Post-mortem analysis of samples from a human victim of a fatal poisoning caused by the xanthid crab, Zosimus aeneus.

After ingestion of a specimen of the crab Zosimus aeneus (Xanthidae), an East Timorese adult male died within several hours. Xanthid crabs are known to harbour paralytic shellfish toxins (PSTs), tetrodotoxin and palytoxin. A post-mortem examination did not find any obvious pathological abnormalities. This absence of pathologies is more often associated with PSTs and tetrodotoxin intoxication. A second, yet uneaten specimen of Z. aeneus from the same meal, contained a significant amount of PSTs and these same toxins were identified in the gut contents, blood, liver and urine of the victim. Metabolism of the PSTs occurred with the ingested crab harbouring gonyautoxin 2, gonyautoxin 3 and saxitoxin (STX) whereas neoSTX, decarbamoylSTX and STX dominated the PSTs in the victim's urine. The PST composition in the gut contents, in both their identity and proportion, was intermediate between the eaten crab and the urine suggesting that toxin conversion commenced in the victim's gut. The dose consumed by the victim was calculated to be between 1 and 2 microg STX equivalents/kg based upon the concentration in the remains of the cooked crab. The victim's meal did not consist solely of the toxic crab eaten and the possibility of other food items acting in a synergistic manner with the consumed PSTs cannot be discounted.

Thyroid hormones in growth and development of fish.

The thyroid hormones (THs), thyroxine (T(4)) and triiodothyronine (T(3)) are products of the thyroid gland in all vertebrates. Their role in early development and metamorphosis is well established in mammals and amphibians, respectively, and recently several studies in fish have highlighted the importance of THs during flatfish metamorphosis. THs are present in high quantities in fish eggs and are presumably of maternal origin. During embryogenesis the concentration of T(4) and T(3) in the eggs decrease until endogenous production starts. Thyroid hormone receptors (TR) have been isolated from several teleosts and in common with tetrapods two receptor isoforms have been identified, TR alpha and TR beta. Both the receptors are expressed in early embryos and larvae of the Japanese flounder (Paralichthys olivaceus), zebrafish (Danio rerio) and seabream (Sparus aurata) although a different temporal pattern is apparent. The role of THs and TRs in fish embryogenesis, larval development and during metamorphosis will be discussed.

Measurement of paralytic shellfish toxins in molluscan extracts: comparison of the microtitre plate saxiphilin and sodium channel radioreceptor assays with mouse bioassay, HPLC analysis and a commercially available cell culture assay.

This study compared five methods of measuring paralytic shellfish toxins (PSTs) including the long-used mouse lethality bioassay, a commercially available cell culture test (MIST Quantification kit), HPLC analysis, and two newly developed radioreceptor assays utilizing mammalian sodium channels and saxiphilin. Methods were challenged with toxic shellfish extracts prepared according to the AOAC official method. The best correlations between predicted toxicity values being 0.9 or better, were those between HPLC analysis when compared with both radioreceptor assays and the mouse lethality bioassay, as well as that between the saxiphilin and the sodium channel radioreceptor assays. In all cases, statistically significant correlations existed between the toxicity measurements of the same extracts. The ratios between some methods were not unitary as measured by the slopes of the regression lines used for correlation analyses. HPLC analysis predicted more toxicity than all of the bioassays. The saxiphilin assay underestimated toxicity relative to the mouse bioassay, the MIST kit determinations and the sodium channel assay. The sodium channel assay predicted there to be less toxicity than the mouse bioassay and the MIST kit. Of all of the techniques used, the MIST kit correlation with the mouse bioassay was nearest to one. Each method possesses differentt virtues and it may be that a multi-method approach would harness the benefits of each method for various aspects of a shellfish testing regime.

Characterization of a sea bream (Sparus aurata) thyroid hormone receptor-beta clone expressed during embryonic and larval development.

