FACIT collagen (1alpha-chain) is expressed by hemocytes and epidermis during the inflammatory response of the ascidian Ciona intestinalis

Based on previous cloning and sequencing study, real-time PCR and in situ hybridization assays of the inflamed body wall of LPS-injected Ciona intestinalis showed the enhanced gene expression of a collagen with FACIT structural features (Ci-type IX-Col 1a-chain). By using specific antibodies raised against an opportunely chosen Ci-type IX-Col synthetic peptide, the fibroblast property of hemocytes challenged in vitro with LPS (at 4 h) was displayed by flow cytometry, while immunocytochemistry identified hemocytes with large granules (morula cells) as collagen-producing cells. Hemocyte lysate supernatant analyzed in immunoblotting contained a 60 kDa band identifiable as 1a-chain-Ci-type IX-C…

Localizzazione di un recettore glucocorticoideo DLGR1 nei tessuti del teleosteo Dicentrarchus labrax

Isolation and characterization of a fish F-type lectin from gilt head bream (Sparus aurata) serum.

A novel fucose-binding lectin, designated SauFBP32, was purified by affinity chromatography on fucose-agarose, from the serum of the gilt head bream Sparus aurata. Electrophoretic mobility of the subunit revealed apparent molecular weights of 35 and 30 kDa under reducing and non-reducing conditions, respectively. Size exclusion analysis suggests that the native lectin is a monomer under the selected experimental conditions. Agglutinating activity towards rabbit erythrocytes was not significantly modified by addition of calcium or EDTA; activity was optimal at 37 degrees C, retained partial activity by treatment at 70 degrees C, and was fully inactivated at 90 degrees C. On western blot anal…

Isolation, characterization and expression analysis of a collectin in Tunicate Ciona intestinalis.

Lectine sieriche di "tipo F" nell'immunità innata dei pesci: aspetti molecolari e funzionali.

Effects of exogenous cortisol on the expression of glucocorticoid receptor (DIGR1) and hsp70 in head-kidney and peritonel cavity cells from Dicentrarchus labrax.

Serum lectins in fish innate immunity: molecular and functional aspects

Fucose-binding lectins (FBL) are present in tissues and fluids from invertebrates and vertebrates. The lectin repertoires in teleost fish are highly diversified and recently has been described the structure of the fucose-binding agglutinin that revealed a novel lectin fold (the “F-type” eel (Anguilla anguilla) fold), which shared a unique fucose-binding sequence motif contained both in carbohydrate-binding proteins and unrelated proteins. In this report, we describe serum FBL from sea bass Dicentrarchus labrax and sea bream Sparus aurata. These lectins were purified, characterized, cloned and sequenced. Studies on structural aspects, biological activity, tissue distribution as well as ontog…

Effetti dell’esposizione a cadmio e rame sull’attività di cellule del sangue di spigola (Dicentrarchus labrax L. Osteichthyes, Moronidae).

Caratterizzazione molecolare di un peptide antimicrobico isolato dai leucociti del teleosteo Dicentrarcus labrax

The prophenoloxidase system is activated during the tunic inflammatory reaction of Ciona intestinalis

Phenoloxidase (PO) activity was examined in the tunic tissue of Ciona intestinalis following lipopolysaccharide (LPS) intratunic injection. Tunic homogenate supernatant (THS), assayed with the Dopa-MBTH reaction, displayed Ca(2+)-independent PO activity that was raised by LPS and further enhanced by proteases. Specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. Assay with soybean trypsin inhibitor showed that, in the tunic, PO activation with trypsin was not significantly inhibited suggesting that proteases diverse from serine proteases were involved. In vivo experiments were carried out by injecting isosmotic medium or LPS, and T…

Inflamed adult pharynx tissues and swimming larva of Ciona intestinalis share CiTNFalfa-producing cells

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

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 immunohi…

Biological activity, tissue distribution and preliminary moleular characterization of a serum fucolectin from the sea bass (Dicentrarchus labrax)

Enhanced expression of a cloned and sequenced Ciona intestinalis TNFa-like (CiTNFa) gene during the LPS-induced inflammatory response.

A tumor necrosis factor-alpha (TNFalpha)-like gene from Ciona intestinalis (CiTNF alpha-like) body wall challenged with bacterial lipopolysaccharide (LPS) was cloned and sequenced 4 h after LPS inoculation. An open reading frame of 936 bp encoding a propeptide of 312 amino acids (35.4 kDa) displaying a transmembrane domain from positions 7 to 29, a TACE cleavage site, and a mature peptide domain of 185 amino acids (20.9 kDa), was determined with a predicted isoelectric point of 9.4. The phylogenetic tree based on deduced amino acid sequences of invertebrate TNF-like protein and vertebrate TNFs supported the divergence between the ascidian and vertebrate TNF families, whereas D. melanogaster…

cDNA sequence and tissue expression of an antimicrobial peptide, dicentracin; a new component of the moronecidin family isolated from head kidney leukocytes of sea bass, Dicentrarchus labrax

A 483-bp cDNA was isolated from sea bass (Dicentrarchus labrax) head kidney leukocytes, dicentracin, using PCR primers designed from conserved moronecidin domains. Gene bank analysis revealed that dicentracin cDNA belongs to the moronecidin family. As deduced from alignment with Morone chrysops moronecidin, the precursor of 79 aa appeared to be composed of a signal peptide of 22 aa, followed by the mature AMP (antimicrobial peptide) of 22 aa named dicentracin, and a C-terminal extension of 35 aa. Dicentracin precursor displayed 3 aa substitutions with other moronecidin sequence but none in the mature peptide sequence. Using in situ hybridization assay, dicentracin gene expression was observ…

Tunicate immunocytes can be cytotoxic toward foreign cells

Espressione e distribuzione del recettore DLGR1 nel cervello del Teleosteo Dicentrarchus labrax.

