Comparative studies on the fish-killing activities of Chattonella marina isolated in 1985 and Chattonella antiqua isolated in 2010, and their possible toxic factors.

Chattonella antiqua isolated in 2010 showed extremely more potent fish-killing activities against red sea bream, Japanese horse mackerel, and blue damselfish than those of Chattonella marina isolated in 1985. Chemiluminescence and electron spin resonance (ESR) analyses suggested greater reactive oxygen species (ROS)-producing activity of C. antiqua than that of C. marina. Sodium benzoate, a hydroxyl radical scavenger, significantly suppressed the fish-killing activity of C. antiqua on blue damselfish. The chlorophyll level in the gill tissue of blue damselfish exposed to flagellate cells increased along with the exposure time, and the cell count of gill-associated C. antiqua estimated with chlorophyll level was higher than that of C. marina. These results suggest that the ROS-producing activity and affinity of Chattonella cells to the gill surface may be important factors influencing the fish-killing activity of Chattonella species.

[A Case of Sentinel Lymph Node Biopsy for Male Breast Cancer].

The patient, a 57-year-old man, observed an elastic hard tumor under his left areola. Ultrasonography showed a circular hypoechoic mass that was 1.5 cm in diameter with a moderately indistinct border. Using fine needle aspiration cytology, the tumor was diagnosed as a ductal carcinoma(T1N0M0, stage I ). The patient underwent a mastectomy and a sentinel lymph node biopsy. We omitted radical axillary lymph node dissection because there were no metastases in the sentinel lymph node according to intraoperative frozen section diagnosis. On histopathology, the patient was diagnosed with papillotubular carcinoma. The tumor was positive for ER and negative for PgR, and the HER2 score was 2+. Postoperatively, oral tamoxifen therapy was administered. There have been no signs of recurrence during 4 years of follow-up. We report the sentinel lymph node biopsy results for the 27 cases of male breast carcinoma in Japan.

Thiobencarb herbicide reduces growth, photosynthetic activity, and amount of Rieske iron-sulfur protein in the diatom Thalassiosira pseudonana.

We investigated the effects of the herbicide thiobencarb on the growth, photosynthetic activity, and expression profile of photosynthesis-related proteins in the marine diatom Thalassiosira pseudonana. Growth rate was suppressed by 50% at a thiobencarb concentration of 1.26 mg/L. Growth and photosystem II activity (Fv /Fm ratio) were drastically decreased at 5 mg/L, at which the expression levels of 13 proteins increased significantly and those of 11 proteins decreased significantly. Among these proteins, the level of the Rieske iron-sulfur protein was decreased to less than half of the control level. This protein is an essential component of the cytochrome b6 f complex in the photosynthetic electron transport chain. Although the mechanism by which thiobencarb decreased the Rieske iron-sulfur protein level is not clear, these results suggest that growth was inhibited by interruption of the photosynthetic electron transport chain by thiobencarb.

Application of LDH-release assay to cellular-level evaluation of the toxic potential of harmful algal species.

Lactate dehydrogenase (LDH)-release assay was applied to estimate the toxic potential of harmful algal species at the cellular level. African green monkey kidney (Vero), yellowtail fin epithelia (MJF), and rainbow trout gill (RTgill-W1) cells were used as target cells. A live cell suspension of Karenia mikimotoi (SUO-1) induced the release of LDH from these cell lines, while the activity of another strain, FUK, was much lower. The cell-free culture supernatants and ruptured cell suspensions of both strains of K. mikimotoi were less effective on LDH-release assay. Exposure experiments against abalone and shrimp revealed that SUO-1 showed much stronger lethal effects on these organisms than FUK. Among six phytoplankton species, three species known to be harmful algal species induced the release of LDH to different extents depending on the cell line, whereas the other three species, known to be non-toxic, showed no effects on any cell lines. These results suggest that LDH-release assay is a useful micro-plate assay for estimation of the toxic potential of harmful phytoplankton.

Growth-phase dependent variation in photosynthetic activity and cellular protein expression profile in the harmful raphidophyte Chattonella antiqua.

