The odor of a nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, is detected by specific olfactory sensory neurons of the green spotted puffers.

Toxic puffers accumulate tetrodotoxin (TTX), a well-known neurotoxin, by feeding on TTX-bearing organisms and using it to defend themselves from predators. Our previous studies have demonstrated that toxic puffers are attracted to 5,6,11-trideoxytetrodotoxin (TDT), a nontoxic TTX analog that is simultaneously accumulated with TTX in toxic puffers and their prey. In addition, activity labeling using immunohistochemistry targeting neuronal activity marker suggests that TDT activates crypt olfactory sensory neurons (OSN) of the green spotted puffer. However, it remains to be determined whether individual crypt OSNs can physiologically respond to TDT. By employing electroporation to express GCaMP6s in OSNs, we successfully identified a distinct group of oval OSNs that exhibited a specific calcium response when exposed to TDT in green spotted puffers. These oval OSNs showed no response to amino acids (AAs), which serve as food odor cues for teleosts. Furthermore, oval morphology and surface positioning of TDT-sensitive OSNs in the olfactory epithelium closely resemble that of crypt OSNs. These findings suggest that TDT is specifically detected by crypt OSNs in green spotted puffer. The TDT odor may act as a chemoattractant for finding conspecific toxic puffers and for feeding TTX-bearing organisms for effective toxification.

5, 6, 11-trideoxy tetrodotoxin attracts tiger puffer Takifugu rubripes.

Tetrodotoxin (TTX) is a potent neurotoxin that binds to voltage-gated sodium channels and blocks the passage of sodium ions. TTX is widely distributed in both terrestrial and marine organisms, and the toxic puffers are believed to accumulate TTX through the food chain. Although pufferfish was previously thought to be attracted by TTX, recent finding from electroolfactogram (EOG) studies have indicated that the olfactory epithelium of T. alboplumbeus responded to 5, 6, 11-trideoxyTTX (TDT), but not to TTX itself. In this study, we examined behavioral experiments for Takifugu rubripes to distinguish between TTX and TDT under static and flow-through conditions. Our data clearly suggested that T. rubripes juveniles were attracted to TDT, not TTX. Moreover, we determined that the minimum effective dose of TDT to attract the puffer was 1-2 nmol of TDT under static conditions and 50-60 nmol of TDT under flow-through conditions. Following the experiments under static conditions, numerous bite marks by the pufferfish were found solely on the agarose gel infused with TDT. Based on these finding, we hypothesize that the pufferfish are attracted to TDT derived from prey, leading them effectively become toxic.

Geographical differences in the composition of tetrodotoxin and 5,6,11-trideoxytetrodotoxin in Japanese pufferfishes and their origins.

Tetrodotoxin (TTX)-bearing fish are thought to accumulate TTXs in their bodies through a food chain that begins with marine bacteria. However, the mechanism of TTXs transfer between prey and predators in the food chain remains unclear and the reasons for regional differences in pufferfish toxicity are also unknown. To investigate these matters, we collected juveniles of four species of pufferfish, Takifugu alboplumbeus, Takifugu flavipterus, Takifugu stictonotus, and Chelonodon patoca, from various locations in the Japanese Islands, and subjected them to liquid chromatography-tandem mass spectrometry analysis for TTX and its analog 5,6,11-trideoxyTTX (TDT). Concentrations of these substances tended to be higher in pufferfish juveniles collected from the Sanriku coastal area (Pacific coast of northern Japan) than in those from other locations. Juveniles had higher concentrations of TTX at all locations than of TDT. Mitochondrial cytochrome c oxidase subunit I (COI) sequences specific to the TTX-bearing flatworm, Planocera multitentaculata, were detected in the intestinal contents of up to 100% of pufferfish juveniles from various sampling sites, suggesting that P. multitentaculata was widely involved in the toxification of the juveniles in the coastal waters of Japan. A toxification experiment was conducted on three species of pufferfish juveniles (T. alboplumbeus, Takifugu rubripes and C. patoca) using TTX-bearing flatworm eggs harboring equal amounts of TTX and TDT. The TTX content of juveniles fed on flatworm eggs was found to be more than twice that of TDT, suggesting that pufferfish preferentially incorporate TTX compared to TDT.

