Browsing by Author "Torreblanca, Amparo"
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- 2-D difference gel electrophoresis approach to assess protein expression profiles in Bathymodiolus azoricus from Mid-Atlantic Ridge hydrothermal ventsPublication . Company, Rui; Antúnez, Oreto; Bebianno, Maria João; Cajaraville, Miren P.; Torreblanca, AmparoHydrothermal vent mussels Bathymodiolus azoricus are naturally exposed to toxic chemical species originated directly from vent chimneys. The amount of toxic elements varies significantly among vent sites along the Mid-Atlantic Ridge and B. azoricus must be able to adapt to changes in hydrothermal fluid composition, temperature and pressure. The aim of this work was to study changes in the proteome in the "gill-bacteria complex" of mussels B. azoricus from three hydrothermal vent sites with distinct environmental characteristics using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE). Results showed that 31 proteins had different expression profiles among vent sites and both cluster and principal component analysis confirm a clear separation of mussels between sites. This suggests the existence of specific parameters grouping individuals from the same hydrothermal site. Protein spots of the more abundant differentially expressed proteins were excised, digested with trypsin and identified by mass spectrometry. All identified proteins (actin, ubiquinone, S-adenosylhomocysteine hydrolase, cysteine peptidases, chaperonin and catalase) have been related previously with oxidative stress conditions and are known to be affected by ROS inducing stressors, including metals. Results point out to specific adaptations at the proteome level of B. azoricus depending on the level of toxicants present in their environment.
- Comparison of thiol subproteome of the vent mussel Bathymodiolus azoricus from different Mid-Atlantic Ridge vent sitesPublication . Company, Rui; Torreblanca, Amparo; Cajaraville, Miren; Bebianno, Maria João; Sheehan, DavidDeep-sea hydrothermal mussels Bathymodiolus azoricus live in the mixing zone where hydrothermal fluid mixes with bottom seawater, creating large gradients in the environmental conditions and are one of the most studied hydrothermal species as a model of adaptation to extreme conditions. Thiol proteins, i.e. proteins containing a thiol or sulfhydryl group (SH) play major roles in intracellular stress defense against reactive oxygen species (ROS) and are especially susceptible to oxidation. However, they are not particularly abundant, representing a small percentage of proteins in the total proteome and therefore are difficult to study by proteomic approaches. Activated thiol sepharose (ATS) was used for the rapid and quantitative selection of proteins comprising thiol- or disulfide-containing subproteomes. This study aims to isolate thiol-containing proteins from the gills of B. azoricus collected in distinct hydrothermal vents and to study the thiol-containing subproteome as a function of site-specific susceptibility to ROS. Results show that ATS is a powerful tool to isolate the thiol-containing sub-proteome and differently-expressed protein spots showed significant differences among the three vent sites, supporting previous findings that specific environmental conditions are crucial for ROS formation and that B. azoricus have different susceptibilities to oxidative stress depending on the vent site they inhabit.
- Metallothionein in the freshwater gastropod Melanopsis dufouri chronically exposed to cadmium: A methodological approachPublication . Ureña, Rocío; Bebianno, Maria João; del Ramo, Jose; Torreblanca, AmparoPrevious studies have demonstrated that the use of differential pulse polarography (DPP) for metallothionein (MT) determination in marine gastropod tissues, particularly the digestive gland, requires taking into account the presence of heat-stable high molecular weight compounds that exhibit polarographic signal. In the present paper, similar compounds were identified in tissues from the freshwater snail Melanopsis dufouri which also interfere with MT determination by DPP and, due to their silver binding capacity, also interfere in the silver assay for MT quantification. Ultrafiltration seems to be effective in removing these high molecular weight compounds from heat-denatured homogenate supernatant allowing direct MT quantification by DPP. A fully validated procedure for metallothionein determination in M. dufouri is described. In spite of a considerable accumulation of cadmium in the visceral complex of M. dufouri following exposure to 100 microg CdL(-1) for 8 weeks (up to 37 microgg(-1)) only a small increase in MT concentration was found.
- Protein expression profiles in Bathymodiolus azoricus exposed to cadmiumPublication . Company, Rui; Antúnez, Oreto; Cosson, Richard P.; Serafim, M. A.; Shillito, Bruce; Cajaraville, Miren; Bebianno, Maria João; Torreblanca, AmparoProteomic changes in the "gill-bacteria complex" of the hydrothermal vent mussel B. azoricus exposed to cadmium in pressurized chambers ((Incubateurs Pressurises pour l'Observation en Culture d'Animaux Marins Profonds - IPOCAMP) were analyzed and compared with the non-exposed control group. 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) showed that less than 1.5% of the proteome of mussels and symbiotic bacteria were affected by a short-term (24 h) Cd exposure. Twelve proteins of the more abundant differentially expressed proteins of which six were up-regulated and six were down-regulated were excised, digested and identified by mass spectrometry. The identified proteins included structural proteins (actin/actin like proteins), metabolic proteins (calreticulin/calnexin, peptidyl-prolyl cis-trans isomerase, aminotransferase class-III, electron transfer flavoprotein, proteasome, alpha-subunit and carbonic anhydrase) and stress response proteins (chaperone protein htpG, selenium-binding protein and glutathione transferases). All differently expressed proteins are tightly connected to Cd exposure and are affected by oxidative stress. It was also demonstrated that B. azoricus was well adapted to Cd contamination therefore B. azoricus from hydrothermal vent areas may be considered a good bioindicator.