Some dinophycean red tide plankton species generate a superoxide scavenging substance

Recent studies indicate that some raphidophycean red tide flagellates produce substances able to scavenge superoxide, whereas there have been no reports on superoxide scavenger production by dinophycean red tide flagellates. In this study, we examined the superoxide-scavenging activity of aqueous extracts from dinophycean red tide flagellates, Gymnodinium spp., Scrippsiella trochoidea, and Karenia sp., by a luminol analog L-012-dependent chemiluminescence (CL) method and an electron spin resonance (ESR)-spin trapping method, and compared the activity to that of raphidophycean red tide flagellates, Chattonella spp., Heterosigma akashiwo, and Fibrocapsa japonica. In the experiment applying the L-012-dependent CL method, only the aqueous extracts from raphidophycean red tide flagellates showed superoxide-scavenging activity. On the other hand, applying the ESR-spin trapping method, we found that the aqueous extracts from dinophycean red tide flagellates also showed superoxide-scavenging activity. This is the first report on the production of a superoxide-scavenger by dinophycean red tide flagellates.     

A discrepancy in superoxide scavenging activity between the ESR-spin trapping method and the luminol chemiluminescence method

In a previous study of ours, the superoxide scavenging activity of aqueous extracts from dinophycean red tide flagellates was detected by an electron spin resonance (ESR)-spin trapping method, but not by an L-012 (luminol analog)-dependent chemiluminescence (CL) method. To investigate the discrepancy between the two methods, the effect of ferric-protein complexes on superoxide scavenging activity was examined. The reduced signal intensity of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO)-OOH due to superoxide dismutase (SOD) did not change with the addition of horseradish peroxidase (HRP), while the reduced CL response due to SOD was restored by the addition of different concentrations of HRP. Since HRP is a ferric-protein complex, the effects of other ferric-protein complexes, catalase and hemoglobin, on the reduced CL response due to SOD were examined, and similar results were obtained. As is the case with SOD, the reduced CL response activity due to an aqueous extract from a raphidophycean red tide flagellate, Chattonella ovata, was also enhanced by HRP, catalase, and hemoglobin. ESR spectra analyzed at 77 K indicated that aqueous extracts of Gymnodinium impudicum and Alexandrium affine, both of which are dinophycean red tide flagellates, contained a ferric-protein complex, and that an extract of C. ovata did not. These results suggest that the presence of such a ferric-protein complex is a causative factor in the discrepancy between the ESR and luminol CL methods when determining superoxide scavenging activity.     

Antioxidant properties of aqueous extracts from red tide plankton cultures

The antioxidant properties of aqueous extracts from the dinophycean flagellates Gymnodinium impudicum and Alexandrium affine and the raphidophycean flagellate Chattonella ovata were examined. An electron spin resonance (ESR)-spin trapping method coupled with steady state kinetic analysis showed that all of the extracts directly scavenge superoxide, and that the superoxide scavenging potential of any of the extracts was comparable to that of L-ascorbic acid. As for hydroxyl radical scavenging, the Fenton reaction and the method of ultraviolet radiation to hydrogen peroxide were used as hydroxyl radical generation systems. All of extracts reduced the level of hydroxyl radicals in both of the systems, indicating that the extracts also directly scavenge hydroxyl radicals. Since the levels of phenolic compounds did not correlate with the antioxidant activities of the extracts, substances other than phenolic compounds also appeared to be attributable to the activities. It is of our interest that the scavenging activities of extract from G. impudicum against superoxide and hydroxyl radicals were increased by heat exposure at 100 degrees C and 200 degrees C respectively. Although the reason for the increased activities of the aqueous extract from G. impudicum is not clear, the heat-resistance of the extract from G. impudicum might make it a desirable antioxidant.     

Antioxidative Activity of Arbutin in a Solution and Liposomal Suspension

Chattonella marina, a raphidophycean flagellate, is one of the most toxic red tide phytoplankton and causes severe damage to fish farming. Recent studies demonstrated that Chattonella sp. generates superoxide (), hydrogen peroxide (H₂ O₂), and hydroxyl radicals (·OH), which may be responsible for the toxicity of C. marina. In this study, we found that other raphidophycean flagellates such as Hetevosigma akashiwo, Otisthodiscus luteus, and Fihrocapsa japonica also produce and H₂O₂ under normal growth condition. Among the flagellate species tested, Chattonella has the highest rates of production of and H₂O₂ as compared on the basis of cell number. This seems to be partly due to differences in their cell sizes, since Chattonella is larger than other flagellate species. The generation of by these flagellate species was also confirmed by a chemiluminescence assay by using 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[l,2-a]pyrazin-3-one (MCLA). All these raphidophycean flagellates inhibited the proliferation of a marine bacterium, Vibrio alginolyticus, in a flagellates/bacteria co-culture system, and their toxic effects were suppressed by the addition of superoxide dismutase (SOD) or catalase. Our results suggest that the generation of reactive oxygen species is a common feature of raphidophycean flagellates.     

