Metallothionein gene family in the sea urchin Paracentrotus lividus: Gene structure, differential expression and phylogenetic analysis

Metallothioneins (MT) are small and cysteine-rich proteins that bind metal ions such as zinc, copper, cadmium, and nickel. In order to shed some light on MT gene structure and evolution, we cloned seven Paracentrotus lividus MT genes, comparing them to Echinodermata and Chordata genes. Moreover, we performed a phylogenetic analysis of 32 MTs from different classes of echinoderms and 13 MTs from the most ancient chordates, highlighting the relationships between them. Since MTs have multiple roles in the cells, we performed RT-qPCR and in situ hybridization experiments to understand better MT functions in sea urchin embryos. Results showed that the expression of MTs is regulated throughout development in a cell type-specific manner and in response to various metals. The MT7 transcript is expressed in all tissues, especially in the stomach and in the intestine of the larva, but it is less metal-responsive. In contrast, MT8 is ectodermic and rises only at relatively high metal doses. MT5 and MT6 expression is highly stimulated by metals in the mesenchyme cells. Our results suggest that the P. lividus MT family originated after the speciation events by gene duplications, evolving developmental and environmental sub-functionalization

An intronic cis-regulatory element is crucial for the alpha tubulin Pl-Tuba1a gene activation in the ciliary band and animal pole neurogenic domains during sea urchin development

In sea urchin development, structures derived from neurogenic territory control the swimming and feeding responses of the pluteus as well as the process of metamorphosis. We have previously isolated an alpha tubulin family member of Paracentrotus lividus (Pl-Tuba1a, formerly known as Pl-Talpha2) that is specifically expressed in the ciliary band and animal pole neurogenic domains of the sea urchin embryo. In order to identify cis-regulatory elements controlling its spatio-temporal expression, we conducted gene transfer experiments, transgene deletions and site specific mutagenesis. Thus, a genomic region of about 2.6 Kb of Pl-Tuba1a, containing four Interspecifically Conserved Regions (ICRs), was identified as responsible for proper gene expression. An enhancer role was ascribed to ICR1 and ICR2, while ICR3 exerted a pivotal role in basal expression, restricting Tuba1a expression to the proper territories of the embryo. Additionally, the mutation of the forkhead box consensus sequence binding site in ICR3 prevented Pl-Tuba1a expression

Occurrence and concentration of PAHs in clams and sediments of the marine coastal lagoon of Ganzirri (Italy). Extraction, GC-MS analysis, distribution and sources

The distribution of selected Polycyclic Aromatic Hydrocarbons (PAHs) in surface sediments and clams of marine coastal lagoons, called Ganzirri, located at the Sicilian coast of the, Messina's strait, has been investigated by GC/MS analysis with Selected Ion Monitoring (SIM) mode. The lagoon is characterized by abundant organic detritus, deriving from aquagenic and anthropogenic inputs. Anoxic/reduced conditions of sediments make them a preferential site for uptake and preservation of PAHs. From an eco-toxicological point of view, the aquatic ecosystem investigated appears to be moderately polluted. The investigations have been performed on the 16 PAHs recommended by US-EPA as priority pollutants to be monitored in the framework of environmental quality control. The sediment PAH concentrations ranged from 135 to 1650 mu g/kg dry matrix. The total concentrations of PAHs in clams ranged from 60 to 1427 mu g/kg d.w. The relative standard deviation (RSD) for all samples of the concentration replicates of individual compounds ranged from 10% to 25%. The resulting distributions and ratios of specific compounds have been discussed in terms of sampling location and origin of contaminants. The results obtained show that levels of contamination are not homogeneous throughout the stations. In the Ganzirri Lagoon, the bioavailability of hydrophobic organic compounds, such as PAHs, seems to be mainly governed by chemical characteristics of the contaminants. Pyrolytic compounds (penta- and hexa-aromatics) are not readily available. In contrast, petroleum hydrocarbons (some tetra-aromatics) are accumulated by clams to a great extent

Fish assemblages cope with ocean acidification in a shallow volcanic CO2 vent benefiting from an adjacent recovery area

Shallow CO2 vents are used to test ecological hypotheses about the effects of ocean acidification (OA). Here, we studied fish assemblages associated with Cymodocea nodosa meadows exposed to high pCO2/low pH conditions at a natural CO2 vent in the Mediterranean Sea. Using underwater visual census, we assessed fish community structure and biodiversity in a low pH site (close to the CO2 vent), a close control site and a far control site, hypothesising a decline in biodiversity and a homogenization of fish assemblages under OA conditions. Our findings revealed that fish diversity did not show a unique spatial pattern, or even significant relationships with pH, but correlated with seagrass leaf canopy. Among-site similarity was found in the abundance of juveniles, contrary to the expected impacts of OA on early life stages. However, pH seems an important driver in structuring fish assemblage in the low pH site, despite its high similarity with the close control site. This unexpected pattern may represent a combined response of fish mobility, enhanced food resources in the acidified site, and a ‘recovery area’ effect of the adjacent control site

Metals distribution in the organic and inorganic fractions of soil: a case study on soils from Sicily

AbstractThe content of Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn, has been determined in the organic and inorganic fraction of eight soil samples from Sicily. A modified type-Tessier sequential extraction procedure has been used to recognize metals in five different fractions categorised as follows: (a) exchangeable metals, (b) metals bound to carbonates, (c) metals bound to iron and manganese oxides, (d) metals bound to organic matter, and (e) metals in residual fraction. The mineralogical composition of soils samples was characterized by X-ray diffraction. Humic substances were extracted by means of an alkaline extraction procedure. It has been found that both the mineralogical composition and the different content of humic fraction of organic matter in the soils investigated are responsible for the observed trace metal distribution. The obtained data show that a significant amount of trace metals is bound to the organic fraction where different binding sites are present simultaneously. In particular, Pb an..

