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K V Ewart

Publications and source records attributed to K V Ewart.

At least 19 recordsLinked to original sources
PubMed2004

Identification of immune-relevant genes from atlantic salmon using suppression subtractive hybridization.

In order to probe the interaction between an invading microorganism and its host, we have investigated differential gene expression in Atlantic salmon (Salmo salar) experimentally infected with the pathogen Aeromonas salmonicida, the causative agent of furunculosis. Subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH) from 3 immune-relevant tissues at 2 time points during the infection process. Both forward- and reverse-subtracted libraries were generated, and approximately 200 clones were sequenced from each library, giving a total of 1778 expressed sequence tags (ESTs), which were annotated according to functional categories and deposited in GenBank (BQ035314-BQ037059). Numerous genes involved in signal transduction, innate immunity, and other processes have been uncovered in the subtractive libraries. These include known acute-phase reactants, along with more novel genes encoding proteins such as tachylectin, hepcidin, precerebellin-like protein, O-methyltransferase, a putative saxitoxin-binding protein, and others. A subset of genes that were represented in the subtracted libraries was further analyzed by virtual Northern, or reverse transcription-polymerase chain reaction (RT-PCR) assays to verify their differential expression as a result of infection.

Aeromonas salmonicida↗
PubMed2003

Cloning and characterization of the Atlantic salmon serum lectin, a long-form C-type lectin expressed in kidney.

We report the cloning of four distinct cDNAs and a genomic sequence encoding a multimeric serum lectin found in the blood of Atlantic salmon (Salmo salar). The sequence variation among the cDNAs as well as genomic Southern blotting analysis revealed a multi-gene family. Expression of the salmon serum lectin (SSL) was specific to kidney, as demonstrated by RT-PCR. Analysis of the 173-amino acid sequence of SSL confirmed that it is a member of the C-type lectin superfamily. Sequence alignments and intron/exon structure of the SSL gene showed it to belong to the type VII C-type lectins, which normally bind to galactose or other ligands, whereas the SSL protein sequence contains the EPN motif of mannose-binding C-type lectins, that bind mannose or related carbohydrates.

Amino Acid Sequence↗
PubMed2002

Molecular cloning of trypsin cDNAs and trypsin gene expression in the salmon louse Lepeophtheirus salmonis (Copepoda: Caligidae).

The salmon louse, Lepeophtheirus salmonis, is a marine ectoparasitic copepod that infects salmonid fishes. We are studying the interactions between this parasite and its salmonid hosts, as it is a common cause of disease in both wild and farmed stocks of salmon. In this paper, we report on the cloning and sequencing of seven trypsin-like enzymes from a cDNA library prepared from whole body preadult female and male L. salmonis. The predicted trypsin activation peptides are 23 or 24 residues in length, considerably longer than previously reported activation peptides of other animals. Differences in the putative signal and activation peptide sequences of the trypsin isoforms suggest that these forms differ in their regulation and function. The calculated molecular weights of the trypsins range from 23.6 to 23.7 kDa. There are eight cysteine residues, which suggest the presence of four disulfide bridges. These trypsins are very similar (>or=46% aa identity) to other crustacean trypsins and insect hypodermins. Using in situ hybridization techniques trypsinogen expression could be identified in all three cell types of the midgut.

Amino Acid Sequence↗
PubMed2001

Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar).

Antimicrobial activity was detected in acid extracts of liver, intestine, and stomach of healthy Atlantic salmon (Salmo salar). An antimicrobial protein was isolated from salmon liver using acid extraction followed by ammonium sulfate precipitation, large-scale gel filtration chromatography, reverse-phase HPLC, and size exclusion HPLC. The salmon antimicrobial (SAM) protein was found to have a molecular mass of 20,734 Da by MALDI TOF mass spectrometry. Peptide mass fingerprinting and partial sequencing by tandem nanoelectrospray mass spectrometry identified the protein as histone H1. The protein had a minimal inhibitory concentration of 31 microg/mL against E. coli D31 in a plate clearing assay. The effect of the SAM protein on bacterial morphology was indistinguishable from that of (Ala-(8,13,18))-magainin II, as shown by scanning electron microscopy, which suggests that the protein disrupts E. coli membranes in a manner similar to that of most antimicrobial peptides. This protein may act as an antimicrobial in vivo through active secretion or by release from cells during infection-related apoptosis.

Animals↗
PubMed2001

Cloning of glycerol-3-phosphate dehydrogenase cDNAs from two fish species and effect of temperature on enzyme expression in rainbow smelt (Osmerus mordax).

