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Cnidium officinalis rhizomes were immersed in 80% MeOH. The extract was fractionated to water, n-butanol, and ethyl acetate fractions (Fr). Open column chromatography was repeatedly carried out on n-butanol and ethyl acetate Fr using silica gel, octadecyl silica gel, and Sephadex LH-20 as the stationary phase affording five phenyl alkanoids 1–5 including two new ones. The molecular structures including stereochemistry were decided based on spectroscopic interpretation of nuclear magnetic resonance, mass spectrometry, and infrared spectroscopy as well as chemical reaction. Three known compounds, coniferyl alcohol methyl ether (1), vanillin (2), and coniferyl aldehyde (3), were reported in the beginning for this plant by authors. Two new phenyl alkanoids were named, 7-methoxyeugenol and cnidiumoside.

Abeliophyllum distichum (Oleaceae), which is the only species in the monotypic genus and is grown only on the Korean peninsula, has a high scarcity value. Its five variants (white, pink, round, blue, and ivory) have different morphological characteristics in terms of the color of petals and sepals or shape of the fruits. Despite its high value, there has been no study on variant classification except in terms of their morphological characteristics. Thus, we performed a volatile component analysis of A. distichum flowers and multivariate data analyses to reveal the relationship between fragments emitted from five variants of A. distichum flowers with their morphological characteristics. As a result, 66 volatile components of this plant were identified by headspace solid-phase microextraction gas chromatography&ndash, mass spectrometry (HS-SPME-GC-MS), showing unique patterns for each set of morphological characteristics, especially the color of the petals. These results suggest that morphological characteristics of each variant are related to the volatile composition.

Because cinnamon is orally ingested, its biological activity is likely to influence the small intestinal epithelium and the microbiota along the intestinal tract. We investigated small intestinal epithelial gene expression associated with immunity and lipid metabolism and measured IgA level in the small intestines and sera of mice fed cinnamon. We also analyzed microbial changes in the small and large intestines and feces using pyrosequencing of the 16 s rRNA gene. Cinnamon increased the gene expression of Muc2, RegIIIγ, and Pigr, which are necessary for defense against gut bacteria. It also increased the gene expression of GATA4 and Slc27a2, which are implicated in lipid absorption. Cinnamon increased luminal IgA level but not serum IgA and decreased the class Gammaproteobacteria (classified within the phylum Proteobacteria), particularly the genus Pseudomonas, in the large intestine. Cinnamon thus affected immunity and lipid absorption in the small intestinal epithelium and microbial composition in the intestines.

A preliminary study revealed that a 10 µg/mL n-BuOH fraction of Malva verticillata aerial parts significantly enhanced splenocyte proliferation and induced significant enhancement of natural-killer (NK) cell activity against tumor cells (YAC-1). This study was initiated to identify the principal components that exhibited these activities, and four glycerides were isolated through repeated SiO2 and ODS column chromatography. Structures of compounds 1–4 were determined to be (2S)-1-O-palmitoyl glyceride, (2S)-1-O-stearoyl glyceride, (2S)-1-O-linolenoyl glyceride, and (2S)-1,2-di-O-linoleoyl glyceride, respectively. Compounds 1–3 showed potential immune-enhancing activity in murine splenocyte and natural-killer (NK) cells at 10 µM. In contrast, compound 4 showed weak activity, indicating the monoacyl glycerides (1–3) are more effective than diacyl glyceride (4). Also, the longer the carbon number of the fatty acid in monoacyl glyceride, the better the activity, and the monoacyl glyceride including an unsaturated fatty acid (3) is more effective than the glycerides including the saturated fatty acids (1–2).

The leaves of eggplant (Solanum melongena L.) were extracted with 80 % aqueous MeOH, and the concentrated extract was partitioned with n-hexane, EtOAc, n-BuOH, and water fractions. From the n-BuOH fraction, five compounds were isolated through the repeated silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatographies. On the basis of physic-chemical and spectroscopic data including mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance, they were identified to be caffeic acid (1), chlorogenic acid (2), cryptochlorogenic acid (3), panasenoside (4), and (6R,7E,9R)-4,7-megastigmadien-3-one-9-β-D-glucopyranoside (5). Compounds 3 and 4 were isolated for the first time from the leaves of S. melongena L. in this study.

