Novel Midkine Inhibitor Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma.

BACKGROUND/AIM: Multiple myeloma (MM), the second most common hematological malignancy, is characterized by the accumulation of malignant plasma cells within the bone marrow. Despite various drug classes for MM treatment, it remains incurable, necessitating novel and efficacious agents. This study aims to explore the anti-cancer activity of a midkine inhibitor, iMDK (C21H13FN2O2S), in myeloma cell lines. MATERIALS AND METHODS: This study assessed the antiproliferative activity using the MTT assay. Cell cycle and apoptosis were evaluated using flow cytometry. To further investigate the inhibitory mechanism, western blotting was used to detect cell cycle-related proteins, pro-apoptotic proteins, and anti-apoptotic proteins. RESULTS: iMDK inhibits MM cell proliferation in a dose- and time-dependent manner, inducing cell cycle arrest and apoptosis. The reduction in Cdc20 expression by iMDK treatment leads to G2/M phase cell cycle arrest. Furthermore, iMDK down-regulates anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1, and c-FLIP), thereby activating both intrinsic and extrinsic apoptosis pathways. CONCLUSION: iMDK could be a potential candidate for MM treatment.

Transcriptional regulation of the Japanese flounder Cu,Zn-SOD (Jfsod1) gene in RAW264.7 cells during oxidative stress caused by causative bacteria of edwardsiellosis.

Edwardsiellosis is one of the most important bacterial diseases in fish, sometimes causing extensive economic losses in the aquaculture industry. Our previous studies demonstrated that the Cu,Zn-SOD (sod1) activity has significantly increased in Japanese flounder, Paralichthys olivaceus, hepatopancreas infected by causative bacteria of edwardsiellosis Edwardsiella tarda NUF251. In this study, NUF251 stimulated intracellular superoxide radical production in mouse macrophage RAW264.7 cells, which was reduced by N-acetylcysteine. This result suggests that NUF251 infection causes oxidative stress. To evaluate the regulatory mechanism of Jfsod1 at transcriptional levels under oxidative stress induced by NUF251 infection, we cloned and determined the nucleotide sequence (1124 bp) of the 5'-flanking region of the Jfsod1 gene. The sequence analysis demonstrated that the binding sites for the transcription factors C/EBPα and NF-IL6 involved in the transcriptional regulation of the mammalian sod1 gene existed. We constructed a luciferase reporter system with the 5'-flanking region (-1124/-1) of the Jfsod1 gene, and a highly increased transcriptional activity of the region was observed in NUF251-infected RAW264.7 cells. Further studies using several mutants indicated that deletion of the recognition region of NF-IL6 (-272/-132) resulted in a significant decrease in the transcriptional activity of the Jfsod1 gene in NUF251-infected RAW264.7 cells. In particular, the binding site (-202/-194) for NF-IL6 might play a major role in upregulating the transcriptional activity of the 5'-flanking region of the Jfsod1 gene in response to oxidative stress induced by NUF251 infection. These results could be provided a new insight to understand the pathogenic mechanism of causative bacteria of edwardsiellosis.

Wheat germ agglutinin affinity chromatography enrichment and glyco-proteomic characterization of tetrodotoxin-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes).

Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125 kDa) and X2 variants (88 and 79 kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2ß, were estimated to have two domains, and X2ß is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed.

Synthesis and structure-activity relationship study of aldose reductase inhibiting marine alkaloid lukianol A and its derivatives.

Lukianol A (1a) and its six derivatives 1b-1g, in which each hydroxyl groups of 1a was individually modified, were synthesized via the common intermediate 7a, which was obtained by condensation of the styryl carbazate 10 with p-hydroxyphenylpyruvic acid and subsequent [3,3]-sigmatropic rearrangement. The synthesized lukianol derivatives were evaluated for their ability to inhibit human aldose reductase. 4'-O-methyl (1b) and 4'-dehydroxy (1g) derivatives showed the same level of inhibitory activity as 1a (IC50 2.2 µm), indicating that the 4'-OH is irrelevant for the activity. In contrast, methylation of the hydroxyl group at the 4″'-position (1d) resulted in the loss of activity at a concentration of 10 µm, and masking the hydroxyl group at the 4″-position (1e) caused a 9-fold decrease in activity compared with that of 1b, suggesting that the 4″-OH is an essential group, and the 4″'-OH is required for higher activity.

