Production of a novel dimeric 4-deoxy-L-erythro-5-hexoseulose uronic acid by a PL-17 exolytic alginate lyase from Hydrogenophaga sp. UMI-18.

Hydrogenopahaga sp. strain UMI-18 is an alginolytic bacterium that can produce poly(3-hydroxybutylate) (PHB) using alginate as its sole carbon source. Genome analysis indicated that this strain harbors both PHB-synthesizing and alginate-assimilating gene clusters. In the present study, we cloned HyAly-I gene that encodes a PL-17 exolytic alginate lyase and investigated its enzymatic properties using recombinant HyAly-I (recHyAly-I) that was produced by Escherichia coli. The recHyAly-I preferably depolymerized poly(ß-D-mannuronate) block of alginate in an exolytic manner at an optimal temperature and a pH at 40 °C and pH 6.0, respectively. It released 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH) from the non-reducing terminus of polymer and oligomer substrates. Interestingly, recHyAly-I was found to produce a novel unsaturated disaccharide, i.e., dimeric DEH (diDEH), along with monomeric DEH. Production of diDEH was prominent in the degradation of trisaccharides.

LIL3, a light-harvesting-like protein, plays an essential role in chlorophyll and tocopherol biosynthesis.

The light-harvesting chlorophyll-binding (LHC) proteins are major constituents of eukaryotic photosynthetic machinery. In plants, six different groups of proteins, LHC-like proteins, share a conserved motif with LHC. Although the evolution of LHC and LHC-like proteins is proposed to be a key for the diversification of modern photosynthetic eukaryotes, our knowledge of the evolution and functions of LHC-like proteins is still limited. In this study, we aimed to understand specifically the function of one type of LHC-like proteins, LIL3 proteins, by analyzing Arabidopsis mutants lacking them. The Arabidopsis genome contains two gene copies for LIL3, LIL3:1 and LIL3:2. In the lil3:1/lil3:2 double mutant, the majority of chlorophyll molecules are conjugated with an unsaturated geranylgeraniol side chain. This mutant is also deficient in α-tocopherol. These results indicate that reduction of both the geranylgeraniol side chain of chlorophyll and geranylgeranyl pyrophosphate, which is also an essential intermediate of tocopherol biosynthesis, is compromised in the lil3 mutants. We found that the content of geranylgeranyl reductase responsible for these reactions was severely reduced in the lil3 double mutant, whereas the mRNA level for this enzyme was not significantly changed. We demonstrated an interaction of geranylgeranyl reductase with both LIL3 isoforms by using a split ubiquitin assay, bimolecular fluorescence complementation, and combined blue-native and SDS polyacrylamide gel electrophoresis. We propose that LIL3 is functionally involved in chlorophyll and tocopherol biosynthesis by stabilizing geranylgeranyl reductase.

Proteomics analysis of heterogeneous flagella in brown algae (stramenopiles).

Flagella are conserved organelles among eukaryotes and they are composed of many proteins, which are necessary for flagellar assembly, maintenance and function. Stramenopiles, which include brown algae, diatoms and oomycetes, possess two laterally inserted flagella. The anterior flagellum (AF) extends forward and bears tripartite mastigonemes, whilst the smooth posterior flagellum (PF) often has a paraflagellar body structure. These heterogeneous flagella have served as crucial structures in algal studies especially from a viewpoint of phylogeny. However, the protein compositions of the flagella are still largely unknown. Here we report a LC-MS/MS based proteomics analysis of brown algal flagella. In total, 495 flagellar proteins were identified. Functional annotation of the proteome data revealed that brown algal flagellar proteins were associated with cell motility, signal transduction and various metabolic activities. We separately isolated AF and PF and analyzed their protein compositions. This analysis led to the identification of several AF- and PF-specific proteins. Among the PF-specific proteins, we found a candidate novel blue light receptor protein involved in phototaxis, and named it HELMCHROME because of the steering function of PF. Immunological analysis revealed that this protein was localized along the whole length of the PF and concentrated in the paraflagellar body.

