Enhanced catalytic and conformational stability of Atlantic cod trypsin upon neoglycosylation.
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The applicability of psychrophilic enzymes is limited because of their lower thermodynamic stability in spite of their higher catalytic rate. In this study, we have shown that the thermodynamic stability of the psychrophilic Atlantic cod trypsin could be enhanced appreciably by covalent chemical modification with oxidized sucrose polymer without affecting its hydrolytic activity. The acquired stability of cod trypsin was found to be on par with the mesophilic porcine trypsin. (+info)
Genetic screening for prey in the gut contents from a giant squid (Architeuthis sp.).
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Giant squids (Architeuthis sp.) remain mysterious; they have evaded observation and are rarely taken from their deep sea habitat. Information on the diet of Architeuthis is scarce due to the limited number of specimens with morphologically recognizable remains in their digestive tracts. We explored the use of polymerase chain reaction (PCR)-based methods for detection of DNA in the prey remains and amorphous slurry from an Architeuthis gut sample. The DNA region amplified varied in size, allowing separation of fish and squid components. Sequence comparisons identified fish prey as Macruronus novaezelandiae. Isolation of Architeuthis DNA from an ingested tentacle and the presence of chitin fragments indicate cannibalism occurs in giant squid. Denaturing gradient gel electrophoresis was used to screen for less common DNA types, revealing a high frequency of PCR-generated false alleles, but no additional prey species. (+info)
Nonbioluminescent strains of Photobacterium phosphoreum produce the cell-to-cell communication signal N-(3-Hydroxyoctanoyl)homoserine lactone.
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Bioluminescence is a common phenotype in marine bacteria, such as Vibrio and Photobacterium species, and can be quorum regulated by N-acylated homoserine lactones (AHLs). We extracted a molecule that induced a bacterial AHL monitor (Agrobacterium tumefaciens NT1 [pZLR4]) from packed cod fillets, which spoil due to growth of Photobacterium phosphoreum. Interestingly, AHLs were produced by 13 nonbioluminescent strains of P. phosphoreum isolated from the product. Of 177 strains of P. phosphoreum (including 18 isolates from this study), none of 74 bioluminescent strains elicited a reaction in the AHL monitor, whereas 48 of 103 nonbioluminescent strains did produce AHLs. AHLs were also detected in Aeromonas spp., but not in Shewanella strains. Thin-layer chromatographic profiles of cod extracts and P. phosphoreum culture supernatants identified a molecule similar in relative mobility (Rf value) and shape to N-(3-hydroxyoctanoyl)homoserine lactone, and the presence of this molecule in culture supernatants from a nonbioluminescent strain of P. phosphoreum was confirmed by high-performance liquid chromatography-positive electrospray high-resolution mass spectrometry. Bioluminescence (in a non-AHL-producing strain of P. phosphoreum) was strongly up-regulated during growth, whereas AHL production in a nonbioluminescent strain of P. phosphoreum appeared constitutive. AHLs apparently did not influence bioluminescence, as the addition of neither synthetic AHLs nor supernatants delayed or reduced this phenotype in luminescent strains of P. phosphoreum. The phenotypes of nonbioluminescent P. phosphoreum strains regulated by AHLs remains to be elucidated. (+info)
Trophic cascades in a formerly cod-dominated ecosystem.
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Removal of top predators from ecosystems can result in cascading effects through the trophic levels below, completely restructuring the food web. Cascades have been observed in small-scale or simple food webs, but not in large, complex, open-ocean ecosystems. Using data spanning many decades from a once cod-dominated northwest Atlantic ecosystem, we demonstrate a trophic cascade in a large marine ecosystem. Several cod stocks in other geographic areas have also collapsed without recovery, suggesting the existence of trophic cascades in these systems. (+info)
Cloning of GLUT3 cDNA from Atlantic cod (Gadus morhua) and expression of GLUT1 and GLUT3 in response to hypoxia.
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A putative facilitative glucose transporter, GLUT3, cDNA was cloned from Atlantic cod. It is ubiquitously expressed, with substantial levels in kidney. The 519 aa protein has the highest sequence identity (66.3%) to grass carp GLUT3. Atlantic cod were exposed to a hypoxic challenge (45% DO2) for 24 h and the effects on GLUT1 and GLUT3 expression assessed. GLUT1 expression in gill is upregulated; however, in spleen, there is a significant decrease in both GLUT1 and GLUT3 expression. The increase in GLUT1 mRNA is considered to be associated with an increased energy demand on gill, whereas, the decrease in gene expression in spleen potentially reflects a general decrease in rates of transcription. (+info)
Isolation and identification of antimicrobial components from the epidermal mucus of Atlantic cod (Gadus morhua).