Midkine inhibitor (iMDK) induces apoptosis of primary effusion lymphoma via G2/M cell cycle arrest.

Primary effusion lymphoma (PEL) is an aggressive B-cell non-Hodgkin lymphoma in immunocompromised individuals such as AIDS patients. PEL shows a poor prognosis (median survival time < 6 months) compared with other AIDS-related lymphomas, and is generally resistant to conventional treatments. Novel drugs for PEL treatment are required. Midkine inhibitor (iMDK) was previously found to suppress midkine protein expression. Interestingly, iMDK suppressed cell proliferation in PEL cell lines in a time- and dose-dependent manner, regardless of midkine gene expression. We examined the mechanism of iMDK on PEL. Importantly, iMDK strongly induced cell cycle arrest at the G2/M phase within 12 h of incubation and suppressed the p-CDK1 protein level, which is associated with the cell cycle checkpoint at G2/M, resulting in mitotic catastrophe with observation of multipolar division. After mitotic catastrophe, iMDK-treated PEL showed apoptosis with caspase-3, - 8, and - 9 activation at 24 h incubation. However, iMDK showed no effects on viral protein-activated signaling pathways such as JAK-STAT, PI3K-Akt and NF-κB, and HHV-8/KSHV gene expression in PEL. These results indicate that iMDK is a novel CDK1 inhibitor and a promising lead compound for PEL chemotherapy treatment.

Generation of Reactive Oxygen Species (ROS) by Harmful Algal Bloom (HAB)-Forming Phytoplankton and Their Potential Impact on Surrounding Living Organisms.

Most marine phytoplankton with relatively high ROS generation rates are categorized as harmful algal bloom (HAB)-forming species, among which Chattonella genera is the highest ROS-producing phytoplankton. In this review, we examined marine microalgae with ROS-producing activities, with focus on Chattonella genera. Several studies suggest that Chattonella produces superoxide via the activities of an enzyme similar to NADPH oxidase located on glycocalyx, a cell surface structure, while hydrogen peroxide is generated inside the cell by different pathways. Additionally, hydroxyl radical has been detected in Chattonella cell suspension. By the physical stimulation, such as passing through between the gill lamellas of fish, the glycocalyx is easily discharged from the flagellate cells and attached on the gill surface, where ROS are continuously produced, which might cause gill tissue damage and fish death. Comparative studies using several strains of Chattonella showed that ROS production rate and ichthyotoxicity of Chattonella is well correlated. Furthermore, significant levels of ROS have been reported in other raphidophytes and dinoflagellates, such as Cochlodinium polykrikoides and Karenia mikimotoi. Chattonella is the most extensively studied phytoplankton in terms of ROS production and its biological functions. Therefore, this review examined the potential ecophysiological roles of extracellular ROS production by marine microalgae in aquatic environment.

Sulfated polysaccharide ascophyllan prevents amyloid fibril formation of human insulin and inhibits amyloid-induced hemolysis and cytotoxicity in PC12 cells.

We found that ascophyllan significantly inhibited the fibrillation of human insulin and was the most effective among the sulfated polysaccharides tested. Gel-filtration analysis suggested that ascophyllan was capable of forming a complex with insulin through a weak interaction. Secondary structure transition from native α-helix to ß-sheet predominant structure of insulin under the fibrillation conditions was suppressed in the presence of ascophyllan. Interestingly, ascophyllan attenuated insulin fibril-induced hemolysis of human erythrocytes. Moreover, ascophyllan attenuated insulin amyloid-induced cytotoxicity on rat pheochromocytoma PC12 cells and reduced the level of intracellular reactive oxygen species. This is the first report indicating that a sulfated polysaccharide, ascophyllan, can suppress the insulin amyloid fibril formation and inhibit the fibril-induced detrimental bioactivities.

