Kinetic Analysis of the Interaction between Yessotoxin and Analogues

Jun 9, 2005 - Andrea Fernández-Araujo , Amparo Alfonso , Mercedes R. Vieytes , Luis M. Botana. Toxicology in Vitro 2015 29 (7), 1545-1554 ...
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Chem. Res. Toxicol. 2005, 18, 1155-1160

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Kinetic Analysis of the Interaction between Yessotoxin and Analogues and Immobilized Phosphodiesterases Using a Resonant Mirror Optical Biosensor Ma Jose´ Pazos,†,‡ Amparo Alfonso,† Mercedes R. Vieytes,§ Takeshi Yasumoto,| and Luis M. Botana*,†,⊥ Departamento de Farmacologı´a, Departamento de Fisiologı´a, y AÄ rea de Apoyo a la Investigacio´ n Agrobiolo´ gica, Facultad de Veterinaria, Universidad de Santiago de Compostela, 27002 Lugo, Spain, Japan Food Research Laboratories, Tama, Tokyo 206-0025 Japan, and Community Reference Laboratory of Marine Biotoxins Received February 9, 2005

Yessotoxin (YTX) is a disulfated polyether toxin produced by phytoplanktonic microalgae from the dinoflagellates group. YTX has structural similarities to maitotoxin or brevetoxins, and the mechanism of action for YTX is specific and related to calcium modulation. Some studies show that YTX decreases adenosine 3′,5′-cyclic monophosphate (cAMP) levels in human lymphocytes. This effect is calcium-dependent, and phosphodiesterases (PDEs) seem to be involved. This point was recently confirmed by studying kinetic constants of YTX-PDEs binding. The aim of this work is to compare the ability of YTX and analogues to bind immobilized PDEs by using a resonant mirror biosensor. These instruments measure biomolecular interactions in real time, without labeling, and allow detailed investigation of the reaction kinetics by analysis of the resultant signals. YTX and the derivatives, 45hydroxyyessotoxin, carboxyyessotoxin, and yessotoxin-45-(S)-a-methoxy-a-trifluormethylphenylacetate, were added over immobilized PDEs. The kinetic constants obtained from these bindings indicate a relationship between toxin structure and affinity. Since the presence of the functional groups in the side chain decreased the toxic YTX effect and the PDEs-YTX association, our results show that the side chain plays an important role in the YTX affinity for PDEs.

Introduction Yessotoxin (YTX)1 and its analogues are disulfated polyether toxins included in extraction procedures with the diarrheic shellfish poison (DSP) group because of their physicochemical nature, although their toxic activities are significantly different; in fact, YTX and its analogues do not induce diarrhea (1) and are not included in the list of DSP toxins (2). YTX was reported to induce cardiotoxic effects in mice after intraperitoneal (ip) and oral exposure of very high doses of YTX (1). YTX is highly toxic toward mice when administered ip, while its oral toxicity is at least 10 times lower (3). YTX was originally isolated from the digestive gland (hepatopancreas) of the scallop Patinopecten yessoensis by Murata et al. (4). Its planar structure was determined in 1987 (4), and the absolute configuration was recently reported (5, 6). The YTX molecule (Scheme 1) has 11 contiguous ether rings and an unsaturated side chain with different functional * Corresponding author: Departamento de Farmacologı´a, Facultad de Veterinaria, 27002 Lugo, Spain. Tel, +34-982 252 242; fax, +34982 252 242; e-mail, [email protected] (L. M. Botana). † Departamento de Farmacologı´a, Universidad de Santiago de Compostela. ‡ A Ä rea de Apoyo a la Investigacio´n Agrobiolo´gica, Universidad de Santiago de Compostela. § Departamento de Fisiologı´a, Universidad de Santiago de Compostela. | Japan Food Research Laboratories. ⊥ Community Reference Laboratory of Marine Biotoxins. 1 Abbreviations: YTX, yessotoxin; PDE, phosphodiesterase.

