Chemistry, biology, and toxicology of domoic acid and its isomers.

The causative agent of toxicity in cultured mussels from a localized area of eastern Prince Edward Island has been identified as domoic acid, a neuroexcitatory amino acid. The toxin was isolated by a number of different bioassay-directed separation techniques including high-performance liquid chromatography (HPLC), high-voltage paper electrophoresis (HVPE), and ion-exchange chromatography, and characterized by a number of spectroscopic techniques including ultraviolet, infrared, mass spectrometry, and nuclear magnetic resonance. The isolation and purification methods are described in detail and some new analytical data for domoic acid are reported. A plankton bloom at the time of the outbreak gave positive mouse bioassays and consisted almost entirely of the pennate diatom, Nitzschia pungens f. multiseries. A positive correlation was found between the number of N. pungens cells and the concentration of domoic acid in the plankton. N. pungens f. multiseries isolated from Cardigan Bay produced domoic acid de novo during stationary phase culture at levels (1 to 10 pg/cell) comparable to values estimated for N. pungens in the plankton samples. We conclude that N. pungens was the major source of the domoic acid in toxic mussels in eastern Prince Edward Island. The recurrence, in November 1988, of a monospecific bloom of N. pungens and the presence of domoic acid in plankton and mussels reinforces this conclusion.

Mussel culture and the accumulation of domoic acid.

The cultured mussel industry of Prince Edward Island had never experienced a toxic disease problem until November of 1987. With the successful use of the long-line culture system, the yearly production of fresh mussels to the gourmet food market had risen to close to 3.2 million pounds (1.46 million kg) of product. The physiology of this sessile bivalve and its method of feeding and location in the estuary leave it prone to the accumulation of a widely distributed biotoxin. Eastern Prince Edward Island became the epicentre of domoic acid-intoxicated mussels as early as 10 November 1987 (retrospective samples) during an intense bloom of the diatom Nitzschia. Mussels were able to accumulate large amounts of the domoic acid with little effect on their own well-being. Despite being in low water temperatures (below 4 degrees C) and under thick ice cover, the levels of the toxin decreased and were undetectable in about 6 weeks. The following year the toxin was detected in much smaller amounts, and the levels of toxin accumulation demonstrated a variable lag time with the increase in concentration of Nitzschia available in the water column. The sales of Prince Edward Island cultured mussels have rebounded to about 140% of the pre-domoic acid crisis.

Accumulation in chick livers of 9-cis versus all-trans beta-carotene.

Previous results indicated that beta-carotene, as present within the unicellular alga Dunaliella, accumulated in the liver of chicks to a much higher degree than did synthetic all-trans beta-carotene. Because the Dunaliella beta-carotene is composed of an approximately equal mixture of two stereoisomers, 9-cis and all-trans, we tested whether a similar mixture of purified beta-carotene isomers would yield comparable results. Indeed, analysis of liver following 2 wk of feeding show a 10-fold higher accumulation of beta-carotene in the chicks fed a mixture of purified 9-cis and all-trans beta-carotene as compared with those fed only all-trans beta-carotene. The ratio of 9-cis to all-trans beta-carotene in the liver was about three times higher than that present in the beta-carotene fed to the chicks. Retinol and retinyl esters accumulated to a similar degree in the livers of chicks given all treatments.

Detection and characterization of hyperfine-shifted resonances in the proton nuclear magnetic resonance spectrum of Anabaena 7120 ferredoxin at high magnetic fields.

