- Email: [email protected]
Detection and some properties of a high molecular weight toxin in the hypobranchial gland of strawberry conch Strombus luhuanus
Toxicon 105 (2015) 1e3
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
Detection and some properties of a high molecular weight toxin in the hypobranchial gland of strawberry conch Strombus luhuanus Keisuke Abiko, Ayane Iwayama, Kazuo Shiomi*, 1 Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo 108-8477, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 May 2015 Received in revised form 22 June 2015 Accepted 5 August 2015 Available online 20 August 2015
The extract from the hypobranchial gland of strawberry conch Strombus luhuanus was found to be lethal to mice. There were no marked regional and seasonal variations in toxicity although a considerable individual variation was recognized. The toxin was thermostable and extractable with aqueous solvents but not with organic solvents. Behaviors in dialysis, ultrafiltration and column chromatography on various adsorbents suggested that the toxin is a high molecular weight acidic substance of 400e500 k. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Hypobranchial gland Marine gastropod Strawberry conch Strombus luhuanus Toxin
Some marine gastropods are known to contain toxins presumably as offensive and/or defensive substances. So far, the following five classes of gastropod toxins have been well characterized: peptide toxins (conotoxins) in the venom of cone shell (genus Conus) (Terlau and Olivera, 2004; Olivera, 2006; Brady et al., 2013), proteinaceous toxins (echotoxins) in the salivary gland of Monoplex echo (Shiomi et al., 2002; Kawashima et al., 2003; Gunji et al., 2010), tetramine in the salivary gland of buccinid gastropods (Anthoni et al., 1989; Kawashima et al., 2002), choline esters in the hypobranchial gland of muricid gastropods (Roseghini et al., 1996; Shiomi et al., 1998) and 6-bromo-2-mercaptotryptamine (potassium channel toxin) in the hypobranchial gland of Calliostoma canaliculatum (Kelley et al., 2003). We recently found that the strawberry conch Strombus luhuanus (Fig. 1) belonging to the family Strombidae possesses a new class of high molecular weight toxin in the hypobranchial gland. This paper deals with the detection and some properties of the hypobranchial gland toxin of S. luhuanus. S. luhuanus is an edible gastropod with a shell height of 5e6 cm that inhabits the tropical and subtropical waters in the Indo-Pacific Ocean. The shell of this gastropod is very similar to but easily distinguishable from that of a cone shell by having an indentation
* Corresponding author. E-mail address: [email protected] (K. Shiomi). 1 Permanent address: 6-10-16-703 Naruse, Machida-shi, Tokyo 194-0044, Japan. http://dx.doi.org/10.1016/j.toxicon.2015.08.011 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
(called stromboid notch) near the anterior end (Fig. 1A). In August 2010, boiled unshelled specimens of S. luhuanus were imported for food from the Philippines to Japan and inspected for paralytic shellfish poison (PSP) at Osaka Quarantine Station. As a result, the extract from the soft tissue was found to be toxic, although the death time of mice was more than 45 min (up to 12 h), being much longer than that (within 15 min) by PSP. In addition, a peculiar symptom, hemoptysis, was observed in dead mice. Pathological examination of the dead mice revealed an extensive hemorrhage per rhexis of pulmonary artery, alveolus capillary and pulmonary vein (personal communication from Professor K. Mitsumori, Tokyo University of Agriculture and Technology). These results indicated that S. luhuanus contains a new class of toxin clearly differing from PSP. To clarify which tissue of S. luhuanus is toxic, foot muscle (11.1 g), digestive gland (2.3 g), gonad (1.0 g), salivary gland (0.05 g), rectum (1.0 g), mantle (2.2 g), style sac (0.2 g) and hypobranchial gland (2.0 g) were collected from five boiled specimens from the Philippines (refer to Fig. 1B for the position of each tissue). Each tissue was homogenized in four volumes (nine volumes only for salivary gland) of PBS (0.15 M NaCle0.01 M phosphate buffer, pH 7.0). After centrifugation, the supernatant obtained was used as an extract. Lethal activity of the extract was assayed using male mice (ddY strain) weighing about 20 g purchased from Sankyo Labo Service (Tokyo, Japan); mouse experiments were performed in compliance with the Regulations for Animal Experiments in Tokyo
2
K. Abiko et al. / Toxicon 105 (2015) 1e3
Fig. 1. Pictures of the shell (A) and various tissues (B) of strawberry conch Strombus luhuanus.
