Ichthyotoxicity of Chattonella marina (Raphidophyceae) to damselfish (Acanthochromis polycanthus): the synergistic role of reactive oxygen species and free fatty acids

This investigation aimed to elucidate the relative roles of putative brevetoxins, reactive oxygen species and free fatty acids as the toxic principle of the raphidophyte Chattonella marina, using damselfish as the bioassay. Our investigations on Australian C. marina demonstrated an absence or only very low concentrations of brevetoxin-like compounds by radio-receptor binding assay and liquid chromatography–mass spectroscopy techniques. Chattonella is unique in its ability to produce levels of reactive oxygen species 100 times higher than most other algal species. However, high levels of superoxide on their own were found not to cause fish mortalities. Lipid analysis revealed this raphidophyte to contain high concentrations of the polyunsaturated fatty acid eicosapentaenoic acid (EPA; 18–23% of fatty acids), which has demonstrated toxic properties to marine organisms. Using damselfish as a model organism, we demonstrated that the free fatty acid (FFA) form of EPA produced a mortality and fish behavioural response similar to fish exposed to C. marina cells. This effect was not apparent when fish were exposed to other lipid fractions including a triglyceride containing fish oil, docosahexaenoate-enriched ethyl ester, or pure brevetoxin standards. The presence of superoxide together with low concentrations of EPA accelerated fish mortality rate threefold. We conclude that the enhancement of ichthyotoxicity of EPA in the presence of superoxide can account for the high C. marina fish killing potential.     

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.     

Life cycle,physiology, ecology and red tide occurrences of the fish-killing raphidophyte Chattonella

The marine fish-killing raphidophytes of the genus Chattonella currently consist of five species, i.e. C. antiqua, C. marina, C. minima, C. ovata and C. subsalasa. The distribution of Chattonella species was confirmed in tropical, subtropical and temperate regions in the world accompanying mass mortalities of fishes in nature and in aquaculture. The fish-killing mechanisms are still unclear, but suffocation is the ultimate cause of fish death. Increasing evidence is pointing towards the generation of reactive oxygen species (ROS, e.g. superoxide), which are responsible for the gill tissue injury and mucus production that leads to death of fishes. A taxonomic revision was proposed based on morphology and genetic diversity that Chattonella antiqua and Chattonella ovata should be varieties of Chattonella marina possessing nomenclatural priority. Optimum temperatures for growth are 25 °C for C. antiqua and C. marina, 25–30 °C for C. ovata and 20–30 °C for Chattonella subsalsa. Adequate ranges of salinity for growth were about 20–30 for Chattonella species. Chattonella cells generally divide once a day. Laboratory culture experiments with artificial synthetic medium demonstrated that C. antiqua, C. marina and C. ovata used only Fe chelated with EDTA for growth, although tested diatoms and dinoflagellates used rather many kinds of chelated Fe. A suitable concentration of humic acid supplied with iron also had enhancing effects on the growth of C. antiqua. Diel vertical migration was observed in Chattonella, and the cells reached 7.5 m deep at night in the case of C. antiqua demonstrated by a mesocosm experiment in the Seto Inland Sea. Chattonella species have diplontic life history and have haploid cyst stage in their life cycle. Encystment was observed through formation of pre-encystment small cells after the depletion of nitrogen, and the small cells sink to the sea bottom to complete cyst formation by attachment to the solid surface such as diatom frustules and sand grains. Newly formed cysts are in the state of spontaneous dormancy and they need cold temperature period of four months or longer for maturation (acquisition of germination ability). Cysts germinate in early summer and resultant vegetative cells play an important role as seed populations in blooming in the summer season. However, relatively small part of cyst populations actually germinate from bottom sediments, and success of red tide formation is dependent on the growth in water columns. Since red tides of Chattonella were observed when diatoms were scarce in seawater, diatoms appear to have a key for the predominance of Chattonella in water columns. Diatom resting stages in sediments need light for germination/rejuvenation, whereas Chattonella cysts can germinate even in the dark, implying the selective germination of Chattonella cysts at the sea bottom under calm oceanographic conditions which contribute to bloom formation of Chattonella. As a mechanism of red tide occurrences of Chattonella in coastal sea, “diatom resting hypothesis” was presented. Biological control using diatoms is proposed through the germination/rejuvenation of resting stages suspending from bottom sediments to euphotic layer by sediment perturbation with submarine tractors or fishing trawling gears. Since diatoms have much higher growth rates, and newly joined diatom vegetative cells grow faster and prevent occurrence of Chattonella red tides as a result. As another prevention strategy for Chattonella red tides, algicidal bacteria inhabiting in seaweed beds and seagrass beds are presented. Co-culture of fish and seaweeds in aquaculture areas, and the developments of seaweed- and seagrass-beds would be practical and ultimately environment-friendly strategies for the prevention of harmful red tides of Chattonella by virtue of natural algicidal bacteria supplied from seaweeds and leaves of seagrass.     