Levels of Tetrodotoxins in Spawning Pufferfish, Takifugu alboplumbeus.

Tetrodotoxin (TTX), also known as pufferfish toxin, is an extremely potent neurotoxin thought to be used as a biological defense compound in organisms bearing it. Although TTX was thought to function as a chemical agent for defense and anti-predation and an attractant for TTX-bearing animals including pufferfish, it has recently been demonstrated that pufferfish were also attracted to 5,6,11-trideoxyTTX, a related compound, rather than TTX alone. In this study, we attempted to estimate the roles of TTXs (TTX and 5,6,11-trideoxyTTX) in the pufferfish, Takifugu alboplumbeus, through examining the location of TTXs in various tissues of spawning pufferfish from Enoshima and Kamogawa, Japan. TTXs levels in the Kamogawa population were higher than those in the Enoshima population, and there was no significant difference in the amount of TTXs between the sexes in either population. Individual differences were greater in females than in males. However, the location of both substances in tissues differed significantly between sexes: male pufferfish accumulated most of their TTX in the skin and liver and most of their 5,6,11-trideoxyTTX in the skin, whereas females accumulated most of their TTX and 5,6,11-trideoxyTTX in the ovaries and skin.

Changes in Tissue Distribution of Tetrodotoxin and Its Analogues in Association with Maturation in the Toxic Flatworm, Planocera multitentaculata.

The toxic flatworm, Planocera multitentaculata, possesses highly concentrated tetrodotoxin (TTX), also known as pufferfish toxin, throughout its life cycle, including the egg and larval stages. Additionally, TTX analogues, 5,6,11-trideoxyTTX and 11-norTTX-6(S)-ol, have also been detected in the flatworm. The high concentration of TTX in the eggs and larvae appears to be for protection against predation, and 11-norTTX-6(S)-ol in the pharyngeal tissue in the adults is likely used to sedate or kill prey during predation. However, information on the role of 5,6,11-trideoxyTTX, a potential important biosynthetic intermediate of TTX, in the toxic flatworm is lacking. Here, we aimed to determine the region of localization of TTX and its analogues in the flatworm body, understand their pharmacokinetics during maturation, and speculate on their function. Flatworm specimens in four stages of maturity, namely juvenile, mating, spawning, and late spawning, were subjected to LC-MS/MS analysis, using the pharyngeal tissue, oocytes in seminal receptacle, sperm, and tissue from 12 other sites. Although TTX was consistently high in the pharyngeal tissue throughout maturation, it was extremely high in the oocytes during the spawning period. Meanwhile, 5,6,11-trideoxyTTX was almost undetectable in the pharyngeal part throughout the maturation but was very abundant in the oocytes during spawning. 11-norTTX-6(S)-ol consistently localized in the pharyngeal tissue. Although the localization of TTX and its analogues was approximately consistent with the MS imaging data, TTX and 11-norTTX-6(S)-ol were found to be highly localized in the parenchyma surrounding the pharynx, which suggests the parenchyma is involved in the accumulation and production of TTXs.

An almost nontoxic tetrodotoxin analog, 5,6,11-trideoxytetrodotoxin, as an odorant for the grass puffer.

Toxic puffers contain the potent neurotoxin, tetrodotoxin (TTX). Although TTX is considered to serve as a defense substance, previous behavioral studies have demonstrated that TTX acts as an attractive pheromone for some toxic puffers. To elucidate the physiological mechanism of putative pheromonal action of TTX, we examined whether grass puffers Takifugu alboplumbeus can detect TTX. Electroolfactogram (EOG) results suggest that the olfactory epithelium (OE) of grass puffers responded to a type of TTX analog (5,6,11-trideoxyTTX), although it did not respond to TTX. We also examined the attractive action of 5,6,11-trideoxyTTX on grass puffers by recording their swimming behavior under dark conditions. Grass puffers preferred to stay on the side of the aquarium where 5,6,11-trideoxyTTX was administered, and their swimming speed decreased. Additionally, odorant-induced labeling of olfactory sensory neurons by immunohistochemistry against neural activity marker (phosphorylated extracellular signal regulated kinase; pERK) revealed that labeled olfactory sensory neurons were localized in the region surrounding "islets" where there was considered as nonsensory epithelium. 5,6,11-trideoxyTTX has been known to accumulate in grass puffers, but its toxicity is much lower (almost nontoxic) than TTX. Our results suggest that toxic puffers may positively use this TTX analog, which has been present in their body with TTX but whose function was unknown, as an odorant for chemical communication or effective TTX accumulation.

