Center for Pharmaceuticals Research and Development

Havana, Cuba

Center for Pharmaceuticals Research and Development

Havana, Cuba
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Rodriguez-Viera L.,University of Habana | Rodriguez-Viera L.,University of Cádiz | Perera E.,Institute Ciencias Marinas Of Andalucia | Perera E.,Institute Of Aquaculture Torre Of La Sal Iats Csic | And 8 more authors.
PLoS ONE | Year: 2016

Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase. © 2016 Rodríguez-Viera et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Lopes A.M.,University of Sao Paulo | Romeu J.S.,Center for Genetic Engineering and Biotechnology | Meireles R.P.,Center for Genetic Engineering and Biotechnology | Perera G.M.,Center for Genetic Engineering and Biotechnology | And 3 more authors.
Chinese Journal of Chromatography (Se Pu) | Year: 2012

Endotoxins (also known as lipopolysaccharides (LPS)) are undesirable by-products of recombinant proteins, purified from Escherichia coli. LPS can be considered stable under a wide range of temperature and pH, making their removal one of the most difficult tasks in downstream processes during protein purification. The inherent toxicity of LPS makes their removal an important step for the application of these proteins in several biological assays and for a safe parenteral administration. Immobilized metal affinity chromatography (IMAC) enables the affinity interactions between the metal ions (immobilized on the support through the chelating compound) and the target molecules, thus enabling high-efficiency separation of the target molecules from other components present in a mixture. Affinity chromatography is applied with Ca2+ -iminodiacetic acid (IDA) to remove most of the LPS contaminants from the end product (more than 90%). In this study, the adsorption of LPS on an IDA-Ca2+ was investigated. The adsorption Freundlich isotherm of LPS-IDA-Ca2+ provides a theoretical basis for LPS removal. It was found that LPS is bound mainly by interactions between the phosphate group in LPS and Ca2+ ligands on the beads. The factors such as pH (4.0 or 5.5) and ionic strength (1.0 mol / L) are essential to obtain effective removal of LPS for contaminant levels between endotoxin' concentration values less than 100 EU / mL and 100 000 EU / mL. This new protocol represents a substantial advantage in time, effort, and production costs.


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.


PubMed | Center for Pharmaceuticals Research and Development, Institute Ciencias Marinas Of Andalucia, University of Habana, University of Cádiz and Institute for Animal Science
Type: Journal Article | Journal: 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 protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other nutrients.


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.


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.


PubMed | Center for Pharmaceuticals Research and Development
Type: Journal Article | Journal: The Journal of biological chemistry | Year: 2013

The melanization reaction promoted by the prophenoloxidase-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.


PubMed | Center for Pharmaceuticals Research and Development
Type: Journal Article | Journal: Fish & 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. -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 -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 -defensins from vertebrates. The phylogenetic study carried out by probabilistic methods confirmed the relation with ancestral -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 -defensins from vertebrates, it is conceivable to further support the finding of a new family of -defensins in invertebrate.


PubMed | Center for Pharmaceuticals Research and Development
Type: Evaluation Studies | Journal: ALTEX | Year: 2011

Disadvantages of the regulatory pyrogen test to assure safety of the end-product Human Serum Albumin (HSA) for parenteral use call for the implementation of an alternative test. In the current study, 16 HSA batches were assayed for pyrogens in parallel with the Rabbit Pyrogen Test, conventional and endotoxin-specific LAL assay and monocyte activation test (MAT). It was found that all HSA batches were contaminated with (1,3)-beta-glucans, which interfere with the conventional LAL. Endotoxin-specific LAL was not suitable to test HSA due to unacceptable endotoxin recovery. Experiments combining polymyxin B and MAT demonstrated that pyrogenic batches were mainly contaminated with endotoxins. However, endotoxin-specific LAL failed to detect one of them. The contaminating (1,3)-beta-glucans enhanced the MAT/IL-6 response to endotoxin, but not that of MAT/IL-1beta. The endotoxin equivalent concentrations obtained using the IL-6 readout were usually higher than those using IL-1beta, probably owing to the direct induction of IL-6 release from monocytes by (1,3)-beta-glucans. The MAT correlates with the rabbit pyrogen test, providing a higher safety level for pyrogenicity testing of HSA and probably other therapeutic proteins.

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