This paper presents previously unobserved signals in the 1H NMR spectra of oxidized and reduced [2Fe-2S]-ferredoxin from Anabaena 7120 detected at 400, 500, and 600 MHz. The signals shifted to low field exhibited longitudinal relaxation (T1) values in the range of 100-400 microseconds and line widths in the range of 1-10 kHz (at 400 MHz), and the chemical shifts of all signals showed strong temperature dependence. Although the line widths were smaller at lower magnetic fields, the resolution was better at higher magnetic fields. In the oxidized state, a broad signal was detected at 37 ppm, which corresponds to at least 6 protons, and whose chemical shift exhibits positive temperature dependence. This signal also was found in oxidized ferredoxin reconstituted in 2H2O, which excludes the signal as arising from solvent-exchangeable amide protons. In the reduced state, four signals detected between 90 and 140 ppm exhibited negative temperature dependence. These consisted of two pairs of signals, each pair having one component with half the linewidth of the other. On the basis of their chemical shifts, linewidths, longitudinal relaxation properties, and temperature dependence we assigned these resonances to four of the beta hydrogens of the ligated cysteines. Two solvent-exchangeable hyperfine-shifted signals were found in the reduced state; these are located upfield of the diamagnetic region. The low-field hyperfine resonances of half-reduced ferredoxin in the presence of sodium dithionite showed a self electron transfer exchange rate that was slow on the NMR scale as observed earlier (Chan, T., and Markley, J. L. (1983) Biochemistry 22, 5982-5987), but the exchange rate was accelerated in the presence of methyl viologen.

Effects of Phaeodactylum tricornutum and Dunaliella tertiolecta extracts on the central nervous system.

We report the effects of aqueous extracts of the microalgae Dunaliella tertiolecta Butcher (Chlorophyceae) and Phaeodactylum tricornutum Bohlin (Bacillariophyceae) on oxotremorin-induced cholinergic symptoms, amphetamine-induced hypermotility, pentylenetetrazole-induced convulsions, and Rota-rod test performance. Both extracts significantly reduced rectal temperature, spontaneous motor activity, and time on the Rota-rod and increased the number of mice falling off the rod before 180 s, but neither protected against pentylenetetrazole-induced convulsions or against oxotremorin-induced tremor, salivation, and diarrhoea.

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 1. Sequence-specific hydrogen-1 resonance assignments and secondary structure in solution of the oxidized form.

Complete sequence-specific assignments were determined for the diamagnetic 1H resonances from Anabaena 7120 ferredoxin (Mr = 11,000). A novel assignment procedure was followed whose first step was the identification of the 13C spin systems of the amino acids by a 13C(13C) double quantum correlation experiment [Oh, B.-H., Westler, M. W., Darba, P., & Markley, J. L. (1988) Science 240, 908-911]. Then, the 1H spin systems of the amino acids were identified from the 13C spin systems by means of direct and relayed 1H(13C) single-bond correlations [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085]. The sequential resonance assignments were based mainly on conventional interresidue 1H alpha i-1HNi + 1 NOE connectivities. Resonances from 18 residues were not resolved in two-dimensional 1H NMR spectra. When these residues were mapped onto the X-ray crystal structure of the homologous ferredoxin from Spirulina platensis [Fukuyama, K., Hase, T., Matsumoto, S., Tsukihara, T., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., & Matsubara, H. (1980) Nature 286, 522-524], it was found that they correspond to amino acids close to the paramagnetic 2Fe.2S* cluster. Cross peaks in two-dimensional homonuclear 1H NMR spectra were not observed for any protons closer than about 7.8 A to both iron atoms. Secondary structural features identified in solution include two antiparallel beta-sheets, one parallel beta-sheet, and one alpha-helix.

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 2. Sequence-specific carbon-13 and nitrogen-15 resonance assignments of the oxidized form.

Multinuclear two-dimensional NMR techniques were used to assign nearly all diamagnetic 13C and 15N resonances of the plant-type 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120. Since a 13C spin system directed strategy had been used to identify the 1H spin systems [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085], the sequence-specific 1H assignments [Oh, B.-H., & Markley, J. L. (1990) Biochemistry (first paper of three in this issue)] also provided sequence-specific 13C assignments. Several resonances from 1H-13C groups were assigned independently of the 1H assignments by considering the distances between these nuclei and the paramagnetic 2Fe.2S* center. A 13C-15N correlation data set was used to assign additional carbonyl carbons and to analyze overlapping regions of the 13C-13C correlation spectrum. Sequence-specific assignments of backbone and side-chain nitrogens were based on 1H-15N and 13C-15N correlations obtained from various two-dimensional NMR experiments.

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 3. Detection and characterization of hyperfine-shifted nitrogen-15 and hydrogen-1 resonances of the oxidized form.

