[show abstract][hide abstract] ABSTRACT: In this study, we constructed and evaluated a target-specific, salt-resistant antimicrobial peptide (AMP) that selectively targeted Streptococcus mutans, a leading cariogenic pathogen. The rationale for creating such a peptide was based on the addition of a targeting domain of S. mutans ComC signaling peptide pheromone (CSP) to a killing domain consisting of a portion of the marine-derived, broad-spectrum AMP pleurocidin to generate a target-specific AMP. Here, we report the results of our assessment of such fusion peptides against S. mutans and two closely related species. The results showed that nearly 95% of S. mutans cells lost viability following exposure to fusion peptide IMB-2 (5.65 μM) for 15 min. In contrast, only 20% of S. sanguinis or S. gordonii cells were killed following the same exposure. Similar results were also observed in dual-species mixed cultures of S. mutans with S. sanguinis or S. gordonii. The peptide-guided killing was further confirmed in S. mutans biofilms and was shown to be dose dependent. An S. mutans mutant defective in the CSP receptor retained 60% survival following exposure to IMB-2, suggesting that the targeted peptide predominantly bound to the CSP receptor to mediate killing in the wild-type strain. Our work confirmed that IMB-2 retained its activity in the presence of physiological or higher salt concentrations. In particular, the fusion peptide showed a synergistic killing effect on S. mutans with a preventive dose of NaF. In addition, IMB-2 was relatively stable in the presence of saliva containing 1 mM EDTA and did not cause any hemolysis. We also found that replacement of serine-14 by histidine improved its activity at lower pH. Because of its effectiveness, salt resistance, and minimal toxicity to host cells, this novel target-specific peptide shows promise for future development as an anticaries agent.
Antimicrobial Agents and Chemotherapy 08/2011; 55(11):5205-13. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: The emergence of multidrug-resistant cancers and the lack of targeted therapies for many cancers underscore an unmet need for new therapeutics with novel modes of action towards cancer cells. Host-defense peptides often exhibit selective cytotoxicity towards cancer cells and show potential as anti-cancer therapeutics. Here, we screen 26 naturally occurring variants of the peptide pleurocidin for cytotoxic and anti-cancer activities, and investigate the underlying mechanism of action. Cytotoxicities were assessed in vitro using cell-based assays and in vivo using zebrafish embryos. Morphological changes were assessed by both transmission and scanning electron microscopy, and functional assays were performed on zebrafish embryos to investigate the mechanism of cell death. A total of 14 peptides were virtually inactive against HL60 human leukemia cells, whereas 12 caused >50% death at ≤32 μg/ml. Morphological changes characteristic of oncosis were evident by electron microscopy after only 1 minute of treatment with 32 μg/ml of variant NRC-03. Only two peptides were hemolytic. Four peptides showed no toxicity towards zebrafish embryos at the highest concentration tested (25 μM; ∼64 μg/ml) and one peptide was highly toxic, killing 4-hour-post-fertilization (hpf) embryos immediately after exposure to 1 μM peptide. Four other peptides killed embryos after 24 hours of exposure at 1 μM. Most peptides caused mortality at one or more developmental stages only after continuous exposure (24 hours) with higher lethal doses (≥5 μM). Pleurocidin NRC-03 bound to embryos and induced the release of superoxide, caused an increase in the number of TUNEL-positive nuclei, and caused membrane damage and the loss of embryonic epithelial integrity, marked by the exclusion of cells from the outer epithelium and the appearance of F-actin within the circumferential cells of the repair site. Our results indicate that specific pleurocidin variants are attractive cancer-selective agents that selectively induce cell death in target cells but leave non-target cells such as erythrocytes and non-transformed cells unaffected.
