[Show abstract][Hide abstract] ABSTRACT: 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: -1.82±0.15, -2.15±0.06, and -2.59±0.48, respectively) in pathological RBCs. The results were validated by western blotting and immunofluorescence microscopy. In addition, beta-actin 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.
PLoS ONE 01/2014; 9(3):e93181. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Inflammation has been advocated as a possible common central mechanism for developmental cognitive impairment. Rett syndrome (RTT) is a devastating neurodevelopmental disorder, mainly caused by de novo loss-of-function mutations in the gene encoding MeCP2. Here, we investigated plasma acute phase response (APR) in stage II (i.e., "pseudo-autistic") RTT patients by routine haematology/clinical chemistry and proteomic 2-DE/MALDI-TOF analyses as a function of four major MECP2 gene mutation types (R306C, T158M, R168X, and large deletions). Elevated erythrocyte sedimentation rate values (median 33.0 mm/h versus 8.0 mm/h, P < 0.0001) were detectable in RTT, whereas C-reactive protein levels were unchanged (P = 0.63). The 2-DE analysis identified significant changes for a total of 17 proteins, the majority of which were categorized as APR proteins, either positive (n = 6 spots) or negative (n = 9 spots), and to a lesser extent as proteins involved in the immune system (n = 2 spots), with some proteins having overlapping functions on metabolism (n = 7 spots). The number of protein changes was proportional to the severity of the mutation. Our findings reveal for the first time the presence of a subclinical chronic inflammatory status related to the "pseudo-autistic" phase of RTT, which is related to the severity carried by the MECP2 gene mutation.
Mediators of Inflammation 01/2014; 2014:480980. · 3.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study mainly aims at examining the erythrocyte membrane fatty acid (FAs) profile in Rett syndrome (RTT), a genetically determined neurodevelopmental disease. Early reports suggest a beneficial effects of omega-3 polyunsaturated fatty acids (ω−3 PUFAs) on disease severity in RTT. A total of 24 RTT patients were assigned to ω−3 PUFAs-containing fish oil for 12 months in a randomized controlled study (average DHA and EPA doses of 72.9, and 117.1 mg/kg b.w./day, respectively). A distinctly altered FAs profile was detectable in RTT, with deficient ω−6 PUFAs, increased saturated FAs and reduced trans 20:4 FAs. FAs changes were found to be related to redox imbalance, subclinical inflammation, and decreased bone density. Supplementation with ω−3 PUFAs led to improved ω−6/ω−3 ratio and serum plasma lipid profile, decreased PUFAs peroxidation end-products, normalization of biochemical markers of inflammation, and reduction of bone hypodensity as compared to the untreated RTT group. Our data indicate that a significant FAs abnormality is detectable in the RTT erythrocyte membranes and is partially rescued by ω−3 PUFAs.
Prostaglandins, Leukotrienes and Essential Fatty Acids (PLEFA). 01/2014;
[Show abstract][Hide abstract] ABSTRACT: Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly linked to mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Respiratory dysfunction, historically credited to brainstem immaturity, represents a major challenge in RTT. Our aim was to characterize the relationships between pulmonary gas exchange abnormality (GEA), upper airway obstruction, and redox status in patients with typical RTT (n = 228) and to examine lung histology in a Mecp2-null mouse model of the disease. GEA was detectable in ~80% (184/228) of patients versus ~18% of healthy controls, with "high" (39.8%) and "low" (34.8%) patterns dominating over "mixed" (19.6%) and "simple mismatch" (5.9%) types. Increased plasma levels of non-protein-bound iron (NPBI), F2-isoprostanes (F2-IsoPs), intraerythrocyte NPBI (IE-NPBI), and reduced and oxidized glutathione (i.e., GSH and GSSG) were evidenced in RTT with consequently decreased GSH/GSSG ratios. Apnea frequency/severity was positively correlated with IE-NPBI, F2-IsoPs, and GSSG and negatively with GSH/GSSG ratio. A diffuse inflammatory infiltrate of the terminal bronchioles and alveoli was evidenced in half of the examined Mecp2-mutant mice, well fitting with the radiological findings previously observed in RTT patients. Our findings indicate that GEA is a key feature of RTT and that terminal bronchioles are a likely major target of the disease.
