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ent-Kaurene is a tetracyclic diterpene hydrocarbon and a biosynthetic intermediate of the plant hormone gibberellins. In flowering plants, ent-kaurene is biosynthesized from geranylgeranyl diphosphate (GGDP) by two distinct cyclases, ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS). Recently, the moss Physcomitrella patens ent-kaurene biosynthetic gene was cloned and functionally characterized. The bifunctional ent-kaurene synthase [P. patens CPS/KS (PpCPS/KS)] produces both ent-kaurene and 16α-hydroxy-ent-kaurane from GGDP via ent-copalyl diphosphate. Here, we cloned and analyzed the function of a cDNA encoding bifunctional ent-kaurene synthase from the liverwort Jungermannia subulata [J. subulata CPS/KS (JsCPS/KS)]. JsCPS/KS catalyzes the cyclization reaction of GGDP to produce ent-kaurene but not 16α-hydroxy-ent-kaurane, even though the PpCPS/KS (881 amino acids) and JsCPS/KS (886 amino acids) sequences share 60% identity. To determine the regions and amino acids involved in 16α-hydroxy-ent-kaurane formation, we analyzed the enzymic functions of JsCPS/KS and PpCPS/KS chimeric proteins. When the C-terminal region of PpCPS/KS was exchanged with the JsCPS/KS C-terminal region, the chimeric cyclases produced only ent-kaurene. The replacement of PpCPS/KS Ala710 with Met or Phe produced a JsCPS/KS-type cyclase that converted GGDP to ent-kaurene as the sole product. In contrast, replacing Ala710 with Gly, Cys or Ser did not affect the PpCPS/KS product profile as much as replacement of Cys of JsCPS/KS by Ala. Thus, the hydrophobicity and size of the side chain residue at the PpCPS/KS amino acid 710 is responsible for quenching the ent-kauranyl cation by the addition of a water molecule.

Due to the inhibition of 5-lipoxygenase-activating protein (FLAP), BAY x1005 is a new selective inhibitor of leukotriene synthesis. The effects of BAY x1005 on the antigen- and bacterial lipopolysaccharide (LPS)-induced airway hyperresponsiveness in guinea pigs were investigated. Six times provocation of aeroantigen caused biphasic increases in airway resistance which peaked at 1 hr (immediate phase reaction) and 4 hrs (late phase reaction). It also caused airway hyperreactivity to acetylcholine. BAY x1005 at doses of 10mg/kg and 30mg/kg significantly inhibited antigen-induced increase in respiratory resistance (Rrs) at 1 and 4 hrs after the last antigen challenge. Simultaneously, BAY x1005 inhibited the antigen-induced airway hyperresponsiveness at doses of 10 and 30mglkg and airway eosinophilia (bronchoalveolar lavage study) at a dose of 30 mg/kg. In addition, BAY x1005 at a dose of 30mg/kg inhibited bacterial LPS-induced airway hyperreactivity to acetylcholine. In this model, BAY x1005 did not affect the increase of the number of leukocytes in bronchoalveolar lavage fluid. These results suggest that BAY x1005 is a potent anti-asthmatic agent with an inhibitory action to airway hyperreactivity.

The effects of a novel leukotriene (LT) C 4 D 4 antagonist, BAY-x-7195 on experimental allergic reactions in airway and skin were compared to that of ONO-1078. BAY-x-7195 showed an antagonistic action to LTD4-induced bronchoconstriction in vitro and in vivo. In in vitro experiments, BAY-x-7195 inhibited LTD4-induced contraction of isolated guinea pig tracheal muscle (pA2=8.03). BAY-x-7195 at doses of 3 – 30 mg/kg clearly inhibited LTD4-induced increases in respiratory resistance (Rrs) in guinea pigs. In contrast, BAY-x-7195 inhibited significantly U-46619-induced increases in Rrs at a dose of 30 mg/kg in guinea pigs. BAY-x-7195 at doses of 3 — 30 mg/kg inhibited the aerosolized antigen-induced biphasic increase in Rrs in guinea pigs. Moreover BAY-x-7195 inhibited repeated aeroantigen-induced airway hyperreactivity in guinea pigs. In mice, aeroantigen-induced airway inflammation were clearly inhibited by BAY-x-7195. These results show the efficacy of BAY-x-7195 against the antigen-induced increase in airway resistance and antigen-induced airway hyperreactivity in guinea pigs and mice, probably due to anti-LTD4 antagonistic action and the inhibition of antigen-induced airway inflammation.

The effect of a novel thromboxane A2 receptor antagonist, BAY-u-3405, on experimental allergic airway and skin reactions was studied in vivo. At doses of 3-30 mg/kg BAY-u-3405 clearly inhibited the U-46619-induced increase in respiratory resistance (Rrs) in guinea pigs. BAY-u-3405 at doses of 3 and 30 mg/kg inhibited the aerosolized antigen-induced biphasic increase in respiratory resistance in guinea pigs. Moreover, BAY-u-3405 inhibited repeated aeroantigen-induced airway hyperactivity and airway inflammation in mice. In IgE antibody-mediated biphasic skin reactions in mice, both immediate and late-phase reactions were inhibited by 10 mg/kg of BAY-u-3405. These results demonstrate the efficacy of BAY-u-3405 on the antigen-induced late-phase reactions in the airway and skin in guinea pigs and mice, and antigen-induced airway hyperactivity in mice.

