The Impact of Metabolic Scion-Rootstock Interactions in Different Grapevine Tissues and Phloem Exudates.

In viticulture, grafting is used to propagate Phylloxera-susceptible European grapevines, thereby using resistant American rootstocks. Although scion-rootstock reciprocal signaling is essential for the formation of a proper vascular union and for coordinated growth, our knowledge of graft partner interactions is very limited. In order to elucidate the scale and the content of scion-rootstock metabolic interactions, we profiled the metabolome of eleven graft combination in leaves, stems, and phloem exudate from both above and below the graft union 5-6 months after grafting. We compared the metabolome of scions vs. rootstocks of homografts vs. heterografts and investigated the reciprocal effect of the rootstock on the scion metabolome. This approach revealed that (1) grafting has a minor impact on the metabolome of grafted grapevines when tissues and genotypes were compared, (2) heterografting affects rootstocks more than scions, (3) the presence of a heterologous grafting partner increases defense-related compounds in both scion and rootstocks in shorter and longer distances from the graft, and (4) leaves were revealed as the best tissue to search for grafting-related metabolic markers. These results will provide a valuable metabolomics resource for scion-rootstock interaction studies and will facilitate future efforts on the identification of metabolic markers for important agronomic traits in grafted grapevines.

Expression Profiling of Castanea Genes during Resistant and Susceptible Interactions with the Oomycete Pathogen Phytophthora cinnamomi Reveal Possible Mechanisms of Immunity.

The most dangerous pathogen affecting the production of chestnuts is Phytophthora cinnamomi a hemibiotrophic that causes root rot, also known as ink disease. Little information has been acquired in chestnut on the molecular defense strategies against this pathogen. The expression of eight candidate genes potentially involved in the defense to P. cinnamomi was quantified by digital PCR in Castanea genotypes showing different susceptibility to the pathogen. Seven of the eight candidate genes displayed differentially expressed levels depending on genotype and time-point after inoculation. Cast_Gnk2-like revealed to be the most expressed gene across all experiments and the one that best discriminates between susceptible and resistant genotypes. Our data suggest that the pre-formed defenses are crucial for the resistance of C. crenata to P. cinnamomi. A lower and delayed expression of the eight studied genes was found in the susceptible Castanea sativa, which may be related with the establishment and spread of the disease in this species. A working model integrating the obtained results is presented.

High-throughput proteomics: a new tool for quality and safety in fishery products.

In order to cope with the increasing demand for fishery products, sensitive technological tools are required to ensure high quality and wholesomeness and to monitor their production process in a sustainable manner while complying with the strict standards imposed by regulatory authorities. Proteomics may assist the industry as it allows an unbiased approach in the discovery of biomarkers that could be used to increase our understanding of different biological, physiological and ecological aspects that may be advantageous in optimizing quality and safety in aquatic species. The aim of this review is to highlight the potential of cost-effective high-throughput technologies, such as those offered by proteomics using "on-line" mass spectrometry to improve the efficiency of the industry in identifying biomarkers relevant for safe high quality products.

Gold nanoparticles and oxidative stress in the blue mussel, Mytilus edulis.

Relatively little is known about how gold nanoparticles (GNP) might interact in vivo with aquatic organisms. Gold nanoparticles (GNP) of defined average diameter may be synthesized and used to challenge test organisms held in aquaria. The blue mussel, Mytilus edulis, is a popular sentinel species in environmental toxicology. This chapter describes how mussels exposed to GNP (average diameter ~5 nm) may be dissected and extracts of digestive gland can be analyzed for oxidative stress. Protein thiols are labeled with 5'-iodoacetamide-fluorescein (IAF), and proteins are separated in one-dimensional electrophoresis. After scanning for IAF-associated fluorescence, gels are stained with colloidal coomassie. A ratio of fluorescence to protein stain is calculated revealing that thiol oxidation increases with GNP treatment.

Proteomic research in bivalves: towards the identification of molecular markers of aquatic pollution.

