Assay for okadaic acid O-acyl transferase using HPLC-FLD.

Dinophysistoxin 1 (DTX1, 1) and okadaic acid (OA, 2), produced by the dinoflagellates Dinophysis spp. and Prorocentrum spp., are primary diarrhetic shellfish toxins (DSTs), which may cause gastric illness in people consuming such as bivalves. Both compounds convert to dinophysistoxin 3 (DTX3, 3; generic name for 1 and 2 with fatty acids conjugated at 7-OH) in bivalves. The enzyme okadaic acid O-acyl transferase (OOAT) is a membrane protein found in the microsomes of the digestive glands of bivalves. In this study, we established an in vitro enzymatic conversion reaction using 4-nitro-2,1,3-benzoxadiazole (NBD)-OA (4), an OA derivative conjugated with (R)-(-)-4-nitro-7-(3-aminopyrrolidin-1-yl)-2,1,3-benzoxadiazole (NBD-APy) on 1-CO2H, as a substrate. We detected the enzymatically produced 3, NBD-7-O-palmitoyl-OA (NBD-Pal-OA), using high-performance liquid chromatography-fluorescence detection. We believe that an OOAT assay using 4 will facilitate the fractionation and isolation of OOAT in the future.

Synthesis and Identification of decarbamoyloxySaxitoxins in Toxic Microalgae and their Reactions with the Oxygenase, SxtT, Reveal Saxitoxin Biosynthesis.

Saxitoxin (STX, 1) is a representative compound of paralytic shellfish toxins (PSTs) that are produced by marine dinoflagellates and freshwater cyanobacteria. Although several pathways have been proposed for the biosynthesis of STX, the order of ring and side chain hydroxylation, and formation of the tricyclic skeleton have not been well established. In this study, 12,12-dideoxy-decarbamoyloxySTX (dd-doSTX, 2), the most reduced STX analogue having the tricyclic skeleton, and its analogues, 12ß-deoxy-doSTX (12ß-d-doSTX, 3), 12α-deoxy-doSTX (12α-d-doSTX, 4), and doSTX (5), were synthesized, and these compounds were screened in the toxic microalgae using high-resolution LCMSMS. dd-doSTX (2) and 12ß-d-doSTX (3) were identified in the PSTs-producing dinoflagellates (Alexandrium catenella, A. pacificum, and/or Gymnodinium catenatum) and in the cyanobacterium Dolichospermum circinale (TA04). doSTX (5), previously isolated from the dinoflagellate G. catenatum, was also identified in D. circinale (TA04). Furthermore, the conversion of 2 to 3, and 4 to 5, by SxtT with VanB, a reported Rieske oxygenase and its redox partner in STX biosynthesis, was confirmed. These results support that 2 is a possible biosynthetic precursor of STX, and that ring and side-chain hydroxylations proceed after cyclization.

The quite low cross-reactivity of Kawatsu's anti-tetrodotoxin monoclonal antibody to 5,6,11-trideoxytetrodotoxin, 11-nortetrodotoxin-6(S)-ol, and 11-oxotetrodotoxin, the major tetrodotoxin analogues in pufferfish.

The monoclonal antibody against tetrodotoxin (TTX), prepared by Kawatsu et al. (1997), has been used in several TTX-related studies. Herein, we confirmed the quite low cross-reactivity of this antibody to three major TTX analogues in pufferfish using competitive ELISA: 5,6,11-trideoxyTTX (<2.2%), 11-norTTX-6(S)-ol (<0.3%), and 11-oxoTTX (<1.5%), with reactivity against TTX being 100%. We further confirmed that the presence of these analogues did not cause a marked overestimation of TTX in pufferfish extracts using competitive ELISA.

State-Dependent Inhibition of Voltage-Gated Sodium Channels in Neuroblastoma Neuro-2A Cells by Arachidonic Acid from Halichondria okadai.