[A case of nonsurgical pneumoperitoneum associated with the incomplete type of intestinal Behçet disease].

A 58-year-old man was admitted with fever and arthralgia. He had some symptoms suggesting the incomplete type of Behçet disease, and a routine chest X-ray films showed the presence of massive pneumoperitoneum (PP). Exploratory laparotomy revealed no evidence of gastrointestinal perforation or peritonitis. Thus, we initially diagnosed it as idiopathic PP. However a 2×1-mm induration located on the antimesentric side of the ileum 50cm proximal to the ileocecal valve. The wedge-shaped pathological specimen showed ulcer perforation and its restoration. Finally we concluded it to be nonsurgical PP. This case provides significant information on the etiology of idiopathic PP.

Effect of the amount of liquid phase in packed column GC on air oxidation product of methyl oleate.

The analysis or preparative isolation of a specimen by a packed column gas chromatograph is affected by the kind and amount (concentration) of the liquid phase coated on the stationary phase. In particular, compounds having the same or similar molecular weight and similar functional groups are largely affected. Methyl oleate, which is a typical component of fats and oils, produces hydroperoxide by air oxidation as a first step. After that, various oxygenated compounds with a carbon number of 19 are produced. In this study, the effect of the concentration of ethylene glycol succinate polyester (EGS) as the liquid phase was examined for these compounds. The retention time of the hydroxides increased with an increase in the EGS concentration. The effect of the EGS concentration on the retention time decreased when these compounds were trimethyl silylated (TMS). The retention time of epoxides and oxo compounds increased with an increase in the EGS concentration independently of trimethyl silylation. Correction values were required for all quantitation.

Bioactive triterpene saponins from the roots of Phytolacca americana.

Five new triterpene saponins named phytolaccasaponins N-1 (1), N-2 (2), N-3 (3) N-4 (4), and N-5 (5) were isolated from the roots of Phytolacca americana together with seven known triterpene saponins (6-12). The structures of the five new saponins were established as shown in structures 1-5 on the basis of their spectroscopic data. The MDR-reversal activity of 1-12 was evaluated on the basis of the amount of calcein accumulated in MDR human ovarian cancer 2780 AD cells in the presence of each compound. The most effective compound was 8 (155% of control at 25 microg/mL).

Bioactive cardenolides from the stems and twigs of Nerium oleander.

Four new cardenolide monoglycosides, cardenolides N-1 (1), N-2 (2), N-3 (3), and N-4 (4), were isolated from Nerium oleander, together with two known cardenolides, 5 and 12, and seven cardenolide monoglycosides, 6-11 and 13. The structures of compounds 1-4 were established on the basis of their spectroscopic data. The in vitro anti-inflammatory activity of compounds 1-13 was examined on the basis of inhibitory activity against the induction of the intercellular adhesion molecule-1 (ICAM-1). Compounds 1, 5, 6, and 11-13 were active at an IC50 value of less than 1 microM. The cytotoxicity of compounds 1-13 was evaluated against three human cell lines, normal human fibroblast cells (WI-38), malignant tumor cells induced from WI-38 (VA-13), and human liver tumor cells (HepG2). Compounds 1, 4, 6, and 11-13 were active toward V-13 cells, and compounds 1, 11, and 12 were active toward HepG2 cells at IC50 values of less than 1 microM. Compounds 4, 5, 10, and 12 showed selective cell growth inhibitory activity toward V-13 tumor cells compared with that of parental normal WI-38 cells. The MDR-reversal activity of compounds 1-13 was evaluated on the basis of the amount of calcein accumulated in MDR human ovarian cancer 2780AD cells in the presence of each compound. Compounds 4, 9, and 10 showed significant effects on calcein accumulation, compound 4 showing stronger activity than that of verapamil.

A new taxoid from a callus culture of Taxus cuspidata as an MDR reversal agent.