Isolation and characterization of light-dependent hemolytic cytotoxin from harmful red tide phytoplankton Chattonella marina

Chattonella marina (C. marina), a raphidophycean flagellate, is a causative organism of red tide, and highly toxic to fish. In this study, we found that the cell-free methanol extract prepared from this flagellate exhibited potent hemolytic activity against rabbit erythrocytes. Interestingly, the hemolytic activity of the extract was absolutely light-dependent, and no hemolytic activity was detected in the dark even at very high concentration. Gel filtration chromatography of the methanol extract on a column of Sephadex LH-20 revealed that the extract contained hemagglutinin as well as hemolytic agents, and the substances responsible for these activities were separately eluted. These results suggest that the hemagglutinating and hemolytic activities were derived from distinct compounds. The hemolytic fraction obtained after gel filtration (F4) caused marked inhibition of the growth of C. marina itself and other species of phytoplanktons. Furthermore, F4 showed a potent cytotoxicity toward various mammalian cultured cell lines including human tumor cells (HeLa cells) in a dose-dependent manner. The cytotoxicity was also light-dependent, and no cytotoxic effect was exhibited in any cell lines tested in the dark. After further purification procedures via preparative thin-layer chromatography and subsequent HPLC, a major hemolytic agent was obtained as highly purified form. Since the methanol extracts prepared from other raphidophycean flagellates such as Heterosigma akashiwo, Olisthodiscus luteus, and Fibrocapsa japonica showed light-dependent hemolytic activity toward rabbit erythrocytes, it was suggested that the light-dependent hemolytic agents commonly exist at least in these raphidophycean flagellates.     

Relationships between dynamics of red tide-causing raphidophycean flagellates and algicidal micro-organisms in the coastal sea of Japan

Temporal fluctuations of algicidal micro-organisms against the red tide causing raphidophycean flagellates Chattonella antiqua (Hada) Ono and Heterosigma akashiwo (Hada) Hada ex Hara et Chihara were investigated using the microplate most probable number (MPN) method in northern Hiroshima Bay and Harima-Nada, the Seto Inland Sea, in 1992 and 1993. In Har-ima-Nada, both flagellates appeared at low levels (< 1 cell mL^?1), and killer micro-organisms against the two flagellates (C-killer for C. antiqua and H-killer for H. akashiwo) also appeared at low densities (< 2 mL^?1). In northern Hiroshima Bay, C. antiqua cells were scarce (< 1 cell mL^?1), and C-killers occurred at a low level (≤ 3.4 mL^?1). Conversely, red tides of H. akashiwo occurred there in June of both years. The dynamics of H-killers revealed a close relationship with that of H. akashiwo populations. H-killers followed the increase of H. akashiwo cells, reached a maximum level after the beginning of decline of H. akashiwo, maintained a high level for at least 1 week after the crash of bloom, and then decreased. C-killers consistently remained at low densities during the period of H. akashiwo red tides in both 1992 and 1993. Hence, algicidal micro-organisms specifically associated with the occurrence and crash of H. akashiwo red tides, and presumably contributed to the rapid termination of the red tides in the coastal seas such as northern Hiroshima Bay.     

Superoxide-scavenging and Tyrosinase-inhibitory Activities of the Extracts of Some Chinese Medicines

The superoxide-scavenging and the tyrosinase-inhibitory activities of 28 kinds of plants used as Chinese medicines were evaluated. Methanol/water extracts were used for the screening tests, and for those which represented high activities, other kinds of extracts were also studied. The extracts of Mallotus japonicus Muell. Arg. scavenged superoxide strongly; the half-inhibiting concentration (IC₅₀) of its 50% methanol/water extract was 10.57 μg of dried material in 1 ml of reaction mixture. The extracts of Fntillaria thunbergii Miq., Carthamus tinctorius L., and Prunus persica (L.) Batsch had strong tyrosinase-inhibitory activities, and the extracts of Scutellariu baicalensis Georgi represented both kinds of activities. These facts suggested that Chinese medicines may be a treasure house of chemical compounds that have the superoxide-scavenging and the tyrosinase-inhibitory activities.     

