case of cardiac surgery, including transplantation.

Protective effect of the phenolic fraction from virgin olive oils against oxidative stress in human cells.

This paper reports the protective effect of the phenolic fraction extracted from extra virgin olive oils (OOPEs) against the cytotoxic effects of reactive oxygen species in human erythrocytes and Caco-2 cells, employed as model systems. Pretreatment of cells with various OOPEs, indeed, provides a remarkable protection against oxidative damages: this effect was strictly dependent on the o-diphenolic content of the extracts. Moreover, the protective effects observable in cellular systems were compared with in vitro antioxidant properties, measured by using the FRAP (ferric reducing/antioxidant power) assay; the reducing ability of OOPEs strictly parallels their o-phenolic content. The linear relationship demonstrated between biological effects and antioxidant capacity measured by the FRAP assay allows us to propose the use of this rapid colorimetric method in assessing and certifying the antioxidant power of extra virgin olive oil.

MCP-1 in psoriatic patients: effect of biological therapy.

BACKGROUND: Monocyte chemoattractant protein-1 (MCP-1) is a chemokine locally and systemically augmented in psoriasis. A single nucleotide polymorphism in MCP-1 promoter region -2518A→G is associated with higher gene expression. OBJECTIVE: The aim was to evaluate MCP-1 plasma level in psoriatic patients and to relate any association in plasmatic and cutaneous MCP-1 with clinical improvement due to biological drugs. METHODS: Blood samples were obtained from: (i) 30 Caucasian patients with psoriasis and 10 controls, for determining MCP-1 plasma concentrations and -2518A→G polymorphism occurrence, (ii) 16 psoriatic patients treated by anti-tumor necrosis factor-α (TNF-α) adalimumab/etanercept or by anti-CD-11 efalizumab, before and after 2 months of treatment. Moreover, biopsies were performed on lesional skin of five patients treated with anti-TNF-α. MCP-1 plasma concentration and cutaneous expression were determined by ELISA and qRT-PCR. RESULTS: MCP-1 plasma level was significantly increased in psoriatic patients. -2518A→G polymorphism was similarly distributed in patients and controls and unrelated to MCP-1 plasma level or to Psoriasis Area and Severity Index. All patients receiving biological drugs showed significant clinical improvement. Anti-TNF-α therapy moderately reduced MCP-1 plasma concentration and robustly decremented MCP-1 expression in lesional skin. CONCLUSION: MCP-1 should be a potential local inflammatory marker in psoriatic patients to assess disease severity and anti-TNF-α treatment efficacy.

Abnormal isoaspartyl residues in erythrocyte membranes from psoriatic patients.

Spontaneous protein deamidation of labile asparagines (Asn), generating abnormal isoaspartyl residues (IsoAsp), is associated with cell aging and enhanced by an oxidative microenvironment. The presence of isopeptide bonds impairs protein structure/function and can trigger autoimmune responses. To minimize the damage, IsoAsp can be "repaired" by a specific L-isoaspartate-(D-aspartate)-protein-O-methyltransferase. The condition of chronic oxidative stress reported in psoriatic patients, and the potential etiological role of unknown self-antigens, prompted us to investigate Asn deamidation in psoriatic tissues. Erythrocytes (RBC) were selected as the model system since, lacking protein synthesis apparatus, they are unable to replace damaged proteins. Blood samples were obtained from 36 patients and 34 controls. L-isoAsp content was highly increased in RBC membrane proteins from psoriatic patients. Deamidated species included ankyrin, band 4.1, band 4.2 and the integral membrane protein band 3. A functional analysis demonstrated that this result was unrelated to a reduced efficiency of the S-adenosylmethionine-dependent repair system suggesting an increased protein instability at Asn sites, responsible for IsoAsp accumulation in psoriatic patients.

Impaired transmethylation potential in Parkinson's disease patients treated with L-Dopa.

Hyperhomocysteinaemia was reported in patients with Parkinson's disease (PD) treated with l-Dopa. The increase in plasma concentration of this sulfur compound arises from the massive methylation of the drug operated by the enzyme catechol-O-methyltransferase (COMT), which acts as a powerful sink of methyl groups. The contemporary occurrence of C677T polymorphism in homozygosity, leading to a temperature-labile variant of the MTHFR enzyme, induces an even more marked increase in tHcy. Here we show that l-Dopa administration in hyperhomocysteinemic PD patients is able to lower intracellular concentration of S-Adenosylmethionine (AdoMet) in erythrocytes (RBC), while the occurrence of hyperhomocysteinaemia causes a significant increase in S-Adenosylhomocysteine (AdoHcy) level. In patients with PD treated with l-Dopa and hyperhomocysteinemic, the remarkable decrease in AdoMet and the concurrent increase in AdoHcy concentration both contribute to significantly lower the transmethylation potential ([AdoMet]/[AdoHcy]), a useful index of the effectiveness of methyl group transfer by methyltransferases. This decrease could indeed contribute to partly attenuate, through a self-limiting kinetic mechanism, the tendency of developing drug resistance, partly mediated in these patients by COMT upregulation. Our results also support the conclusion that COMT inhibitors (entacapone or tolcapone), when administered in PD patients treated with l-Dopa, may potentiate the endogenous AdoHcy-dependent COMT inhibition mechanism already operative in a variable fashion.

Protective effect of polyphenols from Glycyrrhiza glabra against oxidative stress in Caco-2 cells.