Mediators of Inflammation 01/2014; 2014:560120. · 3.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rett syndrome (RTT) is mainly caused by mutations in the X-linked methyl-CpG binding protein (MeCP2) gene. By binding to methylated promoters on CpG islands, MeCP2 protein is able to modulate several genes and important cellular pathways. Therefore, mutations in MeCP2 can seriously affect the cellular phenotype. Today, the pathways that MeCP2 mutations are able to affect in RTT are not clear yet. The aim of our study was to investigate the gene expression profiles in peripheral blood lymphomonocytes (PBMC) isolated from RTT patients to try to evidence new genes and new pathways that are involved in RTT pathophysiology. LIMMA (Linear Models for MicroArray) and SAM (Significance Analysis of Microarrays) analyses on microarray data from 12 RTT patients and 7 control subjects identified 482 genes modulated in RTT, of which 430 were upregulated and 52 were downregulated. Functional clustering of a total of 146 genes in RTT identified key biological pathways related to mitochondrial function and organization, cellular ubiquitination and proteosome degradation, RNA processing, and chromatin folding. Our microarray data reveal an overexpression of genes involved in ATP synthesis suggesting altered energy requirement that parallels with increased activities of protein degradation. In conclusion, these findings suggest that mitochondrial-ATP-proteasome functions are likely to be involved in RTT clinical features.
Mediators of Inflammation 01/2013; 2013:137629. · 3.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 F2-isoprostanes and 4-hydroxynonenal protein adducts. In addition, an oxidative damage of the erythrocyte membrane β -actin protein was evidenced. Therefore, the combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and β -actin alterations constitutes a previously unrecognized triad in classical autism and provides new biological markers in the diagnostic workup of ASDs.
Mediators of Inflammation 01/2013; 2013:432616. · 3.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rett syndrome (RTT) is a progressive neurodevelopmental disorder mainly caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Although over 200 mutations types have been identified so far, nine of which the most frequent ones. A wide phenotypical heterogeneity is a well-known feature of the disease, with different clinical presentations, including the classical form and the preserved speech variant (PSV). Aim of the study was to unveil possible relationships between plasma proteome and phenotypic expression in two cases of familial RTT represented by two pairs of sisters, harbor the same MECP2 gene mutation while being dramatically discrepant in phenotype, that is, classical RTT versus PSV. Plasma proteome was analysed by 2-DE/MALDI-TOF MS. A significant overexpression of six proteins in the classical sisters was detected as compared to the PSV siblings. A total of five out of six (i.e., 83.3%) of the overexpressed proteins were well-known acute phase response (APR) proteins, including alpha-1-microglobulin, haptoglobin, fibrinogen beta chain, alpha-1-antitrypsin, and complement C3. Therefore, the examined RTT siblings pairs proved to be an important benchmark model to test the molecular basis of phenotypical expression variability and to identify potential therapeutic targets of the disease.
Mediators of Inflammation 01/2013; 2013:438653. · 3.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanism of action of omega-3 polyunsaturated fatty acids ( ω -3 PUFAs) is only partially known. Prior reports suggest a partial rescue of clinical symptoms and oxidative stress (OS) alterations following ω -3 PUFAs supplementation in patients with Rett syndrome (RTT), a devastating neurodevelopmental disorder with transient autistic features, affecting almost exclusively females and mainly caused by sporadic mutations in the gene encoding the methyl CpG binding protein 2 (MeCP2) protein. Here, we tested the hypothesis that ω -3 PUFAs may modify the plasma proteome profile in typical RTT patients with MECP2 mutations and classic phenotype. A total of 24 RTT girls at different clinical stages were supplemented with ω -3 PUFAs as fish oil for 12 months and compared to matched healthy controls. The expression of 16 proteins, mainly related to acute phase response (APR), was changed at the baseline in the untreated patients. Following ω -3 PUFAs supplementation, the detected APR was partially rescued, with the expression of 10 out of 16 (62%) proteins being normalized. ω -3 PUFAs have a major impact on the modulation of the APR in RTT, thus providing new insights into the role of inflammation in autistic disorders and paving the way for novel therapeutic strategies.