Detection of Superoxide and Peroxynitrite in Model Systems and Mitochondria by the Luminol Analogue L-012

In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10–100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions.     

Phagotrophic Protist Diversity in the Groundwater of a Karstified Aquifer – Morphological and Molecular Analysis

To clarify the structure of microbial food webs in groundwater, knowledge about the protist diversity and feeding strategies is essential. We applied cultivation‐dependent approaches and molecular methods for further understanding of protist diversity in groundwater. Groundwater was sampled from a karstified aquifer located in the Thuringian Basin (Thuringia, Germany). Cultivable protist abundance estimated up to 8,000 cells/L. Eleven flagellates, 10 naked amoebae, and one ciliate morpho‐species were detected in groundwater enrichment cultures. Most of the flagellates morpho‐species, typically < 10 μm, were sessile or free swimming suspension feeders, e.g., Spumella spp., Monosiga spp., and mobile, surface‐associated forms that grasp biofilms, e.g., Bodo spp. Naked amoebae, typically < 35 μm, that grasp biofilms were represented by, e.g., Vahlkampfia spp., Vannella spp., and Hartmanella spp. The largest fraction of the 18S rRNA gene sequences was affiliated with Spumella‐like Stramenopiles. Besides, also sequences affiliated with fungi and metazoan grazers were detected in clone libraries of the groundwater. We hypothesize that small sized protist species take refuge in the structured surface of the fractures and fissures of the karstified aquifer and mainly feed on biofilm‐associated or suspended bacteria.     

Antioxidant activity of mycosporine-like amino acids isolated from three red macroalgae and one marine lichen

Several standard in vitro assays were performed in order to determine the potential antioxidant capabilities of purified aqueous extracts of the mycosporine-like amino acids (MAAs), porphyra-334 plus shinorine (P-334 + SH), isolated from the red alga Porphyra rosengurttii, asterina-330 plus palythine (AS-330 + PNE), from the red alga Gelidium corneum, shinorine (SH), from the red alga Ahnfeltiopsis devoniensis, and mycosporine -glycine (M-Gly), isolated from the marine lichen Lichina pygmaea. The scavenging potential of hydrosoluble radicals (ABTS⁺ decolorization method), the antioxidant activity in lipid medium (β-carotene/ linoleate bleaching method) and the scavenging capacity of superoxide radicals (pyrogallol autooxidation assay) were evaluated. In terms of scavenging of hydrosoluble radicals, the antioxidant activity of all MAAs studied was dose-dependent and it increased with the alkalinity of the medium (pH 6 to 8.5). M-Gly presented the highest activity in all pH tested; at pH 8.5 its IC₅₀ was 8-fold that of L-ascorbic acid (L-ASC) followed by AS-330 + PNE while P-334 + SH and SH showed scarce activity of scavenging of hydrosoluble free radicals. AS-330 + PNE showed high activity for inhibition of β-carotene oxidation relative to vitamin E and superoxide radical scavenging whilst the activity of P-334 +SH and SH were moderate. According to these results, the potential of MAAs in photoprotection can be considered high due to a double function: (1) UV chemical screening with high efficiency for UVB and UVA regions of the solar spectrum, and (2) their antioxidant capacity.     

Red tide phenomena in Brunei Darussalam — some implications for fisheries

Since the first recorded occurrence in 1976, the coastal waters of Brunei Darussalam have been sporadically subjected to the effects of red tide. The 1976 incident overwhelmed the nation which had no previous experience of red tides. Subsequently, a routine was established to monitor the red tide phenomenon. This included plankton monitoring and shellfish toxicity testing, measures which helped to prevent or mitigate adverse human impacts and economic losses to the fishing industry.The second red tide occurrence was in 1980. However, this time there was greater awareness and with the experience gained from the 1976 incident, the situation was handled effectively.A subsequent incident in 1988 was discovered in a slightly different manner. High densities of Pyrodinium bahamense var. compressum, the causative organism, were found in the stomach contents of a pelagic fish, Sardinella spp. Cats which had fed on contaminated Sardinella suffered sickness and mortality. This led to a ban on the import and sale of Sardinella spp. and the closure of a local shellfish farm for almost a year.Red tide occurrences have had some impact on the fisheries of Brunei Darussalam, mainly due to the need to impose bans on the import, sale and consumption of certain species of fish and shellfish.It is suggested that the effects of red tides on finfish capture fisheries, especially planktivorous fish can be minimised by implementing simple precautions. Shellfish under culture would invariably face losses in the event of a prolonged red tide occurrence and it is proposed that an insurance scheme be implemented to cover such losses.A Red Tide Action Plan is currently in force in Brunei Darussalam. It is a contingency plan for red tide vigilance, monitoring and management, and will serve to reduce the negative impacts of red tides.     