The first confirmed record of the Atlantic blue crab Callinectes sapidus Rathbun, 1896 (Decapoda, Brachyura) from Maltese waters

The invasive portunid species Callinectes sapidus is hereby recorded for the first time from Maltese waters, thus updating the known distribution of this decapod of Atlantic origin within the Mediterranean. Potential introduction pathways of the species to Maltese waters are discussed

Hsp56 protein and mRNA distribution in normal and stressed P.lividus embryos

It was previously demonstrated that Paracentrotus lividus Hsp56 mitochondrial chaperonin is con- stitutively expressed during development, that it increases after heat-shock and cadmium treatment, and that it has a speci\ufb01c territorial distribution, both in normal and heat-shocked embryos, as shown by immunolocalization experiments. In this work, we analyzed by Western blot the territorial distribution of the protein in plutei exposed to heat-shock or sublethal cadmium concentrations, and we found that Hsp56 increases in both ectodermal and en- dodermal cells. Moreover, by \u201cin situ\u201d hybridization, we looked at Hsp56 mRNA during normal development and under stress conditions. We found that the territorial distribution of the messenger changes during development and that its amount is steadily increased in stressed embryos. Finally, by T1 RNase assay, we identi\ufb01ed a cytoplasmic factor that binds to the region of Hsp56 messenger containing the 5\u2019UT

Maintenance of a Protein Structure in the Dynamic Evolution of TIMPs over 600 Million Years

Deciphering the events leading to protein evolution represents a challenge, especially for protein families showing complex evolutionary history. Among them, TIMPs represent an ancient eukaryotic protein family widely distributed in the animal kingdom. They are known to control the turnover of the extracellular matrix and are considered to arise early during metazoan evolution, arguably tuning essential features of tissue and epithelial organization. To probe the structure and molecular evolution of TIMPs within metazoans, we report the mining and structural characterization of a large data set of TIMPs over approximately 600 Myr. The TIMPs repertoire was explored starting from the Cnidaria phylum, coeval with the origins of connective tissue, to great apes and humans. Despite dramatic sequence differences compared with highest metazoans, the ancestral proteins displayed the canonical TIMP fold. Only small structural changes, represented by an α-helix located in the N-domain, have occurred over the evolution. Both the occurrence of such secondary structure elements and the relative solvent accessibility of the corresponding residues in the three-dimensional structures raises the possibility that these sites represent unconserved element prone to accept variations

The major allergen of the Parietaria pollen contains an LPS-binding region with immuno-modulatory activity

Background: The major allergens in Parietaria pollen, Par j 1 and Par j 2, have been identified as lipid transfer proteins. The family of the Par j 1 allergens is composed of two isoforms, which differ by the presence of a 37 amino acid peptide (Par37) exclusive to the Par j 1.0101 isoform. The goal of this study was to elucidate the biological properties of the Par37 peptide. Methods: In silico analysis, spectrofluorimetric experiments and in vitro cell culture assays were used to identify the biological properties of Par37. In addition, a mouse model of sensitization was used to study the influence of Par37 in the murine immune response. Results: In silico analysis predicted that Par37 displays characteristics of a host defence peptide. Spectrofluorimetric analysis, real-time PCR and ELISA assays demonstrated that Par37 possesses an LPS-binding activity influencing cell signalling in vitro. In RAW264.7 cells, LPS-induced IL-6 and TNF-a transcription and translation were inhibited after preincubation with Par37. Consistent with these data, inhibition of IFN-c secretion was observed in murine spleen cells and in human PBMC. Finally, mice immunized with the two Par j 1 isoforms differing in the presence or absence of the Par37 peptide showed different immunological behaviours in vivo. Conclusions: This study demonstrates that the Par j 1.0101 allergen displays LPSbinding activity due to the presence of a 37 amino acid COOH-terminal region and that this region is capable of influencing cytokine and antibody responses in vitro and in vivo

RT-PCR and in situ hybridization analysis of apolipoprotein H expression in rat normal tissues

In this study, by using different techniques (i.e. Northern blot hybridization, RT-PCR and Southern blot hybridization) on various normal rat tissues, we were able to identify liver, kidney, heart, small intestine, brain, spleen, stomach and prostate as tissues in which the ApoH gene is transcribed. Moreover, for some of these tissues, by in situ hybridization, we found a specific localization of apoH transcripts. For instance epithelial cells of the bile ducts in liver and of the proximal tubules in kidney are the major sites of apoH synthesis. Our data suggest that some of the different physiological roles proposed for apoH could correlate with its direct expression, while others could correlate with its absorption from bloodstream or adjacent cells