Purification, structural characterization, and neuroprotective effect of 3,6-diisobutyl-2,5-piperazinedione from Halomonas pacifica CARE-V15 against okadaic acid-induced neurotoxicity in zebrafish model.

Halomonas pacifica CARE-V15 was isolated from the southeastern coast of India to determine its genome sequence. Secondary metabolite gene clusters were identified using an anti-SMASH server. The concentrated crude ethyl acetate extract was evaluated by GC-MS. The bioactive compound from the crude ethyl acetate extract was fractionated by gel column chromatography. HPLC was used to purify the 3,6-diisobutyl-2,5-piperazinedione (DIP), and the structure was determined using FTIR and NMR spectroscopy. Purified DIP was used in an in silico molecular docking analysis. Purified DIP exhibits a stronger affinity for antioxidant genes like glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GSR). Using in silco molecular docking analysis, the protein-ligand binding affinities of GSR (-4.70 kcal/mol), GST (-5.27 kcal/mol), and GPx (-5.37 kcal/mol) were measured. The expression of antioxidant genes were investigated by qRT-PCR. The in vivo reactive oxygen species production, lipid peroxidation, and cell death levels were significantly (p ≤ 0.05) increased in OA-induced group, but all these levels were significantly (p ≤ 0.05) decreased in the purified DIP pretreated group. Purified DIP from halophilic bacteria could thus be a useful treatment for neurological disorders associated with oxidative stress.

Functional Selection of Tau Oligomerization-Inhibiting Aptamers.

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3ß-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

Effects of lipophilic phycotoxin okadaic acid on the early development and transcriptional expression of marine medaka Oryzias melastigma.

The lipophilic okadaic acid (OA)-group toxins produced by some species of Dinophysis spp. and Prorocentrum spp. marine dinoflagellates have been frequently and widely detected in natural seawater environments, e.g. 2.1∼1780 ng/L in Spanish sea and 5.63∼27.29 ng/L in the Yellow Sea of China. The toxicological effects of these toxins dissolved in seawater on marine fish is still unclear. Effects of OA on the embryonic development and 1-month old larvae of marine medaka (Oryzias melastigma) were explored and discussed in this study. Significantly increased mortality and decreased hatching rates occurred for the medaka embryos exposed to OA at 1.0 µg/mL. Diverse malformations including spinal curvature, dysplasia and tail curvature were also observed in the embryos exposed to OA and the heart rates significantly increased at 11 d post fertilization. The 96 h LC50 of OA for 1-month old larvae was calculated at 3.80 µg/mL. The reactive oxygen species (ROS) was significantly accumulated in medaka larvae. Catalase (CAT) enzyme activity was significantly increased in 1-month old larvae. Acetylcholinesterase (AChE) activity significantly increased with a dose-dependent pattern in 1-month old larvae. Differentially expressed genes (DEGs) were enriched in 11 KEGG pathways with Q value < 0.05 in 1-month old medaka larvae exposed to OA at 0.38 µg/mL for 96 h, which were mainly related to cell division and proliferation, and nervous system. Most of DEGs involved in DNA replication, cell cycle, nucleotide excision repair, oocyte meiosis, and mismatch repair pathways were significantly up-regulated, while most of DEGs involved in synaptic vesicle cycle, glutamatergic synapse, and long-term potentiation pathways were markedly down-regulated. This transcriptome analysis demonstrated that a risk of cancer developing was possibly caused by OA due to DNA damage in marine medaka larvae. In addition, the neurotoxicity of OA was also testified for marine fish, which potentially cause major depressive disorder (MDD) via the up-regulated expression of NOS1 gene. The genotoxicity and neurotoxicity of OA to marine fish should be paid attention to and explored further in the future.

Behavioural, genomics and proteomic approach to examine Alzheimer's disease in zebrafish.

