Proteomic profiling of ATM kinase proficient and deficient cell lines upon blockage of proteasome activity

Ataxia Telangiectasia Mutated (ATM) protein kinase is a key effector in the modulation of the functionality of some important stress responses, including DNA damage and oxidative stress response, and its deficiency is the hallmark of Ataxia Telangiectasia (A-T), a rare genetic disorder. ATM modulates the activity of hundreds of target proteins, essential for the correct balance between proliferation and cell death. The aim of this study is to evaluate the phenotypic adaptation at the protein level both in basal condition and in presence of proteasome blockage in order to identify the molecules whose level and stability are modulated through ATM expression. We pursued a comparative analysis of ATM deficient and proficient lymphoblastoid cells by label-free shotgun proteomic experiments comparing the panel of proteins differentially expressed. Through a non-supervised comparative bioinformatic analysis these data provided an insight on the functional role of ATM deficiency in cellular carbohydrate metabolism's regulation. This hypothesis has been demonstrated by targeted metabolic fingerprint analysis SRM (Selected Reaction Monitoring) on specific thermodynamic checkpoints of glycolysis. This article is part of a Special Issue entitled: Translational Proteomics.     

Protein S-nitrosylation and denitrosylation in the mouse spinal cord upon injury of the sciatic nerve

Nitric oxide is a pain signaling molecule and exerts its influence through two primary pathways: by stimulation of soluble guanylylcyclase and by direct S-nitrosylation (SNO) of target proteins. We assessed in the spinal cord the SNO-proteome with two methods, two-dimensional S-nitrosothiol difference gel electrophoresis (2D SNO-DIGE) and SNO-site identification (SNOSID) at baseline and 24h after sciatic nerve injury with/without pretreatment with the nitric oxide synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME). After nerve injury, SNO-DIGE revealed 30 proteins with increased and 23 proteins with decreased S-nitrosylation. SNO-sites were identified for 17 proteins. After sham surgery only 3 proteins were up-nitrosylated. L-NAME pretreatment substantially reduced both constitutive and nerve injury evoked up-S-nitrosylation. For the top candidates S-nitrosylation was confirmed with the biotin switch technique and time course analyses at 1 and 7days showed that SNO modifications of protein disulfide isomerase, glutathione synthase and peroxiredoxin-6 had returned to baseline within 7days whereas S-nitrosylation of mitochondrial aconitase 2 was further increased. The identified SNO modified proteins are involved in mitochondrial function, protein folding and transport, synaptic signaling and redox control. The data show that nitric oxide mediated S-nitrosylation contributes to the nerve injury-evoked pathology in nociceptive signaling pathways.     

Diversity and composition of arboreal beetle assemblages in tropical pasture afforestations: effects of planting schemes and tree species identity

Timber tree plantations are considered for rehabilitating forest biodiversity in the tropics, but knowledge on determinants of faunal diversity patterns in such human-modified forest landscapes is scarce. We quantified the composition of beetle assemblages on three native timber species (Anacardium excelsum, Cedrela odorata and Tabebuia rosea) planted on former pasture to assess effects of tree species identity, tree species diversity, and insecticide treatment on a speciose group of animals in tropical plantations. The beetle assemblage parameters ‘abundance’, ‘species richness’, ‘Chao1 estimated species richness’ and ‘Shannon diversity’ were significantly reduced by insecticide treatment for each tree species. Shannon diversity increased with stand diversification for T. rosea but not for A. excelsum and C. odorata. Species similarity was highest (lowest species turnover) between beetle assemblages on T. rosea, and it was lowest (highest species turnover) for assemblages on insecticide-treated trees of all timber species. Considering trophic guilds, herbivorous beetles dominated on all tree species and in all planting schemes. Herbivores were significantly more dominant on T. rosea and C. odorata than on A. excelsum, suggesting that tree species identity affects beetle guild structure on plantation trees. Insecticide-treated stands harbored less herbivores than untreated stands, but exhibited a high abundance of predator beetle species. Our study revealed that even young pasture-afforestations can host diverse beetle assemblages and thus contribute to biodiversity conservation in the tropics. The magnitude of this contribution, however, may strongly depend on management measures and on the selected tree species.     

Titanium Dioxide Nanoparticles Increase Sensitivity in the Next Generation of the Water Flea Daphnia magna

The nanoparticle industry is expected to become a trillion dollar business in the near future. Therefore, the unintentional introduction of nanoparticles into the environment is increasingly likely. However, currently applied risk-assessment practices require further adaptation to accommodate the intrinsic nature of engineered nanoparticles. Combining a chronic flow-through exposure system with subsequent acute toxicity tests for the standard test organism Daphnia magna, we found that juvenile offspring of adults that were previously exposed to titanium dioxide nanoparticles exhibit a significantly increased sensitivity to titanium dioxide nanoparticles compared with the offspring of unexposed adults, as displayed by lower 96 h-EC₅₀ values. This observation is particularly remarkable because adults exhibited no differences among treatments in terms of typically assessed endpoints, such as sensitivity, number of offspring, or energy reserves. Hence, the present study suggests that ecotoxicological research requires further development to include the assessment of the environmental risks of nanoparticles for the next and hence not directly exposed generation, which is currently not included in standard test protocols.     

Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer

Microtubules can be assembled in vitro from purified alpha/beta tubulin heterodimers in the presence of GTP. Tubulin is routinely obtained from animal brain tissue through repetitive cycles of polymerization-depolymerization, followed by ion-exchange chromatography to remove any contaminating microtubule-associated proteins and motors. Here, we show that only two cycles of polymerization-depolymerization of pig brain tubulin in the presence of a high-molarity PIPES buffer allow the efficient removal of contaminating proteins and production of a high-concentration tubulin solution. The proposed protocol is rapid and yields more active tubulin than the traditional ion-exchange chromatography-based procedures.     