A red pigment produced by Zooshikella ganghwensis, was purified by solvent extraction and fractionation. The pigment was identified to be prodigiosin on the basis of spectroscopic data such as 1H-NMR, 13C-NMR, DEPT, 2D-NMR, IR, and FAB-MS. Prodigiosin inhibited matrix metalloproteinase-1 gene expression and the growth of human cancer cell lines, SK-BR-3, HCT-116, SK-OV-3, HeLa, HepG2, and SK-MEL-5.

Kudzu (Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep) is a perennial leguminous vine, and its root and flower have been used for herbal medicine in Asia for a long time. Most dietary flavonoids are reported to be concentrated in its root, not in its aerial parts including leaves. In this study, we investigated whether kudzu leaf and its major constituent, robinin (kaempferol-3-O-robinoside-7-O-rhanmoside) possessed anti-inflammatory activity. To test this hypothesis, we used peritoneal macrophages isolated from BALB/c mice and stimulated the cells with lipopolysaccharide (LPS) or LPS plus interferon (IFN)-γ. Compared with kudzu root extract, its leaf extract was more potent in inhibiting the production of inducible nitric oxide synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-α, and interleukin-6. Kudzu leaf extract decreased LPS-induced activation of c-Jun N-terminal kinase (JNK) and TANK-binding kinase 1(TBK1) with no effects on nuclear factor-κB and activator protein 1 transcriptional activity. Also, kudzu leaf extract inhibited LPS/IFN-γ-induced signal transducer and activator of transcription 1 (STAT1) activation partly via an altered level of STAT1 expression. Robinin, being present in 0.46% of dry weight of leaf extract, but almost undetected in the root, decreased iNOS protein involving modulation of JNK and STAT1 activation. However, robinin showed no impact on other inflammatory markers. Our data provide evidence that kudzu leaf is an excellent food source of as yet unknown anti-inflammatory constituents.

The sprouts of red kohlrabi (Brassica oleracea var. gongylodes) were extracted with methanol, and the concentrated extracts were successively partitioned with ethyl acetate, n-butyl alcohol (n-BuOH), and H2O. From the n-BuOH fraction, four phenylpropanoids were isolated through repeated silica gel and ODS column chromatographies. On the basis of physico-chemical and spectroscopic data including NMR, MS, and IR, the isolated compounds were identified as 3-(3,4,5-trimethoxyphenyl)-2E-propenoic acid methyl ester (1), (E)-sinapic acid methyl ester (2), (E)-sinapoyl glucoside (3), and lawsoniaside B (4). Moreover, the compounds dose-dependently inhibited NO production in RAW 264.7 cells with IC50 values of 41.22 (1), 38.32 (3), and 22.65 (4) μM, respectively. With further studies, these compounds may be potentially useful in developing new anti-inflammatory agents.

Principal components of aquatic plants responsible for inhibiting the growth of Microcystis aeruginosa, a blue-green alga (cyanobacterium) were determined. Methanol extracts of Brasenia schreberi showed strong inhibitory activity against the growth of the cyanobacterium. Two purified flavonoids were afforded through repeated column chromatographies. The compounds were determined as quercetin (1) and quercetin 3-O-(6″-galloyl)-β-d-glucopyranoside, tellimoside (2) by the interpretation of spectroscopic data including NMR, MS, and IR. Compounds 1 and 2 were first isolated from Brasenia schreberi. In addition, compound 2 showed very strong inhibitory activity against the growth of M. Aeruginosa.

The stem woods of Lindera obtusiloba Blume were extracted in 80% aqueous methanol and the concentrated extract was partitioned with ethyl acetate (EtOAc), butanol (n-BuOH), and H2O, successively. From the EtOAc and n-BuOH fractions, five compounds were isolated through the repeated silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatographies. On the basis of spectroscopic data including mass spectrometry, IR, 1H-NMR, 13C-NMR, distortionless enhancement by polarization transfer, and two-dimensional-NMR gradient correlated spectroscopy (gCOSY), gradient heteronuclear single quantum correlation (gHSQC), gradient heteronuclear multiple bonding connectivity (gHMBC), the chemical structures of the compounds were determined as asarinin (1), (+)-catechin (2), (-)-epicatechin (3), hyperin (4), and nudiposide (5). Compounds 1 and 5 were isolated for the first time from the stem wood of L. obtusiloba Blume.

