Akiyoshi Sawabe

Elucidation of a Carotenoid Biosynthesis Gene Cluster Encoding a Novel Enzyme, 2,2′-β-Hydroxylase, from Brevundimonas sp. Strain SD212 and Combinatorial Biosynthesis of New or Rare Xanthophylls

ABSTRACT A carotenoid biosynthesis gene cluster mediating the production of 2-hydroxyastaxanthin was isolated from the marine bacterium Brevundimonas sp. strain SD212 by using a common crtI sequence as the probe DNA. A sequence analysis revealed this cluster to contain 12 open reading frames (ORFs), including the 7 known genes, crtW, crtY, crtI, crtB, crtE, idi, and crtZ. The individual ORFs were functionally analyzed by complementation studies using Escherichia coli that accumulated various carotenoid precursors due to the presence of other bacterial crt genes. In addition to functionally identifying the known crt genes, we found that one (ORF11, named crtG) coded for a novel enzyme, carotenoid 2,2′-β-hydroxylase, which showed intriguingly partial homology with animal sterol-C5-desaturase. When this crtG gene was introduced into E. coli accumulating zeaxanthin and canthaxanthin, the resulting transformants produced their 2-hydroxylated and 2,2′-dihydroxylated products which were structurally novel or rare xanthophylls, as determined by their nuclear magnetic resonance and high-performance liquid chromatography/photodiode array detector/atmospheric pressure chemical ionization mass spectrometry spectral data. The new carotenoid produced was suggested to have a strong inhibitory effect on lipid peroxidation.

Isolation and characterization of new limonoid glycosides from Citrus unshiu peels.

Three limonoid glycosides were isolated from Citrus unshiu peels, and their structures were determined based on MS and NMR spectroscopic data as nomilinic acid 17-O-beta-D-glucopyranoside (1), methyl nomilinate 17-O-beta-D-glucopyranoside (2), and obacunone 17-O-beta-D-glucopyranoside (3). In particular, the location of the sugar moiety was clearly determined by the B/E constant linked scan FABMS method. No limonoid glycosides obtained here were found to have antitumor activity in NCI-H292 and EL-4 cell lines.

Bioactive glycosides in citrus fruit peels

Abstract Since 1980s we have been investigating bioactive compounds in citrus fruit peels. We have successfully isolated seventy-three glycosides including thirty-four new compounds. They were flavonoid glycosides, phenylpropanoid glycosides, terpenoid glycosides, limonoid glycosides, and alkyl glycosides. The biological activities of the compounds were studied for the utilization as hypotensive and hypertensive drugs. In this manuscript, recent works are briefly reviewed focussing on the isolation and a mutual relationship between the structures of the compounds and their biolosical activities.

Fast atom bombardment mass spectrometry and linked scan analyses at constant B/E in the structural characterization of new polyisoprenepolyols isolated from an edible mushroom (Hypsizigus marmoreus)

Fast atom bombardment (FAB) mass spectrometry is a powerful tool for analyzing the structure of polyisoprenepolyols. Analyses in the positive and negative ion mode are complementary in that the former provides data on the number of hydroxy groups present while the latter provides data on the isoprenoid sequence. Some of this information is already available from routine FAB mass spectra. More detailed information is contained in the linked scan spectra.

The feasibility of using mosquitofish (Gambusia affinis) for detecting endocrine‐disrupting chemicals in the freshwater environment