A new taxoid, 5alpha,13alpha-diacetoxy-10beta-cinnamoyloxy-4(20),11-taxadien-9alpha-ol (1) along with its 9,10-isomer, taxinine NN-11 (2) were isolated from the callus cultures of Taxus cuspidata. The structures were identified by the analyses of the spectral data and chemical method. Their in vitro cytotoxicity against 3 cell lines (HepG2, WI-38 and VA-13) and multidrug resistance (MDR) reversal activity toward 2780AD tumor cells were preliminarily evaluated, the low cytotoxicities and potent MDR reversal activities suggested that they might be good lead compounds of tumor MDR reversal agent.

Bioactive secolignans from Peperomia dindygulensis.

Thirteen secolignans, including eight new ones (1-8), were isolated from the EtOAc extract of Peperomia dindygulensis. The structures were mainly elucidated by 1D and 2D NMR and MS experiments, the relative configurations were determined by NOE correlations, and the absolute configurations were established by the optical rotations and CD spectra. Cytotoxicity and MDR (multidrug resistance) reversal activity of the isolated compounds were examined. Compounds 6 and 7, peperomins B (10) and E (12), showed moderate to strong growth inhibitory activity against a malignant lung tumor cell (VA-13) with IC(50) values of 15.2, 13.5, 13.9, and 1.93 microM, respectively, and also inhibited the growth of a normal lung fibroblast cell (WI-38) at the same levels. Compound 7 and peperomin E (12) exhibited inhibitory activity against a liver tumor cell (HepG2) with IC(50) values of 22.3 and 12.1 microM. Compounds 5 and 7 and peperomins A, B, C, and E (9-12) enhanced calcein accumulation in MDR 2780 cells at 25 microg/mL. Compounds 2, 3, 7, and peperomin E (12) showed inhibitory activity on induction of the intercellular adhesion molecule-1 (ICAM-1).

Taraxasterane- and ursane-type triterpenes from Nerium oleander and their biological activities.

Two new taraxasterane-type triterpenes, 20beta,28-epoxy-28alpha-methoxytaraxasteran-3beta-ol (1) and 20beta,28-epoxytaraxaster-21-en-3beta-ol (2), were isolated from an ethyl acetate extract of the leaves of Nerium oleander, together with ursane-type triterpenes, 28-nor-urs-12-ene-3beta,17beta-diol (3) and 3beta-hydroxyurs-12-en-28-aldehyde (4). The structures of 1 and 2 were established on the basis of their spectroscopic data. Anti-inflammatory activity of 1-4 was examined on the basis of inhibitory activity against the induction of intercellular adhesion molecule-1 (ICAM-1). Cytotoxic activity of 1-4 was evaluated against four human cell lines, A-549, WI-38, VA-13, and HepG2 cells.

Brasilibactin A, a cytotoxic compound from actinomycete Nocardia brasiliensis.

A new cytotoxic compound, brasilibactin A (1), has been isolated from the actinomycete Nocardia brasiliensis IFM 0995, and the structure was elucidated on the basis of spectroscopic data and chemical means.

Three new triterpenes from Nerium oleander and biological activity of the isolated compounds.

New ursane-type triterpene 1, oleanane-type triterpene 2, and dammarane-type triterpene 15 were isolated from the leaves of Nerium oleander together with 12 known triterpenes, 3beta-hydroxy-12-ursen-28-oic acid (ursolic acid, 3), 3beta,27-dihydroxy-12-ursen-28-oic acid (4), 3beta,13beta-dihydroxyurs-11-en-28-oic acid (5), 3beta-hydroxyurs-12-en-28-aldehyde (6), 28-norurs-12-en-3beta-ol (7), urs-12-en-3beta-ol (8), urs-12-ene-3beta,28-diol (9), 3beta-hydroxy-12-oleanen-28-oic acid (oleanolic acid, 10), 3beta,27-dihydroxy-12-oleanen-28-oic acid (11), 3beta-hydroxy-20(29)-lupen-28-oic acid (betulinic acid, 12), 20(29)-lupene-3beta,28-diol (betulin, 13), and (20S,24R)-epoxydammarane-3beta,25-diol (14). On the basis of their spectroscopic data, the structures of the new compounds 1, 2, and 15 were established as 3beta,20alpha-dihydroxyurs-21-en-28-oic acid, 3beta,12alpha-dihydroxyoleanan-28,13beta-olide, and (20S,24S)-epoxydammarane-3beta,25-diol, respectively. The anti-inflammatory activity of the seven isolated compounds and methyl esters of ursolic acid and oleanoic acid in vitro was examined on the basis of inhibitory activity against the induction of the intercellular adhesion molecule-1 (ICAM-1). The anticancer activity of the 14 isolated compounds, including 1, 2, 15, and methyl esters of ursolic acid and oleanolic acid in vitro was examined on the basis of the cell growth inhibitory activities toward three kinds of human cell lines.

