Production of prenylated flavonoids in tomato fruits expressing a prenyltransferase gene from Streptomyces coelicolor A3(2).

Flavonoids are natural compounds found in many plants, including the important fruit crop, tomato. Prenylated flavonoids consist of a large group of compounds, which often exhibit antitumour, antibacterial and/or anti-androgen activities. In this study, we engineered the biosynthesis of prenylated flavonoids using a Streptomyces prenyltransferase HypSc (SCO7190) possessing broad-range substrate specificity, in tomato as a host plant. LC/MS/MS analysis demonstrated the generation of 3'-dimethylallyl naringenin in tomato fruits when recombinant HypSc protein was targeted to the plastids, whereas the recombinant protein hardly produced this compound in vitro. This is the first report confirming the accumulation of a prenylated flavonoid using a bacterial prenyltransferase in transgenic plants, and our results suggest that the product specificities of prenyltransferases can be significantly influenced by the host plant.

Accumulation and membrane transport of plant alkaloids.

Among a large number of plant secondary metabolites, alkaloids comprise one of the most important groups due to their strong and divergent biological activities, and some are applied for clinical use. Alkaloids are often highly accumulated in particular organs of medicinal plants, which are called the 'medicinal part', whereas it is known that some alkaloids are translocated from source organs to such sink organs. The movement of biosynthetic intermediates from specific cells to other types of cells in tissue, and further detailed movement within the organelles in a cell is also suggested. However, little is known how alkaloids are transported across membranes and finally accumulated in specific organelles such as vacuole of the sink organ. To increase the productivity of valuable alkaloids in planta, not only biosynthetic genes of alkaloids but also genes involved in their transport will be important. Recently, the involvement of ABC transporters in the translocation of berberine alkaloid from root to rhizome was reported, while H(+) antiporters were also suggested as the responsible transporters for vacuolar accumulation of the alkaloid. In this review, we describe intra-organ, intra-tissue and intra-cellular transport of the alkaloid via membrane transports. Furthermore, we discuss the possibility of increasing alkaloid production in transgenic plants by using alkaloid transporter genes.

Influence of ascorbic acid on bonding of peroxide-affected dentin and 4-META/MMA-TBB resin.

The purpose of this study was to evaluate the tensile bond strength (TBS) to peroxide-exposed dentin. Furthermore, the effect of ascorbic acid (AA) on the bond strength of peroxide-exposed dentin was investigated. Extracted bovine dentin was exposed to 10% carbamide peroxide, 30% hydrogen peroxide, or distilled water for 30 min, then treated with 10% AA (0, 30, 90, and 180 min), and conditioned with 10% citric acid/3% ferric chloride. The polymethyl-methacrylate (PMMA) rod was bonded to the treated bovine dentin with 4-META/MMA-TBB resin. A minidumbbell-shaped bonded specimen was prepared from these bonded assemblies and the TBS was tested. The fractured surfaces were also observed with a scanning electron microscope. Exposure to peroxide before bonding significantly reduced bond strength. The application of AA to the peroxide-exposed dentin increased bond strength. On the other hand, an adverse effect of AA was found in distilled water-affected dentin. Extended resin fibers were partially seen in the peroxide-exposed dentin. In conclusion, peroxide reduced the bond strength, and the stronger the oxidation, the weaker the obtained bond. Antioxidation with AA recovered the bond strength, and this effect increased the longer the AA was applied.

Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid.

An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem. by heterologous hybridization with cDNA of Glycyrrhiza glabra beta-amyrin synthase. Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene. This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells. The deduced amino-acid sequence of LcIMS1 shows relatively low identity with other triterpene synthases, suggesting that isomultiflorenol synthase should be classified into a new group of triterpene synthases. The levels of isomultiflorenol synthase and cycloartenol synthase mRNAs, which were measured with gene-specific probes, correlated with the accumulation of bryonolic acid and phytosterols over a growth cycle of the Luffa cell cultures. Isomultiflorenol synthase mRNA was low during the early stages of cell growth and accumulated to relatively high levels in the late stages. Induction of this mRNA preceded accumulation of bryonolic acid. In contrast, cycloartenol synthase mRNA accumulated in the early stages of the culture cycle, whereas phytosterols accumulated at the same relative rate throughout the whole growth cycle. These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols.

Growth and annual ring structure of Larix sibirica grown at different carbon dioxide concentrations and nutrient supply rates.