Voltage-gated sodium channels (Nav) are closely associated with epilepsy, cardiac and skeletal muscle diseases, and neuropathic pain. Several toxic compounds have been isolated from the marine sponge Halichondria okadai; however, toxic substances that modulate Nav are yet to be identified. This study aimed to identify Nav inhibitors from two snake venoms and H. okadai using mouse neuroblastoma Neuro-2A cells (N2A), which primarily express the specific Nav subtype Nav1.7, using whole-cell patch-clamp recordings. We successfully isolated arachidonic acid (AA, 1) from the hexane extract of H. okadai, and then the fatty acid-mediated modulation of Nav in N2A was investigated in detail for the first time. Octanoic acid (2), palmitic acid (3), and oleic acid (4) showed no inhibitory activity at 100 µM, whereas AA (1), dihomo-γ-linolenic acid (DGLA, 5), and eicosapentaenoic acid (EPA, 6) showed IC50 values of 6.1 ± 2.0, 58 ± 19, and 25 ± 4.0 µM, respectively (N = 4, mean ± SEM). Structure and activity relationships were investigated for the first time using two ω-3 polyunsaturated fatty acids (PUFAs), EPA (6) and eicosatetraenoic acid (ETA, 7), and two ω-6 PUFAs, AA (1) and DGLA (5), to determine their effects on a resting state, activated state, and inactivated state. Steady-state analysis showed that the half inactivation potential was largely hyperpolarized by 10 µM AA (1), while 50 µM DGLA (5), 50 µM EPA (6), and 10 µM ETA (7) led to a slight change. The percentages of the resting state block were 24 ± 1, 22 ± 1, 34 ± 4, and 38 ± 9% in the presence of AA (1), DGLA (5), EPA (6), and ETA (7), respectively, with EPA (6) and ETA (7) exhibiting a greater inhibition than both AA (1) and DGLA (5), and their inhibitions did not increase in the following depolarization pulses. None of the compounds exhibited the use-dependent block. The half recovery times from the inactivated state for the control, AA (1), DGLA (5), EPA (6), and ETA (7) were 7.67 ± 0.33, 34.3 ± 1.10, 15.5 ± 1.10, 10.7 ± 0.31, and 3.59 ± 0.18 ms, respectively, with AA (1) exhibiting a distinctively large effect. Overall, distributed binding to the resting and the inactivated states of Nav would be significant for the inhibition of Nav, which presumably depends on the active structure of each PUFA.

Isolation and Biological Activity of 9-epiTetrodotoxin and Isolation of Tb-242B, Possible Biosynthetic Shunt Products of Tetrodotoxin from Pufferfish.

Tetrodotoxin (TTX, 1) is a potent voltage-gated sodium channel blocker detected in certain marine and terrestrial organisms. We report here a new TTX analogue, 9-epiTTX (2), and a TTX-related compound, Tb-242B (4), isolated from the pufferfish Takifugu flavipterus and Dichotomyctere ocellatus, respectively. NMR analysis suggested that 2 exists as a mixture of hemilactal and 10,8-lactone forms, whereas other reported TTX analogues are commonly present as an equilibrium mixture of hemilactal and 10,7-lactone forms. Compound 2 and TTX were confirmed not to convert to each other by incubation under neutral and acidic conditions at 37 °C for 24 h. Compound 4 was identified as the 9-epimer of Tb-242A (3), previously reported as a possible biosynthetic precursor of TTX. Compound 4 was partially converted to 3 by incubation in a neutral buffer at 37 °C for 7 days, whereas 3 was not converted to 4 under this condition. Compound 2 was detected in several TTX-containing marine animals and a newt. Mice injected with 600 ng of 2 by intraperitoneal injection did not show any adverse symptoms, suggesting that the C-9 configuration in TTX is critical for its biological activity. Based on the structures, 2 and 4 were predicted to be shunt products for TTX biosynthesis.

Mass spectrometry-guided discovery of new analogs of bicyclic phosphotriester salinipostin and evaluation of their monoacylglycerol lipase inhibitory activity.