All the nitrogen signals from the amino acid side chains and 80 of the total of 98 backbone nitrogen signals of the oxidized form of the 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120 were assigned by means of a series of heteronuclear two-dimensional experiments [Oh, B.-H. Mooberry, E. S., & Markley, J. L. (1990) Biochemistry (second paper of three in this issue )]. Two additional nitrogen signals were observed in the one-dimensional 15N NMR spectrum and classified as backbone amide resonances from residues whose proton resonances experience paramagnetic broadening. The one-dimensional 15N NMR spectrum shows nine resonances that are hyperfine shifted and broadened. From this inventory of diamagnetic nitrogen signals and the available X-ray coordinates of a related ferredoxin [Tsukihara, T., Fukuyama, K., Nakamura, M., Katsube, Y., Tanaka, N., Kakudo, M., Wada, K., Hase, T., & Matsubara, H. (1981) J. Biochem. 90, 1763-1773], the resolved hyperfine-shifted 15N peaks were attributed to backbone amide nitrogens of the nine amino acids that share electrons with the 2Fe.2S* center or to backbone amide nitrogens of two other amino acids that are close to the 2Fe.2S* center. The seven 15N signals that are missing and unaccounted for probably are buried under the envelope of amide signals. 1H NMR signals from all the amide protons directly bonded to the seven missing and nine hyperfine-shifted nitrogens were too broad to be resolved in conventional 2D NMR spectra.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioprotection of hemopoiesis conferred by aqueous extract from chlorococcal algae (Ivastimul) administered to mice before irradiation.

In experiments on mice, single i.p. injections of aqueous extract from chlorococcal freshwater algae (Ivastimul) were found to increase their radioresistance. During the same period the number of spleen colony-forming units (CFUs) in the bone marrow and spleen and their proliferation activity increased. The amount of granulocyte-macrophage colony-forming cells (GM-CFC) in the bone marrow grows and the colony-stimulating activity (CSA) of the blood serum of mice is elevated at an early period after injection of the substance. The recovery of the CFUs and GM-CFC pools in femoral bone marrow after irradiation proceeds at a faster rate in Ivastimul-treated animals than in control groups. The activation of the pool of hemopoietic stem cells and stimulatory effects of Ivastimul on granulocytopoiesis act favorably on the repair of radiation damage and on increased percentage of animals surviving lethal doses of gamma radiation.

Kinetoplastid flagellates: surface-reactive carbohydrates detected by fluorescein-conjugated lectins.

Membrane-associated carbohydrate residues of 3 isolates of Leishmania derived from etiological agents of visceral leishmaniasis (VL), postkala-azar dermal leishmaniasis (PKDL), and cutaneous leishmaniasis (CL), as well as 2 other nonpathogenic insect gut kinetoplastid flagellates, Bodo sp. and Herpetomonas sp., were characterized with the aid of 8 fluorescein-conjugated lectins. Four lectins, concanavalin A, Dolichos biflorus, phytohemagglutinin P, Ricinus communis agglutinin, bound to all kinetoplastid flagellates at different concentrations. All Leishmania promastigotes showed reactions with Ulex agglutinin. Although these lectins were bound to all kinetoplastids, the site and intensity of binding was different. All skin-dwelling Leishmania parasites, viz., Leishmania donovani of PKDL and Leishmania tropica of CL showed unique selectivity toward peanut agglutinin (PNA), soybean agglutinin, and wheatgerm agglutinin (WGA). More interestingly, Herpetomonas showed positive fluorescence with PNA and WGA, whereas Bodo was negative. The results demonstrated that no lectin could distinguish between the pathogenic and nonpathogenic status of kinetoplastid flagellates. Moreover, the antigenic (carbohydrate) profiles of Herpetomonas corresponded more closely to those of L. tropica, whereas Bodo shared some common lectin receptors with L. donovani of VL.

Time trend of PCB concentrations in surface sediments from a hypertrophic, macroalgae populated area of the lagoon of Venice.