Disease Models and Mechanisms 07/2011; 4(5):622-33. · 4.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Aeromonas salmonicida is a fish pathogen that causes furunculosis. Virulent strains of this bacterium are able to infect salmonid macrophages and survive within them, although mechanisms favouring intracellular survival are not completely understood. It is known that A. salmonicida cultured in vivo in the peritoneal cavity of the host undergoes changes in gene expression and surface architecture compared with cultures grown in vitro in broth. Therefore, in this study, the early macrophage responses to A. salmonicida grown in vivo and in vitro were compared. Macrophage-enriched cell preparations from head kidney of Atlantic salmon (Salmo salar) were infected in vitro in 96-well microtitre dishes and changes in gene expression during the infection process were monitored using a custom Atlantic salmon cDNA microarray. A. salmonicida cultures grown in tryptic soy broth and in peritoneal implants were used to infect the macrophages. The macrophages were harvested at 0.5, 1.0 and 2.0h after addition of the bacteria to the medium. Significant changes in gene expression were evident by microarray analysis at 2.0h post-infection in macrophages infected with broth-grown and implant-grown bacteria; however, qPCR analysis revealed earlier up-regulation of JunB and TNF-alpha in macrophages exposed to the implant-grown bacteria. Up-regulation of those genes and others is consistent with the effects of extracellular products of aeromonad bacteria on macrophages and also suggests initiation of the innate immune response.
[show abstract][hide abstract] ABSTRACT: Ghrelin is a conserved vertebrate hormone that affects both GH release and appetite. We have cloned and characterized Atlantic halibut preproghrelin cDNA and examined for the first time preproghrelin expression during fish larval development using quantitative real-time PCR. In addition, cellular sites of expression in larvae and tissue-specific expression in 3-year-old halibut were studied. A full-length cDNA for preproghrelin was isolated from halibut stomach tissue. The 899 bp cDNA encodes an open reading frame of 105 amino acids that is comprised of a signal peptide and two peptides with high similarity to ghrelin and obestatin. The deduced amino acid sequence of halibut ghrelin peptide (GSSFLSPSHKPPKGKPPRA) shows significant conservation relative to other teleostean sequences and is identical to human ghrelin for the first seven amino acids of the sequence. The putative obestatin peptide is well-conserved among fishes but shares limited similarity with its human counterpart. Expression of ghrelin was localized to two different cell types in the stomach of larval halibut by in situ hybridization. However, sensitive PCR assays on tissues collected from 3-year-old fish additionally identified ghrelin transcripts in pyloric caecae, intestine, and in immature ovary and testis. Ontogenetic studies detected ghrelin expression prior to exogenous feeding during larval development (hatching and mouth-opening stages) with increased expression occurring through metamorphosis. This increase was pronounced during climax metamorphosis and coincided with stomach differentiation. Patterns of preproghrelin expression suggest that ghrelin has important roles during and after larval development in halibut, and that ghrelin is associated with digestive and gonadal tissues in this teleost.
Journal of Endocrinology 02/2008; 196(1):181-92. · 4.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Most advanced teleosts lack L-gulono-gamma-lactone oxidase (GULO), a key enzyme required for the biosynthesis of ascorbic acid. However, extant representatives of primitive species including sturgeon and many cartilaginous fishes, are exceptional in their ability to synthesize ascorbic acid de novo. In the present study, full-length GULO cDNAs were isolated from white sturgeon (Acipenser transmontanus) and two shark species belonging to the Triakidae (Triakis scyllium and Mustelus manazo). The open reading frames from all three species contained 440 amino acids and the deduced polypeptides had similar hydropathy profiles, predicted molecular masses and theoretical pI values. These GULO sequences exhibited high amino acid identity (67-97%) with each other, and also shared 61-71% identity with mammalian GULOs. Based on the GULO sequences obtained from these species, we developed degenerate primers for the isolation of partial GULO sequences by RT-PCR from other primitive species including another shark (Mustelus griseus, Triakidae), a spiny dogfish (Squalus acanthias, Squalidae), two ray species (Raja kenojei, Rajidae and Dasyatis akajei, Dasyatidae) and four sturgeons (Acipenser baeri, A. gueldenstaedtii, A. naccarii and A. ruthenus, Acipenseridae). Overall, sequence identities of these amplified GULO segments among primitive species were 63-99% at the nucleotide level and 67-100% at the amino acid level. Considerable numbers of amino acid residues were unique to either fish or mammals, and Acipenseriform species occupied an intermediate position, sharing several residues with either fish or mammalian GULOs. Phylogenetic analyses based on parsimony, distance and likelihood methods of both nucleotide and amino acid sequences resulted in trees that were in agreement with known taxonomy. The transcription and enzyme activity of GULO were kidney-specific when measured by biochemical assay and reverse transcription-PCR.
