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Yuji Nagashima - One of the best experts on this subject based on the ideXlab platform.
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DNA Microarray Analysis on the Genes Differentially Expressed in the Liver of the Pufferfish, Takifugu rubripes, Following an Intramuscular Administration of Tetrodotoxin
MDPI AG, 2014Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hidehiro Kondo, Holger Feroudj, Ryosuke Kikuchi, Yuriko Kawana, Ikuo Hirono, Toshiaki Mochizuki, Gen Kaneko, Hideki UshioAbstract:Pufferfish accumulate tetrodotoxin (TTX) mainly in the liver and ovary. This study aims at investigating the effect of TTX accumulation in the liver of cultured specimens of torafugu Takifugu rubripes on the hepatic gene expression by microarray analysis on Day 5 after the intramuscular administration of 0.25 mg TTX/kg body weight into the caudal muscle. TTX was detected in the liver, skin and ovary in the TTX-administered individuals. The total amount of TTX accumulated in the body was 67 ± 8% of the administered dose on Day 5. Compared with the buffer-administered control group, a total of 59 genes were significantly upregulated more than two-fold in the TTX-administered group, including those encoding chymotrypsin-like elastase family member 2A, transmembrane protein 168 and Rho GTP-activating protein 29. In contrast, a total of 427 genes were downregulated by TTX administration, including those encoding elongation factor G2, R-spondin-3, nuclear receptor activator 2 and fatty acyl-CoA hydrolase precursor. In conclusion, our results demonstrate that the intramuscular administration of TTX changes the expression of hepatic genes involved in various signaling pathways
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differential gene expression profile in the liver of the marine puffer fish Takifugu rubripes induced by intramuscular administration of tetrodotoxin
Toxicon, 2011Co-Authors: Takuya Matsumoto, Shoichiro Ishizaki, Yuji NagashimaAbstract:Marine puffer fish accumulate a high level of tetrodotoxin (TTX) in the liver and ovary, but the underlying mechanism of this toxification is unclear. To elucidate the genes related to toxification of the marine puffer fish, we examined the hepatic gene expression profile of the marine puffer fish Takifugu rubripes by suppression subtractive hybridization in response to the intramuscular administration of 0.50 mg TTX/kg body weight into the caudal muscle. The accumulation of TTX in the liver reached 68 ± 4% that of the administered dose within 12 h of administration. A total of 1048 clones from the subtracted cDNA libraries were successfully sequenced. The nucleotide sequence of 92 of the 1048 clones was identified as a hepcidin precursor. Reverse transcription-polymerase chain reaction experiments revealed that hepcidin precursors were highly expressed in the TTX-administered group. In addition, complement C3 (31 clones), serotransferrin (30 clones), apolipoprotein A-1 (14 clones), high temperature adaptation protein Wap65-2 (14 clones), complement C7 (12 clones), fibrinogen beta chain (12 clones), and 70 kDa heat-shock protein 4 (11 clones) were obtained. This study confirmed that the intramuscular administration of TTX increases the gene expression of the acute-phase response proteins in the liver of puffer fish T. rubripes.
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plasma protein binding of tetrodotoxin in the marine puffer fish Takifugu rubripes
Toxicon, 2010Co-Authors: Takuya Matsumoto, Shoichiro Ishizaki, Daisuke Tanuma, Kazuma Tsutsumi, Joongkyun Jeon, Yuji NagashimaAbstract:Abstract To elucidate the involvement of plasma protein binding in the disposition of tetrodotoxin (TTX) in puffer fish, we used equilibrium dialysis to measure protein binding of TTX in the plasma of the marine puffer fish Takifugu rubripes and the non-toxic greenling Hexagrammos otakii , and in solutions of bovine serum albumin (BSA) and bovine alpha-1-acid glycoprotein (AGP). TTX (100–1000 μg/mL) bound to protein in T. rubripes plasma with low affinity in a non-saturable manner. The amount of bound TTX increased linearly with the TTX concentration, reaching 3.92 ± 0.42 μg TTX/mg protein at 1000 μg TTX/mL. Approximately 80% of the TTX in the plasma of T. rubripes was unbound in the concentration range of TTX examined, indicating that TTX exists predominantly in the unbound form in the circulating blood of T. rubripes at a wide range of TTX concentrations. TTX also bound non-specifically to H. otakii plasma proteins, BSA, and bovine AGP. The amount of the bound TTX in the plasma of H. otakii and BSA, respectively, was 1.86 ± 0.36 and 4.65 ± 0.70 μg TTX/mg protein at 1000 μg TTX/mL, and that in the bovine AGP was 8.78 ± 0.25 μg TTX/mg protein at 200 μg TTX/mL.