Induction of Apoptotic Cell Death in Human Leukemia U937 Cells by C18 Hydroxy Unsaturated Fatty Acid Isolated from Red Alga Tricleocarpa jejuensis.

Our previous studies have found that (±)-(E)-12-hydroxyoctadec-10-enoic acid (HOEA) isolated from the red alga Tricleocarpa jejuensis showed cytotoxic effects on various living organisms including harmful microalgae, Gram-positive bacteria, and mammalian tumor cells. Since natural products with apoptosis-inducing ability can be promising anti-cancer agents, in this study, we investigated the cytotoxic mechanism of HOEA on U937 cells focusing on apoptosis induction. HOEA showed much stronger cytotoxic and cytolytic effects on U937 cells than elaidic acid, which has similar structure but no 12-hydroxy group, suggesting that hydroxy group is important for the cytotoxicity of HOEA. HOEA induced apoptotic nuclear morphological changes, DNA fragmentation, and decrease in mitochondrial membrane potential. Furthermore, time-dependent increase in annexin V+/PI+ cell population in HOEA-treated U937 cells was detected. Among the apoptosis-related reagents, caspase-family inhibitor almost completely inhibited HOEA-induced DNA fragmentation. In the analyses using specific caspase-substrates, extremely high cleavage activity toward caspase-3/7/8 substrate was observed in HOEA-treated U937 cells, and weak activities of caspase-1 and -3 were detected. Analyses using specific caspase inhibitors suggested that caspase-3 and caspase-8 might be predominantly responsible for the cleavage activity. Activation of these caspases were also confirmed by western blotting in which significant levels of cleaved forms of caspase 3, caspase 8, and PARP were detected in HOEA-treated U937 cells. Our results suggest that HOEA is capable of inducing apoptosis in U937 cells in which caspase-3 and caspase-8 might play important roles. Since the cytotoxic effect of HOEA is not strictly specific to tumor cells, development of appropriate drug delivery system for selective tumor targeting is necessary for the clinical applications to reduce the possible side effects.

Cucurbitacin B induces apoptosis of primary effusion lymphoma via disruption of cytoskeletal organization.

BACKGROUND: Primary effusion lymphoma (PEL) is an aggressive B cell non-Hodgkin lymphoma that develops especially in AIDS patients and immunocompromised patients infected with human herpes virus-8 (HHV-8)/Kaposi's sarcoma-associated herpesvirus (KSHV). PEL has a poor prognosis in patients despite conventional chemotherapeutic treatment, and a safe and efficient therapy is required. PURPOSE: To examine the effects on PEL of cucurbitacin B (CuB), a triterpene found in plants of the Cucurbitaceae family that has several anti-cancer activities. STUDY DESIGN: We evaluated the anti-cancer activities of CuB in vitro and in vivo. METHODS: Cell proliferation of PEL cell lines was measured by MTT assay. Cleaved caspases and signaling transduction associated proteins were analyzed by western blotting. Wright and Giemsa staining and immunofluorescence staining were carried out to observe cell morphology. Cell cycles were analyzed by flow cytometry. RT-PCR was performed to detect viral gene expressions. A xenograft mouse model was employed to evaluate the anti-cancer activity of CuB in vivo. RESULTS: CuB inhibited cell proliferation of PEL cell lines (BCBL-1, BC-1, GTO and TY-1) in a dose-dependent manner (0-50 nM) and induced apoptosis of BCBL-1 cells via caspase activation in a dose- and time-dependent manner. In addition, CuB caused cell-shape disruption by inducing actin aggregation and suppressing the p-cofilin level, resulting in BCBL-1 cell arrest at the G2/M phase. In contrast, CuB showed almost no suppression of p-STAT3 and p-Akt activation, which were constitutively activated by KSHV-derived proteins. Furthermore, CuB (0.5 mg/kg) via intraperitoneal injection significantly (p < 0.05) suppressed solid tumor growth in the xenograft mouse model. CONCLUSION: This study suggests that CuB is a promising agent for PEL treatment.

