Benjamin A. Suarez-Isla

Identification and characterization of potentially algal-lytic marine bacteria strongly associated with the toxic dinoflagellate Alexandrium catenella.

Abstract. The toxic dinoflagellate Alexandrium catenella isolated from fjords in Southern Chile produces several analogues of saxitoxin and has been associated with outbreaks of paralytic shellfish poisoning. Three bacterial strains, which remained in close association with this dinoflagellate in culture, were isolated by inoculating the dinoflagellate onto marine agar. The phenotypically different cultivable bacterial colonies were purified. Their genetic identification was done by polymerase chain reaction amplification of the 16S rRNA genes. Partial sequence analysis suggested that the most probable affiliations were to two bacterial phyla: Proteobacteria and the Cytophaga group. The molecular identification was complemented by morphological data and biochemical profiling. The three bacterial species, when grown separately from phytoplankton cells in high‐nutrient media, released algal‐lytic compounds together with aminopeptidase, lipase, glucosaminidase, and alkaline phosphatase. When the same bacteria, free of organic nutrients, were added back to the algal culture they displayed no detrimental effects on the dinoflagellate cells and recovered their symbiotic characteristics. This observation is consistent with phylogenetic analysis that reveals that these bacteria correspond to species distinct from other bacterial strains previously classified as algicidal bacteria. Thus, bacterial‐derived lytic activities are expressed only in the presence of high‐nutrient culture media and it is likely that in situ environmental conditions may modulate their expression.

Activation of single cardiac and skeletal ryanodine receptor channels by flash photolysis of caged Ca2

Single ryanodine-sensitive sarcoplasmic reticulum (SR) Ca2+ release channels isolated from rabbit skeletal and canine cardiac muscle were reconstituted in planar lipid bilayers. Single channel activity was measured in simple solutions (no ATP or Mg2+) with 250 mM symmetrical Cs+ as charge carrier. A laser flash was used to photolyze caged-Ca2+ (DM-nitrophen) in a small volume directly in front of the bilayer. The free [Ca2+] in this small volume and in the bulk solution was monitored with Ca2+ electrodes. This setup allowed fast, calibrated free [Ca2+] stimuli to be applied repetitively to single SR Ca2+ release channels. A standard photolytically induced free [Ca2+] step (pCa 7-->6) was applied to both the cardiac and skeletal release channels. The rate of channel activation was determined by fitting a single exponential to ensemble currents generated from at least 50 single channel sweeps. The time constants of activation were 1.43 +/- 0.65 ms (mean +/- SD; n = 5) and 1.28 +/- 0.61 ms (n = 5) for cardiac and skeletal channels, respectively. This study presents a method for defining the fast Ca2+ regulation kinetics of single SR Ca2+ release channels and shows that the activation rate of skeletal SR Ca2+ release channels is consistent with a role for CICR in skeletal muscle excitation-contraction coupling.

Activation of calcium channels in sarcoplasmic reticulum from frog muscle by nanomolar concentrations of ryanodine.

Sarcoplasmic reticulum vesicles isolated from fast-twitch frog skeletal muscle presented two classes of binding sites for ryanodine, one of high affinity (Kd1 = 1.7 nM, Bmax1 = 3.3 pmol per mg) and a second class with lower affinity (Kd2 = 90 nM, Bmax2 = 7.0 pmol per milligram). The calcium channels present in the sarcoplasmic reticulum membranes were studied in vesicles fused into lipid bilayers. Low concentrations of ryanodine (5 to 10 nM) activated a large conductance calcium channel after a short delay (5 to 10 min). The activation, which could be elicited from conditions of high or low fractional open time, was characterized by an increase in channel fractional open time without a change in conductance. The open and closed dwell time distributions were fitted with the sum of two exponentials in the range of 4 to 800 ms. The activating effect of ryanodine was due to an increase of both open time constants and a concomitant decrease in the closed time constants. Under conditions of low fractional open time (less than 0.1), the time spent in long closed periods (greater than 800 ms) between bursts was not affected by ryanodine. Higher concentrations of ryanodine (250 nM) locked the channel in a lower conductance level (approximately 40%) with a fractional open time near unity. These results suggest that the activating effects of nanomolar concentrations of ryanodine may arise from drug binding to high affinity sites. The expression of the lower conductance state obtained with higher concentrations of ryanodine may be associated with the low affinity binding sites observed in frog sarcoplasmic reticulum.

