Insights into the Adsorption Mechanisms of the Antimicrobial Peptide CIDEM-501 on Membrane Models.

CIDEM-501 is a hybrid antimicrobial peptide rationally designed based on the structure of panusin and panulirin template peptides. The new peptide exhibits significant antibacterial activity against multidrug-resistant pathogens (MIC = 2-4 µM) while conserving no toxicity in human cell lines. We conducted molecular dynamics (MD) simulations using the CHARMM-36 force field to explore the CIDEM-501 adsorption mechanism with different membrane compositions. Several parameters that characterize these interactions were analyzed to elucidate individual residues' structural and thermodynamic contributions. The membrane models were constructed using CHARMM-GUI, mimicking the bacterial and eukaryotic phospholipid compositions. Molecular dynamics simulations were conducted over 500 ns, showing rapid and highly stable peptide adsorption to bacterial lipids components rather than the zwitterionic eucaryotic model membrane. A predominant peptide orientation was observed in all models dominated by an electric dipole. The peptide remained parallel to the membrane surface with the center loop oriented to the lipids. Our findings shed light on the antibacterial activity of CIDEM-501 on bacterial membranes and yield insights valuable for designing potent antimicrobial peptides targeting multi- and extreme drug-resistant bacteria.

The C-Terminus of Panusin, a Lobster ß-Defensin, Is Crucial for Optimal Antimicrobial Activity and Serum Stability.

ß-defensins are one of the most abundant and studied families of antimicrobial peptides (AMPs). Because of their selective toxicity to bacterial membranes and a broad spectrum of microbicidal action, ß-defensins are regarded as potential therapeutic agents. This work focuses on a ß-defensin-like AMP from the spiny lobster Panulirus argus (hereafter referred to as panusin or PaD). This AMP is structurally related to mammalian defensins via the presence of an αß domain stabilized by disulfide bonds. Previous studies of PaD suggest that its C-terminus (Ct_PaD) contains the main structural determinants of antibacterial activity. To confirm this hypothesis, we made synthetic versions of PaD and Ct_PaD to determine the influence of the C-terminus on antimicrobial activity, cytotoxicity, proteolytic stability, and 3D structure. After successful solid-phase synthesis and folding, antibacterial assays of both peptides showed truncated Ct_PaD to be more active than native PaD, confirming the role of the C-terminus in activity and suggesting that cationic residues in that region enhance binding to negatively charged membranes. On the other hand, neither PaD nor Ct_PaD were hemolytic or cytotoxic in human cells. Proteolysis in human serum was also studied, showing high (>24 h) t1/2 values for PaD and lower but still considerable for Ct_PaD, indicating that the missing native disulfide bond in Ct_PaD alters protease resistance, albeit not decisively. NMR-2D experiments in water agree with the results obtained by circular dichroism (CD), where in SDS micelles, CD showed both peptides adopting an increasingly ordered structure in a hydrophobic environment, in tune with their ability to perturb bacterial membrane systems. In conclusion, while the ß-defensin features of PaD are confirmed as advantageous in terms of antimicrobial activity, toxicity, and protease stability, the results of the present work suggest that these same features are preserved, even enhanced, in the structurally simpler Ct_PaD, which must therefore be viewed as a valuable lead for the development of novel anti-infectives.

Crustacean Proteases and Their Application in Debridement.

Digestive proteases from marine organisms have been poorly applied to biomedicine. Exceptions are trypsin and other digestive proteases from a few cold-adapted or temperate fish and crustacean species. These enzymes are more efficient than enzymes from microorganism and higher vertebrates that have been used traditionally. However, the biomedical potential of digestive proteases from warm environment species has received less research attention. This review aims to provide an overview of this unrealised biomedical potential, using the debridement application as a paradigm. Debridement is intended to remove nonviable, necrotic and contaminated tissue, as well as fibrin clots, and is a key step in wound treatment. We discuss the physiological role of enzymes in wound healing, the use of exogenous enzymes in debridement, and the limitations of cold-adapted enzymes such as their poor thermal stability. We show that digestive proteases from tropical crustaceans may have advantages over their cold-adapted counterparts for this and similar uses. Differences in thermal stability, auto-proteolytic stability, and susceptibility to proteinase inhibitors are discussed. Furthermore, it is proposed that the feeding behaviour of the source organism may direct the evaluation of enzymes for particular applications, as digestive proteases have evolved to fill a wide variety of feeding habitats, natural substrates, and environmental conditions. We encourage more research on the biomedical application of digestive enzymes from tropical marine crustaceans.

