IDENTIFICATION AND TOXICITY OF ALEXANDRIUM TAMARENSE (DINOPHYCEAE) IN SCOTTISH WATERS1

Contamination of shellfish with paralytic shellfish poisoning (PSP) toxins produced by Alexandrium species poses a potential threat to the sustainability of the Scottish aquaculture industry. Routine LM analysis of water samples from around the Scottish coast has previously identified Alexandrium (Dinophyceae) as a regular part of the spring and summer phytoplankton communities in Scottish coastal waters. In this study, Alexandrium tamarense (M. Lebour) Balech isolated from sediment and water samples was established in laboratory culture. Species identification of these isolates was confirmed using thecal plate dissections and by molecular characterization based on their LSU and, in some cases, ITS rDNA sequence. Molecular characterization and phylogenetic analysis showed the presence of two ribotypes of A. tamarense: Group I (North American ribotype) and Group III (Western European ribotype). Assessment of PSP toxin production using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) showed that A. tamarense Group I produced a complex array of toxins (～2,000 fg STX equivalents · cell^?1) with the major toxins being C2, neosaxitoxin (NEO), saxitoxin (STX), gonyautoxin‐4 (GTX‐4), and GTX‐3, while A. tamarense Group III did not produce toxins. Historically, it was considered that all Alexandrium species occurring in Scottish waters produce potent PSP toxins. This study has highlighted the presence of both PSP toxin‐producing and benign species of A. tamarense and questions the ecological significance of this finding.     

Effect of Al(III) on surface properties of Bifidobacterium thermophilum as a function of temperature

The effects of Al(III) on surface properties and lactate accumulation by Bifidobacterium thermophilum were investigated. Bacteria were treated with Al(III) at 37°C and 4°C, then exposed to free radicals or nisin. When exposed to Al(III) at 37°C, the organism exhibited spreading on hydrophobic surfaces and showed high susceptibility to free-radical alteration as indicated by Fe(III) binding, but showed little effect on lactate production in the presence or absence of nisin, even after washing with 2 mM EDTA. At 4°C, there was no increased surface spreading or binding of Fe(III), but protection against nisin action was present. This, however, was abolished after washing with EDTA. It was concluded that membrane fluidity is required to affect membrane lipid rearrangement, resulting in surface spreading and increased susceptibility to peroxidation, whereas only loose binding of Al(III) to membrane surfaces is sufficient to prevent transmembrane channel formation by nisin.     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Role of dissimilatory fermentative iron-reducing bacteria in Fe uptake by common bean (Phaseolus vulgaris L.) plants grown in alkaline soil

Iron (Fe) is an essential element for plant growth and development. Some plant growth-promoting rhizobacteria can increase Fe uptake by plants through reduction of Fe(III) to Fe(II) at the root surface. The aim of this work was to identify novel bacterial strains with high Fe(III) reduction ability and to evaluate their role in plant Fe uptake. Four bacterial strains (UMCV1 to UMCV4) showing dissimilatory Fe-reducing activity were isolated from the rhizosphere of bean and maize plants and further identified by 16S rDNA amplification and sequence analysis. From these analyses, UMCV1 and UMCV2 isolates were identified as Bacillus megaterium and Arthrobacter spp., respectively, whereas UMCV3 and UMCV4 were identified as Stenotrophomonas maltophilia. All four isolates showed Fe reduction in a nonflooded soil and when associated with roots of bean plants grown in alkaline soil or in mineral medium. In addition, the bacterial isolates were able to stimulate plant growth in vitro and on a broad level, plants grown in inoculated soil were generally bigger and with higher Fe content than those grown in sterilized soil. These results indicate that bacterial species isolated from the rhizosphere of bean and maize plants contribute significantly to Fe uptake by plants likely through increased Fe(III) reduction in the rhizosphere.     