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change in tetrodotoxin content of the puffer fish Takifugu rubripes during seed production from fertilized eggs to juveniles
Journal of The Food Hygienic Society of Japan (shokuhin Eiseigaku Zasshi), 2010Co-Authors: Yuji Nagashima, Kuniyoshi Shimakura, Isao Mataki, Maho Toyoda, Hiroshi Nakajima, Kingo Tsumoto, Kazuo ShiomiAbstract:Changes in tetrodotoxin (TTX) content of the puffer fish Takifugu rubripes during seed production were examined. Two mature female puffer fish T. rubripes (samples 1 and 2) that contained TTX were used. The toxic eggs were artificially fertilized, and hatchlings were reared in an indoor tank for 50 days and then in a netcage at sea for an additional 48 or 38 days. The TTX content of the fertilized eggs of sample 1 was initially 13.0 microg TTX/g, transiently increased to 67.6 microg TTX/g at 4 days after hatching, and then gradually decreased to 0.28 microg TTX/g at 98 days. In contrast, the total TTX content in an individual was 0.016 microg TTX at the fertilization stage and 0.01-0.03 microg TTX at the larval stage until 30 days after hatching. Thereafter, the total TTX content increased remarkably during culture in the netcage at sea, reaching 4.80 microg TTX at 98 days. Change in the TTX content of sample 2 showed a similar tendency to that of sample 1. The present study showed that the TTX content per gram of puffer fish body weight decreased during progression from fertilized eggs to juveniles, whereas the total TTX content increased.
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evaluation of hepatic uptake clearance of tetrodotoxin in the puffer fish Takifugu rubripes
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Abstract In this study, we investigated the hepatic uptake clearance (CL uptake ) of tetrodotoxin (TTX) in the marine puffer fish Takifugu rubripes by integration plot analysis after a single bolus injection of 0.25 mg TTX/kg body weight into the hepatic vein at 20 °C. The blood concentration of TTX decreased over time after the injection, from 1451 ± 45 ng/mL at 10 min to 364 ± 59 ng/mL at 60 min. TTX concentrations in the spleen and kidney decreased in parallel with the blood concentrations, whereas those in the muscle and skin remained almost the same throughout the experiment. In contrast, the TTX concentration in the liver gradually increased, reaching 1240 ± 90 ng/g liver at 60 min after injection. The amount of TTX that had accumulated in the liver 60 min after injection accounted for 63 ± 5% of the administered dose. Integration plot analysis indicated a CL uptake of 3.1 mL/min/kg body weight in the liver for TTX, a rate far below that of the hepatic portal vein blood flow rate (at most, 9%). This finding is consistent with negligible extraction of TTX by the liver. The results demonstrated conclusively that the liver-specific distribution of TTX in T. rubripes is achieved by removal from the systemic circulation, but not by the hepatic first-pass effect.
Takuya Matsumoto - One of the best experts on this subject based on the ideXlab platform.
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DNA Microarray Analysis on the Genes Differentially Expressed in the Liver of the Pufferfish, Takifugu rubripes, Following an Intramuscular Administration of Tetrodotoxin
MDPI AG, 2014Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hidehiro Kondo, Holger Feroudj, Ryosuke Kikuchi, Yuriko Kawana, Ikuo Hirono, Toshiaki Mochizuki, Gen Kaneko, Hideki UshioAbstract:Pufferfish accumulate tetrodotoxin (TTX) mainly in the liver and ovary. This study aims at investigating the effect of TTX accumulation in the liver of cultured specimens of torafugu Takifugu rubripes on the hepatic gene expression by microarray analysis on Day 5 after the intramuscular administration of 0.25 mg TTX/kg body weight into the caudal muscle. TTX was detected in the liver, skin and ovary in the TTX-administered individuals. The total amount of TTX accumulated in the body was 67 ± 8% of the administered dose on Day 5. Compared with the buffer-administered control group, a total of 59 genes were significantly upregulated more than two-fold in the TTX-administered group, including those encoding chymotrypsin-like elastase family member 2A, transmembrane protein 168 and Rho GTP-activating protein 29. In contrast, a total of 427 genes were downregulated by TTX administration, including those encoding elongation factor G2, R-spondin-3, nuclear receptor activator 2 and fatty acyl-CoA hydrolase precursor. In conclusion, our results demonstrate that the intramuscular administration of TTX changes the expression of hepatic genes involved in various signaling pathways
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differential gene expression profile in the liver of the marine puffer fish Takifugu rubripes induced by intramuscular administration of tetrodotoxin
Toxicon, 2011Co-Authors: Takuya Matsumoto, Shoichiro Ishizaki, Yuji NagashimaAbstract:Marine puffer fish accumulate a high level of tetrodotoxin (TTX) in the liver and ovary, but the underlying mechanism of this toxification is unclear. To elucidate the genes related to toxification of the marine puffer fish, we examined the hepatic gene expression profile of the marine puffer fish Takifugu rubripes by suppression subtractive hybridization in response to the intramuscular administration of 0.50 mg TTX/kg body weight into the caudal muscle. The accumulation of TTX in the liver reached 68 ± 4% that of the administered dose within 12 h of administration. A total of 1048 clones from the subtracted cDNA libraries were successfully sequenced. The nucleotide sequence of 92 of the 1048 clones was identified as a hepcidin precursor. Reverse transcription-polymerase chain reaction experiments revealed that hepcidin precursors were highly expressed in the TTX-administered group. In addition, complement C3 (31 clones), serotransferrin (30 clones), apolipoprotein A-1 (14 clones), high temperature adaptation protein Wap65-2 (14 clones), complement C7 (12 clones), fibrinogen beta chain (12 clones), and 70 kDa heat-shock protein 4 (11 clones) were obtained. This study confirmed that the intramuscular administration of TTX increases the gene expression of the acute-phase response proteins in the liver of puffer fish T. rubripes.