Mechanisms of free-radical induction in relation to fenretinide-induced apoptosis of neuroblastoma

The mechanisms of fenretinide-induced cell death of neuroblastoma cells are complex, involving signaling pathways mediated by free radicals or reactive oxygen species (ROS). The aim of this study was to identify mechanisms generating ROS and apoptosis of neuroblastoma cells in response to fenretinide. Fenretinide-induced ROS or apoptosis of SH-SY5Y or HTLA 230 neuroblastoma cells were not blocked by Nitro l-argenine methyl ester (l-NAME), an inhibitor of nitric oxide synthase. Flavoprotein-dependent superoxide-producing enzymes such as NADPH oxidase were also not involved in fenretinide-induced apoptosis or ROS generation. Similarly, ketoconazole, a cytochrome P450 inhibitor, and inhibitors of cyclooxygenase (COX) were also ineffective. In contrast, inhibition of phospholipase A(2) or lipoxygenases (LOX) blocked the induction of ROS and apoptosis in response to fenretinide. Using specific inhibitors of LOX, blocking 12-LOX but not 5- or 15-LOX inhibited both fenretinide-induced ROS and apoptosis. The effects of eicosatriynoic acid, a specific 12-LOX inhibitor, were reversed by the addition of the 12-LOX products, 12 (S)-hydroperoxyeicosatetraenoic acid and 12 (S)-hydroxyeicosatetraenoic acid. The targeting of 12-LOX in neuroblastoma cells may thus be a novel pathway for the development of drugs inducing apoptosis of neuroblastoma with improved tumor specificity.     

Living-floors and structures from the Lower Paleolithic to the Bronze Age in Italy

New researches have been performed on the analysis of some Italian dwelling structures dating from the Lower Paleolithic to Bronze Age. Different methods have been applied to each study according to the extensions of the areas explored. The following sites have been analyzed: Isernia La Pineta (Molise), Visogliano (Trieste) - Lower Paleolithic; Grotta del Cavallo (Lecce), Grotta Grande and Riparo del Molare (Salerno) - Middle Paleolithic; Grotta di Fumane (Verona), Riparo Tagliente (Verona), Grotta Continenza (Fucino L'Aquila), San Bartolomeo (Maiella Mountain, Abruzzo) - Upper Paleolithic; Mondeval de Sora (Belluno), Alpe Veglia (Verbania) and Grotta Edera (Aurisina, Trieste) -Mesolithic; Cala Giovanna Piano (Pianosa Island, Livorno), Contraguda (Perfugas, Sassari), Colle Santo Stefano (Fucino, L'Aquila), Catignano (Pescara), Settefonti (L'Aquila) - Neolithic; Castellaro Lagusello (Monzambano, Mantua) - Bronze Age.     

Effect of dietary nitrogen level and source on mRNA expression of urea transporters in the rumen epithelium of fattening bulls

This paper aims to study the effect of the dietary treatments on mRNA expression of urea transporter B (UT-B) and some aquaporins (AQP) in rumen epithelium of Italian Simmental young bulls. Eighty animals allocated to 16 pens were fed from about 500 to 650 kg body weight with four experimental diets, which resulted from the combination of two crude protein levels (125 and 110 g/kg dry matter, diets M and L, respectively) and two nitrogen sources (soybean meal (SBM) or SBM partly replaced by an isonitrogenous mixture of corn and urea; diets ?U and +U, respectively). At slaughtering samples of blood and rumen epithelium were collected from six bulls for each diet. Blood samples were analysed for haematological parameters and quantitative PCR was carried out on the mRNA extracted from the rumen epithelium samples. The bulls fed diets M had lower plasma concentrations of aspartate aminotransferase than those receiving diets L (78.9 vs. 88.3 U/l, p = 0.04). Plasma urea was higher (p = 0.03) for diets M and lower for diets +U (2.0 vs. 2.5 and 1.73 vs. 2.00 mmol/l, respectively, in M and L diets, p = 0.04). The effect of dietary treatments on rumen UT expression were limited to AQP3, which was down regulated (p = 0.01) in diets +U. Finally, a high positive correlation (R² = 0.871) between the expressions of AQP7 and AQP10 was found. In conclusion, the AQP3 appears very responsive to dietary treatments and therefore it is a candidate to be further studied in rumen metabolism experiments. The close relationship between mRNA expression of AQP7 and AQP10 indicates a similar function of these two proteins.     

Leaf and canopy photosynthesis of a chlorophyll deficient soybean mutant

The photosynthetic, optical, and morphological characteristics of a chlorophyll‐deficient (Chl‐deficient) “yellow” soybean mutant (MinnGold) were examined in comparison with 2 green varieties (MN0095 and Eiko). Despite the large difference in Chl content, similar leaf photosynthesis rates were maintained in the Chl‐deficient mutant by offsetting the reduced absorption of red photons by a small increase in photochemical efficiency and lower non‐photochemical quenching. When grown in the field, at full canopy cover, the mutants reflected a significantly larger proportion of incoming shortwave radiation, but the total canopy light absorption was only slightly reduced, most likely due to a deeper penetration of light into the canopy space. As a consequence, canopy‐scale gross primary production and ecosystem respiration were comparable between the Chl‐deficient mutant and the green variety. However, total biomass production was lower in the mutant, which indicates that processes other than steady state photosynthesis caused a reduction in biomass accumulation over time. Analysis of non‐photochemical quenching relaxation and gas exchange in Chl‐deficient and green leaves after transitions from high to low light conditions suggested that dynamic photosynthesis might be responsible for the reduced biomass production in the Chl‐deficient mutant under field conditions.