Jeffrey W. Gallant

Identification and expression analysis of hepcidin-like antimicrobial peptides in bony fish

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.

Novel Antimicrobial Peptides Derived from Flatfish Genes

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 μg/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.

Winter Flounder Expressed Sequence Tags: Establishment of an EST Database and Identification of Novel Fish Genes.

Abstract: An EST database of more than 900 sequences has been constructed from complementary DNAs from six different tissues (stomach, intestine, pyloric cecum, liver, spleen, and ovary) of the winter flounder Pleuronectes americanus. Template preparation and automated sequencing were optimized to generate high-quality information in an economic fashion. Using computer scripts developed in our laboratory, the sequences were automatically compared with sequences in the databases via a Web-browser interface, and significant returns were recorded and organized on user-friendly HTML pages. Half (453) of the ESTs had significant matches to database sequences of known function, 33 matched ESTs from other organisms, 34 matched ribosomal RNAs, and 24 matched hypothetical open reading frames of unknown function. Forty-one percent (374) of the ESTs had no matches to sequences in the database and presumably represent previously unidentified cDNAs. Several sequences are the first isolated from teleost fish, and should be of interest for gene mapping and studies of developmental biology.

Molecular analysis of the amylase gene and its expression during development in the winter flounder, Pleuronectes americanus

Abstract Determination of the onset of amylase production in marine fish larvae is difficult due to their small size and the possible presence of exogenous amylases from prey organisms in the diet or from the gut flora. In order to develop a sensitive PCR-based assay for the detection of fish-specific amylase in larvae, a complete cDNA and partial genomic sequence, the first reported from a teleost fish, were determined from winter flounder. The complete cDNA for alpha amylase is 1539 bp and the deduced polypeptide sequence is 512 amino acids, including a putative 15 amino acid signal peptide. The molecular weight of the mature protein is 55,769 Da and the predicted isoelectric point is 6.76. Southern hybridisation analysis showed that the winter flounder amylase cDNA could be used to detect homologs in other species, particularly flatfish, and that there are likely two copies of the gene in the winter flounder genome. The winter flounder genomic sequence corresponding to amino acids 194–404 (including three introns) was amplified by the polymerase chain reaction (PCR) and the sequence used to design primers for PCR-based assays for amylase gene expression in larval and adult fish. The levels of expression of the amylase gene from larvae sampled at 5, 13, 20, 27 and 41 days post-hatch (dph) were determined using the housekeeping gene, actin, as a control. Amylase transcripts were first detected at 5 dph, peaked at 20 dph and then decreased during metamorphosis. The amylase gene is highly expressed in adult winter flounder. This sensitive assay will be useful for investigating amylase gene expression under different feeding conditions and help in the development of optimal diets.

Molecular cloning of three cDNAs that encode cysteine proteinases in the digestive gland of the American lobster (Homarus americanus)

Abstract Three clones were isolated from a lobster digestive gland cDNA library, using oligonucleotide probes based on the partial amino terminal sequence of a digestive cysteine proteinase. The cDNAs, LCP1, LCP2 and LCP3 encode preproenzymes of 322, 323 and 321 amino acid residues, and putative mature enzymes of 217, 216 and 215 residues, respectively. Calculated mature protein molecular masses are 23386 (LCP1), 23093 (LCP2) and 23255 (LCP3) Sequence alignments show that the lobster enzymes are more similar to L (55–62% identity) than H (42–44%) or B (22–24%) cathepsins. Southern analysis indicated as many as eleven genes related to the three cDNAs.Southern analysis indicated as many as eleven genes related to the three cDNAs.

Ontogenetic and tissue-specific expression of preproghrelin in the Atlantic halibut, Hippoglossus hippoglossus L.

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.

A novel target-specific, salt-resistant antimicrobial peptide against the cariogenic pathogen Streptococcus mutans

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.

The zebrafish embryo as a tool for screening and characterizing pleurocidin host-defense peptides as anti-cancer agents

SUMMARY 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.

Serum biomarkers of papillary thyroid cancer

ObjectiveTo identify serum biomarkers of papillary thyroid cancer.MethodsProspective analysis was performed of banked tumor and serum specimens from 99 patients with thyroid masses. Enzyme-linked immunosorbent assay (ELISA) was employed to measure levels of five serum proteins previously demonstrated to be up-regulated in papillary thyroid cancer (PTC): angiopoietin-1 (Ang-1), cytokeratin 19 (CK-19), tissue inhibitor of metalloproteinase-1 (TIMP-1), chitinase 3 like-1 (YKL-40), and galectin-3 (GAL-3). Serum levels were compared between patients with PTC and those with benign tumors.ResultsA total of 99 patients were enrolled in the study (27 men, 72 women), with a median age of 54 years. Forty-three patients had PTC and 58 cases were benign tumors. There were no statistically significant differences when comparing all five different biomarkers between PTC and other benign thyroid tumors. The p-values were 0.94, 0.48, 0.72, 0.48, and 0.90 for YKL-40, Gal-3, CK19, TIMP-1, and Ang-1, respectively.ConclusionSerum levels of four of the five proteins were elevated in patients with thyroid masses relative to normal values. However, the difference between benign and PTC was not significant. Two of the markers (Gal-3 & TIMP-1) displayed a greater potential difference, which may warrant further investigation. This study suggests that other serum markers should be sought. This is the first study to investigate potential serum biomarkers based on over-expressed proteins in thyroid cancer versus benign pathology.

Isolation and transient expression of a cDNA encoding l-gulono-γ-lactone oxidase, a key enzyme for l-ascorbic acid biosynthesis, from the tiger shark Scyliorhinus torazame

Abstract We present the first description of a cDNA encoding the l -gulono-γ-lactone oxidase (GLO) from a fish, a cartilaginous shark species, Scyliorhinus torazame . 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 p I (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 ( Silurus asotus ), 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.