University of Marine Science and Technology and also were approved by the Animal Experiment Committee in Tokyo University of Marine Science and Technology. The tissue extract (or its serial 2-fold dilutions) was injected i.v. into groups of two mice at 10 ml/g of mouse body weight. When both mice of each group were killed, lethal activity was judged to be positive and expressed in terms of titer, which is the reciprocal of the highest dilution giving a positive result. Mouse experiments revealed that only the extract of hypobranchial gland was lethal to mice with a titer of 128. The extract of hypobranchial gland also exhibited mouse lethality (titer 16) on i.p. injection (1 ml/mouse) but did not on p.o. injection (10 ml/g of mouse body weight). As described above, the hypobranchial gland was the sole toxic tissue of S. luhuanus. In addition, the hypobranchial glands of five boiled specimens from the Philippines were all found to be highly toxic (titer 64e512) when their water extracts were individually examined (Table 1). The hypobranchial gland toxin is unlikely to be exogeneous, allowing us to assume that S. luhuanus specimens inhabiting the coast of Japan are also toxic. To confirm this assumption, frozen unshelled specimens collected in Okinawa, Oita and Nagasaki Prefectures were purchased from local retail shops. Each unshelled specimen was heated in a boiling water bath for 10 min to easily take soft tissues out of the shell. Then, the hypobranchial gland from each soft-tissue sample was homogenized in four volumes of distilled water and heated in a boiling water bath for 30 min. After cooling and centrifugation, the supernatant was subjected to mouse assay (i.v. injection). As expected, Japanese specimens were all toxic (Table 1). The results obtained with a total of 35 specimens (including 5 Philippines specimens and 30 Japanese specimens) showed that the toxicity has a large individual variation (titer 8e512) but no marked regional and seasonal variations. The hypobranchial gland toxin of S. luhuanus was effectively extracted with aqueous solvents (distilled water, PBS and 0.1% acetic acid), regardless of heating or non-heating, but not with organic solvents (methanol and acetone), implying that the toxin is thermostable and water-soluble. Some physicochemical properties of the toxin were examined using the water extract (titer 128) prepared from the hypobranchial gland of a boiled sample from the Philippines. Since the hypobranchial gland extract was extremely viscous, it was diluted 1:8 or 1:16 with distilled water (or appropriate buffers) prior to the following experiments. When the extract was diluted 1:8 with 0.1 M glycine-HCl buffer (pH 2.0) and
incubated with pepsin (1 mg/ml) at 37 C for 24 h, no loss of toxicity was recognized. On dialysis using a membrane with a molecular weight cutoff (MWCO) of 3000, the toxin was detected only in the inner solution. Similarly, when the extract was subjected to ultrafiltration using a Vivaspin 20 with an MWCO of 10000 (Sartorius
Table 1 Mouse lethal activity (i.v. injection) of hypobranchial gland of Strombus luhuanus specimens from four different places. Sample Place of collection
Date of collection
No.
Weight of hypobranchial gland (g)
Philippines
August 2010
Okinawa prefecture
September 2010
Oita prefecture
October 2011
Nagasaki prefecture
May 2011
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
0.39 0.38 0.39 0.42 0.51 0.20 0.22 0.14 0.29 0.19 0.12 0.27 0.31 0.30 0.36 0.37 0.39 0.38 0.46 0.58 0.49 0.29 0.39 0.40 0.65 0.34 0.37 0.71 0.61 0.68 0.31 0.47 0.28 0.41 0.48
June 2011
July 2011
October 2011
Mouse lethal activity (titer)
64 128 128 128 512 64 64 128 128 256 8 32 32 128 128 64 64 256 256 256 64 128 256 256 256 32 32 32 128 128 128 128 256 256 256
K. Abiko et al. / Toxicon 105 (2015) 1e3