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plasma protein binding of tetrodotoxin in the marine puffer fish Takifugu rubripes
Toxicon, 2010Co-Authors: Takuya Matsumoto, Shoichiro Ishizaki, Daisuke Tanuma, Kazuma Tsutsumi, Joongkyun Jeon, Yuji NagashimaAbstract:Abstract To elucidate the involvement of plasma protein binding in the disposition of tetrodotoxin (TTX) in puffer fish, we used equilibrium dialysis to measure protein binding of TTX in the plasma of the marine puffer fish Takifugu rubripes and the non-toxic greenling Hexagrammos otakii , and in solutions of bovine serum albumin (BSA) and bovine alpha-1-acid glycoprotein (AGP). TTX (100–1000 μg/mL) bound to protein in T. rubripes plasma with low affinity in a non-saturable manner. The amount of bound TTX increased linearly with the TTX concentration, reaching 3.92 ± 0.42 μg TTX/mg protein at 1000 μg TTX/mL. Approximately 80% of the TTX in the plasma of T. rubripes was unbound in the concentration range of TTX examined, indicating that TTX exists predominantly in the unbound form in the circulating blood of T. rubripes at a wide range of TTX concentrations. TTX also bound non-specifically to H. otakii plasma proteins, BSA, and bovine AGP. The amount of the bound TTX in the plasma of H. otakii and BSA, respectively, was 1.86 ± 0.36 and 4.65 ± 0.70 μg TTX/mg protein at 1000 μg TTX/mL, and that in the bovine AGP was 8.78 ± 0.25 μg TTX/mg protein at 200 μg TTX/mL.
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evaluation of hepatic uptake clearance of tetrodotoxin in the puffer fish Takifugu rubripes
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Abstract In this study, we investigated the hepatic uptake clearance (CL uptake ) of tetrodotoxin (TTX) in the marine puffer fish Takifugu rubripes by integration plot analysis after a single bolus injection of 0.25 mg TTX/kg body weight into the hepatic vein at 20 °C. The blood concentration of TTX decreased over time after the injection, from 1451 ± 45 ng/mL at 10 min to 364 ± 59 ng/mL at 60 min. TTX concentrations in the spleen and kidney decreased in parallel with the blood concentrations, whereas those in the muscle and skin remained almost the same throughout the experiment. In contrast, the TTX concentration in the liver gradually increased, reaching 1240 ± 90 ng/g liver at 60 min after injection. The amount of TTX that had accumulated in the liver 60 min after injection accounted for 63 ± 5% of the administered dose. Integration plot analysis indicated a CL uptake of 3.1 mL/min/kg body weight in the liver for TTX, a rate far below that of the hepatic portal vein blood flow rate (at most, 9%). This finding is consistent with negligible extraction of TTX by the liver. The results demonstrated conclusively that the liver-specific distribution of TTX in T. rubripes is achieved by removal from the systemic circulation, but not by the hepatic first-pass effect.
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pharmacokinetics of tetrodotoxin in puffer fish Takifugu rubripes by a single administration technique
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Abstract Marine puffer fish accumulates tetrodotoxin (TTX) in the liver and ovary. In this study, we examined the pharmacokinetics of TTX in Takifugu rubripes by a single administration under general anesthesia at 20 °C for 300 min. The blood concentration–time profile showed multiple distinct phases after injection into hepatic portal vein. The area under the blood concentration–time curve (AUC) increased linearly at the dosage of 0.25−0.75 mg TTX/kg body weight, and the total body clearance was 2.06±0.17 mL/min/kg body weight. The AUCs following administration into the hepatic portal vein and hepatic vein were closely similar (147±33 versus 141±1 ng·min/μL), indicating negligible hepatic first-pass effect. Comparison of the AUCs following an administration to the hepatic vein and gastrointestinal tract (0.25 mg TTX/kg body weight) elucidated the bioavailability of TTX to be 62%. There was no significant increase in the AUCs following direct injection into the gastrointestinal tract (0.50 versus 1.0 mg TTX/kg body weight). At the dosage of 0.25 mg TTX/kg body weight into the hepatic vein, hepatic portal vein or gastrointestinal tract, TTX amount in the liver accounted for 84±6%, 70±9% or 49±17% of the total TTX amount applied, respectively. These results demonstrate that TTX is absorbed into the systemic circulation from the gastrointestinal tract by saturable mechanism and finally accumulated in the liver within 300 min.
Kazuo Shiomi - One of the best experts on this subject based on the ideXlab platform.
