Masakazu Yamaoka

FATTY ACIDS SELECTIVELY INHIBIT EUKARYOTIC DNA POLYMERASE ACTIVITIES IN VITRO

The in vitro relationship between eukaryotic DNA polymerases and fatty acids was investigated. Some fatty acids strongly inhibited the activities of DNA polymerase alpha and/or beta in vitro. The kinetics of inhibition by linoleic acid showed that DNA polymerase alpha was non-competitively inhibited with respect to the DNA template and substrate (dTTP), while DNA polymerase beta was inhibited competitively with both DNA and substrate.

Improvement of Docosahexaenoic Acid Production in a Culture of Thraustochytrium aureum by Medium Optimization

Growth optimization of a docosahexaenoic acid (DHA) producing marine fungus, Thraustochytrium aureum (ATCC 34304), resulted in cell productivity of 5.7 g/l and 460 mg/l of total lipid in 69 h flask culture, almost twice the level of cell productivity obtained for the strain reported previously. The lipid extracted from the cells was primarily composed of triacylglycerol and contained about 40% DHA. The fermentor culture showed a lower growth rate than the flask culture indicating growth inhibition due to mechanical stirring. Cells grown in the fermentor showed a coagulant tendency. ATCC 34304, in culture, seems to reproduce asexually and the life cycle of the cells consists of a zoospore stage, trophic cell stage and sporangium stage. Zoospore release was observed once during the early growth phase throughout growth period both in flask and fermentor culture.

Efficient accumulation of oleic acid in Saccharomyces cerevisiae caused by expression of rat elongase 2 gene (rELO2) and its contribution to tolerance to alcohols

When the cells of Saccharomyces cerevisiae are exposed to high concentration of ethanol, the content of oleic acid (C18:1n-9) increased as the initial concentration of ethanol increased. Based on this observation, we attempted to confer ethanol tolerance to S. cerevisiae by manipulating fatty acid composition of the cells. Rather than altering OLE1 expression [the desaturase making both C16:1n-7 (palmitoleic acid) and C18:1n-9], we introduced elongase genes. Introduction of rat elongase 1 gene (rELO1) into S. cerevisiae gave cis-vaccenic acid (cis-C18:1n-7) by conversion from C16:1n-7, and the increase in this C18:1 fatty acid did not confer ethanol tolerance to the cells. On the other hand, the introduction of rat elongase 2 gene (rELO2), which elongates C16:0 to C18:0, drastically increased C18:1n-9 content, and the cells acquired ethanol tolerance, emphasizing the specific role of C18:1n-9. Furthermore, the transformant of rELO2 also conferred tolerance to n-butanol, n-propanol, and 2-propanol.

Antioxidative activities of tocotrienols on phospholipid liposomes

Antioxidative activity of tocotrienol (Toc3) was studied in the oxidation of dilinoleoylphosphatidylcholine (DLPC) liposomes. The objective was to measure the differences in the antioxidative activities betweenα-Toc3 andα-tocopherol (α-Toc), and betweenγ-Toc3 andγ-Toc. When each antioxidant was added to the already prepared DLPC liposome solution, the antioxidative activity of Toc3 was larger than that of Toc. However, when incorporated into the liposomal membrane, the antioxidative activities of Toc3 and Toc were the same and were intermediate between those of the added Toc3 and Toc.When added to the liposome solution, the consumption of Toc3 during the induction period was larger than that of Toc. When incorporated into the liposomal membrane, the consumptions of Toc3 and Toc were the same and were intermediate between those of the added Toc3 and Toc.These results suggest that the reactions of Toc3 and Toc with phospholipid peroxide within the membrane are inhibited to a different degree depending on the dosing manner of Toc3 and Toc. Namely, the degree of inhibition decreases in the following order: Toc(added)> Toc(incorporated)= Toc3(incorporated)> Toc3(added).

The effect of 2,4,6,8-tetramethyldecanoic acid on the hydrolysis and acylation of phospholipid by lipase in isooctane

The effect of an unusual natural fatty acid, 2,4,6,8-tetramethyldecanoic acid (TMDA), on the hydrolysis and acylation of phospholipid by lipase in isooctane was studied. Lipases examined were fromRhizopus delemar, Candida cylindracea andPseudomonas sp. The lipase was dispersed in isooctane by dioleoylphosphatidylethanolamine (DOPE) with or without the fatty acid and decanoyl-lysophosphatidylcholine (LPC). The effect of TMDA on both the hydrolysis of DOPE and the acylation of LPC was compared with that of decanoic acid (DA) by varying the water content. At the higher water content, the hydrolysis of DOPE was enhanced or trace amount of phosphatidylcholine was produced. Hydrolysis was enhanced more by the addition of TMDA than by DA. The effect of TMDA on the acylation of LPC was similar to that of DA.