Mediators of Inflammation 01/2013; 2013:723269. · 3.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate aqueous humour protein composition from retinoblastoma patients.
Prospective, hospital-based study.
Eighteen retinoblastoma patients (Reese-Ellsworth stage V or ABC classification group E RB) undergoing ocular enucleation, and 10 normal subjects undergoing cataract surgery. Five of 18 patients presented with associated secondary glaucoma whereas 13 had no secondary glaucoma; 5 of 13 patients with no secondary glaucoma received chemotherapeutical treatment with melphalan.
Aqueous humour samples were collected by limbal paracentesis of the anterior chamber after ocular enucleation in patients and after the stab peripheral corneal incision in controls. Total protein concentration according to Bradford method and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the samples were performed.
Aqueous humour protein concentration.
Aqueous humour protein concentration was significantly higher in retinoblastoma patients than controls (P < 0.01); patients with secondary glaucoma presented the highest values (P < 0.05 vs. controls); patients treated with melphalan presented a significant decrease (P < 0.01) versus non-treated; controls did not significantly differ from treated patients. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis pattern in retinoblastoma patients who did not receive any treatment was very different either from treated or from controls.
This study represents a preliminary step towards a more accurate two dimensional electrophoresis (2DE) pattern, which will be combined with mass spectrometry analysis to clarify the potential role of specific proteins in tumour development and progression; although these results suggest that aqueous humour protein pattern in retinoblastoma is characteristic, several aspects of the study are still under investigation.
Clinical and Experimental Ophthalmology 02/2012; 40(1):e8-e15. · 1.96 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The medicinal plant Mucuna pruriens (MP), with anti-snake properties has been reported to contain kunitz-type trypsin inhibitor. This study was done to further evaluate the protease inhibitory potential of gpMuc, a glycoprotein and other protein fractions from MP seeds against trypsin, chymotrypsin, Echis carinatus snake venom, ecarin and thrombin. The results obtained showed that gpMuc inhibited both trypsin and chymotrypsin activities and was thermally stable, maintaining its trypsin inhibitory activity at temperatures of up to 50°C. Its structural conformation was also maintained at pH ranges of 4-7. Immunoreactivity study confirms that it contains protease recognizing epitope on one of its isoforms. The whole protein extract of MP seeds inhibited prothrombin activation by ecarin and whole Echis carinatus venom, and also thrombin like activity using chromogenic assay.
Phytotherapy Research 01/2012; · 2.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mucuna pruriens (Fabaceae) is an established herbal drug used for the management of male infertility, nervous disorders, and also as an aphrodisiac. It has been shown that its seeds are potentially of substantial medicinal importance. The ancient Indian medical system, Ayurveda, traditionally used M. pruriens, even to treat such things as Parkinson's disease. M. pruriens has been shown to have anti-parkinson and neuroprotective effects, which may be related to its anti-oxidant activity. In addition, anti-oxidant activity of M. pruriens has been also demonstrated in vitro by its ability to scavenge DPPH radicals and reactive oxygen species. In this review the medicinal properties of M. pruriens are summarized, taking in consideration the studies that have used the seeds extracts and the leaves extracts.
Journal of traditional and complementary medicine. 01/2012; 2(4):331-339.