Domestic cat damage to plant leaves containing iridoids enhances chemical repellency to pests.

Catnip (Nepeta cataria) and silver vine (Actinidia polygama) produce iridoids with arthropod-repellent effects. Cats rub and roll against these plants, transferring iridoids to their fur that repels mosquitoes. Cats also lick and chew plant leaves during this response, although the benefit of this additional behavior has remained unknown. Here, we show that feline leaf damage substantially increases iridoid emission from both plants while also diversifying iridoids in silver vine. Cats show an equivalent duration of response to the complex cocktail of iridoids in damaged silver vine and to the much higher level of a single iridoid produced by damaged catnip. The more complex iridoid cocktail produced when silver vine is licked and chewed by cats increases mosquito repellency at low concentration. In conclusion, feline leaf damage contributes by releasing more mosquito-repellent iridoids. Feline olfactory and behavioral sensitivity is fine-tuned to plant-specific iridoid production for maximizing the mosquito repellency gained.

Tetrodotoxins in the flatworm Planocera multitentaculata.

The marine polyclad flatworm Planocera multitentaculata is known to possess high levels of tetrodotoxin (TTX), but the presence of TTX analogues in the species has been unexplored. In this study, TTX and several analogues such as 5,6,11-trideoxyTTX, monodeoxyTTXs, dideoxyTTXs, and 11-norTTX-6(S)-ol were identified in three adults and egg plates of P. multitentaculata using high resolution liquid chromatography-mass spectrometry (HR-LC/MS) for the first time.

Total Syntheses of the Proposed Biosynthetic Intermediates of Tetrodotoxin Tb-210B, Tb-226, Tb-242C, and Tb-258.

The collective synthesis of the four spiro-cyclic guanidines Tb-210B, Tb-226, Tb-242C, and Tb-258, all of which have been isolated from puffer fish and are considered possible biosynthetic intermediates of tetrodotoxin, has been achieved. Our synthesis is based on the stepwise deoxygenation or hydroxylation of a common intermediate, prepared from a known oxazoline.

Green spotted puffers detect a nontoxic TTX analog odor using crypt olfactory sensory neurons.

Toxic puffers accumulate their defense substance (tetrodotoxin; TTX) through the food chain. Although the previous study suggests that 5,6,11-trideoxyTTX, a nontoxic TTX analog detected simultaneously with TTX in toxic puffers or their prey, acts as an olfactory chemoattractant for grass puffers, it is unclear whether toxic puffers are commonly attracted to 5,6,11-trideoxyTTX, and which types of olfactory sensory neurons (OSNs) detect 5,6,11-trideoxyTTX. Here, we demonstrated that green spotted puffer, a phylogenetically distant species from the grass puffer, is attracted to 5,6,11-trideoxyTTX. 5,6,11-TrideoxyTTX administration made green spotted puffers stay longer at the administered site, whereas a food odor (l-Arg) made them actively swim throughout the aquarium. Attractive responses were not observed when TTX or its vehicle was administered, nor when 5,6,11-trideoxyTTX was administered to anosmic fish. Furthermore, double immunohistochemistry with activity marker and crypt OSN marker antibodies labeled oval cells with apical invagination on the olfactory epithelium surface treated with 5,6,11-trideoxyTTX. These results suggest that 5,6,11-trideoxyTTX acts as an olfactory chemoattractant detected by crypt OSNs, and attraction to 5,6,11-trideoxyTTX odor appears to be a trait shared by toxic puffers for social communication or effective toxification.

Local Differences in the Toxin Amount and Composition of Tetrodotoxin and Related Compounds in Pufferfish (Chelonodon patoca) and Toxic Goby (Yongeichthys criniger) Juveniles.