groups. There are several natural derivatives, and only a few structures have been reported. The marine polyether toxin, 45-hydroxyyessotoxin, and carboxyyessotoxin were isolated together with YTX (7, 8). When the 45hydroxy group in the side chain was esterified with (S)and (R)-R-methoxy-R-trifluoromethyphenylacetic acids (MPTA), a new ester of YTX was obtained (9). Routine monitoring of shellfish for DSP toxins is generally carried out using a mouse bioassay. The mouse lethality of YTX by ip injection (0.1 mg/kg) is the strongest among all lipophilic phycotoxins, but its oral toxicity is the weakest as can be deduced by considering that the maximum oral dose of 1 mg/kg does not kill the mouse. The presence of YTX in shellfish may, therefore, lead to a high overestimation of risk of lipophilic phycotoxins in consumers when the standard mouse bioassay is used. Thus, instrumental methods are required to identify the nature of the implied toxins (10). Initial YTX episodes were restricted to specific environments; however, in recent years, they have been found in several marine areas around the world (11). False positives on the mouse bioassay caused by YTX has increased in recent years, arousing an increased interest in studying the YTX effects to elucidate its mechanism of action. The chemical structure of YTX resembles that of brevetoxins, which are known to interfere with the voltage-sensitive sodium channel (12). This finding suggests a possible interaction between YTX and cellular ion channels. However, YTX does not activate sodium chan-

10.1021/tx050035i CCC: $30.25 © 2005 American Chemical Society Published on Web 06/09/2005

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Scheme 1. Structure of Yessotoxin and Various Analogues

nels as brevetoxins and ciguatoxins (13), but it may interact with calcium channels inducing an uptake of calcium in human lymphocytes (14). In addition, YTX modulates the calcium entry induced by maitotoxin in the same cellular model (15). On the other hand, no effect was reported for YTX over the DSP target protein phosphatase PP2A (16). Recently, we reported that the YTX decreases adenosine 3′,5′-cyclic monophosphate (cAMP) by activation of cellular phosphodiesterases (PDEs), which points to the PDE system as the intracellular target of YTX (17). These results were later confirmed with the study of YTX-PDEs interactions in a resonant mirror bionsensor, where kinetic constant values were obtained (18). Recently, biosensor technology has been used to measure, in real time, the binding kinetics between a macromolecule in solution and a receptor immobilized on a sensor surface. Fundamental information that enhances our understanding of biospecific interactions can be obtained using a new analytical system based on biosensor technology. The functional characteristics of biospecific interaction, such as kinetics, affinity, and binding position, are examined by label-free analysis of proteins in free solution binding to an immobilized ligand at a sensor surface (19). To investigate if the action of YTX in PDE is the cause of their in vivo effect, this paper shows the different affinities of YTX analogues on PDEs using resonant mirror technology.

Materials and Methods Chemicals. YTX and the analogues, hydroxy-yessotoxin (OHYTX), carboxy-yessotoxin (carboxy-YTX), and yessotoxin-45-(S)a-methoxy-a-trifluormethylphenylacetate (MTPA-YTX), were purified by Dr. T. Yasumoto. Iasys carboxymethyl dextran (CMD) cuvettes and the coupling kit containing 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC), N-hydroxy-succinimide (NHS), and 1 M ethanolamine were from Labsystems (U.K.). Phosphodiesterase 3,5-cyclic-nucleotide-specific from bovine brain (PDEs), sodium acetate, Tween 20, and the other chemicals were from Sigma Chemical Co. (Madrid, Spain).

The composition of saline solution (PBS/T) used for the biosensor experiments was (in mM): Na+ 145.2, K+ 4.7, HPO428.2, H2PO4- 1.5, Cl- 141.2, and 0.05% v/v Tween 20. The composition of phosphate buffer (PB) was (in mM): Na+ 20 and HPO42- 10. The final pH was adjusted to 7.7. Deionized water (Milli-Q) was used for all experiments. Operation on the Optical Biosensor System. Association measurements were made by using an Iasys Affinity Sensor (Labsystem, U.K.), following the procedures recommended by the manufacturer. The instrument analyzes interactions occurring within a few hundred nanometers from the sensor surface using evanescent waves, an electromagnetic wave that exists at the surface of many forms of optical waveguides, to measure changes in refractive index at the sensor surface. These changes can be measured as shifts in the resonance angle. The units of these shifts, in this equipment, are expressed as arc seconds. The sensor relies upon a biomolecule being attached to the sensor surface. Ligand immobilization can be followed in “realtime” with the instrument producing a plot of response. This was performed using ligands coupled to CMD cuvettes, in a total volume of 60 µL. The stirred cuvette system used in this instrument ensures that mass-transport effects during binding are minimized. The data-sampling interval was 0.3 s, and the stirring rate was 85 rpm controlled by the Iasys software. The temperature experiments were performed at 22 °C. The optical biosensor has dual cuvette surfaces having simultaneous dualchannel monitoring. CMD Surfaces Activation and Ligand Immobilization. The ligand, PDEs, was immobilized directly to dextran surface. Coupling of ligand to the CMD matrix was performed essentially as described before (20). Carboxylic groups on the dextran matrix were activated by addition of EDC/NHS solution for 10 min. Following activation, the EDC/NHS mixture was first replaced by PBS/T for 1 min and finally by 10 mM acetate buffer, pH 5.0, for 1 min to establish a preimmobilization baseline response on the biosensor. After activation, the immobilization of the ligand was then initiated by the addition of 0.1 mg/mL PDEs to the cuvette. After 5 min incubation, the ligand solution was replaced by ethanolamine, pH 8.5, for 3 min to quench residual NHS esters (nonspecific binding), and finally, this solution was replaced by PBS/T. Monitoring the Association/Dissociation of Toxins to Immobilized Ligand. 1. Cuvette Regeneration. A range of

