Papers by Giuseppe Belmonte
Mediators of Inflammation, 2013
Autism spectrum disorders (ASDs) are a complex group of neurodevelopment disorders steadily risin... more Autism spectrum disorders (ASDs) are a complex group of neurodevelopment disorders steadily rising in frequency and treatment refractory, where the search for biological markers is of paramount importance. Although red blood cells (RBCs) membrane lipidomics and rheological variables have been reported to be altered, with some suggestions indicating an increased lipid peroxidation in the erythrocyte membrane, to date no information exists on how the oxidative membrane damage may affect cytoskeletal membrane proteins and, ultimately, RBCs shape in autism. Here, we investigated RBC morphology by scanning electron microscopy in patients with classical autism, that is, the predominant ASDs phenotype (age range: 6-26 years), nonautistic neurodevelopmental disorders (i.e., "positive controls"), and healthy controls (i.e., "negative controls"). A high percentage of altered RBCs shapes, predominantly elliptocytes, was observed in autistic patients, but not in both control groups. The RBCs altered morphology in autistic subjects was related to increased erythrocyte membrane F 2 -isoprostanes and 4-hydroxynonenal protein adducts. In addition, an oxidative damage of the erythrocyte membrane -actin protein was evidenced. Therefore, the combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and -actin alterations constitutes a previously unrecognized triad in classical autism and provides new biological markers in the diagnostic workup of ASDs.
Oxidative Medicine and Cellular Longevity, 2015
Cigarette smoke (CS) contains over 4700 compounds, many of which can affect cellular redox balanc... more Cigarette smoke (CS) contains over 4700 compounds, many of which can affect cellular redox balance through free radicals production or through the modulation of antioxidant enzymes. The respiratory tract is one of the organs directly exposed to CS and it is known that CS can damage the integrity of lung epithelium by affecting cell junctions and increasing epithelium permeability. In this study, we have used a human lung epithelial cell line, Calu-3, to evaluate the effect of CS on lung epithelial cell junctions levels, with special focus on the expression of two proteins involved in intercellular communication: connexins (Cx) 40 and 43. CS exposure increased Cx40 gene expression but not of Cx43. CS also induced NF B activation and the formation of 4HNE-Cxs adducts. Since corilagin, a natural polyphenol, is able to inhibit NF B activation, we have determined whether corilagin could counteract the effect of CS on Cxs expression. Corilagin was able to diminish CS induced Cx40 gene expression, 4HNE-Cx40 adducts formation, and NF B activation. The results of this study demonstrated that CS induced the loss of cellular junctions in lung epithelium, possibly as a consequence of Cx-4HNE adducts formation, and corilagin seems to be able to abolish these CS induced alterations.
Alteration of serum lipid profqile, SRB1 loss and impaired Nrf2 activation in CDKL5 disorder
Free Radical Biology and Medicine, 2015
CDKL5 mutation is associated with an atypical Rett syndrome (RTT) variant. Recently, cholesterol ... more CDKL5 mutation is associated with an atypical Rett syndrome (RTT) variant. Recently, cholesterol homeostasis perturbation and oxidative-mediated loss of HDL receptor SRB1 in typical RTT has been suggested. Here, we demonstrated an altered lipid serum profile also in CDKL5 patients with decreased levels of SRB1 and impaired activation of the defensive system Nrf2. In addition, CDKL5 fibroblasts showed an increase in 4-hydroxy-2-nonenal- and nitrotyrosine-SRB1 adducts that lead to its ubiquitination and probable degradation. This study highlights a possible common denominator between two different RTT variants (MECP2 and CDKL5) and a possible common future therapeutic target.
NADPH oxidase activation and 4-hydroxy-2-nonenal/aquaporin-4 adducts as possible new players in oxidative neuronal damage presents in drug-resistant epilepsy
Biochimica et biophysica acta, 2015
A correlation between epilepsy and cellular redox imbalance has been suggested, although the mech... more A correlation between epilepsy and cellular redox imbalance has been suggested, although the mechanism by which oxidative stress (OS) can be implicated in this disorder is not clear. In the present study several oxidative stress markers and enzymes involved in OS have been determined. In particular, we examined the levels of 4-hydroxy-2-nonenal protein adducts (HNE-PA), a by-product of lipid peroxidation, and the activation of NADPH oxidase 2 (NOX2), as cellular source of superoxide (O2(-)), in surgically resected epileptic tissue from drug-resistant patients (N=50). In addition, we investigated whether oxidative-mediated protein damage can affect aquaporin-4 (AQP4), a water channel implicated in brain excitability and epilepsy. Results showed high levels of HNE-PA in epileptic hippocampus, in both neurons and glial cells and cytoplasmic positivity for p47(phox) and p67(phox) suggesting NOX2 activation. Interestingly, in epileptic tissue immunohistochemical localization of AQP4 was ...