Natural products containing the highly unusual phosphotriester ring are known to be potent serine hydrolase inhibitors. The long-chain bicyclic enol-phosphotriester salinipostins (SPTs) from the marine actinomycete Salinispora have been identified as selective antimalarial agents. A potential regulatory function has been suggested for phosphotriesters based on their structural relationship with actinomycete signaling molecules and the prevalence of spt-like biosynthetic gene clusters across actinomycetes. In this study, we established a mass spectrometry-guided screening method for phosphotriesters focusing on their characteristic fragment ions. Applying this screening method to the SPT producer Salinispora tropica CNB-440, new SPT analogs (4-6) were discovered and their structures were elucidated by spectroscopic analyses. Previously known and herein-identified SPT analogs inhibited the activity of human monoacylglycerol lipase (MAGL), a key serine hydrolase in the endocannabinoid system, in the nanomolar range. Our method could be applied to the screening of phosphotriesters, potential serine hydrolase inhibitors and signaling molecules.

Chemical Ecology of the North American Newt Genera Taricha and Notophthalmus.

The North American newt genera Taricha and Notophthalmus (order Caudata) are well known for the combination of potent toxicity, aposematic coloration, and striking defense postures that protects these animals from predation. This suite of traits is centered around the neurotoxin tetrodotoxin, which causes paralysis and death in metazoans by disrupting the initiation and propagation of electrical signals in the nerves and muscles. Tetrodotoxin defends newts from predation across multiple life history stages and its role in generating arms-race coevolution between Taricha newts and garter snake (genus Thamnophis) predators is well studied. However, understanding the broader picture of chemical defenses in Taricha and Notophthalmus requires an expanded comprehension of the defensive chemical ecology of tetrodotoxin that includes possible coevolutionary interactions with insect egg predators, protection against parasites, as well as mimicry complexes associated with tetrodotoxin and aposematic coloration in both genera. Herein the authors review what is known about the structure, function, and pharmacology of tetrodotoxin to explore its evolution and chemical ecology in the North American newt. Focus is made specifically on the origin and possible biosynthesis of tetrodotoxin in these taxa as well as providing an expanded picture of the web of interactions that contribute to landscape level patterns of toxicity and defense in Taricha and Notophthalmus.

First Identification of 12ß-Deoxygonyautoxin 5 (12α-Gonyautoxinol 5) in the Cyanobacterium Dolichospermum circinale (TA04) and 12ß-Deoxysaxitoxin (12α-Saxitoxinol) in D. circinale (TA04) and the Dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC).

Saxitoxin and its analogues, paralytic shellfish toxins (PSTs), are potent and specific voltage-gated sodium channel blockers. These toxins are produced by some species of freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates of PSTs, as well as new analogues, from such organisms and proposed the biosynthetic and metabolic pathways of PSTs. In this study, 12ß-deoxygonyautoxin 5 (12α-gonyautoxinol 5 = gonyautoxin 5-12(R)-ol) was identified in the freshwater cyanobacterium, Dolichospermum circinale (TA04), and 12ß-deoxysaxitoxin (12α-saxitoxinol = saxitoxin-12(R)-ol) was identified in the same cyanobacterium and in the marine dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC) for the first time from natural sources. The authentic standards of these compounds and 12α-deoxygonyautoxin 5 (12ß-gonyautoxinol 5 = gonyautoxin 5-12(S)-ol) were prepared by chemical derivatization from the major PSTs, C1/C2, produced in D. circinale (TA04). These standards were used to identify the deoxy analogues by comparing the retention times and MS/MS spectra using high-resolution LC-MS/MS. Biosynthetic or metabolic pathways for these analogues have also been proposed based on their structures. The identification of these compounds supports the α-oriented stereoselective oxidation at C12 in the biosynthetic pathway towards PSTs.

