In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti

<p>Abstract</p> <p>Background</p> <p>The major Dengue virus vector <it>Aedes aegypti </it>requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of <it>Ae. aegypti </it>midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes.</p> <p>Results</p> <p>We used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (k_cat/K_M) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin.</p> <p>Conclusions</p> <p>These data show that <it>in vitro </it>activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.</p

Moult cycle specific differential gene expression profiling of the crab Portunus pelagicus

Background: Crustacean moulting is a complex process involving many regulatory pathways. A holistic approach to examine differential gene expression profiles of transcripts relevant to the moulting process, across all moult cycle stages, was used in this study. Custom cDNA microarrays were constructed for Portunus pelagicus. The printed arrays contained 5000 transcripts derived from both the whole organism, and from individual organs such as the brain, eyestalk, mandibular organ and Y-organ from all moult cycle stages.Results: A total of 556 clones were sequenced from the cDNA libraries used to construct the arrays. These cDNAs represented 175 singletons and 62 contigs, resulting in 237 unique putative genes. The gene sequences were classified into the following biological functions: cuticular proteins associated with arthropod exoskeletons, farnesoic acid O-methyltransferase (FaMeT), proteins belonging to the hemocyanin gene family, lectins, proteins relevant to lipid metabolism, mitochondrial proteins, muscle related proteins, phenoloxidase activators and ribosomal proteins. Moult cycle-related differential expression patterns were observed for many transcripts. Of particular interest were those relating to the formation and hardening of the exoskeleton, and genes associated with cell respiration and energy metabolism.Conclusions: The expression data presented here provide a chronological depiction of the molecular events associated with the biological changes that occur during the crustacean moult cycle. Tracing the temporal expression patterns of a large variety of transcripts involved in the moult cycle of P. pelagicus can provide a greater understanding of gene function, interaction, and regulation of both known and new genes with respect to the moulting process

responses to temperature and salinity by white mullet mugil curema: possible explanation for the population decrease in baja california sur

The white mullet Mugil curema is distributed along the Gulf of Mexico and Pacific coasts and constitutes an important food resource in Mexico. However, during recent years the capture of this mugilid has decreased considerably, especially in Bahia and Ensenada de La Paz, Baja California Sur, Mexico. The effects of different levels of temperature (22, 26, and 30degreesC) and salinity (15, 25, and 35) on the instantaneous growth rate, energy content, and condition factor of early juveniles of Mugil curema were evaluated. Fish showed a lower growth rate, lower body energy content, and a less condition factor when exposed to high salinity and temperature. On the basis to the results obtained and considering the unusual extreme temperatures and salinity registered in Ensenada de La Paz, it is expected low functional performance of juvenile mullets beyond the overfishing might contribute to the population decrease of this resource

The role of lysosomal cysteine proteases in crustacean immune response

Over the long course of evolution and under the selective pressure exerted by pathogens and parasites, animals have selectively fixed a number of defense mechanisms against the constant attack of intruders. The immune response represents a key component to optimize the biological fitness of individuals. Two decades ago, prevention and control of diseases in crustacean aquaculture systems were considered priorities in most shrimp-producing countries, but knowledge was scarce and various pathogens have severely affected aquaculture development around the world. Scientific contributions have improved our understanding of the crustacean immune response. Several studies confirm the central role played by proteases in the immune response of animals, and the cooperative interaction of these enzymes in a wide variety of organisms is well known. This review summarizes the current information regarding the role of cysteine proteases in the immune system of Crustacea and points to aspects that are needed to provide a better integration of our knowledge

Marine Viruses: the Beneficial Side of a Threat

© 2014, Springer Science+Business Media New York. Marine viruses are ubiquitous, extremely diverse, and outnumber any form of life in the sea. Despite their ecological importance, viruses in marine environments have been largely ignored by the academic community, and only those that have caused substantial economic losses have received more attention. Fortunately, our current understanding on marine viruses has advanced considerably during the last decades. These advances have opened new and exciting research opportunities as several unique structural and genetic characteristics of marine viruses have shown to possess an immense potential for various biotechnological applications. Here, a condensed overview of the possibilities of using the enormous potential offered by marine viruses to develop innovative products in industries as pharmaceuticals, environmental remediation, cosmetics, material sciences, and several others, is presented. The importance of marine viruses to biotechnology should not be underestimated

Effect of diets containing different levels of highly unsaturated fatty acids on physiological and immune responses in Pacific whiteleg shrimp <i>Litopenaeus vannamei</i> (Boone) exposed to handling stress

Juveniles fed a diet containing a low or a high level of highly unsaturated fatty acids (HUFA) for 38 days were exposed to handling stress. In a first experiment, stress was applied daily for 30 days, after which the physiological and immunological variables were measured, whereas in a second experiment, stress was applied once and samples were obtained 1 and 24 h after the stressor event. Shrimp that were stressed for 30 days showed significantly lower survival, final weight and feed consumption compared with unstressed shrimp. The concentration of the high-density lipoprotein beta-glucan-binding protein was significantly higher in shrimp fed the high-HUFA diet. The glucose concentration in the haemolymph was significantly higher in long-term stressed shrimp compared to controls. The lactate level in the haemolymph was significantly lower in shrimp fed the high-HUFA diet. Lactate and glucose in the haemolymph increased in the 1-h stressed shrimp, but returned to normal levels in 24-h stressed shrimp. A negative effect of repeated-handling stress applied for 30 days was mainly observed on biological performance, whereas the single-stressor event had a more pronounced effect on shrimp physiological and immune responses measured 1 and 24 h after the stressor. A beneficial role of enrichment with HUFA on tolerance to handling stress was observed on immune response capacity

White Spot Syndrome Virus Orf514 Encodes a Bona Fide DNA Polymerase

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome, one of the most devastating diseases in shrimp aquaculture. The genome of WSSV includes a gene that encodes a putative family B DNA polymerase (ORF514), which is 16% identical in amino acid sequence to the Herpes virus 1 DNA polymerase. The aim of this work was to demonstrate the activity of the WSSV ORF514-encoded protein as a DNA polymerase and hence a putative antiviral target. A 3.5 kbp fragment encoding the conserved polymerase and exonuclease domains of ORF514 was overexpressed in bacteria. The recombinant protein showed polymerase activity but with very low level of processivity. Molecular modeling of the catalytic protein core encoded in ORF514 revealed a canonical polymerase fold. Amino acid sequence alignments of ORF514 indicate the presence of a putative PIP box, suggesting that the encoded putative DNA polymerase may use a host processivity factor for optimal activity. We postulate that WSSV ORF514 encodes a bona fide DNA polymerase that requires accessory proteins for activity and maybe target for drugs or compounds that inhibit viral DNA replication