Nutrition research and practice, 2014
Obesity is an epidemic disease characterized by an increased inflammatory state and chronic oxida... more Obesity is an epidemic disease characterized by an increased inflammatory state and chronic oxidative stress with high levels of pro-inflammatory cytokines and lipid peroxidation. Moreover, obesity alters cholesterol metabolism with increases in low-density lipoprotein (LDL) cholesterols and triglycerides and decreases in high-density lipoprotein (HDL) cholesterols. It has been shown that mulberry leaf and fruit ameliorated hyperglycemic and hyperlipidemic conditions in obese and diabetic subjects. We hypothesized that supplementation with mulberry leaf combined with mulberry fruit (MLFE) ameliorate cholesterol transfer proteins accompanied by reduction of oxidative stress in the high fat diet induced obesity. Mice were fed control diet (CON) or high fat diet (HF) for 9 weeks. After obesity was induced, the mice were administered either the HF or the HF with combination of equal amount of mulberry leaf and fruit extract (MLFE) at 500mg/kg/day by gavage for 12 weeks. MLFE treatment a...
International Journal of Molecular Sciences, 2010
Cutaneous tissue is the first barrier against outdoor insults. The outer most layer of the skin, ... more Cutaneous tissue is the first barrier against outdoor insults. The outer most layer of the skin, the stratum corneum (SC), is formed by corneocytes embedded in a lipid matrix (cholesterol, ceramide and fatty acids). Therefore, the regulation of lipids and, in particular, of cholesterol homeostasis in the skin is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a key role in maintaining cellular cholesterol levels. Among the many factors that have been associated with skin diseases, the environmental stressor cigarette smoke has been recently studied. In the present study, we demonstrate that ABCA1 expression in human cells (HaCaT) was increased (both mRNA and protein levels) after CS exposure. This effect was mediated by the inhibition of NFkB (aldehydes adducts formation) that allows the translocation of liver X receptor (LXR). These findings suggest that passive smoking may play a role in skin cholesterol levels and thus affect cutaneous tissues functions.
BioMed Research International, 2015
PLoS ONE, 2012
Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol up... more Scavenger Receptor B1 (SR-B1), also known as HDL receptor, is involved in cellular cholesterol uptake. Stratum corneum (SC), the outermost layer of the skin, is composed of more than 25% cholesterol. Several reports support the view that alteration of SC lipid composition may be the cause of impaired barrier function which gives rise to several skin diseases. For this reason the regulation of the genes involved in cholesterol uptake is of extreme significance for skin health. Being the first shield against external insults, the skin is exposed to several noxious substances and among these is cigarette smoke (CS), which has been recently associated with various skin pathologies. In this study we first have shown the presence of SR-B1 in murine and human skin tissue and then by using immunoblotting, immunoprecipitation, RT-PCR, and confocal microscopy we have demonstrated the translocation and the subsequent lost of SR-B1 in human keratinocytes (cell culture model) after CS exposure is driven by hydrogen peroxide (H 2 O 2 ) that derives not only from the CS gas phase but mainly from the activation of cellular NADPH oxidase (NOX). This effect was reversed when the cells were pretreated with NOX inhibitors or catalase. Furthermore, CS caused the formation of SR-B1-aldheydes adducts (acrolein and 4-hydroxy-2-nonenal) and the increase of its ubiquitination, which could be one of the causes of SR-B1 loss. In conclusion, exposure to CS, through the production of H 2 O 2 , induced post-translational modifications of SR-B1 with the consequence lost of the receptor and this may contribute to the skin physiology alteration as a consequence of the variation of cholesterol uptake.