The concentrations of polychlorinated biphenyls have been determined over a period of 1 year in surface sediments sampled monthly and semi-monthly in the lagoon of Venice. At the sampling station, due to the poor water circulation and an over-abundance of nutrients, macroalgae thrive in spring-summer and standing crops greater than 12 kg m-2 were determined. As a consequence of rapid algal decomposition, sediment PCB concentrations were observed to increase. In particular, on occasions when anoxia occurred at the end of July and all the biomass had decayed, PCB concentrations increased by more than one order of magnitude. In March, when macroalgae started to grow, concentrations were 13 ng g-1 (dry wt); in July-August they exceeded 100 ng g-1. The sediment PCB concentrations were restored to the previous values after algal biomass became negligible, in November. Concentrations of PCBs in young algae fronds were 27 +/- 12 ng g-1, and 121 +/- 4 ng g-1 in older fronds.

Composiçäo primária dos polissacarídeos de Microcystis aeruginosa kutz: emend; Elenkin (Cyanophyceae) / Primary composition of the polysaccharides of Microcystis aeruginosa kutz: emend; Elenkin (Cyanophyceae)

A Microcystis aeruginosa é uma cianofícea característica de ambientes continentais eutrofizados, onde frequentemente constitui floraçöes responsáveis pela liberaçäo de endotoxinas que podem ser nocivas para o homem e para organismos aquáticos. Os polissacarídeos produzidos por essa alga, cuja composiçäo primária, determinada através de cromatografia gasosa, revela a presença de monômetros facilmente metabolizáveis por açäo bacteriana, juntamente com dados sobre a composiçäo química das células, em termos de conteúdo proteico e de carboidratos, sugerem que a cianofícea em questäo possa ser utilizada como fonte indireta de energia pelas espécies zooplanctônicas resistentes à toxicidade algal

Seasonal variation of Sirodotia delicatula skuja (Rhodophyta, Batrachospermaceae) in a small stream from Säo Paulo state, southeastern Brazil

A variaçäo sazonal de Sirodotia delicatula foi estudada num riacho de primeira ordem do Município de Juquitiba, Estado de Säo Paulo, de abril de 1987 a abril de 1988. A sazonalidade foi avaliada em termos de freqüência, porcentagem de cobertura e fenologia, bem como sua relaçäo com algumas variáveis físicas e químicas (velocidade da correnteza, temperatura da água, turbidez, largura, profundidade, vazäo, pH, CO2, O2, saturaçäo de O2, cor da água e duraçäo do dia). A populaçäo da alga näo revelou variaçäo significativa para os valores de freqüência, a qual apresentou valor máximo em junho (33,6%) e mínimo em setembro (25,2%) com pequena variaçäo durante o período de estudo. A porcentagem de cobertura näo apresentou mudança, situando-se no índice 2 (1-10%) da escala de cobertura. Todas as plantas analisadas eram monóicas e estavam férteis. A maioria (90-100%) apresentou-se no estado carposporofítico. A germinaçäo de carpósporos "in situ", reusltando no desenvolvimento de plantas microscópicas de "Chantransia" epífitas sobre o gametófito, foi observada numa porcentagem variável de plantas (5-20%) durante os meses de abril, maio, setembro, outubro e novembro de 1987 e abril de 1988. A alternância entre gametófito e fase "Chantransia", típica dos representantes de Batrachospermales, näo foi observada. A freqüência correlacionou-se positivamente com a profundidade (5 = 0,55, p < 0,05), sugerindo relaçäo com a luminosidade. A relativa estabilidade da populaçäo de S. delicatula parece ...

Biliprotein light-harvesting strategies, phycoerythrin 566.

