Takuro Kisaki

Studies on the production of Digitalis cardenolides by plant tissue culture I. Determination of digitoxin and digoxin contents in first and second passage calli and organ redifferentiating calli of several Digitalis species by radioimmunoassay

With first and second passage calli induced from seedlings of Digitalis purpurea, D. lanata, D. lutea, D. mertonensis, D. ambigua and D. ferruginea Gigantea and those induced from leaf discs of D. purpurea and D. lanata, the contents of digitoxin and digoxin equivalents were assayed and compared with the contents involved in the inocula. Although the total contents of digitoxin and digoxin equivalents in the first passage calli induced from seedlings varied between zero and nine times as high as in the original seedlings, those in the second passage calli were almost undetectable. The total contents of digitoxin equivalents in the first passage calli induced from leaf discs of D. purpurea were approximately equal to those in the original leaf discs, but those in the second passage calli were less than those in the inocula. In the first passage calli induced from leaf discs of D. lanata, the total contents of digitoxin equivalents decreased but those of digoxin equivalents slightly increased. However, in the second passage calli, the amounts of both cardenolides decreased. Root-forming calli accumulated no more digitoxin nor digoxin equivalents than completely dedifferentiated calli. However, shoot-forming calli accumulated considerable amounts of cardenolides, which were assayed as digitoxin and digoxin equivalents by radioimmunoassay.

Metabolism of nicotinic acid in tobacco plants

Abstract 14C from nicotinic acid-6-14C which was administered to tobacco plants was incorporated into nine pyridine compounds during 3 hr incubation; two of these were identified as nicotinic acid-N-glucoside and 6-hydroxynicotinic acid. Nicotinic acid was only incorporated into intermediates of the pyridine nucleotide cycle to a limited extent the main product being the glucoside. Nicotinic acid glucoside was incorporated into nicotine with the same efficiency as nicotinic acid, but the rate of the incorporation was markedly reduced when unlabelled nicotinic acid was also fed, suggesting that the glucoside is not involved in the direct route of nicotine biosynthesis.

Selection of Cultured Tobacco Cell Strains Producing High Levels of Ubiquinone 10 by a Cell Cloning Technique

Strains producing high levels of ubiquinone 10 were isolated from tobacco cell suspension cultures by a cell cloning technique, and the UQ content of these cell strains was analyzed by high performance liquid chromatography. Three strains with high UQ yields in suspension culture were selected and used for investigations on the necessary cultural conditions for UQ production. Sucrose concentration, 2,4-D concentration and culture temperature did not have marked effects on UQ formation. These results differed from those of the original cell lines. Furthermore, the addition of yeast extract to the medium promoted cell growth in these strains remarkably but did not enhance UQ production in the cell strains. According to these investigations, UQ production was increased to 15mg/liter and the UQ level to 1890 μg/g dry wt. of cells after 11 days of incubation.

Phytochemical studies on tobacco alkaloids—XI: A new alkaloid inNicotiana tabacum roots

Abstract A new alkaloid has been isolated from the roots of Nicotiana tabacum . Its chemical structure was confirmed as 2,4-di(3-pyridyl)piperidine ( anatalline ) by elemental analyses, u.v., i.r. and NMR and mass spectroscopy and finally by its dehydrogenation to nicotelline. The biosynthetic route for this compound may be similar to that of anatabine, but it is not formed in a manner similar to anabasine from lysine.

Changes in glycolipids and phospholipids of tobacco leaves during flue-curing.

The changes in the lipid components of tobacco leaves during flue-curing were investigated. The contents of total fatty acid methyl esters and linolenate decreased considerably, though a temporary increase was observed at the early period of the yellowing stage. The amounts of digalactosyl diglyceride (DGDG), monogalactosyl diglyceride (MGDG), sulfoquinovosyl diglyceride and phosphatidyl glycerol decreased markedly while phosphatidyl inositol, phosphatidyl serine and phosphatidyl choline were relatively resistant to the degradation process. MGDG and DGDG were found to have high proportions of linolenate while most phospholipids had high proportions of palmitate. Considerable decrease was observed in the ratio of linolenate in most polar lipids while the ratio of palmitate and stearate showed a significant increase.

Influences of Cultural Conditions on Polyphenol Formation and Growth of Amacha Cells (Hydrangea macrophylla Seringe var. Thunbergii Makino) and Changes of Polyphenol Contents in Leaves of Amacha Plant during Growth

Suitable cultural conditions for polyphenol formation were examined in the amacha cultured cells. Factors tested included; inoculum size, sucrose concentration, various carbon sources, extract (malt extract and yeast extract), kinetin, auxins, initial pH, light exposure and phosphate concentration. Sucrose concentration had marked effects on cell growth and polyphenol production, and phosphate concentration was significant in polyphenol formation, although other investigated factors had no effects on polyphenol productivity. The time course of polyphenol accumulation in cultured cells showed that polyphenol content decreased at the lag and early-exponential phases of growth, and increased remarkably till the stationary phase. Cells at the stationary phase did not accumulate polyphenol and even lost these polyphenols to the external medium as the cells reached the declining phase. Large scale culture of amacha cells was carried out using a 15 liter jar fermentor. Investigations on polyphenol content change...

Phytochemical Studies on the Tobacco Alkaloids: Part VIII. Biosynthesis of the Pyrrolidine Ring of Nornicotine in the Excised Root CulturePart IX. Question for Nornicotine as a Precursor of Nicotine

Duplicate feeding experiments of dl-ornithine-2-14C to the excised tobacco root culture were made, and the radioactive nornicotine was isolated. Approximately two thirds of the radioactivity was located in the 2-position of the pyrrolidine of the nornicotine in these experiments. This fact indicates that there are two modes in nornicotine biosynthesis: exclusive incorporation to the C-2 and equal incorporation to C-2 and C-5 from C-2 of ornithine.On the basis of this finding, biosynthetic route was discussed.dl-Ornithine-2-14C, dl-methionine-14CH3 and partially racemized l-nornicotine-2,5-14C were administered to aseptically grown excised roots (N. rustica var. Brasilia). Incorporation of their radioactivity to nicotine was compared. The extent of their radioactive incorporation to nicotine was high in the order of ornithine, methionine and nornicotine; incorporation of radioactivity of nornicotine to nicotine was extraordinarily low. 15N-Labeled nornicotine was also fed to the same materials and 15N dist...

Phytochemical Studies on Tobacco Alkaloids:Part IV. Stereospecific Demethylation of Nicotine in Tobacco Leaves

From the findings of the feeding experiments of d- and l-nicotine-14CH3 and d-nicotine to the excised tobacco leaves, it was demonstrated that demethylation of nicotine was stereospecific for the d-form in tobacco leaves. Such preferential demethylation to the enantiomer was also observed with N-methylanabasine and 1-(3′-pyridyl)-1-methylaminoethane which are analogous compounds to nicotine.