Stedium Japan, Tokyo), the toxin did not pass through the filter. In gel filtration HPLC on Superdex 75 (separation for 3e70 kDa proteins; GE Healthcare) and Superdex 200 (separation for 10e600 kDa proteins; GE Healthcare), the toxin was eluted at the void volume and immediately after the void volume, respectively. In cation-exchange HPLC on Mono S (GE Healthcare), the toxin was unadsorbed on the column. On the other hand, in anion-exchange HPLC on Mono Q (GE Healthcare), the toxin was adsorbed on the column but could not be eluted from the column even with 1 M NaCl in 0.01 M phosphate buffer (pH 8.0). Furthermore, the toxin exhibited no interaction with a HiTrap Con A 4B column (GE Healthcare). This study established the occurrence of a water-soluble toxin in the hypobranchial gland of strawberry conch S. luhuanus. Behaviors in dialysis, ultrafiltration and column chromatography on various adsorbents suggested that the toxin is a high molecular weight acidic substance (protein, glycoprotein or polysaccharide) of 400e500 k, although it was very stable to heating. The S. luhuanus toxin is the first high molecular weight toxin in the hypobranchial gland of marine gastropods and also is clearly distinguished from proteinaceous toxins (echotoxins) in the salivary gland of Monoplex echo (Shiomi et al., 2002; Kawashima et al., 2003; Gunji et al., 2010) with respect to molecular weight. However, it should be noted that the toxic fractions obtained after dialysis, ultrafiltration and HPLC were highly viscous when concentrated, similar to the crude extract from the hypobranchial gland. In addition, the toxin exhibited unusual behaviors in anion-exchange HPLC. These circumstances hampered further purification and biochemical characterization of the toxin. It is necessary to develop a sophisticated purification method that will lead to the elucidation of detailed properties and primary structure of the S. luhuanus toxin. Finally, it is worth mentioning that the S. luhuanus toxin is not lethal to mice on p.o. injection. Based on this fact, together with no previous records on food poisoning due to ingestion of S. luhuanus, we assume that the S. luhuanus toxin poses no serious risk to human health. Ethical statement This work has not been submitted to any other journal for publication. All authors have read and agreed with the contents of the manuscript and consent to its publication. Animal experiments in the work were performed in compliance with the Regulations for Animal Experiments in Tokyo University of Marine Science and Technology. Also, the experiments were
3
approved by the Animal Experiment Committee in Tokyo University of Marine Science and Technology. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgment This study was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare of Japan (H22-Food-General-011). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2015.08.011. References Anthoni, U., Bohlin, L., Larsen, C., Nielsen, P., Nielsen, N.H., Christophersen, C., 1989. Tetramine: occurrence in marine organisms and pharmacology. Toxicon 27, 707e716. Brady, R.M., Baell, J.B., Norton, R.S., 2013. Strategies for the development of conotoxins as new therapeutic leads. Mar. Drugs 11, 2293e2313. Gunji, K., Ishizaki, S., Shiomi, K., 2010. Cloning of complementary and genomic DNAs encoding echotoxins, proteinaceous toxins from the salivary gland of marine gastropod Monoplex echo. Protein J. 29, 487e492. Kawashima, Y., Nagashima, Y., Shiomi, K., 2002. Toxicity and tetramine contents of salivary glands from carnivorous gastropods. J. Food Hyg. Soc. Jpn. 43, 385e388. Kawashima, Y., Nagai, H., Ishida, M., Nagashima, Y., Shiomi, K., 2003. Primary structure of echotoxin 2, an actinoporin-like hemolytic toxin from the salivary gland of the marine gastropod Monoplex echo. Toxicon 42, 491e497. Kelley, W.P., Wolters, A.M., Sack, J.T., Jockusch, R.A., Jurchen, J.C., Williams, E.R., Sweedler, J.V., Gilly, W.F., 2003. Characterization of a novel gastropod toxin (6bromo-2-mercaptotryptamine) that inhibits shaker K channel activity. J. Biol. Chem. 278, 34934e34942. Olivera, B.M., 2006. Conus peptides: biodiversity-based discovery and exogenomics. J. Biol. Chem. 281, 31173e31177. Roseghini, M., Severini, C., Erspamer, G.F., Vittorio, E., 1996. Choline esters and biogenic amines in the hypobranchial gland of 55 molluscan species of the neogastropod muricoidea superfamily. Toxicon 34, 33e55. Shiomi, K., Ishii, M., Shimakura, K., Nagashima, Y., Chino, M., 1998. Tigloylcholine: a new choline ester toxin from the hypobranchial gland of two species of muricid gastropods (Thais clavigera and Thais bronni). Toxicon 36, 795e798. Shiomi, K., Kawashima, Y., Mizukami, M., Nagashima, Y., 2002. Properties of proteinaceous toxins in the salivary gland of the marine gastropod (Monoplex echo). Toxicon 40, 563e571. Terlau, H., Olivera, B.M., 2004. Conus venoms: a rich source of novel ion channeltargeted peptides. Physiol. Rev. 84, 41e68.
