Two new seco-prezizaane-type sesquiterpenes, 1,2-dehydroneomajucin (1) and jiadifenin (2), were isolated from the methanol extract of the pericarps of Illicium jiadifengpi, indigenous to the southern part of China. Their structures were elucidated on the basis of NMR data. Compound 2, which is an equilibrated mixture of the epimers 2a and 2b on the C-10 acetal carbon, is the first example of a majucin-type seco-prezizaane with an oxo-function at the C-10 position. The proposed structure for 2 was unambiguously confirmed by chemical conversion of the known sesquiterpene (2S)-hydroxy-3,4-dehydroneomajucin (5) to 2. Compounds 2 and 5 were found to significantly promote neurite outgrowth in primary cultures of fetal rat cortical neurons at concentrations from 0.1 to 10 microM.

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An extract of the pericarps of Illicium merrillianum has yielded four new sesquiterpenes: 3 alpha-hydroxycycloparvifloralone (1), 1,2-dehydrocycloparvifloralone (2), (11) 7,14-ortholactone-3 alpha-hydroxyfloridanolide (3), and 11-O-debenzoyltashironin (4) along with cycloparvifloralone (5), merrillianone (6), and tashironin (7). The structures of 1--4 were determined on the basis of spectroscopic analyses. 11-O-Debenzoyltashironin (4) showed neurotrophic activity in primary culture of rat cortical neurons at 0.1--10 microM. However, cycloparvifloralone-type sesquiterpenes (1, 2, 5, and 6) and tashironin (7) had no neurotrophic activity at these concentrations.

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A methanol extract of the pericarps of Illicium minwanense afforded seven new seco-prezizaane-type sesquiterpenes (2-8) and a new abietane-type diterpene (9), together with six previously known compounds (1 and 10-14). The structures of the new compounds, (1S)- and (1R)-minwanenone (2 and 3), 1alpha-hydroxy-6-deoxypseudoanisatin (4), (2S)-hydroxy-6-deoxypseudoanisatin (5), 3-oxopseudoanisatin (6), (3S,6R)-4,7-epoxy-6-deoxypseudoanisatin (7), 7-O-methylpseudomajucin (8), and (+)-8,11,13,15-abietatetraene (9), were elucidated by spectroscopic data analysis and chemical transformations. The absolute configurations of 1 and 5 were established by X-ray crystallographic analysis of their p-bromobenzoyl derivatives.

Previously, we cloned a protease gene of Aeromonas sobria, determined its nucleotide sequence and established a method of purifying its product. In this study, we examined the properties of the purified protease. The protease was temperature‐labile and had an optimal pH of 7.5. Metallo‐protease inhibitors and a cysteine protease inhibitor did not block the proteolytic activity of the enzyme. The treatment with reagents to modify sulfhydryl group did not reduce the activity. But, serine protease inhibitors did, showing that it was a serine protease. Subsequently, we examined the ability of the protease to enhance vascular permeability in dorsal skin. The protease showed activity and the reaction was inhibited by a simultaneously injected antihistaminic agent. Histopathological examination showed that mast cells appeared around the site where the protease was injected. These findings show that the vascular permeability‐enhancing effect of the protease is due to histamine released at the site. Furthermore, we found that a soybean trypsin inhibitor (Kunitz) did not block the proteolytic action of the protease in vitro, but inhibited its vascular permeability‐enhancing activity in skin. This suggests that a trypsin‐like protease from skin mediates the activity of the protease to enhance its vascular permeability.

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We previously reported that the aerolysin-like hemolysin of Aeromonas sobria stimulates T84 cells to produce cyclic AMP, which then emerges in the culture medium. In order to clarify the mechanism of action of the hemolysin, we examined the involvement of adenosine nucleotide. The results show that the hemolysin stimulates T84 cells to release ATP, which is then converted to adenosine by ectonucleotidase. The adenosine generated might stimulate the P1 adenosine receptors of T84 cells to produce cyclic AMP.We purified the enterotoxin from the culture supernatant of Aeromonas sobria and found that it possessed hemolytic activity in addition to enterotoxic activity (5). The analysis of the DNA sequence (GenBank accession number AY157998) showed that the hemolysin is homologous with aerolysin produced by Aeromonas hydrophila. Homology between the two toxins is 68.5% at the amino acid level (5). The aerolysin-like hemolysin and aerolysin bound to cells act to form small pores in the membrane and induce cell damage (5, 11). These results suggest that the mode of action of the hemolysin is identical to that of aerolysin. Since cultured intestinal cells are destroyed by incubation with the hemolysin, we had predicted that the diarrhea caused by the hemolysin was a result of such cytotoxic action of the hemolysin. However, micropathological examination showed that the diarrheic syndrome appeared before the cell damage occurred (5). So, we speculated that the diarrhea was not due to the cytotoxic action of the hemolysin but rather that the hemolysin stimulated the intestinal cells to send some signal(s) to induce diarrhea. Thus, we examined the levels of cyclic nucleotides in T84 cells, i.e., human colonic epithelial cells, after incubation with the hemolysin to obtain a clue as to the signal and found that the level of cyclic AMP in the culture medium increased after the incubation (6). However, it has remained unsolved how the hemolysin induces the elevation of the level in the extracellular cyclic AMP.In the meantime, the action of adenosine existing outside of the cell has been studied extensively and its receptor, P1, has been found (4). At present, P1 receptors are divided into three groups, A 1 to A 3 . A 2 is divided into two subtypes: A 2A and A 2B . These P1 receptors couple to adenylate cyclase, whose activity is regulated by G proteins. A 1 and A 3 function to inhibit the cyclase and A 2 , both A 2A and A 2B , functions to activate the cyclase (3, 4). It was also shown that T84 cells, which we used to examine the activity of the hemolysin, possessed the A 2B receptor (14). Thus, we suspected that the adenosine was involved in the action of the hemolysin.Addition of papaverine to the medium. We reported that the level of cyclic AMP in the medium increased on incubation with the hemolysin (6). The excess cyclic AMP produced by the cells is decomposed by phosphodiesterase. Therefore, the amount of cyclic AMP reported in a previous study should be the residual amount after the attack by phosphodiesterase. To exami...