Suppressive effects of sulfated polysaccharide ascophyllan isolated from Ascophyllum nodosum on the production of NO and ROS in LPS-stimulated RAW264.7 cells.

In this study, we found that a sulfated polysaccharide isolated from the brown alga Ascophyllum nodosum, ascophyllan, showed suppressive effects on stimulated RAW264.7 cells. Ascophyllan significantly inhibited expression of inducible nitric oxide synthase mRNA and excessive production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in a dose-dependent manner without affecting the viability of RAW264.7 cells. Ascophyllan also reduced the elevated level of intracellular reactive oxygen species (ROS) in LPS-stimulated RAW264.7 cells. Furthermore, preincubation with ascophyllan resulted in concentration-dependent decrease in ROS production in phorbol 12-myristate-13-acetate-stimulated RAW264.7 cells. Our results suggest that ascophyllan can exhibit anti-inflammatory effects on stimulated macrophages mainly through the attenuation of NO and ROS productions.

Acidic polysaccharides isolated from marine algae inhibit the early step of viral infection.

We examined the effects of various acidic polysaccharides isolated from marine algae on the infection and replication of human immunodeficiency virus type-1 (HIV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), and human T-cell leukemia virus type-1 (HTLV-1). It was found that sulfated fucan polysaccharides, ascophyllan, and two fucoidans derived from different sources significantly inhibited the early step of HIV-1 (R9 and JR-FL) infection, while they did not affect the late step. The alginate oligomer consisted of uronic acids and sulfated-galactan porphyran showed no significant inhibitory effects. In addition, ascophyllan and two fucoidans inhibited the early step of HBV infection in a dose-dependent manner. Furthermore, these polysaccharides inhibited the early step of HCV infection but had no inhibitory effects on HTLV-1 replication. To further examine the specificity of these polysaccharides in viral infections, we used vesicular stomatitis virus (VSV)-G-pseudotyped HIV-1 infection. Ascophyllan, the two fucoidans, and alginate oligomer also potently inhibited VSV-G-pseudotyped HIV-1 infection in HeLa cells. Taken together, these results suggest that the acidic polysaccharides used in this study are capable of inhibiting the early step of viral infections depending on the polysaccharides but not in a strict species-specific manner.

Apolipoprotein E is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages.

Apolipoprotein E (ApoE) belongs to a class of cellular proteins involved in lipid metabolism. ApoE is a polymorphic protein produced primarily in macrophages and astrocytes. Different isoforms of ApoE have been associated with susceptibility to various diseases including Alzheimer's and cardiovascular diseases. ApoE expression has also been found to affect susceptibility to several viral diseases, including Hepatitis C and E, but its effect on the life cycle of HIV-1 remains obscure. In this study, we initially found that HIV-1 infection selectively up-regulated ApoE in human monocyte-derived macrophages (MDMs). Interestingly, ApoE knockdown in MDMs enhanced the production and infectivity of HIV-1, and was associated with increased localization of viral envelope (Env) proteins to the cell surface. Consistent with this, ApoE over-expression in 293T cells suppressed Env expression and viral infectivity, which was also observed with HIV-2 Env, but not with VSV-G Env. Mechanistic studies revealed that the C-terminal region of ApoE was required for its inhibitory effect on HIV-1 Env expression. Moreover, we found that ApoE and Env co-localized in the cells, and ApoE associated with gp160, the precursor form of Env, and that the suppression of Env expression by ApoE was cancelled by the treatment with lysosomal inhibitors. Overall, our study revealed that ApoE is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages that exerts its anti-HIV-1 activity through association with gp160 Env via the C-terminal region, which results in subsequent degradation of gp160 Env in the lysosomes.

HIV-1 Vpr and p21 restrict LINE-1 mobility.