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change in tetrodotoxin content of the puffer fish Takifugu rubripes during seed production from fertilized eggs to juveniles
Journal of The Food Hygienic Society of Japan (shokuhin Eiseigaku Zasshi), 2010Co-Authors: Yuji Nagashima, Kuniyoshi Shimakura, Isao Mataki, Maho Toyoda, Hiroshi Nakajima, Kingo Tsumoto, Kazuo ShiomiAbstract:Changes in tetrodotoxin (TTX) content of the puffer fish Takifugu rubripes during seed production were examined. Two mature female puffer fish T. rubripes (samples 1 and 2) that contained TTX were used. The toxic eggs were artificially fertilized, and hatchlings were reared in an indoor tank for 50 days and then in a netcage at sea for an additional 48 or 38 days. The TTX content of the fertilized eggs of sample 1 was initially 13.0 microg TTX/g, transiently increased to 67.6 microg TTX/g at 4 days after hatching, and then gradually decreased to 0.28 microg TTX/g at 98 days. In contrast, the total TTX content in an individual was 0.016 microg TTX at the fertilization stage and 0.01-0.03 microg TTX at the larval stage until 30 days after hatching. Thereafter, the total TTX content increased remarkably during culture in the netcage at sea, reaching 4.80 microg TTX at 98 days. Change in the TTX content of sample 2 showed a similar tendency to that of sample 1. The present study showed that the TTX content per gram of puffer fish body weight decreased during progression from fertilized eggs to juveniles, whereas the total TTX content increased.
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evaluation of hepatic uptake clearance of tetrodotoxin in the puffer fish Takifugu rubripes
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Abstract In this study, we investigated the hepatic uptake clearance (CL uptake ) of tetrodotoxin (TTX) in the marine puffer fish Takifugu rubripes by integration plot analysis after a single bolus injection of 0.25 mg TTX/kg body weight into the hepatic vein at 20 °C. The blood concentration of TTX decreased over time after the injection, from 1451 ± 45 ng/mL at 10 min to 364 ± 59 ng/mL at 60 min. TTX concentrations in the spleen and kidney decreased in parallel with the blood concentrations, whereas those in the muscle and skin remained almost the same throughout the experiment. In contrast, the TTX concentration in the liver gradually increased, reaching 1240 ± 90 ng/g liver at 60 min after injection. The amount of TTX that had accumulated in the liver 60 min after injection accounted for 63 ± 5% of the administered dose. Integration plot analysis indicated a CL uptake of 3.1 mL/min/kg body weight in the liver for TTX, a rate far below that of the hepatic portal vein blood flow rate (at most, 9%). This finding is consistent with negligible extraction of TTX by the liver. The results demonstrated conclusively that the liver-specific distribution of TTX in T. rubripes is achieved by removal from the systemic circulation, but not by the hepatic first-pass effect.
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pharmacokinetics of tetrodotoxin in puffer fish Takifugu rubripes by a single administration technique
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Abstract Marine puffer fish accumulates tetrodotoxin (TTX) in the liver and ovary. In this study, we examined the pharmacokinetics of TTX in Takifugu rubripes by a single administration under general anesthesia at 20 °C for 300 min. The blood concentration–time profile showed multiple distinct phases after injection into hepatic portal vein. The area under the blood concentration–time curve (AUC) increased linearly at the dosage of 0.25−0.75 mg TTX/kg body weight, and the total body clearance was 2.06±0.17 mL/min/kg body weight. The AUCs following administration into the hepatic portal vein and hepatic vein were closely similar (147±33 versus 141±1 ng·min/μL), indicating negligible hepatic first-pass effect. Comparison of the AUCs following an administration to the hepatic vein and gastrointestinal tract (0.25 mg TTX/kg body weight) elucidated the bioavailability of TTX to be 62%. There was no significant increase in the AUCs following direct injection into the gastrointestinal tract (0.50 versus 1.0 mg TTX/kg body weight). At the dosage of 0.25 mg TTX/kg body weight into the hepatic vein, hepatic portal vein or gastrointestinal tract, TTX amount in the liver accounted for 84±6%, 70±9% or 49±17% of the total TTX amount applied, respectively. These results demonstrate that TTX is absorbed into the systemic circulation from the gastrointestinal tract by saturable mechanism and finally accumulated in the liver within 300 min.
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pharmacokinetics of tetrodotoxin in puffer fish Takifugu rubripes by a single administration technique
Toxicon, 2008Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Kazuo ShiomiAbstract:Marine puffer fish accumulates tetrodotoxin (TTX) in the liver and ovary. In this study, we examined the pharmacokinetics of TTX in Takifugu rubripes by a single administration under general anesthesia at 20 degrees C for 300 min. The blood concentration-time profile showed multiple distinct phases after injection into hepatic portal vein. The area under the blood concentration-time curve (AUC) increased linearly at the dosage of 0.25-0.75 mg TTX/kg body weight, and the total body clearance was 2.06+/-0.17 mL/min/kg body weight. The AUCs following administration into the hepatic portal vein and hepatic vein were closely similar (147+/-33 versus 141+/-1 ng.min/microL), indicating negligible hepatic first-pass effect. Comparison of the AUCs following an administration to the hepatic vein and gastrointestinal tract (0.25 mg TTX/kg body weight) elucidated the bioavailability of TTX to be 62%. There was no significant increase in the AUCs following direct injection into the gastrointestinal tract (0.50 versus 1.0 mg TTX/kg body weight). At the dosage of 0.25 mg TTX/kg body weight into the hepatic vein, hepatic portal vein or gastrointestinal tract, TTX amount in the liver accounted for 84+/-6%, 70+/-9% or 49+/-17% of the total TTX amount applied, respectively. These results demonstrate that TTX is absorbed into the systemic circulation from the gastrointestinal tract by saturable mechanism and finally accumulated in the liver within 300 min.
