Shawna L. MacKinnon

Comparison of antimicrobial activity in the epidermal mucus extracts of fish

The mucus layer on the surface of fish consists of several antimicrobial agents that provide a first line of defense against invading pathogens. To date, little is known about the antimicrobial properties of the mucus of Arctic char (Salvelinus alpinus), brook trout (S. fontinalis), koi carp (Cyprinus carpio sub sp. koi), striped bass (Morone saxatilis), haddock (Melanogrammus aeglefinus) and hagfish (Myxine glutinosa). The epidermal mucus samples from these fish were extracted with acidic, organic and aqueous solvents to identify potential antimicrobial agents including basic peptides, secondary metabolites, aqueous and acid soluble compounds. Initial screening of the mucus extracts against a susceptible strain of Salmonella enterica C610, showed a significant variation in antimicrobial activity among the fish species examined. The acidic mucus extracts of brook trout, haddock and hagfish exhibited bactericidal activity. The organic mucus extracts of brook trout, striped bass and koi carp showed bacteriostatic activity. There was no detectable activity in the aqueous mucus extracts. Further investigations of the activity of the acidic mucus extracts of brook trout, haddock and hagfish showed that these fish species had specific activity for fish and human pathogens, demonstrating the role of fish mucus in antimicrobial protection. In comparison to brook trout and haddock, the minimum bactericidal concentrations of hagfish acidic mucus extracts were found to be approximately 1.5 to 3.0 times lower against fish pathogens and approximately 1.6 to 6.6 folds lower for human pathogens. This preliminary information suggests that the mucus from these fish species may be a source of novel antimicrobial agents for fish and human health related applications.

Neural Injury Biomarkers of Novel Shellfish Toxins, Spirolides: A Pilot Study Using Immunochemical and Transcriptional Analysis

In 1991, routine biotoxin monitoring of bivalve molluscs at aquaculture sites along the eastern shore of Nova Scotia, Canada revealed a group of novel seafood toxins called spirolides, whose origin was the dinoflagellate Alexandrium ostenfeldii. Result from this preliminary study in rodents demonstrates a highly toxic lethal response in rats and mice after intraperitoneal injections of lipophilic extracts. To elucidate the modes of action and toxicologic pathology, brain and internal organs were examined by histology and various biomarkers of neural injury were monitored by immunohistochemistry (IH) and/or transcriptional analysis. The histological and transcriptional data showed that the effects of spirolides are species dependent for mice and rats. Histopathology showed that in the mouse brain, the hippocampus and brain stem appeared to be the major target regions but no histological changes were observed in the rat. Transcriptional analysis in the mouse brain showed no alterations in the biomarkers whereas in the rat brain there were major changes in the markers of neuronal injury. These biomarkers included the early injury markers HSP-72, c-jun and c-fos which are essential for converting stimuli into intracellular changes within neurons. The potential effects of spirolides were also evaluated with respect to different subtypes of the acetylcholine receptors (AChRs) since earlier reports showed these as putative targets. Both the muscarinic and nicotinic AChRs were found to be upregulated. Hence, transcriptional and immunohistochemical analysis does provide insight to the molecular mechanisms of this novel group of shellfish toxins. No histological changes were observed in other tissues.

Investigations into the Toxicology of Spirolides, a Group of Marine Phycotoxins

Spirolides are marine phycotoxins produced by the dinoflagellates Alexandrium ostenfeldii and A. peruvianum. Here we report that 13-desmethyl spirolide C shows little cytotoxicity when incubated with various cultured mammalian cell lines. When administered to mice by intraperitoneal (ip) injection, however, this substance was highly toxic, with an LD50 value of 6.9 µg/kg body weight (BW), showing that such in vitro cytotoxicity tests are not appropriate for predicting the in vivo toxicity of this toxin. Four other spirolides, A, B, C, and 20-methyl spirolide G, were also toxic to mice by ip injection, with LD50 values of 37, 99, 8.0 and 8.0 µg/kg BW respectively. However, the acute toxicities of these compounds were lower by at least an order of magnitude when administration by gavage and their toxic effects were further diminished when administered with food. These results have implications for future studies of the toxicology of these marine toxins and the risk assessment of human exposure.

Ursolic acid and its esters: occurrence in cranberries and other Vaccinium fruit and effects on matrix metalloproteinase activity in DU145 prostate tumor cells.