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 07/2007; 147(2):178-90. · 2.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: The fish larval stage is a critical step since only those specimens that survive will reach the adult stage in future. Knowledge
related to fish larval nutritional requirements and digestive enzymes capacity is still scarce, although necessary to obtain
satisfactory survival and growth rates. Trypsinogen is the precursor of trypsin, the main proteolytic enzyme acting during
the early larval stage. Bile salt-activated lipase (BAL) is a multi-substrate digestive enzyme that hydrolyzes carboxyl ester
bonds of acylglycerols, cholesterol esters and fat-soluble vitamin esters. The goal of this study was to determine the pattern
of trypsinogen and BAL expression during larval development in red porgy (Pagrus pagrus, Pisces, Sparidae), reared under standard conditions to provide the basis for future experiments testing the possible transcriptional
regulation for this enzyme under different nutritional conditions. Thus, partial cDNAs for trypsinogen and BAL from red porgy
were isolated. The putative aminoacid sequences obtained for both precursors showed around 80% identity to other fish sequences
from GenBank database. Trypsinogen and BAL were expressed from hatching and specifically located in the exocrine pancreas,
revealed by in situ hybridization. The present study shows that this species is being prepared for protein and lipid digestion
before exogenous feeding starts, exhibiting an ontogenetically programmed pattern for trypsinogen and BAL expression during
the yolk-sac stage.
[show abstract][hide abstract] ABSTRACT: The appearance of functionally developed gastric glands is commonly considered as the transition from the larval to the juvenile stage in fish, since it means the switch from the less efficient alkaline digestion to a more efficient acid digestion characteristic of adult specimens. From that moment, fish are supposedly able to better assimilate nutrients from inert diets. Acid digestion takes place by the action of pepsin activity and hydrochloric acid, both secreted by the gastric glands of the stomach. Pepsinogen is the precursor of pepsin which is converted into active enzyme by the action of hydrochloric acid secreted by the proton pump. The goal of this work was to asses the ontogeny of pepsinogen and gastric proton pump expression along larval development of red porgy using RT-PCR and in situ hybridization techniques. Firstly, red porgy specific pepsinogen and proton pump partial sequences were isolated. Amplification products presented 615 and 591 bp and were identified as pepsinogen IIb and the α-subunit of the proton pump (H+/K+-ATPase) by sequencing, respectively. Both sequences were aligned to several predicted pepsinogen and proton pump polypeptides from different vertebrate species showing elevated homologies with them, especially in the case of the proton pump, the identity of which was never less than 90%. Pepsinogen and proton pump expressions were detected from 30 days post-hatching (dph), increasing with development. Proton pump expression was localized in the gastric glands of red porgy larvae as revealed by in situ hybridization, showing increasing signal intensity along the digestive system development. Such results indicated that at 30 dph red porgy starts to acquire the adult digestive capacity and therefore inert diets should be better utilized from that time onwards.
