In the present study, we describe the identified and characterized the gene and the transcript of a novel glutathione peroxidase-7 (GPx7) from the solitary ascidian Ciona robusta, an invertebrate ...chordate widely distributed in temperate shallow seawater. The putative nucleotide and amino acid sequences were compared with those of GPx7 from other metazoans and phylogenetic analysis suggests the presence of a high evolutionary pressure in the contest of neutral evolution. The mRNA of CrGPx7 is located in hemocytes and ovarian follicular cells, as revealed by in situ hybridization. The time course of CrGPx7 mRNA levels in the presence of Cd, Cu and Zn, showed upregulation in the final stages of the experiments, suggesting a role of GPx7 in late protection from oxidative stress. Our in silico analyses of the crgpx7 promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and xenobiotic-responsive elements (XRE), suggesting that the transcription of these genes directly depends on metals. Cell-free extract from C. robusta tissues show the presence of selenium-independent GPx activity that is inhibited by the presence of metals.
► Genes involved in glutathione biosynthesis are well conserved in the ascidian Ciona intestinalis. ► Promoters contain consensus sequences similar to mammalian MRE and ARE. ► Metal exposure results ...in a significant increase in mRNA expression. ► Genes are mostly transcribed in circulating hemocytes.
Exposure to metals is known to generate oxidative stress risk in living organisms, which are able to respond with the induction of antioxidant defenses, both enzymatic and non-enzymatic. Glutathione (GSH) is considered to be an important cellular component involved in protecting cells, both as metal chelating agent and oxygen radical scavenger. In this work we used molecular techniques to analyze the nucleotide and predicted amino acid sequences of genes involved in GSH biosynthesis, γ-glutamyl-cysteine ligase catalytic subunit (ci-gclc), γ-glutamyl-cysteine ligase modifier subunit (ci-gclm) and GSH synthase (ci-gs) in the solitary tunicate Ciona intestinalis. We also studied the transcription of the above genes after in vivo exposure to Cd, Cu and Zn by semiquantitativ RT-PCR to improve our knowledge about the relationship between metal-induced oxidative stress and GSH production and locate mRNA expression by in situ hybridization (ISH). These genes exhibit a good level of sequence conservation with metazoan homologs generally, especially for residues important for the activity of the enzymes. Phylogenetic analyses indicate that the three enzymes evolved in different ways, Ci-GCLC and Ci-GS being mostly correlated with invertebrate proteins, Ci-GCLM being as sister group of vertebrate GCLMs. Our in silico analyses of the ci-gs and ci-gclc promoter regions revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and antioxidant response elements (ARE), indicating that the transcription of these genes may directly depend on metals and/or reactive oxygen species. Results highlight a statistically significant increase in gene transcription, demonstrating that metal treatments have inducible effects on these genes. They can modulate gene transcription not only through MREs but also through AREs, as a consequence of metal-dependent ROS formation. The ISH location of Ci-GS and Ci-GCLC mRNAs shows that the cells most involved in glutathione biosynthesis are circulating hemocytes. The data presented here emphasize the importance of complex metal regulation of ci-gclc, ci-gclm and ci-gs transcription, which can create an efficient detoxification pathway allowing C. intestinalis to survive in continued elevated presence of metals in the environment.
In the present study we describe the molecular characterization of the two paralogous mitochondrial peroxiredoxins from Trematomus bernacchii, a teleost that plays a pivotal role in the Antarctic ...food chain. The two putative amino acid sequences were compared with orthologs from other fish, highlighting a high percentage of identity and similarity with the respective variant, in particular for the residues that are essential for the characteristic peroxidase activity of these enzymes. The temporal expression of Prdx3 and Prdx5 mRNAs in response to short-term thermal stress showed a general upregulation of prdx3, suggesting that this isoform is the most affected by temperature increase. These data, together with the peculiar differences between the molecular structures of the two mitochondrial Prdxs in T. bernacchii as well as in the tropical species Stegastes partitus, suggest an adaptation that allowed these poikilothermic aquatic vertebrates to colonize very different environments, characterized by different temperature ranges.
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•Two new mitochondrial peroxiredoxin genes have been identified in Trematomus bernacchii.•Phylogenetic analyses indicate that of both Prdx3s and Prdx5s probably evolved under purifying selection effect.•Both Prdx3 and Prdx5 are not “cold-adapted” enzymes.•The Antarctic emerald rockcode performs rapid and specific responses to wormer temperature.
Glutathione peroxidases are a family of antioxidant enzymes catalyzing the reduction of H₂O₂ or organic hydroperoxides. In the present study, we report the molecular characterization and gene ...expression analysis of a new GPx-1 from the Antarctic fish Trematomus bernacchii. To expand our knowledge on the GPx-1 s evolution within the group of Antarctic fish, in this work, we also presented the cDNA sequencing of this enzyme in other three species, belonging to two families—Nototheniidae (Trematomus eulepidotus, T. lepidorhinus) and Bathydraconidae (Cygnodraco mawsoni). The deduced amino acid sequences were compared with GPx-1 s of other vertebrates by multiple alignment, in order to evaluate the conservation of amino acids involved in the enzyme activity. The results of phylogenetic analyses indicated that fish GPx-1 s possibly originated from independent duplication events, and Antarctic GPx-1 s evolved according to the molecular and morphological phylogeny of Antarctic fish. Basal GPx-1 mRNA expression analyses in various tissues of T. bernacchii specimens indicated that liver and heart displayed the highest mRNA accumulation; probably a protection of these organs against lipid peroxidation is needed.