A clone encoding thyroid hormone receptor-beta (TR-beta) was isolated from a sea bream (Sparus aurata) ovary cDNA library. Sea bream (sb)TR-beta is closely related to its counterparts from other vertebrates and, like them, preferentially binds T3 rather than T4. However, the putative sbTR-beta protein contains a nine-amino-acid insert that is also present in the corresponding proteins from flounder and salmon but absent in TR-betas from zebrafish and terrestrial vertebrates. Semiquantitative RT-PCR analysis showed that sbTR-beta transcripts begin to accumulate during gastrulation and increase markedly in quantity up to the period around hatch (ca. 40 h postfertilization) before declining slightly. In adult tissues, TR-beta mRNA was present in approximately equal quantities in heart, intestine, brain, kidney, skeletal muscle, liver, and gill. The significance of the relatively strong expression of TR-beta during sea bream embryogenesis is discussed.

Radioreceptor assays for sensitive detection and quantitation of saxitoxin and its analogues from strains of the freshwater cyanobacterium, Anabaena circinalis.

Toxic freshwater cyanobacteria can contaminate water supplies and adversely effect humans, agricultural livestock, and wildlife. Toxicity is strain-specific so morphological observations alone cannot predict the hazard level. Two microtiter plate based bioassays have emerged for measuring saxitoxin (STX) and its derivatives, commonly found in the freshwater cyanobacteria Anabaena and Aphanizomenon. They use radioactively labeled STX binding by sodium channels, STX's pharmacological target, or an unrelated protein, saxiphilin. These bioassays were challenged with extracts of toxic and nontoxic strains of Anabaena circinalis, and the results were compared with HPLC analysis. Both radioreceptor assays had detection limits of 2 microg STX equivalents (STXeq)/L, which is belowthe concentration proposed for a health alert, namely 3 microg STXeq/L. In all cases, statistically significant correlations existed between all toxicity measurements of the same extracts with the methods used herein. Sodium channel and saxiphilin assays however predicted less toxicity relative to HPLC analysis. The only exception to this was the equivalency observed between saxiphilin measurement and HPLC quantitation corrected for mammalian toxicity. Saxiphilin assay predicted toxicity in one strain was 3 orders of magnitude more than by sodium channel assay, and no STX was detected by HPLC. Lack of acetylcholinesterase inhibition showed this bioactivity was not anatoxin-a(S), a toxin also produced by this A. circinalis with some resemblance to the region of STX bound by saxiphilin. Presence of anatoxin-a(S) was predicted for another strain by this same acetylcholinesterase assay that, if confirmed by chemical analysis, would be the first report of anatoxin-a(S) in an Australian cyanobacterium.

Characterisation of inter- and intra-molecular interactions of the dengue virus RNA dependent RNA polymerase as potential drug targets.

Our research is directed towards enhancing the understanding of the molecular biology of dengue virus replication with the ultimate goal being to develop novel antiviral strategies based on preventing critical inter- or intra-molecular interactions required for the normal virus life cycle. The viral RNA-dependent RNA polymerase (NS5) and the viral helicase (NS3) interaction offers a possible target for inhibitors to bind and prevent replication. In this study the yeast-two hybrid system was used to show that a small region of NS5 interacts with NS3, and also with the cellular nuclear transport receptor importin-beta. Furthermore, intramolecular interaction between the two putative domains of NS5 can also be detected by the yeast two-hybrid assay. We have also modified the colony lift assay for the beta-galactosidase reporter activity in intact yeast cells which reflects the strength of interaction between two proteins to a microtiter plate format. This assay offers a unique opportunity to screen for small molecule compounds that block physiologically important interactions.

Microtitre plate assay for paralytic shellfish toxins using saxiphilin: gauging the effects of shellfish extract matrices, salts and pH upon assay performance.