Yessotoxin and Phosphodiesterases Kinetic Analysis YTX or analogues disolved in PB, pH 7.7, were allowed to bind into the immobilized ligand before indicated, and the association curves were monitored for 20-25 min. Subsequently, the cuvette was washed with PB, and the dissociation phase was monitored for 5 min. The cuvette was finally regenerated by 2 min washing in the presence of 0.01 M HCl. In these conditions and after 5 min equilibration with PB, a new toxin addition can be done. Since the instrument has a dual-optical path with two cuvettes, all experiments were performed in one cuvette and, simultaneously, the second cuvette was used as a control to normalize data by subtracting this control cuvette from the response of toxin cuvette. With the subtraction of the control channel (without YTX), the responses caused by nonspecific binding and bulk refractive index change could be corrected. Rate Analysis for the Binding of Toxin-Immobilized Ligand. One of the molecules is immobilized, and the other is in free solution; the assumption made is that the concentration in solution is in excess over the concentration on the surface and therefore considered constant. This is termed a pseudo-firstorder condition. If A is an immobilized species (ligand) and B is the species in solution (ligate), the rate of complex [AB] formation

d[AB]/dt ) kass[A][B] - kdiss[AB] The result of complex (ligand-ligate) formation is a change in response (Rt) which is directly proportional to the complex. And [A] is Rmax - Rt. Therefore, the above equation can be written as

dRt/dt ) [kass(Rmax - Rt)][(B) - (kdissRt)] By integration, the equation can be transformed into a form suitable for application of nonlinear regression

Rt ) Req (1 - e-(kass[B]+kdiss)t) This is a single-exponential equation where Rt is the instrument response time t and Req is the equilibrium response. Using the above equation, we can fit binding data at different concentrations to determine the pseudo-first-order rate constant, where kon ) ((kass[B]) + kdiss). B can be [YTX], and the equation can written as

kon ) ((kass[YTX]) + kdiss) The value kon, with units of s-1, was obtained for each concentration of YTX. Using linear least-squares regression, we derived the association rate constant, kass, with units of M-1 s-1, from the gradient of the plot of kon against YTX concentration, and the dissociation rate constant, kdiss, with units of s-1, from the intercept of the plot. At equilibrium, by definition, the rate of the forward an reverse reactions are equal, so

kass [A][B] ) kdiss[AB]

rearranging kdiss/kass ) [AB]/[A][B] ) KD

KD (M) is the dissociation equilibrium constant. If KD is high, the affinity of the interaction is low. Statistical Analysis. All the experiments were carried out at least three times. Data were normalized. Results were analyzed using the Student’s t-test for unpaired data and the ANOVA test. A probability level of 0.05 or smaller was used for statistical significance. Results were expressed as the means ( SEM.

Results Recently, we have shown that YTX activates cAMP hydrolysis by PDEs activation (17) and that the toxin interacts with cuvette surface-attached PDEs (18). In the

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Figure 1. PDEs immobilization and YTX association. Addition of 0.1 mg/mL PDEs (first arrow) from bovine brain into an activate dextran cuvette surface. After 5 min, the cuvette was washed and the remaining activated sites were blocked with 1 M ethanolamine (second arrow), nonspecific binding. This solution was replaced by PBS/T (third arrow), and finally 0.01 M HCl was added (fourth arrow).