We evaluated the utility of gene-transcriptional responses in the liver of mosquitofish (Gambusia affinis), a species introduced to many countries and therefore widely available, for detecting endocrine-disrupting activity in water. Exposure to β-naphthoflavone, an aryl hydrocarbon receptor (AhR) agonist, significantly increased the transcript of the cytochrome P4501A gene (cyp1a), peaking at 24 h, in both sexes at concentrations of 10 µg/L or more. 17β-Estradiol (E(2) ) at 500 ng/L increased the number of males showing gene transcription of precursors of yolk protein, vitellogenin (Vtga, Vtgb, and Vtgc), at 24, 48, and 72 h. Exposure for 48 h to bisphenol A (BPA), an estrogen mimic, also increased vtg-positive males at 1 mg/L or more. Leachate from a Japanese stable-type landfill significantly increased vtg-positive males after 48 h exposure, and the in vitro activity of the leachate against the estrogen receptor (ER) was estimated as an E(2) equivalent of 240 ng/L by yeast transfected with the ER. Chemical analysis showed that major contributors to the ER activation were BPA and 4-tert-octylphenol. This leachate and drainage water from a control-type landfill had AhR activities, estimated by yeast with the AhR, but had no significant effect on cyp1a transcription. These results showed that mosquitofish are suitable for detecting in vivo AhR and ER effects, but are insensitive to E(2).

Accumulation of Heavy Metals by Japanese Weeds and Their Seasonal Movement

Phytoremediation is a technique for the removal of contaminants in the environment using plants, which is currently is being researched worldwide. We focus attention on certain Japanese weeds, which have a large biomass and high environmental adaptability as a hyperaccumulator. Specifically, we investigated the seasonal metals movement of roots, stems and leaves on Artemisia princeps, a common Japanese weeds, and examined methods of heavy metal removal from the soil. Plants and surface soils were collected at watersides such as a reservoir and adjustment pond around our University at Nara Prefecture, and around the Kizu river of Seika town at Kyoto Prefecture in Japan. The soils and plants were ashed using H2NO3, HCl, and H2O2. Mn, Ni, Cr, Fe, Cu, Zn and Li in the ashes were measured by AAS. Hyperaccumulators have not been found yet at our investigation sites. However, Aster leiophyllus, Artemisia princeps and Stenactis annuus accumulated 2 or 3 times as many metals (Cr, Cu, Mn) as other plants in the same s ites. As to the seasonal metals movement of roots, stems and leaves on Artemisia princeps, plants concentration and accumulation ratio of Cu that compared the concentration between soil and plants are high in autumn. As a result, we think that Cu plays special role different from other metals. Accordingly, this movement is important in examination of the treatment stage after having taken in heavy metal.

Role of GPx4 in human vascular endothelial cells, and the compensatory activity of brown rice on GPx4 ablation condition

Oxidative stress is implicated in the pathologies of vascular endothelial cells. However, the importance of specific antioxidant enzymes in vascular endothelial cells is not fully understood. The purpose of this study was to elucidate the importance of Glutathione peroxidase 4 (GPx4), and the involvement of ferroptosis on cell death induced by GPx4 loss in human vascular endothelial cells. In addition, we examined the compensatory activity of brown rice on GPx4 ablation condition. Human umbilical vein endothelial cells were transfected with GPx4 or scramble control siRNA. GPx4 knockdown caused the increase in the levels of lipid oxidation, and induced cytotoxicity. On the other hand, α-tocopherol (vitamin E) and extract of brown rice, ameliorated lipid peroxidation, cytotoxicity, and delay of proliferation induced by GPx4 knockdown. Furthermore, ferrostatin-1, inhibitor of ferroptosis, also prevented cytotoxicity and delay of proliferation. In conclusion, our data demonstrated that GPx4 is an essential antioxidant enzyme for protecting lipid peroxidation, and is a regulator of ferroptosis in vascular endothelial cells. Furthermore, vitamin E rich food, such as brown rice, can compensate for GPx4 loss by protecting cells against lipid peroxidation.

Investigation of functional molecules in African Celosia argentea L.