Rainbow smelt (Osmerus mordax) can accumulate extreme levels of glycerol in their blood during winter. Low temperatures are required for glycerol accumulation in smelt blood and the enzyme glycerol-3-phosphate dehydrogenase (GPDH) has been suggested to play a role in glycerol production/concentration in this species. In the present study, cDNA sequences encoding glycerol-3-phosphate dehydrogenase (GPDH) from rainbow smelt and Atlantic salmon (Salmo salar) were cloned. The encoded GPDH protein sequences were very similar to one another (88% identity). Using RT-PCR, GPDH mRNA was detected in skin, gill, heart, head kidney, brain and liver from both salmon and smelt obtained in December. However, GPDH was not detected in salmon intestine and spleen or in smelt intestine. Examination of GPDH expression in smelt liver during February by Northern blotting revealed temperature regulation. Elevation of the temperature resulted in a significant decrease in liver GPDH transcript level. Serum glycerol levels decreased concomitantly. These findings suggest a role for GPDH in the accumulation of glycerol in smelt at low temperatures.

Amino Acid Sequence↗
PubMed1999

Studies of a putative ice-binding motif in winter flounder skin-type anti-freeze polypeptide.

Winter flounder contains two distinct anti-freeze protein isoforms, which are the liver-type extracellular anti-freeze proteins and the skin-type intracellular anti-freeze protein. The skin-type anti-freeze proteins exhibit lower anti-freeze activities than the liver-type isoforms and this might be due to their lacking complete ice-binding motifs. One of the skin-type anti-freeze proteins, skin-type anti-freeze protein-3, does contain putative overlapping ice-binding motifs with the sequences '-K-DT-' and '-DT-K-'. Synthetic anti-freezes containing 0-3 repeats of the '-DT-K-' motif were tested for stability and activity. Loss of the single '-DT-K-' of skin-type anti-freeze protein-3 increases the anti-freeze activity and increasing the number of motifs to two or three lowers the activity. The decrease in activity with an increasing frequency of the motif correlates with a decrease in the helical content of these peptides at 0 degrees C.

Animals↗
PubMed1999

Structure, function and evolution of antifreeze proteins.

Antifreeze proteins bind to ice crystals and modify their growth. These proteins show great diversity in structure, and they have been found in a variety of organisms. The ice-binding mechanisms of antifreeze proteins are not completely understood. Recent findings on the evolution of antifreeze proteins and on their structures and mechanisms of action have provided new understanding of these proteins in different contexts. The purpose of this review is to present the developments in contrasting research areas and unite them in order to gain further insight into the structure and function of the antifreeze proteins.

Animals↗
PubMed1999

Identification of a pathogen-binding lectin in salmon serum.

A mannose-binding lectin was isolated from the blood serum of Atlantic salmon (Salmo salar). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and non-reducing conditions revealed a multimeric structure composed of 17000 Mr subunits. Hexosamine analysis and glycosidase digestion showed that the lectin is not glycosylated and amino acid analysis revealed no unusual compositional features. Using ruthenium red staining, the lectin was shown to bind Ca2+ ions. N-terminal sequencing by Edman degradation gave: H2N-TGAKGAEEGVVPAETRNQXPTGWFQFGS. A database search revealed no similarity to protein sequences deposited to date. Binding experiments using biotinylated lectin revealed that it specifically recognizes and binds to mannose on the surfaces of two salmon pathogens, Vibrio anguillarum and Aeromonas salmonicida, implying an immunological role for this lectin in Atlantic salmon.

Aeromonas↗
PubMed1999

Enhancement of anti-Aeromonas salmonicida activity in Atlantic salmon (Salmo salar) macrophages by a mannose-binding lectin.

We investigated the effects of a calcium-dependent mannose-binding lectin isolated from the serum of Atlantic salmon on Aeromonas salmonicida viability and the anti-A. salmonicida activity of Atlantic salmon macrophages. In the absence of other factors, binding of this lectin at concentrations of 0.8, 4.0 and 20.0 ng ml(-1) to virulent A. salmonicida failed to significantly reduce (P> 0.05) cell viability. However, binding of the lectin to A. salmonicida did result in significant (P < or = 0.05) dose-dependent increases in phagocytosis, and bactericidal activity. Significant increases (P < or = 0.05) were also observed in phagocyte respiratory burst activity within the lectin concentration range of 4.0-20.0 ng ml(-1) but the stimulation was not dose dependent at these lectin concentrations. At the lowest lectin concentration tested (0.32 ng ml(-1)), a significant decrease (P < or = 0.05) in respiratory burst was observed. The structure and activity of this lectin are similar to that of mammalian mannose-binding lectins, which are known to play a pivotal role in innate immunity. The presence of this lectin may be an important defense mechanism against Gram-negative bacteria such as A. salmonicida.

Aeromonas↗
PubMed1999

Secretory expression and site-directed mutagenesis studies of the winter flounder skin-type antifreeze polypeptides.

