Hirosuke Oku

Composition of lipids, fatty acids and sterols in Okinawan corals

A survey of lipid composition was made for 15 cnidarians from Okinawa, Japan. Eleven zooxanthellate scleractinian corals, an azooxanthellate scleractinian coral Tubastrea sp., a soft coral Lobophytum crassum, a hydroid coral Millepora murrayi and a sea anemone Boloceroides sp. were examined to elucidate the total lipid content, fatty acid composition for each lipid class and sterol composition. All specimens contained monoalkyldiacylglycerol which migrated between the triacylglycerols and esters on thin layer chromatography (TLC). Analysis by high performance thin layer chromatography (HPTLC) and Gas chromatography-mass spectrometry (GC-MS) revealed that these cnidarians were rich in wax ester and triacylglycerol, and that palmitic acid (16:0) was the most abundant fatty acid component of these lipid classes, followed by stearic (18:0) and oleic (18:1, n-9) acid in order of concentration. Of 11 sterols separated, four sterols were identified. It is suggested that sterol composition may be more useful for the biochemical classification of these cnidarians than fatty acid composition.

Seasonal changes in the content and composition of lipids in the coral Goniastrea aspera

Reef-building corals contain large amounts of storage lipids, which range from 9 to 47% of the dry weight of their tissues (Harland et al. 1993; Yamashiro et al. 1999). These lipids are mainly derived from carbon photosynthetically fixed by symbiotic zooxanthellae (Kelogg and Patton 1983; Harland et al. 1993). Lipid levels in coral tissue appeared to vary with the energy balance between input from zooxanthellae and output as respiration, cell renewal, and release of reproductive materials (Battey and Patton 1984; Rinkevich 1989; Arai et al. 1993; Ward 1995). Lipid levels in corals may show seasonal changes because production of photosynthates varies with light intensity and water temperature (Harland et al. 1992; Al-Moghrabi et al. 1995); however, this has yet to be demonstrated. This study examined the lipid content and composition of the reef-building coral Goniastrea aspera throughout a year. Materials and methods

Coral tumors store reduced level of lipids

The reef building coral Montipora informis (Scleractinia, Acroporidae) found on the colony surface of the fringing reef of Sesoko Island, Okinawa, Japan frequently carries hemispherical protuberances (tumors). We compared the total lipid content and the compositions of lipid and fatty acid between normal and tumorous tissues. The lipid content of tumor was 10.6% of the dry tissue weight, and was much lower than that for normal tissues (32.2%). The decrease in the total lipid content of tumorous tissue accompanied a reduced proportion of wax and triacylglycerol (TG). The major fatty acid component was 16:0 (palmitic) acid, and comprised comparable proportion of polar lipids and free fatty acid from both tumorous and normal tissues. An increasing tendency of polyunsaturated fatty acid (PUFA; C>20) was noted in the free fatty acid and polar lipid fractions from tumorous tissues compared with the normal tissue. Thus, the present study first demonstrated the reduced lipid storage level in the tumorous coral, and discussed the rationale for these observations.

Effect of bleaching on lipid content and composition of Okinawan corals

Lipid contents and compositions of the bleached Okinawan corals were analyzed for the first time. Bleached corals collected at Sesoko Okinawa, Japan (26°38′N, 127°52′E), after the 1998 bleaching event showed decreased lipid content with a concomitant decline of wax in the lipid composition. The reductionin the lipid content of corals showed diversity depending on their colony morphology. On the whole, corals of massive morphology were relatively rich in lipid even after the bleaching event, and hence lesser reduction in the lipid content compared with the cases for those of branching morphology. Furthermore, there was a positive correlation between lipid content and zooxanthellae density in the bleached corals, suggesting again the importance of symbiont for the supply of lipid to the host cells, and hence for the survival of the bleaching event.

Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae

Oleanane‐type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology‐based PCR method. The ORFs of full‐length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of β‐amyrin and lupeol, suggesting that these genes encode β‐amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, β‐amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, β‐amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78–93%) to β‐amyrin synthases, and were located in the same branch as β‐amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the β‐amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to β‐amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves.

Lipid composition of mangrove and its relevance to salt tolerance

Lipid compositions of mangrove trees were studied in relation to the salt-tolerance mechanism. Leaves and roots were obtained from seven mature mangrove trees on Iriomote Island, Okinawa: Bruguiera gymnorrhiza, Rhizophora stylosa, Kandelia candel, Lumnitzera racemosa, Avicennia marina, Pemphis acidula and Sonneratia alba. Lipids of mangrove leaves mainly consisted of 11 lipid classes: polar lipids, unknown (UK) 1–6, sterols, triacyl glycerols, wax ester and sterol ester (UK 3 and 4 were found to be tri-terpenoid alcohol in this study). Of these lipid classes, sterol ester was the main lipid in all species comprising 17.6–33.7% of total lipids. Analysis of the chemical structure found that the sterol esters mainly consisted of fatty acid esters of tri-terpenoid alcohols. One major tri-terpenoid alcohol was identified to be lupeol by interpretation of infrared resonance, nuclear magnetic resonance and mass spectrometry. Because of the unique anatomy of the mangrove root, lipid analyses were made separately for epidermis, cortex and innermost stele, respectively. The concentration of free tri-terpenoid alcohols showed a higher tendency in the outside part than in the inside portion of the roots, suggesting their protective roles. Relevance of lipid composition to salt tolerance was studied with propagules of K. candel and B. gymnorrhiza planted with varied salt concentrations. The proportions of free tri-terpenoids increased with salinity in both leaves and roots of K. candel, and only in roots of B. gymnorrhiza. No salt-dependent changes were noted in the phospholipid and fatty acid compositions in both species. These findings suggested that salt stress specifically modulated the terpenoid concentrations in mangroves.

