Responses of anthocyanin-producing and non-producing cells of Glehnia littoralis to radical generators.

The responses of anthocyanin-producing (violet) and non-producing (white) cells of Glehnia littoralis to radical generators were compared. Cell growth, anthocyanin content, phenylalanine ammonia-lyase (PAL) activity and furanocoumarin production were determined after treatment with H(2)O(2), 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), X-ray and yeast extract, independently. AAPH and H(2)O(2) repressed the growth of both violet and white cells, but violet cells grew better than white cells. On the other hand, the anthocyanin content in violet cells decreased. Neither X-ray nor yeast extract affected cell growth or pigment production. Treatment with H(2)O(2), yeast extract, and X-ray, but not AAPH, induced PAL activity and furanocoumarin production in white cell cultures, whereas violet cell cultures did not produce furanocoumarin following any of the treatment employed.     

Iron availability alters ascorbate-induced stress metabolism in Glehnia littoralis root cultures

Our previous study indicated that formation of furanocoumarin phytoalexins could be induced in Glehnia littoralis root cultures by treatment with 10–40 mM ascorbic acid (AsA). This furanocoumarin production is much less evident when G. littoralis roots are treated with AsA under iron-deficient conditions. Instead, two large unknown peaks appeared in the HPLC chromatogram, whose chemical structures were elucidated by spectroscopic methods as being 6, β-dihydroxyphenethyl ferulate (DF) and 6-hydroxyphenethyl ferulate (HF), respectively. Their maximal level of induction was observed at 20 mM AsA, and the production of DF always exceeded that of HF. This is the first report of these compounds in G. littoralis and of the modulation of the phytoalexin biosynthetic pathway in G. littoralis by iron deficiency.     

Pyranocoumarins from Glehnia littoralis inhibit the LPS-induced NO production in macrophage RAW 264.7 cells

A new dihydropyranocoumarin, (+)-cis-(3′S,4′S)-diisobutyrylkhellactone (1), together with five known compounds, 3′-senecioyl-4′-acetylkhellactone (2), 3′-isovaleryl-4′-acetylkhellactone (3), 3′,4′-disenecioylkhellactone (4), 3′-isovaleryl-4′-senecioylkhellactone (5), and 3′,4′-diisovalerylkhellactone (6), was isolated from Glehnia littoralis. Their chemical structures were elucidated based on the spectroscopic data interpretation, particularly 1D and 2D NMR data including HMQC and HMBC. All the isolated compounds showed the potential to inhibit LPS-induced nitric oxide production in RAW 264.7 cells with IC₅₀ values ranging from 7.4 to 44.3 μM.     

Acetate metabolism in cell suspension cultures

Cell suspension cultures of Paul's Scarlet rose were grown over a 14-day period, during which a 50-fold increase in fresh weight occurred. Three phases could be recognized from weight, DNA determinations, and microscopic examination. From days 0 to 7, cell division was accompanied by cell expansion; from days 7 to 10, only cell expansion occurred; and from days 10 to 14, there was no further growth.     

Solasodine production by immobilized cells and suspension cultures of Solanum surattense

Summary Immobilized cells ofSolanum surattense Burm release far more solasodine into the medium than free cell suspension cultures. This enhancement is probably due to stabilization of cells after immobilization as well as the effect of growth hormones in the medium.     

Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae)

The effects of different levels of Murashige and Skoog (MS) basal medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and sucrose on anthocyanin production and biomass accumulation of cell suspension cultures of Cleome rosea were investigated. Cultures were established in liquid MS medium containing 30 g l⁻¹ sucrose and supplemented with 0.90 μM 2,4-D. Proliferating cell suspension cultures achieved the highest growth capacity, a fourfold increase in biomass accumulation, following subculture at the exponential growth phase, 14–18 days of culture. Moreover, the presence of 2,4-D was essential for anthocyanin production and biomass accumulation. On the other hand, increasing levels of sucrose above 30 g l⁻¹ resulted in a drastic reduction in biomass accumulation. Anthocyanin production was highest in cell suspension cultures grown on half-strength MS medium (1/2 MS), 30 g l⁻¹ sucrose, and 0.45 μM 2,4-D. These cell suspension cultures were mainly composed of small aggregates of spherical cells with similar morphology observed in anthocyanin-producing and non-producing cultures. Moreover, microscopic analysis of anthocyanin-producing cultures showed the presence of mixtures of non-pigmented, low-pigmented, and high-pigmented cells.     

