Examinando por Autor "Novoa, B"

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Hemocitos de mejillón. Producción de radicales de oxígeno y nitrógeno
(Consejo Superior de Investigaciones Científicas, 2007) Novoa, B; Tafalla, C; Ordás, Camino; Figueras, A
Molecular characterisation of a turbot Mx cDNA
(Elsevier, 2005) Abollo, E; Ordás, MC; Dios, S; Figueras, A; Novoa, B
The present work describes the cloning and characterisation of a full-length Mx cDNA from turbot. Expression of this gene in different organs and blood leucocytes was induced by poly I:C, in order to evaluate its capacity to regulate the Mx gene both in vivo and in vitro.
Molecular cloning and expression analysis of interferon regulatory factor 1 (IRF- 1) of turbot and sea bream
(Elsevier, 2006) Ordás, MC; Abollo, E; Costa, M; Figueras, A; Novoa, B
The interferon regulatory factor (IRF) family comprises transcription factors that regulate the expression of interferon and interferon-related cytokines. Using the RACE technique, we have determined the complete cDNA sequence of turbot (Scophthalmus maximus) and sea bream (Sparus aurata) IRFs. These sequences shared characteristics with other IRFs of fish, mammals and birds, and showed high similarity with IRF-1. Indeed, they were included in the IRF-1 cluster of the phylogenetic tree constructed with IRF-1 and IRF-2 sequences of several organisms, and presented a low number of basic amino acid residues in the carboxy-terminal end of the proteins. All of these characteristics led to the identification of turbot and sea bream IRFs as IRF-1. Two IRF-1 sequences were obtained for both turbot and sea bream, and we named them turbot/sea bream IRF-1a and IRF-1b. Turbot IRF-1a differed from turbot IRF-1b in four nucleotides. The presence of both IRF types in cDNA from 45 turbot livers was determined by RFLP, suggesting the duplication of the gene. Sea bream IRF-1b presented a deletion of 121 bp in its ORF compared to sea bream IRF-1a, and since both IRF types were present in all 25 cDNAs analyzed by PCR, we hypothesized that the truncated sea bream IRF-1b was probably an alternative splicing product. Turbot and sea bream IRF-1 expression was constitutive in every analyzed organ, as reported before for other fish species. Poly I:C significantly stimulated turbot IRF-1 expression in muscle, spleen and kidney 24 h post-treatment, while viral haemorrhagic septicemia virus (VHSV) induced a differential expression of this factor in kidney 8 h after infection. These results do not agree with those previously reported for flounder and trout IRF. Other expression experiments with turbot leukocytes stimulated in vitro with poly I:C and with brain and kidney of sea bream infected with nodavirus did not bring out differential IRF expression levels in stimulated samples with respect to controls.
Proteolytic activity of cultured Pseudoperkinsus tapetis extracellular products.
(Elsevier, 2001) Ordás, MC; Novoa, B; Faisal, M; McLaughlin, SM; Figueras, A
Several pathogenic protozoan release proteases are necessary for host invasion and initiation of infection. We have identified proteolytic activities in extracellular proteins secreted by the clam parasite Pseudoperkinsus tapetis (Mesomycetozoa) in vitro. The protein concentration of the P. tapetis extracellular products (ECP) increased only during the first week of culture. The appearance of new proteins of 10 and 157 kDa at the second week sample and of 12 kDa at the third week sample was shown by SDS-PAGE. The protease activity rapidly increased in the first 3 weeks of culture, and five clear bands of 23, 29, 60, 67 and 96 kDa with proteolytic activity were detected in the ECP on gelatin SDS-PAGE. Using inhibitors, the proteases were identified as members of the Ca2+ dependent, serine protease family. Their optimum pH was higher than pH 9.4. The protease activity of the P. tapetis ECP was different than that described for Perkinsus marinus, an oyster pathogen very similar morphologically to the clam parasite and member of the genus in which P. tapetis had been initially included.
Turbot TNFα gene: Molecular characterization and biological activity of the recombinant protein
(Elsevier, 2007) Ordás, MC; Costa, MM; Roca, FJ; López-Castejón, G; Mulero, V; Meseguer, J; Figueras, A; Novoa, B
The tumor necrosis factor (TNF) superfamily is composed by several proteins with similar structure and functions. One of the main representatives of this family is TNF-alpha (TNF ), a proinflammatory cytokine which is produced by different immune cells and presents a wide variety of activities. Using the RACE technique, we have cloned and sequenced the turbot TNF cDNA. The analysis of its sequence showed several conserved motifs characteristic of members of the TNF family. A phylogenetic tree constructed with different TNFs of fish and mammals grouped our sequence within the fish TNF cluster. Therefore, the turbot TNF here studied was identified as TNF . The complete TNF gene was obtained by gene walking, and, similarly to the other known fish TNF genes, presented three introns and four exons. A PCR was designed to study the turbot TNF expression in vivo using as stimulus the bacteria Vibrio pelagius strain Hq222 and virus VHSV. The expression of the cytokine happened early after injection, and it was dependent on the pathogen injected and organ analyzed. Virus induced a higher TNF expression, but this response was shorter in time than that induced by bacteria. In addition, TNF expression was in general higher in kidney than in liver, as expected since the former is the haematopoietic organ of fish. The turbot recombinant TNF (rTNF ) was obtained by IPTG induction of bacteria transformed with the pET15b-TNF construct, and it was purified in native conditions. The recombinant protein was approximately 20 kDa in size, and its biological activity was assessed in vitro. No effect of the rTNF neither alone nor in combination with LPS was observed on the chemiluminescence activity of turbot macrophages at any time tested. However, NO production was enhanced by the recombinant protein alone or with LPS 72 h after the addition of the treatments. Finally, turbot rTNF was able to recruit and activate inflammatory cells when injected in gilthead seabream, although to a lesser extent than gilthead seabream rTNF .