[Show abstract][Hide abstract] ABSTRACT: Purpose Compare the basic protein composition of the aqueous humor from retinoblastoma (RTB) patients with aqueous humor from patients with cataract who served as controls.Methods The study was carried out on 18 hospitalized RTB patients, who had undergone ocular enucleation (Reese-Ellsworth stage V or ABC classification group E RB), and 10 cataract patients. Five out of 18 RTB patients presented with associated secondary glaucoma and 5 out of 13 with no secondary glaucoma received chemotherapeutical treatment with Melphalan. The total protein concentration and electrophoresis pattern (SDS PAGE) of the aqueous humor was analysed.Results The aqueous humor from RTB patients had significantly higher total protein concentrations than controls (p<0.01); patients with secondary glaucoma presented with the highest total protein concentrations, and they were significantly different from controls (p<0.05), while those treated with Melphalan presented a 70% decreasing in protein concentration and, in respect to all non-treated patients, were significantly different (p<0.01). Besides, the protein content of controls was not significantly different from treated patients. The SDS PAGE pattern of RTB patients was very different from controls; yet, after chemotherapeutical treatment, patterns were similar.Conclusion This study represents a preliminary step towards a more accurate 2DE pattern, which will be combined with mass spectrometry analysis to clarify the potential role of specific proteins in tumor development and progression; although this results suggest that aqueous humor protein pattern in RTB is characteristic, several aspects of the study are still under investigation.
[Show abstract][Hide abstract] ABSTRACT: In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein. All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds.
Phytomedicine: international journal of phytotherapy and phytopharmacology 03/2011; 18(10):887-95. · 2.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Westergren method is considered as the reference procedure to measure Erythrocyte Sedimentation Rate (ESR) by the International Council for Standardization in Haematology. However, a closed automated method, VES Matic Cube 80 (DIESSE S.p.A., Siena, Italy), has been introduced as a new ESR measurement instrument. In this article, we report two different studies: first, we compared the two methods (Westergren and VES Matic Cube 80) and second, we correlated the inflammatory state of 248 patients with their ESR values. Total protein, albumin, C-reactive protein, and other inflammatory proteins were detected in each sample. The results obtained using VES Matic Cube 80 demonstrated a good correlation with those obtained using the Westergren method (Ordinary linear regression: y=0.955x-0.205, r(2) =0.816, P<0.05; Passing-Bablock regression equation: y=0.9153x-0.5763; Bland-Altman analysis: bias 1.2; limits of agreement -17.4-19.9) and with the inflammatory protein levels (CRP: r=0.554 and r=0.498 and Fibrinogen: r=0.699 and r=0.663 for Ves Matic Cube 80 and Westergren, respectively), supporting the hypothesis that VES Matic Cube 80 offers a fast and safe ESR determination, ensuring precision and a very good correlation with the reference method.
[Show abstract][Hide abstract] ABSTRACT: Alzheimer disease is the most common degenerative disorders of the brain. High concentrations of homocysteine, a sulfhydryl amino acid produced by the methionine cycle, have been linked to an increased risk of Alzheimer disease. The mechanism by which homocysteine contributes to the pathogenesis of Alzheimer disease is unknown; although it has been proposed that hyperhomocysteinemia produces changes in the structure and function of cerebral blood vessels and neurons and oxidative stress appears to play a major role in mediating these changes. Besides, homocysteine levels are influenced by vitamin B(12) and folate and seem to be modifiable through B vitamin supplementation. We wanted to verify whether some parameters are increased in patients with Alzheimer disease. In our study, we measured the levels of total homocysteine and the main oxidative stress markers (cysteine, thiol groups, malondialdehyde and purine metabolites) in blood samples from patients with Alzheimer disease, taking vitamin B(12) and folate supplements and from age-matched controls in order to evaluate the possible correlation between homocysteine and the main oxidative stress markers. We also measured vitamin B(12) and folate levels in order to verify their influence on homocysteine concentration. Our results show an increase in hypoxanthine, xanthine, homocysteine and cysteine levels in both patients with Alzheimer disease and the control group. The increase seems essentially related to age, rather than to the specific disease.