Winter flounder contains both liver-type, extracellular antifreeze polypeptides (wflAFPs) and less active skin-type, intracellular antifreeze polypeptides (wfsAFPs). The lower activity of wfsAFPs might be due to their lack of complete ice-binding motifs '-K-DT-'. In order to test the functional role of this putative ice-binding motif, mutations were introduced into the N-terminal or C-terminal regions of wfsAFP-2, which lack any presumptive ice-binding motifs. The wild-type and mutant wfsAFP-2 were secreted in Escherichia coli culture media as mature antifreeze proteins and purified to homogeneity. Surprisingly, the antifreeze activity decreased with the introduction of ice-binding motifs. However, there was a corresponding decrease in alpha-helical content as well as thermal stability and this would suggest a compromise in retaining helical structure with the presence of ice-binding motifs. These studies have brought new definitions of the roles of ice-binding motif residues in type I antifreeze proteins.

Amino Acid Sequence↗
PubMed1998

Skin-type antifreeze protein from the shorthorn sculpin, Myoxocephalus scorpius. Expression and characterization of a Mr 9, 700 recombinant protein.

A cDNA clone encoding a presumptive antifreeze protein was isolated from a skin library from shorthorn sculpin, Myoxocephalus scorpius. The clone encodes a 92-residue mature polypeptide (sssAFP-2) without any signal and prosequence, which suggests an intracellular localization. It is the largest alanine-rich, alpha-helical type I antifreeze protein known. A recombinant fusion protein containing an N-terminal-linked His-tag was produced and purified from Escherichia coli. This protein is alpha-helical at 0 degreesC and exhibits significant antifreeze activity. Northern blot and reverse transcription-polymerase chain reaction analyses indicate that sssAFP-2 mRNA has limited tissue distribution and is present in peripheral tissues such as skin and dorsal fin, but is notably absent in the liver. These studies reinforce recent evidence that indicate that the external tissues of cold water marine fishes are major organs for antifreeze protein synthesis and are likely the first line of defense against the threat of freezing.

Amino Acid Sequence↗
PubMed1998

The ice-binding site of Atlantic herring antifreeze protein corresponds to the carbohydrate-binding site of C-type lectins.

The type II antifreeze proteins (AFPs) of smelt and Atlantic herring are homologous to the carbohydrate-recognition domains (CRDs) of Ca2+-dependent (C-type) animal lectins and, like these lectins, acquire a stable and active structure upon binding Ca2+ ions. In the C-type lectin CRD, the carbohydrate-binding site is located at a Ca2+-binding site. Site-directed mutagenesis was used to test the hypothesis that the ice-binding site of the type II AFP corresponds to the carbohydrate-binding site of the lectins. To disrupt this site in the herring AFP without perturbing the Ca2+-dependent protein fold, a double mutant was constructed that changed the Ca2+- and carbohydrate-binding motif from the galactose-type of wild-type AFP containing the sequence Gln-Pro-Asp to a mannose-type that has the sequence Glu-Pro-Asn and is also known to bind Ca2+. The mutant AFP exhibited proper Ca2+ binding, folding, and stability as demonstrated by ruthenium red staining, proteolysis protection assays, and CD spectroscopy. However, it showed no antifreeze activity (thermal hysteresis) and did not alter ice crystal morphology to form bipyramidal crystals as does the active wild-type AFP. These results demonstrate that the ice-binding site of the herring type II AFP corresponds to the carbohydrate-binding site of the C-type lectin CRDs and further suggest that this ice-binding function evolved from the carbohydrate-binding site of a preexisting C-type lectin.

Amino Acid Sequence↗
PubMed1996

An immune response to ice crystals in North Atlantic fishes.

In mammals, the presence of crystals composed of small organic molecules, including urate and related compounds, has been shown to trigger an inflammatory response and the subsequent production of specific immunoglobulins (Ig's). Many fishes that are exposed to ice crystals in cold temperate and polar oceans may harbour ice crystals internally. Here, we report evidence for a specific immune response to ice crystals in cold-ocean marine fishes. Using ice nucleation activity as an assay, anti-ice Ig's were detected in the sera of the cold-ocean marine fish species, ocean pout (Macrozoarces americanus) and Atlantic herring (Clupea harengus harengus), but not in the sera of species that are not exposed to ice. Purified Ig's isolated from ocean pout serum using two different protocols showed ice nucleation activity, thus demonstrating the presence of ice binding specificity among these Ig's.

Animals↗
PubMed1996

Ca2+-dependent antifreeze proteins. Modulation of conformation and activity by divalent metal ions.

