Giuseppina Salerno

Isolation and expression of a novel MBL-like collectin cDNA enhanced by LPS injection in the body wall of the ascidian Ciona intestinalis

Collectins are a family of calcium-dependent lectins that are characterized by their collagen-like domains. Considerable interest has been focused on this class of proteins because of their ability to interact with components of the complement system activating a cascade of events responsible for the activation of the innate immune system. A differential screening between LPS-challenged and naïve Ciona intestinalis has been performed allowing the isolation of a full length cDNA encoding for a 221 AA protein. In silico analysis has shown that this polypeptide displays protein domains with similarities to mannose-binding lectins. A phylogenetic analysis suggested that C. intestinalis MBL has evolved early as a prototype of vertebrate MBL. Real-time PCR assay demonstrated that this gene is strongly activated after LPS injection in the tunica. In situ hybridization performed in LPS-induced animals has shown that this gene is expressed in granular amoebocytes and large granules hemocytes in the inflamed body wall tissue. Finally, an antimicrobial activity of the C. intestinalis MBL has been demonstrated.

Differential expression of two glucocorticoid receptors in seabass (teleost fish) head kidney after exogeneous cortisol inoculation.

Stressful conditions include a prompt release of corticosteroid hormones which can mediate gene expression through glucocorticoid receptors (GR). Since two seabass (Dicentrarchus labrax) GRs have been cloned and sequenced from peritoneal cavity cells (DlGR1) and liver (DlGR2), a comparative amino acid sequence analysis that included Haplochromis burtoni HbGRs, was carried out and homologies disclosed. The DlGR1 and DlGR2 deduced aminoacid sequences showed 61% identity (I) and 70% similarity (S). Moreover, DlGR2 was similar to HbGR2b (69% I, 73% S), and the DlGR1 to HbGR1 (72% I, 78% S). In addition, we examined the expression of the DlGRs after exogeneous cortisol inoculation into the peritoneal cavity, mimicking stress effects. At various times after the administration (3 h, 24 h, 1 week), gene expressions was evaluated in head kidney by real-time PCR. In addition, immunoblotting and densitometry analyses were performed with anti-DlGR1 antibodies. Although sea bass head kidney expressed both DlGR1 and DlGR2 they were differentially modulated by intraperitoneal implant of exogeneous cortisol.

Primary structure and opsonic activity of an F-lectin from serum of the gilt head bream Sparus aurata (Pisces, Sparidae)

Abstract The recently described fucose-binding agglutinin from the European eel revealed a novel lectin fold (the ‘F-type’ fold) that is shared with other carbohydrate-binding proteins and proteins from prokaryotes to vertebrates clustered under the newly established F-type lectin (FTL) family. We previously reported the purification and biochemical characterization of a fucose-binding protein (FBP) isolated from serum of the gilt head bream (Sparus aurata, SauFBP). In the present article, the complete coding sequence of SauFBP revealed that it is a member of the FTL family, consisting of two tandem carbohydrate recognition domains (CRD) that display the F-type sequence motif. In vitro opsonization assays showed that the isolated SauFBP binds to formalin-killed Escherichia coli and enhances their phagocytosis by peritoneal macrophages.

Effect of in vitro exposure to cadmium and copper on sea bass blood cells

Abstract Blood cells freshly collected from sea bass (Dicentrarchus labrax) were exposed in vitro to different concentrations of cadmium (Cd) and copper (Cu) at 10-7 M, 10-5 M, 10-3 M, and examined for neutral red retention capacity and for cell vitality with MTT assay. A relationship between heavy metal exposure and alteration in responses of blood cells in a dose-time-dependent was found. Our results showed that fish blood cells may constitute an interesting biological model for experimental and applied toxicology, especially in the case of environmental pollution.

Expression and distribution of the glucocorticoid receptor DlGR1 in the teleost Dicentrarchus labrax brain.

Abstract Cortisol is the main corticosteroid secreted by the interrenal cells of the head kidney and it exerts a role in mantaining the omeostatic status in fish. In teleosts its effects are mediated through intracellular receptors expressed in several tissues, that are ligand-dependent transcription factors by binding to specific tissue DNA sequences. In Dicentrarchus labrax we previously cloned and sequenced a glucocorticoid receptor, DlGR1, isolated from leukocytes of peritoneal cavity. In this work we showed mRNA expression and tissue immunohistochemical localization of brain DlGR1 by in situ hybridization assays, with a riboprobe with DlGR1 cDNA trascriptional activation domain, and by immunohistochemical methods, using a specific antibody for a selected sequence of the receptor transcriptional domain. The mRNA and the protein are expressed in pyramidal cells of the optic lobe and in the small globular neurons of the diencephalon.