Marina Marini

Oxidative Stress and Erythrocyte Membrane Alterations in Children with Autism: Correlation with Clinical Features

It has been suggested that oxidative stress may play a role in the pathogenesis of Autism Spectrum Disorders (ASD), but the literature reports somewhat contradictory results. To further investigate the issue, we evaluated a high number of peripheral oxidative stress parameters, and some related issues such as erythrocyte membrane functional features and lipid composition. Twenty-one autistic children (Au) aged 5 to 12 years, were gender and age-matched with 20 typically developing children (TD). Erythrocyte thiobarbituric acid reactive substances, urinary isoprostane and hexanoyl-lysine adduct levels were elevated in Au, thus confirming the occurrence of an imbalance of the redox status of Au, whilst other oxidative stress markers or associated parameters (urinary 8-oxo-dG, plasma radical absorbance capacity and carbonyl groups, erythrocyte superoxide dismutase and catalase activities) were unchanged. A very significant reduction of Na+/K+-ATPase activity (−66%, p<0.0001), a reduction of erythrocyte membrane fluidity and alteration in erythrocyte fatty acid membrane profile (increase in monounsaturated fatty acids, decrease in EPA and DHA-ω3 with a consequent increase in ω6/ω3 ratio) were found in Au compared to TD, without change in membrane sialic acid content. Some Au clinical features appear to be correlated with these findings; in particular, hyperactivity score appears to be related with some parameters of the lipidomic profile and membrane fluidity. Oxidative stress and erythrocyte membrane alterations may play a role in the pathogenesis of ASD and prompt the development of palliative therapeutic protocols. Moreover, the marked decrease in NKA could be potentially utilized as a peripheral biomarker of ASD.

Moderate exercise training induces ROS-related adaptations to skeletal muscles.

Aim of the present work was the evaluation of the effects of moderate exercise training on 2 skeletal muscles differing in fibre-type composition, Tibialis Anterior (TA) and Soleus (SOL). Fibre adaptations, including their metabolic shift and mechanisms underlying proliferation and differentiation, oxidative stress markers, antioxidant and cytoprotective molecules, activity of Ca2+-handling molecules were examined. 6 male 2-month-old rats trained on a treadmill for 1 h/day, 3 days/week, for 14 weeks, reaching 30 m/min at the end of training. 6 age-matched sedentary rats served as controls. Rats were sacrificed 24 h after the last training session. Muscle regulatory factors increased in both muscles, activating satellite cell proliferation, which led to moderate hypertrophy in SOL and to moderate hyperplasia in TA, where the upregulation of desmin and TNFR2 expression suggests that myotube formation by proliferating myoblasts is somehow delayed. Changes leading to a more oxidative metabolism together with the upregulation of a number of antioxidant enzymes occurred in TA. HSP70i protein was upregulated in both SOL and TA, while oxidative stress markers increased in SOL alone. The status of ionic channels and pumps was preserved. We suggest that the increase in ROS, known to be associated with exercise, underlies most observed results.

Proteomic analysis and protein carbonylation profile in trained and untrained rat muscles

Understanding the relationship between physical exercise, reactive oxygen species and skeletal muscle modification is important in order to better identify the benefits or the damages that appropriate or inappropriate exercise can induce. Unbalanced ROS levels can lead to oxidation of cellular macromolecules and a major class of protein oxidative modification is carbonylation. The aim of this investigation was to study muscle protein expression and carbonylation patterns in trained and untrained animal models. We analyzed two muscles characterized by different metabolisms: tibialis anterior and soleus. Whilst tibialis anterior is mostly composed of fast-twitch fibers, the soleus muscle is mostly composed of slow-twitch fibers. By a proteomic approach we identified 15 protein spots whose expression is influenced by training. Among them in tibialis anterior we observed a down-regulation of several glycolitic enzymes. Concerning carbonylation, we observed the existence of a high basal level of protein carbonylation. Although this level shows some variation among individual animals, several proteins (mostly involved in energy metabolism, muscle contraction, and stress response) appear carbonylated in all animals and in both types of skeletal muscle. Moreover we identified 13 spots whose carbonylation increases after training.