Long interspersed element-1 (LINE-1, L1) composes ∼17% of the human genome. However, genetic interactions between L1 and human immunodeficiency virus type 1 (HIV-1) remain poorly understood. In this study, we found that HIV-1 suppresses L1 retrotransposition. Notably, HIV-1 Vpr strongly inhibited retrotransposition without inhibiting L1 promoter activity. Since Vpr is known to regulate host cell cycle, we examined the possibility whether Vpr suppresses L1 retrotransposition in a cell cycle dependent manner. We showed that the inhibitory effect of a mutant Vpr (H71R), which is unable to arrest the cell cycle, was significantly relieved compared with that of wild-type Vpr, suggesting that Vpr suppresses L1 mobility in a cell cycle dependent manner. Furthermore, a host cell cycle regulator p21Waf1 strongly suppressed L1 retrotransposition. The N-terminal kinase inhibitory domain (KID) of p21 was required for this inhibitory effect. Another KID-containing host cell cycle regulator p27Kip1 also strongly suppressed L1 retrotransposition. We showed that Vpr and p21 coimmunoprecipitated with L1 ORF2p and they suppressed the L1 reverse transcriptase activity in LEAP assay, suggesting that Vpr and p21 inhibit ORF2p-mediated reverse transcription. Altogether, our results suggest that viral and host cell cycle regulatory machinery limit L1 mobility in cultured cells.

Increase in anti-inflammatory activities of radical-degraded porphyrans isolated from discolored nori (Pyropia yezoensis).

The anti-inflammatory properties of porphyrans (D1-D4) obtained from four discolored nori (Pyropia yezoensis) with different growth backgrounds were studied to examine possible variations in their bioactivities. Elution profiles of the porphyrans on Sepharose 4B indicated that D2-porphyran had relatively lower-molecular-size porphyrans than the other porphyrans. Inhibitory activities of the four porphyrans against nitric oxide (NO) and tumor necrosis factor-α (TNF-α) secretion by lipopolysaccharide (LPS)-stimulated RAW264.7 cells were different, whereas no significant differences were observed in the sulfate and anhydrogalactose levels. D2-porphyran showed the highest inhibitory activity against NO and TNF-α secretion by LPS-stimulated RAW264.7 cells, whereas D3- and D4-porphyrans had almost no activity. All porphyrans were efficiently degraded by free radical generated with ascorbate and hydrogen peroxide. The free-radical degradation resulted in a significant increase in the inhibitory activities of the four porphyrans against NO and TNF-α secretion, with varying rates depending on the porphyrans. The ability of D2-porphyran to suppress the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells was also significantly enhanced after degradation. Our results suggest that molecular size is an important factor affecting the anti-inflammatory activity of porphyrans, and radical degradation might be a promising procedure to obtain active low-molecular-size porphyrans.

Inhibitory effect of sulphated polysaccharide porphyran (isolated from Porphyra yezoensis) on RANKL-induced differentiation of RAW264.7 cells into osteoclasts.

Safe and efficient therapeutic agents for bone diseases are required in natural sources. We previously found that edible seaweed-derived polysaccharide porphyran exhibited anti-inflammatory effects through the down regulation of nuclear factor-κB. The aim of this study was to investigate the availability of porphyran as a therapeutic agent for bone diseases. The effects of porphyran on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells were examined. Porphyran suppressed RANKL-induced osteoclast formation in a concentration-dependent manner (6.25-50 µg/ml) without any cytotoxic effects. Furthermore, real-time polymerase chain reaction analyses indicated that porphyran at 50 µg/ml significantly attenuated the RANKL-induced increase in the mRNA levels of osteoclastogenesis-related marker genes such as nuclear factor of activated T cells, tartrate-resistant acid phosphatase, cathepsin K, and matrix metalloproteinase-9 in RAW264.7 cells. To our knowledge, this is the first report showing that edible-seaweed-derived polysaccharide porphyran can suppress RANKL-induced osteoclastogenesis. Our results suggest that porphyran can be used as a safe therapeutic agent to improve osteoclast-related pathological conditions.