Bioactive tetrahydrofuran lignans from Peperomia dindygulensis.

Five new tetrahydrofuran lignans (1-5), accompanied by four known compounds, were isolated from the ethyl acetate extract of Peperomia dindygulensis. Structures were elucidated mainly using 1D NMR, 2D NMR, and mass spectroscopic studies. The relative configurations of 1-5 were determined by NOE correlations. Several of the compounds showed weak growth inhibitory activity against three cell lines (WI-38, VA-13, and HepG2). Compound 5 exhibited stronger MDR (multidrug resistance) reversal activity than verapamil at 2.5 microg/mL in a cellular calcein accumulation assay. Compounds 4 and 5 showed weak inhibitory activity against induction of the intercellular adhesion molecule-1 (ICAM-1) in anti-inflammatory activity experiments.

Comparison of the compositions of phospholipid-associated fatty acids in wild-type and extracellular matrix tenascin-X-deficient mice.

Tenascin-X (TNX) is a member of the tenascin family of glycoproteins of the extracellular matrix. We previously showed that TNX regulates the synthesis of triglyceride and the composition of triglyceride-associated fatty acids. The aim of the present study was to determine whether TNX controls the synthesis of phospholipids and the composition of phospholipid-associated fatty acids by using TNX-deficient (TNX-/-) mice and TNX-overexpressing fibroblast cell lines. Thin-layer chromatography of total lipids of the skin and sciatic nerves from wild-type and TNX-/- mice revealed that the amounts of major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in wild-type and TNX-/- mice are not different. Gas chromatography-mass spectrometry showed that the major fatty acid compositions of PC and PE in wild-type and TNX-/- mice are almost the same. Fibroblast cells stably overexpressing TNX also showed almost the same amounts of PC and PE and almost the same fatty acid compositions of PC and PE as those in mock-transfected cells. These results suggest that TNX regulates the amount of triglyceride and the composition of triglyceride-associated fatty acids but not the amount and species of phospholipids or the composition of phospholipid-associated fatty acids.

Triglyceride accumulation and altered composition of triglyceride-associated fatty acids in the skin of tenascin-X-deficient mice.

Tenascin-X (TNX) is a member of the tenascin family of glycoproteins of the extracellular matrix. Here, we observed abnormalities in the skin of TNX-deficient mice in comparison with that of wild-type mice. Histological analysis with Oil Red O staining demonstrated that there was considerable accumulation of lipid in the skin of TNX-deficient (TNX-/-) mice. By thin-layer chromatography of total lipids, it was found that the level of triglyceride was significantly increased in TNX-/- mice. The mRNA levels of most of the lipogenic enzyme genes examined were remarkably increased in TNX-/- mice. By gas chromatography-mass spectrometry analysis of triglyceride-associated fatty acids in the skin, saturated fatty acid palmitoic acid was decreased, whereas unsaturated fatty acids palmitoleic acid and oleic acid were increased in TNX-/- mice compared with those in wild-type mice. Conversely, fibroblast cell lines transfected with TNX showed a significant decrease in the amount of triglyceride. An increase in the saturated fatty acid stearic acid and decreases in the unsaturated fatty acids palmitoleic acid, oleic acid and linoleic acid, compared to those in mock-transfected cells were also caused by over-expression of TNX. These results indicate that TNX is involved in the regulation of triglyceride synthesis and the regulation of composition of triglyceride-associated fatty acids.