Tetrodotoxin (TTX)-bearing fish ingest TTX from their preys through the food chain and accumulate TTX in their bodies. Although a wide variety of TTX-bearing organisms have been reported, the missing link in the TTX supply chain has not been elucidated completely. Here, we investigated the composition of TTX and 5,6,11-trideoxyTTX in juveniles of the pufferfish, Chelonodon patoca, and toxic goby, Yongeichthys criniger, using LC-MS/MS, to resolve the missing link in the TTX supply chain. The TTX concentration varied among samples from different localities, sampling periods and fish species. In the samples from the same locality, the TTX concentration was significantly higher in the toxic goby juveniles than in the pufferfish juveniles. The concentration of TTX in all the pufferfish juveniles was significantly higher than that of 5,6,11-trideoxyTTX, whereas the compositional ratio of TTX and 5,6,11-trideoxyTTX in the goby was different among sampling localities. However, the TTX/5,6,11-trideoxyTTX ratio in the goby was not different among samples collected from the same locality at different periods. Based on a species-specific PCR, the detection rate of the toxic flatworm (Planocera multitentaculata)-specific sequence (cytochrome c oxidase subunit I) also varied between the intestinal contents of the pufferfish and toxic goby collected at different localities and periods. These results suggest that although the larvae of the toxic flatworm are likely to be responsible for the toxification of the pufferfish and toxic goby juveniles by TTX, these fish juveniles are also likely to feed on other TTX-bearing organisms depending on their habitat, and they also possess different accumulation mechanisms of TTX and 5,6,11-trideoxyTTX.

Synthesis of the 8-Deoxy Analogue of 4,9-Anhydro-10-hemiketal-5-deoxy-tetrodotoxin, a Proposed Biosynthetic Precursor of Tetrodotoxin.

Despite decades of research, the biosynthesis of tetrodotoxin, also known as a puffer fish toxin, remains an unsolved mystery. We disclose a synthesis of the 8-deoxy analogue of 4,9-anhydro-10-hemiketal-5-deoxy-tetrodotoxin, a possible biosynthetic precursor of tetrodotoxin isolated from the Japanese toxic newt, by an intramolecular radical cyclization of a known starting material.

JBIR-155, a Specific Class D ß-Lactamase Inhibitor of Microbial Origin.

We discovered JBIR-155 as a novel specific class D ß-lactamase inhibitor from Streptomyces polymachus SoB100815Hv02. JBIR-155 consists of a 6-oxabicyclo[3.2.0]heptan-7-one skeleton and a long unsaturated alkyl chain moiety of which absolute configuration was determined by spectroscopic data, modified Mosher's method, and analyses of the relative configuration of chemically modified derivative. JBIR-155 specifically exhibited inhibitory activity against the class D ß-lactamase, with an IC50 value of 0.36 µM.

The characteristic response of domestic cats to plant iridoids allows them to gain chemical defense against mosquitoes.

Domestic cats and other felids rub their faces and heads against catnip (Nepeta cataria) and silver vine (Actinidia polygama) and roll on the ground as a characteristic response. While this response is well known, its biological function and underlying mechanism remain undetermined. Here, we uncover the neurophysiological mechanism and functional outcome of this feline response. We found that the iridoid nepetalactol is the major component of silver vine that elicits this potent response in cats and other felids. Nepetalactol increased plasma ß-endorphin levels in cats, while pharmacological inhibition of µ-opioid receptors suppressed the classic rubbing response. Rubbing behavior transfers nepetalactol onto the faces and heads of respondents where it repels the mosquito, Aedes albopictus Thus, self-anointing behavior helps to protect cats against mosquito bites. The characteristic response of cats to nepetalactol via the µ-opioid system provides an important example of chemical pest defense using plant metabolites in nonhuman mammals.

First Detection of Tetrodotoxins in the Cotylean Flatworm Prosthiostomum trilineatum.

Several polyclad flatworm species are known to contain high levels of tetrodotoxin (TTX), but currently TTX-bearing flatworms seem to be restricted to specific Planocera lineages belonging to the suborder Acotylea. During our ongoing study of flatworm toxins, high concentrations of TTXs were detected for the first time in the flatworm Prosthiostomum trilineatum, suborder Cotylea, from the coastal area of Hayama, Kanagawa, Japan. Toxin levels were investigated by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing that this species contains comparable concentrations of toxins as seen in planocerid flatworms such as Planocera multitentaculata. This finding indicated that there may be other species with significant levels of TTXs. The distribution of TTXs among other flatworm species is thus of great interest.