[show abstract][hide abstract] ABSTRACT: A partial alpha-amylase cDNA was isolated from red porgy (Pagrus pagrus, Teleostei: Sparidae) and its tissue specific expression during larval development was examined. The cDNA was 949 bp long and showed 90% identity with other fish amylases. A 545 bp fragment was used to study amylase expression using in situ hybridization and RT-PCR techniques. Both methods showed a similar pattern: high and relatively constant expression for the first 30 days after hatching (dah), subsequently decreasing until the end of the experiment at 60 dah. The goal of this work was to extend the existing knowledge of the functionality of larval fish digestive systems and to provide new information about alpha-amylase gene expression.
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 03/2006; 143(2):209-18. · 2.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Using biochemical and molecular biological techniques, we describe the expression of several key digestive enzymes throughout the ontogeny of larval haddock and Atlantic cod. The pattern of activity of general proteases (GP), trypsin-like enzymes (TLE), pepsin-like enzymes (PLE), general lipase (GL), bile salt-activated lipase (BAL), alkaline phosphatase (AP) and α-amylase were studied in larvae of both species using colourometric techniques. All enzymes were detected as early as hatch, except for α-amylase. Activity of GP generally increased from 0 to 144 degree-days (DD), decreased until 333 DD and then increased again in the oldest larvae of both species. Activity of TLE in haddock was high at hatch and generally showed an inverse pattern to that of Atlantic cod. Using zymography, we detected serine proteases, particularly trypsin, in the protein digestion of both species. Using RT-PCR, trypsinogen transcripts were detected as early as hatch. For both species, in situ hybridization analysis showed localization of trypsinogen expression to the pancreas during larval development. Although activity of PLE was detected as early as hatch in both species, pepsinogen transcripts were detected by RT-PCR only in the oldest larvae sampled, and after the gastric glands were identified morphologically. Atlantic cod BAL activity showed no significant differences over time. Activity of GL remained constant over time in larvae of both species. Levels of AP activity in haddock larvae showed no significant differences from hatch to 333 DD, except at 66 and 401 DD when there was a significant increase compared to other time points. In Atlantic cod, AP activity remained relatively constant until 476 DD, when a significant increase was detected. α-Amylase activity could not be detected in either species, except at 43 and 60 DD for Atlantic cod and 144 DD for haddock when large numbers of live prey were present in the gut. The estimated contribution of Pavlova lutheri-enriched rotifers to the total larval digestion was negligible, except for α-amylase, for which their contribution was estimated to be 100%. Our results suggest that both Atlantic cod and haddock larvae are capable of digesting protein and lipids at the time of mouth opening and that they have a limited capacity to digest carbohydrates.
[show abstract][hide abstract] ABSTRACT: The objective of the present study was to describe the histological and physiological development of the gastrointestinal system in Atlantic halibut from the time of first-feeding until metamorphosis. At first-feeding, the gastrointestinal (GI) tract is divided into anterior, mid and hindgut regions. The liver is present, as is the pancreas. During development the pancreas changes from a compact organ to a diffuse tissue interspersed through much of the mesentery surrounding the GI tract. Functional gastric glands are not present until approximately 66 days post-hatch (dph). Using primers based upon winter flounder digestive enzyme gene sequences, we were able to amplify partial sequences for bile salt-activated lipase (BAL), trypsinogen (TRP), and pepsinogen (PEP) from RNA extracted from whole larvae and juveniles using Reverse Transcription-PCR (RT-PCR). PCR products were sequenced and the sequences used to design halibut gene-specific primers for BAL and PEP. RT-PCR analysis revealed that BAL and TRP gene expression was evident at least from the time of first-feeding but PEP gene expression was not detectable until 80 dph. In situ hybridization using molecular probes from winter flounder sequences localized expression of BAL and TRP to the exocrine pancreas. PEP expression was only localized to the glandular regions of the stomach. These data provide a first step toward understanding the molecular events governing the ontogeny of digestive capacity in Atlantic halibut.
