Isolation and characterisation of Indian Ocean ciguatoxin

We report the isolation and initial characterisation of Indian Ocean ciguatoxin (I-CTX) present in toxic lipid soluble extracts isolated from ciguateric fishes collected off the Republic of Mauritius in the Indian Ocean. Following i.p. injection of this extract, mice displayed symptoms that were similar, though not identical, to those produced by Pacific and Caribbean ciguatoxins (P-CTXs and C-CTXs). Using a radiolabelled brevetoxin (PbTx) binding assay and mouse bioassay guided fractionation, I-CTX was purified by Florisil, Sephadex LH-20 and TSK HW-40S chromatography with good recovery. Isolation to purity was not possible by preparative reversed phase high-performance liquid chromatography (HPLC) due to significant losses of toxicity. However, analytical reversed phase HPLC coupled to an electrospray mass spectrometry detector identified a [M + H](+) ion at m/z 1141.58 which co-eluted with activity that displaced [3 H]-PbTx binding to rat brain. This mass corresponded to C-CTX-1, but the fragmentation pattern of I-CTX showed a different ratio of pseudo molecular and product ions. I-CTX was found to elute later than P-CTX-1 but was practically indistinguishable from C-CTX-1 on reversed phase HPLC, while the TSK HW-40S column chromatography differentiated I-CTX from the later eluting C-CTX-1. Taken together, these results indicate that I-CTX is a new ciguatoxin (CTX) responsible for ciguatera caused by reef fish in the Indian Ocean. (C) 2002 Elsevier Science Ltd. All rights reserved.

Analysis of toxin profiles in three different fish species causing ciguatera fish poisoning in Guadeloupe, French West Indies

A grey snapper (Lutjanus griseus), a grouper (Serranidae) and a blackjack (Caranx lugubris) were implicated in three different ciguatera poisonings in Guadeloupe, French West Indies. A mouse bioassay indicated toxicity for each specimens: 0.5-1, greater than or equal to 1 and > 1 M Ug g(-1), respectively. After purification by gel filtration chromatography, the samples were analysed by high-performance liquid chromatography coupled to mass spectrometry (LC-MS). The toxin profiles differ from one fish to another. C-CTX-1 was detected at 0.24, 0.90 and 13.8 ng g(-1) flesh in the snapper, grouper and jack, respectively. It contributed only to part of the whole toxicity determined by the mouse bioassay. Other toxins identified were C-CTX-2 (a C-CTX-1 epimer), three additional isomers of C-CTX-1 or -2, and five ciguatoxin congeners (C-CTX-1127, C-CTX-1143 and its isomer C-CTX-1143a, and C-CTX-1157 and its isomer C-CTX-1157b). Putative hydroxy-polyether-like compounds were also detected in the flesh of the grouper with [M+ + H](+) ions at m/z 851.51, 857.50, 875.51, 875.49 and 895.54 Da. Some of these compounds have the same mass range as some known dinoflagellate toxins. In conclusion, this study confirms the usefulness of LC-MS analysis to determine the ciguatoxins levels and the toxin profile in fish flesh hazardous to humans.

Effect of organic amendments and solarisation on Fusarium wilt in susceptible banana plantlets, transplanted into naturally infested soil

Despite extensive research since pathogenicity was first established in 1919, no cultural or chemical control strategy has proven effective against Fusarium wilt of bananas. The efficacy of cultural control is attributed to the suppression of pathogen activity. Yet, amending naturally infested soil with aged chicken manure has been shown to enhance disease severity, without any change in the activity of the pathogen Fusarium oxysporum f. sp. cubense (Foc) in the soil. In this study, the effect of amending soil with composted sawdust, and of solarising soil, was compared with the effect of amending soil with chicken manure. Bioassays comparing the activity of Foc in the soil with the extent of invasion of banana pseudostem tissue by Foc were used to investigate why strategies targetting pathogen survival have not proven successful in controlling this disease. The enhancement of Foc invasion of the banana plantlets was reproduced with the addition of chicken manure to the naturally infested soil. However, changes in the activity of Foc in the soil were not associated with changes in the frequency of invasion of the plantlets. Invasion of banana pseudostems in the sawdust and solarisation treatments was not significantly different from invasion in the respective control treatments, despite a reduction in the activity of Foc in the sawdust-amended soil and an enhancement in the solarised soil. Moreover, the increase in Foc activity in the solarised soil recorded during the bioassays occurred despite the effectiveness of solarisation in reducing the survival of Foc in pre-colonised banana root tips buried in the soil. Changes in the frequency of invasion were associated with changes in the availability of mineral nitrogen, particularly ammonium N. These results suggest that the physiological response of banana cultivars to ammonium N may be associated with their susceptibility to Fusarium wilt. Accordingly, cultural strategies for controlling Panama disease will only be effective if they enhance the ability of the host to resist invasion.

