Flavan-3-ol Biosynthesis : The Conversion of (+)-Dihydroquercetin and Flavan-3,4-cis-Diol (Leucocyanidin) to (+)-Catechin by Reductases Extracted from Cell Suspension Cultures of Douglas Fir

Extracts of cell suspension cultures from Douglas fir (Pseudotsuga menziesii) needles catalyzed the production of (+)-catechin (2,3-trans flavan-3-o1) from the 2,3-trans-flavan,3,4-cis-diol (leucocyanidin) in a NADPH-dependent reductase reaction at pH 7.4. Catechin was also produced, along with the 3,4-cis-diol, in a double step reduction from (+)-dihydroquercetin. It was necessary to eliminate any thiol such as 2-mercaptoethanol or dithiothreitol from the extract or assay mixture because these thiols apparently formed thioethers with the 3,4-diols.     

Glucosylation of Capsaicin by Cell Suspension Cultures of Coffea arabica

Capsaicin was converted into the corresponding glucoside when administered to cell suspension cultures of Coffea arabica cultured in a modified Murashige and Skoog’s medium with 5 μM 2,4-dichlorophenoxyacetic acid and 0.5 μM kinetin. The glucoside was identified as capsaicin-β-D-glucopyranoside by FAB-MS, ¹H-NMR, and hydrolysis with α- and β-glucosidases. The pungency of the glucoside was approximately 1/100 of that of capsaicin.     

长春花悬浮培养细胞对天麻素的生物转化

应用长春花(Catharanthus roseus (L.) G. Don)悬浮细胞培养体系对天麻素进行了生物转化反应研究.经过8 d培养形成一个转化产物,应用光谱方法鉴定转化产物的结构为对羟基苯甲醇,为天麻素水解后形成的甙元.     

Effects of Glyphosate on the Shikimate Pathway and Regulation of Phenylalanine Ammonia-lyase in Cryptomeria and Perilla Cell Suspension Cultures

Treatment of Cryptomeria and Perilla cell suspension cultureswith glyphosate resulted in a marked suppression of the formationof flavans and caffeic acid derivatives, respectively, whileit caused only a slight decline in the cell growth. In contrastwith 3-deoxy-D-arabino-heptulosonate (DAHP) synthase-Mn isozyme,DAHP synthase-Co isozyme from Cryptomeria and Perilla cellswas much more sensitive to inhibition by glyphosate. The additionof 1 to 2 mM glyphosate caused an accumulation of shikimateand quinate and a reduction of L-phenylalanine in both cellcultures. The inhibition of phenylalanine ammonia-lyase (PAL)activity by glyphosate was reversed by exogenously suppliedL-phenylalanine to near the control level. Cycloheximide andactinomycin D nullified the recovery by exogenous L-phenylalanineon PAL activity. L-Phenylalanine itself promoted PAL activityto some extent. No recovery of PAL activity in L--aminooxy-ß-phenylpropionate(L-AOPP)-treated cell cultures could be observed by the additionof L-phenylalanine. Therefore, L-AOPP seems to inhibit the formationof PAL, though it has been considered a competitive inhibitor. ³Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan. (Received October 28, 1985; Accepted March 13, 1986)     

镉胁迫引起烟草悬浮细胞程序性死亡

镉胁迫会造成烟草悬浮细胞大规模死亡。通过TUNEL技术和琼脂糖凝胶电泳技术的检测发现,这种细胞死亡伴随有典型的DNA“梯形带”出现,表明这种由Cd胁迫引起的细胞死亡是一种程序性死亡。受胁迫细胞氧化性增强及细胞中丙二醛(MDA)水平升高,说明Cd胁迫时会在细胞中造成大量活性氧(ROS),暗示烟草细胞的程序性死亡可能与ROS有关。     

Photoautotrophic Cell Suspension Cultures from Mesembryanthemum crystallinum and their Response to Salt Stress

For the first time, photoautotrophic cell suspension cultures of Mesembryanthemum crystallinum have been established. The cells are growing in a sugar-free culture medium in the presence of 2 % (v/v) CO₂ as the sole carbon source. A 16 h light photoperiod is applied. Increase in fresh and dry weight during a 21 days growth cycle was more than 3-fold. Treatment of the cells with 200 mM NaCl from day 10 to day 21 of subculture stimulated cell culture growth, enhanced CO₂ fixation and elicited an increase in the extractable activities of enzymes related to CO₂ fixation (RubisCO; PEP carboxylase) and malic acid metabolism (NAD / NADP dependent malic enzyme and malic acid dehydrogenase). The cells performed osmotic adjustment to high salinity by uptake of K⁺, Na⁺, Cl^? and formation of proline as well as by a reduction in cell size. Although sugar and starch content of the cells changed during light/dark transition, a CAM-related diurnal fluctuation of malic acid was not observed.     

