Okadaic Acid, an Apoptogenic Toxin for Symbiotic/Parasitic Annelids in the Demosponge Suberites domuncula

The role of okadaic acid (OA) in the defense system of the marine demosponge Suberites domuncula against symbiotic/parasitic annelids was examined. Bacteria within the mesohyl produced okadaic acid at concentrations between 32 ng/g and 58 ng/g of tissue (wet weight). By immunocytochemical methods and by use of antibodies against OA, we showed that the toxin was intracellularly stored in vesicles. Western blotting experiments demonstrated that OA also existed bound to a protein with a molecular weight of 35,000 which was tentatively identified as a galectin (by application of antigalectin antibodies). Annelids that are found in S. domuncula undergo apoptotic cell death. OA is one candidate inducer molecule of this process, since this toxin accumulated in these symbionts/parasites. Furthermore, we identified the cDNA encoding the multifunctional prosurvival molecule BAG-1 in S. domuncula; it undergoes strong expression in the presence of the annelid. Our data suggest that sponges use toxins (here, OA) produced from bacteria to eliminate metazoan symbionts/parasites by apoptosis.     

ELISA FOR DETECTING OKADAIC ACED IN MODEL SYSTEMS USING PURIFIED POLYCLONAL ANTIBODIES

Polyclonal antibodies were produced in rabbits against okadaic acid (OA) following its coupling to bovine serum albumin using standard carbodiimide condensation procedure to form the immunogen. The immunogen was dialyzed against Tris buffer pH 7.45 at 4C and used to immunize two rabbits. Each rabbit received (at four sites) 0.5 mL immunogen (i.d.) together with adjuvant followed by three repeated injections of 0.3 mL emulsified immunogen-adjuvant mixture at weekly intervals. The last injection was made 60 days after the fourth. The antiserum was collected, at intervals, the immunoglobulin fraction (IgG) isolated, purified and used in an ELISA system to capture the okadaic moiety of the immunogen. Antibody titers increased following repeated immunization and the IgG recognized low levels of OA. However, ELISA was more sensitive for detecting the immunogen than for pure OA, but using the biotinylated-IgG enhanced the titration to both and the detection limit for OA was 0.63 ng per 0.1 mL buffer-methanol and the assay linearity ranged from 0.63 to 5.0 ng OA.     

A screening lateral flow immunochromatographic assay for on-site detection of okadaic acid in shellfish products

A lateral flow immunochromatographic (LFIC) test strip based on a colloidal gold-monoclonal antibody (McAb) conjugate was developed for on-site rapid detection of okadaic acid (OA) in shellfish. It applies a competitive format using an immobilized toxin conjugate and free toxin present in samples. The McAb against OA was conjugated with 20-nm colloidal gold as detector reagent. The toxin in the sample competed with the immobilized toxin to bind to the gold conjugated with McAb. The colloidal gold/McAb/toxin mobile complex was not captured by OA-bovine serum albumin (BSA) on the test line, but it was captured by goat anti-mouse immunoglobulin G (IgG) on the control line. The color density of the test line correlated with the concentration of toxin in the range of 10-50 ng ml(-1). The qualitative detection limit of 150 μg kg(-1) sample was close to the European Union (EU) regulatory limit (160 μg kg(-1)). Therefore, these strips were able to directly and qualitatively estimate the consuming safety of shellfish. They require no equipment because of available visual results, and they screened numerous samples within 10 min. The results were further confirmed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). As a food safety screening tool, the test strips are convenient and useful to rapidly on-site test the presence of OA in shellfish products.     

Development of an immunochromatographic strip test for the rapid detection of okadaic acid in shellfish sample

A rapid detection technology for okadaic acid (OA) in shellfish with one-step immunochromatographic assay using colloidal gold-labeled monoclonal antibody (Mab) probe was developed. OA is one of the diarrhetic shellfish toxins. Firstly, OA was conjugated to bovine serum albumin, and the conjugations as immunogen were injected into mice to raise the polyclonal antibody against OA. Hybridoma cells fused between spleen cells from immunized mouse and myeloma cells (Sp2/0) were prepared and injected into mice intraperitoneally at 1?×?10⁶?cells to produce monoclonal antibody in the ascitic fluid. With the monoclonal antibody against OA, the idc-ELISA assay was established to detect OA. The calibration curve for OA was linear over the concentration range of 0.31–50 ng mL^?1, and the detection limit for OA was 0.45 ng mL^?1. On that basis, paper test strips for detecting OA were prepared, and a fast detection method for okadaic acid using gold-labeled immunological assay was established. With the paper test strips, the detection limit was 6.25 ng mL^?1, and whole detection process for OA in shellfish samples needed only about 40 min.     

