Ken'ichi Takeda

Effect on ergosterol biosynthesis of a fungicide, SSF-109, in Botrytis cinerea

Abstract Treatment of Botrytis cinerea with a novel fungicide SSF-109, (dl)-cis-1-(4-chlorophenyl)-2-(1H-1,2,4-triazol-1-yl)cyclo-heptanol (0.45μgml−1), gave five 14α-methyl sterols, 24-methylene-24(25)-dihydrolanosterol, 24-methylene-24(25)-dihydrolanosten-3-one, obtusifoliol, obtusifolione, and 14α-methylfecosterol, together with ergosterol and ergosta-5,8,22-trien-3β-ol. SSF-109 was found to inhibit the biosynthesis of ergosterol at the 14α-demethylation step.

Bile acid metabolism in partially hepatectomized rats

The bile flow and the bile acid secretion, calculated on liver weight basis, increased 12 H and 24 H after 60-70% hepatectomy and returned to the initial levels thereafter. The biliary phospholipid secretion much more increased than bile acids, but the cholesterol secretion decreased. Bile acid composition changed with an increase of the cholic acid group and a decrease of the chenodeoxycholic acid group in both bile and feces. These changes almost disappeared on Day 14. The pool size of bile acid decreased maximally on Day 4 to about 40% of the initial, but the distribution of bile acids in the enterohepatic circulation was not changed. The fecal cholesterol and coprostanol markedly decreased on Day 2 but gradually returned to the initial levels according to the recovery of diet intake. The fecal bile acids decreased on Day 2, increased on Day 4, and returned to the normal range after Day 7. In conclusion, the regenerating liver secretes more bile, bile acids and phospholipids, and less cholesterol than the normal liver. Cholic acid predominates in the bile acids. These changes restored to the initial levels by about one week after the operation.

Biosynthesis of campesterol and dihydrobrassicasterol in cultured cells of Amsonia elliptica

Abstract Reversed phase HPLC analysis of sterols of cultured cells of Amsonia elliptica showed that campesterol is the major sterol. Campesterol and its 24-epimer, dihydrobrassicasterol, were isolated from suspension cultures of A. elliptica incubated with l -[ Me - 2 H 3 ]methionine. Mass spectral analysis showed that both 24-epimeric 24-methylcholesterols retained two deuterium atoms. This result indicates that campesterol and dihydrobrassicasterol are biosynthesized via an intermediate with the 24-methylene side-chain.

Biosynthesis of isofucosterol from [2-13C2H3]acetate and [1,2-13C2]acetate in tissue cultures of Physalis peruviana—the stereochemistry of the hydride shift from C-24 to C-25

Migration of the hydrogen atom at C-24 of cycloartenol to C-25 from the Re-face of the double bond occurred in the biosynthesis of isofucosterol in cell cultures of Physalis peruviana fed [2-13C2H3]acetate and [1,2-13C2] acetate. This was verified by 13C n.m.r. spectroscopy.

Effect of sterol biosynthesis inhibitor, SSF-109, on cholesterol synthesis in isolated rat hepatocytes.

The inhibitory effect of SSF-109 on cholesterol synthesis in isolated hepatocytes was studied using a radio-high-performance liquid chromatography system, and the results were compared with those of other inhibitors, triparanol and AMO-1618. SSF-109 caused accumulation of two trimethylsterols: lanosterol and 24-dihydrolanosterol. The distribution profile of [2-14C]mevalonate-originated radioactivity in cholesterol, lanosterol, dihydrolanosterol, 2,3-oxidosqualene, and squalene indicates that SSF-109 inhibits the 14 alpha-methyl demethylase activity. Triparanol accumulated the radioactivity of [2-14C]mevalonate in desmosterol and 2,3-oxidosqualene suggesting that triparanol inhibits sterol delta 24-reductase and 2,3-oxidosqualene cyclase. AMO-1618 caused enrichment of the radioisotope from [2-14C]mevalonate in 2,3-oxidosqualene but reduced it in squalene, suggesting that AMO-1618 acts on 2,3-oxidosqualene cyclase and some enzyme that catalizes a metabolic pathway between mevalonate and squalene.

24β-Ethylsterol biosynthesis: Incorporation of l-[ME-2H3]methionine in tissue cultures of Trichosanthes kirilowii

Abstract A maximum of five deuterium atoms was incorporated into the 24β-ethylsterols, 22-dihydro-25-dehydrochondrillasterol and 22-dihydrochondrillasterol, in tissue cultures of Trichosanthes kirilowii incubated with l -[Me- 2 H 3 ]methionine. The results proved that a 24-ethylidene intermediate is not involved in the biosynthesis of 24β-ethylsterols in T. kirilowii .