We compared effects of ambient (360 vpm) and elevated (720 vpm) carbon dioxide concentration ([CO2]) and high and low nutrient supply rates on stem growth, annual ring structure and tracheid anatomy of Siberian larch (Larix sibirica Ledeb.) seedlings over two growing seasons. Elevated [CO2] had no significant effect on either stem height or diameter growth; however, both stem height and diameter growth were enhanced by the high nutrient supply rate, and these increases were stimulated by elevated [CO2]. Elevated [CO2] tended to increase the width of the annual xylem ring, the number of cells in a radial file spanning the ring, and tracheid lumen diameter, whereas it tended to reduce cell wall thickness, although there were no statistically significant CO2 effects on tracheid anatomy. Changes in tracheid cell morphology seemed to be dependent on changes in shoot elongation rates.

A novel Coptis japonica multidrug-resistant protein preferentially expressed in the alkaloid-accumulating rhizome.

A full-length cDNA, Cjmdr1, which belongs to the multidrug-resistant (mdr) gene family, was isolated by nested RT-PCR from alkaloid-producing cultured cells of Coptis japonica. The cDNA is 4192 nucleotides long and has an ORF of 1289 amino acids. Northern analysis of the intact plant showed a clear preference in its expression in the rhizome, where alkaloids are highly accumulated compared to other organs.

Increases of secondary metabolite production in various plant cell cultures by co-cultivation with cork.

Cork tissues increased secondary metabolite production of various plant cell cultures in a different manner from those of conventional elicitors. In Sophora flavescens and Glycyrrhiza glabra cultured cells, cork tissues increased the amounts of both lipophilic and hydrophilic flavonoids without affecting the cell growth, although elicitors such as copper ion and yeast extracts showed a clear inhibition of cell growth with the increasing amount of these lipophilic ones. The validity of this effect of cork tissues covered a wide range of aromatic compounds produced by suspension cell cultures derived from diverse plant species. Woody tissues of Japanese cypress had a very similar effect to that of cork. Partial purification of cork tissues suggested that the production-stimulating factor was present in the hemicellulose B fraction that was not included in the dedifferentiated cultured tissues.

A novel dark-inducible protein, LeDI-2, and its involvement in root-specific secondary metabolism in Lithospermum erythrorhizon.

Lithospermum erythrorhizon produces red naphthoquinone pigments that are shikonin derivatives. They are accumulated exclusively in the roots of this plant. The biosynthesis of shikonin is strongly inhibited by light, even though other environmental conditions are optimized. Thus, L. erythrorhizon dark-inducible genes (LeDIs) were isolated to investigate the regulatory mechanism of shikonin biosynthesis. LeDI-2, showing the strict dark-specific expression, was further characterized by use of cell suspension cultures and hairy root cultures as model systems. Its mRNA accumulation showed a similar pattern with that of shikonin. In the intact plants LeDI-2 expression was observed solely in the root, and the longitudinal distribution of its mRNA was also in accordance to that of shikonin. LeDI-2 encoded a very hydrophobic polypeptide of 114 amino acids that shared significant similarities with some root-specific polypeptides such as ZRP3 (maize) and RcC3 (rice). Reduction of LeDI-2 expression by its antisense DNA in hairy roots of L. erythrorhizon decreased the shikonin accumulation, whereas other biosynthetic enzymes, e.g. p-hydroxybenzoic acid:geranyltransferase, which catalyzed a critical biosynthetic step, showed similar activity as the wild-type clone. This is the first report of the gene that is involved in production of secondary metabolites without affecting biosynthetic enzyme activities.

The mouse putative pheromone receptor was specifically activated by stimulation with male mouse urine.

To detect the biological activity of mammalian putative pheromone receptors (V1Rs and V2Rs), the mouse V1R gene was introduced into a primary culture of vomeronasal cells using the adenovirus expression system, and the response of these cells to mouse urine was analyzed by calcium imaging. These cells specifically responded to male but not female mouse urine. This response was attenuated by pertussis toxin, a specific inhibitor of G-protein G(ialpha)/G(oalpha) coupling from receptors. Our findings indicate that a putative pheromone receptor was specifically activated by mouse urine, a major source of mouse pheromones, and suggest that G(i)/G(o) are functionally coupled with the receptor.

Polysomes of eukaryotic cells observed by electron microscopy.

Shapes of polysomes of eukaryotic cells were determined by surface spreading of cells. We examined unicellular protozoan Tetrahymena cells, rabbit reticulocytes, and cultured MDCK, BHK and HeLa cells. Polysomes had ring-, 8- and caterpillar-shaped forms, indicating that eukaryotic cells contain fundamentally circular polysomes. Isolated and partially purified polysomes from Tetrahymena cells had features of polysomes similar to those of spread cells. These findings indicate that circular polysomes are not an effect of the spreading, but are actual features of the cells.