Effect of norleucine on the metabolism of tobacco cells in suspension cultures

The phenolic content of tobacco cells in suspension cultures rose during the initial stages of growth and then fell again. Norleucine treatment prevented this initial rise in production of phenolics, but a steady level of the compounds was maintained. No significant changes in the type of phenolics produced was observed. Norleucine had a slight effect on the amino acid composition of proteins probably by changing the relative amounts of soluble proteins. The free amino acid pool was changed drastically and the amount of free glutamine especially was reduced. A slight increase of ethylene production was noted, perhaps due to a sparing action of norleucine on methionine. The results are discussed in terms of the possible use of an amino acid antagonist such as norleucine to change cell metabolism and cell composition without changing cell growth.     

Somatic embryogenesis in mature zygotic embryo culture of Glehnia littoralis

In vitro somatic embryogenesis of Glehnia littoralis Fr. schm. was observed when zygotic embryos were cultured on a medium containing 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid (0.01-10 μM), with 1 μM being the optimum. Microscopic observations revealed globular, heart-shaped and torpedo-shaped embryo formations and plantlet regeneration. These somatic embryos seemed to be produced directly from cells of the zygotic embryos used as explants. Of seven types of media tested, Nitsch's medium showed the highest rate of somatic embryogenesis. Somatic embryos developed into normal plantlets and were able to be potted. This revised version was published online in June 2006 with corrections to the Cover Date.     

Growth, metabolism and baculovirus production in suspension cultures of an Anticarsia gemmatalis cell line

The UFL-AG-286 cell line, established from embryonic tissue of the lepidopteran insect Anticarsia gemmatalis, has been identified as a good candidate to be used as a cellular substrate in the development of a process for in vitro production of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, a baculovirus widely used as bioinsecticide. In order to characterize the technological properties of this cell line and evaluate its feasibility to use it for the large-scale production of Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, UFL-AG-286 cells were adapted to grow as agitated suspension cultures in spinner-flasks. Batch suspension cultures of adapted cells in serum-supplemented TC-100 medium grew with a doubling time of about 29 h and reached a maximum cell density higher than 3.5 × 10⁶ viable cells ml⁻¹. At the end of the growth period glucose was completely depleted from the culture medium, but l-lactate was not produced. Amino acids, with the exception of glutamine, were only negligibly consumed or produced. In contrast to other insect cell lines, UFL-AG-286 cells appeared to be unable to synthesize alanine as a metabolic way to dispose the by-product ammonia. The synchronous infection of suspension cultures with Anticarsia gemmatalis multicapsid nucleopolyhedrovirus in the early to medium exponential growth phase yielded high amounts of both viral progenies per cell and reduced the specific demands of UFL-AG-286 cells for the main nutrients.     

珊瑚菜植株分泌道发育和分布的解剖学观察

利用植物解剖学方法对珊瑚菜（Glehnia littoralis Fr．Schmidt ex Miq．）体内分泌道的发育和分布进行了观察。结果表明,珊瑚菜的分泌道有分枝,为溶生型,由1层分泌细胞围绕腔道而成。珊瑚菜叶片的分泌道发育较早,在幼叶阶段即发育成形。在根的次生韧皮部、根状茎的皮层和靠近初生木质部的髓部、叶脉的薄壁组织、叶柄维管束周围和厚角组织内侧的薄壁组织、花序轴正对维管束的皮层薄壁组织中以及果实的果壁维管束内外侧的薄壁组织中均分布有分泌道,分泌道在珊瑚菜体内分布广泛。     

江苏野生珊瑚菜生存现状及其灭绝原因探析

主要介绍了江苏濒危植物珊瑚菜的特征特性、生存现状.江苏沿海野生珊瑚菜种群被证实已灭绝,并探析了其灭绝的原因,为内在因素和外在因素的结合,但人为的外在因素对其生存的影响是致命性的.呼吁保护环境以拯救其它濒临灭绝的物种.     