Globally around 24 million elderly population are dealing with dementia, and this pathological characteristic is commonly seen in people suffering from Alzheimer's disease (AD). Despite having multiple treatment options that can mitigate AD symptoms, there is an imperative call to advance our understanding of the disease pathogenesis to unfold disease-modifying treatments/therapies. To explore the driving mechanisms of AD development, we stretch out further to study time-dependant changes after Okadaic acid (OKA)-induced AD-like conditions in zebrafish. We evaluated the pharmacodynamics of OKA at two-time points, i.e., after 4-days and 10-days exposure to zebrafish. T-Maze was utilized to observe the learning and cognitive behaviour, and inflammatory gene expressions such as 5-Lox, Gfap, Actin, APP, and Mapt were performed in zebrafish brains. To scoop everything out from the brain tissue, protein profiling was performed using LCMS/MS. Both time course OKA-induced AD models have shown significant memory impairment, as evident from T-Maze. Gene expression studies of both groups have reported an overexpression of 5-Lox, GFAP, Actin, APP, and OKA 10D group has shown remarkable upregulation of Mapt in zebrafish brains. In the case of protein expression, the heatmap suggested an important role of some common proteins identified in both groups, which can be explored further to investigate their mechanism in OKA-induced AD pathology. Presently, the preclinical models available to understand AD-like conditions are not completely understood. Hence, utilizing OKA in the zebrafish model can be of great importance in understanding the pathology of AD progression and as a screening tool for drug discovery.

An inhibitor with GSK3ß and DYRK1A dual inhibitory properties reduces Tau hyperphosphorylation and ameliorates disease in models of Alzheimer's disease.

Since Alzheimer's disease (AD) is a complex and multifactorial neuropathology, the discovery of multi-targeted inhibitors has gradually demonstrated greater therapeutic potential. Neurofibrillary tangles (NFTs), the main neuropathologic hallmarks of AD, are mainly associated with hyperphosphorylation of the microtubule-associated protein Tau. The overexpression of GSK3ß and DYRK1A has been recognized as an important contributor to hyperphosphorylation of Tau, leading to the strategy of using dual-targets inhibitors for the treatment of this disorder. ZDWX-12 and ZDWX-25, as harmine derivatives, were found good inhibition on dual targets in our previous study. Here, we firstly evaluated the inhibition effect of Tau hyperphosphorylation using two compounds by HEK293-Tau P301L cell-based model and okadaic acid (OKA)-induced mouse model. We found that ZDWX-25 was more effective than ZDWX-12. Then, based on comprehensively investigations on ZDWX-25 in vitro and in vivo, 1) the capability of ZDWX-25 to show a reduction in phosphorylation of multiple Tau epitopes in OKA-induced neurodegeneration cell models, and 2) the effect of reduction on NFTs by 3xTg-AD mouse model under administration of ZDWX-25, an orally bioavailable, brain-penetrant dual-targets inhibitor with low toxicity. Our data highlight that ZDWX-25 is a promising drug for treating AD.

C-glycosides analogues of the okadaic acid central fragment exert neuroprotection via restoration of PP2A-phosphatase activity: A rational design of potential drugs for Alzheimer's disease targeting tauopathies.

Protein phosphatase 2A (PP2A) is an important Ser/Thr phosphatase that participates in the regulation of multiple cellular processes. This implies that any deficient activity of PP2A is the responsible of severe pathologies. For instance, one of the main histopathological features of Alzheimer's disease is neurofibrillary tangles, which are mainly comprised by hyperphosphorylated forms of tau protein. This altered rate of tau phosphorylation has been correlated with PP2A depression AD patients. With the goal of preventing PP2A inactivation in neurodegeneration scenarios, we have aimed to design, synthesize and evaluate new ligands of PP2A capable of preventing its inhibition. To achieve this goal, the new PP2A ligands present structural similarities with the central fragment C19-C27 of the well-established PP2A inhibitor okadaic acid (OA). Indeed, this central moiety of OA does not exert inhibitory actions. Hence, these compounds lack PP2A-inhibiting structural motifs but, in contrast, compete with PP2A inhibitors, thus recovering phosphatase activity. Proving this hypothesis, most compounds showed a good neuroprotective profile in neurodegeneration models related to PP2A impairment, highlighting derivative 10, named ITH12711, as the most promising one. This compound (1) restored in vitro and cellular PP2A catalytic activity, measured on a phospho-peptide substrate and by western-blot analyses, (2) proved good brain penetration measured by PAMPA, and (3) prevented LPS-induced memory impairment of mice in the object recognition test. Thus, the promising outcomes of the compound 10 validate our rational approach to design new PP2A-activating drugs based on OA central fragment.