BACKGROUND Ursolic acid and its cis- and trans-3-O-p-hydroxycinnamoyl esters have been identified as constituents of American cranberries (Vaccinium macrocarpon), which inhibit tumor cell proliferation. Since the compounds may contribute to berry anticancer properties, their content in cranberries, selected cranberry products, and three other Vaccinium species (V. oxycoccus, V. vitis-idaea and V. angustifolium) was determined by liquid chromatography-mass spectroscopy. The ability of these compounds to inhibit growth in a panel of tumor cell lines and inhibit matrix metalloproteinase (MMP) activity associated with tumor invasion and metastasis was determined in DU145 prostate tumor cells. RESULTS The highest content of ursolic acid and esters was found in V. macrocarpon berries (0.460-1.090 g ursolic acid and 0.040-0.160 g each ester kg(-1) fresh weight). V. vitis-idaea and V. angustifolium contained ursolic acid (0.230-0.260 g kg(-1) ), but the esters were not detected. V. oxycoccus was lowest (0.129 g ursolic acid and esters per kg). Ursolic acid content was highest in cranberry products prepared from whole fruit. Ursolic acid and its esters inhibited tumor cell growth at micromolar concentrations, and inhibited MMP-2 and MMP-9 activity at concentrations below those previously reported for cranberry polyphenolics. CONCLUSION Cranberries (V. macrocarpon) were the best source of ursolic acid and its esters among the fruit and products tested. These compounds may limit prostate carcinogenesis through matrix metalloproteinase inhibition.

Lipophilic components of the brown seaweed, Ascophyllum nodosum, enhance freezing tolerance in Arabidopsis thaliana

Extracts of the brown seaweed Ascophyllum nodosum enhance plant tolerance against environmental stresses such as drought, salinity, and frost. However, the molecular mechanisms underlying this improved stress tolerance and the nature of the bioactive compounds present in the seaweed extracts that elicits stress tolerance remain largely unknown. We investigated the effect of A. nodosum extracts and its organic sub-fractions on freezing tolerance of Arabidopsis thaliana. Ascophyllum nodosum extracts and its lipophilic fraction significantly increased tolerance to freezing temperatures in in vitro and in vivo assays. Untreated plants exhibited severe chlorosis, tissue damage, and failed to recover from freezing treatments while the extract-treated plants recovered from freezing temperature of −7.5°C in in vitro and −5.5°C in in vivo assays. Electrolyte leakage measurements revealed that the LT50 value was lowered by 3°C while cell viability staining demonstrated a 30–40% reduction in area of damaged tissue in extract treated plants as compared to water controls. Moreover, histological observations of leaf sections revealed that extracts have a significant effect on maintaining membrane integrity during freezing stress. Treated plants exhibited 70% less chlorophyll damage during freezing recovery as compared to the controls, and this correlated with reduced expression of the chlorphyllase genes AtCHL1 and AtCHL2. Further, the A. nodosum extract treatment modulated the expression of the cold response genes, COR15A, RD29A, and CBF3, resulting in enhanced tolerance to freezing temperatures. More than 2.6-fold increase in expression of RD29A, 1.8-fold increase of CBF3 and two-fold increase in the transcript level of COR15A was observed in plants treated with lipophilic fraction of A. nodosum at −2°C. Taken together, the results suggest that chemical components in A. nodosum extracts protect membrane integrity and affect the expression of stress response genes leading to freezing stress tolerance in A. thaliana.

Characterization of a Dispiroketal Spirolide Subclass from Alexandrium ostenfeldii

A new subclass of spirolide marine toxins, represented by spirolides H (1) and I (2), were isolated from the marine dinoflagellate Alexandrium ostenfeldii. Spirolides H and I are structurally distinct from other spirolides in that they contain a 5:6 dispiroketal ring system rather than the trispiroketal ring system characteristic of previously isolated spirolides. The structures were assigned using a combination of spectrometric and spectroscopic techniques. Previously isolated spirolides containing a cyclic imine moiety showed toxicity in the mouse bioassay. Spirolide H contains this cyclic imine moiety but does not show toxicity in the mouse assay, suggesting that the presence of the cyclic imine moiety is not the only structural requirement for toxicity.

Profiling phlorotannins in brown macroalgae by liquid chromatography-high resolution mass spectrometry.