Saxiphilin is a hydrophilic protein with a high affinity and specificity for paralytic shellfish toxins (PSTs) found in the circulatory fluid of many invertebrates and ectothermic vertebrates. Saxiphilin has been found to be closely related to the iron binding transferrins, a group of proteins that range in molecular weight between 70 and 90 kDa. One saxiphilin isoform, that from the centipede Ethmostigmus rubripes, has been used to develop a microtitre plate assay for PSTs which relies upon detection of bound tritiated saxitoxin (STX). In this study, this assay was challenged with differing conditions of salt concentration and identity, pH and addition of non-toxic extracts of commercial shellfish prepared following the Association of Official Analytical Chemists endorsed procedure, elements the assay would encounter if used for PST monitoring and may compromise assay performance. The assay tolerated up to 15% of the total reaction, volume being shellfish extract before assay signal started to diminish. The presence of these extract matrices had little effect upon assay accuracy and precision when measuring STX, as a typical PST. Also, the detection of STX in the presence of shellfish matrices could be confidently reproduced on different days by different experimenters. The elements present in the shellfish extracts were measured by inductively coupled plasma spectroscopy, with the most common cationic elements being Na followed by K, Mg and Ca. Only trace amounts of other cationic elements were also present. From these results, the effects upon the assay by the four most common salts of these elements, NaCl, KCl, CaCl(2) and MgCl(2) were measured. Both NaCl and KCl did not impair assay performance at concentrations as high as 550 mM. It should be noted, however, that greater than 80 mM of either of these salts must be present in the assay for it to achieve the maximal signal. Adding CaCl(2) and MgCl(2) to the assay had dramatic effects upon performance. In the case of CaCl(2), the NaCl that was present in standard assay conditions enhanced its negative impact upon the assay. With MgCl(2), NaCl counteracted its inhibitory effect to some extent. After taking into account sample dilutions of shellfish extracts however, the potential for an interfering effect by Ca or Mg is minimal. A pH of 5.4 or less is necessary for there to be any significant impact upon the assay, with the assay signal being stable up to an alkaline pH as high as 9. Using the conditions herein, this assay can be used to reliably detect 1.3 microg STXeq/100 g shellfish tissue if it were to be used for monitoring for PST contaminated shellfish. These results demonstrate that this assay is a highly robust diagnostic tool for the measurement of PSTs in shellfish extracts.

Cloning of the Atlantic salmon (Salmo salar) estrogen receptor-alpha gene.

A cDNA clone encoding most of an Atlantic salmon (Salmo salar) estrogen receptor (ER) was obtained from a liver cDNA library and the remainder of the coding sequence from the gene was isolated from a genomic library. Sequence comparisons showed that the cloned gene represents ER-alpha. Expression of the ER-alpha gene in male and female salmon parr was analysed by RT-PCR. Highest expression was found in brain and liver, with lower levels of ER-alpha mRNA present in all other tissues tested. There was little difference in expression of ER-alpha between male and female.

Cloning and Expression of an Elongation Factor-1alpha in Sea Bream (Sparus aurata) Larvae and Adult Tissue.

A clone encoding the polypeptide elongation factor EF-1alpha was isolated from a complementary DNA library prepared from sea bream (Spartus aurata) larvae 1 to 10 days after hatching. The deduced amino acid sequence is between 82% and 95% similar to EF-1alpha in other animal species. EF-1alpha messenger RNA is present at low abundance in sea bream embryos prior to gastrulation, but at around 15 hours postfertilization, there is a 10-fold increase in transcript levels. This increase presumably reflects midblastula transition in this species. In adult sea bream, EF-1alpha appeared to have a relatively uniform distribution across all the tissues analyzed.

Community care. Home service.

A nurse-led scheme to prevent hospital admissions by providing emergency services in the community for up to 72 hours cared for 155 people in its first six months. The scheme operated well below full capacity partly because of initial opposition from GPs. The mean age of service users was 79 and the main reasons for referral were chest infections, pneumonia and the need for support following an injury. More than three quarters of those cared for were fit to be discharged from the scheme within 72 hours.

Cloning, characterisation and expression of the apolipoprotein A-I gene in the sea bream (Sparus aurata).

A full length cDNA clone representing apolipoprotein A-I was isolated from a sea bream (Sparus aurata) liver library. The clone encodes a 261 amino acid protein which shows highest amino acid identity (38%) with salmon apolipoprotein A-I. Northern blot analysis showed strong expression of a 1.4 kb transcript in liver with lower expression in intestine. Expression of apolipoprotein A-I in intestine was markedly reduced by treatment with triiodothyronine (T3).

A high-throughput, microtiter plate assay for paralytic shellfish poisons using the saxitoxin-specific receptor, saxiphilin.

