Giovanni Principato

Glutathione, glutathione-dependent and antioxidant enzymes in mussel, Mytilus galloprovincialis, exposed to metals under field and laboratory conditions: implications for the use of biochemical biomarkers

Abstract The effects of exposure to metals under and laboratory conditions were investigated in the Mediterranean mussel Mytilus galloprovincialis. The examined biological responses included the concentrations of heavy metals, the level of glutathione, and the activity of several enzymes selected among glutathione-dependent oxidoreductases and hydrolases: glutathione reductase. EC1.6.4.2; glyoxalase I, EC4.4.1.5; glyoxalase II, EC3.1.2.6; glutathione S-transferases, EC2.5.1.18; Se-dependent, EC1 11.1.9 and Se-independent, EC2.5.1.18 glutathione peroxidases; catalase, EC1.11.1.6; superoxide dismutase, EC1.15.1.1; alkaline phosphatase, EC3.1.3.1; cholinesterase, EC3.1.1.8; arylesterases, EC3.1.1.2. The analyses were carried out on digestive gland and gills of mussels from two populations, respectively from a polluted and a clean site. The same parameters were measured in control mussels transplanted to the polluted environment, and in molluscs exposed to copper under laboratory conditions. The comparison between different field and laboratory exposures was expected to give useful indications for a proper use of biochemical responses as biomarkers in monitoring trace metal pollution. Compared to control mussels, the polluted ones (native, transplanted and copper-exposed) showed significantly lower levels of glutathione and higher activities of the glyoxalases (which detoxify reactive α-ketoaldehydes formed in cellular oxidative processes). On the other hand, native mussels from both the polluted and control populations exhibited similar enzymatic activities of glutathione reductase, glutathione peroxidases, catalase, superoxide dismutase and alkaline phosphatase, whereas, in both transplanted and copper-exposed mussels, these enzymes showed significant variations. This finding could suggest the occurrence of some adaptation or compensatory mechanism in chronically polluted organisms. No clear results were obtained with glutathione S-transferases, whereas arylesterases and cholinesterases appeared not to be affected by metal pollution. From the results, three different kind of biological responses were identified and the implications for ecotoxicological studies discussed.

SpliceAid: a database of experimental RNA target motifs bound by splicing proteins in humans

UNLABELLED The correct post-transcriptional RNA processing is finely regulated by RNA-binding proteins. Unfortunately, there is little experimental information on target RNA sequences of RNA-binding proteins and moreover such experimentally derived target sequences are annotated in a compact form by the score matrices that overestimate the number of possible recognized sequences. We carried out an exhaustive hand curated literature search to create a database, SpliceAid, collecting all the experimentally assessed target RNA sequences that are bound by splicing proteins in humans. We built a web resource, database driven, to easy query SpliceAid and give back the results by an accurate and dynamic graphic representation. AVAILABILITY SpliceAid database is freely accessible at http://www.introni.it/splicing.html.

Mitochondrial dysfunctions during aging: vitamin E deficiency or caloric restriction--two different ways of modulating stress.

Caloric restriction (CR), which has been demonstrated to offset the age-associated accrual of oxidative injury, involves a reduction in calory intake while maintaining adequate nutrition, preserves the activities of antioxidant enzymes in postmitotic tissues, maintains organ function, opposes the development of spontaneous diseases, and prolongs maximum life span in laboratory rodents. It has been proposed that reductions in Reactive Oxygen Species (ROS) production and cellular oxidative injury are central to the positive effects of CR. In the present investigation we studied the effect of CR and of a vitamin E deprived diet on mitochondrial structure and features in the liver of rats during aging, in order to ascertain the extent of modifications induced by these experimental conditions. CR rats displayed structural and functional mitochondrial properties (fatty acid pattern, respiratory chain activities, antioxidant levels, and hydroperoxide contents) similar to those of younger rats whilst vitamin E deficient rats appeared older than their own age. The mitochondria of the former, together with those of young rats, possessed the lowest Coenzyme Q9, hydroperoxide, and cytochrome contents as well as a suitable fatty acid membrane composition. Our study confirms that CR is a valuable tool in limiting aging-related free-radical damage also at mitochondrial liver level.