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involvement of carrier mediated transport system in uptake of tetrodotoxin into liver tissue slices of puffer fish Takifugu rubripes
Toxicon, 2007Co-Authors: Takuya Matsumoto, Yuji Nagashima, Hiroyuki Kusuhara, Shoichiro Ishizaki, Kuniyoshi Shimakura, Yuichi Sugiyama, Kazuo ShiomiAbstract:Although puffer fish contain tetrodotoxin (TTX) at a high concentration mainly in liver, the underlying mechanism remains to be elucidated. In the present study, uptake of TTX into the liver tissue slices of puffer fish Takifugu rubripes was investigated by in vitro incubation experiment. When T. rubripes liver slices were incubated with 0-2000microM TTX at 20 degrees C for 60min, the uptake rates exhibited non-linearity, suggesting that the TTX uptake into T. rubripes liver is carrier-mediated. The TTX uptake was composed of a saturable component (V(max) 47.7+/-5.9pmol/min/mg protein and K(m) 249+/-47microM) and a non-saturable component (P(dif) 0.0335+/-0.0041microL/min/mg protein). The uptake of TTX was significantly decreased to 0.4 and 0.6 fold by the incubation at 5 degrees C and the replacement of sodium-ion by choline in the buffer, respectively, while it was not affected by the presence of 1mM l-carnitine, p-aminohippurate, taurocholate or tetraethylammonium. The TTX uptake by black scraper Thamnaconus modestus liver slices was much lower than that of T. rubripes and independent of the incubation temperature, unlike T. rubripes. These results reveal the involvement of carrier-mediated transport system in the TTX uptake by puffer fish T. rubripes liver slices.
Osamu Arakawa - One of the best experts on this subject based on the ideXlab platform.
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transcriptome analysis of tetrodotoxin sensing and tetrodotoxin action in the central nervous system of tiger puffer Takifugu rubripes juveniles
Fisheries Science, 2017Co-Authors: Kogen Okita, Shuichi Asakawa, Tomohiro Takatani, Shigeharu Kinoshita, Engkong Tan, Hina Satone, Daisuke Ojima, Hideki Yamazaki, Kazutaka Sakiyama, Osamu ArakawaAbstract:To reveal the sensing of tetrodotoxin (TTX) by tiger puffer Takifugu rubripes juveniles and its action in the central nervous system (CNS), we conducted transcriptome analysis using next-generation sequencing for the olfactory system and brain of non-toxic cultured juveniles administered TTX. Sixty-seven million reads from the nasal region (olfactory epithelium and skin) and the brain of each of three individuals of the control, TTX-sensing and TTX-administered juveniles were assembled into 153,958 contigs. Mapping raw reads from each sample onto the nucleotide sequences of predicted transcripts in the T. rubripes genome (FUGU version 4) and the de novo assembled contigs to investigate their frequency of expression revealed that the expression of 21 and 81 known genes significantly changed in TTX-sensing and TTX-administered juveniles in comparison with control juveniles, respectively. These genes included those related to feeding regulation and a reward system, and indicated that TTX ingestion of T. rubripes juveniles is controlled in the feeding center in the brain, that T. rubripes may sense TTX as a reward, and that accumulated TTX directly acts on the central nervous system to adjust TTX ingestion.
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change in the transfer profile of orally administered tetrodotoxin to non toxic cultured pufferfish Takifugu rubripes depending of its development stage
Toxicon, 2013Co-Authors: Ryohei Tatsuno, Tomohiro Takatani, Junjie Wang, Miwako Shikina, Yoshiyuki Shirai, Kiyoshi Soyano, Gregory N Nishihara, Osamu ArakawaAbstract:To investigate the effects of growth (organ development) on tetrodotoxin (TTX) dynamics in the pufferfish body, TTX-containing feed homogenate was administered to 6- and 15-month old non-toxic cultured specimens of the pufferfish Takifugu rubripes at a dose of 40 mouse units (MU) (8.8 μg)/20 g body weight by oral gavage. After 24 h, the specimens were killed and the skin tissues (dorsal and ventral), muscle, liver, digestive tract, and gonads were separated. TTX content (μg/g) in each tissue, determined by liquid chromatography/mass spectrometry, revealed that the TTX distribution profile, particularly the TTX content of the liver, greatly differed between the two ages; the TTX score of 15-month old fish (3.3 μg/g) was nearly 5-fold that of 6-month old fish (0.68 μg/g). The total remaining TTX amount per individual (relative amount to the given dose) was 31% in 6-month old fish, of which 71% was in the skin, and 84% in 15-month old fish, of which 83% was in the liver. The gonadosomatic index (GSI) and hepatosomatic index (HSI) scores, and histologic observations of the gonads and liver suggest that although there is little difference in maturation stage between these two ages, there are clear distinctions in the developmental stage of the liver. The results suggest that the TTX dynamics in T. rubripes are linked to the development of the liver, i.e., the TTX taken up into the pufferfish body via food organisms is eliminated or transferred mainly to the skin in young fish with an undeveloped liver, but as the fish grow and the liver continues to develop, most of the TTX is transferred to and accumulated in the liver.
