Spiro Bicyclic Guanidino Compounds from Pufferfish: Possible Biosynthetic Intermediates of Tetrodotoxin in Marine Environments.

Tetrodotoxin (TTX, 1) is a potent neurotoxin that is widely found in both terrestrial and marine animals; however, the biosynthetic pathway and genes for TTX have not yet been elucidated. Previously, we proposed that TTX originated from a monoterpene; this hypothesis was based on the structures of cyclic guanidino compounds that are commonly found in toxic newts. However, these compounds have not been detected in marine organisms. Instead, a series of deoxy analogues of TTX were found in toxic marine animals; thus, we further screened for TTX-related compounds in marine animals. Herein, we report seven novel spiro bicyclic guanidino compounds 2-8 that were isolated from the pufferfish Tetraodon biocellatus. In compounds 2-5 and 7-8, a six-membered cyclic guanidino amide is spiro-fused with 2,4-dimethyl cyclohexane, whereas in compound 6, the same cyclic guanidino amide is spiro-fused with 2,3,5-trimethylcyclopentane. Compounds 2-5 and 7-8 have the same carbon skeleton and relative configuration as TTX. Thus, we proposed that compounds 2-8 are biosynthetic intermediates of TTX in marine environments. TTX could be biosynthetically derived from compound 7 via intermediates 2-5 through several oxidations, amide hydrolysis, and formation of the hemiaminal and lactone found in 5,6,11-trideoxyTTX, the major TTX analogue, whereas compounds 6 and 8 might be shunt products. LC-MS analysis confirmed the wide distribution of compounds 2, 3, or both in TTX-containing marine animals, namely pufferfish, crab, octopus, and flatworm, but compounds 2-8 were not detected in newts.

Recursive Additive Complement Networks for Cell Membrane Segmentation in Histological Images.

A recursive additive complement network (RacNet) is introduced to segment cell membranes in histological images as closed lines. Segmenting cell membranes as closed lines is necessary to calculate cell areas and to estimate N/C ratio, which is useful to diagnose early hepatocellular carcinoma. The RacNet is composed of a complement network and an element-wise maximization (EWM) process and is recursively applied to the network output. The complement network complements the lacking parts of cell membranes. The network, however, has a tendency to mistakenly delete some parts of the segmented cell membranes. The EWM process eliminates this unwanted effect.Experiments carried out using unstained hepatic sections showed that the accuracy for segmenting cell membranes as closed lines was significantly improved by using the RacNet.Three imaging methods, bright-field, dark-field, and phase-contrast, were used, as unstained sections show very low contrast in the bright-field imaging commonly used in pathological diagnosis. These imaging methods are available in optical microscopes used by pathologists. Among the three methods, phase-contrast imaging showed the highest accuracy.

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.