Emergence and disappearance of an immune molecule,an antimicrobial lectin,in basal metazoa. A tachylectin-related protein in the sponge Suberites domuncula

Sponges (phylum Porifera) represent the evolutionarily oldest metazoans that comprise already a complex immune system and are related to the crown taxa of the protostomians and the deuterostomians. Here, we demonstrate the existence of a tachylectin-related protein in the demosponge Suberites domuncula, termed Suberites lectin. The MAPK pathway was activated in response to lipopolysaccharide treatment of the three-dimensional cell aggregates, the primmorphs; this process was abolished by the monosaccharide D-GlcNAc. The cDNA encoding the S. domuncula lectin was identified and cloned; it comprises 238 amino acids (26 kDa) in the open reading frame. The deduced protein has one potential transmembrane region, three characteristic Cys residues, and six internal tandem repeats; it shares the highest sequence similarity with lectins from the horseshoe crab Tachypleus trunculus. The steady-state level of expression of the Suberites lectin rises in primmorphs in response to lipopolysaccharide, an effect that was prevented by co-incubation with D-GlcNAc. The natural sponge lectin was purified by affinity chromatography; it has a size of 27 kDa and displays antibacterial activity against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus. The putative protein, deduced from the cloned gene, is identical/similar to the purified natural protein, as demonstrated by immunological cross-reactivity with specific antibodies. We conclude that the S. domuncula lectin acts as an antibacterial molecule involved in immune defense against bacterial invaders.     

Myosin degradation fragments in skeletal muscle

Myosin heavy chain degradation fragments produced in vivo have been identified in chicken pectoralis muscle. The fragments were identified by electrophoresis of unfractionated extracts of chicken pectoralis muscle on sodium dodecyl sulfate/polyacrylamide gels followed by immunoblotting on nitrocellulose sheets. Monoclonal antibodies directed against the S2 and light meromyosin subfragments as well as type II myosin-specific polyclonal antibodies directed against the entire myosin heavy chain were used to characterize the fragments, which range in molecular weight from approximately 80,000 to 180,000. All fragments contain the extreme carboxy-terminal portion of the molecule and are distinct from the classical proteolytic fragments such as heavy and light meromyosin, S1, S2 or rod. These fragments appear to be produced in vivo by proteolytic cleavage of peptides from the amino-terminal (S1) end of the heavy chain while the myosin molecule is still embedded in the thick filament. Fragment concentrations are estimated to be approximately 5 to 10% of that of the intact myosin heavy chain. These fragments are not the result of artifactual damage to myosin, e.g. proteolysis or hydrodynamic shear. The techniques described in this paper provide a probe into the early stages of myosin and thick filament degradation in vivo.     

Hyperphosphorylation of N-60, a protein structurally and immunologically related to nucleolin after tumour-promoter treatment.

Okadaic acid, a non-TPA-type tumour promoter, induces hyperphosphorylation of a 60-kd protein in primary human fibroblasts. Treatment with TPA-type tumour promoters (e.g. TPA and teleocidin) did not cause this hyperphosphorylation. Phosphorylation of this protein was not seen at times earlier than 90 min after the addition of 75 ng/ml okadaic acid to the proliferating cell cultures. The presence of inhibitors such as actinomycin D and cycloheximide, did not significantly influence the level of hyperphosphorylation induced by okadaic acid treatment. By immunoblotting using an antibody anti-nucleolin, the 60-kd protein was identified as a fragment of nucleolar protein, nucleolin. Similarly, antibodies against the 60-kd protein cross-reacted with nucleolin. Furthermore peptide mapping, using staphylococcal V8 protease, showed that the 60-kd protein phosphorylated by casein kinase II in vitro and the okadaic-acid-induced hyperphosphorylated 60-kd protein exhibited identical phosphopeptide maps, indicating that there is also structural relatedness between N-60 and nucleolin. Hyperphosphorylation of the nucleolin fragment (N-60) was suppressed by anti-tumour promoter retinoic acid.     