present paper, we used the biosensor to measure the binding of YTX or analogues in solution to PDEs. PDEs were used as the ligand and YTX or analogues were used as the ligate. To study this binding, the enzymes were first immobilized on a CMD cuvette surface. As Figure 1 shows, when 0.1 mg/mL PDEs from bovine brain, dissolved in sodium acetate, were applied for 5 min over an activated cuvette surface, a typical curve of covalent binding was obtained. Then, the cuvette was washed with 1 M ethanolamine, pH 8.5, for 3 min to deactivate the remaining activated sites, then PBS/T was added, and finally, the surface was washed with HCl to remove any remaining noncovalently bound PDEs. This solution was finally replaced by PBS/T. The difference between the signal after HCL washing and the signal before PDE addition indicates the level of immobilized PDEs on the cuvette sensor surface. After PDEs immobilization, different concentrations of YTX were added. As Figure 2A shows, the response after YTX addition increases with the toxin dose and follows a typical association curve profile. In the presence of 1 µM YTX, the response reached 4.81 arc s and, after the addition of 10 µM YTX, the response was 20.01 arc seconds. The data from Figure 2A were analyzed to determine the kinetic constants of YTX-PDEs binding, namely, the apparent association rate constant (kon), the association rate constant (kass), the dissociation rate constant (kdiss), and the kinetic equilibrium dissociation constant (KD). The fitting was satisfactory, as judged by the reduced chi-square values and randomness of the residuals (data not shown). Figure 2B shows a representation of each kon against the corresponding YTX concentration. This plot follows a lineal regression with a correlation coefficient of r ) 0.98. From this representation, the kass (slope of the curve) and the kdiss (y-axis intersection) were obtained with values of 367 ( 152 M-1 s-1 and (6.85 ( 1.98) × 10-4 s-1, respectively. From these constant values, the kinetic equilibrium dissociation constant (KD) for the YTX-PDEs binding was calculated. The value obtained for this constant was 2.80 ( 1.21 µM. The structure-affinity relationship in YTX-PDEs association was then studied by using three YTX analogues: OH-YTX, carboxy-YTX and MTPA-YTX. The OH-

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Figure 3. PDEs-OH-YTX association: (A) Association curves after addition of different amounts of OH-YTX over immobilized PDEs; (B) analysis of ligand binding. Kinetic plot of kon obtained from plot in Figure 3A versus OH-YTX concentration. Representative experiment of N ) 3. Figure 2. Analysis of ligand binding: (A) association curves after addition of different amounts of YTX over the immobilized PDEs; (B) kinetic plot of apparent association rate constant (kon) obtained from plot in Figure 1B versus YTX concentration. Representative experiment of N ) 4.

YTX derivative has a hydroxy group in C45 that may play an important role in biological activity (9). As Figure 3A shows, in the presence of different amounts of OH-YTX, the response was increased from 2 arc s in the presence of 1 µM OH-YTX to 25 arc s in the presence of 10 µM OH-YTX. As Figure 3B shows, the representation of the apparent rate constant obtained from Figure 3A plots versus OH-YTX concentration also follows a lineal regression (r ) 0.96). The values of kinetic constants calculated in this last plot were kass ) 327 ( 52.18 M-1 s-1, a kdiss ) 2.31 × 10-3 ( 9.22 × 10-4 s-1, and KD ) 7.36 ( 0.6 µM OH-YTX, which means a lower affinity of OH-YTX than YTX for PDEs. The carboxy-YTX analogue has a COOH group in C44. In the presence of this derivative, the response was again increased with toxin concentration. As Figure 4A shows, the response increases from 1 arc s with 1 µM carboxyYTX to 22 arc s with 12.5 µM toxin. The representation of kon versus toxin concentration, Figure 4B, follows also a lineal regression, and the kinetic constants in these conditions were kass ) 131.05 ( 25.50 M-1 s-1, kdiss ) 2.89 × 10-3 ( 6.6 × 10-4 s-1, and this gave a KD value of 23 ( 5.13 µM carboxy-YTX, which is indicative of a lower affinity of carboxy-YTX than OH-YTX or YTX.

Finally, the C45 ester MTPA-YTX was used. In this case, Figure 5A, the response was increased from 3 arc s in the presence of 1 µM MTPA-YTX to 40 arc s in the presence of 15 µM MTPA-YTX. The analysis of kon versus MTPA-YTX concentration, Figure 5B, also follows a lineal regression (r ) 0.99), and the kinetic constants values were found to be kass ) 183 ( 37.18 M-1 s-1, a kdiss ) 2.09 × 10-3 ( 3.12 × 10-4 s-1, and KD ) 13 ( 3.81 µM MTPA-YTX. These results are shown in the Table 1. In summary, KD values indicate that MTPA-YTX has a lower affinity than YTX and OH-YTX for PDEs, but a higher affinity than the carboxy derivative.