Three new gymnomitrane sesquiterpenoids have been isolated from the liverwort Marsupella emarginata var. patens together with the known (−)-gymnomitrene and (−)-ar-curcumene. Their structures and absolute configurations have been determined on the basis of chemical and spectroscopic evidence as well as from the results of an X-ray analysis of a derivative. We also describe the ring expansion reaction of the gymnomitrane framework and the chemosystematics of the genus Marsupella.

Momilactones A (1) and B (2), which have been identified as phytoalexins in rice, were isolated from extracts of the moss Hypnum plumaeforme. This is the first isolation and identification of momilactones as allelochemicals from a bryophyte. H. plumaeforme produces considerable amounts of momilactones (isolated yield: 8.4 mg/Kg plant for 1; 4.2 mg/Kg for 2). EtOAc extracts from H. plumaeforme and 2 showed growth inhibitory activity against angiosperms, moss, and liverwort plants. On the other hand, the growth of H. plumaeforme was insensitive to its extract and 2. Our finding suggests that momilactones play an important role as allelochemicals in this moss.

A suspension culture of the liverwort Plagiochila ovalifolia was established from callus tissue induced by culturing spores. From the cultured cells, four phaeophytins were isolated as major components and their structures determined by spectroscopic methods. The phaeophytin derivatives showed antibacterial activity. A major sesquiterpenoid, ovalifoliene, found in the mother plant, was detected in the cultures by GC-mass spectrometry.

The sterols of 16 mosses were analysed by GLC and GC-MS. Cholest-5-en-3β-ol, 24-methylchloesta-5,22-dien-3β-ol, 24-methylcholest-5-en-3gb-ol, 24-ethylcholesta-5,22-dien-3β-ol, 24-ethylcholest-5-en-3β-ol, were detected in all samples but there were differences in the relative amounts present. The C29-sterols were predominant constituents in most of the mosses examined.

Five new dolabellane diterpenoids have been isolated from the liverwort Odontoschisma denudatum, and their structures and absolute configurations have been determined on the basis of 1H and 13C NMR evidence and chemical correlation.

Three labdane type diterpene glycosides ( 1 , 2 and 3 ) with 6-deoxy-L-idose have been isolated from Aster spathulifolius Maxim. and their structures have been determined on the basis of chemical and spectral data.

The diterpene acid content in 10 species of Helianthus has been investigated. Ent-12,16-cyclokauranoic acid, isolated from H. annuus, is converted into a series of 12,16-cyclogibberellins by cultures of Gibberella fujikuroi, mutant B1-41a, and 12,16-cyclogibberellins A9, and A12 have been isolated. Ent-12β-acetoxykaurenoic acid and ent-13(S)-angeloxyatisenoic acid have been isolated from H. decapetalus; the metabolism of ent-13(S)-hydroxyatisenoic acid and atisenoic acid by B1-41a is also described.

Two enantiomeric cuparene-type sesquiterpenoids, (R)-(−)-cuparene ( 1 ) and (R)-(−)-δ-cuparenol ( 2 ), have been isolated from the liverwort, Bazzania pompeana . The structures and absolute configurations of the two compounds have been determined.

13C NMR spectra of several 2,3-seco-alloaromadendrane-type acetyl hemiacetals were analysed and the structure of an additional plant growth-inhibitor was determined to be (+)-9α-acetoxyovalifoliene. The biological activity of the new compound is also described.

A series of ent-sesquiterpenoids corresponding to the optical antipodes of those in higher plants have been isolated from the liverwort Marchantia polymorpha. These sesquiterpenoids provide yet another example of the peculiar stereospecificity of the biogenesis of the liverwort sesquiterpenoids and suggest a special taxonomic position of the liverworts in the plant kingdom.

A natural precursor (L) of streptomycin which had no antibiotic potency was obtained from mycelium suspension of Streptomyces griseus in glucose solution and was transformed to streptomycin by H enzyme obtained from mycelium of the organism. This transforming reaction was carried out most effectively at slightly alkaline pH and inhibited by inorganic phosphate and ethylenediaminetetraacetate. L component was considered to be a phosphorylated compound and liberation of the phosphoric acid was essential for L component to be transformed to streptomycin. This transformation was performed not only by H enzyme but also by intestinal alkaline phosphatase, although some difference in the reaction mechanism was supposed to be between those two enzymes.

Two homologous series of n-paraffins and sec-alcohols from C20 to C34 or C35 were isolated a wax constituents of a liverwort, Isotachis japonica. In both series, odd-members were predominant compared with even-members: the ratio of the total amount of odd- to total amounts of even-members was 3·9 in the n-paraffin homologues, and 16·5 in the sec-alcohol homologues.