Life cycle of Chattonella marina (Raphidophyceae) inferred from analysis of microsatellite marker genotypes

Red tides of Chattonella spp. have caused continuous damage to Japanese aquaculture, however, the life cycle of this organism remains incompletely understood. To further investigate this matter, we assessed genotypes at 14 microsatellite markers in three varieties of Chattonella marina, viz., C. marina var. antiqua, C. marina var. marina, and C. marina var. ovata, to establish whether Chattonella undergoes asexual diploidization or sexual reproduction. After genotyping 287 strains of C. marina, all but one of these strains was shown to be heterozygous for at least some loci, and thus, in the diploid state, suggesting that Chattonella strains undergo sexual reproduction. In addition, we performed single‐cell amplification on ‘small cells’ that are derived from vegetative cells under dark and low‐nutrient conditions. The results indicated the existence of two types of small cells. The ‘Small cell Type 1’ was found to be heterozygous, genotypically equivalent to the vegetative cells, and is therefore diploid. These small cells may change to resting cells (cysts) directly. The ‘Small cell Type 2’ was homozygous at all analyzed loci, suggesting that these small cells are haploid and may be derived by meiosis. As fusion between small cells has previously been observed, the ‘Small cell Type 2’ may be the gamete of Chattonella. We present a construct of the full life cycle of Chattonella marina based on our own and previous results.     

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.     

Iron uptake by the ichthyotoxic Chattonella marina (Raphidophyceae): impact of superoxide generation1

Significant production of superoxide, a known reductant of both inorganic and organically complexed iron(III), occurs in natural systems by both biotic and abiotic pathways. We have investigated the generation of superoxide by Chattonella marina (Subrahman.) Y. Hara et Chihara, a phytoplankton taxon known to produce high levels of this reactive oxygen species, and examined the role of superoxide in the acquisition of iron by this organism. Additionally, a generalized model for iron acquisition by C. marina has been developed, which includes three pathways of iron acquisition from organically complexed iron(III): nondissociative reductive uptake, dissociative reductive uptake, and nonreductive dissociative uptake. The model is shown to be particularly useful in ascertaining the relative importance of these various iron‐uptake pathways as a function of solution parameters including concentration and iron‐binding strength of the organic ligand and superoxide concentration. Our results suggest that superoxide can participate in the C. marina iron‐uptake process when iron is complexed to weak ligands, such as citrate, but plays only a minor role when iron is bound to a strong ligand. It thus appears that facilitation of iron acquisition is not the sole purpose of superoxide production by these organisms.     

Chemotaxonomic survey of sterols and fatty acids in six marine raphidophyte algae

Fatty acid and sterol profiles allowed for clear discrimination betweentheraphidophyte genera Chattonella,Heterosigma, Fibrocapsa andOlisthodiscus, but exhibited little differentiation forindividual Chattonella species(C.marina, C. antiqua and C.subsalsa). Sterol and fatty acid profiles do not support theseparation of Chattonella antiqua and C.marina as distinct species. Ecophenotypic variations in lipidprofiles were also observed between Chattonella strainsfromdifferent geographic locations. Sterol signatures which may be useful aschemotaxonomic markers were: the absence of C₂₇ sterols (cholesteroland 24-dihydrozymosterol) in Heterosigma akashiwo; thepresence of isofucosterol in Chattonella; and theoccurrence of brassicasterol, poriferasterol and fucosterol inOlisthodiscus luteus. High levels of eicosapentaenoic acid(EPA; 17-27% of fatty acids) were present in all raphidophyte species. Lipidcomposition correlated more closely to recent molecular classification ofraphidophytes than carotenoid pigments.     

