Laura Maria Gaeta

Axonal degeneration in paraplegin-deficient mice is associated with abnormal mitochondria and impairment of axonal transport

In several neurodegenerative diseases, axonal degeneration occurs before neuronal death and contributes significantly to patients disability. Hereditary spastic paraplegia (HSP) is a genetically heterogeneous condition characterized by selective degeneration of axons of the corticospinal tracts and fasciculus gracilis. HSP may therefore be considered an exemplary disease to study the local programs mediating axonal degeneration. We have developed a mouse model for autosomal recessive HSP due to mutations in the SPG7 gene encoding the mitochondrial ATPase paraplegin. Paraplegin-deficient mice are affected by a distal axonopathy of spinal and peripheral axons, characterized by axonal swelling and degeneration. We found that mitochondrial morphological abnormalities occurred in synaptic terminals and in distal regions of axons long before the first signs of swelling and degeneration and correlated with onset of motor impairment during a rotarod test. Axonal swellings occur through massive accumulation of organelles and neurofilaments, suggesting impairment of anterograde axonal transport. Retrograde axonal transport is delayed in symptomatic mice. We speculate that local failure of mitochondrial function may affect axonal transport and cause axonal degeneration. Our data suggest that a timely therapeutic intervention may prevent the loss of axons.

Friedreich's ataxia: Oxidative stress and cytoskeletal abnormalities

Friedreichs ataxia (FRDA) is an autosomal recessive disorder caused by mutations in the gene encoding frataxin, a mitochondrial protein implicated in iron metabolism. Current evidence suggests that loss of frataxin causes iron overload in tissues, and increase in free-radical production leading to oxidation and inactivation of mitochondrial respiratory chain enzymes, particularly Complexes I, II, III and aconitase. Glutathione plays an important role in the detoxification of ROS in the Central Nervous System (CNS), where it also provides regulation of protein function by glutathionylation. The cytoskeletal proteins are particularly susceptible to oxidation and appear constitutively glutathionylated in the human CNS. Previously, we showed loss of cytoskeletal organization in fibroblasts of patients with FRDA found to be associated with increased levels of glutathione bound to cytoskeletal proteins. In this study, we analysed the glutathionylation of proteins in the spinal cord of patients with FRDA and the distribution of tubulin and neurofilaments in the same area. We found, for the first time, a significant rise of the dynamic pool of tubulin as well as abnormal distribution of the phosphorylated forms of human neurofilaments in FRDA motor neurons. In the same cells, the cytoskeletal abnormalities co-localized with an increase in protein glutathionylation and the mitochondrial proteins were normally expressed by immunocytochemistry. Our results suggest that in FRDA oxidative stress causes abnormally increased protein glutathionylation leading to prominent abnormalities of the neuronal cytoskeleton.

Protein glutathionylation in human central nervous system: Potential role in redox regulation of neuronal defense against free radicals

Neuronal defense against free radicals is mediated primarily by the glutathione system. A cerebral defect of this system gives rise to the oxidative stress occurring in some neurological diseases. Glutathione provides a means of regulating protein function by glutathionylation, consisting of the formation of mixed disulfides between cysteines and glutathione. The glutathionylation of proteins, during both constitutive metabolism and oxidative stress, represents for the cell a mechanism to link physiological processes, and/or adaptive stress responses, to changes in intracellular redox states. In this study, we analyzed the topographic distribution of the protein glutathionylation normally occurring in human central nervous system. Constitutively glutathionylated proteins appeared uniformly distributed throughout all cortical layers of the cerebral and cerebellar cortex as well as throughout the gray matter of the spinal cord. The degree of immunocytochemical staining was clear in neurons, mild in oligodendrocytes, and weaker in astrocytes. The proteins preferentially glutathionylated were cytoskeletal proteins. Our results suggest a potential role of glutathionylation in the redox regulation of neuronal survival and in the control of axon/dendrite stability.