Biomonitoring of aquatic environment and assessment of ecosystem health play essential roles in the development of effective strategies for the protection of the environment, human health and sustainable development. Biomarkers of pollution exposure have been extensively utilized in the last few decades to monitor the health of organisms and hence assess environmental status. However, the use of single biomarkers against biotic or abiotic stressors may be limited by the lack of sensitivity and specificity. Therefore, more recently, the search for novel biomarkers has been focused on the application of OMICS methodologies. Environmental proteomics focuses on the analysis of an organism's proteome and the detection of changes in the level of individual proteins/peptides in response to environmental stressors. Proteomics can provide a more robust approach for the assessment of environmental stress and therefore exposure to pollutants. This review aims to summarize the proteomic research in bivalves, a group of sessile and filter feeding organisms that play an important function as "sentinels" of the aquatic environment. A description of the main proteomic methodologies is provided. The current knowledge in bivalves' toxicology, achieved with proteomics, is reported describing the main biochemical markers identified. A brief discussion regarding future challenges in this area of research emphasizing the development of more descriptive gene/protein databases that could support the OMICs approaches is presented.

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.

Oxidative stress and toxicity of gold nanoparticles in Mytilus edulis.

Gold nanoparticles (AuNP) have potential applications in drug delivery, cancer diagnosis and therapy, food industry and environment remediation. However, little is known about their potential toxicity or fate in the environment. Mytilus edulis was exposed in tanks to 750 ppb AuNP (average diameter 5.3 ± 1 nm) for 24h to study in vivo biological effects of nanoparticles. Traditional biomarkers and an affinity procedure selective for thiol-containing proteins followed by two-dimensional electrophoresis (2DE) separations were used to study toxicity and oxidative stress responses. Results were compared to those obtained for treatment with cadmium chloride, a well known pro-oxidant. M. edulis mainly accumulated AuNP in digestive gland which also showed higher lipid peroxidation. One-dimensional SDS/PAGE (1DE) and 2DE analysis of digestive gland samples revealed decreased thiol-containing proteins for AuNP. Lysosomal membrane stability measured in haemolymph gave lower values for neutral red retention time (NRRT) in both treatments but was greater in AuNP. Oxidative stress occurred within 24h of AuNP exposure in M. edulis. Previously we showed that larger diameter AuNP caused modest effects, indicating that nanoparticle size is a key factor in biological responses to nanoparticles. This study suggests that M. edulis is a suitable model animal for environmental toxicology studies of nanoparticles.

Covalent selection of the thiol proteome on activated thiol sepharose: a robust tool for redox proteomics.

Protein thiols contribute significantly to antioxidant defence and selective oxidation of cysteines is important in signal transduction even in sub-stress scenarios. However, cysteine is the second rarest residue in proteins and it can be difficult to target low-abundance thiol (-SH)-containing proteins in proteomic separations. Activated thiol sepharose (ATS) allows covalent selection of -SH-containing proteins which can then be recovered by reduction with mercaptoethanol or dithiothreitol. This is a robust method for enriching -SH-containing proteins. We have used ATS to estimate the percentage (by weight) of thiol-containing proteins in cell extracts from a range of biological sources: a bacterium, Escherichia coli; a fungus, Trichoderma harzianum; and a bivalve mollusc Mytilus edulis. -SH-containing proteins account for 2.52% (E. coli), 1.4% (T. harzianum) and 1.4% (M. edulis) of total protein. Exposure to pro-oxidants did not materially alter these values. On removal of low M(r) thiols such as glutathione, the values for M. edulis did not significantly change but those for T. harzianum increased threefold. The two-dimensional electrophoresis profiles of ATS-selected proteins for each organism were compared in control and pro-oxidant-exposed preparations. This revealed that some proteins present in controls were absent in pro-oxidant-treated extracts which we attribute to thiol oxidation. ATS has significant potential in enrichment for -SH-containing proteins in redox proteomics.

Selection of thiol- and disulfide-containing proteins of Escherichia coli on activated thiol-Sepharose.