Gene expression profile of rat left ventricles reveals persisting changes following chronic mild exercise protocol: implications for cardioprotection

BackgroundEpidemiological studies showed that physical exercise, specifically moderate lifelong training, is protective against cardiovascular morbidity and mortality. Most experimental work has focused into the effects and molecular mechanisms underlying intense, rather than mild exercise, by exploring the acute effect of training. Our study aims at investigating the cardioprotective effect of mild chronic exercise training and the gene expression profile changes at 48 hrs after the exercise cessation. Rats were trained at mild intensity on a treadmill: 25 m/min, 10%incline, 1 h/day, 3 days/week, 10 weeks; about 60% of the maximum aerobic power. By Affymetrix technology, we investigated the gene expression profile induced by exercise training in the left ventricle (LV) of trained (n = 10) and control (n = 10) rats. Cardioprotection was investigated by ischemia/reperfusion experiments (n = 10 trained vs. n = 10 control rats).ResultsMild exercise did not induce cardiac hypertrophy and was cardioprotective as demonstrated by the decreased infarct size (p = 0.02) after ischemia/reperfusion experiments in trained with respect to control rats. Ten genes and 2 gene sets (two pathways) resulted altered in LV of exercised animals with respect to controls. We validated by real-time PCR the increased expression of four genes: similar to C11orf17 protein (RGD1306959), caveolin 3, enolase 3, and hypoxia inducible factor 1 alpha. Moreover, caveolin 3 protein levels were higher in exercised than control rats by immunohistochemistry and Western Blot analysis. Interestingly, the predicted gene similar to C11orf17 protein (RGD1306959) was significantly increased by exercise. This gene has a high homology with the human C11orf17 (alias: protein kinase-A interacting protein 1 or breast cancer associated gene 3). This is the first evidence that this gene is involved in the response to the exercise training.ConclusionOur data indicated that few, but significant, genes characterize the gene expression profile of the rat LV, when examined 48 hrs since the last training section and that mild exercise training determines cardioprotection without the induction of hypertrophy.

Effect of training and sudden detraining on the patellar tendon and its enthesis in rats

BackgroundDifferent conditions may alter tendon characteristics. Clinical evidence suggests that tendon injuries are more frequent in athletes that change type, intensity and duration of training. Aim of the study was the assessment of training and especially detraining on the patellar tendon (PT) and its enthesis.Methods27 male adult Sprague-Dawley rats were divided into 3 groups: 20 rats were trained on a treadmill for 10 weeks. Of these, 10 rats were euthanized immediately after training (trained group), and 10 were caged without exercise for 4 weeks before being euthanized (de-trained group). The remaining 7 rats were used as controls (untrained rats). PT insertion, structure (collagen fiber organization and proteoglycan, PG, content), PT thickness, enthesis area, and subchondral bone volume at the enthesis were measured by histomorphometry and microtomography.ResultsBoth PG content and collagen fiber organization were significantly lower in untrained and detrained animals than in trained ones (p < 0.05 and p < 0.0001). In the detrained group, fiber organization and PG content were worse than that of the untrained groups and the untrained group showed a significantly higher score than the detrained group (p < 0.05). In the trained group, the PT was significantly thicker than in untrained group (p < 0.05). No significant differences in the enthesis area and subchondral bone volume among the three groups were seen.ConclusionsModerate exercise exerts a protective effect on the PT structure while sudden discontinuation of physical activity has a negative effect on tendons. The present results suggest that after a period of sudden de-training (such as after an injury) physical activity should be restarted with caution and with appropriate rehabilitation programs.

Modulation of paraoxonase 1 and 3 expression after moderate exercise training in the rat

Paraoxonases (PONs) are a small family of antioxidant enzymes whose antiatherogenic activity is well known. The aim of the present study was the evaluation of the effects of moderate aerobic training on their expression using a rat model. In order to discriminate between PON1 and PON3 enzymatic activity, we took advantage of some differences in their substrate preferences. PON1 and PON3 enzymatic activities and their protein levels were analyzed in plasma and in liver microsomes, and their mRNA levels in the liver. Exercise training did not affect PON1 expression or enzymatic activity but increased PON3 mRNA, protein levels, and enzymatic activity. Training also induced variations in plasma membrane composition, including an increase in polyunsaturated and a decrease in mono- and di-unsaturated fatty acids. On the other hand, acute exercise inhibited PON activities while increasing PON3 protein content in liver microsomes and reversing the relative composition in mono-, di-, and poly-unsaturated fatty acids, suggesting that physical stress, by altering membrane composition, may impair PON release from liver membranes. In conclusion, we documented, for the first time, the presence of PON3 in rat serum and, notably, found that the upregulation of PON3, rather than PON1, appears to be associated with physical training.