Increase in stearidonic acid by increasing the supply of histidine to oleaginous Saccharomyces cerevisiae.

Increasing concentration of histidine significantly increased stearidonic acid production and cell growth in oleaginous Saccharomyces cerevisiae that has been genetically modified by Δsnf2 disruption, DGA1 and Δ6 desaturase gene overexpression, and LEU2 expression. High concentration of histidine in wild-type transformant and HIS3 expression in Δsnf2 transformant also increased stearidonic acid.

[29] Antioxidative activity of tocotrienol in heterogeneous system: Indication of restriction within membrane by fluorescence measurement

Publisher Summary This chapter discusses the comparison of antioxidative activities of the vitamin E homologs tocotrienol (Toc3) and tocopherol (Toc) in both homogeneous and heterogeneous systems. In a homogeneous system below 60 ° C, no significant difference is observed in antioxidative activities. This can be explained by the similar chemical structures of Toc3 and Toc, except for the unsaturated side chain of Toc3, and their similar intrinsic reactivities with peroxyl radicals. In a heterogeneous system at physiological temperature, however, Toc3 shows a greater antioxidative activity than the corresponding Toc. The results exhibit that the antioxidative activity of Toc3 is greater than that of Toc when Toc3 and Toc are distributed from water to the model membrane, while both are the same when the compounds are in the membrane from the beginning. It is also observed that both Toc3 and Toc in a model membrane are not completely consumed during the induction period and that the longer the induction period, the greater is the consumption.

Modulation of Lipid Body Size and Protein Profiles in the Oleaginous Fungus by Changing Nitrogen Concentration in Culture Medium

Mortierella ramanniana var. angulispora,an oleaginous fungus, accumulates large amounts of triacylglycerol (TG) and lipid bodies [1,2]. Lipid bodies in this fungus have a diameter of about 1 dun at the initial culture phase and then enlarge into a diameter of about 2–3 μm during lipid accumulation [2]. To form matured lipid bodies, TG and/or its precursors have to transport to lipid bodies. We have characterized the lipid transport into lipid bodies using fluorescent phospholipid analogues to find several transport pathways for the lipid body formation [2,3]. The question arises how the lipid transport into lipid bodies is regulated. To address the question, we looked for culture conditions to change the size of lipid bodies and characterized the protein profile of lipid bodies with different size. We found that changing nitrogen concentration represented as carbon to nitrogen ratio (C/N ratio) in culture medium affected the lipid body size in addition to lipid content. Comparing the lipid body fraction obtained from fungal cells with different lipid body size revealed that tyrosine phosphorylation was enhanced in the lipid body fraction when fungal cells were cultured with a lower C/N ratio (higher concentration of nitrogen).

Production of Fatty Acids from 1-Propanol in Pseudomonas sp

Pseudomonas sp (ATCC 12085) grown in a medium containing 1-propanol produced pentadecanoic (C15), heptadecanoic (C17), heptadecenoic (C17 : 1), and methyleneheptadecanoic acids (C18cyc), a cyclopropane fatty acid. The ratio of these fatty acids except for heptadecenoic acid to all fatty acids increased with the growth of Pseudomonas. Maximum concentrations of these fatty acids were obtained at 0.5 % 1-propanol. The fatty acids were also produced in the presence of sodium propionate instead of 1-propanol. The production of odd-numbered fatty acids and methylene-heptadecanoic acid by Pseudomonas sp can be explained based on the intermediary propionyl-CoA from 1-propanol and propionicacid.

4-Desmethylsterol composition of steryl glycoside in oil palm leaf

4-Desmethylsterols of steryl glycoside (SG) of oil palm leaves were separated and analyzed by GLC on both OV-1 (0.5 μm) and OV-17 (0.25 μm) capillary columns of the same size (25 m × 0.25 mm i.d.) under the same conditions. The extent of separation of each 4-desmethylsterol of SG was essentially the same on both columns. Campesterol, stigmasterol, sitosterol, 7-stigmastenol and avenasterol were found, in addition to a traces of cholesterol and brassicasterol. The compositions of these sterols by the OV-17 column were 4.8, 8.9, 84.2, 1.1, and 1.0 %, respectively