Accumulation of altered aspartyl residues in erythrocyte membrane proteins from patients with sporadic amyotrophic lateral sclerosis.

Spontaneous protein deamidation of labile asparagines (Asn), generating abnormal l-isoaspartyl residues (IsoAsp), is associated with cell aging and enhanced by an oxidative microenvironment. The presence of isopeptide bonds impairs protein structure/function. To minimize the damage, IsoAsp can be "repaired" by the protein l-isoaspartyl/d-aspartyl O-methyltransferase (PIMT) and S-adenosylmethionine (AdoMet) is the methyl donor of this reaction. PIMT is a repair enzyme that initiates the conversion of l-isoAsp (or d-Asp) residues to l-Asp residues. Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease principally affecting motor neurons. The condition of oxidative stress reported in familial and sporadic forms of ALS prompted us to investigate Asn deamidation in ALS tissue. Erythrocytes (RBCs) were selected as a model system since they are unable to replace damaged proteins and protein methylesterification is virtually the only AdoMet-consuming reaction operating in these cells. Our data show that, in vitro assay, abnormal IsoAsp residues were significantly higher in ALS patients erythrocyte membrane proteins with an increased methyl accepting capability relative to controls (p<0.05). Moreover, we observed a reduction in AdoMet levels, while AdoHcy concentration was comparable to that detected in the control, resulting in a lower [AdoMet]/[AdoHcy] ratio. Then, the accumulation of altered aspartyl residues in ALS patients is probably related to a reduced efficiency of the S-adenosylmethionine (AdoMet)-dependent repair system causing increased protein instability at Asn sites. The increase of abnormal residues represents a new protein alteration that may be present not only in red blood cells but also in other cell types of patients suffering from ALS.

Effect of Annurca apple polyphenols on human HaCaT keratinocytes proliferation.

Polyphenols have been demonstrated to have clear antioxidant activities in vitro. However, in complex biological systems, they exhibit additional properties, which are yet poorly understood. The 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. The present study aimed at evaluating the Annurca apple polyphenol extract (APE) effects both proliferation and apoptosis on HaCaT cells. The data indicate that apple polyphenolic compounds had significant antiproliferative action on HaCaT cells. The fluorescence-activated cell-sorting analysis showed that APE induced cell apoptosis in a dose-dependent manner. Moreover, apple polyphenols induced apoptosis in epithelial cells by triggering a death receptor-associated extrinsic pathway p53-independent. APE was also capable of inducing morphological changes as evidenced by nuclear condensation. The cellular, morphological, and molecular data unequivocally demonstrated that induction of cellular apoptosis was mainly responsible for the previously observed antiproliferation-induced APE on HaCaT keratinocytes. Our experimental results suggest that apple polyphenols are a promising source from which a natural-based topical agent could be developed for skin diseases treatment.

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.

C-reactive protein is released in the coronary circulation and causes endothelial dysfunction in patients with acute coronary syndromes.

BACKGROUND: C-reactive protein (CRP) plasma levels correlate with cardiovascular events. Although a direct role for CRP in atherothrombosis has been suggested, at the moment little is known about its involvement in the pathophysiology of acute coronary syndromes (ACS). Thus, the aim of this study was to determine whether CRP is produced in the culprit lesion and released within the coronary circulation of patients with ACS and whether it may affect coronary endothelial function. METHODS: Blood samples were simultaneously obtained from the aorta (Ao) and the coronary sinus (CS) of patients with normal coronary artery (n=16), stable angina (n=30), and ACS (n=29) for later measurement of plasma CRP levels. Endothelium-dependent and -independent coronary vasodilation were evaluated by means of a Doppler Flow Wire in response to the increasing intracoronary doses of acetylcholine and adenosine, respectively. RESULTS: CRP plasma levels were significantly higher across the coronary circulation only in ACS patients with the culprit lesion located in the left coronary artery, while no differences between CS and Ao CRP plasma levels were observed in all other groups. Transcardiac CRP levels were correlated with impairment in coronary endothelium-dependent vasodilation. In six additional patients (SA=3 and ACS=3), subjected to coronary atherectomy, real-time quantitative PCR revealed presence of CRP mRNA only in unstable plaques. CONCLUSIONS: Thus, CRP is produced and released within the coronary circulation of patients with ACS; this is associated with impairment of endothelial function, suggesting a new pathophysiological link between CRP and ACS.

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.

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.

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.

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.

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.

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.

Matrix metalloproteinases and their inhibitors as biomarkers for metal toxicity in vitro.