PRODUCTION OF 7-DEOXY-OKADAIC ACID BY A NEW CALEDONIAN STRAIN OF PROROCENTRUM LIMA (DINOPHYCEAE)

7-Deoxy-okadaic acid and okadaic acid were identified as the major diarrhetic shellfish poisoning (DSP) toxins produced by a New Caledonian strain of Prorocentrum lima Ehrenberg. Dinophysistoxin-1 was not produced by this strain. The cellular concentrations of 7-deoxy-okadaic acid were about one tenth that of okadaic acid and were maximal (∼1.4 pg·cell ^{− 1}) during the stationary growth phase of batch culture. Autolytic hydrolysis of cell extracts did not increase the concentrations of 7-deoxy-okadaic acid, whereas okadaic acid production increased more than 4-fold, indicating that 7-deoxy-okadaic acid, unlike okadaic acid, is not directly derived from large sulfated precursors. 7-Deoxy-okadaic acid could be detected by liquid chromatography-selected reaction monitoring mass spectrometry, HPLC-fluorescence detection after derivatization with 9-anthryldiazomethane (ADAM), and inhibition of protein phosphatases. The solvent washes currently used for solid-phase clean-up of ADAM-derivatized DSP samples elute derivatized 7-deoxy-okadaic acid, indicating that the current sample clean-up protocol for HPLC-fluorescence detection would miss any contamination by this toxin.     

Bone morphogenetic proteins and neurotrophins provide complementary protection of septal cholinergic function during phosphatase inhibitor-induced stress

Cultures of embryonic rat septum were exposed for 24-48 h to 2-5 nm okadaic acid (OA), an inhibitor of pp1A and pp2A phosphatases. This stress killed approximately 75% of neurons. A neurotrophin (NT) combination (nerve growth factor and brain-derived neurotrophic factor, each 100 ng/mL) plus a bone morphogenetic protein (BMP6 or BMP7, 5 nm) reduced the death of both cholinergic and non-cholinergic neurons, and preserved choline acetyltransferase (ChAT) activity assayed 2-6 days post-stress. This NT + BMP combination preserved ChAT activity better than either NTs or BMPs alone, and was effective even if trophic factor addition was delayed until 12 h after stress onset. A general caspase inhibitor (qVD-OPH, 10 micro g/mL) also increased survival of stressed cholinergic neurons, but its protection of ChAT activity was shorter lived than that produced by the NT + BMP combination. Neither the NT + BMP combination nor the caspase inhibitor reduced the OA-induced increase in tau phosphorylation. These findings indicate that NTs and BMPs have synergistic protective effects against an OA stress, and suggest that at least some of these protective effects occur upstream of caspase activation.     

A new model of the tautomycin-PP1 complex that is not analogous to the corresponding okadaic acid structure

A revised model of PP1-tautomycin (TM) complex suggests that this toxin does not bind in a conformation analogous to its structural cousin okadaic acid (OA), as has been assumed, but instead more resembles the mode of binding adopted by calyculin. This model rationalizes the unexpected potency of a truncated TM analogue lacking the bicyclic ketal common to TM and OA.     

Molecular markers for germ cell differentiation in the demosponge Suberites domuncula

Sponges (phylum Porifera) are simple metazoans for which no molecular information on gametogenesis and larval development is available. To support the current study, it was confirmed by histology that oocytes and larvae were produced by the demosponge Suberites domuncula. Three genes/expressed products from S. domuncula whose expression correlated with sexual reproduction were identified and characterized (they are used here as marker genes): i) a receptor tyrosine kinase (RTK) with sequence similarity in the tyrosine kinase domain to fibroblast growth factor receptors; ii) the sex-determining protein FEM1 and iii) the sperm associated antigen (SAA) of triploblasts. Antibodies against the extracellular domain of the RTK specifically stained oocytes and larvae in S. domuncula tissue sections. Induction of these three genes was successful at elevated temperature, a factor which also promotes natural gametogenesis. In situ hybridization analyses revealed that FEM1 and SAA were expressed in those areas in which gametogenesis begins. Our results indicate that genes which play a role in sex determination may be present in Porifera.     