This study investigated temporal variations in the potential maximum quantum yield of photosystem II (F(v)/F(m) ratio) and growth-phase dependent cellular protein expressions of Chattonella antiqua under laboratory conditions. Despite the culture conditions, significant positive correlations between the F(v)/F(m) ratio and daily growth rate were observed. Threshold F(v)/F(m) ratios associated with positive cell growth were calculated to be >0.44, >0.44, and >0.37, and those associated with active cell growth (growth rate >0.5 div. d(-1)) were >0.58, >0.60, and >0.49 under control culture, low nutrient and intense light conditions, respectively. Proteome profiles obtained by two-dimensional gel electrophoresis (2-DE) indicated that 42 protein spots were differentially expressed at various growth phases of C. antiqua, which indicates changes in cellular physiological status throughout the growth cycle, and suggests that oxygen evolving enhancer 1 and 2-cysteine peroxiredoxin play roles in maintaining the positive growth of C. antiqua.

Transitional reactive oxygen species (ROS) production in fertilized egg embryos of devil stinger (Inimicus japonicus), a marine fish species.

A time-course analysis of reactive oxygen species (ROS) generation in fertilized eggs of the devil stinger (Inimicus japonicus) from 0 h post-fertilization (hpf) to the early larval stage indicated that the ROS level was highest in the 22 hpf embryo, and declined thereafter. Phorbol myristate acetate (PMA) had no effect on ROS generation by the 22 hpf embryo, whereas PMA significantly increased larval ROS generation, suggesting that the ROS generation mechanisms of the 22 hpf embryo and larva are different at least in terms of PMA-responsiveness. Our results suggest the presence of a specific ROS generation system in devil stinger embryo which can be transitionally activated during embryogenesis.

Comparative study on the toxic effects of red tide flagellates Heterocapsa circularisquama and Chattonella marina on the short-necked clam (Ruditapes philippinarum).

Heterocapsa circularisquama showed much higher toxic effects on short-necked clams than Chattonella marina. Clams exposed to H. circularisquama exhibited morphological changes concomitant with an accumulation of mucus-like substances in the gills, a profound reduction in filtration activity, and lysosomal destabilization in hemocytes. Chattonella marina was less effective than H. circularisquama, and Heterocapsa triquetra was almost harmless in all these criteria. These results suggest that H. circularisquama exerted its lethal effect on short-necked clams through gill tissue damage and subsequent induction of physiological stress.

Comparative study on modeccin- and phytohemagglutinin (PHA)-induced secretion of cytokines and nitric oxide (NO) in RAW264.7 cells.

The effects of cytotoxic lectins, modeccin and phytohemagglutinin (PHA) on mouse macrophage cell line RAW264.7 was studied by detecting the induction of inflammatory mediators. Results showed that modeccin induced the release of tumor necrosis factor-α (TNF-α) from RAW264.7 cells with a bell-shape concentration-dependent profile. PHA that showed no significant cytotoxicity on RAW264.7 cells up to 100,000 ng/ml induced much higher level of TNF-α than modeccin. PHA simultaneously induced the secretion of granulocyte colony stimulation factor (G-CSF) from RAW264.7 cells with even much higher level than that of TNF-α, whereas modeccin did not. Furthermore, PHA induced the secretion of nitric oxide (NO) in RAW264.7 cells, while no significant level of NO was detected in the modeccin-treated cells. NH4Cl (a lysomotoropic agent) and cycloheximide (a ribosome inhibitor) strongly inhibited modeccin-induced TNF-α secretion, but no significant inhibitory effects of these reagents on the PHA-induced TNF-α secretion were observed. Contrary to modeccin-induced TNF-α secretion, even slightly increased TNF-α secretion was observed in PHA-treated cells in the presence of 10 mM NH4Cl. In addition, the inhibition profiles of modeccin-induced TNF-α secretion by various kinase inhibitors were different from those of PHA. These results suggested that the action mode of modeccin to stimulate RAW264.7 cells leading to the secretion of inflammatory molecules, including TNF-α, is distinct from that of PHA. On the other hand, significantly increased translocation of activator protein-1 (AP-1), a crucial transcription factor involved in expression of inflammatory molecules, into nucleus was observed in RAW264.7 cells treated with PHA and modeccin.