Reproduction, 2014
In studies carried out previously, we demonstrated that small ubiquitin-like modifier 1 (SUMO1) i... more In studies carried out previously, we demonstrated that small ubiquitin-like modifier 1 (SUMO1) is associated with poor sperm motility when evaluated with a protocol that reveals mostly SUMO1-ylated live sperm. Recently, with another protocol, it has been demonstrated that SUMO is expressed in most sperm and is related to poor morphology and motility, suggesting that sumoylation may have multiple roles depending on its localisation and targets. We show herein, by confocal microscopy and co-immunoprecipitation, that dynaminrelated protein 1 (DRP1), Ran GTPase-activating protein 1 (RanGAP1) and Topoisomerase IIa, SUMO1 targets in somatic and/or germ cells, are SUMO1-ylated in mature human spermatozoa. DRP1 co-localises with SUMO1 in the mid-piece, whereas RanGAP1 and Topoisomerase IIa in the post-acrosomal region of the head. Both SUMO1 expression and co-localisation with the three proteins were significantly higher in morphologically abnormal sperm, suggesting that sumoylation represents a marker of defective sperm. DRP1 sumoylation at the mid-piece level was higher in the sperm of asthenospermic men. As in somatic cells, DRP1 sumoylation is associated with mitochondrial alterations, this protein may represent the link between SUMO and poor motility. As SUMO pathways are involved in responses to DNA damage, another aim of our study was to investigate the relationship between sumoylation and sperm DNA fragmentation (SDF). By flow cytometry, we demonstrated that SUMO1-ylation and SDF are correlated (rZ0.4, P!0.02, nZ37) and most sumoylated sperm shows DNA damage in co-localisation analysis. When SDF was induced by stressful conditions (freezing and thawing and oxidative stress), SUMO1-ylation increased. Following freezing and thawing, SUMO1-Topoisomerase IIa co-localisation and co-immunoprecipitation increased, suggesting an involvement in the formation/repair of DNA breakage.
Oxidative Medicine and Cellular Longevity, 2015
Cigarette smoke (CS) contains over 4700 compounds, many of which can affect cellular redox balanc... more Cigarette smoke (CS) contains over 4700 compounds, many of which can affect cellular redox balance through free radicals production or through the modulation of antioxidant enzymes. The respiratory tract is one of the organs directly exposed to CS and it is known that CS can damage the integrity of lung epithelium by affecting cell junctions and increasing epithelium permeability. In this study, we have used a human lung epithelial cell line, Calu-3, to evaluate the effect of CS on lung epithelial cell junctions levels, with special focus on the expression of two proteins involved in intercellular communication: connexins (Cx) 40 and 43. CS exposure increased Cx40 gene expression but not of Cx43. CS also induced NF B activation and the formation of 4HNE-Cxs adducts. Since corilagin, a natural polyphenol, is able to inhibit NF B activation, we have determined whether corilagin could counteract the effect of CS on Cxs expression. Corilagin was able to diminish CS induced Cx40 gene expression, 4HNE-Cx40 adducts formation, and NF B activation. The results of this study demonstrated that CS induced the loss of cellular junctions in lung epithelium, possibly as a consequence of Cx-4HNE adducts formation, and corilagin seems to be able to abolish these CS induced alterations.
Modulation of scavenger receptor B1 (SRB1) levels in human keratynocites by cigarette smoke The role of H202
Aging and oxidative stress affect lung vitamin E tissue uptake
Effect of Cigarette Smoke on Keratineocites Scavenger Receptor B1 exspression. Aldehydes vs H202
Evaluation of three ozonated oil effect in wound healing using the SKH1 mice as a model
S7-4-Scavenger Receptor B1 oxidative post-translational modifications are responsible for its loss in Rett syndrome
The modulation of the HDL receptor scavenger receptor B1 (SRB1) was evaluated in skin fibroblasts... more The modulation of the HDL receptor scavenger receptor B1 (SRB1) was evaluated in skin fibroblasts isolated from Rett syndrome (RTT) patients, a rare neurodevelopmental disorder affecting almost exclusively females associated in up to 95% of cases to de novo loss-of-function mutations in the X-chromosome-linked gene encoding the methyl-CpG-binding protein 2 (MeCP2). Patients showed an altered plasma lipid profile, while their skin fibroblasts showed a dramatic reduction in SRB1 (immunogold, Western blot and immunohistochemistry). The decreased SRB1 levels were demonstrated to be the consequence of its binding with 4-hydroxy-2-nonenal (4HNE), a product of lipid peroxidation, and its increased ubiquitination. Therefore the loss of SRB1 in RTT cells is a consequence of the chronic oxidative stress status present in RTT. In addition RTT fibroblast presented high intracellular levels of H2O2 and 4HNE protein adducts. This finding was correlated with the constitutive activation of NADPH oxidase (NOX) and was reverted by DPI (NOX inhibitor) or Desferal (Iron chelator) pre-treatment. To confirm the alteration of status redox in RTT cells, the activity of several enzymes involved in protecting the cell from OS was also evaluated. Glutathione peroxidase (GPx), Supeoxide dismutase and Glucose-6-phosphate dehydrogenase (G6PDH) activity were decreased respect to control. These data paralleled with a constitutive activation of NRF2 and elevated gene expression of Heme oxigenase-1 (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO-1). Of note, when NRF2 pathway was stimulated via exogenous oxidants, RTT fibroblast did not respond as the control cells.