Characterization of microRNA expression in B cells derived from Japanese black cattle naturally infected with bovine leukemia virus by deep sequencing.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a malignant B cell lymphoma. However, the mechanisms of BLV-associated lymphomagenesis remain poorly understood. Here, after deep sequencing, we performed comparative analyses of B cell microRNAs (miRNAs) in cattle infected with BLV and those without BLV. In BLV-infected cattle, BLV-derived miRNAs (blv-miRNAs) accounted for 38% of all miRNAs in B cells. Four of these blv-miRNAs (blv-miR-B1-5p, blv-miR-B2-5p, blv-miR-B4-3p, and blv-miR-B5-5p) had highly significant positive correlations with BLV proviral load (PVL). The read counts of 90 host-derived miRNAs (bta-miRNAs) were significantly down-regulated in BLV-infected cattle compared to those in uninfected cattle. Only bta-miR-375 had a positive correlation with PVL in BLV-infected cattle and was highly expressed in the B cell lymphoma tissue of EBL cattle. There were a few bta-miRNAs that correlated with BLV tax/rex gene expression; however, BLV AS1 expression had a significant negative correlation with many of the down-regulated bta-miRNAs that are important for tumor development and/or tumor suppression. These results suggest that BLV promotes lymphomagenesis via AS1 and blv-miRNAs, rather than tax/rex, by down-regulating the expression of bta-miRNAs that have a tumor-suppressing function, and this downregulation is linked to increased PVL.

Identification of Tricyclic Guanidino Compounds from the Tetrodotoxin-Bearing Newt Taricha granulosa.

The biosynthesis of the potent neurotoxin tetrodotoxin (TTX, 1) is still unresolved. We used MS-guided screening and nuclear magnetic resonance analyses including long-range HSQMBC to characterize two novel skeletal tricyclic guanidino compounds, Tgr-288 (2a and 2b) and Tgr-210 (3), from the TTX-bearing newt, Taricha granulosa. The presence of these compounds in toxic newts is congruent with a previously proposed pathway for TTX biosynthesis in terrestrial organisms that includes a monoterpene precursor and the production of structurally diversified guanidino compounds.

Two new skeletal analogues of saxitoxin found in the scallop, Patinopecten yessoensis, as possible metabolites of paralytic shellfish toxins.

The scallop, Patinopecten yessoensis, was screened for new saxitoxin analogues to study the metabolism of paralytic shellfish toxins (PSTs), and this resulted in the discovery of two new analogues: M5-hemiaminal (HA) and M6-HA. M5-HA was isolated and its structure was determined by using NMR spectroscopy. It contains hydrogen at C-4 with opposite stereochemistry to that in saxitoxin, and a hemiaminal was formed between 9-NH2 and the hydrated ketone at C-12 in α-orientation. This is the first reported structural feature in a natural saxitoxin analogue, whereas the same ring system has previously been reported in a synthetic saxitoxin analogue, FD-saxitoxin. Acid hydrolysis of the carbamoyl N-sulfate in M5-HA produced M6-HA which was also identified in P. yessoensis by using LC-MSMS. M5-HA was not synthetically produced from M1 (11-hydroxy gonyautoxin-5) and M3 (11,11-dihydroxy gonyautoxin-5) through incubation in aqueous buffers. Furthermore, PSTs in the hepatopancreas of P. yessoensis, cultured in a bay located in northeastern Japan, were chronologically analyzed in 2018. The highest concentrations of M1/M3/M5-HA were observed two weeks after C-toxins had reached their highest concentrations, which provides evidence that M1/M3/M5-HA are metabolites of C-toxins. The voltage-gated sodium channel blockage activity of M6-HA was not detected at the concentration of 140 nM by using the Neuro-2A veratridine/ouabain assay.

Expansion of Gamma-Butyrolactone Signaling Molecule Biosynthesis to Phosphotriester Natural Products.

