Francisco Vargas-Albores

An anticoagulant solution for haemolymph collection and prophenoloxidase studies of penaeid shrimp (Penaeus californiensis)

Abstract 1. 1. An anticoagulant solution was designed from data on osmolality, ionic concentration and pH to resemble shrimp haemolymph. 2. 2. This Shrimp Salt Solution (SSS) prevents coagulation and prophenoloxidase system activation during the extraction of shrimp haemolymph. 3. 3. The location of the the proPO system in the brown shrimp (Penaeus californiensis) was determined using this anticoagulant solution.

Beta glucan binding protein and its role in shrimp immune response

Abstract Despite their relatively short life and assumed lesser complexity, crustaceans have mechanisms to detect foreign matter. In particular, they appear to recognize common characteristics present in bacteria and fungi, such as lipopolysaccharides (LPS) and β-glucans. Although these microbial components can directly activate defensive cellular functions such as phagocytosis, melanization, encapsulation and coagulation, plasma recognition proteins amplify these stimuli. Beta glucan binding protein (BGBP) reacts with β-glucans and the glucan–BGBP complex induces degranulation and the activation of prophenoloxidase (proPO). This protein is present in all crustaceans studied so far and is highly conserved. Together with LPS-binding agglutinin, BGBP stimulates cellular function only after its reaction with LPS or β-glucans, resembling the secondary activities of vertebrate antibodies.

A lipopolysaccharide-binding agglutinin isolated from brown shrimp (Penaeus californiensis Holmes) haemolymph

Abstract 1. 1. Haemolymph from the brown shrimp (Penaeus californiensis Holmes) agglutinates erythrocytes of different vertebrate species. 2. 2. A fraction containing haemagglutinating activity was isolated by affinity chromatography. The corresponding protein showed a molecular weight of 175 kDa, formed by four subunits. 3. 3. The haemagglutinin is inhibited by GalNAc, NANA, fetuin and bacterial lipopolysaccharide. 4. 4. Here we demonstrate the ability of this agglutinin to react with different Vibrio strains, and the inhibitory capacity of LPS in this reaction.

A plasma protein isolated from brown shrimp (Penaeus californiensis) which enhances the activation of prophenoloxidase system by β-1,3-glucan

A beta-glucan-binding protein (BGBP) has been identified in brown shrimp plasma by using a polyclonal antiserum against a BGBP from the freshwater crayfish. The protein was purified by immunoaffinity chromatography, and its molecular and biological properties described. Brown shrimp BGBP is a monomeric protein with a molecular mass of 100 kDa, similar to those described for other crustacean BGBPs. This protein is capable of enhancing the prophenoloxidase (proPO) system activation induced by laminarin. Both amino acid composition and N-terminal sequence are markedly similar to those of crayfish BGBP.

cDNA cloning of the lysozyme of the white shrimp Penaeus vannamei.

Lysozyme, an antibacterial protein, has been implicated in innate immunity in invertebrates, but its activity in shrimp remained to be determined. We cloned the white shrimp lysozyme cDNA using a PCR strategy and detected its activity in haemocytes using a lytic-zone assay against Micrococcus luteus. The cloning was based on a reported EST (dbEST BE18831). The deduced amino acid sequence resulted in 150 amino with 46% identity to hen egg white lysozyme. RT-PCR was used to detect lysozyme mRNA in haemocytes. Analysis of the amino acid sequence of the shrimp lysozyme showed that it belongs to the C-type family of lysozymes. Furthermore, the lysozyme amino acid sequence contained extra residues at its C-terminus, which are characteristic of marine invertebrates. This information will be useful in future studies on the molecular mechanisms of immunity in marine invertebrates.

Hemolymph metabolic variables and immune response in Litopenaeus setiferus adult males: the effect of acclimation

Abstract Massive nauplii production in Litopenaeus setiferus by means of natural matings has not been reported. The main reason is the low spermatophore attachment rate that has been associated with male sterilization because of male reproductive tract degenerative and the male reproductive melanization syndromes (MRTDS and MRMS). This information indicated both syndromes could be related to the captivity and management stress that affects the immune system and the physiological state of shrimp. We used some blood metabolic variables, sperm quality, and immune response as indicators of captivity stress in adult males of L. setiferus . A comparison between freshly captured shrimp with shrimp maintained in the laboratory for 7 days at two temperatures were examined. Glucose and calcium were not different between fresh and acclimated shrimp at either temperature ( P >0.05). A reduction in triacylglycerol, proteins, and cholesterol was observed in acclimated shrimp in comparison with base line shrimp ( P L. setiferus was proposed.