The clotting system in decapod crustaceans: History, current knowledge and what we need to know beyond the models.

Hemolymph coagulation is among the major arms of the humoral immune response in crustaceans. According to the current model, hemolymph clotting in decapod crustacean relies mostly on the polymerization of the plasmatic clotting protein (CP) which is directly promoted by calcium-depended transglutaminase (TGase) released from hemocytes upon microbial stimulus or injury. However, the type of hemocytes containing TGase, and hence how the TGase is released, might vary among species. Thus, we discourse here about possible mechanisms for clotting initiation. On the other hand, the initiation of coagulation reaction in the absence of microbial elicitors is poorly understood and seems to involve hemocytes lability, yet the mechanism remains unknown. A cellular clottable protein called coagulogen, different to the plasma CP, occurs in several species and could be related with the immune response, but the biological relevance of this protein is unknown. It is also demonstrated that the clotting response is actively involved in defense against pathogens. In addition, both TGase and the CP show pleiotropic functions, and although both proteins are relatively conserved, some of their physic-chemical properties vary significantly. The occurrence of differences in the clotting system in crustaceans is conceivable given the high number of species and their diverse ecology. Results from still non-studied decapods may provide explanation for some of the issues presented here from an evolutionary perspective.

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.

Panusin represents a new family of ß-defensin-like peptides in invertebrates.

Beta_defensin have been solely found in vertebrates until ß-defensin-like peptides were described as transcript isoforms in two species of Panulirus genus. They were considered as putative antimicrobials since their biological activity have not been demonstrated. Here we purified and characterized a defensin-like peptide from the hemocytes of spiny lobster P. argus, hereafter named panusin. Structurally, panusin presents a cysteine-stabilized α/ß motif, and is prone to form homodimers. Biological activity of panusin showed broad-spectrum antimicrobial activity, characterized for being strikingly salt-resistant. Panusin did not showed hemolytic activity but was demonstrated its binding capacity to different lipid membrane models, indicating amphipathicity of ß-sheet core as driving force for its antimicrobial activity. Panusin is considered a new kind of arthropod defensin which share structural and biological features with beta-defensin from vertebrates. The presence of beta-defensin like peptides in crustacean might suggest the emergence of the evolutionary relationship of ß-defensins from vertebrates.

Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus.

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.

Soluble ß-(1,3)-glucans enhance LPS-induced response in the monocyte activation test, but inhibit LPS-mediated febrile response in rabbits: Implications for pyrogenicity tests.

In the present study, we aimed to determine the influence of ß-(1,3)-d-glucans on the LPS-induced pro-inflammatory cytokine response in the Monocyte Activation Test (MAT) for pyrogens, and on the LPS-induced febrile response in the Rabbit Pyrogen Test (RPT), thus evaluating the resulting effect in the outcome of each test. It was found that ß-(1,3)-d-glucans elicited the production of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α, also known as endogenous pyrogens, but not enough to classify them as pyrogenic according to MAT. The same ß-(1,3)-d-glucans samples were non-pyrogenic by RPT. However, ß-(1,3)-d-glucans significantly enhanced the LPS-induced pro-inflammatory cytokines response in MAT, insomuch that samples containing non-pyrogenic concentrations of LPS become pyrogenic. On the other hand, ß-(1,3)-d-glucans had no effect on sub-pyrogenic LPS doses in the RPT, but surprisingly, inhibited the LPS-induced febrile response of pyrogenic LPS concentrations. Thus, while ß-(1,3)-d-glucans could mask the LPS pyrogenic activity in the RPT, they exerted an overstimulation of pro-inflammatory cytokines in the MAT. Hence, MAT provides higher safety since it evidences an unwanted biological response, which is not completely controlled and is overlooked by the RPT.

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.

The trypsin inhibitor panulirin regulates the prophenoloxidase-activating system in the spiny lobster Panulirus argus.

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.