Exposure to nickel and chromium, and their compounds, has been associated with adverse health effects. These metals are two human carcinogens whose pathogenesis involves active extracellular matrix degradation and remodelling. In this work we have compared the effects of in vitro exposure to nickel and chromium of a keratinocyte cell line (HaCat). The modulation of matrix metalloproteinase genes was used as biomarker of chemical damage. Confluent cells were constantly exposed to subtoxic chromium and nickel concentrations (10(-5) and 10(-7)M) up to 72 h. Total RNA was extracted and specific matrix metalloproteinase, and inhibitor, gene expression was analyzed by RT-PCR. Moreover, cell cycle alterations were evaluated by flow cytometry. Nickel and chromium showed different results, with an upregulation of MMP-2 mRNA production in nickel-treated cells while chromium exposure down-regulated MMP-2 mRNA production. This result could be correlated to the precocious (6h) over-expression of tissue inhibitor-1 (TIMP-1) mRNA in chromium-treated cells. Cell cycle analysis showed and increase of cells with 4N DNA. These results could be explained as a survival response of cells that escape metal induced apoptosis through the anti-apoptotic effects of TIMP-1. These cells that encompass the genotoxic insult may have a selective proliferation advantage, and therefore represent the precursor pool from which degenerating variants may emerge. To study if the chemical damage was reversible, subconfluent cells were stimulated only for 24 h, then the medium was replaced without metal. Cells were able to recover from nickel exposure, showing only weak alterations in specific mRNA expression and cell cycle alteration respect to control. Chromium-induced damage was irreversible. Our results demonstrated that there is an association between metal toxicity and expression of MMPs and their inhibitors. These biomarkers could be potentially useful to elaborate a prediction model of chemical toxicity.

Diverse effects of natural antioxidants on cyclosporin cytotoxicity in rat renal tubular cells.

BACKGROUND: As is well known, the use of the immunosuppressive drug cyclosporin A (CsA) is partially restricted by its nephrotoxic effects, which include early changes in haemodynamics followed by irreversible injuries to the renal tubules. Although the mechanisms responsible for these side effects are poorly understood, an involvement of reactive oxygen species (ROS) has been suggested. In this study, we selected three natural antioxidants, resveratrol, hydroxytyrosol and vitamin E, on the basis of their scavenging capabilities, and tested their protective effects against CsA toxicity. METHODS: Immortalized rat tubular cells (RPTc) were used as the model system. Cell viability was checked with trypan blue assay, and free radical formation was measured using the fluorescent probe 2,7-dichlorofluorescein (DCF). We evaluated several oxidative stress parameters, including phospholipid peroxidation products, glutathione levels and oxygenase expression. RESULTS: Incubation of RPTc with 25 muM CsA induced a significant decrease in cell viability paralleled by intracellular ROS formation and alterations in lipid peroxidation. There was also an imbalance of glutathione redox state as well as upregulation of heme oxygenase-1 (HO-1). The three antioxidants, at micromolar concentration, quantitatively prevented the ROS-activated DCF fluorescent signal and membrane lipid peroxidation. Both hydroxytyrosol and resveratrol strengthened the CsA induction of HO-1 expression. Moreover, vitamin E and resveratrol counteracted CsA-induced changes in the glutathione redox state via different mechanisms, whereas hydroxytyrosol was completely ineffective. Similarly, CsA-dependent nephrotoxicity was prevented by vitamin E, while resveratrol only exerted partial protection, and hydroxytyrosol showed no protective effects. CONCLUSION: Our results indicate that the diverse cytoprotective effects of the antioxidants tested in these studies were not directly related to their scavenging capabilities. These findings confirm a key role for glutathione in protecting cells from CsA-induced adverse effects and do not support a direct link between CsA-mediated ROS generation and adverse renal effects.

Hydroxytyrosol, a natural antioxidant from olive oil, prevents protein damage induced by long-wave ultraviolet radiation in melanoma cells.

Previous studies showed that long-wave ultraviolet (UVA) radiation induces severe skin damage through the generation of reactive oxygen species and the depletion of endogenous antioxidant systems. Recent results from our laboratory indicate a dramatic increase of both lipid peroxidation products (TBARS) and abnormal L-isoaspartyl residues, marker of protein damage, in UVA-irradiated human melanoma cells. In this study, the effects of hydroxytyrosol (DOPET), the major antioxidant compound present in olive oil, on UVA-induced cell damages, have been investigated, using a human melanoma cell line (M14) as a model system. In UVA-irradiated M14 cells, a protective effect of DOPET in preventing the uprise of typical markers of oxidative stress, such as TBARS and 2'7'-dichlorofluorescein (DCF) fluorescence intensity, was observed. In addition, DOPET prevents the increase of altered L-isoAsp residues induced by UVA irradiation. These protective effects are dose dependent, reaching the maximum at 400 microM DOPET. At higher concentrations, DOPET causes an arrest of M14 cell proliferation and acts as a proapoptotic stimulus by activating caspase-3 activity. In the investigated model system, DOPET is quantitatively converted into its methylated derivative, endowed with a radical scavenging ability comparable to that of its parent compound. These findings are in line with the hypothesis that the oxidative stress plays a major role in mediating the UVA-induced protein damage. Results suggest that DOPET may exerts differential effects on melanoma cells according to the dose employed and this must always be taken into account when olive oil-derived large consumer products, including cosmetics and functional foods, are employed.

Plasma protein aspartyl damage is increased in hemodialysis patients: studies on causes and consequences.