Revisiting the multisite phosphorylation that produces the M-phase supershift of key mitotic regulators.

The term M-phase supershift denotes the phosphorylation-dependent substantial increase in the apparent molecular weight of numerous proteins of varied biological functions during M-phase induction. Although the M-phase supershift of multiple key mitotic regulators has been attributed to the multisite phosphorylation catalyzed by the Cdk1/cyclin B/Cks complex, this view is challenged by multiple lines of paradoxical observations. To solve this problem, we reconstituted the M-phase supershift of Xenopus Cdc25C, Myt1, Wee1A, APC3, and Greatwall in Xenopus egg extracts and characterized the supershift-producing phosphorylations. Our results demonstrate that their M-phase supershifts are each due to simultaneous phosphorylation of a considerable portion of S/T/Y residues in a long intrinsically disordered region that is enriched in both S/T residues and S/TP motifs. Although the major mitotic kinases in Xenopus egg extracts, Cdk1, MAPK, Plx1, and RSK2, are able to phosphorylate the five mitotic regulators, they are neither sufficient nor required to produce the M-phase supershift. Accordingly, inhibition of the four major mitotic kinase activities in Xenopus oocytes did not inhibit the M-phase supershift in okadaic acid-induced oocyte maturation. These findings indicate that the M-phase supershift is produced by a previously unrecognized category of mitotic phosphorylation that likely plays important roles in M-phase induction.

Toxic Responses of Different Shellfish Species after Exposure to Prorocentrum lima, a DSP Toxins Producing Dinoflagellate.

Prorocentrum lima is a global benthic dinoflagellate that produces diarrhetic shellfish poisoning (DSP) toxins, which can be ingested by filter-feeding bivalves, and eventually pose a great threat to human health through food chain. After being exposed to P. lima, different bivalves may accumulate various levels of DSP toxins and display different toxic responses. However, the underlying mechanism remains unclear. Here, we found that the content of okadaic acid-equivalents (OA-eq) varied in the digestive glands of the three bivalves including Crassostrea gigas, Mytilus coruscus and Tegillarca granosa after P. lima exposure. The degree of esterification of OA-eq in the three bivalves were opposite to the accumulation of OA-eq. The digestive gland tissues of the three bivalve species were damaged to different degrees. The transcriptional induction of Nrf2 targeted genes such as ABCB1 and GPx indicates the functionality of Nrf2 pathway against DSP toxins in bivalves. The oyster could protect against DSP toxins mainly through ABC transporters and esterification, while the mussel and clam reduce the damage induced by DSP toxins mainly by regulating the expression of antioxidant genes. Our findings may provide some explanations for the difference in toxic response to DSP toxins in different shellfish.

Discovery of novel α-carboline derivatives as glycogen synthase kinase-3ß inhibitors for the treatment of Alzheimer's disease.