[show abstract][hide abstract] ABSTRACT: Clones for two forms of pepsinogen A differing in isoelectric point as well as both and β subunits of the proton pump were isolated from the stomach of winter flounder Pleuronectes americanus. All clones gave positive hybridization signals with DNA from other flatfish species. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the two forms of pepsinogen were sequentially expressed. Expression of pepsinogen IIa was first detected in 13 days post-hatch (dph) larvae and increased towards the beginning of metamorphosis at 20 dph. Expression of pepsinogen IIb and proton pump genes was first detected at 20 dph and coincided with the appearance of gastric glands and the increase in pepsin activity. The levels of expression of pepsinogen IIb as well as of pepsin activity in 20-dph larvae were similar to those recorded in metamorphosed larvae (27 dph) and juveniles (41 dph). High pepsin activity and levels of expression of the pepsinogen and proton pump genes observed in 20-dph larvae indicate an advanced level of development and functionality of the winter flounder stomach at this stage of ontogeny. This advanced level of the stomach development at 1 week before the currently practised time of weaning suggests that feeding formulated feeds may be initiated earlier.
Journal of Fish Biology 03/2005; 55(5):897 - 915. · 1.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Abstract: In the present work, we report the characterization of the partial cDNA sequence of a bile salt-activated lipase (BAL) cDNA from haddock. The predicted polypeptide encoded by the cDNA sequence contains the bile salt-binding site characteristic of all BALs at amino acid positions 3645, and the lipid-binding site thus far only reported in fish BALs starting at position 345. Other features of BAL are also present including: the active site serine motif at positions 111117, the catalytic triad formed by the residues S114, D239 and H358, and an N-glycosylation site at position 107. The relative levels of BAL gene expression were determined in haddock larvae during ontogeny by reverse transcriptionpolymerase chain reaction (RTPCR) with the earliest detectable transcript levels identified at hatch. Using in situ hybridization, the BAL transcripts were localized consistently in the pancreas of haddock larvae from mouth opening until 401 degree days (DD). Using biochemical techniques, the specific activity of BAL was found to decline significantly over time. Our results also suggest that haddock larvae are capable of digesting lipids at the time of mouth opening. yes
[show abstract][hide abstract] ABSTRACT: Histological, biochemical and molecular techniques were used to describe the functional development of the pancreas in winter flounder (Pleuronectes americanus) with specific reference to the expression of three trypsinogen genes. The pancreas was identified shortly following hatch, appearing as a compact structure situated dorsal and slightly posterior to the liver. As the larval fish approached metamorphosis, the pancreas became diffuse, spreading throughout the mesentery surrounding the stomach, the upper intestine and the pyloric caecae. Trypsin 2 expression was detected from 5 days post-hatch (dph). Two other related trypsinogen genes isolated from the pyloric caecae (Trypsin 1) and the intestine (Trypsin 3) showed contrasting results. Trypsin 1 showed very low levels of expression and only in late larval stages and metamorphosis. Trypsin 3 showed expression only after 20 dph. In order to determine tissue-specific expression of the three trypsinogen genes, the RNA from seven gastrointestinal-associated tissues was examined. Trypsin 1 and Trypsin 2 expression was most notably associated with the pyloric caecae, cardiac stomach, pyloric stomach and the rectum, although some variation in expression level between tissues was observed. Trypsin 3 expression had a narrower tissue distribution and was only associated with the pyloric caecae and the rectum. The tissue expression patterns observed here are likely due in part to the diffuse nature of the pancreas. Trypsin-like activity was evident from hatch and continued at significant levels through to at least 25 dph.