Plasma proteins in hemodialysis patients display a significant increase in deamidated/isomerized Asx (asparagine and aspartic acid) content, a marker of protein fatigue damage. This has been linked to the toxic effects of hyperhomocysteinemia in uremic erythrocytes; however, treatment aimed at abating homocysteine levels did not lead to significant reductions in plasma protein damage. The hypothesis that lack of reduction in protein damage could be due to protein increased intrinsic instability, as result of interference with the uremic milieu rather than to hyperhomocysteinemia, was put forward. The deamidated/isomerized Asx content of normal plasma incubated with several uremic toxins for 24 h, 72 h, and 7 d was measured, identifying a group of toxins that were able to elicit this kind of damage. Uremic toxins were also incubated with purified human albumin, and dose-response experiments with the two most toxic agents in terms of protein damage (guanidine and guanidinopropionic acid) were carried out. The effect of the hemodialysis procedure on protein damage was evaluated. For investigating also the consequences of these alterations, human albumin was treated in vitro to produce an increase in deamidated/isomerized Asx residues, and the effects of albumin deamidation on protein binding were evaluated. Among the uremic toxins that are able to elicit protein damage, guanidine produced a dose-dependent increase in protein damage. No difference was found after a hemodialysis session. Deamidated albumin shows normal binding capacity to warfarin, salicylic acid, or diazepam but reduced binding to homocysteine. In conclusion, uremic toxins, especially guanidine, display an ability to induce significant protein damage, which can in turn have functional consequences.

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.

Hyperhomocysteinemia and protein damage in chronic renal failure and kidney transplant pediatric patients--Italian initiative on uremic hyperhomocysteinemia (IIUH).

BACKGROUND: Plasma homocysteine, a new cardiovascular risk factor in both children and adults, is higher in chronic renal failure or kidney transplant patients. This alteration has been linked, in chronic renal failure, to plasma protein damage, represented by increased L-isoaspartyl residues. We measured plasma homocysteine levels and plasma protein damage in pediatric patients from four different Italian regions with conservatively treated renal failure; hemodialysis, continuous ambulatory peritoneal dialysis (CAPD), or transplants, to establish the presence of protein damage and the relative role of hyperhomocysteinemia. METHODS: High performance liquid chromatography (HPLC) separation measured total plasma homocysteine levels, using precolumn derivatization with ammonium 7-fluorobenzo-2-oxa-1, 3-diazole-4-sulphonate (SBD-F). Plasma protein L-isoaspartyl residues were quantitated using human recombinant protein carboxyl methyl transferase (PCMT). RESULTS: In all patient groups, homocysteine levels were significantly higher with respect to the control (Control: 6.87 +/- 0.73 microM) conservatively treated, 14.19 +/- 1.73 microM; hemodialysis, 27.03 +/- 4.32 microM; CAPD, 22.38 +/- 3.73 microM; transplanted, 20.22 +/- 2.27 microM, p < 0.001 vs. control]. Plasma protein damage was significantly higher in conservatively treated, hemodialysis (HD) and CAPD patients, while in transplant patients it was no different from the control. CONCLUSIONS: We concluded that in pediatric patients of different Italian geographical origin, plasma homocysteine levels were significantly higher in all groups with respect to healthy children; therefore contributing to the elevated cardiovascular risk present in these patients. Plasma protein L-isoaspartyl content was higher in renal failure patients, but kidney transplant patients had normal levels, indicating that this kind of protein damage relates more to the toxic action of uremic retention solutes, than to plasma homocysteine levels.

Folate treatment and unbalanced methylation and changes of allelic expression induced by hyperhomocysteinaemia in patients with uraemia.

BACKGROUND: Hyperhomocysteinaemia occurs in several genetically determined and acquired disorders and is highly prevalent in patients with uraemia. In these disorders, homocysteine precursor S-adenosylhomocysteine, a powerful competitive inhibitor of S-adenosylmethionine-dependent methyltransferases, is increased, suggesting unbalanced methylation. We aimed to investigate whether DNA hypomethylation is present in patients with uraemia who also have hyperhomocysteinaemia and whether regulation of specific classes of genes, dependent on DNA methylation, is compromised. METHODS: We selected men with hyperhomocysteinaemia and uraemia who were having standard haemodialysis treatment, and compared them with healthy male controls. We measured the homocysteine concentration from plasma samples and obtained DNA and RNA samples from peripheral mononuclear cells. DNA methylation was assessed by cytosine extension assay and by Southern blotting. Allelic expression of pseudoautosomal and imprinted genes was investigated by analysis of suitable restriction fragment length polymorphisms. FINDINGS: Total DNA hypomethylation was higher in patients than in controls (z score -4.593, p=0.0006) and allelic expression was changed in both sex-linked and imprinted genes. The shift from monoallelic to biallelic expression was dependent on homocysteine concentrations. Folate therapy, a common method to reduce hyperhomocysteinaemia, restored DNA methylation to normal levels and corrected the patterns of gene expression. INTERPRETATION: Our results suggest that hyperhomocysteinaemia affects epigenetic control of gene expression, which can be reverted by folate treatment. Our data support the hypothesis that the toxic action of homocysteine can be mediated by macromolecule hypomethylation.

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.