Macrophage-stimulating activities of newly isolated complex polysaccharides from Parachlorella kessleri strain KNK-A001.

Our previous studies demonstrated that the microalga Parachlorella kessleri (KNK-A001) has immunostimulatory activities, which were observed as an increase in natural killer (NK) cell activity in mice after intraperitoneal injection or as a protective effect on a virus-infected model shrimp after oral administration. In this study, we attempted to gain insight into the constituent substances of KNK-A001 that are responsible for the immunostimulatory activity. First, we obtained five polysaccharide fractions from KNK-A001 by DEAE anion exchange chromatography. Among the fractions, F5 showed the most potent induction of nitric oxide (NO) secretion in RAW264.7 cells, and both mRNA and protein expression levels of inducible NO synthase (iNOS) were increased in F5-treated RAW264.7 cells. A significant increase in the nuclear translocation of the p65 subunit of nuclear factor-kappa B (NF-κB) was observed in F5-treated RAW264.7 cells. F5 also induced the secretion of tumor necrosis factor (TNF)-α in RAW264.7 cells. Analysis using mitogen-activated protein (MAP) kinase inhibitors suggested that c-Jun N-terminal kinase (JNK) and p38 MAP kinase were mainly involved in F5-induced NO and TNF-α productions. The compositional analysis of F5 identified the main constituents as galactose, glucose, galacturonic acid, and mannose. Gel-filtration analysis suggested that molecular mass of F5 was approximately 400kDa.

Effects of alginate oligomer on the expression of cell cycle- and stress-related genes in Chlamydomonas reinhardtii.

Enzymatically prepared alginate oligomer (AO) promoted the growth of Chlamydomonas reinhardtii in a concentration-dependent manner. AO at 2.5 mg/mL induced increase in expression levels of cyclin A, cyclin B, and cyclin D in C. reinhardtii. CuSO4 at 100 µM suppressed the growth of C. reinhardtiin, and AO at 2.5 mg/mL significantly alleviated the toxicity of CuSO4. Increased intracellular reactive oxygen species level in C. reinhardtii induced by CuSO4 was reduced by AO. After cultivation with CuSO4 at 100 µM, expression levels of ascorbate peroxidase and superoxide dismutase in C. reinhardtii were increased, and AO reduced the increased levels of these enzymes. These results suggest that AO exhibits beneficial effects on C. reinhardtii through influencing the expression of various genes not only at normal growth condition but also under CuSO4 stress.

Protective effect of porphyran isolated from discolored nori (Porphyra yezoensis) on lipopolysaccharide-induced endotoxin shock in mice.

Porphyran, a sulfated polysaccharide, isolated from discolored nori (Porphyra yezoensis) (dc-porphyran) and one fraction (F1) purified from dc-porphyran by DEAE-chromatography showed the protective effects on LPS-induced endotoxin shock in mice. Intraperitoneal (i.p.) treatment with dc-porphyran or F1 (100mg/kg) 60min prior to i.p. injection of LPS (30mg/kg) completely protected mice from LPS lethality. At 10mg/kg concentration, F1 demonstrated more protection than dc-porphyran. Intravenous (i.v.) challenge of LPS, even at 20mg/kg, was more lethal than i.p. administration; i.v. injection of F1 (100mg/kg) with LPS significantly improved the survival rate. However, i.v. dc-porphyran (100mg/kg) produced an even lower survival rate than that of LPS alone. We examined pro-inflammatory mediators such as NO and TNF-α in serum. F1 significantly reduced the levels of these markers. Additionally, F1 significantly decreased the malondialdehyde level in the liver, a marker of oxidative stress, while dc-porphyran had almost no effect. Furthermore, F1 significantly decreased the production of TNF-α and NO in peritoneal exudate cells harvested from LPS-challenged mice, while dc-porphyran treatment showed a lesser decrease. Our results suggest that porphyran isolated from discolored nori, especially F1, is capable of suppressing LPS-induced endotoxin shock in vivo.

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.

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.