Impact of the Phosphatidylinositide 3-Kinase Signaling Pathway on the Cardioprotection Induced by Intermittent Hypoxia

Background Exposure to intermittent hypoxia (IH) may enhance cardiac function and protects heart against ischemia-reperfusion (I/R) injury. To elucidate the underlying mechanisms, we developed a cardioprotective IH model that was characterized at hemodynamic, biochemical and molecular levels. Methods Mice were exposed to 4 daily IH cycles (each composed of 2-min at 6-8% O2 followed by 3-min reoxygenation for 5 times) for 14 days, with normoxic mice as controls. Mice were then anesthetized and subdivided in various subgroups for analysis of contractility (pressure-volume loop), morphology, biochemistry or resistance to I/R (30-min occlusion of the left anterior descending coronary artery (LAD) followed by reperfusion and measurement of the area at risk and infarct size). In some mice, the phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin was administered (24 µg/kg ip) 15 min before LAD. Results We found that IH did not induce myocardial hypertrophy; rather both contractility and cardiac function improved with greater number of capillaries per unit volume and greater expression of VEGF-R2, but not of VEGF. Besides increasing the phosphorylation of protein kinase B (Akt) and the endothelial isoform of NO synthase with respect to control, IH reduced the infarct size and post-LAD proteins carbonylation, index of oxidative damage. Administration of wortmannin reduced the level of Akt phosphorylation and worsened the infarct size. Conclusion We conclude that the PI3K/Akt pathway is crucial for IH-induced cardioprotection and may represent a viable target to reduce myocardial I/R injury.

Perspective Biological Markers for Autism Spectrum Disorders: Advantages of the Use of Receiver Operating Characteristic Curves in Evaluating Marker Sensitivity and Specificity.

Autism Spectrum Disorders (ASD) are a heterogeneous group of neurodevelopmental disorders. Recognized causes of ASD include genetic factors, metabolic diseases, toxic and environmental factors, and a combination of these. Available tests fail to recognize genetic abnormalities in about 70% of ASD children, where diagnosis is solely based on behavioral signs and symptoms, which are difficult to evaluate in very young children. Although it is advisable that specific psychotherapeutic and pedagogic interventions are initiated as early as possible, early diagnosis is hampered by the lack of nongenetic specific biological markers. In the past ten years, the scientific literature has reported dozens of neurophysiological and biochemical alterations in ASD children; however no real biomarker has emerged. Such literature is here reviewed in the light of Receiver Operating Characteristic (ROC) analysis, a very valuable statistical tool, which evaluates the sensitivity and the specificity of biomarkers to be used in diagnostic decision making. We also apply ROC analysis to some of our previously published data and discuss the increased diagnostic value of combining more variables in one ROC curve analysis. We also discuss the use of biomarkers as a tool for advancing our understanding of nonsyndromic ASD.

Aerobic training affects fatty acid composition of erythrocyte membranes

The effect of exercise training on the fatty acid composition of erythrocyte membranes was evaluated in an experimental animal model where rats were subjected to a ten-wk aerobic training. Five groups of rats were compared: sedentary rats at 19 or 23 wks of age, rats trained at moderate or high intensity sacrificed at 19 wks of age, and rats trained at high intensity, and sacrificed following 4 weeks of sedentary life. We had already demonstrated that cardioprotection correlates with training intensity and partially persists in detrained rats. Main findings are that rats trained at higher intensity display consistent signs of lipid peroxidation but a lower ω6/ω3 ratio and a lower content of trans fatty acids when compared to rats trained at lower intensity and to older sedentary rats. Trans fatty acids negatively affect cell membrane fluidity and permeability. Detrained rats showed intermediate values. Gene expression evaluation of selected enzymes involved in lipid biosynthesis revealed some of the adaptive mechanisms leading to the maintenance of membrane fatty acid homeostasis following exercise. The decrease in the amount of trans fatty and in the inflammatory pathways (i.e. ω6/ω3 ratio) in high-intensity trained rats underscores the protective effect of high intensity aerobic training.

Proteomic and Carbonylation Profile Analysis of Rat Skeletal Muscles following Acute Swimming Exercise

Previous studies by us and other groups characterized protein expression variation following long-term moderate training, whereas the effects of single bursts of exercise are less known. Making use of a proteomic approach, we investigated the effects of acute swimming exercise (ASE) on protein expression and carbonylation patterns in two hind limb muscles: the Extensor Digitorum Longus (EDL) and the Soleus, mostly composed of fast-twitch and slow-twitch fibres, respectively. Carbonylation is one of the most common oxidative modifications of proteins and a marker of oxidative stress. In fact, several studies suggest that physical activity and the consequent increase in oxygen consumption can lead to increase in reactive oxygen and nitrogen species (RONS) production, hence the interest in examining the impact of RONS on skeletal muscle proteins following ASE. Results indicate that protein expression is unaffected by ASE in both muscle types. Unexpectedly, the protein carbonylation level was reduced following ASE. In particular, the analysis found 31 and 5 spots, in Soleus and EDL muscles respectively, whose carbonylation is reduced after ASE. Lipid peroxidation levels in Soleus were markedly reduced as well. Most of the decarbonylated proteins are involved either in the regulation of muscle contractions or in the regulation of energy metabolism. A number of hypotheses may be advanced to account for such results, which will be addressed in future studies.