Strawberry intake increases blood fluid, erythrocyte and mononuclear cell defenses against oxidative challenge

The health promoting effects of a regular consumption of strawberries deserve attention, and a direct or indirect antioxidant role of strawberry bioactive compounds is among the most probable mechanisms underlying their beneficial properties. In the present study, we evaluated the overall effects of a 2-week daily consumption of strawberries on plasma antioxidant status, membrane lipid susceptibility to ex vivo-induced oxidation, and erythrocyte and mononuclear cell resistance to oxidative damage in apparently healthy volunteers. After strawberry intake, a moderate increase in fasting plasma antioxidant capacity and vitamin C was observed, together with a significant increase in the lag phase preceding plasma lipid oxidation. A significantly enhanced resistance to oxidative hemolysis was confirmed in red blood cells, while no significant changes were found in the extent of their membrane lipid peroxidation. For the first time, increased intake of strawberries for only 2weeks was shown to be sufficient to attenuate mononuclear cell mortality after ex vivo exposure to a single acuteoxidative challenge, but the analysis of DNA oxidative damage gave conflicting results. These findings suggest that a regular consumption of strawberries may enhance body defences against oxidative challenges.

An improved in silico selection of phenotype affecting polymorphisms in SLC6A4, HTR1A and HTR2A genes

Among the experimentally assessed DNA variations in serotonin related genes, some influence physiological expression of personality and mental disorders, others alter the responses to pharmacological and/or psychotherapeutic treatments. Because of the huge number of polymorphisms lying in genes and of the great length of time necessary to perform association studies, a selection of the variations being studied is a necessary and crucial step.

Total antioxidant capacity and nuclear DNA damage in keratinocytes after exposure to H2O2.

Abstract Studies of oxidative stress have classically been performed by analyzing specific, single antioxidants. In this study, susceptibility to oxidative stress in the human keratinocyte cell line NCTC2544 exposed to hydrogen peroxide (H2 O2) was measured by the TOSC (total oxyradical scavenging capacity) assay, which discriminates between the antioxidant capacity toward peroxyl radicals and hydroxyl radical. The generation of H2 O2-induced DNA damage, total antioxidant capacity and levels of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, glutathione Stransferase, glutathione peroxidase) were studied. Exposure to H2 O2-induced DNA damage that was gradually restored while a significant reduction in cellular TOSC values was obtained independently of stressor concentrations and the degree of DNA repair. Whereas TOSC values and cell resistance to H2 O2 showed a good relationship, the extent of DNA damage is independent from cellular total antioxidant capacity. Indeed, maximum DNA damage and cell mortality were observed in the first 4 h, whereas TOSC remained persistently low until 48 h. Catalase levels were significantly lower in exposed cells after 24 and 48 h. Keratinocytes exposed after 48 h to a second H2 O2 treatment exhibited massive cell death. A possible linkage was observed between TOSC values and NCTC2544 resistance to H2 O2 challenge. The TOSC assay appears to be a useful tool for evaluating cellular resistance to oxidative stress.

Glyoxalase II and glutathione levels in rat liver mitochondria during cold storage in Euro-Collins and university of Wisconsin solutions

Glutathione and glyoxalase II levels were evaluated in cytosolic and mitochondrial compartments of rat liver after 7 and 24 hr of cold storage in University of Wisconsin (UW) and Euro-Collins solutions. 1-4 A similar time-dependent depletion of cytosolic glutathione up to about 60% of control values was observed in both Euro-Collins and UW solutions. Cytosolic glyoxalase II showed activity oscillations in livers stored in Euro-Collins but not in UW. Mitochondrial glutathione and glyoxalase II were severely depleted soon after 7 hr of cold storage in Euro-Collins, whereas the same parameters did not change in liver stored in UW after 24 hr. UW is confirmed to be the most suitable solution for liver cold storage and we conclude that mitochondrial glutathione and glyoxalase II can be important parameters in assessing mitochondrial and cell function.