Discussion Recently, we have shown that YTX activates cAMP hydrolysis by PDEs activation (17) and that YTX binds to PDEs (18). In the present study, we used a cuvettebased resonant mirror biosensor to study the association of YTX and analogues with PDEs immobilized on the sensor surface. This technique allows the monitoring of the binding interactions in real time without the need of radiolabeled ligand. Thus, problems associated with the competitive binding assay, such as the interference of the binding interactions by radioactive label and incomplete separation of bound and free ligand, could be overcome by using the biosensor technique. In addition, because the biosensor method provides real-time recording of the binding interactions, both the equilibrium affinity constant and kinetic rate constants for binding pairs could be derived directly from the measured sensograms.

Yessotoxin and Phosphodiesterases Kinetic Analysis

Figure 4. PDEs-carboxy-YTX association: (A) association curves after addition of different amounts of carboxy-YTX over immobilized PDEs; (B) analysis of ligand binding. Kinetic plot of kon obtained from plot in Figure 4A versus carboxy-YTX concentration. Representative experiment of N ) 3.

The first step in this kind of studies is the ligand immobilization to the cuvette sensor surface. In the case of PDEs, CMD cuvettes are suitable surfaces for immobilization (21). The PDEs immobilization shows a high response, around 150 arc s. This signal did not fall when the cuvette was washed, indicating a good immobilization. The YTX interaction to immobilized PDEs is significantly increased with YTX concentration ,and the kinetic equilibrium dissociation constant of PDEs-YTX binding indicates a high affinity between these two components. The value of this constant 2.80 ( 1.21 µM is in the range of KD values for unions between biological active species (10-11 and 10-4 M). This KD value is similar to the result obtained when PDEs were attached on an aminosilane surface and different amounts of YTX were added (18). The KD value increases when the YTX molecule is modified, indicating a structure-activity relationship. When C44 and C45 are free of functional groups, as for the YTX molecule, the KD value is lower than in the presence of functional groups on these carbons as in the case of the YTX derivatives. The presence in C45 of a hydroxy group significantly increases the kinetic equilibrium dissociation constant value, but this value is even higher when the 45-hydroxy group is esterified with

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Figure 5. PDEs-MTPA-YTX association: (A) association curves after addition of different amounts of MTPA-YTX over immobilized PDEs; (B) analysis of ligand binding. Kinetic plot of kon obtained from plot in Figure 5A versus carboxy-YTX concentration. Representative experiment of N ) 4.

MTPA. In addition, when C44 has a carboxylic group, the KD value is even higher. The increase in KD value indicates a lower affinity of YTX derivatives than YTX for PDEs. All these results indicate that the side chain plays an important role in the YTX activity on PDEs. In fact, we checked the activity of MTPA-YTX over cAMP levels and we found a lower activity than YTX (results not shown). The structure-activity relationship is an important observation, since the absolute configuration of the side chain is not elucidated and there are several natural YTX analogues. The importance of molecule configuration in the biological activity of YTXs has not been extensively studied. Some toxicological studies were done with YTX and the desulfonated derivative. After ip mice injection, it has been reported that YTX desulfonation resulted in damages in liver and pancreas but not in heart, while YTX induced acute heart failure (22). On the other hand, it has been recently described that YTX and homoYTX did show similar lethality, while 45-OHhomoYTX did not cause death (23). Therefore, it might be possible that the functional groups in the side chain decreased the toxic effect of YTX and the PDEs-YTX association.

Table 1. Summary of Kinetic Values Obtained from the Union of PDEs-YTX and Analoguesa kass (M-1 s-1) kdiss (s-1) KD (µM) a

YTX

OH-YTX

carboxy-YTX

MPTA-YTX

367 ( 152 (6.85 ( 1.98) × 10-4 2.80 ( 1.21

327 ( 52.18 2.31 × 10-3 ( 9.22 × 10-4 7.36 ( 0.6

131.05 ( 25.50 2.89 × 10-3 ( 6.6 × 10-4 23 ( 5.13

183 ( 37.18 2.09 × 10-3 ( 3.12 × 10-4 13 ( 3.81

Mean ( SEM of three or four experiments.