ANTIOXIDANT ENZYME RESPONSE AND REACTIVE OXYGEN SPECIES PRODUCTION IN MARINE RAPHIDOPHYTES1

Raphidophytes (class Raphidophyceae) produce high levels of reactive oxygen species (ROS), yet little is known regarding cellular scavenging mechanisms needed for protection against these radicals. Enzymatic activities of the antioxidants superoxide dismutase (SOD) and catalase (CAT) were measured in conjunction with the production of superoxide (O₂^??) and hydrogen peroxide (H₂O₂) in batch cultures of five different raphidophytes species during early exponential, late‐exponential, and stationary growth phases. The greatest concentrations of O₂^?? per cell were detected during exponential growth with reduced levels in stationary phases in raphidophytes Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara, Chattonella marina (Subrahman.) Y. Hara et Chihara, and Chattonella antiqua (Hada) Ono (strain 18). Decreasing trends from exponential to stationary phases for SOD activity and H₂O₂ per cell were observed in all species tested. Significant correlations between O₂^?? per cell and SOD activity per cell over growth phase were only observed in three raphidophytes (Heterosigma akashiwo, Chattonella marina, and Chattonella antiqua strain 18), likely due to different cellular locations of externally released O₂^?? radicals and intracellular SOD enzymes measured in this study. CAT activity was greatest at early exponential phase for several raphidophytes, but correlations between H₂O₂ per cell and CAT activity per cell were only observed for Fibrocapsa japonica Toriumi et Takano, Chattonella antiqua (strain 18), and Chattonella subsalsa Biecheler. Our results suggest that SOD and CAT play important protective roles against ROS during exponential growth of several raphidophytes, while other antioxidant pathways may play a larger role for scavenging ROS during later growth.     

RAPHIDOPHYCEAE [CHADEFAUD EX SILVA] SYSTEMATICS AND RAPID IDENTIFICATION: SEQUENCE ANALYSES AND REAL‐TIME PCR ASSAYS1

Species within the class Raphidophyceae were associated with fish kill events in Japanese, European, Canadian, and U.S. coastal waters. Fish mortality was attributable to gill damage with exposure to reactive oxygen species (peroxide, superoxide, and hydroxide radicals), neurotoxins, physical clogging, and hemolytic substances. Morphological identification of these organisms in environmental water samples is difficult, particularly when fixatives are used. Because of this difficulty and the continued global emergence of these species in coastal estuarine waters, we initiated the development and validation of a suite of real‐time polymerase chain reaction (PCR) assays. Sequencing was used to generate complete data sets for nuclear encoded small‐subunit ribosomal RNA (SSU rRNA; 18S); internal transcribed spacers 1 and 2, 5.8S; and plastid encoded SSU rRNA (16S) for confirmed raphidophyte cultures from various geographic locations. Sequences for several Chattonella species (C. antiqua, C. marina, C. ovata, C. subsalsa, and C. verruculosa), Heterosigma akashiwo, and Fibrocapsa japonica were generated and used to design rapid and specific PCR assays for several species including C. verruculosa Hara et Chihara, C. subsalsa Biecheler, the complex comprised of C. marina Hara et Chihara, C. antiqua Ono and C. ovata, H. akashiwo Ono, and F. japonica Toriumi et Takano using appropriate loci. With this comprehensive data set, we were also able to perform phylogenetic analyses to determine the relationship between these species.     

Dynamics of seagrass bed microbial communities in artificial Chattonella blooms: A laboratory microcosm study

The influence of algicidal and growth-inhibiting bacteria in a seagrass (Zostera marina) bed, and their capability of controlling blooms of the fish-killing raphidophyte flagellate, Chattonella antiqua, were examined in laboratory microcosm experiments. Bacterial communities in seawater collected from the seagrass bed and Z. marina biofilm suppressed artificial Chattonella blooms in the presence of their natural competitors and predators. Phylogenetic analysis suggest that considerable numbers of bacteria that suppress Chattonella, including algicidal or growth-inhibiting bacteria isolated from seagrass biofilm and seawater from the seagrass bed, are members of Proteobacteria that can decompose lignocellulosic compounds. A direct comparison of partial 16S rRNA gene sequences (500 bp) revealed that the growth-limiting bacterium (strain ZM101) isolated from Z. marina biofilm belonged to the genus Phaeobacter (Alphaproteobacteria) showed 100% similarity with strains of growth-limiting bacteria isolated from seawater of both the seagrass bed and nearshore region, suggesting that the origin of these growth-limiting bacteria are the seagrass biofilm or seawater surrounding the seagrass bed. This study demonstrates that Chattonella growth-limiting bacteria living on seagrass biofilm and in the adjacent seawater can suppress Chattonella blooms, suggesting the possibility of Chattonella bloom prevention through restoration, protection, or introduction of seagrass in coastal areas.     