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Transfer profile of intramuscularly administered tetrodotoxin to artificial hybrid specimens of pufferfish, Takifugu rubripes and Takifugu niphobles.
Toxicon, 2011Co-Authors: Junjie Wang, Koichi Ikeda, Tomohiro Takatani, Ryohei Tatsuno, Masaomi Hamasaki, Taiichiro Araki, Shinya Nina, Yoshitaka Sakakura, Osamu ArakawaAbstract:Tetrodotoxin (TTX) was intramuscularly administered to artificially hybridized specimens of the pufferfish Takifugu rubripes and Takifugu niphobles to investigate toxin accumulation in hybrids, and TTX transfer/accumulation profiles in the pufferfish body. In the test fish administered 146 MU TTX in physiologic saline, TTX rapidly transferred from the muscle via the blood to other organs. Toxin transfer to the ovary rapidly increased to 53.5 MU/g tissue at the end of the 72-h test period. The TTX content in the liver and skin was, at most, around 4-6 MU/g tissue, and in the testis it was less than 0.01 MU/g tissue. On the other hand, based on the total amount of toxin per individual (% of the administered toxin), the skin and the liver contained higher amounts (20-54% and 2-24%, respectively), but the amount in the liver rapidly decreased after 8-12 h, and fell below the level in the ovary after 48 h. These findings suggest that part of the TTX is first taken up in the liver and then transferred/accumulated in the skin in male specimens and in the ovary in female specimens.
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transfer profile of intramuscularly administered tetrodotoxin to non toxic cultured specimens of the pufferfish Takifugu rubripes
Toxicon, 2009Co-Authors: Koichi Ikeda, Tomohiro Takatani, Yumi Murakami, Yu Emoto, Laymithuna Ngy, Shigeto Taniyama, Motoaki Yagi, Osamu ArakawaAbstract:Tetrodotoxin (TTX) was intramuscularly administered to non-toxic cultured specimens of the pufferfish Takifugu rubripes to investigate TTX transfer/accumulation profiles in the pufferfish body. In two groups of test fish administered either 50MU/individual of TTX standard (purified TTX; PTTX) or crude extract of toxic pufferfish ovary (crude TTX; CTTX), TTX rapidly transferred from the muscle via the blood to other organs. The toxin transfer profiles differed between groups, however, from 4 to 72h. In the PTTX group, little TTX was retained in the liver, and most (>96%) of the toxin remaining in the body transferred/accumulated in the skin after 12h, whereas in the CTTX group, a considerable amount of toxin (15%-23% of the administered toxin or 28%-58% of the remaining toxin) was transferred/retained in the liver for up to 24h, despite the fact that 89% of the remaining toxin transferred/accumulated in the skin at the end of rearing period (168h). The total amount of toxin remaining in the entire body at 1-4h was approximately 60% of the administered toxin in both groups, which decreased at 8-12h, and then increased again to approximately 60%-80% at 24-168h. Immunohistochemical observation revealed that the toxin accumulated in the skin was localized at the basal cells of the epidermal layer.
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toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land
Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 2006Co-Authors: Tamao Noguchi, Osamu Arakawa, Tomohiro TakataniAbstract:Marine pufferfish (family Tetraodontidae) are believed to accumulate tetrodotoxin (TTX) mainly in liver and ovary through the food chain by ingesting TTX-bearing organisms such as starfish, gastropods, crustacean, flatworms, ribbonworms, etc. Consequently, it is hypothesized that non-toxic pufferfish can be produced if they are cultured with TTX-free diets in netcages at sea or aquaria on land, where the invasion of TTX-bearing organisms is completely shut off. To confirm this hypothesis, more than 5000 specimens of the pufferfish ("torafugu", Takifugu rubripes) cultured in such manners for 1-3 years were collected from several locations in Japan during 2001-2004, and toxicity of their livers and some other parts was examined according to the Japanese official mouse assay method for TTX. In addition, typical specimens were submitted to LC/MS analysis. The results showed that all the livers and other parts tested were 'non-toxic' in both of the mouse assay (less than 2 MU/g) and LC/MS analysis (less than 0.1 MU/g). Thus, it is undoubtedly confirmed that pufferfish are intoxicated through the food chain, and non-toxic pufferfish can be successfully produced by netcage or land culture. The livers from these fish can be used with safety as a Japanese traditional food "fugu-kimo" (puffer liver).