A Comparative Study of the Biodesulfurization Efficiency of Acidithiobacillus ferrooxidans LY01 Cells Domesticated with Ferrous Iron and Pyrite

The high-sulfur coal desulfurization process completed by A. ferrooxidans LY01 cells domesticated with either ferrous iron [Fe(II)] or pyrite (FeS₂) was investigated in this article. The desulfurization rate for 13 d was as high as 67.8% for the LY01 cells domesticated with pyrite but was only 45.6% for the LY01 cells domesticated with Fe(II). Bacterial adsorption experiments indicated that the bacterial adsorption quantity onto the pyrite particles was similar to the desulfurization efficiency. FTIR analysis showed that chemical composition of the two cell types was similar, but the LY01 cells domesticated with pyrite had higher levels of hydrophobic aromatic R-O groups than cells domesticated with Fe(II). The amount of extracellular polymeric substances (EPS) from the pyrite-domesticated LY01 cells was 1820 μg C/10¹⁰ cells, which was five times more than the amount of EPS in the Fe(II)-domesticated cells; the EPS readily bound Fe(III) with a maximum binding capacity of 0.21 mg Fe(III) per mg C EPS. Strains of pyrite-domesticated LY01 with a high amount of Fe(III) in their EPS possess greater oxidation activity than Fe(II)-domesticated strains with fewer Fe(III). These experiments showed the importance of the substrate-specific differences in the oxidative activity of A. ferrooxidans LY01. In addition, this study provides theoretical guidance for the future optimization of the biodesulfurization process.     

Temperature Elevation Regulates Iron Protoporphyrin IX and Hemoglobin Binding by Porphyromonas gingivalis

Porphyromonas gingivalis, an obligate anerobe with a growth requirement for iron protoporphyrin IX (FePPIX), is exposed to increased temperatures in the inflamed periodontal pocket. In this study, P. gingivalis was grown in a chemostat at 37°C (control), 39°C, and 41°C, and examined for hemagglutinating (HA) activity, hemoglobin binding and degrading activity, and iron protoporphyrin IX binding. HA activity decreased in cells as the growth temperature increased. Binding of μ-oxo bishaem (dimeric haem), and Fe(II)- and Fe(III)-monomeric forms was increased in 39°C-grown cells but decreased in 41°C-grown cells compared with controls. Cellular hemoglobin binding and degradation decreased with increased growth temperature. The decrease in cellular hemagglutination and hemoglobin degradation occurring with increased growth temperature would limit the potential overproduction of toxic monomeric haem molecules. The increased binding of μ-oxo bishaem and monomeric forms of FePPIX at 39°C may reflect a defense strategy against reactive oxidants and a mechanism of dampening down the inflammatory response to maintain an ecological balance. Received: 24 April 2000 / Accepted: 30 May 2000     

Induction of iron(III) and copper(II) reduction in pea (Pisum sativum L.) roots by Fe and Cu status: Does the root-cell plasmalemma Fe(III)-chelate reductase perform a general role in regulating cation uptake?

We investigated the effects of Fe and Cu status of pea (Pisum sativum L.) seedlings on the regulation of the putative root plasma-membrane Fe(III)-chelate reductase that is involved in Fe(III)-chelate reduction and Fe²⁺ absorption in dicotyledons and nongraminaceous monocotyledons. Additionally, we investigated the ability of this reductase system to reduce Cu(II)-chelates as well as Fe(III)-chelates. Pea seedlings were grown in full nutrient solutions under control, -Fe, and -Cu conditions for up to 18 d. Iron(III) and Cu(II) reductase activity was visualized by placing roots in an agarose gel containing either Fe(III)-EDTA and the Fe(II) chelate, Na₂bathophenanthrolinedisulfonic acid (BPDS), for Fe(III) reduction, or CuSO₄, Na₃citrate, and Na₂-2,9-dimethyl-4,7-diphenyl-1, 10-phenanthrolinedisulfonic acid (BCDS) for Cu(II) reduction. Rates of root Fe(III) and Cu(II) reduction were determined via spectrophotometric assay of the Fe(II)-BPDS or the Cu(I)-BCDS chromophore. Reductase activity was induced or stimulated by either Fe deficiency or Cu depletion of the seedlings. Roots from both Fe-deficient and Cu-depleted plants were able to reduce exogenous Cu(II)-chelate as well as Fe(III)-chelate. When this reductase was induced by Fe deficiency, the accumulation of a number of mineral cations (i.e., Cu, Mn, Fe, Mg, and K) in leaves of pea seedlings was significantly increased. We suggest that, in addition to playing a critical role in Fe absorption, this plasma-membrane reductase system also plays a more general role in the regulation of cation absorption by root cells, possibly via the reduction of critical sulfhydryl groups in transport proteins involved in divalent-cation transport (divalent-cation channels?) across the root-cell plasmalemma.     