Determination of superoxide dismutase and glutathione peroxidase activities in blood of healthy pediatric subjects

BACKGROUNDnGiven the growing requirement of antioxidant enzymes measurements in laboratory and the increasing role of SOD/GPx ratio in the balance of reactive oxygen species (ROS), the aim of our study was to contribute to define reference values of enzyme activities in Italian healthy children, by determining SOD in erythrocytes and GPx in whole blood.nnnMETHODSnSOD (E.C.1.15.1.1) and GPx (E.C.1.11.1.9) activities were spectrophotometrically assayed in erythrocytes with commercial kits. SOD activity was expressed as the amount of protein causing a 50% inhibition of formazan dye (505 nm), employing xanthine and xanthine oxidase to generate superoxide radicals. Units of GPx activity were calculated following NADPH oxidation at 340 nm using cumene hydroperoxide as the substrate.nnnRESULTSnSuperoxide dismutase (SOD) and glutathione peroxidase (GPx) activities were assayed in blood of 45 healthy pediatric subjects (26 males and 19 females between 0 and 14 years of age). We found no significant differences in blood antioxidant enzymes both in all population and when we distributed the subjects for age classes and sex.nnnCONCLUSIONnthe definition of pediatric values of antioxidant enzyme activities in blood of healthy children may be useful in monitoring SOD and GPx in physiologic and pathologic conditions, also for future therapeutic trials.

Myosin as a potential redox-sensor: an in vitro study

A balanced redox status is necessary to optimize force production in contractile apparatus, where free radicals generated by skeletal muscle are involved in some basic physiological processes like excitation–contraction coupling. Protein glutathionylation has a key role in redox regulation of proteins and signal transduction. Here we show that myosin is sensitive to inxa0vitro glutathionylation and MALDI-TOF analysis identified three potential sites of glutathione binding, two of them locating on the myosin head. Glutathionylation of myosin has an important impact on the protein structure, as documented by the lower fluorescence quantum yield of glutathionylated myosin and its increased susceptibility to the proteolytic cleavage. Myosin function is also sensitive to glutathionylation, which modulates its ATPase activity depending on GSSG redox balance. Thus, like the phosphorylation/dephosphorylation cycle, glutathionylation may represent a mechanism by which glutathione modulates sarcomere functions depending on the tissue redox state, and myosin may constitute a muscle redox-sensor.

Protein glutathionylation increases in the liver of patients with non‐alcoholic fatty liver disease

Background and Aim:u2002 Oxidative stress is an important pathophysiological mechanism in non‐alcoholic steatohepatitis, where hepatocyte apoptosis is significantly increased correlating with disease severity. Protein glutathionylation occurs as a response to oxidative stress, where an increased concentration of oxidized glutathione modifies post‐translational proteins by thiol disulfide exchange. In this study, we analyzed the protein glutathionylation in non‐alcoholic fatty liver disease (NAFLD) and evaluated a potential association between glutathionylation, fibrosis, and vitamin E treatment.

Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography

Since glutathionyl-hemoglobin has been suggested to be a clinical marker of oxidative stress in human blood and given the growing biological relevance of oxidative stress as a pathogenic factor in several diseases, we describe a method to measure glutathionyl-hemoglobin concentration in erythrocytes, by using cation-exchange high-pressure liquid chromatography with UV detection. The glutathionyl-hemoglobin peak has been identified on the basis of the following findings: (a) the peak increased when the sample was incubated with oxidized glutathione; (b) the peak disappeared when the sample was reduced with dithiothreitol, with the simultaneous increase of that corresponding to hemoglobin A(0); (c) the peak could be detected by incubating hemoglobin A(0) with reduced glutathione; (e) deconvoluted mass spectrum of the glutathionyl-hemoglobin peak showed a 16172.0-Da molecular mass, corresponding to hemoglobin beta bound to glutathione. Glutathionyl-hemoglobin concentration has been determined in erythrocytes of 40 healthy subjects, with a mean value of 2.58+/-0.7%, calculated as the percentage of its peak area ratio to that of total hemoglobin (HbA(0)+HbA(2)+HbA(1C)+glutathionyl-hemoglobin). The availability of a simple and reproducible method to detect glutathionyl-hemoglobin concentration in blood could be useful in monitoring oxidative stress, and for investigating the efficacy of antioxidant therapies in clinical trials.

M1116 Levofloxacin-Based Sequential Therapy vs Clarithromycin-Based Sequential Therapy for Eradication of H. pylori Infection

s / Digestive and Liver Disease 42S (2010) S61–S192 S113 (ITT) analysis, respectively; 3) Eradication rate in the CLA-ST group was 62/73 (84.9%) and 62/75(82.6%) in the PP and ITT analysis, respectively; 4) The difference in the eradication rate between LEV-ST and CLA-ST was statistically significant (p<0.001) by χ2 test in both PP and ITT analysis; 5) No significant differences in the incidence of adverse events was observed. Conclusions: This study indicates that in an area of high prevalence of CLA-resistant H. pylori strains, a modified, LEV-based ST is well tolerated and significantly superior to standard, CLA-based ST for eradication of H. pylori infection. # B. Gastric diseases 3. H. pylori basic/diagnosis/therapy