Biosynthesis of sitosterol from [methyl-2H3]methionine in tissue cultures of Physalis peruviana

Abstract Tissue cultures of Physalis peruviana incubated with l -[Me- 2 H 3 ]methionine incorporated a maximum of four deuterium atoms into sitosterol, and thus confirmed that sitosterol is biosynthesized through a 24-ethylidene intermediate in this plant. Isofucosterol and 24-methylenecholesterol incorporated four and two deuterium atoms, respectively.

Biosynthesis of the 24-methylcholesterols dihydrobrassicasterol and campesterol in cultured cells of Amsonia elliptica: incorporation of [1,2-13C2]acetate and [2-13C,2H3]acetate

Feeding experiments with [1,2-13C2]acetate and [2-13C,2H3]acetate in suspension cultures of A. elliptica demonstrate that campesterol has C-26 (the pro-R methyl group at C-25) and C-27 (the pro-S methyl group at C-25) arising from C-6 and C-2 of mevalonate (MVA), respectively. In contrast, dihydrobrassicasterol has C-26 and C-27 derived from C-2 and C-6 of MVA, respectively. In both 24-methylcholesterols, the methyl group derived fron C-2 and C-6 of MVA retained two and three deuterium atoms, respectively. Furthermore, no signal due to a deuterium atom at C-24 or C-25 originating from [2-13C,2H3]acetate was found for campesterol and dihydrobrassicasterol. These data suggest that both campesterol and dihydrobrassicasterol are biosynthesized by regio-facial specific reduction of the 24(25)-double bond of the 24-methyl-24(25)-ene side-chain which is formed by double-bond isomerization of Δ24(28)-sterol.

Maintenance of androgen-, glucocorticoid- or estrogen-responsive growth in shionogi carcinoma 115 subline sustained in castrated mice with high dose of estrogen for 30 generations (3 years).

Shionogi carcinoma 115 (SC115), an androgen‐dependent mouse mammary tumor, rapidly loses its androgen responsiveness after androgen withdrawal. The growth of this tumor can also be stimulated by high doses of estrogen or glucocorticoid. In the present study, the maintenance of hormone‐responsive growth of SC115 tumors with a high dose of estrogen was examined in castrated male mice using an SCI 15 subline obtained by serial transplantations of SCI 15 tumors in estrogen‐treated castrated mice for 3 years (30 generations) (subline E2). Seed tumors from both SC115 and subline E2 could rapidly grow in castrated mice given daily injections of testosterone propionate (TP), 17β‐estradiol (E2), or dexamethasone (Dex) (100 μg/mouse/day) but not in those given vehicle alone. Although SCI 15 and subline‐E2 tumors grown with TP or Dex showed temporary regression after steroid withdrawal, the tumors grown with E2 did not show such temporary regression. The TP‐, E2‐, or Dex‐induced growth of subline‐E2 tumors was almost the same as that of the original SCI 15 tumors. However, responsiveness to androgen, estrogen or glucocorticoid of both tumors disappeared within one passage in steroid‐depleted castrated mice. The present findings demonstrate that the loss of responsiveness to androgen as well as to high doses of estrogen or glucocorticoid of SCI 15 tumors can be prevented in castrated mice not only with androgen but also with high doses of estrogen.

Structure Elucidation of Furostanol Glycosides Using Liquid Secondary Ion Mass Spectrometry

Abstract The structures of five genuine furostanol glycosides isolated from Metanarthecium luteo-viride MAXIM. (Liliaceae) were determined on the basis of liquid secondary ion mass spectrometric (LSIMS) analysis including liquid secondary ion mass spectrometry/mass spectrometry (LSIMS/MS). These glycosides were elucidated as bisdesmosides of furostanols (i.e. 2-0-acetyl-furometagenin, furometagenin, furonogiragenin, 2-0-acetyl- furometanarthogenin and 2-O-acetyl-3-oxo-furometagenin) as aglycons. which have cyclic hemiacetal moieties. bearing 2,3,4-tri-O-acetyl arabinopyranose at 11−C1) and glucopyranose at 26-C. In the LSIMS of these compounds, the protonated molecular ion [M + H]+ was not observed but the fragment ion [M - OH]+ corresponding to the loss of the hydroxyl group at 22-C was observed. By addition of NaCI to the sample matrix, the ion peaks for [M + Na]+ appeared in the spectra, which were used to determine the molecular formulae. Molecular orbital calculation of a model compound indicated that the ions [M+Na]+ were stabilized by formation of a four-membered ring structure bonding two oxygen atoms at the hemiacetal moiety and a sodium ion. Since the energy required for sodium ion addition to the neutral molecule was less than for proton addition, the sodium ion addition is more favorable. In addition, the repulsion energy of the protonated hemiacetal hydroxyl group is only 4.25 kcal/mol, and the potential energy of the fragmentation products ([M - OH]+ + H2 0) is 18.13 kcal/mol less than that of [M + H]+. These data rationalized the easy dehydration from the protonated furostanol glycoside.