Arabidopsis thaliana sterol glycosyltransferase (SGT), UGT80A2, was expressed from stably transformed Drosophila melanogaster Schneider 2 (S2) cells. Recombinant SGT was detected in both intracellular and extracellular fractions with a molecular mass of approximately 76 kDa. Secreted recombinant SGT accounted for approximately 60% of the total recombinant SGT production. Recombinant SGT in the extracellular fractions was purified to homogeneity using a simple one-step Ni-NTA affinity fractionation. Radiometrical assay using uridine diphospho-d-[U-14C]glucose (UDP-14C-glucose) as a sugar donor and sterols, β-sitosterol and stigmasterol, as sugar acceptors showed that the purified recombinant SGT contained UDP-glycosyltransferase activity and could attach 14C-glucose to β-sitosterol and stigmasterol. Recombinant SGT contained higher catalytic activity with β-sitosterol, which was similar to the recombinant SGT produced by a bacterial expression system. The transfer of 14C-glucose by recombinant SGT was further determined by gas chromatography-mass spectrometry (GC-MS) analysis of cellulase-treated 14C-glucosetransferred β-sitosterol and stigmasterol reactants.

The sprouts of Brassica oleracea var. gongylodes were extracted with 100% MeOH and the concentrated extract was partitioned with EtOAc, n-BuOH and , successively. From the EtOAc fraction, three sterols were isolated through the repeated silica gel and ODS column chromatographies. On the basis of physico-chemical and spectroscopic data including NMR, MS, and IR, the chemical structures of the sterols were determined as -sitosterol (1), brassicasterol (2), and 7-ketobrassicasterol (3). Compound 1 is usually observed in plant. Compounds 2 is observed in Brassica sp., and compounds 3 have very rarely occurred in natural source including plant.

β-Secretase (β-SEC) is one of the prime targets for therapeutic intervention of Alzheimer’s disease. We previously described the expression of human β-SEC in Trichoplusia ni BTI Tn5B1-4 cells transformed with cDNAs encoding β1,4-galactosyltransferase (GalT) and Galβ1,4-GlcNAc α2,6-sialyltransferase (ST). To extend this work we investigated the kinetics of recombinant β-secretase and the feasibility of recombinant β-secretase for its inhibitor screening application. Recombinant β-SECs were purified from cultures of Tn5B1-4βSEC and Tn5B1-4βSEC/GalT-ST cells. Recombinant β-SEC from Tn5B1-4βSEC/GalT-ST cells exhibited a higher kcat/KM value compared to the control Tn5B1-4βSEC cells. We also attempted to screen the compounds inhibiting the activity of recombinant β-secretase among the metabolites isolated from Chrysanthemum coronarium L. Based on the inhibition analysis using purified β-SEC from Tn5B1-4βSEC/GalT-ST cells, 3-O-[β-D-galactopyranosyl (1→6)-α-D-galactopyranosyl]-glyceride was the best inhibitor among the compounds tested. The concentration at half-maximum inhibition values (IC50) of this compound was estimated to be 2.8 μM.

Expression of the human cyclooxygenase 1 (COX-1) In Trichoplusia ni BTI Tn 5B1-4 cells transformed with cDNAs encoding β1,4-galactosyltransferase (GalT) and Galβ1,4-GlcNAc α2,6-sialyltransferase (ST6Gal) was examined. Southern blot analysis indicated that the glycosyl-transferase genes were integrated into the Tn 5B1-4 cell genome. A lectin blot analysis also indicated that the recombinant COX-1s from the Tn 5B1-4/COX-1/GalT-ST cells contained the glycan residues of β1,4-linked galactose and α2,6-linked sialic acid. The specific peroxidase activity of the recombinant sialylated COX-1 from Tn 5B1-4/COX-1/GalT-ST cells was 23,930 units/mg, indicating an increase of approximately 17% compared with the non-sialylated control (20,500 units/mg) from the Tn 5B1-4/COX-1 cells.