Alzheimer's disease (AD) is a chronic and progressive neurodegenerative disease, characterized by irreversible cognitive impairment, memory loss, and behavioral disturbances, ultimately resulting in death. The critical roles of glycogen synthase kinase-3ß (GSK-3ß) in tau pathology have also received considerable attention. Based on molecular docking studies, a series of novel α-carboline derivatives were designed, synthesized, and evaluated as GSK-3ß inhibitors for their various biological activities. Among them, compound ZCH-9 showed the most potent inhibitory activity against GSK-3ß, with an IC50 value of 1.71 ± 0.09 µM. The cytotoxicity assay showed that ZCH-9 had low cytotoxicity toward the cell lines SH-SY5Y, HepG2, and HL-7702. Moreover, Western blot analysis indicated that ZCH-9 effectively inhibited hyperphosphorylation of the tau protein in okadaic acid-treated SH-SY5Y cells. The binding mode between ZCH-9 and GSK-3ß was analyzed and further clarified throughout the molecular dynamics simulations. In general, these results suggested that the α-carboline-based small-molecule compounds could serve as potential candidates targeting GSK-3ß for the treatment of AD.

The early molecular events leading to COFILIN phosphorylation during mouse sperm capacitation are essential for acrosomal exocytosis.

The actin cytoskeleton reorganization during sperm capacitation is essential for the occurrence of acrosomal exocytosis (AR) in several mammalian species. Here, we demonstrate that in mouse sperm, within the first minutes of exposure upon capacitating conditions, the activity of RHOA/C and RAC1 is essential for LIMK1 and COFILIN phosphorylation. However, we observed that the signaling pathway involving RAC1 and PAK4 is the main player in controlling actin polymerization in the sperm head necessary for the occurrence of AR. Moreover, we show that the transient phosphorylation of COFILIN is also influenced by the Slingshot family of protein phosphatases (SSH1). The activity of SSH1 is regulated by the dual action of two pathways. On one hand, RHOA/C and RAC1 activity promotes SSH1 phosphorylation (inactivation). On the other hand, the activating dephosphorylation is driven by okadaic acid-sensitive phosphatases. This regulatory mechanism is independent of the commonly observed activating mechanisms involving PP2B and emerges as a new finely tuned modulation that is, so far, exclusively observed in mouse sperm. However, persistent phosphorylation of COFILIN by SSH1 inhibition or okadaic acid did not altered actin polymerization and the AR. Altogether, our results highlight the role of small GTPases in modulating actin dynamics required for AR.

Okadaic Acid Depuration from the Cockle Cerastoderma edule.

The cockle Cerastoderma edule is a commercially important species in many European Countries. It can accumulate okadaic acid (OA) and other toxins in its group, which makes it unsuitable for human consumption, producing harvesting bans to avoid intoxications. The duration of those bans depends in part on the depuration kinetics of the toxin in this species. In this work, this kinetics was studied by means of fitting different models to depuration data experimentally obtained, using naturally contaminated cockles. Cockles depurated OA faster than most other bivalve species studied. Models that include Michaelis-Menten kinetics describe the depuration better than those using a first order exponential decrease to describe the first (or the only) compartment. One-compartment models were not able to describe the final part of the depuration curve, in which OA was depurated very slowly. Therefore, two-compartment models were needed. Esters were depurated at a much faster rate than the free form of the toxin; however, no significant esterification was detected during the process. The slow depuration rate suggests that other bivalve species could be used as sentinels to monitor cockle populations, but caution should be taken when toxin concentrations are very high.

Multi-omics analysis reveals metabolism of okadaic acid in gut lumen of rat.