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding similar to42 and 36 mg EPF from 300 ml bacterial and I L Sf9 cultures, respectively. The preparations were highly purified ( greater than or equal to99% purity on SDS-PAGE for the bacterial products and greater than or equal to97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immuno suppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. (C) 2003 Elsevier Inc. All rights reserved.

Ultraviolet signals in birds are special

Recent behavioural experiments have shown that birds use ultraviolet (UV)-reflective and fluorescent plumage as cues in mate choice. It remains controversial, however, whether such UV signals play a special role in sexual communication, or whether they are part of general plumage coloration. We use a comparative approach to test for a general association between sexual signalling and either UV-reflective or fluorescent plumage. Among the species surveyed, 72% have UV colours and there is a significant positive association between UV reflectance and courtship displays. Among parrots (Psittaciformes), 68% of surveyed species have fluorescent plumage, and again there is a strong positive association between courtship displays and fluorescence. These associations are not artefacts of the plumage used in courtship displays, being generally more 'colourful' because there is no association between display and colours lacking UV reflectance or fluorescence. Equally, these associations are not phylogenetic artefacts because all results remain unchanged when families or genera, rather than species, are used as independent data points. We also find that, in parrots, fluorescent plumage is usually found adjacent to UV-reflective plumage. Using a simple visual model to examine one parrot, the budgerigar Melopsittacus undulatus, we show that the juxtaposition of UV-reflective and fluorescent plumage leads to a 25-fold increase in chromatic contrast to the budgerigar's visual system. Taken together, these results suggest that signals based on UV contrast are of special importance in the context of active sexual displays. We review briefly six hypotheses on why this may be the case: suitability for short-range signalling; high contrast with backgrounds; invisibility to predators; exploitation of pre-existing sensory biases; advertisement of feather structure; and amplification of behavioural signals.

Ichthyotoxicity of Chattonella marina (Raphidophyceae) to damselfish (Acanthochromis polycanthus): the synergistic role of reactive oxygen species and free fatty acids

This investigation aimed to elucidate the relative roles of putative brevetoxins, reactive oxygen species and free fatty acids as the toxic principle of the raphidophyte Chattonella marina, using damselfish as the bioassay. Our investigations on Australian C. marina demonstrated an absence or only very low concentrations of brevetoxin-like compounds by radio-receptor binding assay and liquid chromatography-mass spectroscopy techniques. Chattonella is unique in its ability to produce levels of reactive oxygen species 100 times higher than most other algal species. However, high levels of superoxide on their own were found not to cause fish mortalities. Lipid analysis revealed this raphidophyte to contain high concentrations of the polyunsaturated fatty acid eicosapentaenoic acid (EPA; 18-23% of fatty acids), which has demonstrated toxic properties to marine organisms. Using damselfish as a model organism, we demonstrated that the free fatty acid (FFA) form of EPA produced a mortality and fish behavioural response similar to fish exposed to C. marina cells. This effect was not apparent when fish were exposed to other lipid fractions including a triglyceride containing fish oil, docosahexaenoate-enriched ethyl ester, or pure brevetoxin standards. The presence of superoxide together with low concentrations of EPA accelerated fish mortality rate threefold. We conclude that the enhancement of ichthyotoxicity of EPA in the presence of superoxide can account for the high C. marina fish killing potential. (C) 2003 Elsevier B.V All rights reserved.

Identification of slow and fast-acting toxins in a highly ciguatoxic barracuda (Sphyraena barracuda) by HPLC/MS and radiolabelled ligand binding

A barracuda implicated in ciguatera fish poisoning in Guadeloupe was estimated to have an overall flesh toxicity of 15 MUg/g using mouse bioassay. A lipid soluble extract was separated into two toxic fractions, FrA and FrB, on a LH20 Sephadex column eluted with dichloromethane/methanol (1:1). When intraperitoneal injected into mice, FrA provoked symptoms characteristic of slow-acting ciguatoxins, whereas FrB produced symptoms indicative of fast-acting toxins (FAT). High performance liquid chromatography/mass spectrometry/radio-ligand binding (HPLC/MS/RLB) analysis confirmed the two fractions were distinct, because only a weak overlap of some compounds was observed. HPLC/MS/RLB analysis revealed C-CTX-1 as the potent toxin present in FrA, and two coeluting active compounds at m/z 809.43 and 857.42 in FrB, all displaying the characteristic pattern of ion formation for hydroxy-polyethers. Other C-CTX congeners and putative hydroxy-polyether-like compounds were detected in both fractions, however, the RLB found them inactive. C-CTX-1 accounted for >90% of total toxicity in this barracuda and was confirmed to be a competitive inhibitor of brevetoxin binding to voltage-sensitive sodium channels (VSSCs) with a potency two-times lower than P-CTX-1. However, FAT active on VSSCs and

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental antoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-gamma mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP. (C) 2003 Elsevier B.V. All rights reserved.