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The overall conclusion to this paper is that the agreement between known YTX analogues and their PDE binding, supports our theory that PDE is indeed the functional target of this group of compounds and explains their in vivo potency and effect.

Acknowledgment. This work was funded with grants MCYT BMC2000-0441, SAF2003-08765-C03-02, REN2001-2959-C04-03, REN2003-06598-C02-01, AGL200408268-02-O2/ALI, INIA CAL01-068, Xunta PGIDT99INN26101, PGIDIT03AL26101PR, and PGIDIT04TAL261005PR, FISS REMA-G03-007 and EU VIth Frame Program FOOD-CT-2004-06988 (BIOCOP) and FOOD-CT-2004514055 (DETECTOX).

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Pazos et al. (10) Aune, T., Sorby, R., Yasumoto, T., Ramstad, H., and Landsverk, T. (2001) Comparasion of oral and intraperitoneal toxicity of yessotoxin towards mice. Toxicon 40, 77-82. (11) Draisci, R., Lucentini, L., and Mascioni, A. (2000) Pectenotoxins and yessotoxins: chemistry, toxicology, pharmacology, and analysis. In Seafood and Freshwater Toxins: Pharmacology, Physiology and Detection (Botana, L. M., Ed.) pp 289-324, Marcel Dekker: New York. (12) Huang, J. M., Wu, C. H., and Baden, D. G. (1984) Depolarization action of a red-tide dinoflagellate brevetoxin on axonal membranes. J. Pharmacol. Exp. Ther. 229, 615-621. (13) Inoue, M., Hirama, M., Satake, M. S., Ugiyama, K., and Yasumoto, T. (2003) Inhibition of brevetoxin binding to the voltage-gated sodium channel by gambierol and gambieric acid-A. Toxicon 41, 469-474. (14) De La Rosa, L. A., Alfonso, A., Vilarin˜o, N., Vieytes, M. R., Yasumoto, T., and Botana, L. M. (2001) Modulation of cytosolic calcium levels of human lymphocytes by yessotoxin, a novel marine phycotoxin. Biochem. Pharmacol. 61, 827-833. (15) De La Rosa, L. A., Alfonso, A., Vilarin˜o, N., Vieytes, M. R., Yasumoto, T., and Botana, L. M. (2001) Maitotoxin-induced calcium entry in human lymphocytes-modulation by yessotoxin, Ca+2 channel blockers and kinases. Cell. Signalling 13, 711716. (16) Ramstad, H., Hovgaard, P., Yasumoto, T., Larsen, S., and Aune, T. (2001) Monthly variations in diarrhetic toxins and yessotoxin in shellfish from coast to the inner part of the Sognefjord, Norway. Toxicon 39, 1035-1043. (17) Alfonso, A., De La Rosa, L. A., Vieytes, M. R., Yasumoto, T., and Botana, L. M. (2003) Yessotoxin a novel phycotoxin, activates phosphodiesterase activity. Effect of yessotoxin on cAMP levels in human lymphocytes. Biochem. Pharmacol. 65, 193-208. (18) Pazos, M., Alfonso, A., Vieytes, M., Yasumoto, T., Vieites, J., and Botana, L. (2004) Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions. Anal. Biochem. 335, 112-118. (19) Malmqvist, M. (1993) Biospecific interaction analysis using biosensor technology. Nature 361, 186-187. (20) Davies, R. J., Edwards, P. R., Watts, H. J., Lowe, C. R., Buckle, P. E., Yeung, D. K. T. M., and Pollard-Knight, D. V. (1994) The resonant mirror: a versatile tool for the study of biomolecular interactions. In Techniques in Protein Chemestry V (Crabb, J. B., Ed.) pp 285-292, Academic Press: San Diego, CA. (21) Iasys (1997) Immobilization protocols. A guide to the selection of sensor surfaces. Affinity Sensors, U.K. (22) Terao, K., Ito, E., Yanagi, T., and Yasumoto, T. (1986) Histopathological studies on experimental marine toxin poisoning. Ultrastructural changer in the small intestine and liver of suckling mice induced by dinophysistoxin-1 and pectenotoxin-1. Toxicon 24, 1141-1151. (23) Tubaro, A., Sosa, S., Carbonatto, M., Altinier, G., Vita, F., Melato, M., Satake, M., and Yasumoto, T. (2003) Oral and intraperitoneal acute toxicity studies of yessotoxin and homoyessotoxins in mice. Toxicon 41, 783-792.

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