Chattonella marina (Raphidophyceae), a potentially toxic alga in the Salton Sea, California

Chattonella marina was found in high abundance in the phytoplankton of the Salton Sea from April to November, 1997. Maximum mean density was over 600 cells ml^–1 mid-lake. It was not detected from January 1997 to March 1997 or in January and February 1998. Transmission electron microscope studies show the typical Chattonella features: a teardrop-shaped nucleus, numerous radially arranged chloroplasts and lack of a cell wall. Gills of fish collected at the height of the Chattonella bloom showed damage to the primary and secondary lamellae and increased numbers of mucus cells. To our knowledge this is the first report of a species of Chattonella in a salt lake.     

Strain variability in fatty acid composition of Chattonella marina (Raphidophyceae) and its relation to differing ichthyotoxicity toward rainbow trout gill cells

Lipid profiles of three strains (Mexico, Australia, Japan) of Chattonella marina (Subrahmanyan) Hara et Chihara were studied under defined growth (phosphate, light, and growth phase) and harvest (intact and ruptured cells) conditions. Triacylglycerol levels were always <2%, sterols <7%, free fatty acids varied between 2 and 33%, and polar lipids were the most abundant lipid class (>51% of total lipids). The major fatty acids in C. marina were palmitic (16:0), eicosapentaenoic (EPA, 20:5ω3), octadecatetraenoic (18:4ω3), myristic (14:0), and palmitoleic (16:1ω7c) acids. Higher levels of EPA were found in ruptured cells (21.4–29.4%) compared to intact cells (8.5–25.3%). In general, Japanese N‐118 C. marina was the highest producer of EPA (14.3–29.4%), and Mexican CMCV‐1 the lowest producer (7.9–27.1%). Algal cultures, free fatty acids from C. marina, and the two aldehydes 2E,4E‐decadienal and 2E,4E‐heptadienal (suspected fatty acid‐derived products) were tested against the rainbow trout fish gill cell line RTgill‐W1. The configuration of fatty acids plays an important role in ichthyotoxicity. Free fatty acid fractions, obtained by base saponification of total lipids from C. marina showed a potent toxicity toward gill cells (median lethal concentration, LC₅₀ (at 1 h) of 0.44 μg · mL^?1 in light conditions, with a complete loss of viability at >3.2 μg · mL^?1). Live cultures of Mexican C. marina were less toxic than Japanese and Australian strains. This difference could be related to differing EPA content, superoxide anion production, and cell fragility. The aldehydes 2E,4E‐decadienal and 2E,4E‐heptadienal also showed high impact on gill cell viability, with LC₅₀ (at 1 h) of 0.34 and 0.36 μg · mL^?1, respectively. Superoxide anion production was highest in Australian strain CMPL01, followed by Japanese N‐118 and Mexican CMCV‐1 strains. Ruptured cells showed higher production of superoxide anion compared to intact cells (e.g., 19 vs. 9.5 pmol · cell^?1 · hr^?1 for CMPL01, respectively). Our results indicate that C. marina is more ichthyotoxic after cell disruption and when switching from dark to light conditions, possibly associated with a higher production of superoxide anion and EPA, which may be quickly oxidized to produce more toxic derivates, such as aldehydes.     

Carnivorous pitcher plant uses free radicals in the digestion of prey

Abstract

A study of the involvement of free oxygen radicals in trapping and digestion of insects by carnivorous plants was the main goal of the present investigation. We showed that the generation of oxygen free radicals by pitcher fluid of Nepenthes is the first step of the digestion process, as seen by EPR spin trapping assay and gel-electrophoresis. The EPR spectrum of N. gracilis fluid in the presence of DMPO spin trap showed the superposition of the hydroxyl radical spin adduct signal and of the ascorbyl radical signal. Catalase addition decreased the generation of hydroxyl radicals showing that hydroxyl radicals are generated from hydrogen peroxide, which can be derived from superoxide radicals. Gel-electrophoresis data showed that myosin, an abundant protein component of insects, can be rapidly broken down by free radicals and protease inhibitors do not inhibit this process. Addition of myoglobin to the pitcher plant fluid decreased the concentration of detectable radicals. Based on these observations, we conclude that oxygen free radicals produced by the pitcher plant aid in the digestion of the insect prey.     