Prophenoloxidase from brown shrimp (Penaeus californiensis) hemocytes

Prophenoloxidase (proPO) was purified from blood cells of the brown shrimp Penaeus californiensis by ultracentrifugation and dye affinity chromatography. The isolated proPO is a 114-kDa monomeric protein as determined by SDS-PAGE. This protein can be hydrolyzed by proteinases, producing a 107-kDa active phenoloxidase (PO). The isoelectric point for both protein forms was 7.35. The PO reaction using L-DOPA as substrate, has an optimum pH of 8, and was poorly inhibited by sodium azide, thiourea and EDTA, but strongly inhibited by diethyl thiocarbamate. According to the substrate affinity and inhibition characteristics, this phenoloxidase was classified as a tyrosinase-like phenoloxidase. Purified proPO was not activated by bacterial lipopolysaccharides or beta-glucans.

Haemolymph metabolic variables and immune response in Litopenaeus setiferus adult males: the effect of an extreme temperature

Abstract The stress of captivity could be the main reason for the loss of sperm quality of Litopenaeus setiferus (L) males in captivity. The aim of this study was focused on the physiological and immunological response of adult male shrimp exposed to a temperature extreme (33 °C) in order to understand how the stress modulates the physiological, immunological and reproductive behavior of L. setiferus males. We used some blood metabolic variables, sperm quality and immune response as indicators of stress measured daily during the 10-day experiment. The high temperature provoked loss of osmotic capacity (OC), a reduction in blood metabolites at day 5, a reduction in hemocyte pro Phenoloxidase (proPO) activity and a dramatic reduction in sperm quality in comparison to that observed in freshly captured wild shrimp populations. The ratio between proPO and granular cells (small granular and large granular cells) (proPO/GC) showed a cyclic behavior with lower peaks at days 3, 7 and 10 and high peaks at days 0, 5–6 and 8–9, evidencing migration and recovery of cells and proPO every 48–96 h, depending on the intensity of the stress. Such hemocyte migration was directed to the ampule and spermatophore where bacterial growth could be enhanced by abnormal and dead sperm cells. In such circumstances, a rapid mobilization of reserves could help to promote tissue regeneration in male testes and vas deferens. Such use of reserves could explain the reduction in blood metabolites observed at day 5 in present study. Although blood metabolites recovered after day 5, the loss of OC suggests that shrimp lose their osmotic homeostasis, possibly associated with the entrance of water due to inflammation of tissues provoked by melanization.

Proteins and amino acids in beers, their contents and relationships with other analytical data

Abstract Fluorometry, ion-exchange chromatography, electrophoretic separations and Fourier transform–infrared (FT–IR) spectra were used to determine and characterize amino acids and proteins in 15 different beer samples. Proteins precipitated by ammonium sulfate yielded complex electrophoretic patterns. The major bands corresponded to 45–40 kDa as determined by a two-dimensional gel electrophoresis (2-DE). Proteins and some amino acids are partially responsible for nutritional value and stability of beer. Therefore, electrophoretic analysis revealed that protein characterization of beer during all technological stages might be useful in its quality. FT–IR protein spectra showed the presence of I, II and III amide bands. Protein distribution and amino acid composition of beer differ significantly, depending on the raw materials and enzymatic reactions used in beer technology. Concentrations of histamine (3.02–3.23 mg/l), proline (1.60–3.13 mg/l) and tyramine (3.61–7.4 mg/l) increased during beer fermentation. Statistically significant change was registered in the protein content of the final product, which was less than that in wort ( p p

Purification and characterization of α2-macroglobulin from the white shrimp (Penaeus vannamei)

Abstract α2-Macroglobulin (α2M) is a broad-spectrum protease-binding protein abundant in plasma from vertebrates and several invertebrate phyla. This protein was purified from cell-free hemolymph of the white shrimp, Penaeus vannamei, using Blue-Sepharose and Phenyl-Sepharose chromatography. The shrimp α2M is a 380 kDa protein, a homodimer of two apparently identical subunits of ∼180 kDa linked by disulphide bridges. The amino acid sequence of the N-terminus is similar to the Limulus α2M counterpart. The shrimp α2M has a wide inhibition spectrum against different proteinase types including trypsin, leucine amino peptidase, chymotrypsin, elastase and papain. The secondary structure of shrimp α2M is mainly beta-sheet (36%), with a characteristic minimum elipticity at 217 nm. Evidence for a thiolester-mediated inhibition mechanism of proteases by α2M was provided by inactivation with methylamine.