Tumor-selective cytotoxicity of benzo[c]phenanthridine derivatives from Toddalia asiatica Lam.

PurposeTo develop a novel anti-cancer drug of low side effect against lung adenocarcinoma, the authors screened the bioresources of Okinawa Island, Japan. The medicinal plant Toddalia asiatica Lam. contained three benzo[c]phenanthridine derivatives: dihydronitidine (DHN), nitidine (NTD) and demethylnitidine (DMN). Of the three derivatives, DHN had been shown to selectively inhibit the growth of cancer cells in our previous study. Because of similar molecular topology of NTD or DMN to DHN, it can be expected that NTD and DMN also show selective cytotoxicity. The aim of the present study was therefore to examine the selective cytotoxicity of these two compounds in vitro and in vivo.MethodsBenzo[c]phenanthridine derivatives were isolated from T. asiatica Lam., and their chemical structures were identified by interpretation of NMR and MS spectrum. Of the isolated compounds, NTD and DMN were evaluated for cytotoxicity in vitro or in vivo.ResultsNTD as well as DHN selectively reduced the growth of murine and human lung adenocarcinoma in vitro with selective intracellular accumulation. NTD has also been proven to be highly effective in vivo to inhibit the growth of both murine and human lung adenocarcinoma in a subcutaneous xenograft model without any deteriorating side effect. In contrast, DMN had no selective cytotoxicity suggesting that 8-methoxy group of NTD is the critical structural feature for the selective cytotoxicity.ConclusionsThis study thus proves the effectiveness of benzo[c]phenanthridine derivatives as anti-cancer agent in vivo for the first time, and discusses the mechanisms responsible for the selective cytotoxicity.

Tumor specific cytotoxicity of arctigenin isolated from herbal plant Arctium lappa L.

The effectiveness of cancer chemotherapy is often limited by the toxicity to other tissues in the body. Therefore, the identification of non-toxic chemotherapeutics from herbal medicines remains to be an attractive goal to advance cancer treatments. This study evaluated the cytotoxicity profiles of 364 herbal plant extracts, using various cancer and normal cell lines. The screening found occurrence of A549 (human lung adenocarcinoma) specific cytotoxicity in nine species of herbal plants, especially in the extract of Arctium lappa L. Moreover, purification of the selective cytotoxicity in the extract of Arctium lappa L. resulted in the identification of arctigenin as tumor specific agent that showed cytotoxicity to lung cancer (A549), liver cancer (HepG2) and stomach cancer (KATO III) cells, while no cytotoxicity to several normal cell lines. Arctigenin specifically inhibited the proliferation of cancer cells, which might consequently lead to the induction of apoptosis. In conclusion, this study found that arctigenin was one of cancer specific phytochemicals, and in part responsible for the tumor selective cytotoxicity of the herbal medicine.

Conjugated Linoleic Acid (CLA) Inhibits Fatty Acid Synthetase Activity in Vitro

This paper describes the in vitro effect of conjugated linoleic acid (CLA) on fatty acid biosynthesis. Among the rat liver enzymes involved in fatty acid biosynthesis, fatty acid synthetase (FAS) showed the largest activity fluctuation with the types of fatty acids. Of the fatty acids, CLA was the most potent inhibitor of FAS, and the 9c, 11t-rather than the 10t, 12c-isomer showed greater inhibition. CLA also significantly lowered the incorporation of [14C]-acetate into phospholipid in breast cancer cells, supporting the view that CLA inhibits fatty acid biosynthesis through the interaction with FAS.

Coordinated Actions of Glyoxalase and Antioxidant Defense Systems in Conferring Abiotic Stress Tolerance in Plants

Being sessile organisms, plants are frequently exposed to various environmental stresses that cause several physiological disorders and even death. Oxidative stress is one of the common consequences of abiotic stress in plants, which is caused by excess generation of reactive oxygen species (ROS). Sometimes ROS production exceeds the capacity of antioxidant defense systems, which leads to oxidative stress. In line with ROS, plants also produce a high amount of methylglyoxal (MG), which is an α-oxoaldehyde compound, highly reactive, cytotoxic, and produced via different enzymatic and non-enzymatic reactions. This MG can impair cells or cell components and can even destroy DNA or cause mutation. Under stress conditions, MG concentration in plants can be increased 2- to 6-fold compared with normal conditions depending on the plant species. However, plants have a system developed to detoxify this MG consisting of two major enzymes: glyoxalase I (Gly I) and glyoxalase II (Gly II), and hence known as the glyoxalase system. Recently, a novel glyoxalase enzyme, named glyoxalase III (Gly III), has been detected in plants, providing a shorter pathway for MG detoxification, which is also a signpost in the research of abiotic stress tolerance. Glutathione (GSH) acts as a co-factor for this system. Therefore, this system not only detoxifies MG but also plays a role in maintaining GSH homeostasis and subsequent ROS detoxification. Upregulation of both Gly I and Gly II as well as their overexpression in plant species showed enhanced tolerance to various abiotic stresses including salinity, drought, metal toxicity, and extreme temperature. In the past few decades, a considerable amount of reports have indicated that both antioxidant defense and glyoxalase systems have strong interactions in conferring abiotic stress tolerance in plants through the detoxification of ROS and MG. In this review, we will focus on the mechanisms of these interactions and the coordinated action of these systems towards stress tolerance.