Shugo Watabe - One of the best experts on this subject based on the ideXlab platform.
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ultrahigh density linkage map construction using low coverage whole genome sequencing of a doubled haploid population case study of torafugu Takifugu rubripes
Genes, 2018Co-Authors: Xiang Zhang, Hong Zhang, Shigeharu Kinoshita, Kazuyoshi Saito, Engkong Tan, Yutaka Suzuki, Misaki Mizukoshi, Yoji Igarashi, Susumu Mitsuyama, Shugo WatabeAbstract:Next-generation sequencing enables genome-wide genotyping of a large population and further facilitates the construction of a genetic linkage map. Low-coverage whole-genome sequencing has been employed for genetic linkage map construction in several species. However, this strategy generally requires available high-quality reference genomes and/or designed inbred pedigree lines, which restrict the scope of application for non-model and unsequenced species. Here, using torafugu (Takifugu rubripes) as a test model, we propose a new strategy for ultrahigh-density genetic linkage map construction using low-coverage whole-genome sequencing of a haploid/doubled haploid (H/DH) population without above requirements. Low-coverage (≈1×) whole-genome sequencing data of 165 DH individuals were used for de novo assembly and further performed single nucleotide polymorphisms (SNPs) calling, resulting in the identification of 1,070,601 SNPs. Based on SNP genotypes and de novo assembly, genotypes were associated with short DNA segments and an ultrahigh-density linkage map was constructed containing information of 802,277 SNPs in 3090 unique positions. Comparative analyses showed near-perfect concordance between the present linkage map and the latest published torafugu genome (FUGU5). This strategy would facilitate ultrahigh-density linkage map construction in various sexually reproducing organisms for which H/DH populations can be generated.
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characterization of pax3 and pax7 genes and their expression patterns during different development and growth stages of japanese pufferfish Takifugu rubripes
Gene, 2016Co-Authors: Dadasaheb B Akolkar, Shugo Watabe, Shuichi Asakawa, M Asaduzzaman, Shigeharu KinoshitaAbstract:Pax3 and Pax7 are the regulators and markers of muscle progenitors and satellite cells that contribute to the embryonic development and postembryonic growth of skeletal muscle in vertebrates, as well as to its repair and regeneration. However, information regarding them in vertebrate genome model, torafugu Takifugu rubripes, has remained unknown. Therefore, as an initial step, here we characterized Pax3 and Pax7 from torafugu and investigated their expression patterns during different developmental stages by RT-PCR. In silico analysis with the Fugu genome database (ver. 4.0) yielded two distinct genes each for Pax3 (Pax3a and Pax3b) and Pax7 (Pax7a and Pax7b). The 75th amino acid, glutamine (Gln75), from the N-terminus was replaced by proline in the paired box domain (PD) of Pax3a. One single cDNA clone encoding Pax3a had deletion of Gln75 in PD, suggesting the presence of alternatively spliced variants (Q+/Q-). This was further supported by identification of two adjacent alternative 3' splice acceptor sites which produce Pax3b Q+ (aagCAGGGA) and Q- (aagcagGGA) variants. Interestingly, torafugu Pax7a, but not Pax7b, had an insert encoding five amino acid residues (SGEAS) in a C-terminal region of PD in two out of three cDNA clones. Genomic analysis showed two alternate splice donor sites at exon 4 of Pax7a. In synteny analysis, torafugu Pax3a showed syntenic relationship with the corresponding regions in other teleosts only, whereas Pax3b and Pax7b showed high syntenic relationship with the corresponding regions of both mammals and other teleosts. RT-PCR revealed that expression of Pax3a and Pax3b transcripts was restricted to embryonic stages only, whereas those of Pax7a and Pax7b was continued to be expressed in larvae and importantly those of Pax7a were found in adult skeletal muscles. Therefore, Pax3 appears to be most important for primary myogenesis and Pax7 for secondary myogenesis and growth by hyperplasia in fish. In this regard, the transcripts of torafugu Pax3 and Pax7 genes might be used for further investigation as a marker for identification of muscle precursor cells during different phases of growth, and this ambiguity is the next target of our research.
