Comprehensive identification of terpene synthase genes and organ-dependent accumulation of terpenoid volatiles in a traditional medicinal plant Angelica archangelica L.

Angelica archangelica L. is a traditional medicinal plant of Nordic origin that produces an unusual amount and variety of terpenoids. The unique terpenoid composition of A. archangelica likely arises from the involvement of terpene synthases (TPSs) with different specificities, none of which has been identified. As the first step in identifying TPSs responsible for terpenoid chemodiversity in A. archangelica, we produced a transcriptome catalogue using the mRNAs extracted from the leaves, tap roots, and dry seeds of the plant; 11 putative TPS genes were identified (AaTPS1-AaTPS11). Phylogenetic analysis predicted that AaTPS1-AaTPS5, AaTPS6-AaTPS10, and AaTPS11 belong to the monoterpene synthase (monoTPS), sesquiterpene synthase (sesquiTPS), and diterpene synthase clusters, respectively. We then performed in vivo enzyme assays of the AaTPSs using recombinant Escherichia coli systems to examine their enzymatic activities and specificities. Nine recombinant enzymes (AaTPS2-AaTPS10) displayed TPS activities with specificities consistent with their phylogenetics; however, AaTPS5 exhibited a strong sesquiTPS activity along with a weak monoTPS activity. We also analyzed terpenoid volatiles in the flowers, immature and mature seeds, leaves, and tap roots of A. archangelica using gas chromatography-mass spectrometry; 14 monoterpenoids and 13 sesquiterpenoids were identified. The mature seeds accumulated the highest levels of monoterpenoids, with ß-phellandrene being the most prominent. α-Pinene and ß-myrcene were abundant in all organs examined. The in vivo assay results suggest that the AaTPSs functionally identified in this study are at least partly involved in the chemodiversity of terpenoid volatiles in A. archangelica.

Paenibacillus relictisesami sp. nov., isolated from sesame oil cake.

A facultatively anaerobic, Gram-stain-positive, rod-shaped bacterium, designated strain KB0549T, was isolated from sesame oil cake. Cells were motile, round-ended rods, and produced central or terminal spores. The cell wall peptidoglycan contained meso-diaminopimelic acid as the diamino acid. The major fatty acids were anteiso-C15:0 and anteiso-C17:0. The DNA G+C content of strain KB0549T was 51.9 mol%. On the basis of 16S rRNA gene sequence phylogeny, strain KB0549T was affiliated with the genus Paenibacillus in the phylum Firmicutes and was most closely related to Paenibacillus cookii with 97.4% sequence similarity. Strain KB0549T was physiologically differentiated from P. cookii by the high content of anteiso-C17:0, inability to grow at 50 °C, spore position, and negative Voges-Proskauer reaction. Based on these unique physiological and phylogenetic characteristics, it is proposed that the isolate represents a novel species, Paenibacillus relictisesami sp. nov.; the type strain is KB0549T (=JCM 18068T=DSM 25385T).

Synthesis of 3,5,3',4'-tetrahydroxy-trans-stilbene-4'-O-beta-D-glucopyranoside by glucosyltransferases from Phytolacca americana.

Two glucosyltransferase isozymes from Phytolacca americana, PaGT3 and PaGT2, catalyzed stereo- and regio-selective monoglucosylation of 3,5,3',4'-tetrahydroxy-trans-stilbene to yield 3,5,3',4'-tetrahydroxy-trans-stilbene-4'-O-beta-D-glucopyranoside.

Hyperbaric oxygen induces basic fibroblast growth factor and hepatocyte growth factor expression, and enhances blood perfusion and muscle regeneration in mouse ischemic hind limbs.

BACKGROUND: It is not clear how hyperbaric oxygen therapy (HBO) affects ischemia-induced pathophysiological responses such as angiogenesis and skeletal muscle regeneration. In the present study the effects of HBO on the functional and morphological recovery of ischemic hind limbs, blood perfusion and the local production of angiogenic growth factors were studied in a mouse model. METHODS AND RESULTS: Mice were placed in pure oxygen under 3 atm for 1 h/day for 14 days after the removal of a segment of the left femoral artery. HBO-treated mice showed better functional recovery and greater blood flow in the ischemic hind limb than untreated mice. Histological examination revealed unatrophied muscle fibers with islands of small regenerating muscle cells only in HBO-treated mice. Regeneration of muscle was confirmed by the increase in myf5 mRNA. The amount of mRNA for vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) was slightly increased in the ischemic hind limbs. HBO eliminated the increase in VEGF mRNA. In contrast, the amount of mRNA for bFGF and HGF was further increased by HBO treatment. HBO transiently increased early growth response protein 1 (Egr-1) in the ischemic hind limbs. CONCLUSIONS: HBO accelerates the recovery of ischemic hind limbs by increasing the production of bFGF and HGF and by promoting muscle regeneration in mice.

UDP-glucuronic acid:anthocyanin glucuronosyltransferase from red daisy (Bellis perennis) flowers. Enzymology and phylogenetics of a novel glucuronosyltransferase involved in flower pigment biosynthesis.

In contrast to the wealth of biochemical and genetic information on vertebrate glucuronosyltransferases (UGATs), only limited information is available on the role and phylogenetics of plant UGATs. Here we report on the purification, characterization, and cDNA cloning of a novel UGAT involved in the biosynthesis of flower pigments in the red daisy (Bellis perennis). The purified enzyme, BpUGAT, was a soluble monomeric enzyme with a molecular mass of 54 kDa and catalyzed the regiospecific transfer of a glucuronosyl unit from UDP-glucuronate to the 2''-hydroxyl group of the 3-glucosyl moiety of cyanidin 3-O-6''-O-malonylglucoside with a kcat value of 34 s(-1) at pH 7.0 and 30 degrees C. BpUGAT was highlyspecific for cyanidin 3-O-glucosides (e.g. Km for cyanidin 3-O-6''-O-malonylglucoside, 19 microM) and UDP-glucuronate (Km, 476 microM). The BpUGAT cDNA was isolated on the basis of the amino acid sequence of the purified enzyme. Quantitative PCR analysis showed that transcripts of BpUGAT could be specifically detected in red petals, consistent with the temporal and spatial distributions of enzyme activity in the plant and also consistent with the role of the enzyme in pigment biosynthesis. A sequence analysis revealed that BpUGAT is related to the glycosyltransferase 1 (GT1) family of the glycosyltransferase superfamily (according to the Carbohydrate-Active Enzymes (CAZy) data base). Among GT1 family members that encompass vertebrate UGATs and plant secondary product glycosyltransferases, the highest sequence similarity was found with flavonoid rhamnosyltransferases of plants (28-40% identity). Although the biological role (pigment biosynthesis) and enzymatic properties of BpUGAT are significantly different from those of vertebrate UGATs, both of these UGATs share a similarity in that the products produced by these enzymes are more water-soluble, thus facilitating their accumulation in vacuoles (in BpUGAT) or their excretion from cells (in vertebrate UGATs), corroborating the proposed general significance of GT1 family members in the metabolism of small lipophilic molecules.