The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL 3,4-dihydroxyphenyllactate - DHPP 3,4-dihydroxyphenylpyruvate - pHPL 4-hydroxyphenyllactate - pHPP 4-hydroxyphenylpyruvate - RA rosmarinic acid     

Correlation of betacyanin synthesis with cell division in cell suspension cultures of Phytolacca americana

In suspension cultures of Phytolacca americana , betacyanin accumulation was reduced when cell division was inhibited by treatment with various inhibitors of DNA synthesis or anti-microtubule drugs. Aphidicolin (APC), an inhibitor of DNA synthesis, reduced the incorporation of radioactivity from labeled tyrosine into betacyanin, but the incorporation of radioactivity from labeled 3,4-dihydroxyphenylalanine (DOPA) into betacyanin was not affected by similar treatments. Propyzamide, another anti-microtubule drug, reduced incorporation of radioactivity from tyrosine and DOPA into betacyanin. However, the rate of incorporation from DOPA was higher than that from tyrosine. The results suggest that inhibition of betacyanin accumulation in Phytolacca americana cells by APC and propyzamide is due to suppression of the reaction converting tyrosine to DOPA, which may be closely related to cell division.     

从水稻根部悬浮培养细胞分离原生质体及植株再生

以长香稻(Oryza sativa L．)(糯稻)的幼嫩种子根为材料,在Ms和N6培养基上诱导产生结构松软而分散的愈伤组织,经过AA液体培养基振荡悬浮培养,悬浮培养细胞经酶解后得到了活性较高的原生质体,试验结果表明这是分离原生质体较理想的材料。在附加2,4-D lmg／L(以下单位同)、KT(激动素)0．2、各氨酰胺876、天门冬酰胺266的Ms琼脂糖培养基中,诱导出了大量愈伤组织,在含KT2、NAA(α-萘乙酸)0．2、zT(玉米素)0．2、cH(水解酪蛋白)1000和4％椰子汁的N6培养基中成功地诱导出了由原生质体再生的植株。当代原生质体再生植株能正常开花、结实、产生种子;染色体数均为二倍性(2n＝24),最显著的特点是结实率低,穗粒数减步、生育期延迟。     

人参寡糖素对三七悬浮培养细胞生长的效应

从人参培养细胞的细胞壁中分离纯化到不同分子量的单体人参寡糖素。试验结果表明命名为人参寡糖素Ⅶ和人参寡糖素Ⅷ的两种寡糖素对三七悬浮培养细胞的生长具有明显的促进作用,其增长率分别为１９．３４％和１０．５８％,人参寡糖素Ⅶ的适宜浓度为５—１０ｍｇ／ｌ。在高浓度下（大于２５ｍｇ／ｌ）稍抑制培养细胞生长。在细胞培养２２天（指数生长期）后．加入１０ｍｇ／ｌ的人参寡糖素Ⅶ．然后再培养２天。其生长速率即提高,加入人参寡糖素Ⅷ后．缩短了三七细胞悬浮培养生长的延缓期．提前进入对数生长期和指数生长期,并在对数生长期和指数生长期作用最明显,因而最终收获时培养细胞的产率增加。     

HPLC and NMR methods for the quantitation of the (R)-enantiomer in (−)-(S)-timolol maleate drug raw materials

HPLC and ¹H-NMR methods for the quantitation of the (R)-enantiomer in (?)-(S)-timolol maleate were developed and validated. The HPLC method requires a 25 cm × 4.6 mm 5 μm Chiracel OD-H (cellulose tris-3,5-dimethylphenylcarbamate) column, a mobile phase of 0.2% (v/v) diethylamine and 4% (v/v) isopropanol in hexane at a flow rate of 1 ml/min and UV detection at 297 nm. A system suitability test was devised to verify the separation of the (R)- and (S)-enantiomers of timolol from other drug-related impurities. The NMR method requires the use of a high-field NMR spectrometer (>360 MHz) and a chiral solvating agent, (?)-(R)-2,2,2-trifluoro-1-(9-anthrylethanol) (R-TFAE). The limits of quantitation were 0.05% and 0.2% (m/m) for HPLC and NMR, respectively. The methods were applied to the determination of the (R)-enantiomer in eight lots of raw material. The results for the two methods were in very good agreement, with results ranging from 0.1 to 4.1% (m/m) by HPLC and none detected to 4.3% (m/m) by NMR. The USP method for specific rotation was found to be unsuitable for detecting the presence of low levels of the (R)-enantiomer in (?)-(S)-timolol maleate. © 1994 Wiley-Liss, Inc.     

Chiral HPLC with carbohydrate carbamates: Influence of support structure on enantioselectivity

The effect of particle size and pore size of the aminopropylated silica support for cellulose tris(phenylcarbamate) and tris(3,5-dimethylphenylcarbamate) chiral HPLC phases was investigated. It was necessary to reduce phase loading below 20% w/w as pore size and particle size were reduced, but high efficiency columns could be prepared at a 15% w/w loading on 5 and 2.5 μm supports with 120-Å-diameter pores. The 2.5 μm phase permits the use of relatively high flow rates and very efficient enantioselective separations of a range of chiral compounds could be achieved in less than 3 min. © 1994 Wiley-Liss, Inc.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

On the inducibility of nitrate transport by tobacco cells

The question as to whether the nitrate transport system is induced by nitrate was addressed using a cell suspension of the XD line of Nicotiana tabacum L. cv. Xanthi as an experimental system. The cells were grown on area as the sole nitrogen source, and tungstate was used to render nitrate reductase non-functional. To avoid shock due to vacuum filtration, the cells, were harvested by gravity filtration. Nitrate uptake by cells, which were harvested, transferred to fresh medium, and immediately exposed to nitrate (freshly harvested cells), displayed a lag period of about 3 h.
In cells which were given incubation periods in fresh medium before exposure to nitrate (preincubated cells), the lag period was considerably shortened. After 3 h of preincubation in the absence of nitrate (recovered cells), the lag period was almost completely eliminated. Cycloheximide inhibited nitrate uptake by recovered cells within minutes, and prevented the development of nitrate uptake in freshly harvested cells. Cycloheximide did not affect uptake of α-aminoisobutyric acid (AIB) within the first 2 h after its addition. Recovery of the membrane potential from a low value just after the harvest of the cells to a maximal value 3 h later, was observed using the lipophilic cation methyltriphenylphosphonium (MTPP⁺), supplied at low concentrations, as a probe. Depolarization of the membrane potential by MTPP⁺, at the millimolar range, caused a rapid inhibition of nitrate uptake by recovered cells. The results indicate that nitrate transport by the XD cells depends on the membrane potential and on protein components with short half life. In addition, it requires a continuous protein synthesis. The effects of physical manipulation on nitrate uptake are discussed.     

Regulation of nitrate uptake by amino acids in maize cell suspension culture and intact roots

The effect of amino acids on nitrate transport was studied in Zea mays cell suspension cultures and in Zea mays excised roots. The inclusion of aspartic acid, arginine, glutamine and glycine (15mM total amino acids) in a complete cell-culture media containing 1.0 mM NO₃ ^- strongly inhibited nitrate uptake and the induction of accelerated uptake rates. The nitrate uptake rate increased sharply once solution amino acid levels fell below detection limits. Glutamine alone inhibited induction in the cell suspension culture. Maize seedlings germinated and grown for 7 days in a 15 mM mixture of amino acids also had lower nitrate uptake rates than seedlings grown in 0.5 mM Ca(NO₃)₂ or 1 mM CaCl₂. As amino acids are the end product of nitrate assimilation, the results suggest an end-product feed-back mechanism for the regulation of nitrate uptake.