Purificación de isoformas de proteasas tipo tripsina de crustáceos

Las tripsinas son las principales peptidasas digestivas de crustáceos. En los últimos años se han producido revisiones sobre las tripsinas de invertebrados, así como de crustáceos, e incluso de las presentes en determinada especie de crustáceo. Sin embargo, los aspectos relacionados con la purificación de estas enzimas (y sus isoformas) no se han abordado en estas revisiones. En la presente revisión se exponen los procedimientos más empleados en la purificación de tripsinas de crustáceos, las dificultades en la separación de isoformas, y se discuten las experiencias de los autores en la purificación de isoformas de tripsina en la langosta espinosa Panulirus argus, las cuales pudieran ser de interés para los que se inicien en el campo de la purificación de enzimas tipo tripsina de crustáceos. Se excluyen los aspectos básicos relacionados con los principios de cada método y con la purificación de proteínas en general, los cuales pueden ser encontrados en la vasta literatura publicada tanto en revisiones como en libros especializados

Using the In vitro pyrogen test in the validation of depyrogenation process by Dry-Heat

In the present study, the feasibility to employ the in vitro pyrogen test (IPT) in the validation of depyrogenation process is presented. As one of the main advantages of IPT is its ability to determine pyrogen absorbed to the container surface, direct incubation of diluted whole blood with the endotoxin indicator was first attempted. It was not possible to quantify the endotoxin in control indicators due to the high content, which is discussed. However, it was possible to demonstrate that indicators subjected to the depyrogenation process were indeed pyrogen free, a quality that is difficult to assure when the LAL assay is employed in extract of indicators or medical devices. On the other hand, IPT performed as well as LAL when endotoxin was previously extracted from the indicator surface. Finally, some conditions for incubation of whole blood with the test surface and to dilute the supernatant obtained from the incubation are presented.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Trypsin isozymes in the lobster Panulirus argus (Latreille, 1804): from molecules to physiology

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.Most of the work outlined on this topic was supported by the International Foundation for Science (No. A/4306-1 and No. A/4306-2) granted to EP, the Agencia Española de Cooperación Internacional/Asociación Universitaria Iberoamericana de Postgrado (AUIP/AECI) from Spain, and the Program ʻDoctorado Iberoamericano en Ciencias’ at the University of Cadiz, Spain.Peer reviewe

Carbohydrates digestion and metabolism in the spiny lobster (Panulirus argus): biochemical indication for limited carbohydrate utilization

As other spiny lobsters, Panulirus argus is supposed to use preferentially proteins and lipids in energy metabolism, while carbohydrates are well digested but poorly utilized. The aim of this study was to evaluate the effect of dietary carbohydrate level on digestion and metabolism in the spiny lobster P. argus. We used complementary methodologies such as post-feeding flux of nutrients and metabolites, as well as measurements of α-amylase expression and activity in the digestive tract. Lobsters readily digested and absorbed carbohydrates with a time-course that is dependent on their content in diet. Lobster showed higher levels of free glucose and stored glycogen in different tissues as the inclusion of wheat flour increased. Modifications in intermediary metabolism revealed a decrease in amino acids catabolism coupled with a higher use of free glucose as carbohydrates rise up to 20%. However, this effect seems to be limited by the metabolic capacity of lobsters to use more than 20% of carbohydrates in diets. Lobsters were not able to tightly regulate α-amylase expression according to dietary carbohydrate level but exhibited a marked difference in secretion of this enzyme into the gut. Results are discussed to highlight the limitations to increasing carbohydrate utilization by lobsters. Further growout trials are needed to link the presented metabolic profiles with phenotypic outcomes.This work was partially supported by Ministry of Economy and Competitiveness through Project AGL2013-48835-C2-R and AGL2016-76069-C2-1-R granted to Juan M. Mancera, UCA-International fellowship granted to Leandro Rodriguez-Viera, and AUIP grant in the framework of the Academic Mobility Program between Andalusian and Ibero-American Universities associated to the AUIP granted to Leandro Rodríguez-Viera.Peer reviewe