Production of neutralizing human monoclonal antibody directed to tetanus toxin in CHO cell.

By the fusion of lymphocytes from hyperimmunized people with heteromyeloma cells, 600 human hybridoma cell lines were generated. Even though seven cell lines produced antibodies against tetanus toxoid, only two antibodies from hybrid CH8 and CH5 only neutralized the tetanus toxin and completely protected the mice that had been challenged with the toxin even at the level of 90 mean lethal dose. The cDNA of light (L) chain and heavy (H) chain variable region was isolated, and then inserted into expression vectors containing human IgG constant regions. After transfection of the recombinant human IgG gene into Chinese Hamster Ovary (CHO) cells, transformants secreting the complete human antibody were selected. The recombinant human antibodies produced from CHO cells possessed neutralizing activity against tetanus toxin just like the original human antibodies produced from human hybridoma cell lines. Western blot analysis showed that rCH8 and rCH5 antibodies recognized the H chain of tetanus toxin and did not bind to its L chain. The neutralizing test showed that HmAb rCH5 had 4.55IU and HmAb rCH8 had 1.09IU/100 micro g of IgG, respectively. Mixing of the two HmAbs resulted in synergistic effects. On a weight basis (IU/100 micro g IgG), the highest potency values were obtained when the two HmAbs were combined in equal quantity. The neutralizing activity of rCH8 and rCH5 mixture was 6.94IU/100 micro g IgG.     

Methodological improvement of the protein phosphatase inhibition assay for the detection of okadaic acid in mussels

A simplified procedure for the enzyme inhibition assay to measure okadaic acid and DTX-1 in mussels, based on the use of a commercially available enzyme preparation, is presented. The detection limit is 10 ng of toxin per g of digestive glands. Using Certified Reference Material (MUS-2), high accuracy and good precision is demonstrated for contamination levels higher than 32 ng g(-1). Twenty samples can be processed in about 9 h by one operator, at the cost of US$ 10 per sample. Some possibilities for further enhancing the sensitivity and reducing the processing time are discussed and a monitoring example is presented.     

Okadaic acid, a potent inhibitor of type 1 and type 2A protein phosphatases, activates cdc2/H1 kinase and transiently induces a premature mitosis-like state in BHK21 cells.

When BHK21 cells synchronized in early S phase were exposed to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, mitosis specific events such as premature chromosome condensation, the production of MPM-2 antigens, dispersion of nuclear lamins and the appearance of mitotic asters were induced, and then disappeared upon further incubation. These mitosis specific events occurred even in the presence of cycloheximide. Within 1 h of exposure to OA, cdc2/histone H1 kinase activity rose 10-fold compared with untreated controls, but returned to the control level upon further incubation. Using antibodies against either p34cdc2 or cyclin B it was found that p34cdc2 complexed with cyclin B was dephosphorylated after OA treatment concomitant with the activation of cdc2 kinase, and that cyclin B was subsequently degraded concomitant with a decrease in cdc2 kinase activity, as in normal mitosis. In contrast, when cells in G1 phase were treated with OA no increase in cdc2 kinase activity was observed. Moreover when cells in pseudo-metaphase induced by nocodazole were treated with OA, cdc2 kinase was inactivated. These results suggest that OA sensitive protein phosphatases control both the activation and inactivation of the p34cdc2 kinase.     