An isoform of the paralytic shellfish poison (PSP)-specific receptor saxiphilin, from the tropical centipede Ethmostigmus rubripes, was used as the basis for a radiometric, high-throughput, microtiter plate assay for this group of toxins. Characterization of the assay revealed that it was able to detect several representatives from the various structural PSP subgroups and yet was insensitive toward tetrodotoxin. To test the utility of the assay as a seafood toxin-monitoring tool, the assay was subjected to a variety of marine organism extracts, some of which were known to contain PSPs, and whole extract toxicity expressed as STX equivalents (STXeq) was measured by two methods: First, by comparison of values from a screening assay with a standard STX inhibition curve and, second, for highly active extracts, by calculation using the IC50 from a full inhibition curve of the extract. For extracts which could be quantified by both methods, there was almost 100% correlation between the derived values. STXeq derived by both methods from the bioassay highly correlated with absolute toxin quantities from HPLC analysis.

Use of sequential case-control studies to investigate a community Salmonella outbreak in Wales.

STUDY OBJECTIVE: To establish the source of a community outbreak of Salmonella typhimurium definitive type 124. DESIGN: Two stage case-control study. SETTING: Three districts in south east Wales. SUBJECTS: Cases of salmonella food poisoning and community controls. MAIN RESULTS: An initial case-control study identified an association between illness and eating ham (odds ratio 4.50, 95% confidence intervals 1.10, 21.8) and also found a possible association between illness and food bought from delicatessen stores (odds ratio 5.03, 95% confidence intervals 1.01, 32.3). However, only after a second stage case-control study was a single common ham producer identified as the source (odds ratio 25.0, 95% confidence intervals 2.33, 1155). CONCLUSION: Sequential case-control studies are an important and underused tool in the investigation of community outbreaks.

Comparative analyses by HPLC and the sodium channel and saxiphilin 3H-saxitoxin receptor assays for paralytic shellfish toxins in crustaceans and molluscs from tropical North West Australia.

The increased frequency and distribution of red tides requires the development of high-throughput detection methods for paralytic shellfish toxins (PST). Community ethics also requires that there be a reduced reliance upon the standard mouse bioassay. A biomolecular assay such as the sodium channel 3H-saxitoxin binding assay can satisfy both of these requirements but may be compromised by cross-reactivity with the structurally unrelated tetrodotoxins (TTX). This study utilised the sodium channel assay but also an alternative 3H-saxitoxin binding assay based upon a saxiphilin isoform from the centipede Ethmostigmus rubripes to screen for PSTs. Saxiphilin is a novel transferrin which binds saxitoxin (STX) but differs from the sodium channel in not having any measurable affinity for TTX. A detailed analysis of toxin composition was achieved by high performance liquid chromatography (HPLC). Various crustaceans and molluscs accumulate PSTs and TTX, thus proving useful biomarkers for these toxins in their immediate environment and an ideal challenge to the detection and analysis of PSTs in this presumptive screening program. Also, there has been little investigation of PSTs in invertebrates from the Indian Ocean so this region was selected to extend our knowledge of the distribution of these toxins. 190 crabs and shellfish encompassing 31 species were collected from reefs along the North-West Australian coast and tested for PSTs and TTX by sodium channel and saxiphilin bioassays as well as HPLC. PSTs were detected in 18 species of crabs and shellfish of the 31 species tested. Eight of these species have not been previously described as toxic, these being the crabs Euzanthus exsculptus, Lophozozymus octodentatus, Metopograpsus frontalis, Pilumnus pulcher, Platypodia pseudogranulosa and Portunus pelagicus, and the molluscs Tectus fenestratus and Trochus hanleyanus. By HPLC, only one or both of STX and decarbamoyl-STX was detected in any extract. Some extracts markedly inhibited 3H-saxitoxin binding by the sodium channel but not by saxiphilin. The close agreement between toxin quantification by the PST specific methods of HPLC and the saxiphilin bioassay is indicative that the additional toxicity detected by the sodium channel assay is TTX.

Cloning and unusual expression profile of the aldolase B gene from Atlantic salmon.

A full-length clone of the aldolase B gene has been isolated from a cDNA library constructed from liver of Atlantic salmon (Salmo salar). Sequencing showed that the clone encodes a typical aldolase B, possessing a number of amino acid residues which are seen in aldolase B, but not in other aldolase isoforms. RT-PCR analysis showed that the gene is expressed in liver, kidney and intestine as expected. However, in contrast to mammalian and avian aldolase B, expression was also found in a number of other tissues. Levels of aldolase B mRNA in liver and kidney were not significantly altered during smoltification, the transformation of freshwater-dwelling salmon (parr) into saltwater-adapted salmon (smolts).