Cell Apoptosis Induced by Oligosaccharide in Suspension Cultures of Taxus chinenesis

Taxol production of Taxus chinensis（Pilger） Rehd. var.mairei (Lemeeet Lévl.) Cheng et L. K. Fu induced by oligosaccharide from Fusarium oxysporum f.vasinfectum (Atkinson) Snyder et Hansen was studied in suspension cultures, and it was found that oligosaccharide triggered cell apoptosis. Under transmission electron microscope the following morphological changes were observed: cell shrinkage, condensation of cytoplasm, nuclear fragmentation, and the increase of high electron density bodies in vacuole in great quantity. In oligosaccharide treated cells, agarose gel electrophoresis revealed that DNA was digested into oligonucleosomal fragments that were times of 200 bp appearing as DNA ladders. Control cells were in normal physiological state, they were intact, abundant in organelle and with integral nucleus DNA, and the rate of taxol biosynthesis in these cells was very low. After the oligosaccharide to the culture system, the defense system of cells was elicited and the secondary metabolism was strengthened, i.e. phenolics were accumulated in the medium, the activity of polyphenol oxidase (PPO) was increased quickly and secondary wall of cells was thickened. The activity of L phenylalanine ammonia lyase (PAL), the critical enzyme of the phenylpropanoid pathway, was increased promptly 1 h after elicitation. The rate of taxol production was improved sharply and the maximal taxol concentration at 72 h was six times that of control. Appearance of cell apoptosis was accompanied with the highest concentration of taxol in suspension cultures.     

寡聚糖诱导悬浮培养南方红豆杉细胞的凋亡(英)

在真菌 (Fusariumoxysporumf.vasinfectum (Atkinson)SnyderetHansen)寡聚糖诱导悬浮培养南方红豆杉(Taxuschinensis (Pilger)Rehd .var.mairei (LemeeetL啨ｖｌ.)ChengetL .K .Fu)细胞生产紫杉醇的体系中发现细胞出现凋亡 ,次生代谢增强。电镜观察到细胞核质和原生质出现凝集现象 ,液泡内出现大量的高电子致密体。核DNA经琼脂糖凝胶电泳 ,呈 2 0 0bp的整数倍的梯状条带 (ladders) ;而对照组细胞核DNA完整 ,呈大片段 ,细胞完整 ,细胞器发达 ,但紫杉醇合成速率很低。加入寡聚糖后 ,细胞防御系统开启 ,细胞生长停止 ,次生代谢物酚类物质大量积累且次生壁加厚 ,多酚氧化酶活性迅速提高 ,苯丙烷类代谢途径的关键酶苯丙氨酸解氨酶的活性在 1h后急速提高 ,目的产物紫杉醇在诱导后 72h达到峰值 ,比对照组提高了 6倍 ,且细胞凋亡的出现与紫杉醇合成的峰值具有时间上的一致性。     

寡聚糖诱导悬浮培养南方红豆杉细胞的凋亡

在真菌(Fusarium oxysporum f.vasinfectum (Atkinson) Snyder et Hansen)寡聚糖诱导悬浮培养南方红豆杉(Taxus chinensis (Pilger) Rehd.var.mairei (Lemee et Lévl.) Cheng et L.K.Fu)细胞生产紫杉醇的体系中发现细胞出现凋亡,次生代谢增强.电镜观察到细胞核质和原生质出现凝集现象,液泡内出现大量的高电子致密体.核DNA经琼脂糖凝胶电泳,呈200 bp的整数倍的梯状条带(ladders);而对照组细胞核DNA完整,呈大片段,细胞完整,细胞器发达,但紫杉醇合成速率很低.加入寡聚糖后,细胞防御系统开启,细胞生长停止,次生代谢物酚类物质大量积累且次生壁加厚,多酚氧化酶活性迅速提高,苯丙烷类代谢途径的关键酶苯丙氨酸解氨酶的活性在1 h后急速提高,目的产物紫杉醇在诱导后72 h达到峰值,比对照组提高了6倍,且细胞凋亡的出现与紫杉醇合成的峰值具有时间上的一致性.     

Purification and Interconversion of Homoserine Dehydrogenase from Daucus carota Cell Suspension Cultures

Homoserine dehydrogenase from cell suspension cultures of carrot (Daucus carota L.) has been purified to apparent homogeneity by a combination of selective heat denaturation, ion exchange and gel filtration chromatographies, and preparative gel electrophoresis. Carrot homoserine dehydrogenase is composed of subunits of equal molecular weight (85,000 ± 5,000). During purification, the enzyme exists predominantly in two molecular weight forms, 180,000 and 240,000. The enzyme can be reversibly converted from one form to the other, and each has different regulatory properties. When the enzyme is dialyzed in the presence of 5 millimolar threonine, the purified enzyme is converted into its trimeric form (240,000), which is completely inhibited by 5 millimolar threonine and is stimulated 2.6-fold by K⁺. When the enzyme is dialyzed in the presence of K⁺ and absence of threonine, the purified enzyme is converted into a dimer (180,000), which is not inhibited by threonine and is only stimulated 1.5-fold by K⁺. The enzyme also can polymerize under certain conditions to form higher molecular weight aggregates ranging in size up to 720,000, which also are catalytically active. This interconversion of homoserine dehydrogenase conformations may reflect the daily stream of events occurring in vivo. When light stimulates protein synthesis, the threonine pool decreases in the chloroplast, while K⁺ concentrations increase. The change in threonine and K⁺ concentrations shift the homoserine dehydrogenase from the threonine-sensitive to the threonine-insensitive conformation resulting in increased production of threonine, which would meet the demands of protein synthesis. The reverse process would occur in the dark.     