A natural precursor (L) of streptomycin which had no antibiotic potency was obtained from mycelium suspension of Streptomyces griseus in glucose solution and was transformed to streptomycin by H enzyme obtained from mycelium of the organism. This transforming reaction was carried out most effectively at slightly alkaline pH and inhibited by inorganic phosphate and ethylenediaminetetraacetate. L component was considered to be a phosphorylated compound and liberation of the phosphoric acid was essential for L component to be transformed to streptomycin. This transformation was performed not only by H enzyme but also by intestinal alkaline phosphatase, although some difference in the reaction mechanism was supposed to be between those two enzymes.

Investigation was carried out on demonstration of two substances constructing a precursor system located at a late stage of streptomycin biosynthesis by Streptomyces griseus. One of them is thought to be a natural precursor of Streptomycin(L) and the other is suggested as an enzymatic substance(H) transforming L to streptomycin. Both substances had no antibiotic potency and H was inactivated at low pH. L was obtained from a cell-free supernatant (active supernatant) prepared from suspension of young mycelium of Streptomyces griseus in glucose solution. H was obtained not only from active supernatant but also from cell-free extract of the organism.Two ways of isolation were established for L. Active supernatant was adsorbed on a CM-cellulose column equilibrated with 0.05 m Tris-maleate buffer (pH 8.0). Elution of this column with the same buffer as was used for equilibration gave L-containing fraction separated from streptomycin which was eluted with the buffer including 1% of sodium chloride. L was adsorb...

ent-Kaurene is the key intermediate in biosynthesis of gibberellins (GAs), plant hormones. In higher plants, ent-kaurene is synthesized successively by copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) from geranylgeranyl diphosphate (GGDP). On the other hand, fungal ent-kaurene synthases are bifunctional cyclases with both CPS and KS activity in a single polypeptide. The moss Physcomitrella patens is a model organism for the study of genetics and development in an early land plant. We identified ent-kaurene synthase (PpCPS/KS) from P. patens and analyzed its function. PpCPS/KS cDNA encodes a 101-kDa polypeptide, and shows high similarity with CPSs and abietadiene synthase from higher plants. PpCPS/KS is a bifunctional cyclase and, like fungal CPS/KS, directly synthesizes the ent-kaurene skeleton from GGDP. PpCPS/KS has two aspartate-rich DVDD and DDYFD motifs observed in CPS and KS, respectively. The mutational analysis of two conserved motifs in PpCPS/KS indicated that the DVDD motif is responsible for CPS activity (GGDP to CDP) and the DDYFD motif for KS activity (CDP to ent-kaurene and ent-16α-hydroxykaurene).

The structure of a new ent-longipinane type of sesquiterpenoid, (−)-ent-12β-acetoxylongipin- 2(10)-en-3-one from the liverwort Marsupella aquatica, has been elucidated by spectroscopic and chemical methods.

Five soluble carbohydrates, (−)- l -bornesitol, (−)- d -mannitol, (+)- d -glucose, (+)-sucrose and (+)-trehalose, have been isolated as their acetyl derivatives from the liverwort, Mylia taylorii. The absolute configuration of these carbohydrates is the same as that of sugars found in vascular plants, although the liverwort sesquiterpenoids are usually enantiomeric relative to those of vascular plants. The soluble carbohydrates in seven other liverwort species have also been analysed by HPLC.

Three new diterpenoids of the verrucosane class were isolated as minor constituents of the methanol extract of the liverwort Mylia verrucosa and their structures shown to be 2β,9α,13β-trihydroxyverrucosane,9α-acetoxy-2β,13β-dihydroxyverrucosane and 2β,13β-dihydroxy-9-oxoverrucosane.

The sterol fractions of eight leafy liverworts were analyzed by GLC and GC-MS. Five 3β-sterols, cholest-5-en-3β-ol, 24-methylcholest-5,22-dien-3β-ol, 24-methylcholest-5-en-3p-ol, 24-ethylcholest-5,22-dien-3β-ol and 24-ethylcholest-5-en-3β-ol, were detected in all samples but there were differences in the relative amounts present.

Methyl decanoate, undecanoate, laurate, tridecanoate, myristate, pentadecanoate, palmitate, oleate and stearate were isolated as steam-volatile components from this liverwort. Total amounts of these methyl esters accounted for 75·3 per cent of the steam-volatile substances and methyl palmitate (60%) was the major constituent.

A series of fatty acid ethyl esters ranging from C 14 to C 24 was isolated from a hexane extract of the liverwort Conocephalum conicum , these esters accounted for 77% of the extract. The ethyl esters consisting of even-numbered fatty acids were predominant and ethyl palmitate was the major constituent.

12,16-Cyclogibborellins A9 and A12 have been isolated from the microbiological transformation of trachylobanic acid by Gibberella fujikuroi, mutant B1-41a, thus providing a notable example of the non-specificity of enzymes, catalysing the biosynthesis of fungal gibberellins; the 12,16-cyclogibberellin A9 retains gibberellin-like biological activity.