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Short communication
Detection and some properties of a high molecular weight toxin in the hypobranchial gland of strawberry conch Strombus luhuanus Keisuke Abiko, Ayane Iwayama, Kazuo Shiomi*, 1 Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Konan-4, Minato-ku, Tokyo 108-8477, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 May 2015 Received in revised form 22 June 2015 Accepted 5 August 2015 Available online 20 August 2015
The extract from the hypobranchial gland of strawberry conch Strombus luhuanus was found to be lethal to mice. There were no marked regional and seasonal variations in toxicity although a considerable individual variation was recognized. The toxin was thermostable and extractable with aqueous solvents but not with organic solvents. Behaviors in dialysis, ultrafiltration and column chromatography on various adsorbents suggested that the toxin is a high molecular weight acidic substance of 400e500 k. © 2015 Elsevier Ltd. All rights reserved.
Keywords: Hypobranchial gland Marine gastropod Strawberry conch Strombus luhuanus Toxin
Some marine gastropods are known to contain toxins presumably as offensive and/or defensive substances. So far, the following five classes of gastropod toxins have been well characterized: peptide toxins (conotoxins) in the venom of cone shell (genus Conus) (Terlau and Olivera, 2004; Olivera, 2006; Brady et al., 2013), proteinaceous toxins (echotoxins) in the salivary gland of Monoplex echo (Shiomi et al., 2002; Kawashima et al., 2003; Gunji et al., 2010), tetramine in the salivary gland of buccinid gastropods (Anthoni et al., 1989; Kawashima et al., 2002), choline esters in the hypobranchial gland of muricid gastropods (Roseghini et al., 1996; Shiomi et al., 1998) and 6-bromo-2-mercaptotryptamine (potassium channel toxin) in the hypobranchial gland of Calliostoma canaliculatum (Kelley et al., 2003). We recently found that the strawberry conch Strombus luhuanus (Fig. 1) belonging to the family Strombidae possesses a new class of high molecular weight toxin in the hypobranchial gland. This paper deals with the detection and some properties of the hypobranchial gland toxin of S. luhuanus. S. luhuanus is an edible gastropod with a shell height of 5e6 cm that inhabits the tropical and subtropical waters in the Indo-Pacific Ocean. The shell of this gastropod is very similar to but easily distinguishable from that of a cone shell by having an indentation
* Corresponding author. E-mail address: [email protected] (K. Shiomi). 1 Permanent address: 6-10-16-703 Naruse, Machida-shi, Tokyo 194-0044, Japan. http://dx.doi.org/10.1016/j.toxicon.2015.08.011 0041-0101/© 2015 Elsevier Ltd. All rights reserved.
(called stromboid notch) near the anterior end (Fig. 1A). In August 2010, boiled unshelled specimens of S. luhuanus were imported for food from the Philippines to Japan and inspected for paralytic shellfish poison (PSP) at Osaka Quarantine Station. As a result, the extract from the soft tissue was found to be toxic, although the death time of mice was more than 45 min (up to 12 h), being much longer than that (within 15 min) by PSP. In addition, a peculiar symptom, hemoptysis, was observed in dead mice. Pathological examination of the dead mice revealed an extensive hemorrhage per rhexis of pulmonary artery, alveolus capillary and pulmonary vein (personal communication from Professor K. Mitsumori, Tokyo University of Agriculture and Technology). These results indicated that S. luhuanus contains a new class of toxin clearly differing from PSP. To clarify which tissue of S. luhuanus is toxic, foot muscle (11.1 g), digestive gland (2.3 g), gonad (1.0 g), salivary gland (0.05 g), rectum (1.0 g), mantle (2.2 g), style sac (0.2 g) and hypobranchial gland (2.0 g) were collected from five boiled specimens from the Philippines (refer to Fig. 1B for the position of each tissue). Each tissue was homogenized in four volumes (nine volumes only for salivary gland) of PBS (0.15 M NaCle0.01 M phosphate buffer, pH 7.0). After centrifugation, the supernatant obtained was used as an extract. Lethal activity of the extract was assayed using male mice (ddY strain) weighing about 20 g purchased from Sankyo Labo Service (Tokyo, Japan); mouse experiments were performed in compliance with the Regulations for Animal Experiments in Tokyo
2
K. Abiko et al. / Toxicon 105 (2015) 1e3
Fig. 1. Pictures of the shell (A) and various tissues (B) of strawberry conch Strombus luhuanus.
