Cysteinylation and homocysteinylation of plasma protein thiols during ageing of healthy human beings

The purpose of the present study was to determine the relative amount of S-thiolated proteins (i.e. S-homocysteinylated, S-cysteinylglycinylated, S-glutathionylated and S-cysteinylated proteins) to the total protein thiols (i.e. the sum of reduced protein sulphydryl groups (PSHs) and protein mixed disulphides with homocysteine [HcySH], cysteinylglycine, cysteine [CysSH] and glutathione) in the plasma of healthy individuals aged 20 to 93. After plasma separation, total protein thiols, S-thiolated proteins, as well as CysSH, cystine, HcySH and homocystine were measured by high-performance liquid chromatography (HPLC) with fluorescence determination of the thiol-monobromobimane conjugate. Determination of plasma levels of protein thiols was performed by spectrophotometry with 5,5′-dithiobis(2-nitrobenzoic acid) as a titrating agent. The present study demonstrates an age-dependent reduction in the amount of PSHs, and an age-dependent increase in cysteinylated and homocysteinylated plasma proteins in healthy human beings. This indicates that the efficiency of the reduced protein thiol pool as an antioxidant defence system decreases with age, possibly causing an increased risk of irreversible oxidation (i.e. further oxidation to sulphinic and sulphonic acids, which are usually not reducible by thiol reducing agents) of sulphydryl groups of plasma proteins. The drop in the plasma level of protein sulphydryl groups suggests depletion and/or impairment of the antioxidant capacity of plasma, likely related to an alteration of the delicate balance between the different redox forms of thiols

Single Silver Nanoparticle instillation induced early and persisting moderate cortical damage in rat kidney

The potential toxic effects of silver nanoparticles (AgNPs), administered by a single intratracheal instillation (i.t), was assessed in a rat model using commercial physico-chemical characterized nanosilver. Histopathological changes, overall toxic response and oxidative stress (kidney and plasma protein carbonylation), paralleled by ultrastructural observations (TEM), were evaluated to examine renal responses 7 and 28 days after i.t. application of a low AgNP dose (50 g/rat), compared to an equivalent dose of ionic silver (7 g AgNO3/rat). The AgNPs caused moderate renal histopathological and ultrastructural alteration, in a region-specific manner, being the cortex the most affected area. Notably, the bulk AgNO3, caused similar adverse effects with a slightly more marked extent, also triggering apoptotic phenomena. Specifically, 7 days after exposure to both AgNPs and AgNO3, dilatation of the intercapillary and peripheral Bowman\u2019s space was observed, together with glomerular shrinkage. At day 28, these effects still persisted after both treatments, accompanied by an additional injury involving the vascular component of the mesangium, with interstitial micro-hemorrhages. Neither AgNPs nor AgNO3 induced oxidative stress effects in kidneys and plasma, at either time point. The AgNP-induced moderate renal effects indicate that, despite their benefits, novel AgNPs employed in consumer products need exhaustive investigation to ensure public health safety

Protective CD8+ T-cell responses to cytomegalovirus driven by rAAV/GFP/IE1 loading of dendritic cells

Background: Recent studies demonstrate that recombinant adeno-associated virus (rAAV)-based antigen loading of dendritic cells (DCs) generates in vitro, significant and rapid cytotoxic T-lymphocyte (CTL) responses against viral antigens. Methods: We used the rAAV system to induce specific CTLs against CVM antigens for the development of cytomegalovirus HCMV) gene therapy. As an extension of the versatility of the rAAV system, we incorporated immediate-early 1 (IE1), expressed in HCMV. Our rAAV vector induced a strong stimulation of CTLs directed against the HCMV antigen IE1. We then investigated the efficiency of the CTLs in killing IE1 targeted cells. Results: A significant MHC Class I-restricted, anti-IE1-specificCTL killing was demonstrated against IE1 positive peripheral blood mononuclear cells (PBMC) after one, in vitro, stimulation. Conclusion: In summary, single PBMC stimulation with rAAV/IE1 pulsed DCs induces strong antigen specific-CTL generation. CTLs were capable to lyse low doses of peptides pulsed into target cells. These data suggest that AAV-based antigen loading of DCs is highly effective for generating human CTL responses against HCMV antigens

Viability is associated with melanin-based coloration in the barn swallow (Hirundo rustica)