Increase in the production of allelopathic substances by Prymnesium parvum cells grown under N- or P-deficient conditions

The haptophyte Prymnesium parvum is known to produce a set of highly potent exotoxins, commonly called prymnesins. These toxins have been shown to have several biological activities, including ichthyotoxic, neurotoxic, cytotoxic, hepatotoxic and hemolytic activity towards a range of marine organisms. In addition, recent studies have shown that the toxicity of P. parvum is enhanced when the cells are grown under N- or P-deficient conditions. In this study, the influence of prymnesium toxins on the growth of other phytoplankton species was investigated by addition of cell-free filtrate of P. parvum cultures grown under nutrient-deficient (N or P) or non-deficient conditions. Addition of cell-free filtrate from P. parvum cultures grown under N or P limitation inhibited the growth of Thalassiosira weissflogii, Prorocentrum minimum and Rhodomonas cf. baltica. In contrast, a strain of Prymnesium patelliferum known to produce prymnesium toxins was not negatively affected under any conditions. Furthermore, addition of filtrates from nutrient-sufficient P. parvum cultures did not negatively influence the growth of any of the tested species. These findings suggest that prymnesium toxins may play an allelopathic role, and that the production of allelopathic substances is regulated by the availability of nutrients.     

Whole -root iron(III)-reductase activity throughout the life cycle of iron-grown Pisum sativum L. (Fabaceae): relevance to the iron nutrition of developing seeds

To understand the whole-plant processes which influence the Fe nutrition of developing seeds, we have characterized root Fe(III)-reductase activity and quantified whole-plant Fe balance throughout the complete 10-week (10-wk) life cycle of pea (Pisum sativum L., cv. Sparkle). Plants were grown hydroponically in complete nutrient solution with a continuous supply of chelated Fe; all side shoots were removed at first appearance to yield plants with one main shoot. Root Fe(III)-reductase activity was assayed with Fe(III)-EDTA. Flowering of the experimental plants began on wk 4 and continued until wk 6; seed growth and active seed import occurred during wks 5–10. Vegetative growth terminated at wk 6. Iron(III) reduction in whole-root systems was found to be dynamically modulated throughout the plant's life cycle, even though the plants were maintained on an Fe source. Iron(III)-reductase activity ranged from 1–3 mol Fe reduced · g ^–1 DW · h^–1 at early and late stages of the life cycle to 9.5 mol Fe reduced · ^g–1 DW · h^–1 at wk 6. Visual assays demonstrated that Fe(III)-reductase activity was localized to extensive regions of secondary and tertiary lateral roots during this peak activity. At midstages of growth (wks 6–7), root Fe(III)-reductase activity could be altered by changes in internal shoot Fe demand or external root Fe supply: removal of all pods or interruption of phloem transport from the reproductive portion of the shoot (to the roots) resulted in lowered root Fe(III)-reductase activity, while removal of Fe from the nutrient solution resulted in a stimulation of this activity. Total shoot Fe content increased throughout the 10-wk growth period, with Fe content in the non-seed tissues of the shoot declining by 50% of their maximal level and accounting for 35% of final seed Fe content. At maturity, total seed Fe represented 74% of total shoot Fe; total Fe in the roots (apoplasmic and symplasmic Fe combined) was minimal. These studies demonstrate that the root Fe(III)-reductase system responds to Fe status and/or Fe requirements of the shoot, apparently through shoot-to-root communication involving a phloem-mobile signal. During active seed-fill, enhanced root Fe(III)-reductase activity is necessary to generate sufficient Fe²⁺ for continued root Fe acquisition. This continuing Fe supply to the shoot is essential for the developing seeds to attain their Fe-content potential. Increased rates of root Fe(III) reduction would be necessary for seed Fe content to be enhanced in Pisum sativum.Abbreviations BPDS bathophenanthrolinedisulfonic acid - DAF days after flowering - DW dry weight - EDDHA N,N-ethylenebis[2-(2-hydroxyphenyl)-glycine] - wk week This project has been funded in part with federal funds from the U.S. Department of Agriculture, Agricultural Research Service under Cooperative Agreement number 58-6250-1-003. The contents of this publication do not necessarily reflect the views or policies of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The author wishes to acknowledge S. Pezeshgi and K. Koch for their excellent technical assistance, L. Loddeke for editorial comments, and A. Gillum for assistance with the figures.     