Effect of exposure cadmium on the echinoderm Paracentrotus lividus (Echinoidea)

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 perit…

Inducible galectins are expressed in the inflamed pharynx of the ascidian Ciona intestinalis

Although ascidians belong to a key group in chordate phylogenesis, amino acid sequences of Ciona intestinalis galectin-CRDs (CiLgals-a and -b) have been retained too divergent from vertebrate galectins. In the present paper, to contribute in disclosing Bi-CRD galectin evolution a novel attempt was carried out on CiLgals-a and -b CRDs phylogenetic analysis, and their involvement in ascidian inflammatory responses was shown. CiLgals resulted aligned with Bi-CRD galectins from vertebrates (Xenopus tropicalis, Gallus gallus, Mus musculus, Homo sapiens), cephalochordates (Branchiostoma floridae), echinoderms (Strongylocentrotus purpuratus) and a mono-CRD galectin from the ascidian Clavelina pict…

F-type lectin from the sea bass (Dicentrarchus labrax): purification, cDNA cloning, tissue expression and localization, and opsonic activity.

Recently described biochemical and structural aspects of fucose-binding lectins from the European eel (Anguilla anguilla) and striped bass (Morone saxatilis) led to the identification of a novel lectin family ("F-type" lectins) characterized by a unique sequence motif and a characteristic structural fold. The F-type fold is shared not only with other members of this lectin family, but also with apparently unrelated proteins ranging from prokaryotes to vertebrates. Here we describe the purification, biochemical and molecular properties, and the opsonic activity of an F-type lectin (DlFBL) isolated from sea bass (Dicentrarchus labrax) serum. DlFBL exhibits two tandemly arranged carbohydrate-r…

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

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 exam- ined 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 experimen- tal and applied toxicology, especially in the case of environmental pollution.

Inflamed adult pharynx tissues and swimming larva of Ciona intestinalis share CiTNFalpha-producing cells.

In situ hybridisation and immunohistochemistry analyses have shown that the Ciona intestinalis tumour necrosis factor alpha gene (CiTNFalpha), which has been previously cloned and sequenced, is expressed either during the inflammatory pharynx response to lipopolysaccharide (LPS) or during the swimming larval phase of development. Granulocytes with large granules and compartment/morula cells are CiTNFalpha-producing cells in both inflamed pharynx and larvae. Pharynx vessel endothelium also takes part in the inflammatory response. Haemocyte nodules in the vessel lumen or associated with the endothelium suggest the involvement of CiTNFalpha in recruiting lymphocyte-like cells and promoting the…

Localizzazione di una lectina di “tipo F” nell’adulto e nell’ontogenesi di Dicentrarchus labrax

The purification, cloning, sequencing, molecular properties and expression of a fucose-binding lectin from the serum of Dicentrarchus labrax (DlFBL) have been previously reported. We now describe the distribution and expression of DlFBL during fish ontogeny. Immunohistochemistry and in situ hybridization assays were carried out at various developmental stages (from 10 days posthatching larvae to juveniles). Another fucose-binding lectin, similar to DlFBL in biochemical, immunochemical and agglutinating properties, was extracted and purified from eggs and appeared to be localized in the embryo yolk sack residual. DlFBL was found in columnar and goblet cells of the intestinal epithelium of la…

Expression of a glucocorticoid receptor (D1GR1) in several tissues of the teleost fish Dicentrarchus labrax

Since glucocorticoids have a role in maintaining the homeostatic status in fish, in the present paper mRNA expression (in situ hybridization) and tissue immunohistochemical localization of a glucocorticoid receptor (DlGR1) in several Dicentrarchus labrax organs are reported. Riboprobe and specific antibodies were prepared by using the DlGR1 that has been previously cloned and sequenced from peritoneal cavity leukocytes. Both mRNA and receptor were identified in head kidney, spleen, gills, intestine, heart and liver tissues. The functional roles of DlGR1 localization are discussed.

Expression of a type IX-like collagen in tissue injury of the ascidian Ciona intestinalis durino inflammatory process

Espressione e distribuzione del recettore DLGR1 nel cervello del teleosteo Dicentrarchus labrax (L. 1758)

Effetti modulanti del cadmio e del rame sulle attività cellulari del teleosteo Dicentrarchus labrax

Effetti del cadmio sulle attività cellulari dell’echinoderma Paracentrotus lividus (Echinoidea)

Attività biologica, distribuzione tissutale e caratterizzazione molecolare della fucolectina sierica di spigola (Dicentrarchus labrax).