Wound Repair and Regeneration, 2011
Ozone is well recognized as a bactericidal agent and its beneficial effect on wound healing could... more Ozone is well recognized as a bactericidal agent and its beneficial effect on wound healing could be a consequence of this property. Because ozone itself does not penetrate the cells but immediately reacts with polyunsaturated fatty acids, its effects should be the results of oxidative reaction. For this reason, ozonated oils could be a way to deliver ozone messengers to the skin. This paper evaluated the therapeutic effects of three different grades of ozonated sesame oil in acute cutaneous wounds made in the skin of SKH1 mice. Specifically, wound closure rate, histological parameters, and the level of key proteins such as vascular endothelial growth factors and cyclin D1 have been analyzed in relation to the peroxide level present in the ozonated oil. Treatment with moderately ozonated sesame oilexpressed as peroxide value about 1,500)-has a faster wound closure rate in the first 7 days than treatment with oil containing either lower or higher peroxide value, and even with controls. Moreover, under the same treatment, an earlier and higher response of cells involved in wound repair, a higher angiogenesis, as well as an enhanced vascular endothelial growth factors and cyclin D1 expression were observed. The present study shows the validity of ozonated sesame oil in cutaneous wound healing and emphasizes the importance of the ozonation grade.
Wound tissue repair is a complex and dynamic process of restoring cellular structures and tissue ... more Wound tissue repair is a complex and dynamic process of restoring cellular structures and tissue layers. Improvement of this process is crucial for several pathologies characterized by chronic delayed wound closure such as diabetes, and the investigation of new approaches aimed to ameliorate the wound healing process is under continuous evolution. Recently, the usage of vegetable matrices in the form of ozonated oils has been proposed and several researchers have shown a positive effect in the wound, based on their bactericidal, antiviral, and antifungal properties. The present study was undertaken to compare the effect that different ozonated oils (olive, sesame and linseed) with the same level of ozonation have on wound healing rate in SKH1 mice. Several histological parameters and the level of key proteins such as VEGF and PCNA have been analyzed. Only treatment with ozonated sesame oil shows a faster wound closure in the first 7 days. This effect paralleled with the increased VEGF and PCNA levels, NFB nuclear translocation and 4-HNE formation. The present study shows that not only the ozonation grade is of importance for the improvement of wound healing process but also the typical composition of the oil.
PLoS ONE, 2014
Beta-actin, a critical player in cellular functions ranging from cell motility and the maintenanc... more Beta-actin, a critical player in cellular functions ranging from cell motility and the maintenance of cell shape to transcription regulation, was evaluated in the erythrocyte membranes from patients with typical Rett syndrome (RTT) and methyl CpG binding protein 2 (MECP2) gene mutations. RTT, affecting almost exclusively females with an average frequency of 1:10,000 female live births, is considered the second commonest cause of severe cognitive impairment in the female gender. Evaluation of beta-actin was carried out in a comparative cohort study on red blood cells (RBCs), drawn from healthy control subjects and RTT patients using mass spectrometry-based quantitative analysis. We observed a decreased expression of the beta-actin isoforms (relative fold changes for spots 1, 2 and 3: 21.8260. 15, 22.1560.06, and 22.5960.48, respectively) in pathological RBCs. The results were validated by western blotting and immunofluorescence microscopy. In addition, betaactin from RTT patients also showed a dramatic increase in oxidative posttranslational modifications (PTMs) as the result of its binding with the lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE). Our findings demonstrate, for the first time, a beta-actin down-regulation and oxidative PTMs for RBCs of RTT patients, thus indicating an altered cytoskeletal organization.
Oxidative Medicine and Cellular Longevity, 2014
Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT),... more Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16) we investigated four different oxidative stress markers, F 2 -Isoprostanes (F 2 -IsoPs), F 4 -Neuroprostanes (F 4 -NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F 4 -NeuroPs (12-folds), F 2 -IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (−43.6%) and GSH/GSSG ratio (−3.05 folds). A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients.
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Papers by Giuseppe Belmonte