Abstract Five glycosides were isolated from the leaves of Celosia argentea L. and their structures were determined based on UV, MS, 1 H NMR and 13 C NMR spectroscopic data as 1-(4- O -β-glucopyranosyl-3-methoxyphenyl) propane-2-ene (citrusin C, 1 ), 3- O -β-glucopyranosyl-1 H -indole (indican, 2 ), (3 Z )-hexenyl-(1- O -α-rhamnopyranosyl-β-glucopyranoside) ( 3 ), (3 Z )-hexenyl-1- O -β-glucopyranoside ( 4 ), and (7 E )-6,9-dihydromegastigma-7-ene-3-one-9- O -[β-glucopyranoside ( 5 ). On bioassay on the germination of lettuce, compound 1 , the aglycone of 1 ( 1a ), and the aglycone of 3 (3a) exhibited growth inhibitory activity, whereas compound 2 and 3 exhibited growth promotive activity. Compounds 1 –5 were detected in the leaves of Celosia argentea L. for the first time in this study.

Structural analyses of glycolipids from edible mushroom by fast atom bombardment mass spectrometry

Abstract The structures of glycolipids isolated from Hypsizigus marmoreus (Bunashimeji, a mushroom) and Pleurotus citrinopileatus (Nireouma, a mushroom) were determined to be (4E, 8E)-N-2-hydroxyhexadecanoyl-1-O-β-glucopyranosyl-9-methyl-C 18 -sphinga-4, 8-dienine ( 1 ), phosphodihexose N-(2-hydroxyoctadecanoyl)-4-hydroxy-C 18 -sphinganine ( 2 ), phosphodihexose N-(2-hydroxyhexadecanoyl)-4-hydroxy-C 18 -sphinganine ( 3 ), phosphodihexose N-(2-hydroxytetracosanoyl)-4-hydroxy-C 18 -sphinganine ( 4 ), and phosphodihexose N-(2-hydroxydocosanoyl)-4-hydroxy-C 18 -sphinganine ( 5 ). In particular, location of the double bonds in the long-chain base of 1 was clearly determined by the B/E constant linked scan method. The structure of cerebroside having a long-chain base of 9-methyl-C 18 -sphinga-4,8-dienine could be determined in general by the presence of characteristic fragment ions of [C 19 -sphingadienine + H-H 2 0]+ at m/z 276 and [C 19 -sphingadienine + H] + at m/z 294, and the fatty acid carbon number could be calculated from the characteristic fragment ion of [ceramide-180] + ([MH -GlcOH-180] + ) in positive ion mode FAB mass spectrometry. In the structural determination of 2–5, the ions of m/z 421 and 720 in the negative ion mode analyses are assigned to be characteristic of phosphodihexose and phytosphingosine containing phosphodihexose, respectively. This is a useful methodology for the structural determination of other unstable natural products such as lipids.

Evaluation of RAR binding activity materials in plant with yeast two hybrid assay

Extended Abstract A great variety of chemicals are present in environment. In particular effect to give the human, an animal and an ecosystem with hormone-disrupting chemicals is concerned. For example, the substance called 17β-estradiol is a kind of the female sex hormone called estrogen. The abnormality of the generative organ is found excessively by binding to an estrogen receptor. Also, it has been reported the retinoic acid produces malformation for frame formation by binding to a receptor excessively. Resemblance active substance binding to these receptors, an unknown active agent, unexpected product are present in environment, and there is multiplex exposure. It is difficult to distinguish these by instrumental analysis. Therefore it is an organism reply, the bioassay that can evaluate complex effect comprehensively to be used. Not only we performed environmental monitoring using the yeast twohybrid method which is one of the bioassay, but also we applied it to the component analysis of the plant. Yeast Two-Hybrid Assay is bioassay technique to use the recombination yeast which introduced a fusion protein of GAL4DNA binding domain (GAL4 DBD) of ligand binding domain (LBD) of nuclear receptors such as sex hormone or the thyroid hormone (Shiraishi et al., 2000, Kamata et al., 2008). The yeast Two-hybrid Assay method which we used in this study was carried out by the method that improved a traditional approach in our laboratory. In a previous study, we investigated Azolla [Azolla cristata × filiculoides] of the aquatic fern plant which showed RAR binding activity (Sawabe et al., 2012). Red and green leaves of Azolla were extracted with methanol for one week, respectively. The methanol extract was treated with organic solvents, and the extracts examined RAR binding activity. Remarkable activity was separated over silica gel column chromatography.