Pigmentation of body surface in animals can have multiple determinants and accomplish diverse functions. Eumelanin and pheomelanin are the main animal pigments, being responsible of yellow, brownish-red and black hues, and have partly common biosynthetic pathways. Many populations of vertebrates show individual variation in melanism, putatively with large heritable component. Genes responsible for eu- or pheomelanogenesis have pleiotropic but contrasting effects on life-history traits, explaining the patterns of covariation observed between melanization and physiology (e.g. immunity and stress response), sexual behavior and other characters in diverse taxa. Yet, very few studies in the wild have investigated if eu- and pheomelanization predict major fitness traits like viability or fecundity. In this correlative study, by contrasting adult barn swallows (Hirundo rustica) matched for age, sex, breeding site, and year and date of sampling, we show that males but not females that survived until the next year had paler, relatively more eu- than pheomelanic pigmentation of ventral body feathers. Better performance of individuals that allocate relatively more to eumelanogenesis was expected based on previous evidence on covariation between eumelanic pigmentation and specific traits related to immunity and susceptibility to stress. However, together with the evidence of no covariation between viability and melanization among females, this finding raises the question of the mechanisms that maintain variation in genes for melanogenesis. We discuss the possibility that eu- and pheomelanization are under contrasting viability and sexual selection, as suggested by larger breeding and sperm competition success of darker males from other barn swallow subspecies

Yolk vitamin E positively affects prenatal growth but not oxidative status in yellow-legged gull embryos

Parental effects occur whenever the phenotype of parents or the environment that they experience influences the phenotype and fitness of their offspring. In birds, parental effects are often mediated by the size and biochemical quality of the eggs in terms of maternally transferred components. Exogenous antioxidants are key egg components that accomplish crucial physiological functions during early life. Among these, vitamin E plays a vital role during prenatal development when the intense metabolism accompanying rapid embryo growth results in overproduction of pro-oxidant molecules. Studies of captive birds have demonstrated the positive effect of vitamin E supplementation on diverse phenotypic traits of hatchling and adult individuals, but its effects on embryo phenotype has never been investigated neither in captivity nor under a natural selection regime. In the present study, we experimentally tested the effect of the in ovo supplementation of vitamin E on morphological traits and oxidative status of yellow-legged gull (Larus michahellis) embryos. The supplementation of vitamin E promoted somatic growth in embryos soon before hatching, but did not affect their oxidative status. Our results suggest that maternally transferred vitamin E concentrations are optimized to prevent imbalances of oxidative status and the consequent raise of oxidative damage in yellow-legged gull embryos during prenatal development

Red blood cells protect albumin from cigarette smoke\u2013induced oxidation

Different studies reported the presence of oxidized (carbonylated) albumin in the extravascular pool, but not in the intravascular one of cigarette smokers. In this study we attempted to explain this apparent discrepancy exposing human serum albumin (HSA) to aqueous cigarette smoke extract (CSE). CSE induces HSA carbonylation and oxidation of the HSA Cys34 sulfhydryl group. An antioxidant action of glutathione, cysteine, and its synthetic derivative N-acetylcysteine was observed only at supra-physiological concentrations, suggesting that physiological (plasma) concentrations of glutathione and cysteine in the low micromolar range are ineffective in preventing cigarette smoke-induced oxidation of HSA. Differently, human erythrocytes resulted to be protective towards CSE-induced oxidation (carbonylation and thiol oxidation) of both HSA and total human plasma proteins

Plasma Protein Carbonylation in Haemodialysed Patients : focus on Diabetes and Gender

Patients with end-stage renal disease (ESRD) undergoing haemodialysis (HD) experience oxidative/carbonyl stress, which is postulated to increase after the HD session. The influence of diabetes mellitus and sex on oxidation of plasma proteins in ESRD has not yet been clarified despite that diabetic nephropathy is the most common cause of ESRD in developed and developing countries and despite the increasingly emerging differences between males and females in epidemiology, pathophysiology, clinical manifestations, and outcomes for several diseases. Therefore, this study aimed to evaluate the possible effect of type 2 diabetes mellitus, gender, and dialysis filter on plasma level of protein carbonyls (PCO) in ESRD patients at the beginning and at the end of a single HD session. Results show that mean post-HD plasma PCO levels are significantly higher than mean preHD plasma PCO levels and that the type of dialysis filter and dialysis technique are unrelated to plasma PCO levels. The mean level of plasma PCO after a HD session increases slightly but significantly in nondiabetic ESRD patients compared to diabetic ones, whereas it increases more markedly in women than in men. These novel findings suggest that women with ESRD are more susceptible than men to oxidative/carbonyl stress induced by HD

Sex-Related effects of reproduction on biomarkers of oxidative damage in free-living barn swallows (Hirundo rustica)

