Hideo Naoki

Two analogs of azaspiracid isolated from mussels, Mytilus edulis, involved in human intoxication in Ireland.

Two new analogs of azaspiracid, azaspiracid-2 and azaspiracid-3, were isolated from mussels collected at Arranmore Island, Ireland in 1997 as additional causes of human intoxication. Their structures were determined to be 8-methylazaspiracid and 22-demethylazaspiracid, respectively by NMR and negative ion FAB CID MS/MS experiments.

Identification of putative palytoxin as the cause of clupeotoxism

In 1994 in Madagascar a woman died after eating a sardine, Herklotsichthys quadrimaculatus. Two heads removed, respectively, from a toxic and a nontoxic fish before cooking were retrieved, kept frozen, and used for toxin analysis. The causative toxin was identified as palytoxin or its analogs based on its cytotoxicity, delayed hemolysis, neutralization with an anti-palytoxin antibody, chromatographic properties on different columns, and MS data. The gill and esophagus of the fish contained large amount of bottom sediments indicating that the fish had fed on the bottom and thus probably obtained the toxin from a benthic organism. The benthic dinoflagellate Ostreopsis siamensis that produces palytoxin and its analogs was inferred as the probable toxin source. This is the first study to shed light on clupeotoxism, a highly fatal form of human intoxication due to ingestion of clupeoid fish.

Production of tetrodotoxin and its derivatives by Pseudomonas sp. isolated from the skin of a pufferfish

Bacteria isolated from the skin of the pufferfish Fugu poecilonotus were screened for tetrodotoxin production. Tetrodotoxin and its derivatives were detected by HPLC analyses in broth cultures of a Pseudomonas sp. Upon alkali treatment the compounds yielded, as do tetrodotoxin and its derivatives, 2-amino-6-hydroxymethyl-8-hydroxyquinazoline, the identity of which was confirmed, after methylation or trimethylsilylation, by HPLC, mass spectrometry and gas chromatography - mass spectrometry.

Gymnocin-a, a cytotoxic polyether from the notorious red tide dinoflagellate, gymnodinium mikimotoi

A new cytotoxic polyether, gymnocin-A, was isolated from the notorious red tide dinoflagellate, Gymnodinium mikimotoi, and its structure consisting of 14 contiguous ether rings and a 2-methyl-2-butenal side-chain was determined by NMR and CID MS/MS experiments.

Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.

Location of functional groups in mycobacterial meromycolate chains; the recognition of new structural principles in mycolic acids.

Mycobacterial alpha-, methoxy- and keto-mycolic acid methyl esters were separated by argentation chromatography into mycolates with no double bond, with one trans double bond or with one cis double bond. Meromycolic acids were prepared from each methyl mycolate fraction by pyrolysis, followed by silver oxide oxidation, and analysed by high-energy collision-induced dissociation/fast atom bombardment MS to reveal the exact locations of the functional groups within the meromycolate chain. The locations of cis and trans double bonds, cis and trans cyclopropane rings, methoxy and keto groups, and methyl branches within the meromycolate chain were determined from their characteristic fragment ion profiles, and the structures of the meromycolic acids, including those with three functional groups extracted from Mycobacterium tuberculosis H37Ra, Mycobacterium bovis BCG and Mycobacterium microti, were established. Meromycolic acids with one cis double bond were structurally closely related to those with one cis cyclopropane ring, whereas the meromycolic acids with one trans cyclopropane ring were closely related to the corresponding meromycolic acids with one cis cyclopropane ring. A close relationship between methoxy- and keto-meromycolic acids was also implied. The relationship between the meromycolic acids with a trans double bond and the other meromycolic acids was not clearly revealed, and they did not appear to be immediate substrates for trans cyclopropanation.

Purification, structure-function analysis, and molecular characterization of novel linear peptides from scorpion Opisthacanthus madagascariensis.

In the previous report [Biochem. Biophys. Res. Commun. 286 (2001) 820], we described a novel short linear peptide, IsCT, with cytolytic activity isolated from the venom of scorpion Opisthacanthus madagascariensis. From the same scorpion venom, we further purified and characterized three short linear peptides named IsCT2, IsCTf, and IsCT2f that shared high homology with IsCT, while with different C-terminal areas between IsCT/IsCT2 and IsCTf/IsCT2f. Structure-activity relationship was analyzed by performing vivo and vitro assays and circular dichroism spectroscopy. Like IsCT, IsCT2 showed broad activity spectra against microbes (Gram positive and negative bacteria as well as fungi) and relatively weak hemolytic activity against sheep red blood cells. It adopts an amphipathic alpha-helical structure in aqueous TFE and is able to disrupt the artificial membrane. However, the other two peptides IsCTf and IsCT2f showed no activity in antimicrobial or hemolytic assay. Furthermore, IsCTf and IsCT2f cannot form amphipathic alpha-helix while demonstrating random coil structure in aqueous TFE, which might result in their lost cytolytic activity. IsCTf and IsCT2f both exist in the crude venom and are proved to be enzymatic products from IsCT and IsCT2. Whether they have some other biological activity is still unclear. In addition, we got the cDNAs encoding the precursors of IsCT and IsCT2. Besides the signal peptide, they still contain an unusual acidic pro-peptide at the C-terminal that was quite different from other known precursors of scorpion venom peptides. The novel structure and biological activity of these peptides proposed them to be a new class in scorpion venom.

Studies on palytoxins

Abstract The complete structure of palytoxin ( 1 ) was elucidated by us in 1982. 1 Our continuous interests in palytoxin led us to examine minor constituents of Okinawan Palythoa tuberculosa . In this paper, we describe successful isolation and structural elucidation of four minor toxins, which were named homopalytoxin ( 2 ), bishomopalytoxin ( 3 ), neopalytoxin ( 4 ) and deoxypalytoxin ( 5 ).

Brevetoxin B3, a new brevetoxin analog isolated from the greenshell mussel perna canaliculus involved in neurotoxic shellfish poisoning in new zealand

Abstract A new brevetoxin analog, brevetoxm B3 (BTXB3 1 ). was isolated from the greenshell mussel, Perna canaliculus from New Zealand. In BTXB3 the brevetoxin B skeleton is modified by cleavage of ring D. esterification of the resulting alcohol, and oxidation of the aldehydic terminus.

Isolation and structure of a new brevetoxin analog, brevetoxin B2, from greenshell mussels from New Zealand

Abstract A new brevetoxin analog, brevetoxin B2 (BTXB2), was isolated from greenshell mussels, Perna canaliculus , collected at the time of the neurotoxic shellfish poisoning incident in New Zealand. The structure was elucidated based on NMR, CAD FAB MS/MS, and chemical degradation experiments.