Superoxide Production by the Red Tide-Producing Chattonella marina Complex (Raphidophyceae) Correlates with Toxicity to Aquacultured Fishes.

The marine raphidophyte Chattonella marina complex forms red tides, causing heavy mortalities of aquacultured fishes in temperate coastal waters worldwide. The mechanism for Chattonella fish mortality remains unresolved. Although several toxic chemicals have been proposed as responsible for fish mortality, the cause is still unclear. In this study, we performed toxicity bioassays with red sea bream and yellowtail. We also measured biological parameters potentially related to ichthyotoxicity, such as cell size, superoxide (O2•-) production, and compositions of fatty acids and sugars, in up to eight Chattonella strains to investigate possible correlations with toxicity. There were significant differences in moribundity rates of fish and in all biological parameters among strains. One strain displayed no ichthyotoxicity even at high cell densities. Strains were categorized into three groups based on cell length, but this classification did not significantly correlate with ichthyotoxicity. O2•- production differed by a factor of more than 13 between strains at the late exponential growth phase. O2•- production was significantly correlated with ichthyotoxicity. Differences in fatty acid and sugar contents were not related to ichthyotoxicity. Our study supports the hypothesis that superoxide can directly or indirectly play an important role in the Chattonella-related mortality of aquacultured fishes.

Mitogenic activity of CEL-I, an N-acetylgalactosamine (GalNAc)-specific C-type lectin, isolated from the marine invertebrate Cucumaria echinata (Holothuroidea).

An N-acetylgalactosamine (GalNAc)-specific Ca(2+)-dependent lectin (C-type lectin), isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), CEL-I, showed potent mitogenic activity toward normal mouse spleen cells. The mitogenic activity of CEL-I, which reached a maximum at 100 microg/ml, was inhibited by GalNAc in a concentration-dependent manner. The mitogenic effect of CEL-I at 10 microg/ml on T cell- enriched splenocytes was at a similar level due to a well-known T cell mitogen, concanavalin A (Con A), at 10 microg/ml. Furthermore, CEL-I evoked a mitogenic response from nude mouse spleen cells, while no significant effects of Con A on this cell population were observed over a wide range of concentrations. These results suggest that CEL-I is a potent mitogenic lectin with the ability to stimulate both T and B cells.

Extracellular secretion of superoxide is regulated by photosynthetic electron transport in the noxious red-tide-forming raphidophyte Chattonella antiqua.

The raphidophyte Chattonella antiqua is a noxious red-tide-forming alga that harms fish culture and the aquatic environment. Chattonella antiqua produces and secretes superoxide anions (O2-), and excessive secretion of O2- into the water has been associated with fish mortality. It is known that strong light stimulates the production of O2- in Chattonella spp. but the mechanism of the light-induced production of O2- remains to be clarified. In the present study, we examined the effects of light on extracellular levels of O2- and photosynthesis in C. antiqua. Extracellular levels of O2- rose during growth under high-intensity light, and the level of O2- was correlated with the photosynthetic parameter qP, which reflects the rate of transport of electrons downstream of photosystem II. The production of O2- was inhibited in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosynthetic electron transport, suggesting that reducing power derived from electron transport might be required for the production of O2-. By contrast, the production of O2- was enhanced in the presence of glycolaldehyde, an inhibitor of the Calvin-Benson cycle, suggesting that the accumulation of NADPH might stimulate the production of O2-. Thus, it is likely that the production of O2- is regulated by photosynthesis in C. antiqua.

Optimization of stereospecific targeting technique for selective production of monoclonal antibodies against native ephrin type-A receptor 2.