The Arabidopsis (Arabidopsis thaliana) hexokinase 1 (AtHXK1) is recognized as an important glucose (Glc) sensor. However, the function of hexokinases as Glc sensors has not been clearly demonstrated in other plant species, including rice (Oryza sativa). To investigate the functions of rice hexokinase isoforms, we characterized OsHXK5 and OsHXK6, which are evolutionarily related to AtHXK1. Transient expression analyses using GFP fusion constructs revealed that OsHXK5 and OsHXK6 are associated with mitochondria. Interestingly, the OsHXK5ΔmTP-GFP and OsHXK6ΔmTP-GFP fusion proteins, which lack N-terminal mitochondrial targeting peptides, were present mainly in the nucleus with a small amount of the proteins seen in the cytosol. In addition, the OsHXK5NLS-GFP and OsHXK6NLS-GFP fusion proteins harboring nuclear localization signals were targeted predominantly in the nucleus, suggesting that these OsHXKs retain a dual-targeting ability to mitochondria and nuclei. In transient expression assays using promoter∷luciferase fusion constructs, these two OsHXKs and their catalytically inactive alleles dramatically enhanced the Glc-dependent repression of the maize (Zea mays) Rubisco small subunit (RbcS) and rice α-amylase genes in mesophyll protoplasts of maize and rice. Notably, the expression of OsHXK5, OsHXK6, or their mutant alleles complemented the Arabidopsis glucose insensitive2-1 mutant, thereby resulting in wild-type characteristics in seedling development, Glc-dependent gene expression, and plant growth. Furthermore, transgenic rice plants overexpressing OsHXK5 or OsHXK6 exhibited hypersensitive plant growth retardation and enhanced repression of the photosynthetic gene RbcS in response to Glc treatment. These results provide evidence that rice OsHXK5 and OsHXK6 can function as Glc sensors.

Flowers of Erigeron annuus L. were extracted with 80% aqueous MeOH, and the concentrated extract was partitioned with EtOAc, n-BuOH, and H2O. Repeated silica gel and ODS column chromatography of the EtOAc fraction led to the isolation of a sterol, through activity-guided fractionation, using ACAT inhibitory activity measurements. From the physico-chemical data, including NMR, MS, and IR, the chemical structure of the compound was determined to be an ergosterol peroxide (1), which has been isolated for the first time from this plant. This compound exhibited hACAT-1 and Lp-PLA2 inhibitory effects, with inhibitory values of 51.6 +/- 0.9 and 51.7 +/- 1.2%, at a treatment concentration of 0.23 mM.

Cell-wall invertase (CIN) catalyzes the hydrolysis of sucrose into glucose and fructose for the supply of carbohydrates to sink organs via an apoplastic pathway. To study the CIN genes in rice (Oryza sativa L.), we isolated cDNA clones showing amino acid similarity to the plant cell wall invertase proteins from a search of rice sequence databases. Profile analyses revealed that the cloned genes are expressed in unique patterns in various organs. For example, transcripts of OsCIN1, OsCIN2, OsCIN4, and OsCIN7 were detected in immature seeds whereas OsCIN3 gene expression was flower-specific. Further transcript analysis of these genes expressed in developing seeds indicated that OsCIN1, OsCIN2, and OsCIN7 might play an important role involving sucrose partitioning to the embryo and endosperm. Sucrose, a substrate of CINs, induced the accumulation of OsCIN1 transcripts in excised leaves and OsCIN2 in immature seeds, while the level of OsCIN5 was significantly down-regulated in excised leaves treated with sucrose. Infecting the tissues with rice blast (Magnaporthe grisea) as a biotic stressor increased the expression of OsCIN1, OsCIN4, and OsCIN5, suggesting that these genes may participate in a switch in metabolism to resist pathogen invasion. These results demonstrate that OsCIN genes play diverse roles involving the regulation of metabolism, growth, development, and stress responses.

To investigate the effects of sucrose-phosphate synthase (SPS) on carbon partitioning, transgenicArabidopsis plants transformed withSynechocystis SPS were constructed. The integration, copy number and expression level were confirmed by Southern and Northern blot analyses. SPS activity in leaves from the transgenic and wild type plants was not significantly different. The level of sucrose and starch in the leaves of transgenic plant were slightly decreased compared to wild type. The glucose and fructose contents were increased up to two-fold compared to wild type during the light period. It is our speculation that the decreased sucrose level of the transgenic plant might be caused by the high acid invertase.

Recombinant human cyclooxygenase 2 (Cox 2) was expressed in stably transformed Drosophila melanogaster S2 cells, and was present primarily in the cellular fraction at a molecular weight of 70 to 74 kDa. Recombinant Cox 2 was purified using Ni2+-affinity fractionation to a specific activity of 24 800 U mg−1 protein. The peak level of recombinant Cox 2 production was 1.6 μg (107 cells)−1, seven days after induction with 0.5 mM CuSO4. Supplementing the cultures with dimethylsulfoxide or sodium butyrate increased recombinant Cox 2 production by 170% and 86%, respectively.