Activated thiol-Sepharose (ATS) facilitates selection of thiol-containing proteins. In control- and menadione-treated Escherichia coli, batch selection performed under denaturing conditions revealed distinct two-dimensional electrophoresis (2DE) patterns. Using shotgun proteomics, 183 thiol-containing proteins were identified in control ATS-selected extracts and 126 were identified in menadione-treated E. coli, with 85 proteins being common to both. More than 90% of identified proteins contained one or more cysteines. Blocking with N-ethyl maleimide followed by reduction facilitated ATS-based selection of disulfide-containing proteins. In total, 62 proteins were unique to control cells and 164 were identified in menadione-treated E. coli cells, with 29 proteins being common to both. Proteins from menadione-treated cells were excised from 2DE gels, digested with trypsin, and identified by peptide mass fingerprinting. This revealed 19 unique proteins, 14 of which were identified by shotgun proteomics. Outer membrane proteins A, C, W, and X and 30S ribosomal protein S1 were found in 2DE but not by shotgun proteomics. Foldases, ribosomal proteins, aminoacyl transfer RNA (tRNA) synthetases, and metabolic and antioxidant enzymes were prominent among identified proteins, and many had previously been found to respond to, and be targets for, oxidative stress in E. coli. ATS provides a convenient and rapid way to select thiol-containing proteins.

Shotgun redox proteomics in sub-proteomes trapped on functionalised beads: Identification of proteins targeted by oxidative stress.

If reactive oxygen species (ROS) levels exceed antioxidant defences, oxidative stress occurs; a common response to environmental pollutants. Proteins absorb ∼70% of ROS, altering amino acid side-chains. Cys (-SH) oxidises to sulphenic (-SOH), sulphinic (-SO(2)H), cysteic (-SO(3)H) acids and disulphide bridges (-S-S-). Two-dimensional electrophoresis (2DE) under-selects certain protein categories (e.g. extreme pI, small proteins) so activated thiol sepharose (ATS) was used to select sub-proteomes of thiol-containing proteins in menadione-exposed Escherichia coli. ATS bound thiol-containing proteins (but not oxidised thiols) via mixed disulphides. Tryptic digestion of bead-bound proteins was followed by LC-tandem MS. Many proteins were identified in controls with significantly fewer in menadione-treated cells (e.g. chaperonins, transcription/translation-related and ribosomal proteins; aminoacyl tRNA synthetases and metabolic enzymes. Non-denaturing ATS capture (followed by reduction) demonstrated lower specific activities of key enzymes which is attributed to thiol oxidation. This method may be generally useful in ecotoxicology for identification of oxidative stress targets.

Exposure of the blue mussel, Mytilus edulis, to gold nanoparticles and the pro-oxidant menadione.

Relatively little is known about how gold nanoparticles (GNP) might interact in vivo with marine organisms. Mytilus edulis was exposed (24h) to approximately 15 nm GNP, menadione and both compounds simultaneously (GNP/menadione). GNP was detected by inductively coupled plasma-optical emission spectroscopy mainly in digestive gland of samples exposed to GNP though not GNP/menadione, perhaps due to impaired feeding. Thioredoxin reductase activity and malondialdehyde levels were determined in all tissues. Thioredoxin reductase inhibition was detected only in digestive gland exposed to menadione whilst malondialdehyde levels did not vary in response to treatment in all tissues. GNP caused a decrease in the reduced/oxidized glutathione ratio in digestive gland, but no difference was found in other tissues or for other treatments. One dimensional electrophoresis of proteins containing thiol groups was performed in all tissues and revealed a reduction in protein thiols for all treatments in digestive gland. Two dimensional electrophoresis of digestive gland extracts, from GNP and control groups, showed decreased levels of thiol proteins in response to GNP which we attribute to oxidation. Our results suggest that GNP causes a modest level of oxidative stress sufficient to oxidize thiols in glutathione and proteins but without causing lipid peroxidation or induction of thioredoxin reductase activity.

Use of the land snail Helix aspersa as sentinel organism for monitoring ecotoxicologic effects of urban pollution: an integrated approach.