High-Temperature Microbial Sulfate Reduction Can Be Accompanied by Magnetite Formation

The hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was found to be capable of lithoautotrophic growth on medium containing molecular hydrogen, sulfate, and amorphous Fe(III) oxide. During the growth of this microorganism, amorphous Fe(III) oxide was transformed into black strongly magnetic precipitate rich in magnetite, as shown by Moessbauer studies. Experiments involving inhibition of microbial sulfate reduction and abiotic controls revealed that magnetite production resulted from chemical reactions proceeding at elevated temperatures (83°C) between molecular hydrogen, amorphous Fe(III) oxide, and sulfide formed enzymatically in the course of dissimilatory sulfate reduction. It follows that magnetite production in this system can be characterized as biologically mediated mineralization. This is the first report on magnetite formation as a result of activity of sulfate-reducing microorganisms.     

Enzyme production by the nematode-trapping fungus,Duddingtonia flagrans

Duddingtonia flagrans degrades peptides, proteins, starch, pectin, lipase, lecithin and oils when grown on agar medium. Serine proteases with optimal activity at pH 8.5 to 10.5 were produced when it was grown in submerged culture. It also produced phospholipase C with optimal activity at pH 8.5, lipases with high activity at pH 3.5 and at 7.5 to 8.5 and pectin-degrading enzymes with pH optima of 3 and 8. The pH of the culture medium affected the types of lipases and pectin degrading enzymes produced but not the proteases or phospholipase C.     

Lipid peroxidation and superoxide dismutase activity in relation to photoinhibition induced by chilling in moderate light

The effect of a chilling stress, at a moderate photon flux density for a few hours, on the peroxidation of membrane lipids and on superoxide dismutase (SOD) activity was compared in leaf slices of chilling-sensitive and chilling-insensitive plants. The aim was to determine if susceptibility to chill-temperature photoinhibition could be related to either damage to membrane lipids by superoxide and-or a decrease in activity of chloroplast SOD. Plants used were Nerium oleander L., grown at 45° C, and Cucumis sativus L., both susceptible to chill-temperature photoinhibition, and N. oleander, grown at 20° C and Spinacia oleracea L., both insensitive to chill-temperature photoinhibition. Lipid peroxidation was assessed by measuring the concentration of malondialdehyde (MDA). Leaf slices from all plants showed a basal level of MDA which decreased by about 15% when the leaf slices were chilled in the light. The level of MDA was not increased by the addition of either KHCO₃ or methyl viologen during chilling but it was increased, up to threefold, by the addition of Rose Bengal, which produces singlet oxygen. Chloroplast SOD activity was assessed in leaf extracts as the cyanide-sensitive production of H₂O₂ in a system which produced superoxide. Activity of SOD was similar in all the plants and was altered little by chilling. The results show that for the plants tested, chilling at a moderate photon flux density for 5 h does not increase the susceptibility of cell membranes to peroxidative damage nor does it decrease the activity of SOD. It was concluded that the susceptibility of chilling-sensitive plants to chill-temperature photoinhibition cannot be explained on the basis of differences in the vulnerability of membrane lipids to damage by superoxide or differences in SOD activity.Abbreviations Chl chlorophyll - MDA malondialdehyde - MV methyl viologen - O ₂ ^- superoxide - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density - SOD superoxide dismutase Deceased     