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Trichoplusia ni BTI Tn 5B1-4 (Tn 5B1-4) cells. Recombinant endostatin expressed in the stably transformed Tn 5B1-4 cells was secreted into the medium. Recombinant endostatin was also purified to homogeneity using a simple one-step Ni2+ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition (ED50) for recombinant endostatin was approximately 0.35 μg ml−1. In a T-flask, the stably transformed Tn 5B1-4 cells produced 14.3 mg recombinant endostatin l−1 at 6 days of cultivation.

Recombinant rotavirus VP6 production was 0.49 mg l−1 when 10 mM sodium butyrate was added to the suspension culture of transgenic tomato cells after 6 days' incubation. Recombinant VP6 production increased to 0.73 mg l−1, when the culture was supplemented with 10 mM sodium butyrate on day 6 and 9 after incubation. Total protein extracts of transgenic tomato cells from sodium butyrate-supplemented culture were examined by two-dimensional electrophoresis: 14 proteins were up-regulated and 7 down-regulated.

Graduate School of Biotechnology & Plant Metabolism Research Center, Kyung Hee University, Suwon 446-701, Korea *E-mail: nibaek@khu.ac.kr Department of Pharmacy, Woosuk University, Jeonbuk 565-701, Korea Graduate School of East-West Medical Science, Kyung Hee University, Suwon 446-701, Korea Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea Erom Life Co. Ltd., Sungnam 463-828, Korea Received June 20, 2007

Fucosterol (2), oxygenated fucosterol, 24-ethylcholesta-4,24(28)-dien-3,6-dione (3), β-sitosterol (1), and daucosterol (4) were isolated from ethyl acetate (EtOAc) fraction of Osmanthus fragrans var. aurantiacus flower by repeated silica gel, octadecyl silica gel, and Sephadex LH-20 column chromatography. The sterol structures were determined based on spectroscopic methods including nuclear magnetic resonance, infrared spectroscopy, fast atom bombardment mass spectrometry, and electron ionization mass spectrometry. Fucosterol (2), oxygenated fucosterol, and 24-ethylcholesta-4,24(28)-dien-3,6-dione (3) are representative sterols mainly isolated from marine algae, but rarely found in land plants. This is the first reported isolation of these sterols from O. fragrans var. aurantiacus. Interestingly, the oxygenated fucosterol (3) isolated inhibited the in vitro growth of the HCT-116 human colon cancer cell line with higher cytotoxic effects than doxorubicin, a well-known cytotoxic drug.

We generated transgenic lines of Arabidopsis thaliana with an RNA interference construct that expressed hairpin double-stranded RNA for DET2:DWF4:SMT2 to induce sequence-specific RNA silencing. In transgenic plants, expressions of DET2, DWF4, and SMT2 were simultaneously reduced, and the campesterol content was increased by up to 420% compared to the level in the wild-type plant. Triple knock-down of the DET2, DWF4, and SMT2 enzymes also resulted in reduction of brassinosteroid (BR)-specific biosynthesis intermediates. Transgenic plants harboring the RNA interference construct displayed a semi-dwarf phenotype due to altered development. Our findings indicate that redesigning of plant architecture is possible through simultaneous suppression of multiple genes involved in BR biosynthesis.

Methoxychlor is considered as pro-estrogen, while some of its metabolites are more potent endocrine disruptors than the parent insecticide. Major activation of methoxychlor is through cytochrome P450-catalyzed demethylation to bisphenol A-like metabolites. Cunninghamella elegans is a well-known fungal species with its strong resemblance of the xenobiotic metabolism of the mammalian system. In this study, the metabolism of methoxychlor was investigated with the corresponding organism. Methoxychlor was rapidly transformed to approximately 11 metabolites in phase I metabolism, including oxidation, hydroxylation, and dechlorination. Concentrations of phase I metabolites reached a maximum at 4-6 days and gradually decreased until the end of the experiments. Most metabolites from the phase I reaction were further transformed to sugar conjugates. Approximately 11 or more glucose conjugates were found in culture supernatants and gradually increased, while no glucuronides were observed throughout the experiments. Piperonyl butoxide and chlorpyrifos strongly inhibit the degradation of methoxychlor and concomitant accumulation of metabolites, indicating cytochrome P450 mediated metabolism. Little or no glycosides were detected in chlorpyrifos- and piperonyl butoxide-treated cultures. From the results, Cunninghamella elegans has shown strong similarities of the phase I metabolism of methoxychlor, while the conjugation reaction is different from those of animal metabolism.