An Attempt to re-examine the immune role of Ciona intestinalis hemocytes

Molecular characterization of an antimicrobial peptide isolated from leucocytes of the teleostean Dicentrarchus labrax.

Enhanced CiTNFα expression is an inflammatory response of the ascidian Ciona intestinalis

The Ciona intestinalis prophenoloxidase activating system during LPS inflammatory reaction

Cloning and expression of a novel component of the CAP superfamily enhanced in the inflammatory response to LPS of the ascidian Ciona intestinalis.

The CAP superfamily is a group of proteins that have been linked to several biological functions such as reproduction, cancer, and immune defense. A differential screening between lipopolysaccharide (LPS)-challenged and naive Ciona intestinalis has been performed to identify LPS-induced genes. This strategy has allowed the isolation of a full-length 1471-bp cDNA encoding for a 413-amino-acid protein (CiCAP). In silico analysis has shown that this polypeptide displays a modular structure with similarities to vertebrate CAP-superfamily proteins and to a collagen-binding adhesin of Streptococcus mutans. Domain organization analysis and alignment of CiCAP to other vertebrate CAP proteins have r…

FACIT collagen (1α-chain) is expressed by hemocytes and epidermis during the inflammatory response of the ascidian Ciona intestinalis

Based on previous cloning and sequencing study, real-time PCR and in situ hybridization assays of the inflamed body wall of LPS-injected Ciona intestinalis showed the enhanced gene expression of a collagen with FACIT structural features (Ci-type IX-Col 1alpha-chain). By using specific antibodies raised against an opportunely chosen Ci-type IX-Col synthetic peptide, the fibroblast property of hemocytes challenged in vitro with LPS (at 4h) was displayed by flow cytometry, while immunocytochemistry identified hemocytes with large granules (morula cells) as collagen-producing cells. Hemocyte lysate supernatant analyzed in immunoblotting contained a 60 kDa band identifiable as 1alpha-chain-Ci-ty…

Primary structure and opsonic activity of an F-lectin from serum of the gilt head breamSparus 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 opso…

Expression of a glucocorticoid receptor (DlGR1) in several tissues of the teleost fish Dicentrarchus labrax

Since glucocorticoids have a role in maintaining the homeostatic status in fish, in the present paper mRNA expression (in situ hybridization) and tissue immunohistochemical localization of a glucocorticoid receptor (DlGR1) in several Dicentrarchus labrax organs are reported. Riboprobe and specific antibodies were prepared by using the DlGR1 that has been previously cloned and sequenced from peritoneal cavity leukocytes. Both mRNA and receptor were identified in head kidney, spleen, gills, intestine, heart and liver tissues. The functional roles of DlGR1 localization are discussed.

Comparative analysis of fucose binding lectins isolated and characterized from different teleost species, and distribution of a F-Lectin during Dicentrarchus labrax ontogenesis

Effetti del cadmio sulla fagocitosi e sull'attività di formazione di placche di lisi dei celomociti di Parcentrotus lividus

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 …

Localization of glucocorticoid receptor DIGR1 in tissues pf teleost Dicentrarchus labrax.

A serum fucose-binding lectin (D1FBL) from adult Didentrarchus labrax is expressed in larva and juvenile tissue and contained in eggs

The purification, cloning, sequencing, molecular properties and expression of a fucose-binding lectin from the serum of Dicentrarchus labrax (DlFBL) have been previously reported. We now describe the distribution and expression of DlFBL during fish ontogeny. Immunohistochemistry and in situ hybridization assays were carried out at various developmental stages (from 10 days posthatching larvae to juveniles). Another fucose-binding lectin, similar to DlFBL in biochemical, immunochemical and agglutinating properties, was extracted and purified from eggs and appeared to be localized in the embryo yolk sack residual. DlFBL was found in columnar and goblet cells of the intestinal epithelium of la…

Espression of a type IX-like collagen in tissue injury of the ascidian Ciona intestinalis during inflammatory process

Enhanced expression of a CinTNF gene in the LPS challenged inflammatiry responses of the ascidian Ciona intestinalis

Effects of cadmium on expression of the hsp70 in sea bass (Dicentrarchus labrax L., Osteichthyes. Moronidae) blood cells

Abstract To gain further insight into the role of cortisol in Wsh innate immune responses, we cloned and sequenced a 2592 bp cDNA from sea bass (Dicentrarchus labrax) peritoneal leukocytes (PCLs) encoding a glucocorticoid receptor (DlGR1). The deduced aminoacid sequence displayed that DlGR1 belong to a multigenic family of steroid hormone receptors, and exhibited high homology (80%) to the Burton’s mouth breeder (Haplochromis burtoni) HbGR1. The DlGR1 functional domains presented homologies with those of several vertebrate species. In situ hybridization assay revealed that DlGR1 was expressed in macrophages and neutrophils from the peritoneal cavity. Since in a previous paper, sea bass PCL …

Effect of cadmium exposure on phagocytosis and plaque lysis activity of Paracentrotus lividus coelomocyte