A mutualistic interaction between the bacterium Pseudomonas asplenii and the harmful algal species Chattonella marina (Raphidophyceae)

Several studies on various Chattonella species have reported that bacteria may play an important role in Chattonella bloom initiation, however, no studies have described how these bacteria promote the growth of C. marina. The interaction between C. marina and bacteria was investigated for identification and characterization of potential growth-promoting bacteria. In preliminary tests, the growth promoting effect of Pseudomonas species (25 strains) was investigated and P. asplenii (≥2.27) was determined as a growth-promoting bacteria for both C. marina strains (CCMP 2049 and 2050). This bacterium exerted optimal growth-promoting effects on C. marina, causing an increase in the initial density of P. asplenii to approximately 1 × 10⁷ cells mL⁻¹, which was used as the initial density in this study. To determine whether the growth-promoting activity was direct or indirect, P. asplenii was incubated in the algal media and then a filtrate of this culture was added to both C. marina strains. The P. asplenii filtrate stimulated the growth of C. marina and maintained the growth-promoting effects after high temperature (121 °C for 20 min) and pressure (15 psi) treatment. Thus, P. asplenii is able to promote C. marina growth through the release of a heat-resistant substance, such as inorganic nutrients. A nutrient analysis indicated that this bacterium elevated the phosphate concentration. Interestingly, P. asplenii was unable to survive in phosphate-limited media but could grow in phosphate-limited media incubating C. marina. Moreover, this bacterium could secrete significantly more phosphate in the presence of C. marina (p < 0.0001). These results suggested that P. asplenii and C. marina may have a mutualistic interaction.     

Nitric oxide synthase-like enzyme mediated nitric oxide generation by harmful red tide phytoplankton, Chattonella marina

The unicellular marine phytoplankton Chattonella marina is knownto exhibit potent fish-killing activity. Previous studies havedemonstrated that C. marina produces reactive oxygen species(ROS), and ROS-mediated ichthyotoxic mechanism has been postulated.However, the exact toxic mechanism is still controversial. Inthis study, we obtained evidence that C. marina produces nitricoxide (NO) under normal growth conditions. We utilized chemiluminescence(CL) reaction between NO and luminol–H₂O₂ to detect NOin C. marina cell suspensions. In this assay, significant CLwas observed in C. marina in a cell-number-dependent manner,and this was diminished by the addition of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide(carboxy-PTIO), a specific NO scavenger. The NO generation byC. marina was also confirmed by a spectrophotometric assay basedon the measurement of the diazo-reaction-positive substances(NO_x) and by fluorometric assay using highly specific fluorescentindicator of NO. The NO level in C. marina was significantlydecreased by N^G-nitro-L-arginine methyl ester (L-NAME), a specificNO synthase (NOS) inhibitor. The addition of L-arginine resultedin the increased NO level, whereas NaNO₂ had no effect. Theseresults suggest that a NOS-like enzyme is mainly responsiblefor NO generation in C. marina.     

Effects of Hyperthermic Conditions on the Reactivity of Oxygen Radicals

Generation and reactivity of superoxide (0₂^?) and hydroxyl (OH') radicals in enzymatic and radiolytic systems were investigated over the temperature range from 20^o-50^oC. The generation rate and reaction kinetics of both enzymatically and radiolytically produced superoxide radicals were determined by a cytochrome c reduction assay. For OH' radical reaction studies the degradation of hyaluronic acid was assayed. An increase in temperature leads to a greater reactivity of both radicals, but in the case of an enzymatic source a disproportionate increase in the rate of generation is observed. In the pulse radiolysis system, the reactivity of superoxide radicals was found to be stimulated 15-fold over the temperature range from 20^oC to 60^oC, although the activity of superoxide dismutase was only minimally increased (about 1.6-fold). The results are discussed with respect to the possible importance of active oxygen species to the biological effects of hyperthermia.     

Molecular Analysis of Ribosomal RNA Gene of Red Tide Algae Obtained from the Seto Inland Sea

Eleven clones from five species of the planktonic microalgae, (Chattonella antiqua, Chattonella marina, Heterosigma akashiwo, Alexandrium catenella, and Scrippsiella trochoidea), which were collected from the Seto Inland Sea in Japan and from Thailand, were subjected to nucleotide sequence analysis of the D1/D2 domain of the large subunit (LSU) of their ribosomal RNA genes. After amplification by polymerase chain reaction using degenerated primers, whole-nucleotide sequences for the D1/D2 domains of the LSU rRNA gene of 11 microalgae were analyzed. Phylogenic tree analysis using these nucleotide sequences showed each species located in a cluster corresponding to its morphological classification. The nucleotide sequence data for Chattonella spp. suggest that multiple clones of both Chattonella antiqua and Chattonella marina are present in the Seto Inland Sea and that red tide blooms of Chattonella spp. in different years may have contained different clones. Received September 6, 1999; accepted December 16, 1999.     