Bacterial hormones, such as the iconic gamma-butyrolactone A-factor, are essential signaling molecules that regulate diverse physiological processes, including specialized metabolism. These low molecular weight compounds are common in Streptomyces species and display species-specific structural differences. Recently, unusual gamma-butyrolactone natural products called salinipostins were isolated from the marine actinomycete genus Salinispora based on their antimalarial properties. As the salinipostins possess a rare phosphotriester motif of unknown biosynthetic origin, we set out to explore its construction by the widely conserved 9-gene spt operon in Salinispora species. We show through a series of in vivo and in vitro studies that the spt gene cluster dually encodes the salinipostins and newly identified natural A-factor-like gamma-butyrolactones (Sal-GBLs). Remarkably, homologous biosynthetic gene clusters are widely distributed among many actinomycete genera, including Streptomyces, suggesting the significance of this operon in bacteria.

Structures of N-Hydroxy-Type Tetrodotoxin Analogues and Bicyclic Guanidinium Compounds Found in Toxic Newts.

The biosynthesis of tetrodotoxin (TTX, 1), a potent neurotoxin widely distributed in marine and terrestrial metazoans, remains unresolved. A significant issue has been identifying intermediates and shunt products associated with the biosynthetic pathway of TTX. We investigated TTX biosynthesis by screening and identifying new TTX-related compounds from Cynops ensicauda popei and Taricha granulosa. Mass spectrometry (MS)-guided screening identified two new N-hydroxy TTX analogues in newts: 1-hydroxy-8-epiTTX (2) and 1-hydroxy-8-epi-5,11-dideoxyTTX (3, previously reported as 1-hydroxy-5,11-dideoxyTTX). We prepared a new analogue, 8-epi-5,11-dideoxyTTX (4), from 3 via N-OH reduction and confirmed the presence of 4 in T. granulosa using hydrophilic interaction liquid chromatography (HILIC)-LCMS. The presence of 8-epi-type TTX analogues in both Cynops and Taricha supports a branched biosynthetic pathway of terrestrial TTX, which produces 6- and 8-epimers. In addition, new bicyclic guanidinium compounds Tgr-238 (5) and Tgr-240 (6) were identified as putative shunt products of our proposed TTX biosynthesis pathway. A structural analysis of Cep-228A (7), another bicyclic compound, was performed using NMR. Based on the structures of 5-7 and their analogues, we propose a model of the shunt and metabolic pathways of the terrestrial TTX biosynthesis.

Simple relation between frustration and transition points in diluted spin glasses.

We investigate a possible relation between frustration and phase-transition points in two-dimensional spin glasses at zero temperature. The relation consists of a condition on the average number of frustrated plaquettes and was reported to provide very good predictions for the critical points at zero temperature, for several two-dimensional lattices. Although there has been no proof of the relation, the good correspondence in several lattices suggests the validity of the relation and an important role of frustration in the phase transitions. To examine the relation further, we present a natural extension of the relation to diluted lattices and verify its effectiveness for bond-diluted square lattices. We then confirm that the resulting points are in good agreement with the phase-transition points in a wide range of dilution rate. Our result supports the suggestion from R. Miyazaki [J. Phys. Soc. Jpn. 82, 094001 (2013)JUPSAU0031-901510.7566/JPSJ.82.094001] for nondiluted lattices on the importance of frustration to the phase transition of two-dimensional spin glasses at zero temperature.

Design and Characterization of an EEG-Hat for Reliable EEG Measurements.

In this study, a new hat-type electroencephalogram (EEG) device with candle-like microneedle electrodes (CMEs), called an EEG-Hat, was designed and fabricated. CMEs are dry EEG electrodes that can measure high-quality EEG signals without skin treatment or conductive gels. One of the challenges in the measurement of high-quality EEG signals is the fixation of electrodes to the skin, i.e., the design of a good EEG headset. The CMEs were able to achieve good contact with the scalp for heads of different sizes and shapes, and the EEG-Hat has a shutter mechanism to separate the hair and ensure good contact between the CMEs and the scalp. Simultaneous measurement of EEG signals from five measurement points on the scalp was successfully conducted after a simple and brief setup process. The EEG-Hat is expected to contribute to the advancement of EEG research.

Cariogenic Streptococcus mutans Produces Tetramic Acid Strain-Specific Antibiotics That Impair Commensal Colonization.