University of Marine Science and Technology and also were approved by the Animal Experiment Committee in Tokyo University of Marine Science and Technology. The tissue extract (or its serial 2-fold dilutions) was injected i.v. into groups of two mice at 10 ml/g of mouse body weight. When both mice of each group were killed, lethal activity was judged to be positive and expressed in terms of titer, which is the reciprocal of the highest dilution giving a positive result. Mouse experiments revealed that only the extract of hypobranchial gland was lethal to mice with a titer of 128. The extract of hypobranchial gland also exhibited mouse lethality (titer 16) on i.p. injection (1 ml/mouse) but did not on p.o. injection (10 ml/g of mouse body weight). As described above, the hypobranchial gland was the sole toxic tissue of S. luhuanus. In addition, the hypobranchial glands of five boiled specimens from the Philippines were all found to be highly toxic (titer 64e512) when their water extracts were individually examined (Table 1). The hypobranchial gland toxin is unlikely to be exogeneous, allowing us to assume that S. luhuanus specimens inhabiting the coast of Japan are also toxic. To confirm this assumption, frozen unshelled specimens collected in Okinawa, Oita and Nagasaki Prefectures were purchased from local retail shops. Each unshelled specimen was heated in a boiling water bath for 10 min to easily take soft tissues out of the shell. Then, the hypobranchial gland from each soft-tissue sample was homogenized in four volumes of distilled water and heated in a boiling water bath for 30 min. After cooling and centrifugation, the supernatant was subjected to mouse assay (i.v. injection). As expected, Japanese specimens were all toxic (Table 1). The results obtained with a total of 35 specimens (including 5 Philippines specimens and 30 Japanese specimens) showed that the toxicity has a large individual variation (titer 8e512) but no marked regional and seasonal variations. The hypobranchial gland toxin of S. luhuanus was effectively extracted with aqueous solvents (distilled water, PBS and 0.1% acetic acid), regardless of heating or non-heating, but not with organic solvents (methanol and acetone), implying that the toxin is thermostable and water-soluble. Some physicochemical properties of the toxin were examined using the water extract (titer 128) prepared from the hypobranchial gland of a boiled sample from the Philippines. Since the hypobranchial gland extract was extremely viscous, it was diluted 1:8 or 1:16 with distilled water (or appropriate buffers) prior to the following experiments. When the extract was diluted 1:8 with 0.1 M glycine-HCl buffer (pH 2.0) and
incubated with pepsin (1 mg/ml) at 37 C for 24 h, no loss of toxicity was recognized. On dialysis using a membrane with a molecular weight cutoff (MWCO) of 3000, the toxin was detected only in the inner solution. Similarly, when the extract was subjected to ultrafiltration using a Vivaspin 20 with an MWCO of 10000 (Sartorius
Table 1 Mouse lethal activity (i.v. injection) of hypobranchial gland of Strombus luhuanus specimens from four different places. Sample Place of collection
Date of collection
No.
Weight of hypobranchial gland (g)
Philippines
August 2010
Okinawa prefecture
September 2010
Oita prefecture
October 2011
Nagasaki prefecture
May 2011
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
0.39 0.38 0.39 0.42 0.51 0.20 0.22 0.14 0.29 0.19 0.12 0.27 0.31 0.30 0.36 0.37 0.39 0.38 0.46 0.58 0.49 0.29 0.39 0.40 0.65 0.34 0.37 0.71 0.61 0.68 0.31 0.47 0.28 0.41 0.48
June 2011
July 2011
October 2011
Mouse lethal activity (titer)
64 128 128 128 512 64 64 128 128 256 8 32 32 128 128 64 64 256 256 256 64 128 256 256 256 32 32 32 128 128 128 128 256 256 256
K. Abiko et al. / Toxicon 105 (2015) 1e3