Reduction of Fe(III), Mn(III), and Cu(II) chelates by roots of pea (Pisum sativum L.) or soybean (Glycine max)

Neither the reduction of Fe(III) to Fe(II) by roots nor its induction by Fe-deficiency are unique characteristics of the reductive activities of roots. We show that chelated Mn(III) or chelated Cu(II), as well as chelated Fe(III), may be reduced by Fe-stressed roots of pea (Pisum sativum L.). Deficiency of Fe stimulated the reduction of Fe(III)EDTA about 20-fold, the reduction of Mn(III)CDTA about 11-fold, the reduction of Cu(II)(BPDS)₂ about 5-fold, and the reduction of Fe(III)(CN)₆ by only about 50%. Not only are metals other than Fe reduced as part of the Fe-stress response, but deficiencies of metals other than Fe stimulate the reductive activity of roots. We show that depriving peas or soybeans (Glycine max) of Cu or Zn stimulates the reduction of Fe(III).     

Synergistic iron reduction and citrate dissimilation by Shewanella alga and Aeromonas veronii

Two bacterial isolates from Great Bay Estuary, New Hampshire, in co-culture carried out anaerobic dissimilation of citric acid with Fe(III) as the terminal electron acceptor. Neither isolate oxidized citrate with Fe(III) anaerobically in axenic culture. The Fe(III) reducer, Shewanella alga strain BrY, did not grow anaerobically with citrate as an energy source. The citrate utilizer, Aeromonas veronii, did not reduce iron axenically with a variety of electron donors including citrate. The onset of iron reduction by the co-culture occurred after initiation of citrate dissimilation and just prior to initiation of growth by either organism (as measured by viable plate counts). Anaerobic culture growth rates and final cell densities of each bacterial strain were greater in co-culture than in axenic cultures. By 48 h of growth, the co-culture had consumed 27 mM citrate as compared with 12 mM dissimilated by the axenic culture of A. veronii. By 48 h the co-culture produced half as much formate (6 mM) and twice as much acetate (40 mM) as did A. veronii grown axenically (12 mM and 20 mM, respectively). Formate produced from citrate by A. veronii appeared to have supported growth and Fe(III) reduction by S. alga.Although not obligatory, nutrient coupling between these two organisms illustrates that fermentative (A. veronii-type) organisms can convert organic compounds such as citrate to those used as substrates by dissimilatory Fe(III) reducers, including S. alga. This synergism broadens the range of substrates available for iron reduction, stimulates the extent and rate of organic electron donor degradation (and that of iron reduction) and enhances the growth of each participant. Received: 11 December 1995 / Accepted: 19 June 1996     

Regulation of nitrogen-fixation by different nitrogen sources in the marine non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067

The effect of various nitrogen sources on the synthesis and activity of nitrogenase was studied in the marine, non-heterocystous cyanobacterium Trichodesmium sp. NIBB1067 grown under defined culture conditions. Cells grown with N₂ as the sole inorganic nitrogen source showed light-dependent nitrogenase activity (acetylene reduction). Nitrogenase activity in cells grown on N₂ was not suppressed after 7 h incubation with 2 mM NaNO₃ or 0.02 mM NH₄Cl. However, after 3 h of exposure to 0.5 mM of urea, nitrogenase was inactivated. Cells grown in medium containing 2 mM NaNO₃, 0.5 mM urea or 0.02 mM NH₄Cl completely lacked the ability to reduce acetylene. Western immunoblots tested with polyclonal antisera against the Fe-protein and the Mo–Fe protein, revealed the following: (1) both the Fe-protein and the Mo–Fe protein were synthesized in cells grown with N₂ as well as in cells grown with NaNO₃ or low concentration of NH₄Cl; (2) two bands (apparent molecular mass of 38 000 and 40 000) which cross-reacted with the antiserum to the Fe-protein, were found in nitrogen-fixing cells; (3) only one protein band, corresponding to the high molecular mass form of the Fe-protein, was found in cells grown with NaNO₃ or low concentration of NH₄Cl; (4) neither the Fe-protein nor the Mo–Fe protein was found in cells grown with urea; (5) the apparent molecular mass of the Fe-protein of Trichodesmium sp. NIBB1067 was about 5000 dalton higher than that of the heterocystous cyanobacterium, Anabaena cylindrica IAM-M1.     