Okadaic acid (OA) is an important marine lipophilic phycotoxin with various pathological properties, responsible for diarrheal shellfish poisoning events in human beings over the world. However, to date no mechanism can well explain the toxicity and symptom of OA, even diarrhea. Here, to reveal the toxic mechanism of OA to mammals, we analyzed the metabolism of OA in rat and the effects of OA exposure on the composition and function of gut bacteria using a multi-omics strategy and rRNA high-throughput technology. We found that OA exerted great effects on gut bacteria, mainly featured in heavy fluctuation of dominant genera and significant changes in the mapped bacterial function genes, including not only virulence genes of pathogenic bacteria, but also bacterial metabolism genes. In the feces of the OA-exposed group, we detected dinophysistoxin-2 (DTX-2), lespedezaflavanone F and tolytoxin, suggesting that OA could be transformed into other metabolites like DTX-2. Other metabolic biomarkers such as N-Acetyl-a-neuraminic acid, N,N-dihydroxy-L-tyrosine, nalbuphine, and coproporphyrin I and III were also highly correlated with OA content, which made the toxicity of OA more complicated and confusing. Spearman correlation test demonstrated that Bacteroides and Romboutsia were the genera most related to OA transformation, suggesting that Bacteroides and Romboutsia might play a key role in the complicated and confusing toxicity of OA. In this study, we found for the first time that OA may be converted into other metabolites in gut, especially DTX-2. This finding could not only help to reveal the complex toxicity of OA, but also have important significance for clarifying the transportation, metabolism, and environmental fate of OA in the food chain.

The effects of okadaic acid-treated SH-SY5Y cells on microglia activation and phagocytosis.

The activation of microglia is found to be associated with neurodegenerative disorders including Alzheimer's disease (AD). Several studies have shown that okadaic acid (OA) induced deposition of tau hyperphosphorylation, and subsequent neuronal degeneration, loss of synapses, and memory impairment, all of which resemble the pathology of AD. Although OA is a powerful tool available for mechanisms of the neurotoxicity associated with AD, the exact mechanism underlying the activation of microglial cells remains unrevealed. The aim of this study was to determine the effect of both OA and OA-treated neuroblastoma SH-SY5Y cells on microglial HAPI cell viability, activation, and phagocytosis. The results showed that both OA and OA-treated neurons did not induce any detectable cytotoxicity of microglial cells. Furthermore, incubation with OA-treated SH-SY5Y cells could increase the expression of ionized calcium-binding adapter molecule 1 (Iba1) on microglial HAPI cells. This result indicated that OA may induce microglial activation through the toxicity of neurons. Moreover, we also demonstrated that OA-treated SH-SY5Y cells were engulfed by CD11b/c-labeled microglial HAPI cells, which were abolished after treatment with 10 mM O-phospho- l-serine ( L-SOP) for 30 min before co-culture with OA-treated SH-SY5Y cells, indicating cells experiencing phagocytic activity. We also confirmed that OA treatment for 24 h significantly increased tau hyperphosphorylation at S396 in SH-SY5Y cells. In conclusion, our findings indicate that OA is a potential toxic inducer underlying the role of microglia in AD pathogenesis.

Inhibition of Diarrheal Shellfish Toxins Accumulation in the Mussel Perna viridis by Curcumin and Underlying Mechanisms.

Diarrheal shellfish toxins (DSTs) are among the most widely distributed phytotoxins, and are associated with diarrheal shellfish poisoning (DSP) events in human beings all over the world. Therefore, it is urgent and necessary to identify an effective method for toxin removal in bivalves. In this paper, we found that curcumin (CUR), a phytopolylphenol pigment, can inhibit the accumulation of DSTs (okadaic acid-eq) in the digestive gland of Perna viridis after Prorocentrum lima exposure. qPCR results demonstrated that CUR inhibited the induction of DSTs on the aryl hydrocarbon receptor (AhR), hormone receptor 96 (HR96) and CYP3A4 mRNA, indicating that the CUR-induced reduction in DSTs may be correlated with the inhibition of transcriptional induction of AhR, HR96 and CYP3A4. The histological examination showed that P. lima cells caused severe damage to the digestive gland of P. viridis, and the addition of curcumin effectively alleviated the damage induced by P. lima. In conclusion, our findings provide a potential method for the effective removal of toxins from DST-contaminated shellfish.

Response of fatty acids and lipid metabolism enzymes during accumulation, depuration and esterification of diarrhetic shellfish toxins in mussels (Mytilus galloprovincialis).