A series of experiments on the light-harvesting properties of the cryptomonad biliprotein phycoerythrin 566 has been carried out on purified protein isolated from Cryptomonas ovata. Although this pigment has an absorption maximum at 566 nm, a property very close to that of other phycoerythrins, it was found to have a totally unique set of chromophores. The chromophores (bilins) responsible for its absorption spectrum were analyzed by a number of approaches. Chromophore-containing peptides were produced by trypsin treatment and purified in order to isolate the individual peptide-bound bilins free of overlapping absorption. These chromopeptides, after comparison with appropriate controls, showed that three spectrally distinct bilins occurred on the purified oligomeric protein. Two of the bilins were the well-known phycoerythrobilin and cryptoviolin, but the third was previously undiscovered and had an absorption spectrum between that of cryptoviolin and phycocyanobilin. Since the spectral diversity of the three bilins was fully maintained in solvents that minimize the effects of apoprotein on the spectra of the bilins, it is likely that the three bilins are also structurally dissimilar. The alpha and beta subunits, which constitute the protein, were separated by ion-exchange chromatography, and the new bilin was found to be the sole chromophore on the alpha subunit. It was also found that at least two alpha subunits could be separated and they both had this unusual bilin (cryptobilin 596). The beta subunit, therefore, contained both phycoerythrobilin and cryptoviolin. On the basis of the spectra of the three chromopeptides, the absorption spectrum of the protein was modeled using the known absorptivities of cryptoviolin and phycoerythrobilin.

Causitive toxin(s) in the death of two Atlantic dolphins.

Following the death of two Atlantic dolphins in a lagoon in March of 1989, the Hawaiian fishes in the lagoon were examined as a potential source of toxin(s). This study reports the findings of the causitive toxin(s) involved, utilizing the stick enzyme immunoassay (S-EIA) and the mouse and guinea pig atrium assays. The S-EIA proved effective in screening the toxic fishes (mullet, wrasse, manini, and aholehole). Following extraction, the major toxin was found in the viscera of these fishes, as confirmed in the mouse assay. The most toxic level was shown in the viscera of the mullet (13.2 mouse units/mg of extract). The viscera of the wrasse, aholehole, and manini also showed high levels of the toxic substance. The guinea pig atrium assay showed the presence of a potent Na+ channel inhibitor, characteristic of tetrodotoxin and saxitoxin. The toxin was also demonstrated in low levels in the dolphin liver and gut content and in the sand and algae extracts from the lagoon. This is the first report of this type of toxin in Hawaii.

[The use of ribonucleic acids as markers for the measuring of microbial protein yield in the rumen. 2. The effect of sample treatment, the time of sampling and the composition of the ration on the RNA:N ratio in rumen microbes]. / Untersuchungen zur Nutzung von Ribonukleinsäure als Marker für die Messung des mikrobiellen Proteinertrages im Pansen. 2. Mitteilung. Einfluss der Probenbehandlung, des Probenahmezeitpunktes und der Rationszusammensetzung auf das RNS: N-Verhältnis in den Pansenmikroben.

In two experiments the influence of the treatment of samples, the sampling time and the composition of the rations on the RNA: N ratio in the rumen microbes was checked. Experiment I proved that freezing (-21 degrees C), thawing and freeze-drying of isolated bacteria and protozoa from the rumen fluid and from the duodenal content did not result in a change of the RNA content and the RNA-N: total N relation. If, however, the rumen fluid is stored deep-frozen before the isolation of the bacteria the N content in the DM of the bacteria decreases by 17% and that of RNA by 30%. This results in a change of the RNA: N relation of 16%. In conclusion, the bacteria are to be isolated immediately after rumen fluid sampling. Isolated bacteria can be stored deep-frozen before RNA determination and then freeze-dried. Experiment II showed that the RNA content of the rumen protozoa varies according to the period after feeding. The RNA: N relation was 0.50, 0.92, 0.70 and 0.58 on average 0, 3, 6 and 8 h after feeding, in which the 3rd hour after feeding can obviously be considered the time of increased microbial activity. The conclusion from this variation is that more than one isolation of microbes must be carried out in the course of the day in order to achieve representative samples. These statements apply to easily and not easily fermentable protein as N source in the feed. It could also be proved that no essential variation is to be expected in the RNA: N relation in the microbes isolated from the rumen fluid in the range of 8-21% crude protein in the DM of the ration (roughage: concentrate = 55: 45). On average the rumen microbes contained 1.7 g RNA-N/16 g N, essential differences between bacteria and protozoa could not be ascertained. From the slight variation of the RNA-N: N relation in the isolated bacteria from various cows one can conclude that there is no need to isolate the microbes of each individual animal.