INTRODUCTION Phlorotannins, phenolic compounds produced exclusively by Phaeophyceae (brown algae), have recently been associated with a wide variety of beneficial bioactivities. Several studies have measured the total phenolic content in extracts from various species, but little characterisation of individual phlorotannin components has been demonstrated. OBJECTIVE The purpose of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) based method for rapid profiling of phlorotannins in brown algae. METHODOLOGY Phlorotannin-enriched extracts from five phaeophyceaen species were analysed by ultrahigh-pressure liquid chromatography (UHPLC) operating in hydrophilic interaction liquid chromatography (HILIC) mode combined with high resolution mass spectrometry (HRMS). The method was optimised using an extract of Fucus vesiculosus; separation was achieved in less than 15 min. The basic mobile phase enhanced negative-ion electrospray ionisation (ESI), and generated multiply charged ions that allowed detection of high molecular weight phlorotannins. RESULTS The phlorotannin profiles of Pelvetia canaliculata, Fucus spiralis, F. vesiculosus, Ascophyllum nodosum and Saccharina longicruris differed significantly. Fucus vesiculosus yielded a high abundance of low molecular weight (< 1200 Da) phlorotannins, while P. canaliculata exhibited a more evenly distributed profile, with moderate degrees of polymerisation ranging from 3 to 49. HRMS enabled the identification of phlorotannins with masses up to 6000 Da using a combination of accurate mass and ¹³C isotopic patterns. CONCLUSION The UHPLC-HRMS method described was successful in rapidly profiling phlorotannins in brown seaweeds based on their degree of polymerisation. HILIC was demonstrated to be an effective separation mode, particularly for low molecular weight phlorotannins.

Extracts of the Brown Seaweed Ascophyllum nodosum Induce Gibberellic Acid (GA3)-independent Amylase Activity in Barley

Extracts of the brown seaweed Ascophyllum nodosum have been used as a biostimulant to promote growth and productivity in a number of agricultural production systems. Although the extracts have been shown to improve seedling emergence and vigor in a variety of plants, including barley, the mechanism(s) of this growth-promoting effect is(are) largely unknown. In our study, A. nodosum extract induced amylase activity in barley seed-halves; a significant difference in amylase activity was observed in seeds without an embryo. The addition of activated charcoal to the treatment media negated the bioactivity of the extracts suggesting the organic nature of bioactive compounds in A. nodosum extracts. The extracts induced amylase activity in a gibberellic acid (GA)-deficient barley mutant (grd2). LC-MS-MS analysis failed to detect the presence of GA3 in the extracts. ABA supplementation of the medium caused a significant reduction of amylase activity in GA-treated seeds compared with those treated with the A. nodosum extract. Taken together, our results suggest that the organic components of A. nodosum extract induce amylase activity independent of GA3 and might act in concert with GA-dependent amylase production leading to enhanced germination and seedling vigor in barley. Being derived from a renewable resource, the bioactive compounds from A. nodosum could be used to improve crop productivity in sustainable agricultural systems.

Comparison of the biochemical composition of normal epidermal mucus and extruded slime of hagfish (Myxine glutinosa L.)

Hagfish (Myxine glutinosa) secrete normal epidermal mucus and extruded slime. The epidermal mucus is produced continuously to prevent pathogen adherence while the extruded slime is observed predominantly during feeding, provocation or stress. To date little is known about the involvement of extruded slime in the physiological functions of hagfish. In this preliminary study, innate immune enzymes and the protein composition of hagfish normal epidermal mucus and extruded slime were analysed and compared. The lysozyme specific activity of hagfish was observed approximately two-fold higher in extruded slime than that of epidermal mucus. The extruded slime had approximately 3.5-5.0 fold increased levels of alkaline phosphatase, cathepsin B and proteases in comparison to epidermal mucus. Protease characterization using specific inhibitors showed that the extruded slime had higher levels of serine trypsin-like proteases compared to metalloproteases whereas epidermal mucus showed equal proportion of both serine and metalloproteases. SDS-PAGE analysis showed high levels of three proteins with molecular masses in the range of 13-16kDa in the extruded slime. The LC/MS/MS analysis of protein bands 1, 2 and 3 showed closest matches to hemoglobulin-3, histone H3 and H2B proteins, respectively. The observation of elevated levels of innate immune parameters in the extruded slime suggested that the extruded slime has a significant role in innate immunity of hagfish against infectious pathogens.

Improved methods of analysis for betaines in Ascophyllum nodosum and its commercial seaweed extracts

Beneficial effects of seaweeds and their extracts on crop performance have been attributed to a variety of compounds, including the betaines which are quaternary ammonium betaines. Methods of analysis of betaines published thus far suffer from low sensitivity, lack of baseline separation of individual betaines and from interference from other sample constituents. A rapid cleanup protocol and a sensitive LC-MS/MS method of analysis were developed to afford baseline separation of four betaines in the brown alga Ascophyllum nodosum and its commercial seaweed extract. Using this method, the presence of glycine betaine, δ-aminovaleric acid betaine, γ-aminobutyric acid betaine and laminine in A. nodosum, and commercial extracts derived from A. nodosum, were confirmed and quantified. The major betaine present was γ-aminobutyric acid betaine accounting for 0.008–0.014% of the dry weight of the seaweed and 0.014–0.027% of the dry weight of the commercial extracts. Seasonal variation in betaine content was observed. Differences in the total betaine content were observed between A. nodosum of the yellow (0.011–0.017% dry weight) and the olive green (0.017–0.021% dry weight) coloured morphologies.