Bivalve mollusks accumulate diarrhetic shellfish toxins (DSTs) from toxigenic microalgae, thus posing a threat to human health by acting as a vector of toxins to consumers. In bivalves, free forms of DSTs can be esterified with fatty acids at the C-7 site to form acyl esters (DTX3), presumably a detoxification mechanism for bivalves. However, the effects of esterification of DSTs on fatty acid metabolism in mollusks remain poorly understood. In this study, mussels (Mytilus galloprovincialis) were fed the DST-producing dinoflagellate Prorocentrum lima for 10 days followed by an additional 10-days depuration in filtered seawater to track the variation in quantity and composition of DST acyl esters and fatty acids. A variety of esters of okadaic acid (OA) and dinophysistoxin-1 (DTX1) were mainly formed in the digestive gland (DG), although trace amounts of esters also appeared in muscle tissue. A large relative amount of OA (60%-84%) and DTX1 (80%-92%) was esterified to DTX3 in the visceral mass (referred to as digestive gland, DG), and the major ester acyl chains were C16:0, C16:1, C18:0, C18:1, C20:1 and C20:2. The DG and muscle tissues showed pronounced differences in fatty acid content and composition during both feeding and depuration periods. In the DG, fatty acid content gradually decreased in parallel with increasing accumulation and esterification of DSTs. The decline in fatty acids was accelerated during depuration without food. This reduction in the content of important polyunsaturated fatty acids, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), would lead to a reduction in the nutritional value of mussels. Enzymes involved in lipid metabolism, including acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), lipoprotein lipase (LPL) and hepatic lipase (HL), were actively involved in the metabolism of fatty acids in the DG, whereas their activities were weak in muscle tissue during the feeding period. This study helps to improve the understanding of interactions between the esterification of DSTs and fatty acid dynamics in bivalve mollusks.

In vitro biotransformation of OA-group and PTX-group toxins in visceral and non-visceral tissues of Mytilus chilensis and Ameghinomya antiqua.

Lipophilic marine toxins (LMTs) are made up of multiple groups of toxic analogues, which are characterised by different levels of cellular and toxic action. The most prevalent groups in the southern Pacific zone are: a) okadaic acid group (OA-group) which consists of okadaic acid (OA) and dinophysistoxin-1 (DTX-1); and, b) pectenotoxin-2 (PTX2) group which consists of pectenotoxin-2 (PTX-2). The main objective of our study was to examine in vitro biotransformation of OA-group and PTX-group in the tissues of two endemic species of bivalves from southern Chile; blue mussels (Mytilus chilensis) and clams (Ameghinomya antiqua). The biotransformation processes of both groups were only detected in the digestive glands of both species using LC-MS/MS. The most frequently detected analogues were acyl derivatives (≈2.0 ± 0.1 µg ml-1) for OA-group and PTX-2SA (≈1.4 ± 0.1 µg ml-1) for PTX-group, with a higher percentage of biotransformation for OA-group (p < .001). In addition, simultaneous incubations of the different analogues (OA/PTX-2; DTX-1/PTX-2 and OA/DTX-1/PTX-2) did not show any interaction between the biotransformation processes. These results show that the toxicological variability of endemic species leads to biotransformation of the profile of toxins, so that these new analogues may affect people's health.

Polycystin-1 Inhibits Cell Proliferation through Phosphatase PP2A/B56α.