Atmospheric pollution from vehicular traffic is a matter of growing interest, often leading to temporary restrictions in urban areas. Although guidelines indicate limits for several parameters, the real toxicologic impacts remain largely unexplored in field conditions. In this study our aim was to validate an ecotoxicologic approach to evaluate both bioaccumulation and toxicologic effects caused by airborne pollutants. Specimens of the land snail Helix aspersa were caged in five sites in the urban area of Ancona, Italy. After 4 weeks, trace metals (cadmium, chromium, copper, iron, manganese, nickel, lead, and zinc) and polycyclic aromatic hydrocarbons (PAHs) were measured and these data integrated with the analyses of molecular and biochemical responses. Such biomarkers reflected the induction of detoxification pathways or the onset of cellular toxicity caused by pollutants. Biomarkers that correlated with contaminant accumulation included levels of metallothioneins, activity of biotransformation enzymes (ethoxyresorufin O-deethylase, ethoxycoumarin O-deethylase), and peroxisomal proliferation. More general responses were investigated as oxidative stress variations, including efficiency of antioxidant defenses (catalase, glutathione reductase, glutathione S-transferases, glutathione peroxidases, and total glutathione) and total oxyradical scavenging capacity toward peroxyl and hydroxyl radicals, onset of cellular damages (i.e., lysosomal destabilization), and loss of DNA integrity. Results revealed a marked accumulation of metals and PAHs in digestive tissues of organisms maintained in more traffic-congested sites. The contemporary appearance of several alterations confirmed the cellular reactivity of these chemicals with toxicologic effects of potential concern for human health. The overall results of this exploratory study suggest the utility of H. aspersa as a sentinel organism for biomonitoring the biologic impact of atmospheric pollution in urban areas. Key words: atmospheric pollutants, bioindicators, biomarkers, DNA integrity, lysosomes, metallothioneins, oxidative stress, peroxisomes, polycyclic aromatic hydrocarbons, trace metals.

Pro-oxidant effects of extremely low frequency electromagnetic fields in the land snail Helix aspersa.

Pro-oxidant effects of extremely low frequency (ELF) 50-Hz magnetic fields were investigated in the land snail Helix aspersa exposed both in short-term laboratory treatments and under field conditions by maintaining the organisms in the proximity of a power line for up to 2 months. Oxidative perturbations were investigated as individual antioxidants (catalase, glutathione reductase, glutathione S-transferases, and total glutathione) and total scavenging capacity toward peroxyl radicals and hydroxyl radicals. Accumulation of lipid peroxidation products, destabilization of lysosomal membranes, and loss of DNA integrity were also evaluated as markers of cell damage. The overall results indicated an oxidative challenge caused by ELF magnetic fields with particularly prompt and sensitive responses for catalase, glutathione reductase, and the overall capability to neutralize peroxyl radicals. Cell injuries occurred to different extents according to duration and intensity of electromagnetic exposure and confirmed complex cause-effect relationships between pro-oxidant factors, efficiency of antioxidant defenses, and the onset of oxidative toxicity. This study highlights the importance of a multimarker approach for detecting a wide panel of biological responses, the necessity of investigating the long-term effects of early oxidative responses, and the role of ELF in enhancing susceptibility to other forms of pathologies or diseases.

Antioxidant efficiency and detoxification enzymes in spotted dogfish Scyliorhinus canicula.

Although elasmobranchs are widely distributed species, commonly found on sandy, gravely or muddy bottoms, several biological aspects of their metabolism still remain poorly investigated. In this work the efficiency of antioxidant system and detoxification enzymes were investigated in the coastal spotted dogfish Scyliorhinus canicula and in the red mullet Mullus barbatus for comparison with a teleost species. Organisms were sampled during a bottom trawl survey and analyzed for the biotransformation activity (EROD), levels of metallothioneins, catalase, glutathione S-transferases and total oxyradical scavenging capacity (TOSC) toward peroxyl radicals and hydroxyl radicals. EROD activity in the elasmobranchs was more than one order of magnitude lower than in the red mullets, while similar levels of metallothioneins were measured in these species. S. canicula showed significantly lower antioxidant enzymes and a more reduced efficiency in neutralizing *OH; on the other hand the scavenging capability toward ROO* was comparable in S. canicula and M. barbatus.