The antifreeze proteins (AFPs) are structurally diverse molecules that share an ability to bind to ice crystals and inhibit their growth. The type II fish AFPs of Atlantic herring and smelt are unique among known AFPs in their requirement of a cofactor for antifreeze activity. These AFPs are homologous with the carbohydrate-recognition domains of Ca2+-dependent (C-type) lectins and require Ca2+ for their activity. To investigate the role of metal ions in the structure and function of type II AFPs, the binding of Ca2+ and other divalent cations to herring AFP was investigated. Binding studies using 45Ca2+ demonstrated that the AFP has a single Ca2+-binding site with a Kd of 9 microM. Proteolysis protection studies and measurement of antifreeze activity revealed a conformational change from a protease-sensitive and inactive apoAFP to a protease-resistant active AFP upon Ca2+ binding. Other divalent metal ions including Mn2+, Ba2+, and Zn2+ bind at the Ca2+-binding site and induce a similar change. A saturatable increase in tryptophan emission intensity at 340 nm also occurred upon Ca2+ addition. Whereas antifreeze activity appeared normal when Ca2+ or Mn2+ were bound, it was much lower in the presence of other metal ions. When Ba2+ was bound to the AFP, ice crystals showed a distinct difference in morphology. These studies demonstrate that herring AFP specifically binds Ca2+ and, consequently, adopts a conformation that is essential for its ice-binding activity.

Amino Acid Sequence↗
PubMed1996

Skin antifreeze protein genes of the winter flounder, Pleuronectes americanus, encode distinct and active polypeptides without the secretory signal and prosequences.

Distinct antifreeze polypeptides (AFP) were isolated from the skin of the winter flounder, Pleuronectes americanus, by gel filtration and reverse phase high performance liquid chromatography. In parallel, several cDNA clones were isolated from a skin cDNA library using a liver AFP cDNA probe. Both protein and DNA sequence analyses indicate that flounder skin contains several distinct but homologous alanine-rich AFPs. Although the skin type AFPs contain 11 similar amino acid repeats found in the secretory liver type AFPs, the skin type AFPs are mature polypeptides lacking both the signal and prosequences, indicating that they may function intracellularly. The skin type AFP is significantly less active in thermal hysteretic activity than the liver type AFP. Genomic Southern analysis indicates that like the liver type AFP genes, there are multiple copies (30-40 copies) of skin type AFP. Although the liver type AFP genes are specifically expressed in the liver and to a lesser extent in intestine, the skin type AFP genes are expressed in all tissues examined including the liver and abundantly in exterior tissues, i.e. skin, scales, fin, and gills, suggesting an important protecting role in these exterior tissues.

Alanine↗
PubMed1995

Antifreeze proteins and their potential use in frozen foods.

Antifreeze proteins (AFPs) are proteins that have the ability to modify the growth of ice, resulting in the stabilization of ice crystals over a defined temperature range and in the inhibition of the recrystallization of ice. AFPs are found in a wide range of organisms, including bacteria, fungi, plants, invertebrates and fish. Moreover, multiple forms of AFPs are synthesized within each organism. As a result, it should be possible to select an AFP with appropriate characteristics and a suitable level of activity for a particular food product. Antifreeze proteins may improve the quality of foods that are eaten while frozen by inhibiting recrystallization and maintaining a smooth texture. In foods that are frozen only for preservation, AFPs may inhibit recrystallization during freezing, storage, transport and thawing, thus preserving food texture by reducing cellular damage and also minimizing the loss of nutrients by reducing drip. Antifreeze proteins are naturally present in many foods consumed as part of the human diet. However, AFPs may be introduced into other food products either by physical processes, such as mixing and soaking, or by gene transfer.

Journal Article↗
PubMed1993

Herring antifreeze protein: primary structure and evidence for a C-type lectin evolutionary origin.

A complementary DNA (cDNA) for a type II antifreeze protein (AFP) was isolated from an Atlantic herring liver cDNA library and sequenced. The predicted protein sequence is homologous to those of the type II fish AFP from smelt and sea raven; it is also homologous to the carbohydrate recognition domains (CRD) of calcium-dependent (C-type) lectins and similar domains in lectin-like proteins. Herring belong to the infradivision Clupeomorpha, which is distinct from the Euteleostei to which all other AFP-producing fish belong. The occurrence of type II AFP in widely divergent fish groups and their homology to C-type lectin CRDs suggest that type II AFPs evolved from these lectins. Amino acid residues forming the hydrophobic cores of rat mannose-binding protein A (MBP-A) that are conserved in character among C-type lectins are also conserved in the herring AFP. The herring AFP also requires Ca2+ for thermal hysteresis activity. These results suggest that herring AFP is structurally and functionally similar to the CRDs of C-type lectins and related domains in other proteins.

Amino Acid Sequence↗