Biosynthesis of Indole Diterpene Lolitrems: Radical-Induced Cyclization of an Epoxyalcohol Affording a Characteristic Lolitremane Skeleton.

Lolitrems are tremorgenic indole diterpenes that exhibit a unique 5/6 bicyclic system of the indole moiety. Although genetic analysis has indicated that the prenyltransferase LtmE and the cytochrome P450 LtmJ are involved in the construction of this unique structure, the detailed mechanism remains to be elucidated. Herein, we report the reconstitution of the biosynthetic pathway for lolitrems employing a recently established genome-editing technique for the expression host Aspergillus oryzae. Heterologous expression and bioconversion of the various intermediates revealed that LtmJ catalyzes multistep oxidation to furnish the lolitrem core. We also isolated the key reaction intermediate with an epoxyalcohol moiety. This observation allowed us to establish the mechanism of radical-induced cyclization, which was firmly supported by density functional theory calculations and a model experiment with a synthetic analogue.

Asymmetric Synthesis of the Aromatic Fragment of Sespendole.

Sespendole is an indole sesquiterpene alkaloid bearing two isoprenyl groups, one of which is highly oxidized. Herein, we disclose an eight-step synthesis of the aromatic fragment of sespendole in an optically pure form, starting from 4-bromo-2-fluoronitrobenzene. The key steps were a Claisen rearrangement at room temperature for introduction of the prenyl group and a coupling between the dianion generated from prenylated bromo-N-tosylanilide and a chiral epoxy aldehyde.

Total Syntheses and Determination of Absolute Configurations of Cep-212 and Cep-210, Predicted Biosynthetic Intermediates of Tetrodotoxin Isolated from Toxic Newt.

Total syntheses of Cep-212 and Cep-210, predicted biosynthetic intermediates of tetrodotoxin isolated from the Japanese toxic newt, have been accomplished from geraniol by an intramolecular hetero Diels-Alder reaction as a key step in a highly stereoselective manner. The success of these syntheses enabled us to determine their absolute configurations by using a chiral normal-phase HPLC/MS analysis of the bis-dinitrobenzene derivative of natural Cep-212 and reference derivatives prepared from chemically synthesized enantiomers.

Differential binding of tetrodotoxin and its derivatives to voltage-sensitive sodium channel subtypes (Nav 1.1 to Nav 1.7).

BACKGROUND AND PURPOSE: The development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC subtypes Nav 1.1-Nav 1.7. EXPERIMENTAL APPROACH: Each VSSC subtype was transiently expressed in HEK293T cells. The inhibitory effects of TTX analogues on each subtype were assessed using whole-cell patch-clamp recordings. KEY RESULTS: The inhibitory effects of TTX on Nav 1.1-Nav 1.7 were observed in accordance with those reported in the literature; however, the 5-deoxy-10,7-lactone-type analogues and 4,9-anhydro-type analogues did not cause inhibition. Chiriquitoxin showed less binding to Nav 1.7 compared to the other TTX-sensitive subtypes. Two amino acid residues in the TTX binding site of Nav 1.7, Thr1425 and Ile1426 were mutated to Met and Asp, respectively, because these residues were found at the same positions in other subtypes. The two mutants, Nav 1.7 T1425M and Nav 1.7 I1426D, had a 16-fold and 5-fold increase in binding affinity for chiriquitoxin, respectively. CONCLUSIONS AND IMPLICATIONS: The reduced binding of chiriquitoxin to Nav 1.7 was attributed to its C11-OH and/or C12-NH2 , based on reported models for the TTX-VSSC complex. Chiriquitoxin is a useful tool for probing the configuration of the TTX binding site until a crystal structure for the mammalian VSSC is solved.

One-Step Transformation of Trichloroacetamide into Isonitrile.

A one-step transformation of trichloroacetamide, a protective group for the amine function, into isonitrile was successfully developed. The substrate scope and functional group tolerance of this procedure are also described.