Comparison of AOAC 2005.06 LC official method with other methodologies for the quantitation of paralytic shellfish poisoning toxins in UK shellfish species

AbstractA refined version of the pre-column oxidation liquid chromatography with fluorescence detection (ox-LC-FLD) official method AOAC 2005.06 was developed in the UK and validated for the determination of paralytic shellfish poisoning toxins in UK shellfish. Analysis was undertaken here for the comparison of PSP toxicities determined using the LC method for a range of UK bivalve shellfish species against the official European reference method, the PSP mouse bioassay (MBA, AOAC 959.08). Comparative results indicated a good correlation in results for some species (mussels, cockles and clams) but a poor correlation for two species of oysters (Pacific oysters and native oysters), where the LC results in terms of total saxitoxin equivalents were found to be on average more than double the values determined by MBA. With the potential for either LC over-estimation or MBA under-estimation, additional oyster and mussel samples were analysed using MBA and ox-LC-FLD together with further analytical and functional methodologies: a post-column oxidation LC method (LC-ox-FLD), an electrophysiological assay and hydrophilic interaction liquid chromatography with tandem mass spectrometric detection. Results highlighted a good correlation among non-bioassay results, indicating a likely cause of difference was the under-estimation in the MBA, rather than an over-estimation in the LC results. FigureTotal saxitoxin equivalents in oysters (Pacific and native) quantified by ox-LC-FLD, LC-ox-FLD, HPLC-MS/MS and electrophysiological assay as compared with the MBA PSP toxicity reference method

Investigations into matrix components affecting the performance of the official bioassay reference method for quantitation of paralytic shellfish poisoning toxins in oysters

Significant differences previously observed in the determination of paralytic shellfish poisoning toxins (PSTs) in oysters using official method AOAC 2005.06 and 959.08 were investigated in detail with regard to possible matrix effects. Method AOAC 2005.06 gave results 2-3 times higher than the mouse bioassay method, 959.08, differences thought to be due to underestimation of PSTs by the mouse bioassay. In order to prove the cause of these large differences, work was conducted here to examine the presence and effects of matrix components on the performance of each of the two assays. A range of oyster, cockle and mussel samples were extracted using the AOAC 959.08 hydrochloric acid (HCl) extraction method and analysed for PSP by both MBA and LC-FLD. In addition, extracts were analysed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for metals as well as being subjected to a range of nutritional testing methods. Whilst there was no evidence for effect of nutritional components on either assay, ICP-MS analysis revealed a relationship between samples exhibiting the largest differences in relative method performance, specifically those with the largest LC-FLD/MBA toxicity ratio, and samples containing the highest concentrations of zinc and manganese. In order to prove the potential effect of the metals on either the LC-FLD and/or MBA assays, HCl extracts of a range of shellfish were subjected to a number of matrix modifications. Firstly, a number of PSP-positive oyster samples were processed to reduce the concentrations of metals within the extracts, without significantly reducing the concentrations of PSTs. Secondly, a range of mussel and cockle extracts, plus a standard solution of saxitoxin di-hydrochloride were spiked at variable concentrations of zinc. All treated and non-treated extracts, plus a number of controls were subjected to ICP-MS, LC-FLD and MBA testing. Results proved the absence of any effect of metals on the performance of the LC-FLD, whilst showing a large suppressive effect of the metals on the MBA. As such, the results show the performance of the official MBA is potentially unsafe for application to the routine monitoring of PSP toxicity in oysters or in any other shellfish found to contain high concentrations of metal ions.

Activation of inositol trisphosphate‐sensitive Ca2+ channels of sarcoplasmic reticulum from frog skeletal muscle.

1. The modulation by Ca2+ of the activation by inositol 1,4,5‐trisphosphate (IP3) of Ca2+ channels present in native sarcoplasmic reticulum membranes from frog skeletal muscle was studied after channel incorporation into planar phospholipid bilayers in the presence of Ca2+ or Ba2+ as current carrier species. 2. Channel activity expressed as fractional open time (Po) was low (less than or equal to 0.15) in the presence of varying free Ca2+ concentrations bathing the myoplasmic face of the channel (cis side), and did not increase significantly between 0.01 and 30 microM‐Ca2+. 3. Channel activation mediated by IP3 could be elicited from free Ca2+ levels similar to those of resting skeletal muscle (about 0.1 microM) and was found to be strongly regulated by the free Ca2+ concentration present at the myoplasmic moiety of the channel. 4. Channel activation by 10 microM‐IP3 depended on the Ca2+ concentration on the cis side. Po reached a maximum between pCa 7.0 and 6.0, but decreased at higher concentrations of free Ca2+. Thus, Ca2+ exerted a modulatory influence on IP3‐mediated activation in a concentration range where the channel was insensitive to Ca2+. 5. The results indicate that Ca2+ ions act as modulators of IP3 efficacy to open the channel. This could arise from an interaction of Ca2+ with the channel gating mechanism or with the agonist binding site.