Pharmacological characterization of an 18-kDa protein associated with the peripheral-type benzodiazepine receptor in salivary glands.

Pharmacological characterization of peripheral type benzodiazepine receptors in rat, rabbit, mouse and human salivary glands was determined by receptor binding and photoaffinity labeling analysis using [3H]PK14105 (1-(2-fluoro-5-nitrophenyl)-3-isoquinolinecarboxylic acid). [3H]PK14105 bound to the membranes of salivary glands in rats, rabbits, mice and humans with high affinity at the nanomolar level. The rank order of receptor density in submandibular glands among several species was as follows: human > or = rat > or = mouse > rabbit. Competitive potency of receptor ligands against [3H]PK14105 was as follows: PK1195 > or = Ro5-4864 > diazepam > clonazepam > Ro15-1788. The rank order of potency against calcium channel ligands and co-transport inhibitors was as follows: nitrendipine > BAY K 8644 > bumetanide > furosemide. Pretreatment with nitrendipine or BAY K 8644 decreased the affinity of [3H]PK14105 binding to rat parotid gland membranes, without changing the density. The photoaffinity labeling with [3H]PK14105 indicated the presence of the 18-kDa protein in all salivary glands of our experiment. The inhibition of photolabeling by some receptor ligands was the same results as the receptor binding assay. In conclusion, the peripheral type benzodiazepine receptors include the 18-kDa protein photolabeled with [3H]PK14105 in salivary glands of rat, mouse, rabbit and human.

Simultaneous analysis of shikimate-derived secondary metabolites in Lithospermum erythrorhizon cell suspension cultures by high-performance liquid chromatography.

A high-performance liquid chromatography (HPLC) analysis system based on a water-acetonitrile gradient program was established for simultaneous quantification of shikimate-derived secondary metabolites in cultured cells of Lithospermum erythrorhizon. The cells cultured in pigment production medium (M-9) are capable of producing five highly hydrophilic compounds such as p-hydroxybenzoic acid-O-glucoside and lithospermic acid B, as well as eleven lipophilic compounds including echinofuran B and acetylshikonin. In addition to the wide polarities of those compounds, many of them are unstable under light, dryness, oxygen and heating. Thus, a new extraction procedure for all these compounds was also established by use of ultrasonication under ice-water chilling with MeOH as the solvent. This procedure was applied to the quantitative analyses of these compounds in cell cultures and hairy root cultures of Lithospermum, and in the intact plants as well.

Caffeic acid oligomers in Lithospermum erythrorhizon cell suspension cultures.

Lithospermum erythrorhizon cells cultured in pigment production (M-9) medium produced lithospermic acid B, a dimerized caffeic acid ester derivative, in quantities similar to the production of shikonin. The cells also produced a related dimer, (+)-rabdosiin. In Linsmaier-Skoog liquid medium, which suppresses shikonin production, both lithospermic acid B and (+)-rabdosiin were still formed. Lithospermic acid, a caffeic acid-rosmarinic acid conjugate, was isolated as a main constituent in Lithospermum hairy root cultures. In the aerial parts of L. erythrorhizon, the content of these phenylpropanoid oligomers was relatively low compared to that of rosmarinic acid.

Effects of anticonvulsants on local anaesthetic-induced neurotoxicity in rats.

The effects of various anticonvulsants on local anaesthetics procaine- and lidocaine-induced convulsions were investigated in rats. Pretreatment with diazepam (2.5-5 mg/kg, intraperitoneally) and clonazepam (5-10 mg/kg, intraperitoneally) completely protected the rats against both local anaesthetic-induced convulsions. Phenobarbital (12.5-50 mg/kg, subcutaneously) also significantly decreased the incidence of both convulsions and prolonged their latencies. Carbabazepine (10-40 mg/kg, intraperitoneally) did not completely repress both convulsions, but it prolonged their latencies. Phenytoin (5-20 mg/kg, intraperitoneally) and primidone (30-60 mg/kg, intraperitoneally) markedly enhanced both local anaesthetic-induced convulsions, as shown by shortening of latency and increase in mortality. Valproate (100-200 mg/kg, intraperitoneally) produced a protective effect against procaine-induced convulsions, while it strongly enhanced lidocaine-induced convulsions. These results suggest that the benzodiazepines are effective drugs to prevent neurotoxicity induced by local anaesthetics, while phenytoin and primidone potentiate them.

