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Acosta J.,Center for Genetic Engineering and Biotechnology | Montero V.,Center for Pharmaceuticals Research and Development | Carpio Y.,Center for Genetic Engineering and Biotechnology | Velazquez J.,Center for Genetic Engineering and Biotechnology | And 6 more authors.
Aquaculture | Year: 2013

Antimicrobial peptides constitute an important component of the innate immune system. Teleost fish represent a potentially fruitful resource for novel antimicrobial peptide discovery since these organisms rely significantly on their innate immune systems to combat the constant threat of infections in the aquatic environment. In the present study, we isolated three antimicrobial peptides-like transcripts from tilapia (Oreochromis niloticus) gills based on EST reported sequences. These peptides were named oreochromicins (Oreoch-1, Oreoch-2 and Oreoch-3). The cDNA sequences for these putative AMPs encode three pre-pro-peptides with the highest similarity with the members of the piscidin family from teleost fish. The predicted three pre-pro-peptides consist of a signal peptide, a highly cationic mature peptide of 23, 25, and 32 amino acids, respectively and a carboxy terminal pro-domain. The synthetic peptides displayed a broad-spectrum of antimicrobial activity against Gram-negative, Gram-positive bacteria and fungi. These peptides are constitutively expressed in the brain, heart, head kidney, spleen and gut. Additionally, their binding properties to lipopolysaccharide and cytotoxic activity in mammalian and fish cells were assayed. © 2012 Elsevier B.V.

Rodriguez-Viera L.,University of Habana | Rodriguez-Viera L.,University of Cadiz | Perera E.,Institute Ciencias Marinas Of Andalucia | Casuso A.,University of Habana | And 7 more authors.
PLoS ONE | Year: 2014

Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2-6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6-12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a proteinsparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other nutrients. © 2014 Rodriguez-Viera et al.

Perdomo-Morales R.,Center for Pharmaceuticals Research and Development | Montero-Alejo V.,Center for Pharmaceuticals Research and Development | Corzo G.,National Autonomous University of Mexico | Besada V.,Center for Genetic Engineering and Biotechnology | And 4 more authors.
Journal of Biological Chemistry | Year: 2013

The melanization reaction promoted by the prophenoloxi-dase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (Ki = 8.6 nM) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Montero-Alejo V.,Center for Pharmaceuticals Research and Development | Acosta-Alba J.,Center for Genetic Engineering and Biotechnology | Perdomo-Morales R.,Center for Pharmaceuticals Research and Development | Perera E.,University of Habana | And 3 more authors.
Fish and Shellfish Immunology | Year: 2012

Naturally occurring antimicrobial peptides take place in the first line of host defense against pathogen as part of the humoral innate immune response. β-defensins are among the most abundant antimicrobial peptides in mammals, and thought to be solely found in vertebrates until a recent report describing the cloning and sequencing of defensin like peptides in the spiny lobster Panulirus japonicus. In the current study, we cloned and sequenced two genes from the hemocytes of the spiny lobster Panulirus argus encoding for two isoforms of defensin-like peptides, thus confirming the presence of this protein in the Panulirus genus. The 44 amino acids mature peptides showed the conservation of cysteine pattern characterizing the β-defensins, as well as known amino acids residues critical to exert their antimicrobial activity. They are also amphipathics, hydrophobics, and display an overall positive charge (+1) located at the C-terminus. The tertiary structure obtained by homology modeling indicated that likely conformations of lobster peptides are highly similar to β-defensins from vertebrates. The phylogenetic study carried out by probabilistic methods confirmed the relation with ancestral β-defensin from vertebrates. The finding of a putative defensin-like peptide in the expressed sequence tag (EST) of the lobster Homarus americanus with high homology with those of P. argus described in this study, would indicate the presence of this peptides in Palinuridae family. Taking into account all similarities between these peptides with β-defensins from vertebrates, it is conceivable to further support the finding of a new family of β-defensins in invertebrate. © 2012 Elsevier Ltd.

Perera E.,University of Habana | Rodriguez-Viera L.,University of Habana | Perdomo-Morales R.,Center for Pharmaceuticals Research and Development | Montero-Alejo V.,Center for Pharmaceuticals Research and Development | And 3 more authors.
Journal of Comparative Physiology B: Biochemical, Systemic, and Environmental Physiology | Year: 2015

Trypsin enzymes have been studied in a wide variety of animal taxa due to their central role in protein digestion as well as in other important physiological and biotechnological processes. Crustacean trypsins exhibit a high number of isoforms. However, while differences in properties of isoenzymes are known to play important roles in regulating different physiological processes, there is little information on this aspect for decapod trypsins. The aim of this review is to integrate recent findings at the molecular level on trypsin enzymes of the spiny lobster Panulirus argus, into higher levels of organization (biochemical, organism) and to interpret those findings in relation to the feeding ecology of these crustaceans. Trypsin in lobster is a polymorphic enzyme, showing isoforms that differ in their biochemical features and catalytic efficiencies. Molecular studies suggest that polymorphism in lobster trypsins may be non-neutral. Trypsin isoenzymes are differentially regulated by dietary proteins, and it seems that some isoenzymes have undergone adaptive evolution coupled with a divergence in expression rate to increase fitness. This review highlights important but poorly studied issues in crustaceans in general, such as the relation among trypsin polymorphism, phenotypic (digestive) flexibility, digestion efficiency, and feeding ecology. © 2014, Springer-Verlag Berlin Heidelberg.

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