According to life-history theory, the allocation of limiting resources to one trait has negative consequences for other traits requiring the same resource, resulting in trade-offs among life-history traits, such as reproduction and survival. In vertebrates, oxidative stress is increasingly being considered among the physiological mechanisms forming the currency of life-history trade-offs. In this study of the barn swallow (Hirundo rustica), we focus on the oxidative costs of reproduction, especially egg laying, by investigating the effects of breeding stage (pre- vs. post-laying) and progression of the season on three biomarkers of oxidative damage (OD) to plasma proteins, namely the concentration of malondialdehyde (MDA)-protein adducts and of protein thiol groups (PSH), and the protein carbonyl (PCO) content. Moreover, we investigated whether males and females differed in plasma OD levels, because the inherent sex differences in reproductive roles and physiology may originate sex-specific patterns of OD during breeding. We found that MDA-protein adduct levels were higher in the pre-laying than in the post-laying phase, that males had lower levels of MDA-modified proteins than females, and that the decline of MDA-protein adduct concentration between the pre- and the post-laying phase was more marked for females than males. In addition, MDA-protein adduct levels declined with sampling date, but only during the pre-laying phase. On the other hand, plasma PCO levels increased from the pre- to the post-laying phase in both sexes, and females had higher levels of PCO than males. PSH concentration was unaffected by breeding stage, sex or sampling date. On the whole, our findings indicate that biomarkers of protein oxidation closely track the short-term variation in breeding stage of both male and female barn swallows. Moreover, the higher protein OD levels observed among females compared to males suggest that egg laying entails oxidative costs, which might negatively affect female residual reproductive value

Sulforaphane Cannot Protect Human Fibroblasts From Repeated, Short and Sublethal Treatments with Hydrogen Peroxide

A delicate balance of reactive oxygen species (ROS) exists inside the cell: when the mechanisms that control the level of ROS fail, the cell is in an oxidative stress state, a condition that can accelerate aging processes. To contrast the pro-aging effect of ROS, the supplementation of antioxidants has been recently proposed. Sulforaphane (SFN) is an isothiocyanate isolated from Brassica plants that has been shown to modulate many critical factors inside the cells helping to counteract aging processes. In the present work, we exposed human dermal fibroblast to short, sublethal and repeated treatments with hydrogen peroxide for eight days, without or in combination with low concentration of SFN. Hydrogen peroxide treatments did not affect the oxidative status of the cells, without any significant change of the intracellular ROS levels or the number of mitochondria or thiols in total proteins. However, our regime promoted cell cycle progression and cell viability, increased the anti-apoptotic factor survivin and increased DNA damage, measured as number of foci positive for -H2AX. On the other hand, the treatment with SFN alone seemed to exert a protective effect, increasing the level of p53, which can block the expansion of possible DNA damaged cells. However, continued exposure to SFN at this concentration could not protect the cells from stress induced by hydrogen peroxide

Early cytotoxic effects of ochratoxin A in rat liver: a morphological, biochemical and molecular study

We characterized the overall early effect of chronic ochratoxin A (OTA) treatment on rat liver, analyzing different aspects related to: (i) fibrosis, by measuring collagen content and turnover, and alpha-smooth muscle actin (alphaSMA); (ii) oxidative stress and stress response, by analyzing protein carbonylation, superoxide dismutase (SOD) and heat shock protein (HSP70) gene expression; (iii) the possible tumor promoter effect, evaluating cadherin and connexin (CX) mRNA levels. Light microscopy analysis showed no histological differences in OTA-treated and control (CT) rats. Collagen content, determined by computer analysis of Sirius red-stained liver sections, was similar in both groups. In liver homogenates COL-I, COL-III, TIMP-1 and TGF-beta1 mRNA levels and alphaSMA were unaffected by OTA. Matrix metalloproteinase (MMP)-1, MMP-2 and MMP-9 protein levels were also similar in the two groups. Protein carbonylation, a marker of severe oxidative stress, was not evident in the homogenates of OTA-treated livers; superoxide dismutase (SOD) mRNA tended to be lower and HSP70 was strongly down-regulated. OTA reduced E-cadherin and DSC-2 transcription, and down-regulated liver CX26, CX32 and CX43. In conclusion, these in vivo results show that OTA-induced liver injury involves a reduction in the ability to counterbalance oxidative stress, maybe leading to altered gap junction intercellular communication and loss of cell adhesion and polarity. This suggests that mild oxidative damage might be a key factor, in combination with other cytotoxic effects, in triggering the promotion of liver tumors after exposure to OTA