Innate immune defense of the sponge Suberites domuncula against bacteria involves a MyD88-dependent signaling pathway. Induction of a perforin-like molecule

Sponges (phylum Porifera) are the phylogenetically oldest metazoa; as filter feeders, they are abundantly exposed to marine microorganisms. Here we present data indicating that the demosponge Suberites domuncula is provided with a recognition system for gram-negative bacteria. The lipopolysaccharide (LPS)-interacting protein was identified as a receptor on the sponge cell surface, which recognizes the bacterial endotoxin LPS. The cDNA was isolated, and the protein (Mr 49,937) was expressed. During binding to LPS, the protein dimerizes and interacts with MyD88, which was also identified and cloned. The sponge MyD88 (Mr 28,441) is composed of two protein interaction domains, a Toll/interleukin-1 receptor domain (found in MyD88 and in Toll-like receptors) and a death domain (present in MyD88 and interleukin-1 receptor-associated kinase). Northern blot experiments and in situ hybridization studies showed that after LPS treatment, the level of the LPS-interacting protein remains unchanged, whereas MyD88 is strongly up-regulated. A perforin-like molecule (Mr 74,171), the macrophage-expressed protein, was identified as an executing molecule of this pathway. This gene is highly expressed after LPS treatment, especially at the surfaces of the animals. The recombinant protein possesses biological activity and eliminates gram-negative bacteria; it is inactive against gram-positive bacteria. These data indicate that S. domuncula is provided with an innate immune system against gram-negative bacteria; the ligand LPS (a pathogen-associated molecular pattern) is recognized by the pattern recognition receptor (LPS-interacting protein), which interacts with MyD88. A signal transduction is established, which results in an elevated expression of MyD88 as well as of the macrophage-expressed protein as an executing protein.     

Cold stress defense in the freshwater sponge Lubomirskia baicalensis. Role of okadaic acid produced by symbiotic dinoflagellates

The endemic freshwater sponge Lubomirskia baicalensis lives in Lake Baikal in winter (samples from March have been studied) under complete ice cover at near 0 degrees C, and in summer in open water at 17 degrees C (September). In March, specimens show high metabolic activity as reflected by the production of gametes. L. baicalensis lives in symbiosis with green dinoflagellates, which are related to Gymnodinium sanguineum. Here we show that these dinoflagellates produce the toxin okadaic acid (OA), which is present as a free molecule as well as in a protein-bound state. In metazoans OA inhibits both protein phosphatase-2A and protein phosphatase-1 (PP1). Only cDNA corresponding to PP1 could be identified in L. baicalensis and subsequently isolated from a L. baicalensis cDNA library. The deduced polypeptide has a molecular mass of 36 802 Da and shares the characteristic domains known from other protein phosphatases. As determined by western blot analysis, the relative amount of PP1 is almost the same in March (under ice) and September (summer). PP1 is not inhibited by low OA concentrations (100 nm); concentrations above 300 nm are required for inhibition. A sponge cell culture system (primmorphs) was used to show that at low temperatures (4 degrees C) expression of hsp70 is strongly induced and hsp70 synthesis is augmented after incubation with 100 nm OA to levels measured at 17 degrees C. In the enriched extract, PP1 activity at 4 degrees C is close to that measured at 17 degrees C. Immunoabsorption experiments revealed that hsp70 contributes to the high protein phosphatase activity at 4 degrees C. From these data we conclude that the toxin OA is required for the expression of hsp70 at low temperature, and therefore contributes to the cold resistance of the sponge.     

Characterization and comparison of toxin‐producing isolates of Dinophysis acuminata from New England and Canada

Following the identification of the first toxic isolate of Dinophysis acuminata from the northwestern Atlantic, we conducted detailed investigations into the morphology, phylogeny, physiology, and toxigenicity of three isolates from three sites within the northeastern U.S./Canada region: Eel Pond and Martha's Vineyard, Massachusetts, and the Bay of Fundy. Another isolate, collected from the Gulf of Mexico, was grown under the same light, temperature, and prey conditions for comparison. Despite observed phenotypic heterogeneity, morphometrics and molecular evidence classified the three northwestern Atlantic isolates as D. acuminata Claparède & Lachmann, whereas the isolate from the Gulf of Mexico was morphologically identified as D. cf. ovum. Physiological and toxin analyses supported these classifications, with the three northwestern Atlantic isolates being more similar to each other with respect to growth rate, toxin profile, and diarrhetic shellfish poisoning (DSP) toxin content (okadaic acid + dinophysistoxin 1/cell) than they were to the isolate from the Gulf of Mexico, which had toxin profiles similar to those published for D. cf. ovum F. Schütt. The DSP toxin content, 0.01–1.8 pg okadaic acid (OA) + dinophysistoxin (DTX1) per cell, of the three northwestern Atlantic isolates was low relative to other D. acuminata strains from elsewhere in the world, consistent with the relative scarcity of shellfish harvesting closures due to DSP toxins in the northeastern U.S. and Canada. If this pattern is repeated with the analyses of more geographically and temporally dispersed isolates from the region, it would appear that the risk of significant DSP toxin outbreaks in the northwestern Atlantic is low to moderate. Finally, the morphological, physiological, and toxicological variability within D. acuminata may reflect spatial (and/or temporal) population structure, and suggests that sub‐specific resolution may be helpful in characterizing bloom dynamics and predicting toxicity.     