Identification of vibriocidal compounds from medicinal plants using chromatographic fingerprinting

The aim of the present study was to investigate the vibriocidal activity of bark of Syzygium cumini, leaves of Lawsonia inermis, fruits of Terminalia bellerica and identify the bioactive compounds. The vibriocidal activity of plant extracts was determined in aqueous and organic solvents, and the minimum inhibitory concentration (MIC) against Vibrio spp. using the disk diffusion method was established. The chemical constituents of the plant extracts were analysed by thin layer chromatography (TLC), the vibriocidal compounds were determined by TLC-bioautography and were further confirmed by high performance liquid chromatography (HPLC). Significant inhibitory activity was observed with ethanol extract of plants against the test bacteria while less antibacterial activity was observed in acetone, methanol and aqueous extracts. The MIC of the plant extracts ranged between 2.5 and 20 mg/ml. The TLC, TLC-bioautography and HPLC analysis showed that gallic acid and tannin present in ethanol extracts of S. cumini, tannin present in L. inermis and gallic acid present in T. bellerica may be responsible for the vibriocidal activity. S. cumini, L. inermis and T. bellerica can be used for the treatment of gastroenteritis, diarrhoea and cholera diseases after detailed investigations. We also conclude that the plants rich in gallic acid and tannin can be used as an alternative to search for new vibriocidal drugs.     

Flagellate grazing on bacteria in a small dystrophic lake

Fluorescent beads were used to determine the grazing on bacteria by heterotrophic and mixotrophic flagellates in a highly humic (water colour 300–600 mg Pt l^–1) small lake. In summer phagotrophic flagellates constituted about three quarters of the numbers of phytoplankton (including heterotrophic or mixotrophic flagellates) in the uppermost epilimnion. Due to their small size their respective contribution to the biomass was about one quarter. The most important phagotrophic species were Ochromonas sp., and Chromulina spp. which ingested 75–203% of their body carbon per day from bacteria.In view of the abundance and biomass of phagotrophic and mixotrophic flagellates and their very high growth potential, they clearly play a significant role in the food chains of this lake. In addition to providing energy, bacteriovory also represents an important supply of inorganic and organic nutrients under nutrient limiting conditions.     

Free radical scavenging and hepatoprotective actions of Quercus aliena acorn extract against CCl4-induced liver

In the present study, we investigated the protective effect of Quercus aliena acorn extracts against CCl₄-induced hepatotoxicity in rats, and the mechanism underlying the protective effects. Aqueous extracts of Quercus aliena acorn had higher superoxide radical scavenging activity than other types of extracts. The Quercus aliena acorn extracts displayed dose-dependent superoxide radical scavenging activity (IC₅₀ = 4.92 μg/ml), as assayed by the electron spin resonance (ESR) spin-trapping technique. Pretreatment with Quercus aliena acorn extracts reduced the increase in serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels. The hepatoprotective action was confirmed by histological observation. The aqueous extracts reversed CCl₄-induced liver injury and had an antioxidant action in assays of FeCl₂- ascorbic acid induced lipid peroxidation in rats. Expression of cytochrome P450 2E1 (CYP2E1) mRNA, as measured by RT-PCR, was significantly decreased in the livers of Quercus aliena acorn-pretreated rats compared with the livers of the control group. These results suggest that the hepatoprotective effects of Quercus aliena acorn extract are related to its antioxidative activity and effect on the expression of CYP2E1.     

Important Roles of Minor Peroxide Components in the Autoxidation of Methyl Linoleate

Superoxide dismutase (SOD)-like compounds and activators of SOD were screened for in the extracts of fruits, vegetables, and mushrooms by measuring their effects on pyrogallol autoxidation, which is catalyzed by superoxide anion. SOD-like activity was high in aqueous extract of nameko, garlic, broccoli, and oriental lettuce. Ethanolic extracts of onion and watermelon could enhance human SOD activity more than 40%.     