Autosomal dominant polycystic kidney disease (ADPKD) is associated with a number of cellular defects such as hyperproliferation, apoptosis, and dedifferentiation. Mutations in polycystin-1 (PC1) account for ∼85% of ADPKD. Here, we showed that wild-type (WT) or mutant PC1 composed of the last five transmembrane (TM) domains and the C-terminus (termed PC1-5TMC) inhibits cell proliferation and protein translation, as well as the downstream effectors of mTOR, consistent with previous reports. Knockdown of B56α, a subunit of the protein phosphatase 2A (PP2A) complex, or application of PP2A inhibitor okadaic acid or calyculin A, abolished the inhibitory effect of PC1 and PC1-5TMC on proliferation, indicating that PP2A/B56α mediates the regulation of cell proliferation by PC1. In addition to the phosphorylated S6 and 4EBP1, B56α was also downregulated by PC1 and PC1-5TMC. Furthermore, the downregulation of B56α, which may be mediated by mTOR but not AKT, can account for the dependence of PC1-inhibited proliferation on PP2A.

Morphology and Phylogenetics of Benthic Prorocentrum Species (Dinophyceae) from Tropical Northwestern Australia.

Approximately 70 species of Prorocentrum are known, of which around 30 species are associated with benthic habitats. Some produce okadaic acid (OA), dinophysistoxin (DTX) and their derivatives, which are involved in diarrhetic shellfish poisoning. In this study, we isolated and characterized Prorocentrum concavum and P. malayense from Broome in north Western Australia using light and scanning electron microscopy as well as molecular sequences of large subunit regions of ribosomal DNA, marking the first record of these species from Australian waters. The morphology of the motile cells of P. malayense was similar to P. concavum in the light microscopy, but differed by the smooth thecal surface, the pore pattern and the production of mucous stalk-like structures and a hyaline sheath around the non-motile cells. P. malayense could also be differentiated from other closely related species, P. leve and P. foraminosum, despite the similarity in thecal surface and pore pattern, by its platelet formula and morphologies. We tested the production of OA and DTXs from both species, but found that they did not produce detectable levels of these toxins in the given culturing conditions. This study aids in establishing more effective monitoring of potential harmful algal taxa in Australian waters for aquaculture and recreational purposes.

Simple Diffusion as the Mechanism of Okadaic Acid Uptake by the Mussel Digestive Gland.

Okadaic acid (OA) and other toxins of the diarrheic shellfish poisoning (DSP) group are accumulated and transformed mainly in many bivalves, inside the digestive gland cells. In this work the absorption of okadaic acid by those cells has been studied by supplying the toxin dissolved in water and including it in oil droplets given to primary cell cultures, and by checking if the uptake is saturable and/or energy-dependent. Okadaic acid was found to be absorbed preferentially from the dissolved phase, and the uptake from oil droplets was substantially lower. The process did not require energy and was non-saturable, indicating that it involved a simple diffusion across the cellular membrane. Some apparent saturation was found due to the quick biotransformation of OA to 7-O-acyl esters.

ABC Transporters in Prorocentrum lima and Their Expression Under Different Environmental Conditions Including Okadaic Acid Production.

Prorocentrum lima is a typical benthic toxic dinoflagellate, which can produce phycotoxins such as okadaic acid (OA). In this study, we identified three ABC transporter genes (ABCB1, ABCC1 and ABCG2) and characterized their expression patterns, as well as OA production under different environmental conditions in P. lima. We found that the three ABC transporters all showed high identity with related ABC proteins from other species, and contained classical features of ABC transport proteins. Among them, ABCG2 was a half size transporter. The three ABC transporter genes displayed various expression profiles under different conditions. The high concentration of Cu2+ could up-regulate ABCB1, ABCC1 and ABCG2 transcripts in P. lima, suggesting the potential defensive role of ABC transporters against metal ions in surrounding waters. Cu2+, in some concentration, could induce OA production; meanwhile, tributyltin inhibited OA accumulation. The grazer Artemia salina could induce OA production, and P. lima displayed some toxicity to the grazer, indicating the possibility of OA as an anti-grazing chemical. Collectively, our results revealed intriguing data about OA production and the expression patterns of three ABC transporter genes. However, we could not find any significant correlation between OA production and expression pattern of the three ABC transporters in P. lima. Our results might provide new molecular insights on the defensive responses of P. lima to the surrounding environment.