Efecto neuroprotector de una formulación nasal de eritropoyetina con bajo contenido de ácido siálico / Neuroprotective effect of a nasal formulation of erythropoietin with low sialic acid content

La administración intranasal de eritropoyetina humana recombinante con bajo contenido de ácido siálico (rHu-EPOb) puede ser empleada como un agente neuroprotector eficaz en el tratamiento de la isquemia cerebral. En el presente trabajo fueron administradas 10 µg de rHu-EPOb 3 veces al día durante 2 días, comenzando inmediatamente antes de la inducción de la isquemia focal transitoria en ratas. Los resultados demostraron que la administración nasal de rHu-EPOb mejoró significativamente el comportamiento neurológico en animales sometidos a isquemia focal transitoria y disminuyó el área de infarto en las diferentes zonas cerebrales estudiadas. La evaluación histopatológica de la zona del hipocampo de los animales sometidos al daño neuronal, evidenció ligeras alteraciones caracterizadas por la presencia de neuronas picnóticas. Los resultados mostraron el efecto neuroprotector de la administración intranasal de una formulación a partir de rHu-EPOb en ratas sometidas a isquemia focal transitoria y confirman la necesidad de realizar estudios clínicos para evaluar el posible efecto anti-isquémico de esta formulación(AU)

Intranasal administration of recombinant human erythropoietin (rHu-EPOb) may be used lake an effective neuroprotective agent in treatment of cerebral ischemia. In present paper 10 µg of rHu-EPOb three times/day during 2 days were administered, starting immediately before induction of a transitory focal ischemia in rats. Results showed that nasal administration of rHu-EPOb improved significantly the neurologic behavior in animals underwent to transitory focal ischemia, and decreased infarction area in the different cerebral zones studied. Histopathological assessment of hippocampus zone of animal underwent to neuronal damage, showed slight alterations characterized by presence of pycnotic neurons. Results showed the neuroprotective effect of intranasal administration of a formula from rHu-EPOb in rats underwent to transitory focal ischemia, and confirm the need of clinical studies to assess the possible anti-ischemic effect of this formula(AU)

Efecto neuroprotector de una formulación nasal de eritropoyetina con bajo contenido de ácido siálico / Neuroprotective effect of a nasal formulation of erythropoietin with low sialic acid content

La administración intranasal de eritropoyetina humana recombinante con bajo contenido de ácido siálico (rHu-EPOb) puede ser empleada como un agente neuroprotector eficaz en el tratamiento de la isquemia cerebral. En el presente trabajo fueron administradas 10 µg de rHu-EPOb 3 veces al día durante 2 días, comenzando inmediatamente antes de la inducción de la isquemia focal transitoria en ratas. Los resultados demostraron que la administración nasal de rHu-EPOb mejoró significativamente el comportamiento neurológico en animales sometidos a isquemia focal transitoria y disminuyó el área de infarto en las diferentes zonas cerebrales estudiadas. La evaluación histopatológica de la zona del hipocampo de los animales sometidos al daño neuronal, evidenció ligeras alteraciones caracterizadas por la presencia de neuronas picnóticas. Los resultados mostraron el efecto neuroprotector de la administración intranasal de una formulación a partir de rHu-EPOb en ratas sometidas a isquemia focal transitoria y confirman la necesidad de realizar estudios clínicos para evaluar el posible efecto anti-isquémico de esta formulación.

Intranasal administration of recombinant human erythropoietin (rHu-EPOb) may be used lake an effective neuroprotective agent in treatment of cerebral ischemia. In present paper 10 µg of rHu-EPOb three times/day during 2 days were administered, starting immediately before induction of a transitory focal ischemia in rats. Results showed that nasal administration of rHu-EPOb improved significantly the neurologic behavior in animals underwent to transitory focal ischemia, and decreased infarction area in the different cerebral zones studied. Histopathological assessment of hippocampus zone of animal underwent to neuronal damage, showed slight alterations characterized by presence of pycnotic neurons. Results showed the neuroprotective effect of intranasal administration of a formula from rHu-EPOb in rats underwent to transitory focal ischemia, and confirm the need of clinical studies to assess the possible anti-ischemic effect of this formula.

Hemocyanin-derived phenoloxidase activity in the spiny lobster Panulirus argus (Latreille, 1804).

Hemocyanin and phenoloxidase belong to the type-3 copper protein family, sharing a similar active center whereas performing different roles. In this study, we demonstrated that purified hemocyanin (450 kDa) from the spiny lobster Panulirus argus shows phenoloxidase activity in vitro after treatment with trypsin, chymotrypsin and SDS (0.1% optimal concentration), but it is not activated by sodium perchlorate or isopropanol. The optimal pHs of the SDS-activated hemocyanin were 5.5 and 7.0. Hemocyanin from spiny lobster behaves as a catecholoxidase. Kinetic characterization using dopamine, L-DOPA and catechol shows that dopamine is the most specific substrate. Catechol and dopamine produced substrate inhibition above 16 and 2 mM respectively. Mechanism-based inhibition was also evidenced for the three substrates, being less significant for L-DOPA. SDS-activated phenoloxidase activity is produced by the hexameric hemocyanin. Zymographic analysis demonstrated that incubation of native hemocyanin with trypsin and chymotrypsin, produced bands of 170 and 190 kDa respectively, with intense phenoloxidase activity. Three polypeptide chains of 77, 80 and 89 kDa of hemocyanin monomers were identified by SDS-PAGE. Monomers did not show phenoloxidase activity induced by SDS or partial proteolysis.