High priority stereospecific targeting (SST) featuring selective production of conformation-specific monoclonal antibodies was directed against a native receptor, EphA2 (ephrin type-A receptor 2). A critical point for this technology is selection of sensitized B lymphocytes by antigen-expressing myeloma cells through their B-cell receptors (BCRs). The essential point is that antigens expressed on myeloma cells retain their original three dimensional structures and only these are recognized. Immunization with recombinant plasmid vectors as well as antigen-expressing CHO cells elicits enhanced sensitization of target B lymphocytes generating stereospecific antibodies. More than 24% of hybridoma-positive wells were identified to be cell-ELISA positive, confirming high efficiency. IgG-typed conformation-specific monoclonal antibodies could be also produced by the SST technique. Immunofluorescence analysis confirmed specific binding of sensitized B lymphocytes to antigen-expressing myeloma cells. Furthermore, stereospecific monoclonal antibodies to EphA2 specifically recognized EphA2-expressing cancer cells as demonstrated by Cell-ELISA. In the present study, we were able to develop priority technology for selective production of conformation-specific monoclonal antibodies against an intact receptor EphA2, known to be overexpressed by epithelial tumor cells of multiple cancer types.

Growth-promoting effect of alginate oligosaccharides on a unicellular marine microalga, Nannochloropsis oculata.

Effects of an alginate oligosaccharide mixture (AOM) on Nannochloropsis oculata, a unicellular marine microalga, were investigated. The growth of N. oculata was significantly promoted by AOM in a concentration-dependent manner. The maximum effect was attained at 20 mg/ml, at which the growth rate of the alga became nearly 5 times higher than that of control without AOM. The growth-promoting effect of AOM decreased slightly at 40 mg/ml. Furthermore, the algicidal effect of Cu(2+) was profoundly alleviated by the addition of AOM. These results suggest that AOM is useful for promoting and/or improving the growth of N. oculata.

The possibility of reactive oxygen species (ROS)-independent toxic effects of Cochlodinium polykrikoides on damselfish (Chromis caerulea).

To elucidate the ichthyotoxic mechanism of the harmful dinoflagellate Cochlodinium polykrikoides, a bioassay using damselfish was conducted. After exposure to a live-cell suspension of C. polykrikoides, all the fish were died within 90 min. In the presence of catalase and superoxide dismutase (SOD), no significant reduction in the toxicity of C. polykrikoides on the fish was observed. Furthermore, no significant levels of reactive oxygen species (ROS) were detected in five strains of C. polykrikoides isolated in Japan. Our results support the idea that certain toxic substances, rather than ROS, are mainly responsible for the fish-killing activity of C. polykrikoides.

RNA-Seq Analysis Reveals Genes Related to Photoreception, Nutrient Uptake, and Toxicity in a Noxious Red-Tide Raphidophyte Chattonella antiqua.

Aquaculture industries are under threat from noxious red tides, but harm can be mitigated by precautions such as early harvesting and restricting fish feeding to just before the outbreak of a red tide. Therefore, accurate techniques for forecasting red-tide outbreaks are strongly needed. Omics analyses have the potential to expand our understanding of the eco-physiology of these organisms at the molecular level, and to facilitate identification of molecular markers for forecasting their population dynamics and occurrence of damages to fisheries. Red tides of marine raphidophytes, especially Chattonella species, often extensively harm aquaculture industries in regions with a temperate climate around the world. A red tide of Chattonella tends to develop just after an input of nutrients along the coast. Chattonella displays diurnal vertical migration regulated by a weak blue light, so it photosynthesizes in the surface layer during the daytime and takes up nutrients in the bottom layer during the nighttime. Superoxide produced by Chattonella cells is a strong candidate for the cause of its toxicity to bacteria and fishes. Here we conducted mRNA-seq of Chattonella antiqua to identify genes with functions closely related to the dynamics of the noxious red tide, such as photosynthesis, photoreception, nutrient uptake, and superoxide production. The genes related to photosynthetic pigment biosynthesis and nutrient uptake had high similarity with those of model organisms of plants and algae and other red-tide microalgae. We identified orthologous genes of photoreceptors such as aureochrome (newly five genes), the cryptochrome/photolyase (CRY/PHR) family (6-4PHR, plant CRY or cyclobutane pyrimidine dimer [CPD] Class III, CPD Class II, and CRY-DASH), and phytochrome (four genes), which regulate various physiological processes such as flagellar motion and cell cycle in model organisms. Six orthologous genes of NADPH oxidase, which produces superoxide on the cell membrane, were found and divided into two types: one with 5-6 transmembrane domains and another with 11 transmembrane domains. The present study should open the way for analyzing the eco-physiological features of marine raphidophytes at the molecular level.