Stedium Japan, Tokyo), the toxin did not pass through the filter. In gel filtration HPLC on Superdex 75 (separation for 3e70 kDa proteins; GE Healthcare) and Superdex 200 (separation for 10e600 kDa proteins; GE Healthcare), the toxin was eluted at the void volume and immediately after the void volume, respectively. In cation-exchange HPLC on Mono S (GE Healthcare), the toxin was unadsorbed on the column. On the other hand, in anion-exchange HPLC on Mono Q (GE Healthcare), the toxin was adsorbed on the column but could not be eluted from the column even with 1 M NaCl in 0.01 M phosphate buffer (pH 8.0). Furthermore, the toxin exhibited no interaction with a HiTrap Con A 4B column (GE Healthcare). This study established the occurrence of a water-soluble toxin in the hypobranchial gland of strawberry conch S. luhuanus. Behaviors in dialysis, ultrafiltration and column chromatography on various adsorbents suggested that the toxin is a high molecular weight acidic substance (protein, glycoprotein or polysaccharide) of 400e500 k, although it was very stable to heating. The S. luhuanus toxin is the first high molecular weight toxin in the hypobranchial gland of marine gastropods and also is clearly distinguished from proteinaceous toxins (echotoxins) in the salivary gland of Monoplex echo (Shiomi et al., 2002; Kawashima et al., 2003; Gunji et al., 2010) with respect to molecular weight. However, it should be noted that the toxic fractions obtained after dialysis, ultrafiltration and HPLC were highly viscous when concentrated, similar to the crude extract from the hypobranchial gland. In addition, the toxin exhibited unusual behaviors in anion-exchange HPLC. These circumstances hampered further purification and biochemical characterization of the toxin. It is necessary to develop a sophisticated purification method that will lead to the elucidation of detailed properties and primary structure of the S. luhuanus toxin. Finally, it is worth mentioning that the S. luhuanus toxin is not lethal to mice on p.o. injection. Based on this fact, together with no previous records on food poisoning due to ingestion of S. luhuanus, we assume that the S. luhuanus toxin poses no serious risk to human health. Ethical statement This work has not been submitted to any other journal for publication. All authors have read and agreed with the contents of the manuscript and consent to its publication. Animal experiments in the work were performed in compliance with the Regulations for Animal Experiments in Tokyo University of Marine Science and Technology. Also, the experiments were
3
approved by the Animal Experiment Committee in Tokyo University of Marine Science and Technology. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgment This study was partly supported by a Grant-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare of Japan (H22-Food-General-011). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.toxicon.2015.08.011. References Anthoni, U., Bohlin, L., Larsen, C., Nielsen, P., Nielsen, N.H., Christophersen, C., 1989. Tetramine: occurrence in marine organisms and pharmacology. Toxicon 27, 707e716. Brady, R.M., Baell, J.B., Norton, R.S., 2013. Strategies for the development of conotoxins as new therapeutic leads. Mar. Drugs 11, 2293e2313. Gunji, K., Ishizaki, S., Shiomi, K., 2010. Cloning of complementary and genomic DNAs encoding echotoxins, proteinaceous toxins from the salivary gland of marine gastropod Monoplex echo. Protein J. 29, 487e492. Kawashima, Y., Nagashima, Y., Shiomi, K., 2002. Toxicity and tetramine contents of salivary glands from carnivorous gastropods. J. Food Hyg. Soc. Jpn. 43, 385e388. Kawashima, Y., Nagai, H., Ishida, M., Nagashima, Y., Shiomi, K., 2003. Primary structure of echotoxin 2, an actinoporin-like hemolytic toxin from the salivary gland of the marine gastropod Monoplex echo. Toxicon 42, 491e497. Kelley, W.P., Wolters, A.M., Sack, J.T., Jockusch, R.A., Jurchen, J.C., Williams, E.R., Sweedler, J.V., Gilly, W.F., 2003. Characterization of a novel gastropod toxin (6bromo-2-mercaptotryptamine) that inhibits shaker K channel activity. J. Biol. Chem. 278, 34934e34942. Olivera, B.M., 2006. Conus peptides: biodiversity-based discovery and exogenomics. J. Biol. Chem. 281, 31173e31177. Roseghini, M., Severini, C., Erspamer, G.F., Vittorio, E., 1996. Choline esters and biogenic amines in the hypobranchial gland of 55 molluscan species of the neogastropod muricoidea superfamily. Toxicon 34, 33e55. Shiomi, K., Ishii, M., Shimakura, K., Nagashima, Y., Chino, M., 1998. Tigloylcholine: a new choline ester toxin from the hypobranchial gland of two species of muricid gastropods (Thais clavigera and Thais bronni). Toxicon 36, 795e798. Shiomi, K., Kawashima, Y., Mizukami, M., Nagashima, Y., 2002. Properties of proteinaceous toxins in the salivary gland of the marine gastropod (Monoplex echo). Toxicon 40, 563e571. Terlau, H., Olivera, B.M., 2004. Conus venoms: a rich source of novel ion channeltargeted peptides. Physiol. Rev. 84, 41e68.