In the present study, we describe the purification and molecular characterization of two peroxiredoxins (Prdxs), referred to as Prdx6A and Prdx6B, from
Trematomus bernacchii
, a teleost widely ...distributed in many areas of Antarctica, that plays a pivotal role in the Antarctic food chain. The two putative amino acid sequences were compared with Prdx6 orthologs from other fish, highlighting a high percentage of identity and similarity with the respective variant, in particular for the residues that are essential for the characteristic peroxidase and phospholipase activities of these enzymes. Phylogenetic analyses suggest the appearance of the two
prdx6
genes through a duplication event before the speciation that led to the differentiation of fish families and that the evolution of the two gene variants seems to proceed together with the evolution of fish orders and families. The temporal expression of Prdx6 mRNA in response to short-term thermal stress showed a general upregulation of
prdx6b
and inhibition of
prdx6a
, suggesting that the latter is the variant most affected by temperature increase. The variations of mRNA accumulation are more conspicuous in heart than the liver, probably related to behavioral changes of the specimens in response to elevated temperature. These data, together with the peculiar differences between the molecular structures of the two Prdx6s in
T. bernacchii
as well as in the tropical species
Stegastes partitus
, suggest an adaptation that allowed these poikilothermic aquatic vertebrates to colonize very different environments, characterized by different temperature ranges.
Orphan receptor GPR103, a pyroglutamylated RFamide peptide receptor (QRFPR), is a class A G protein-coupled receptor (GPCR) and it is coupled to a Gi alpha subunit (Gi/0) and/or to a Gq protein. ...Synteny analysis revealed the existence of paralogous genes of QRFPR in mouse, zebrafish and coelacanth. These paralogous genes emerged along with species-specific gene or genome duplications that occurred during vertebrate evolution. 26RFa/QRFP is the high-affinity endogenous ligand for QRFPR and in fish it has been suggested as an orexigenic action of 26RFa/QRFP. The structure, tissue-specific expression and biochemical activity of the 26RFa/QRFP–QRFPR system are conserved across the Chordata phylum, from fish to mammals. In order to study the molecular evolution of mammal QRFPR, we searched for the presence of natural selection on the qrfpr genes using a bioinformatic approach. Overall, the results clearly indicate that mammal QRFPRs are under positive selection, but the majority of positively selected amino acids did not alter the biochemical properties of these proteins.
Orphan receptor GPR103, a pyroglutamylated RFamide peptide receptor (QRFPR), is a class-A G protein-coupled receptor (GPCR) and it is coupled to a Gi alpha subunit (Gi/0) and/or to a Gq protein. ...Synteny analysis revealed the existence of qrfpr paralogous genes in mouse, zebrafish and coelacanth. These paralogous genes emerged along with the species-specific gene or genome duplications that occurred during vertebrate evolution. Neuropeptide 26RFa (also termed QRFP) is the latest member of the RFamide peptide family to be discovered in the hypothalamus of vertebrates. 26RFa/QRFP is a 26-amino acid residue peptide that was originally identified from the frog brain. It has been shown to exert orexigenic activity in mammals and to be a ligand of the previously identified orphan G protein-coupled receptor, QRFPR. The structure, tissue-specific expression and biochemical activity of the 26RFa/QRFP–QRFPR system are conserved across the Chordata phylum, from fish to mammals. In order to study the molecular evolution of fish QRFPRs, we investigated the presence of natural selection on the QRFPR family using a bioinformatic approach. Overall, the obtained results clearly indicate that fish QRFPRs are under positive selection, but the positively selected amino acids did not significantly alter the biochemical properties of these proteins.
Heavy metal pollution is known to be widespread in the sediments of the Lagoon of Venice. Therefore, physiological parameters influenced by this form of contamination were examined. The bivalve ...molluscs blue mussel (Mytilus galloprovincialis), ark clam (Scapharca inaequivalvis), and Japanese littleneck (Tapes philippinarum) were sampled in two sites (Marghera, Chioggia) every 3 months for 1 year. The digestive gland and gills of each bivalve were analyzed. The concentrations of essential and nonessential metals (including chromium, manganese, iron, cobalt, nickel, copper, zinc, and cadmium) were determined. Because glutathione and metallothioneins (MTs) are involved in metal homeostasis and detoxification, their concentrations were evaluated in relation to metal concentrations. Results show that (1) all three studied species accumulate metals to a considerable extent, with some species-specific differences between the digestive gland and gills; (2) gills are a good tissue to evaluate pollution by examining the MT content. In particular, the correlation between Zn and MT levels in the gills indicates that M. galloprovincialis and S. inaequivalvis are sentinel organisms and can be used specifically for Zn pollution; (3) T. philippinarum accumulates Cu in the digestive gland more readily than the other two bivalves and therefore has the highest MT.
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