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deep sequencing profiling and detailed annotation of micrornas in Takifugu rubripes
BMC Genomics, 2015Co-Authors: Chaninya Wongwarangkana, Shugo Watabe, Shigeharu Kinoshita, Kazuhiro E Fujimori, Masaki Akiba, Morimi Teruya, Maiko Nezuo, Tsukahara Masatoshi, Shuichi AsakawaAbstract:microRNAs (miRNAs) in fish have not been as extensively studied as those in mammals. The fish species Takifugu rubripes is an intensively studied model organism whose genome has been sequenced. The T. rubripes genome is approximately eight times smaller than the human genome, but has a similar repertoire of protein-coding genes. Therefore, it is useful for identifying non-coding genes, including miRNA genes. To identify miRNA expression patterns in different organs of T. rubripes and give fundamental information to aid understanding of miRNA populations in this species, we extracted small RNAs from tissues and performed deep sequencing analysis to profile T. rubripes miRNAs. These data will be of assistance in functional studies of miRNAs in T. rubripes. After analyzing a total of 139 million reads, we found miRNA species in nine tissues (fast and slow muscles, heart, eye, brain, intestine, liver, ovaries, and testes). We identified 1420 known miRNAs, many of which were strongly expressed in certain tissues with expression patterns similar to those described for other animals in previous reports. Most miRNAs were expressed in tissues other than the ovaries or testes. However, some miRNA families were highly abundant in the gonads, but expressed only at low levels in somatic tissue, suggesting specific function in germ cells. The most abundant isomiRs (miRNA variants) of many miRNAs had identical sequences in the 5′ region. However, isomiRs of some miRNAs, including fru-miR-462-5p, varied in the 5′ region in some tissues, suggesting that they may target different mRNA transcripts. Longer small RNAs (26–31 nt), which were abundant in the gonads, may be putative piRNAs because of their length and their origin from repetitive elements. Additionally, our data include possible novel classes of small RNAs. We elucidated miRNA expression patterns in various organs of T. rubripes. Most miRNA sequences are conserved in vertebrates, indicating that the basic functions of vertebrate miRNAs share a common evolution. Some miRNA species exhibit different distributions of isomiRs between tissues, suggesting that they have a broad range of functions.
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Characterization of the torafugu (Takifugu rubripes) immunoglobulin heavy chain gene locus
Immunogenetics, 2015Co-Authors: Xi Fu, Hong Zhang, Shugo Watabe, Shuichi AsakawaAbstract:In this study, we investigated the immunoglobulin heavy (IGH) gene locus of torafugu ( Takifugu rubripes ) from publicly available assembly sequences and presented an annotated locus map, including the IGHV genes, pseudogenes, and IGHC genes. Three new IGHV gene families (IGHV3–IGHV5) were discovered. We observed the interspersion of IGHV1 and IGHV2 family members and that they often intermingled with each other, while other family members were further interspersed. Conservation of the promoter and recombination signal sequences (RSS) was observed in a family-specific manner. In addition to known variable region genes present on chromosome 5 (current torafugu genome assembly), we found 34 additional IGHV genes on scaffold 287 and three novel potentially functional IGHD genes on scaffold 483. In total, the variable region of the torafugu IGH locus consists of at least 48 IGHV genes, seven IGHD genes, and six IGHJ genes. IGHC genes have also been mapped in this study, with three genes encoding immunoglobulin classes: IgT, IgM, and IgD. We confirmed the expression of newly identified IGHV3 family sequences in the spleen and kidney of adult torafugu and found a favorable IGHV segment usage by IgM and IgT. Possible structural variation in the IGHδ locus was observed based on the current torafugu assembly. The complete characterization of the torafugu IGH locus will facilitate detailed studies of large-scale mechanisms associated with the recombination of the variable region genes and will offer insights into the genetic basis of the potential diversity in the antibody response observed in torafugu.
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the expression of multiple myosin heavy chain genes during skeletal muscle development of torafugu Takifugu rubripes embryos and larvae
Gene, 2013Co-Authors: M Asaduzzaman, Shugo Watabe, Dadasaheb B Akolkar, Shigeharu KinoshitaAbstract:Abstract In vertebrates, the development-dependent and tissue-specific expression of myosin heavy chain (MYH) genes ( MYH s) contributes to the formation of diverged muscle fiber types. The expression patterns of developmentally regulated MYH s have been investigated in certain species of fish. However, the expression profiles of MYH s during torafugu Takifugu rubripes development, although extensively studied in adult tissues, have not been sufficiently studied, and also the expression orders of MYH s during development have remained unclear. In the present study, we comprehensively cloned four MYH s ( MYH M743-2 , MYH M86-2 , MYH M5 and MYH M2126-1 ) from embryos, and two MYH s ( MYH M2528-1 and MYH M1034 ) from larvae, and characterized their expression pattern in relation to developmental stages of torafugu by reverse transcription (RT)-PCR and in situ hybridization. The expression of MYHs from torafugu embryos and larvae appeared sequentially and varied largely in relation to the developmental stage-dependent and fibers-type-specific manners. The transcripts of MYH M743-2 appeared first in embryos at 3 days post fertilization (dpf) and were localized in the epaxial and hypaxial domains of fast muscle fibers of larval myotome, whereas those of MYH M5 and MYH M86-2 in 3 dpf and 4 dpf, respectively, and both were localized in superficial slow and horizontal myoseptum regions. The expression of MYH M1034 and MYH M2126-1 was quite low and mostly undetectable. Different MYH s from torafugu embryos and larvae have also been found to be expressed differentially in pectoral fin and craniofacial muscles. Interestingly, the transcripts of MYH M2528-1 first appeared at 6 dpf and were distinctly expressed at the dorsal and ventral extremes of larval myotome, suggesting its involvement in stratified hyperplasia. The novel involvement of MYH M2528-1 in mosaic hyperplasia was further confirmed in juvenile torafugu, where the transcripts were expressed in fast fibers with small diameters as well as the inner part of superficial slow fibers.