Solubilization and Characterization of a Membrane-Bound Auxin-Binding Protein from Cell Suspension Cultures of Nicotiana tabacum

A membrane-bound auxin-binding protein (MABP) was solubilized by Triton X-100 from cell suspension cultures of Nicotiana tabacum L. Solubilization of MABP was dependent on the detergent concentration and more than 80% of naphthalene-1-acetic acid (NAA)-binding activity was recovered by an optimum concentration of 0.2%. The solubilized MABP was highly heat-unstable and sensitive to protease. The properties of MABP (affinity, temperature dependence, pH optimum, and analog specificity for auxin binding) did not significantly change after solubilization, e.g. the solubilized MABP showed no or very low levels of NAA-binding at 0 to 4°C but showed a high-affinity binding (dissociation constant K_d = 2.7 ± 0.3 × 10⁻⁷m) at 25°C at an optimum pH of 5.0. NAA-binding of the solubilized MABP proceeded very slowly, i.e. a time of half-maximum binding was at least 15 minutes, although the solubilized MABP showed higher rates of association (k₁ = 1.3 versus 0.9 × 10⁵m⁻¹ min⁻¹) and dissociation (k₋₁ = 2.2 versus 1.6 × 10⁻² min⁻¹) with NAA than the bound MABP. These results show that specific, saturable, and reversible auxin binding to MABP from dicotyledonous N. tabacum differs from that from monocotyledonous Zea mays, and confirm that MABP is distinct from a soluble auxin-binding protein which also is present in N. tabacum.     

The Conversion of d-Tryptophan to l-Tryptophan in Cell Cultures of Tobacco

d-Tryptophan was converted to l-tryptophan in tissue cultures of tobacco, in whole cells treated with dimethylsulfoxide, and in cell-free extracts treated by Sephadex G-25 filtration. Evidence was obtained that tryptophanase, tryptophan pyrrolase, and transaminase activities were not involved. The data were best explained by the presence of a tryptophan racemase as the enzyme catalyzing the reaction. The possible role of d-tryptophan in the biosynthesis of indoleacetic acid is discussed.     

A diterpene quinone from the bark of Cryptomeria japonica

A diterpene, cryptoquinone, was isolated from the bark of Cryptomeria japonica, the structure, 7,11,14-trioxoabieta-8,12-diene, was established by spectral analyses and X-ray crystallography. This diterpene quinone showed moderate antifungal activities against Pyricularia orizae and Alternaria alternata, and cytotoxic activity against mouse lymphoid neoplasm (P388) cells with an IC(50) of 0.26 microg/ml.     

In Vitro DNA Synthesis by Chloroplasts Isolated from Marchantia polymorpha L. Cell Suspension Cultures

Lysate of chloroplasts prepared from liverwort Marchantia polymorpha L. cell suspension cultures incorporated [³H]-dTTP into acid insoluble materials when DNA was added exogenously as a template. The incorporation was highly dependent on the addition of template DNA, four deoxynucleoside triphosphates and magnesium ions (maximum incorporation at 5mM). Magnesium ions could be replaced by manganese ions. DNA synthesis inhibitors, N-ethylmaleimide (NEM) and ethidium bromide (EtBr), strongly inhibited the incorporation. Dideoxythymidine triphosphate (ddTTP), an inhibitor of DNA polymerases β and γ, inhibited the incorporation at the concentration of 50 μM (molar ratio of ddTTP/dTTP = 17). On the other hand, the incorporation by the chloroplast lysate was resistant to arabinofuranosyl cytosine triphosphate (araCTP) and aphidicolin as well as the RNA polymerase inhibitors, rifampicin and α-amanitin. The chloroplast lysate highly utilized denatured calf thymus DNA and bacteriophage ?X174 single-stranded DNA as templates when added exogenously, while a synthetic homopolymer, poly(rA)-oligo(dT)₁₂ ~ ₁₈, did not stimulate the incorporation at all. Autoradiographic analysis of DNA synthesized in isolated chloroplasts showed that the chloroplast DNA synthesis took place at several specific sites on the chloroplast DNA from cells of the liverwort, Marchantia polymorpha.     

Two new sesquarterpenoids from the bark of Cryptomeria japonica

Two new sesquarterpenoids, i.e. ferrugicadinol A (1) and ferrugicryptomeridiol (3), and one known sesquarterpenoid, ferrugicadinol (2) were isolated from the bark of Cryptomeria japonica D. Don. Their structures were identified by extensive spectral analysis and comparison with the data of known analogues.