Taxol production in suspension cultures of Taxus baccata

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25°C on a modified B5 medium with two-week transfers. Under these conditions, no taxol^® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1^–1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1^–1) and low (<0.1 mg 1^–1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1^–1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1^–1.®|Taxol is a registered trademark of Bristol Meyer Squibb for paclitaxel     

Paclitaxel production in suspension cell cultures of Taxus

Five separate cell lines, three of Taxus canadensis Marsh. and two of Taxus cuspidata Sieb. et Zucc., were used to test the effect of carbohydrates and plant growth regulators on the growth of cells and production of paclitaxel in culture. There was no significant correlation between growth of cells and paclitaxel production. While no single medium was developed that was optimal for all cell lines, it was possible to develop a medium for each species that represented a superior combination of growth and paclitaxel production. A combination of NAA and thidiazuron produced the best combination of growth and paclitaxel production in cell lines of T. canadensis, while IAA and BA produced the best results in cell lines of T. cuspidata. A mixture of sucrose and fructose gave the best combination of growth and paclitaxel production. The addition of carbohydrates midway through the growth cycle increased the rate at which paclitaxel accumulated in the culture medium. The highest paclitaxel concentration obtained was 14.78±0.86 mg 1^–1 (n=3).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2ip 6-(,-dimethylamino)-purine - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA -napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - thidiazuron 1-phenyl-3 (1,2,3-thiadiazol-5-yl)urea     

珊瑚菜不同部位香豆素含量的研究

应用高效液相色谱技术,对珊瑚菜（Glehnia littoralis Fr.Schmidt ex Miq.）的果实、叶和根以及采后去皮入药的根和根皮中的补骨脂素、欧前胡素和异欧前胡素3种香豆素的含量进行了测定。结果显示：（1）3种香豆素在果实、叶与根、根皮中均有积累,总含量在果实中最高,为0.6364mg·g-1,根中为0.0657mg·g-1,根皮中为0.0312mg·g-1,叶中为0.0151mg·g-1,采后去皮处理的根中最低,仅为0.0081mg·g-1。（2）珊瑚菜根经水烫去皮处理后香豆素含量急剧下降,与同期采收未去皮处理的根相比,去皮后根内香豆素总含量、补骨脂素、欧前胡素、异欧前胡素含量分别下降了87.7%、100%、82.76%、85.25%。（3）与未去皮的根相比,处理后的根皮中香豆素总量、补骨脂素、欧前胡素和异欧前胡素含量分别是未去皮处理的珊瑚菜根中的47.42%、31.37%、51.54%和53.28%。研究表明：从充分利用香豆素成分的角度出发,根入药时应带根皮使用,另外,叶和根皮不应丢弃,均可收集作为提取香豆素新的植物资源,果实中香豆素含量很高,亦可作为香豆素资源。     

Low-glutamine fed-batch cultures of 293-HEK serum-free suspension cells for adenovirus production

Recent developments in gene therapy using adenoviral (Ad) vectors have fueled renewed interest in the 293 human embryonic kidney cell line traditionally used to produce these vectors. Low-glutamine fed-batch cultures of serum-free, suspension cells in a 5-L bioreactor were conducted. Our aim was to tighten the control on glutamine metabolism and hence reduce ammonia and lactate accumulation. Online direct measurement of glutamine was effected via a continuous cell-exclusion system that allows for aseptic, cell-free sampling of the culture broth. A feedback control algorithm was used to maintain the glutamine concentration at a level as low as 0.1 mM with a concentrated glucose-free feed medium. This was tested in two media: a commercial formulation (SFM II) and a chemically defined DMEM/F12 formulation. The fed-batch and batch cultures were started at the same glucose concentration, and it was not controlled at any point in the fed-batch cultures. In all cases, fed-batch cultures with double the cell density and extended viable culture time compared to the batch cultures were achieved. An infection study on the high density fed-batch culture using adenovirus-green fluorescent protein (Ad-GFP) construct was also done to ascertain the production capacity of the culture. Virus titers from the infected fed-batch culture showed that there is an approximately 10-fold improvement over a batch infection culture. The results have shown that the control of glutamine at low levels in cultures is sufficient to yield significant improvements in both cell densities and viral production. The applicability of this fed-batch system to cultures in different media and also infected cultures suggests its potential for application to generic mammalian cell cultures.     