Localization of the Site of Oxygen Radical Generation inside the Complex I of Heart and Nonsynaptic Brain Mammalian Mitochondria

Mitochondrial production of oxygen radicals seems to be involved in many diseases and aging. Recent studies clearly showed that a substantial part of the free radical generation of rodent mitochondria comes from complex I. It is thus important to further localize the free radical generator site within this respiratory complex. In this study, superoxide production by heart and nonsynaptic brain submitochondrial particles from up to seven mammalian species, showing different longevities, were studied under different conditions. The results, taking together, show that rotenone stimulates NADH-supported superoxide generation, confirming that complex I is a source of oxygen radicals in mammals, in general. The rotenone-stimulated NADH-supported superoxide production of the heart and nonsynaptic brain mammalian submitochondrial particles was inhibited both by p-chloromercuribenzoate and by ethoxyformic anhydride. These results localize the complex I oxygen radical generator between the ferricyanide and the ubiquinone reduction site, making iron—sulfur centers possible candidates, although unstable semiquinones can not be discarded. The results also indicate that the previously described inverse correlation between rates of mitochondrial oxygen radical generation and mammalian longevity operates through mechanisms dependent on the presence of intact functional mitochondria.     

Cross-talk of NO,superoxide and molecular oxygen,a majesty of aerobic life

Because nitric oxide (NO) reacts with various molecules, such as hemeproteins, superoxide and thiols including glutathione (GSH) and cysteine residues in proteins, biological effects and metabolic fate of this gaseous radical are affected by these reactants. Although the lifetime of NO is short particularly under air atmospheric conditions (where the oxygen tension is unphysiologically high), it increases significantly under physiologically low oxygen concentrations. Because oxygen tensions in human body differ from one tissue to another and change depending on their metabolism, biological activity of NO in various tissues might be affected by local oxygen tensions. To elucidate the role of NO and related radicals in the regulation of circulation and energy metabolism, their effects on arterial resistance and energy metabolism in mitochondria, mammalian cells and enteric bacteria were studied under different oxygen tensions. Kinetic analysis revealed that NO-dependent generation of cGMP in resistance arteries and their relaxation were strongly enhanced by lowering oxygen tensions in the medium. NO reversibly suppressed the respiration and ATP synthesis of isolated mitochondria and intact cells particularly under low oxygen tensions. Kinetic analysis revealed that cross-talk between NO and superoxide generated in and around endothelial cells regulates arterial resistance particularly under physiologically low oxygen tensions. NO also inhibited the respiration and ATP synthesis of E. coli particularly under low oxygen tensions. Because concentrations of NO and H⁺ in gastric juice are high, most ingested bacteria are effectively killed in the stomach. However, the inhibitory effects of NO on the respiration and ATP synthesis of H. pylori are extremely small. Kinetic analysis revealed that H. pylori generates the superoxide radical thereby inhibiting the bactericidal action of NO in gastric juice. Based on such observations, critical roles of the cross-talk of NO, superoxide and molecular oxygen in the regulation of energy metabolism and survival of aerobic and microaerophilic organisms are discussed.     

Differences in photoprotective pigment production between Japanese and Australian strains of Chattonella marina (Raphidophyceae)

Previous studies have shown that isolates of Chattonella marina from Australia and Japan exhibit differences in tolerance to high intensities of visible light. Here we show that the Australian strain of C. marina produces around five times more UV-absorbing mycosporine amino acids (MAAs) than the Japanese strain. This corresponds with 66% increased growth by the Australian strain under UVB exposure compared to no UV exposure. The MAA mycosporine-glycine, which reportedly acts as an antioxidant, was found in high quantity (110 fg cell^-1) in the Australian but was absent in the Japanese strain. In contrast, changes in the concentration of violaxanthin and zeaxanthin per cell were 4.7-4.8 times greater in the Japanese relative to the Australian strain suggesting that the Japanese strain uses a xanthophyll cycle to moderate inhibition by high photosynthetically active radiation (PAR) irradiance. Increased MAA production under high irradiance was also observed in other Australian strains of Chattonella, but not noted in other Japanese strains suggesting ecophenotypic adaptation due to differing environmental conditions.