Ajuga Δ24-Sterol Reductase Catalyzes the Direct Reductive Conversion of 24-Methylenecholesterol to Campesterol.

Dimunito/Dwarf1 (DWF1) is an oxidoreductase enzyme that is responsible for the conversion of C28- and C29-Δ(24(28))-olefinic sterols to 24-methyl- and 24-ethylcholesterols. Generally, the reaction proceeds in two steps via the Δ(24(25))intermediate. In this study, we characterized theArDWF1gene from an expression sequence tag library ofAjuga reptansvar.atropurpureahairy roots. The gene was functionally expressed in the yeast T21 strain. Thein vivoandin vitrostudy of the transformed yeast indicated that ArDWF1 catalyzes the conversion of 24-methylenecholesterol to campesterol. A labeling study followed by GC-MS analysis suggested that the reaction proceeded with retention of the C-25 hydrogen. The 25-H retention was established by the incubation of the enzyme with (23,23,25-(2)H3,28-(13)C)-24-methylenecholesterol, followed by(13)C NMR analysis of the resulting campesterol. Thus, it has been concluded that ArDWF1 directly reduces 24-methylenecholesterol to produce campesterol without passing through a Δ(24(25))intermediate. This is the first characterization of such a unique DWF1 enzyme. For comparison purposes,Oryza sativa DWF1(OsDWF1) was similarly expressed in yeast. Anin vivoassay of OsDWF1 supported the generally accepted two-step mechanism because the C-25 hydrogen of 24-methylenecholesterol was eliminated during its conversion to 24-methylcholesterol. As expected, the 24-methylcholesterol produced by OsDWF1 was a mixture of campesterol and dihydrobrassicasterol. Furthermore, the 24-methylcholesterol contained in theAjugahairy roots was determined to be solely campesterol through its analysis using chiral GC-MS. Therefore, ArDWF1 has another unique property in that only campesterol is formed by the direct reduction catalyzed by the enzyme.

Functional characterization of CYP71D443, a cytochrome P450 catalyzing C-22 hydroxylation in the 20-hydroxyecdysone biosynthesis of Ajuga hairy roots.

20-Hydroxyecdysone (20HE), a molting hormone of insects, is also distributed among a variety of plant families. 20HE is thought to play a role in protecting plants from insect herbivores. In insects, biosynthesis of 20HE from cholesterol proceeds via 7-dehydrocholesterol and 3ß,14α-dihydroxy-5ß-cholest-7-en-6-one (5ß-ketodiol), the latter being converted to 20HE through sequential hydroxylation catalyzed by four P450 enzymes, which have been cloned and identified. In contrast, little is known about plant 20HE biosynthesis, and no biosynthetic 20HE gene has been reported thus far. We recently proposed involvement of 3ß-hydroxy-5ß-cholestan-6-one (5ß-ketone) in 20HE biosynthesis in the hairy roots of Ajuga reptans var. atropurpurea (Lamiaceae). In this study, an Ajuga EST library was generated from the hairy roots and P450 genes were deduced from the library. Five genes with a high expression level (CYP71D443, CYP76AH19, CYP76AH20, CYP76AH21 and CYP716D27) were screened for a possible involvement in 20HE biosynthesis. As a result, CYP71D443 was shown to have C-22 hydroxylation activity for the 5ß-ketone substrate using a yeast expression system. The hydroxylated product, 22-hydroxy-5ß-ketone, had a 22R configuration in agreement with that of 20HE. Furthermore, labeling experiments indicated that (22R)-22-hydroxy-5ß-ketone was converted to 20HE in Ajuga hairy roots. Based on the present results, a possible 20HE biosynthetic pathway in Ajuga plants involved CYP71D443 is proposed.

Constituents of twig bark of pear cultivars (Pyrus species).

Organic solvent extracts from fresh twig bark of Japanese pear cultivars (Pyrus serotina) Shinko and Nijisseiki, and European pear cultivar (P. communis) Le Lectier were obtained by maceration with n-hexane and EtOAc, and analyzed in GC-EIMS experiments. In these two Japanese cultivars, the lupeol, betulin, epifriedelinol, friedelin and arbutin contents of Nijisseiki were higher than those of Shinko. In the case of the lupane-type triterpenes, lupeol and betulin, the lupeol content of Japanese pears Shinko and Nijisseiki was higher than that of European pear Le Lectier. The betulin content of Le Lectier was higher than those of Shinko and Nijisseiki. Friedelane-type triterpenes, epifriedelinol and friedelin, were not detected in twig bark of Le Lectier. Quantitative and qualitative differences in the constituents of these three pear cultivars were observed.

[Study of the voxel-based specific regional analysis system for Alzheimer's disease imaging sequence after magnetic resonance apparatus replacement].

In our institute, an MR apparatus, MAGNETOM VISION (Siemens) was replaced by ECHELON Vega (HITACHI). Z-score data acquired by MPRAGE (VISION) was compared with those by radio frequency-spoiled steady-state acquisition with rewinded gradient echo (RSSG) and gradient echo inversion recovery (GEIR) (ECHELON). For this study, ten normal volunteers were recruited and their data ware obtained within two months using both apparatuses. In addition, the difference of the contrasts of the images of these apparatuses was compared. There was a significant difference between Z-scores of MPRAGE and RSSG while there was no difference between MPRAGE and GEIR. As for the contrast, data of MPRAGE were similar to those of GEIR. To compare Z-scores acquired with MAGNTOM VISION (Siemens), it seems appropriate to use GEIR in ECHELON Vega.

Structural determination of montanacin D by total synthesis.

The first total syntheses of acetogenin 3 and its 4 S,8 R-isomer are described. The key step involves intermolecular metathesis of an alpha,beta-unsaturated ketone carrying a tetrahydropyranyl lactone with a tetrahydrofuran derivative. Compound 3 has spectroscopic and physical data consistent with those of natural montanacin D, suggesting that the absolute configuration of the natural product is as shown in 3.