High-level production of arbutin from hydroquinone in suspension cultures of Catharanthus roseus plant cells

Summary Plant cell suspensions of Catharanthus roseus efficiently converted exogenously supplied hydroquinone (HQ) into arbutin. Arbutin productivity of the cells was strongly influenced by the growth stage of the cultivated cells and by the manner of the addition of HQ. We have developed two methods: (i) cultivating suitable cells for producing arbutin at high density; (ii) efficiently adding toxic HQ to the cells. The yield of arbutin could be increased up to 9.2 g/l (45% of cell dry weight), which is the highest yield in the field of plant biotechnology. Repeated examinations and scaling up to a 20-l jar fermentor suggested that C. roseus cells stably produce arbutin in large amounts under the established conditions. Offprint requests to: S. Inomata     

不同采收期珊瑚菜中香豆素含量的比较

应用高效液相色谱技术,分析了当年生珊瑚菜栽培后期4个采收时期的根、叶以及采后根去皮处理对香豆素含量的影响.结果显示:(1)珊瑚菜的根中,香豆素的含量和产量在10月15日均达到最高,分别为0.077 2 mg*g-1和1.087 8 mg/株,且显著高于其他采收期,其中,补骨脂素在栽培后期含量相差不大,欧前胡素和异欧前胡素均在10月15日含量最高.(2)珊瑚菜的叶中,不同采收期均有一定量的香豆素存在,其中在9月15日采收时,香豆素的含量和产量均极显著高于其他采收期,分别达到0.206 5 mg*g-1和1.317 5 mg/株,为同期根中香豆素含量和产量的4.4倍和2.0倍.(3)珊瑚菜根经水烫去皮处理后香豆素总量急剧下降到0.008 1 mg*g-1,与同期采收未去皮处理的根相比,去皮后根内香豆素总含量、补骨脂素、欧前胡素、异欧前胡素含量分别下降了87.7%、100%、82.76%、85.25%.(4)9月15日采收的珊瑚菜单株(根+叶)香豆素总产量(根+叶)极显著高于其他采收期,达1.980 2 mg/株,其次为10月15日1.157 5 mg/株.研究表明:珊瑚菜叶应作为提取香豆素的重要原材料,采收时应一起收获;对珊瑚菜根去皮处理会导致根的药用质量大幅度下降,生产中应明确禁止; 珊瑚菜的采收期应依据其用途决定,以根直接入药应选择10月15日采收,作为提取香豆素生产原料应选择9月15日采收.     

Verbascoside production in callus and suspension cultures of Hygrophila erecta

Cell suspension cultures of Hygrophila erecta obtained from hypocotyl explants accumulate up to 2.6% of their dry wt as only one caffeic glycoside ester, verbascoside. On the contrary, different callus lines initiated from stem explants contain not more than 1 % of verbascoside and a few of them produced other caffeoyl glycosidic esters.     

Elicitor-enhanced syringin production in suspension cultures of Saussurea medusa

Syringin production and related secondary metabolism enzyme activities in suspension cultures of Saussurea medusa treated with different elicitors (yeast extract, chitosan and Ag⁺) were investigated. All elicitors enhanced syringin production, and the optimal feeding protocol was the combined addition of 1.5% (v/v) yeast extract, 0.2 g l⁻¹ chitosan and 75 μM Ag⁺ at the 15th day of the cell culture. The highest syringin production reached 741.9 mg l⁻¹, which was 3.6−fold that of the control. The glucose−6-phosphate dehydrogenase (EC 1.1.1.49), phenylalanine ammonia lyase (EC 4.3.1.5) and peroxidase (EC 1.11.1.7) activities increased significantly after elicitor treatment. The maximum enzyme activities were obtained when the treatment time was 6 h.