Comparative Biochemistry and Physiology - Part A Molecular & Integrative Physiology 06/2004; 138(1):53-9. · 2.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: Antimicrobial peptides form one of the first lines of defense against invading pathogens by killing the microorganisms and/or mobilizing the host innate immune system. Although over 800 antimicrobial peptides have been isolated from many different species, especially insects, few have been reported from marine fish. Sequence analysis of two genomic clones (15.6 and 12.5 kb) from the winter flounder, Pseudopleuronectes americanus (Walbaum) resulted in the identification of multiple clustered genes for novel pleurocidin-like antimicrobial peptides. Four genes and three pseudogenes (Psi) are encoded in these clusters, all of which have similar intron/exon boundaries but specify putative antimicrobial peptides differing in sequence. Pseudogenes are easily detectable but have incorrect initiator codons (ACG) and often contain a frameshift(s). Potential promoters and binding sites for transcription factors implicated in regulation of expression of immune-related genes have been identified in upstream regions by comparative genomics. Using reverse transcription-PCR assays, we have shown for the first time that each gene is expressed in a tissue-specific and developmental stage-specific manner. In addition, synthetic peptides based on the sequences of both genes and pseudogenes have been produced and tested for antimicrobial activity. These data can be used as a basis for prediction of antimicrobial peptide candidates for both human and nonhuman therapeutants from genomic sequences and will aid in understanding the evolution and transcriptional regulation of expression of these peptides.
European Journal of Biochemistry 10/2003; 270(18):3720-30. · 3.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: We report on the identification of active novel antimicrobials determined by screening both the genomic information and the mRNA transcripts from a number of different flatfish for sequences encoding antimicrobial peptides, predicting the sequences of active peptides from the genetic information, producing the predicted peptides chemically, and testing them for their activities. We amplified 35 sequences from various species of flatfish using primers whose sequences are based on conserved flanking regions of a known antimicrobial peptide from winter flounder, pleurocidin. We analyzed the sequences of the amplified products and predicted which sequences were likely to encode functional antimicrobial peptides on the basis of charge, hydrophobicity, relation to flanking sequences, and similarity to known active peptides. Twenty peptides were then produced synthetically and tested for their activities against gram-positive and gram-negative bacteria and the yeast Candida albicans. The most active peptide (with the carboxy-terminus amidated sequence GWRTLLKKAEVKTVGKLALKHYL, derived from American plaice) showed inhibitory activity over a concentration range of 1 to 8 micro g/ml against a test panel of pathogens, including the intrinsically antibiotic-resistant organism Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and C. albicans. The methods described here will be useful for the identification of novel peptides with good antimicrobial activities.
Antimicrobial Agents and Chemotherapy 09/2003; 47(8):2464-70. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: The partial sequencing of two lipases from winter flounder Pseudopleuronectes americanus, one most closely related to gastric, lingual and lysosomal acid lipase from other vertebrates and one most closely related to bile salt-activated lipase, is reported. Biochemical analyses of enzymatic activity demonstrated the greater contribution made by bile salt-activated lipase relative to neutral bile salt-independent lipase. Using molecular techniques, the tissue-specific expression of bile salt-activated lipase in pancreatic tissue and acid triacylglycerol lipase in a wide variety of organs was demonstrated. Furthermore, the developmental expression of these types of lipase in larval fish was established.
Journal of Fish Biology 05/2003; 62(4):816 - 833. · 1.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Antimicrobial peptides play a crucial role as the first line of defense against invading pathogens. Several types of antimicrobial peptides have been isolated from fish, mostly of the cationic alpha-helical variety. Here, we present the cDNA sequences of five highly disulphide-bonded hepcidin-like peptides from winter flounder, Pseudopleuronectes americanus (Walbaum) and two from Atlantic salmon, Salmo salar (L.). These hepcidin-like molecules consist of a 24 amino acid signal peptide and an acidic propiece of 38-40 amino acids in addition to the mature processed peptide of 19-27 amino acids. Exhaustive data mining of GenBank with these sequences revealed that similar peptides are encoded in the genomes of Japanese flounder, rainbow trout, hybrid striped bass and medaka, indicating that they are widespread among fish. Southern hybridization analysis suggests that closely related hepcidin-like genes are present in other flatfish species, and that they exist as a multigene family clustered on the winter flounder genome. Hepcidin variants are differentially expressed during bacterial challenge, during larval development of P. americanus and in different tissues of adult fish.