The respiratory inhibitor antimycin A specifically binds Fe(III) ions and mediates utilization of iron by the halotolerant alga Dunaliella salina (Chlorophyta)

It is demonstrated that Antimycin A (AA), a respiratory inhibitor produced by Streptomyces bacteria, forms lipophylic complexes with Fe(III) ions. Spectroscopic titration indicates that Fe(III) ions interact with 2AA molecules. At growth-limiting Fe concentrations, AA mediates Fe uptake and promotes growth and chlorophyll synthesis better than other Fe chelators in the halotolerant alga Dunaliella salina. It is proposed that AA enhances Fe bioavailability in hypersaline solutions by formation of lipophylic Fe-AA complexes which are taken-up and utilized by the algae. The results suggest that the respiratory inhibitor AA can affect Fe metabolism in microorganisms.     

Structural determinants of the affinity of saxitoxin for neuronal sodium channels. Electrophysiological studies on frog peripheral nerve

The potencies of saxitoxin (STX) and of five structurally related toxins were determined by their ability to block impulses at equilibrium in frog sciatic nerve. The order of potency, with values relative to STX potency in parentheses, was: neo-STX (4.5) greater than gonyautoxin (GTX) III (1.4) greater than STX (1.0) greater than GTXII (0.22) greater than 12 alpha-dihydroSTX (0.050) greater than 12 beta-dihydroSTX (0.0014). When equipotent solutions of STX and neo-STX were exchanged, impulses in the treated nerve were transiently overblocked or underblocked, thus kinetically distinguishing neo-STX from STX. Similar phenomena occurred with exchanges of STX and GTXIII. No consistent evidence was found for any blocking activity of STX molecules that were not protonated at the C8 guanidinium, but the pH dependence of STX potency cannot be described simply by the titration of this guanidinium group. The effects of pH and of various substituents on STX potency are accounted for by changes in the molecular forms of STX and by alterations in specific electrical charges on STX and at the receptor. The results support a model in which toxin molecules bind in two steps; initial binding of the C8 guanidinium to an anionic group induces the loss of water from the normally hydrated ketone (at carbon 12), which then forms a weak covalent bond with a nucleophilic group on the receptor.     

In vitro transformation of paralytic shellfish toxins in the clams Mya arenaria and Protothaca staminea

Dissected tissues of two clam species, the Pacific littleneck, Protothaca staminea, and soft-shell, Mya arenaria, were evaluated for in vitro conversion of paralytic shellfish poisoning (PSP) toxins. Tissue homogenates were incubated with purified PSP toxins to determine the time-course of toxin conversion. The effects of boiling and addition of a natural reductant (glutathione) on toxin conversion were also assessed. For P. staminea, the digestive gland showed the greatest capacity for biotransformation, followed by gill, but mantle, adductor muscle, and siphon tissues exhibited very low conversion. In this species, the production of decarbamoyl derivatives was much greater from low potency N-sulfocarbamoyl toxins than from carbamate analogues. Decarbamolyation exhibited apparent specificity for α-epimers of all toxin substrates and this reaction was inhibited by boiling. Glutathione-mediated desulfation was tissue specific and had apparent specificity for β-epimers. These observations on P. staminea suggest that the above reactions are enzyme-mediated. In contrast, there was little toxin conversion in M. arenaria homogenates, but even this low activity was heat-labile and thus likely enzyme-mediated.