Streptococcus mutans is a common constituent of dental plaque and a major etiologic agent of dental caries (tooth decay). In this study, we elucidated the biosynthetic pathway encoded by muc, a hybrid polyketide synthase and nonribosomal peptide synthetase (PKS/NRPS) biosynthetic gene cluster (BGC), present in a number of globally distributed S. mutans strains. The natural products synthesized by muc included three N-acyl tetramic acid compounds (reutericyclin and two novel analogues) and an unacylated tetramic acid (mutanocyclin). Furthermore, the enzyme encoded by mucF was identified as a novel class of membrane-associated aminoacylases and was responsible for the deacylation of reutericyclin to mutanocyclin. A large number of hypothetical proteins across a broad diversity of bacteria were homologous to MucF, suggesting that this may represent a large family of unexplored acylases. Finally, S. mutans utilized the reutericyclin produced by muc to impair the growth of neighboring oral commensal bacteria. Since S. mutans must be able to out-compete these health-associated organisms to persist in the oral microbiota and cause disease, the competitive advantage conferred by muc suggests that this BGC is likely to be involved in S. mutans ecology and therefore dental plaque dysbiosis and the resulting caries pathogenesis.

Temporal Variation of the Profile and Concentrations of Paralytic Shellfish Toxins and Tetrodotoxin in the Scallop, Patinopecten yessoensis, Cultured in a Bay of East Japan.

Paralytic shellfish toxins (PSTs) are the major neurotoxic contaminants of edible bivalves in Japan. Tetrodotoxin (TTX) was recently detected in bivalve shellfish around the world, drawing widespread attention. In Japan, high levels of TTX were reported in the digestive gland of the scallop, Patinopecten yessoensis, in 1993; however, no new data have emerged since then. In this study, we simultaneously analyzed PSTs and TTX in scallops cultured in a bay of east Japan using hydrophilic interaction chromatography (HILIC)-MS/MS. These scallops were temporally collected from April to December 2017. The highest concentration of PSTs (182 µmol/kg, total congeners) in the hepatopancreas was detected in samples collected on May 23, lined to the cell density of the dinoflagellate, Alexandrium tamarense, in seawater around the scallops, whereas the highest concentration of TTX (421 nmol/kg) was detected in samples collected on August 22. Contrary to the previous report, temporal variation of the PSTs and TTX concentrations did not coincide. The highest concentration of TTX in the entire edible tissues was 7.3 µg/kg (23 nmol/kg) in samples obtained on August 22, which was lower than the European Food Safety Authority (EFSA)-proposed threshold, 44 µg TTX equivalents/kg shellfish meat. In addition, 12ß-deoxygonyautoxin 3 was firstly identified in scallops.

Isolation and Biological Activity of 8- Epitetrodotoxin and the Structure of a Possible Biosynthetic Shunt Product of Tetrodotoxin, Cep-226A, from the Newt Cynops ensicauda popei.

Tetrodotoxin (TTX, 1), a potent neurotoxin, has been found in various animal species in both marine and terrestrial environments. In this study, a new TTX analogue, 8- epiTTX (2), and a possible biosynthetic shunt compound of TTX, Cep-226A (3), were isolated from the newt Cynops ensicauda popei. The voltage-gated sodium ion channel (Nav) blocking activity of 2 and 6- epiTTX (4), a known analogue, were investigated by a colorimetric cell-based assay and compared with that of 1. The EC50 values for 2 and 4 were determined to be 110 ± 40 and 33 ± 11 nM, respectively, which were larger than that of 1 (1.9 ± 0.7 nM). The results indicated that the equatorial hydroxy group at C-8 in TTX significantly contributes to its Nav blocking activity, whereas the 6-epimer of TTX retains substantial activity, consistent with its previously reported toxicity in mice and binding affinity to rat brain membrane preparations. The presence of these epimers of TTX (2 and 4) and Cep-226A (3) in newts supports our hypothesis that TTX is derived from a monoterpene in terrestrial environments.

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.