RIBOSOMAL RNA HETEROGENEITY AND IDENTIFICATION OF TOXIC DINOFLAGELLATE CULTURES BY HETERODUPLEX MOBILITY ASSAY

The heteroduplex mobility assay (HMA) reveals sequence dissimilarity between DNA by measuring the retarded migration of the hybrid or heteroduplex using polyacrylamide gel electrophoresis. Heterogeneity in some cultures of toxic dinoflagellates of the genus Alexandrium (Halim) Balech was observed during comparison of the amplified D1–D2 region of the large subunit rRNA gene (rDNA) using this method. HMA also allowed grouping of clones obtained from toxic bloom events in the Chilean, southernmost Pacific within the Asian Southern Pacific lineage of A. catenella (Whedon et Kofoid) Balech. The applied methodology provides a rapid and simple tool for use in assessing heterogeneity as well as for molecular grouping of strains among the genus Alexandrium.

A Roadmap for Hazard Monitoring and Risk Assessment of Marine Biotoxins on the Basis of Chemical and Biological Test Systems

Aquatic food accounts for over 40% of global animal food products, and the potential contamination with toxins of algal origin--marine biotoxins--poses a health threat for consumers. The gold standards to assess toxins in aquatic food have traditionally been in vivo methods, i.e., the mouse as well as the rat bioassay. Besides ethical concerns, there is also a need for more reliable test methods because of low inter-species comparability, high intra-species variability, the high number of false positive and negative results as well as questionable extrapolation of quantitative risk to humans. For this reason, a transatlantic group of experts in the field of marine biotoxins was convened from academia and regulatory safety authorities to discuss future approaches to marine biotoxin testing. In this report they provide a background on the toxin classes, on their chemical characterization, the epidemiology, on risk assessment and management, as well as on their assumed mode of action. Most importantly, physiological functional assays such as in vitro bioassays and also analytical techniques, e.g., liquid chromatography coupled mass spectrometry (LC-MS), as substitutes for the rodent bioassay are reviewed. This forms the basis for recommendations on methodologies for hazard monitoring and risk assessment, establishment of causality of intoxications in human cases, a roadmap for research and development of human-relevant functional assays, as well as new approaches for a consumer directed safety concept.

Functionally Heterogenous Ryanodine Receptors in Avian Cerebellum

The functional heterogeneity of the ryanodine receptor (RyR) channels in avian cerebellum was defined. Heavy endoplasmic reticulum microsomes had significant levels of ryanodine and inositol 1,4,5-trisphosphate binding. Scatchard analysis and kinetic studies indicated the existence of at least two distinct ryanodine binding sites. Ryanodine binding was calcium-dependent but was not significantly enhanced by caffeine. Incorporation of microsomes into planar lipid bilayers revealed ion channels with pharmacological features (calcium, magnesium, ATP, and caffeine sensitivity) similar to the RyR channels found in mammalian striated muscle. Despite a wide range of unitary conductances (220-500 picosiemens, symmetrical cesium methanesulfonate), ryanodine locked both channels into a characteristic slow gating subconductance state, positively identifying them as RyR channels. Two populations of avian RyR channels were functionally distinguished by single channel calcium sensitivity. One population was defined by a bell-shaped calcium sensitivity analogous to the skeletal muscle RyR isoform (type I). The calcium sensitivity of the second RyR population was sigmoidal and analogous to the cardiac muscle RyR isoform (type II). These data show that there are at least two functionally distinct RyR channel populations in avian cerebellum. This leads to the possibility that these functionally distinct RyR channels are involved in different intracellular calcium signaling pathways.

Contrasting physiological responses of two populations of the razor clam Tagelus dombeii with different histories of exposure to paralytic shellfish poisoning (PSP).

This study describes the physiological performance of two populations of the razor clam Tagelus dombeii from two geographic areas with different histories of exposure to paralytic shellfish poisoning (PSP) linked to the toxic dinoflagellate Alexandrium catenella. Clams from Melinka-Aysén, which are frequently exposed to PSP, were not affected by the presence of toxins in the diet. However, clams from Corral-Valdivia, which have never been exposed to PSP, exhibited significantly reduced filtration activity and absorption, affecting the energy allocated to scope for growth (SFG). Ammonia excretion and oxygen uptake were not affected significantly by the presence of A. catenella in the diet. Measurements of energy acquisition and expenditure were performed during a 12-day intoxication period. According to three-way repeated measure ANOVAs, the origin of the clams had a highly significant effect on all physiological variables, and the interaction between diet and origin was significant for the clearance and absorption rates and for the scope for growth. The scope for growth index showed similar positive values for both the toxic and non-toxic individuals from the Melinka-Aysén population. However, it was significantly reduced in individuals from Corral-Valdivia when exposed to the diet containing A. catenella. The absence of differences between the physiological response of the toxic and non-toxic clams from Melinka-Aysén may be related to the frequent presence of A. catenella in the environment, indicating that this bivalve does not suffer negative consequences from PSP. By contrast, A. catenella has a negative effect on the physiological performance, primarily on the energy gained from the environment, on T. dombeii from Corral-Valdivia. This study supports the hypothesis that the history of PSP exposure plays an important role in the physiological performance and fitness of filter feeding bivalves.