Glucose uptake in enterocytes: a test for molecular targets of okadaic acid

The main diarrheic shellfish poisoning (DSP) toxin is okadaic acid (OA). Although OA is a protein phosphatase 1 and 2A inhibitor less is known about the involvement of the toxin in diarrhea. The initial statement was that OA, by altering the phosphorylation state of proteins, might modify glucose uptake and consequently ionic and water reabsorption across the small intestine. This report presents studies of glucose transport in isolated rabbit enterocytes by using a fluorescent derivative of D-glucose. The dye allowed examining the relation between the toxic effect of OA and cellular mechanisms involved in glucose transport. The central findings are: (i) OA potentiates decrease on glucose uptake due to protein kinase A (PKA) inhibitors such as H89; and (ii) the increase of sugar uptake induced by the protein kinase C (PKC) inhibitor chelerythrine is enhanced by OA. Importance of this work is justified by the need to determine molecular targets of diarrheic toxins in intestinal cells.     

Mutation induction by okadaic acid, a protein phosphatase inhibitor, in CHL cells, but not in S. typhimurium.

Okadaic acid (OA) is a specific and strong inhibitor of protein phosphatase 1 and 2A present in eukaryotes, and a potent promoter of carcinogenesis in mouse skin. In this study, we examined the mutagenicity of OA. OA did not induce mutations in S. typhimurium TA100 and TA98, with or without a microsomal metabolic activation system. However, it was strongly mutagenic to Chinese hamster lung (CHL) cells without a microsomal activation system, as shown using diphtheria toxin (DT) resistance (DT^r) as a selective marker. Treatment of CHL cells with OA at 17.5 ng/ml induced 164 DT^r mutants per 10⁶ survivors. A plot of the mutation frequency against the OA concentration gave a concave curve, and the mutant frequency was calculated to be 5500/10⁶ survivors/μg, with OA in the dose range of 10–15 ng/ml. This value was about 680 times that of ethyl methanesulfonate (EMS), and comparable to that of 2-amino-N⁶-hydroxyadenine, one of the strongest knowon mutgens. Elongation factor 2 (EF-2) obtained from 4 DT^r clones was not ADP-ribosylated by DT fragment A. PCR-direct sequencing revealed that the hot spot of EF-2 for EMS mutagenesis in CHO-K1 cells, the first letter of codon 717, was not a t spot for OA mutagenesis in CHL cells.     

Evaluation of rapid methods for the determination of okadaic acid in mussels

AIMS: Two different screening methods, a Buffalo Green Monkey cytotoxicity test and a biosensor test, have been considered to replace the official mouse bioassay in monitoring for okadaic acid (OA) levels in mussels. METHODS AND RESULTS: Diarrhoetic shellfish poison-contaminated mussels from the Adriatic Sea were assayed in parallel by means of the mouse bioassay and both alternative methods. Both the cytotoxicity test and the biosensor test showed high sensitivity (OA 0.01 mg g-1 hepatopancreas and 0.002 mg g-1 hepatopancreas, respectively) and a high correlation with the mouse bioassay (r=0.932, P < 0.001 and r=- 0.850, P < 0.001, respectively). CONCLUSION: Both methods are efficacious, quick, inexpensive and provide data on the amount of toxin present in mussels. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods, besides allowing the simultaneous assay of a great number of samples, comply with the ethical need to reduce the use of animals in the laboratory.