Several bioactive metabolites, including pyrrolnitrin, Nacylhomoserine lactones, and polyhydroxyalkanoates were isolated from Burkholderia sp. O33. Effects of various nutrients, including sugars, gluconolactone, glycerol, tryptophan, chloride, and zinc were investigated in relation to the production of these metabolites. Logarithmic increase of pyrrolnitrin was observed between 2-5 days and reached a maximum at 7-10 days. Tryptophan concentration reached the maximum at 3 days, whereas 7-chlorotryptophan was gradually increased throughout the studies. Among various carbon sources, gluconolactone, trehalose, and glycerol enhanced pyrrolnitrin production, whereas strong inhibitory effects were found with glucose. Relative concentrations of pyrrolnitrin and its precursors were in the order of pyrrolnitrin >> dechloroaminopyrrolnitrin or aminopyrrolnitrin throughout the experiments. Among three N-acylhomoserine lactones, the N-octanoyl analog was the most abundant quorum sensing signal, of which the concentrations reached the maximum in 2-3 days, followed by a rapid dissipation to trace level. No significant changes in pyrrolnitrin biosynthesis were observed by external addition of N-acylhomoserine lactones. Polyhydroxyalkanoates accumulated up to 3-4 days and decreased slowly thereafter. According to the kinetic analyses, no strong correlations were found between the levels of pyrrolnitrin, N-acylhomoserine lactones,and Polyhydroxyalkanoates.

Graduate School of Biotechnology & Plant Metabolism Research Center, Kyung Hee University, Suwon 446-701, Korea *E-mail: nibaek@khu.ac.kr Department of Pharmacy, Woosuk University, Jeonbuk 565-701, Korea Graduate School of East-West Medical Science, Kyung Hee University, Suwon 446-701, Korea Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-333, Korea Erom Life Co. Ltd., Sungnam 463-828, Korea Received June 20, 2007

Extraction and fractionation of Pulsatilla koreana flowers followed by, repeated open column chromatography for EtOAc and n-BuOH fractions yielded four flavonoid glycosides, namely, astragalin (1), tiliroside (2), buddlenoide A (3), and apigenin-7-O-(3``-E-p-coumaroyl)-glucopyranoside (4). The chemical structures of these flavonoid glycosides were elucidated on the basis of various spectroscopic methods including electronic ionization mass spectrometry (EI-MS), 1D NMR ((1)H, (13)C, DEPT), 2D NMR (gCOSY, gHSQC, gHMBC), and infrared (IR) spectrometry. This study represents the first report of the isolation of the flavonoid glycosides from the flowers of P. koreana.

Phytochrome-interacting proteins have been extensively studied to elucidate light-signaling pathway in plants. However, most of these proteins have been identified by yeast two-hybrid screening using the C-terminal domain of phytochromes. We used co-immunoprecipitation followed by proteomic analysis in plant cell extracts in an attempt to screen for proteins interacting either directly or indirectly with native holophytochromes including the N-terminal domain as well as C-terminal domain. A total of 16 protein candidates were identified, and were selected from 2-DE experiments. Using MALDI-TOF MS analysis, 7 of these candidates were predicted to be putative phytochrome A-interacting proteins and the remaining ones to be phytochrome B-interacting proteins. Among these putative interacting proteins, protein phosphatase type 2C (PP2C) and a 66-kDa protein were strong candidates as novel phytochrome-interacting proteins, as knockout mutants for the genes encoding these two proteins had impaired light-signaling functions. A transgenic knockout Arabidopsis study showed that a 66-kDa protein candidate regulates hypocotyl elongation in a light-specific manner, and altered cotyledon development under white light during early developmental stages. The PP2C knockout plants also displayed light-specific changes in hypocotyl elongation. These results suggest that co-immunoprecipitation, followed by proteomic analysis, is a useful method for identifying novel interacting proteins and determining real protein-protein interactions in the cell.