Estandarización del ensayo del lisado de amebocitos de Limulus (LAL): método de gelificación / Standardization of the Limulus amebocyte lysate (LAL): gelification method

Entre las principales aplicaciones del método del lisado de amebocitos de Limulus (LAL) está el control de endotoxinas en producto final de drogas parenterales. Para la aplicación exitosa del ensayo se requiere del dominio de un conjunto de habilidades, por lo que usualmente se corre en laboratorios especializados que ya cuentan con una basta experiencia. Aunque ya es un método ampliamente establecido y conocido, la aplicación del ensayo puede ser laborioso, consumir tiempo y reactivos hasta la obtención de resultados correctos. En el presente trabajo se describe la estandarización del ensayo del LAL por el método de gelificación. Se evalúan varias estrategias con la finalidad de optimizar y/o reducir el tiempo total de ensayo, el empleo de materiales como puntas certificadas libres de endotoxinas, tubos de ensayo y la sustitución de agua libre de endotoxinas suministrada por los fabricantes de reactivo LAL por agua para inyección. El procedimiento estandarizado produce resultados válidos según los criterios de la Farmacopea de EE.UU(AU)

Validación de un ciclo de despirogenización por calor seco con el empleo del ensayo del lisado de amebocitos de limulus / Validation of a depyrogenation cycle by dry heat with the utilization of the limulus amoebocytes lysate assay

Para el cumplimiento de las buenas prácticas de laboratorio y de fabricación de parenterales, una de las exigencias es la validación de los ciclos de despirogenización que se emplean. En el presente trabajo se describe la validación de un ciclo de despirogenización por calor seco en un horno de convección. En la primera etapa se estudiaron características físicas del equipo como el tiempo requerido para alcanzar la temperatura establecida, su distribución, estabilidad, y la influencia de la carga en el patrón de calentamiento. Considerando los resultados de esta fase se estableció un proceso total de 7 h a 180 ºC. La segunda etapa consistió en la determinación del grado de despirogenización retando el proceso con bioindicadores de endotoxinas. La concentración de endotoxinas en los bioindicadores control y sometidos al proceso de despirogenización se cuantificó con el empleo del ensayo del lisado de amebocitos de limulus método de gelificación. La diferencia entre el contenido de endotoxinas en el control y los tratamientos fue de 2 400 U de endotoxinas, por lo que el proceso rindió una reducción logarítmica mínima de 4,6 log, la cual es mayor que el límite de 3 log establecido por la Farmacopea de los Estados Unidos(AU)

Validación de un ciclo de despirogenización por calor seco con el empleo del ensayo del lisado de amebocitos de limulus / Validation of a depyrogenation cycle by dry heat with the utilization of the limulus amoebocytes lysate assay

Para el cumplimiento de las buenas prácticas de laboratorio y de fabricación de parenterales, una de las exigencias es la validación de los ciclos de despirogenización que se emplean. En el presente trabajo se describe la validación de un ciclo de despirogenización por calor seco en un horno de convección. En la primera etapa se estudiaron características físicas del equipo como el tiempo requerido para alcanzar la temperatura establecida, su distribución, estabilidad, y la influencia de la carga en el patrón de calentamiento. Considerando los resultados de esta fase se estableció un proceso total de 7 h a 180 ºC. La segunda etapa consistió en la determinación del grado de despirogenización retando el proceso con bioindicadores de endotoxinas. La concentración de endotoxinas en los bioindicadores control y sometidos al proceso de despirogenización se cuantificó con el empleo del ensayo del lisado de amebocitos de limulus método de gelificación. La diferencia entre el contenido de endotoxinas en el control y los tratamientos fue de 2 400 U de endotoxinas, por lo que el proceso rindió una reducción logarítmica mínima de 4,6 log, la cual es mayor que el límite de 3 log establecido por la Farmacopea de los Estados Unidos