Galloylglucoses and riccionidin A in Rhus javanica adventitious root cultures.

Adventitious root cultures of Rhus javanica L. produced large amounts of galloylglucoses (gallotannins) and an anthocyanidin, riccionidin A, formerly found only in liverworts. Production of both galloylglucoses and riccionidin A in the adventitious root culture system was suppressed by light. The Rhus root culture showed the highest productivity for those secondary metabolites in a modified Linsmaier-Skoog (LS) liquid medium containing 30 mM NH4+ and 30 mM NO3- as nitrogen sources in the presence of 10(-6) M 3-indoleacetic acid (IAA).

Shikonin derivative formation on the stem of cultured shoots in Lithospermum erythrorhizon.

LITHOSPERMUM ERYTHRORHIZON: , which are capable of producing red pigments, have been established. The red pigments were formed on the stems of L. erythrorhizon shoots cultured both on solid and in liquid media without phytohormones at 25 °C in the dark. Thin-layer chromatography, high-performance liquid chromatography and 1 H nuclear magnetic resonance analyses revealed that the red pigments which accumulated on the cultured shoots were shikonin derivatives. The effects of various basal media and phytohormones (indole-3-acetic acid, indole-3-butyric acid and kinetin) on the growth and the formation of shikonin derivatives were investigated. When the shoots were cultured on Murashige and Skoog solid medium, the addition of kinetin remarkably enhanced shikonin derivative accumulation in the shoots. However, these effects of kinetin were not observed in the liquid culture when cultured in Gamborg B5 medium. The maximum content of shikonin derivatives (2.3% as dry weight, ca. 1.5 mg/100 ml flask) was observed in the shoots cultured in phytohormone-free B5 liquid medium for 5 weeks.

Nrk: a murine X-linked NIK (Nck-interacting kinase)-related kinase gene expressed in skeletal muscle.

We report the cloning and expression pattern of a novel Ste20-type kinase gene, NIK-related kinase (Nrk), located on the mouse X chromosome. The full-length Nrk cDNA encodes a 1455-amino-acid polypeptide characterized by a N-terminal Ste20-type catalytic domain and a C-terminal regulatory domain characteristic of the group I GCK subfamily. The overall structure of the NRK protein is closely related to that of Nck-interacting kinase (Nik). In situ hybridization revealed that Nrk was predominantly expressed in skeletal muscle during mouse embryogenesis. Nrk gene expression was detected in the myotome at 10.5 dpc and, thereafter, was observed in developing skeletal musculature from 11.5 to 13.5 dpc. However, expression in skeletal muscle was not observed in adults.

Genetic engineering of shikonin biosynthesis hairy root cultures of Lithospermum erythrorhizon transformed with the bacterial ubiC gene.

The biosynthetic pathway to 4-hydroxybenzoate (4HB), a precursor of the naphthoquinone pigment shikonin, was modified in Lithospermum erythrorhizon hairy root cultures by introduction of the bacterial gene ubiC. This gene of Escherichia coli encodes chorismate pyruvate-lyase (CPL), an enzyme that converts chorismate into 4HB and is not normally present in plants. The ubiC gene was fused to the sequence for a chloroplast transit peptide and placed under control of a constitutive plant promoter. This construct was introduced into L. erythrorhizon by Agrobacterium rhizogenes-mediated transformation. The resulting hairy root cultures showed high CPL activity. 4HB produced by the CPL reaction was utilized for shikonin biosynthesis, as shown by in vivo inhibition of the native pathway to 4HB with 2-aminoindan-2-phosphonic acid (AIP), an inhibitor of phenylalanine ammonia-lyase. A feeding experiment with [1,7-13C2]shikimate showed that in the absence of AIP the artificially introduced CPL reaction contributed ca. 20% of the overall 4HB biosynthesis in the transgenic cultures. ubiC transformation did not lead to a statistically significant increase of shikonin formation, but to a 5-fold increase of the accumulation of menisdaurin, a nitrile glucoside which is presumably related to aromatic amino acid metabolism.

Expression of brain-2 in the developing olfactory bulb.

The expression of Brain-2, a POU domain transcription factor, was examined in the developing olfactory bulb. Brain-2 was expressed mainly in the output neurons, mitral cell and tufted cells in the main olfactory bulb (MOB), and mitral/tufted cells (MT cells) in the accessory olfactory bulb (AOB). It was not expressed in granular cells in either the MOB or the AOB. Our results suggest that Brain-2 was specifically expressed in output neurons but not in interneurons in the developing olfactory bulb. Brain-2 may play a role in the development of these output neurons.