[show abstract][hide abstract] ABSTRACT: We present the first description of a cDNA encoding the l-gulono-γ-lactone oxidase (GLO) from a fish, a cartilaginous shark species, Scyliorhinustorazame. An expressed sequence tag (EST) from the shark kidney, which showed high similarity with a rat GLO gene, was isolated and its full-length sequence (1752 bp) was determined. The putative shark GLO (sGLO) cDNA sequence contained 98 bp of 5′-untranslated region, an open reading frame consisting 440 amino acids, and 334 bp of 3′-untranslated region including the poly(A+) tail. The deduced amino acid sequence was 63% identical to the rat GLO sequence and showed high conservation in the flavine adenine dinucleotide (FAD)-binding region. In addition, the calculated molecular mass (50,976 Da), theoretical pI (7.17) and hydropathy profile were similar to those of the rat GLO. Using both reverse transcription-PCR (RT-PCR) assays and the sGLO cDNA as a probe in Northern hybridisation experiments, expression was demonstrated in the shark kidney but not in any other tissues (brain, intestine, liver, muscle, pituitary and spleen). Biologically functional GLO activity was demonstrated by direct delivery of an expression vector containing the sGLO cDNA into kidney of far eastern catfish (Silurusasotus), which lacks endogenous GLO activity. Transient expression of GLO activity was dependent on the amount of plasmid injected (up to 120 μg of DNA), and persisted for 12 days post injection, as demonstrated by RT-PCR and biochemical assays.
[show abstract][hide abstract] ABSTRACT: The primary structure of a polypeptide with significant similarity to human aminopeptidase N (EC 188.8.131.52) was deduced from the sequence of two overlapping cDNA clones derived from winter flounder (Pleuronectes americanus, Walbaum) intestinal RNA and a partial genomic clone derived from winter flounder DNA. The deduced amino acid sequence (975 amino acids) is comprised of an N-terminal cytosolic portion of seven amino acid residues, followed by a 24-residue transmembrane anchor region, a 38-residue serine/threonine-rich junction and a 906-residue enzymatic domain that protrudes from the apical membrane of the enterocyte. A highly conserved signature sequence characteristic of zinc-dependent metallopeptidases (HExxH) is present, as well as seven putative glycosylation sites (NxS/T). Fourteen introns are present in the 7.53 kb portion of the gene that was cloned and sequenced. Reverse transcriptase polymerase chain reaction assays using primers spanning intron/exon boundaries were used to determine the timing of expression of this gene in larval winter flounder. This represents the first aminopeptidase N sequence to be determined from a teleost fish and underscores the utility of molecular biological information in the investigation of larval fish digestion.
[show abstract][hide abstract] ABSTRACT: Low molecular weight antimicrobial peptides are an important component of the innate immune system in animals, yet they have not been examined widely in fish. Of particular interest is their expression during development and in response to environmental conditions and disease. Here, we report the isolation of four genomic sequences encoding putative antimicrobial peptides from the winter flounder, Pleuronectes americanus (Walbaum), as well as reverse transcription-PCR products from two tissues that form the first defensive barrier to microbes - skin and intestine. Alignment of the predicted polypeptide sequences shows a conserved hydrophobic signal peptide of 22 amino acids followed by 25 amino acids that are identical (WF2) or homologous to the amino acid sequence of pleurocidin, followed by a conserved acidic portion. Southern hybridisation analysis indicates that related peptides are encoded in the genomes of other flatfish species. Northern and RT-PCR analyses of RNA from multiple tissues show that two of the pleurocidin genes are expressed predominantly in the skin whereas two other genes are expressed mainly in the intestine. RT-PCR assays of total RNA from larvae of different ages provide the first evidence of developmental expression of antimicrobial peptides in fish and indicate that the pleurocidin gene is first expressed at 13 days post-hatch in winter flounder.