In the present article, we have investigated the antioxidant properties of methanolic liquorice polyphenol extracts (LPE(s)). Polyphenol extraction was performed with 60% and 100% methanol. Analysis of LPE(s) by thin-layer chromatography revealed that a higher amount of polyphenols was recovered by extraction with 60% methanol. Antioxidant activity measurement of the reducing power, scavenging effect on 2,2'-diphenyl-1-picrylhydrazyl free radical, and hydrogen peroxide scavenging capability have been taken as the parameters for assessment of antioxidant potential of LPE(s). Results have been compared with both natural and synthetic antioxidants. All experimental data have indicated that LPE(s) possess strong antioxidant power proportional to their o-diphenolic and total polyphenolic content, independently from the assay used. Therefore, the LPE(s) antioxidant property was examined against the cytotoxic effects of reactive oxygen species in human colon carcinoma cells. Pretreatment of Caco-2 cells with liquorice polyphenolic extracts provided a remarkable protection against oxidative damage induced by H(2)O(2). The highest oxidative stress protection (72% of cell vitality) was measured in cells pretreated with 0.54 mM polyphenols. This effect seems to be associated to the antioxidant activity of liquorice polyphenolic compounds. Our data suggest that polyphenols from Glycyrrhiza glabra could exert a beneficial action in the prevention of intestinal pathologies related to production of reactive oxygen species.

Olive oil phenolic compounds inhibit homocysteine-induced endothelial cell adhesion regardless of their different antioxidant activity.

In this study, we examine the effect of extra virgin olive oil phenolic compounds on homocysteine-induced endothelial dysfunction and whether the protective effects are related to their different scavenging activities. Structurally related compounds have been assayed for their ability to reduce homocysteine-induced monocyte adhesion as well as the cell surface expression of intercellular adhesion molecule-1 (ICAM-1) in EA.hy.926 cells. As well-known, among the selected phenolic compounds, hydroxytyrosol, homovanillyl alcohol, and the hydroxycinnamic acid derivatives caffeic and ferulic acid display high scavenging activities, while tyrosol and p-coumaric acid are poorly active. All of the tested compounds, approaching potential in vivo concentrations, significantly reduce homocysteine-induced cell adhesion and ICAM-1 expression. Interestingly, we report the first evidence that monophenols tyrosol and p-coumaric acid are selectively protective only in homocysteine-activated cells, while they are ineffective in reducing ICAM-1 expression induced by TNFalpha. Finally, we report the synergistic effect of o-diphenolic and monophenolic compounds.

Protein isoaspartate methyltransferase prevents apoptosis induced by oxidative stress in endothelial cells: role of Bcl-Xl deamidation and methylation.

BACKGROUND: Natural proteins undergo in vivo spontaneous post-biosynthetic deamidation of specific asparagine residues with isoaspartyl formation. Deamidated-isomerized molecules are both structurally and functionally altered. The enzyme isoaspartyl protein carboxyl-O-methyltransferase (PCMT; EC 2.1.1.77) has peculiar substrate specificity towards these deamidated proteins. It catalyzes methyl esterification of the free alpha-carboxyl group at the isoaspartyl site, thus initiating the repair of these abnormal proteins through the conversion of the isopeptide bond into a normal alpha-peptide bond. Deamidation occurs slowly during cellular and molecular aging, being accelerated by physical-chemical stresses brought to the living cells. Previous evidence supports a role of protein deamidation in the acquisition of susceptibility to apoptosis. Aim of this work was to shed a light on the role of PCMT in apoptosis clarifying the relevant mechanism(s). METHODOLOGY/PRINCIPAL FINDINGS: Endothelial cells transiently transfected with various constructs of PCMT, i.e. overexpressing wild type PCMT or negative dominants, were used to investigate the role of protein methylation during apoptosis induced by oxidative stress (H(2)O(2); 0.1-0.5 mM range). Results show that A) Cells overexpressing "wild type" human PCMT were resistant to apoptosis, whereas overexpression of antisense PCMT induces high sensitivity to apoptosis even at low H(2)O(2) concentrations. B) PCMT protective effect is specifically due to its methyltransferase activity rather than to any other non-enzymatic interactions. In fact negative dominants, overexpressing PCMT mutants devoid of catalytic activity do not prevent apoptosis. C) Cells transfected with antisense PCMT, or overexpressing a PCMT mutant, accumulate isoaspartyl-containing damaged proteins upon H(2)O(2) treatment. Proteomics allowed the identification of proteins, which are both PCMT substrates and apoptosis effectors, whose deamidation occurs under oxidative stress conditions leading to programmed cell death. These proteins, including Hsp70, Hsp90, actin, and Bcl-xL, are recognized and methylated by PCMT, according to the general repair mechanism of this methyltransferase. CONCLUSION/SIGNIFICANCE: Apoptosis can be modulated by "on/off" switch partitioning the amount of specific protein effectors, which are either in their active (native) or inactive (deamidated) molecular forms. Deamidated proteins can also be functionally restored through methylation. Bcl-xL provides a case for the role of PCMT in the maintenance of functional stability of this antiapoptotic protein.

Resveratrol: from basic science to the clinic.

Plants produce an extraordinary array of low molecular mass natural products endowed with biological activity. Among these molecules, resveratrol (3,5,4'-trihydroxystilbene) has been identified as an inhibitor of carcinogenesis with a pleiotropic mode of action. Extensive literature on its anticancer activity, performed in cellular models, suggests a potential antiproliferative and apoptogenic use of the stilbene. Similarly, studies on implanted cancers and chemical-induced tumors confirm a potential chemotherapeutical interest of the compound. Moreover, recent intriguing studies have demonstrated, in mice, that the negative effects (insulin resistance and hyperglycemia) of a high-fat diet might be prevented by resveratrol treatment. Despite these promising observations, only few clinical trials have been performed on the compound due to the scarce interest of pharmaceutical industry. We suggest that resveratrol might be considered an interesting compound in association with more specific target-oriented drugs.