Purification and characterization of a novel high molecular weight exotoxin produced by red tide phytoplankton, Alexandrium tamarense.

Our recent studies have demonstrated that the aqueous extract prepared from Alexandrium tamarense, a harmful red tide phytoplankton, showed cytotoxicity on Vero cells. In this study, the toxic substance was purified from the culture supernatant of A. tamarense. Based on the gel-filtration profile, the molecular mass of a purified toxin was estimated to be about 1,000 kDa. On sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, a main band with molecular mass of 1,000 kDa was detected with periodic acid-Schiff (PAS) staining, but no protein bands were detected by Coomassie brilliant blue (CBB) protein staining. Sugar composition analysis of the toxin suggested that the toxin contains galactose, fucose, mannose, N-acetylglucosamine, xylose, and other minor saccharides, whereas no significant levels of amino acids were detected by amino acid analysis. These results suggest that the toxin is a polysaccharide-based compound. The toxin showed cytotoxic effects on various cell lines in a concentration-dependent manner. Among the cell lines tested, U937 cells were the most susceptible to the toxin. In U937 cells treated with the toxin, a typical apoptotic nuclear morphological change and DNA fragmentation were observed. This is the first report demonstrating that a polysaccharide-based toxin isolated from red tide phytoplankton can induce apoptotic cell death.

Photo-induced antibacterial activity of a porphyrin derivative isolated from the harmful dinoflagellate Heterocapsa circularisquama.

The dinoflagellate Heterocapsa circularisquama is highly toxic to bivalves. However, significant toxicity to finfish species has not been reported. We previously found that H. circularisquama has light-dependent haemolytic agents. Purification and chemical structural analyses revealed that the haemolytic agent H2-a is a porphyrin derivative, which exhibits light-dependent cytotoxicity toward tumour cells. To clarify the biological activity of H2-a further, its antibacterial activities against Gram-positive and Gram-negative bacteria were investigated in this study. A fraction (F5) equivalent to H2-a purified from the methanol extract of H. circularisquama showed potent light-dependent bactericidal activity toward Staphylococcus aureus, and the activity was concentration- and light illumination time-dependent; however, Escherichia coli was highly resistant to F5. Electron microscopic observation suggested that F5 induces morphological changes in S. aureus in a light-dependent manner. Further analysis using other bacterial species showed that the Gram-positive bacterium Bacillus subtilis was more sensitive than the Gram-negative bacteria Pseudomonas aeruginosa and Vibrio alginolyticus. These results indicate that F5 is a photo-induced antibacterial agent with relatively higher specificity to Gram-positive bacteria. Iodometric assay suggested that singlet oxygen was generated from light-illuminated F5. Histidine, a specific singlet oxygen scavenger, markedly inhibited the photosensitising antibacterial activity of F5 against S. aureus, suggesting the involvement of singlet oxygen in antibacterial activity. The antibacterial spectrum of F5 was evidently different from that of 5,10,15,20-tetra (N,N,N-trimethylanilinium) porphyrin tetratosylate, a commercially available porphyrin compound with antibacterial activity. Our results demonstrate that H. circularisquama has a novel antibacterial photosensitiser, a porphyrin derivative, with relatively higher specificity to Gram-positive bacteria. To the best of our knowledge, this is the first study to discover a porphyrin derivative with antibacterial activity in marine microalga.

A metabolic profile in Ruditapes philippinarum associated with growth-promoting effects of alginate hydrolysates.