Cloning and sequencing of a full-length sea bream (Sparus aurata) beta-actin cDNA.

A full-length cDNA clone encoding beta-actin (beta-actin) was isolated from a sea bream (Sparus aurata) liver cDNA library. Sequencing of this clone reveals an open reading frame encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The sea bream beta-actin sequence showed 98% identity to carp and human beta-actin and 95% and 94% identity to sea squirt and Dictyostelium cytoplasmic actins, respectively.

Phylogenetic survey of soluble saxitoxin-binding activity in pursuit of the function and molecular evolution of saxiphilin, a relative of transferrin.

Saxiphilin is a soluble protein of unknown function which binds the neurotoxin, saxitoxin (STX), with high affinity. Molecular characterization of saxiphilin from the North American bullfrog, Rana catesbeiana, has previously shown that it is a member of the transferrin family. In this study we surveyed various animal species to investigate the phylogenetic distribution of saxiphilin, as detected by the presence of soluble [3H]STX binding activity in plasma, haemolymph or tissue extracts. We found that saxiphilin activity is readily detectable in a wide variety of arthropods, fish, amphibians, and reptiles. The pharmacological characteristics of [3H]STX binding activity in phylogenetically diverse species indicates that a protein homologous to bullfrog saxiphilin is likely to be constitutively expressed in many ectothermic animals. The results suggest that the saxiphilin gene is evolutionarily as old as an ancestral gene encoding bilobed transferrin, an Fe(2+)-binding and transport protein which has been identified in several arthropods and all the vertebrates which have been studied.

Expression of saxiphilin in insect cells and localization of the saxitoxin-binding site to the C-terminal domain homologous to the C-lobe of transferrins.

Saxiphilin is a plasma protein from the bullfrog (Rana catesbeiana) that is homologous to transferrin. Most known transferrins contain two binding sites for Fe3+/HCO3-, one in each of two homologous domains called the N-lobe and C-lobe. However, native saxiphilin does not bind Fe3+ but stoichiometrically binds one molecule of the neurotoxin saxitoxin (STX) with a dissociation constant (KD) of approximately 0.2 nM. To pursue structural analysis of the STX binding sites, cDNA encoding saxiphilin was used to construct a baculovirus expression vector that directs synthesis and secretion of a approximately 92-kDa recombinant saxiphilin protein (R-sax) in cultured insect cells. Culture medium harvested from infected cells contained 25-67 pmol of [3H]STX binding sites/mL with a KD of 0.22 nM. The kinetics and pH dependence (pK0.5 = 5.4) of [3H]STX binding to R-sax are similar to native saxiphilin, implying proper folding and functional activity. Another baculovirus expression vector was constructed to encode a deletion mutant of saxiphilin consisting of the first 20 N-terminal residues containing the secretory signal sequence spliced to the C-terminal, 361-residue fragment homologous to the C-lobe domain of transferrins. This vector directed the secretion of a approximately 38-kDa derivative of saxiphilin (C-sax) that was recognized by antisaxiphilin antibody. C-sax also exhibited [3H]STX binding activity with a lower affinity KD of approximately 0.9 nM, a 4-fold faster dissociation rate for [3H]STX than native saxiphilin, and a pH dependence (pK0.5 = 5.7) similar to R-sax (pK0.5 = 5.4).(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and characterisation of a fish aldolase B gene.

A full length cDNA clone representing an aldolase mRNA was isolated from a sea bream (Sparus aurata) liver cDNA library. Sequencing of this clone revealed it to encode a 364 amino acid protein with 74% amino acid identity to human aldolase B and slightly lower similarity to human aldolase A and C. In view of the sequence data and of Northern blot analysis showing strong expression of a 1.6 kb transcript in liver it was concluded that the cloned gene represents aldolase B. This clone represents the first aldolase gene to be sequenced from any fish species thus providing new data on the evolution of the vertebrate aldolase gene family.