To understand the transcriptional regulatory mechanism of host genes during the activation of defense responses in rice, we isolated WRKY transcription factors whose expressions were altered upon attack of the fungal pathogen Magnaporthe grisea, the causal agent of the devastating rice blast disease. A systematic expression analysis of OsWRKYs (Oryza sativa L. WRKYs) revealed that among 45 tested genes the expression of 15 genes was increased remarkably in an incompatible interaction between rice and M. grisea. Twelve of the M. grisea-inducible OsWRKY genes were also differentially regulated in rice plants infected with the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). In experiments with defense signaling molecules, the expression of two genes, OsWRKY45 and OsWRKY62, was increased in salicylic acid (SA)-treated leaves and the expression of three genes, OsWRKY10, OsWRKY82, and OsWRKY85 was increased by jasmonic acid (JA) treatment. OsWRKY30 and OsWRKY83 responded to both SA- and JA treatments. The expression profiles suggest that a large number of WRKY DNA-binding proteins are involved in the transcriptional activation of defense-related genes in response to rice pathogens.

Two new triterpenoids along with three known ones, 3-oxoolean-12-en-27-oic acid (1), 3alpha-hydroxyolean-12-en-27-oic acid (2) and 3beta-hydroxyolean-12-en-27-oic acid (3), were isolated from Aceriphyllum rossii. The structures of the new compounds were determined to be a 3alpha,23-dihydroxyolean-12-en-27-oic acid (4) and a 3alpha,23-dihydroxyolean-12-en-29-oic acid (5) by spectroscopic and chemical methods; they were designated aceriphyllic acids A and B, respectively. Compounds 2, 3 and 4 remarkably inhibited the activity of ACAT.

Over evolutionary time RNA sequences which are successfully fixed in a population are selected from among those that satisfy the structural and chemical requirements imposed by the function of the RNA. These sequences together comprise the structure space of the RNA. In principle, a comprehensive understanding of RNA structure and function would make it possible to enumerate which specific RNA sequences belong to a particular structure space and which do not. We are using bacterial 5S rRNA as a model system to attempt to identify principles that can be used to predict which sequences do or do not belong to the 5S rRNA structure space. One promising idea is the very intuitive notion that frequently seen sequence changes in an aligned data set of naturally occurring 5S rRNAs would be widely accepted in many other 5S rRNA sequence contexts. To test this hypothesis, we first developed well-defined operational definitions for a Vibrio region of the 5S rRNA structure space and what is meant by a highly variable position. Fourteen sequence variants (10 point changes and 4 base-pair changes) were identified in this way, which, by the hypothesis, would be expected to incorporate successfully in any of the known sequences in the Vibrio region. All 14 of these changes were constructed and separately introduced into the Vibrio proteolyticus 5S rRNA sequence where they are not normally found. Each variant was evaluated for its ability to function as a valid 5S rRNA in an E. coli cellular context. It was found that 93% (13/14) of the variants tested are likely valid 5S rRNAs in this context. In addition, seven variants were constructed that, although present in the Vibrio region, did not meet the stringent criteria for a highly variable position. In this case, 86% (6/7) are likely valid. As a control we also examined seven variants that are seldom or never seen in the Vibrio region of 5S rRNA sequence space. In this case only two of seven were found to be potentially valid. The results demonstrate that changes that occur multiple times in a local region of RNA sequence space in fact usually will be accepted in any sequence context in that same local region.

The genes encoding body-wall-specific glyceraldehyde-3-phosphate dehydrogenase from Caenorhabditis briggsae were sequenced and compared to the homologous genes from Caenorhabditis elegans. The direct tandem organization of these genes, gpd-2 and gpd-3, and the size and location of the two introns m each gene are the same in C. elegans and C. briggsae. Primer-extension studies demonstrated that the two genes in C. briggsae are trans-spliced differentially with the same splice leader (SL) RNAs as are observed in C. elegans. The gpd-2 gene is trans-spliced with SL1 while gpd-3 is trans-spliced with SL2. Significant sequence conservation was observed within the promoter regions of each species and may indicate those regions responsible for body-wall-muscle-specific gene expression and/or differential trans-splicing. Comparisons of the sequences suggest that the tandem repeat of the genes has been subjected to concerted evolution and that C. briggsae and C. elegans diverged much earlier than would be anticipated based on morphological similarities alone. Finally, an open reading frame found several hundred nucleotides upstream from gpd-2, in both species, appears to be homologous to the ATP synthase subunit, ATPase inhibitor protein, from bovine mitochondria.