The aim of this study is to demonstrate the growth-promoting effect of alginate hydrolysates (AHs) on the Manila clam Ruditapes philippinarum, and to verify the physiological change occurring within a living R. philippinarum stimulated by AHs. We show that growth of clams was dramatically promoted by supplementing a diet of the diatom Chaetoceros neogracile with AHs at 4 mg/L. Furthermore, metabolomics indicates that each state of starvation, food satiation, and sexual maturation have a characteristic pattern. In the groups given AHs in addition to C. neogracile in particular, excess carbohydrate was actively utilized for the development of reproductive tissue. In contrast, it appeared that clams in the groups given C. neogracile only were actively growing, utilizing their adequate carbohydrate resources. Meanwhile, the unfed groups have slowed growth because of the lack of an energy source. Hence, supplementation of AHs in addition to the algal diet may be an inexpensive way to shorten the rearing period of R. philippinarum. Moreover, metabolomics can evaluate the growth condition of R. philippinarum in a comprehensive way, and this approach is crucially important for not only the development of a mass culture method but also for the conservation of the clam resource in the field.

Intracellular haemolytic agents of Heterocapsa circularisquama exhibit toxic effects on H. circularisquama cells themselves and suppress both cell-mediated haemolytic activity and toxicity to rotifers (Brachionus plicatilis).

A harmful dinoflagellate, Heterocapsa circularisquama, is highly toxic to shellfish and the zooplankton rotifer Brachionus plicatilis. A previous study found that H. circularisquama has both light-dependent and -independent haemolytic agents, which might be responsible for its toxicity. Detailed analysis of the haemolytic activity of H. circularisquama suggested that light-independent haemolytic activity was mediated mainly through intact cells, whereas light-dependent haemolytic activity was mediated by intracellular agents which can be discharged from ruptured cells. Because H. circularisquama showed similar toxicity to rotifers regardless of the light conditions, and because ultrasonic ruptured H. circularisquama cells showed no significant toxicity to rotifers, it was suggested that live cell-mediated light-independent haemolytic activity is a major factor responsible for the observed toxicity to rotifers. Interestingly, the ultrasonic-ruptured cells of H. circularisquama suppressed their own lethal effect on the rotifers. Analysis of samples of the cell contents (supernatant) and cell fragments (precipitate) prepared from the ruptured H. circularisquama cells indicated that the cell contents contain inhibitors for the light-independent cell-mediated haemolytic activity, toxins affecting H. circularisquama cells themselves, as well as light-dependent haemolytic agents. Ethanol extract prepared from H. circularisquama, which is supposed to contain a porphyrin derivative that displays photosensitising haemolytic activity, showed potent toxicity to Chattonella marina, Chattonella antiqua, and Karenia mikimotoi, as well as to H. circularisquama at the concentration range at which no significant toxicity to rotifers was observed. Analysis on a column of Sephadex LH-20 revealed that light-dependent haemolytic activity and inhibitory activity on cell-mediated light-independent haemolytic activity existed in two separate fractions (f-2 and f-3), suggesting that both activities might be derived from common compounds. Our results suggest that the photosensitising haemolytic toxin discharged from ruptured H. circularisquama cells has a relatively broad spectrum of phytoplankton toxicity, and that physical collapse of H. circularisquama cells can lead not only to the disappearance of its own toxicity, but also to mitigation of the effects of other HABs.

Production of superoxide anion and hydrogen peroxide by the red tide dinoflagellate Karenia mikimotoi.

We found that the red tide dinoflagellate Karenia mikimotoi (Gymnodinium mikimotoi) generates reactive oxygen species (ROS). In chemiluminescence analysis using an O2- specific probe, a slightly lower but significant level of O2- was detected in a K. mikimotoi cell suspension as compared to one containing Chattonella marina, a well-known ROS-producing red tide phytoplankton. Interestingly, the chemiluminescence response pattern caused by K. mikimotoi was different from that of C. marina, although both responses were almost completely abolished in the presence of superoxide dismutase (SOD). By fluorescence spectrophotometric analysis, H2O2 was also detected in the K. mikimotoi cell suspension, but the level of H2O2 was approximately 10% of that in the C. marina suspension based on the cell number. No significant increase in O2- generation by K. mikimotoi was observed in the presence of lectins such as concanavalin A (Con A) and wheat germ agglutinin (WGA) or fish mucus substances prepared from the skin and gills of yellowtail, whereas C. marina generated an increased level of O2- in response to these stimuli. Furthermore, O2- production in C. marina, but not in K. mikimotoi, was inhibited by treatment with proteinase K.