Evidence for a molecular polymorphis of cholinesterase in Sepia officinalis (cephalopoda: decapoda)

1. 1. Three forms of cholinesterase were sequentially extracted from head and tentacles of Sepia officinalis and noted as low-salt (LSS), detergent (DS) and high-salt (HSS) soluble. They represent about 24, 30 and 46% of total activity. 2. 2. All enzyme forms seem to be amphiphilic proteins with hydrophobic domains interacting with non-ionic detergent (Triton X-100) and giving self-aggregation (LSS form). 3. 3. The DS form is membrane-anchored by a phosphatidylinositol, while the HSS form is likely linked to some proteoglycan molecule of the extracellular matrix by ionic interactions. 4. 4. According to Vmax/Km values, all the enzymes are acetylcholinesterases, even if hydrolyze propionylthiocoline at the highest rate. 5. 5. Some kinetic and molecular properties of the studied enzymes are compared with those of other cholinesterases from vertebrates and invertebrates. Possible phylogenic and adaptive features are discussed.

Mitochondrial contribution to metabolic changes in the digestive gland of Mytilus galloprovincialis during anaerobiosis

The activities of several enzymes and the levels of metabolites have been measured in the digestive gland of Mytilus galloprovincialis exposed to increasing hypoxia from 7 to 168 hr. A sharp decrease of pyruvate kinase was observed after 7 hr. The anoxic enzyme showed increased Km for phosphoenolpyruvate and decreased apparent Ki for alanine. Glyoxalase I was constant after up to 72 hr of exposure and then decreased. Phosphoenolpyruvate carboxykinase and alanopine dehydrogenase decreased. The metabolites alanine and succinate increased with hypoxia time, whereas D- and L-lactic and aspartic acids were undetectable and constant respectively. Mitochondrial formation of pyruvate from D-lactate was demonstrated in intact mitochondria isolated from the digestive gland of Mytilus galloprovincialis. The significance of the observed enzyme and metabolite changes in hypoxia is discussed in comparison with other invertebrate organisms. The role of mitochondria in the overall adaptive strategy of Mytilus galloprovincialis is discussed. J. Exp. Zool. 286:107-113, 2000.

Insights into the influence of 5-HT2c aminoacidic variants with the inhibitory action of serotonin inverse agonists and antagonists

AbstractSpecific modulation of serotonin 5-HT2C G protein-coupled receptors may be therapeutic for obesity and neuropsychiatric disorders. The different efficacy of drugs targeting these receptors are due to the presence of genetic variants in population and this variability is still hard to predict. Therefore, in order to administer the more suitable drug, taking into account patient genotype, it is necessary to know the molecular effects of its gene nucleotide variations. In this work, starting from an accurate 3D model of 5-HT2C, we focus on the prediction of the possible effect of some single nucleotide polymorphisms (SNPs) producing amino acidic changes in proximity of the 5-HT2C ligand binding site. Particularly we chose a set of 5-HT2C inverse agonists and antagonists which have high inhibitory activity. After prediction of the structures of the receptor-ligand complexes using molecular docking tools, we performed full atom molecular dynamics simulations in explicit lipid bilayer monitoring the interactions between ligands and trans-membrane helices of the receptor, trying to infer relations with their biological activity. Serotonin, as the natural ligand was chosen as reference compound to advance a hypothesis able to explain the receptor inhibition mechanism. Indeed we observed a different behavior between the antagonists and inverse agonist with respect to serotonin or unbounded receptor, which could be responsible, even if not directly, of receptor’s inactivation. Furthermore, we analyzed five aminoacidic variants of 5HT2C receptor observing alterations in the interactions between ligands and receptor which give rise to changes of free energy values for every complex considered. FigureAntagonist and inverse agonist binding sites in 5-HT2C wild type and its SNP variants