[Show abstract][Hide abstract] ABSTRACT: Echis carinatus venom (EV) is a complex mixture of toxins that contribute to its lethality. EV proteolytic activity was analyzed by zymography, chromogenic assays, and SDS-PAGE. To understand the molecular mechanism of the envenomation, we investigated the in vitro effect of EV on human plasma proteins. We looked for EV protein substrates and their proteolytic fragments. We analyzed EV proteolytic activity on standard proteins such as prothrombin or fibrinogen. To set up the optimal EV:plasma protein ratio conditions, plasma was incubated with EV (treated plasma), depleted of abundant proteins, and subjected to SDS-PAGE. Samples from control and treated plasma were also analyzed by 2-DE/MALDI-TOF MS, leading to the identification of four classes of plasma proteins cleaved by EV: proteases, protease inhibitors, binding proteins, and transporters. EV mainly proteolyzes entire proteins but can also act on physiological fragments. In summary, the physiological effects of EV proteases involve other important processes in addition to blood coagulation; complement activation and hemoglobin metabolism are also affected. In particular, the cleavage of protease inhibitors appears to be the mechanism through which the venom neutralizes the body's defenses.
[Show abstract][Hide abstract] ABSTRACT: Proteomic approach is an effective method to study changes in human plasma proteome. Coagulopathies are commonly encountered in victims of viper envenomation which were treated with an administration of immunoglobulin. Unfortunately, this treatment shows significant risk to the patient due to an anaphylactic reaction. Since Echis carinatus Venom (EV) toxins mainly acts both directly and indirectly on fibrinogen, we planned to establish a suitable analysis of its beta (FIBB) e gamma (FIBG) chains. This study will help us to understand the mechanism of envenomation and to find alternative treatments other than the common treatment with the administration of IgG.
We evaluated the EV proteolytic activity on whole human plasma proteome from the blood of an healthy volunteer. Two-dimensional electrophoresis (2-DE) using mini-gel was performed to analyse EV effects on the differents fibrinogen chains.
Changes in whole plasma proteome were focused on fibrinogen beta and gamma chains after EV incubation. Protein spots were detected and analyzed using ImageMaster 2D Platinum software. Results were represented as mean +/- standard deviation (mean+/-SD) with p<0.05 as a statistically significant value. 2-DE gel analysis showed that some spots of FIBB disappeared and some spots of FIBG decreased.
We found that the proteomic approach is a valid method in studying in-depth causes of different diseases, in particular those are involved in coagulopathies linked with proteins like fibrinogen from victims of viper envenomation.
[Show abstract][Hide abstract] ABSTRACT: Haptoglobin (Hpt) is an acute phase protein characterized by three major phenotypes (Hpt 1-1, Hpt 2-1 and Hpt 2-2). The Hpt 2-2 phenotype is associated with increased prevalence of various systemic diseases, including autoimmune disorders. Moreover, the Hpt 2-2 phenotype induces a shift from Th1 to Th2 response and increases fibrotic processes. On this basis, we performed serum proteomic analysis of patients with Systemic Sclerosis (SSc), a connective tissue disorder associated with Th2-type immune response and characterized by interstitial and perivascular fibrosis due to different factors (including genetic, environmental, immunological and microchimeric factors). Serum of 23 SSc outpatients patients (4 males, 19 females, mean age 54+-5.3 years) diagnosed according to the American Rheumatism Association (ARA) criteria, were considered for the proteomic analysis and compared to serum of 21 control subjects. Serum depleted of HAP was analyzed by 2-DE, and Hpt chain spots were identified by WB. The expression frequency of each Hpt α chain in SSc patients and controls was compared and quantitative analysis of spot expression (percent Vol) was performed. Above all,, our study amplifies the limited data in the literature on proteomic analysis in SSc, also confirming previous data that revealed a significant increase of haptoglobin type 2-2 and a concomitant decrease of the 1-1 phenotype in SSc patients. Moreover, our results demonstrate that c spots are more prevalent in SSc patients than in controls (91.3% vs 55.5%, p<0.05), while the expression frequency of a and b spots does not change. In patients Hpt 2-1 or Hpt 1-1 e spot is less abundant. According to our results, the c and e spots can be considered markers for SSc and thus be of use for the early diagnosis of connective tissue disorders and in establishing appropriate treatment.
International journal of immunopathology and pharmacology 01/2010; 23(3):901-9. · 2.99 Impact Factor