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The epidermal mucus of fish species has been found to contain antimicrobial proteins and peptides, which is of interest in regard to fish immunity. An acidic extract from the epidermal mucus of the Atlantic cod (Gadus morhua) was found to exhibit antimicrobial activity against Bacillus megaterium, Escherichia coli and Candida albicans. This activity varied significantly when salt was added to the antimicrobial assay, and was eliminated by pepsin digestion. No lysozyme activity was detected in the extract. By using weak cationic exchange chromatography together with reversed-phase chromatography, and monitoring the antimicrobial activity, we have isolated four cationic proteins from the mucus extract. Using N-terminal and C-terminal amino acid sequence analysis, together with MS, the antimicrobial proteins were identified as histone H2B (13 565 Da), ribosomal protein L40 (6397 Da), ribosomal protein L36A (12 340 Da) and ribosomal protein L35 (14 215 Da). The broad spectra of antimicrobial activities in the cod mucus and the characterization of four antimicrobial polypeptides suggest that mucus compounds contribute to the innate host defence of cod. (+info)
Conformation and stability of elastase from Atlantic cod, Gadus morhua.
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Metal binding and conformational stability characteristics of psychrophilic elastase (ACE) from Atlantic cod (Gadus morhua) has been investigated. Chelation to Ca(2+) was found to be important for maintaining the biologically active conformation and for the thermal stability of the enzyme. However, presence of metal ions such as Zn(2+), Fe(3+) and Cu(2+) was found to inhibit its hydrolytic activity and so did the chelating agent EDTA. Both pH and guanidinium chloride induced denaturation of the enzyme was followed by monitoring the changes in the tryptophan fluorescence. ACE exhibited a simple two-state unfolding pattern in both acidic and basic conditions with the midpoint of transition at pH values 4.08 and 10.29, respectively. Guanidinium chloride and heat induced denaturation of the enzyme was investigated at two pH values, 5.50 and 8.00, wherein the enzyme possesses similar tertiary structure but differ in its hydrolytic activity. Guanidinium chloride induced denaturation indicated that the enzyme unfolds with a C(m) of 1.53 M at pH 8.0 and a DeltaG(H2O) of 6.91 kJ mol(-1) (28.65 J mol(-1) residue(-1)) which is the lowest reported for psychrophilic enzymes investigated till-date. However, at pH 5.50, DeltaG(H2O) value is slightly lowered by 0.65 kJ mol(-1) consistent with the observed increase in the apparent quenching constant obtained with acrylamide. On the other hand, increase in T(m) by 38.45 degrees C was observed for the enzyme at acid pH (5.50) in comparison to the heat induced unfolding at pH 8.0. The increase in the apparent T(m) has been attributed to the possible weak intermolecular association of the enzyme molecules at moderately high temperatures that is favoured by the increase in the accessible surface area / dynamics under acidic conditions. The stability characteristics of ACE have been compared with the available data for mesophilic porcine pancreatic elastase and possible mechanism for the low temperature adaptation of ACE has been proposed. (+info)
Reversible inactivation of alkaline phosphatase from Atlantic cod (Gadus morhua) in urea.
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Alkaline phosphatase (AP) from Atlantic cod (Gadus morhua) is a zinc and magnesium containing homodimer that requires the oligomeric state for activity. Its kinetic properties are indicative of cold-adaptation. Here, the effect of urea on the structural stability was studied in order to correlate the activity with metal content, the microenvironment around tryptophan residues, and events at the subunit interface. At the lowest concentrations of urea, the first detected alteration in properties was an increase in the activity of the enzyme. This was followed by inactivation, and the release of half of the zinc content when the amount of urea reached levels of 2 M. Intrinsic tryptophan fluorescence and circular dichroism ellipticity changed in the range 2.5 to 8 M urea, signaling dimer dissociation, followed by one major monomer unfolding transition at 6-8 M urea as indicated by ANS fluorescence and KI fluorescence quenching. Gibbs free energy was estimated by the linear extrapolation method using a three-state model as 8.6 kcal/mol for dimer stability and 11.6 kcal/mol for monomer unfolding giving a total of 31.8 kcal/mol. Dimer association had a very small ionic contribution. Dimers were stable in relatively high concentration of urea, whereas the immediate vicinity around the active site was vulnerable to low concentrations of urea. Thus, inactivation did not coincide with dimer dissociation, suggesting that the active site is the most dynamic part of the molecule and closest related to cold-adaptation of its enzymatic activity. (+info)