Antifungal potentiality of some plant extracts against Fusarium sp.

Abstract

Aqueous extracts of 46 plants belonging to 32 different families of the plant kingdom were screened for antifungal activity against eight important species of Fusarium viz., Fusarium equiseti, F. moniliforme, F. semitectum, F. graminearum, F. oxysporum, F. proliferatum, F. solani and F. lateritium. The test fungi were isolated from maize, paddy and sorghum seeds collected from Mysore district, Mysore, India. Among the several plants screened only 12 plants have recorded significant antifungal activity. The antifungal activity of aqueous extracts varied among the test pathogens and was compared with that of the synthetic fungicides Blitox, Captan, Dithane M-45 and Thiram. F. proliferatum, which showed high susceptibility for the aqueous extracts, was tested using different solvent extracts viz., petroleum ether, benzene, chloroform, methanol and ethanol extracts of all the 12 plants. The results revealed that these plants could be exploited for ecofriendly management of the diseases caused by the test fungal pathogens and seed biodeterioration during storage.     

In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012

Production of reactive oxygen and nitrogen species (ROS/RNS) is an important part of the inflammatory response, but prolonged elevated levels of ROS/RNS as under chronic inflammation can contribute to the development of disease. Monitoring ROS/RNS in living animals is challenging due to the rapid turnover of ROS/RNS and the limited sensitivity and specificity of ROS/RNS probes. We have explored the use of the chemiluminescent probe L-012 for noninvasive imaging of ROS/RNS production during inflammation in living mice. Various inflammatory conditions were induced, and L-012-dependent luminescence was recorded with an ultrasensitive CCD camera. Strong luminescent signals were observed from different regions of the body corresponding to inflammation. The signal was reduced by administration of the SOD mimetic tempol, the NADPH oxidase inhibitor apocynin, and the inhibitor of nitric oxide synthesis L-NAME, signifying the requirement for the presence of ROS/RNS. Additionally, the L-012 signal was abolished in mice with a mutation in the Ncf1 gene, encoding a protein in the NADPH oxidase complex 2, which generates ROS/RNS during inflammation. In conclusion, L-012 is well distributed in the mouse body and mediates a strong ROS/RNS-dependent luminescent signal in vivo and is useful for monitoring the development and regulation of inflammation in living organisms.     

Characterization of oxygen free radicals generated during vanadate-stimulated NADH oxidation

The oxidation of NADH and accompanying reduction of oxygen to H₂O₂ stimulated by polyvanadate was markedly inhibited by SOD and cytochrome c. The presence of decavanadate, the polymeric form, is necessary for obtaining the microsomal enzyme-catalyzed activity. The accompanying activity of reduction of cytochrome c was found to be SOD-insensitive and therefore does not represent superoxide formation. The reduction of cytochrome c by vanadyl sulfate was also SOD-insensitive. In the presence of H₂O₂ all the forms of vanadate were able to oxidize reduced cytochrome c, which was sensitive to mannitol, tris and also catalase, indicating H₂0₂-dependent generation of hydroxyl radicals. Using ESR and spin trapping technique only hydroxyl radicals, but not superoxide anion radicals, were detected during polyvanadate-dependent NADH oxidation.     

Winter phytoplankton community structure in three shallow temperate lakes during ice cover

The general model of seasonal phytoplankton succession in temperate lakes suggests that winter phytoplankton growth is minimal under ice-cover. However, some studies have found diverse phytoplankton communities during winter. The primary objectives of this study were to determine the species composition and the changes in the winter phytoplankton community structure under the ice. For 2 consecutive winters, phytoplankton samples were collected under ice-cover at 4 sites on 3 lakes in Arrowwood National Wildlife Refuge (ANWR), near Pingree, North Dakota. Ninety taxa were identified and enumerated. Densities of several of these taxa frequently exceeded 10⁶ cells l^–1. The winter phytoplankton communities in these lakes were dominated by flagellates, principally cryptomonads, a synurophyte (Synura uvella), small chrysophytes (Chrysococcus spp., Kephyrion spp.) and a dinoflagellate (Peridinium aciculiferum), as well as non-flagellate microchlorophytes (Monoraphidium spp., Ankistrodesmus spp., and Pseudodictyosphaerium sp.), a cyanobacterium (Gloeocapsa aeruginosa) and centric diatoms (Stephanodiscus minutulus, S. parvus and Cyclotella meneghiniana).