Effect of chitosan elicitation and media components on the production of anthraquinone colorants in madder (Rubia akane Nakai) cell culture

To increase the production of anthraquinone colorants in madder (Rubia akane Nakai) cell culture, the effects of elicitation on the colorant production were investigated. Chitosan was the best biotic elicitor among nine plant derived and microbial derived polysaccharides. When elicited with 25 mg/L chitosan, the total production was increased approximately two times in a seven-day culture as compared to that in the unelicited cells. Anthraquinone production was increased in proportion to the contact period up to day 3. Maximum anthraquinone colorants were obtained with 3-day treatment of chitosan. During chitosan elicitation, the total production was increased 1.3 times in MS medium containing galactose as compared to that containing sucrose. The degree of deacetylation in chitosan and the use of growth regulator or addition of precursor did not affect the production of anthraquinone colorants. When madder cells were elicited at optimum condition, anthraquinone concentration and specific anthraquinone content increased 1.3 times (0.69 g/L) and 2.2 times (0.32 g/g DCW), respectively.     

Permeabilization of the plasmalemma and wall of soybean root cells to macromolecules

A technique has been developed that results in the reversible permeabilization of the cell wall and plasmalemma of soybean (Glycine max (L.) Merr.) root cells grown in suspension and callus culture. Cells in culture are treated with saponin (0.1 mg/ml) for 15 min at room temperature. They are then coincubated in separate experiments with fluorescent-derivatized dextrans (20–70 kDa) or fluorescein-conjugated goat anti-rabbit immunoglobulin G to ascertain the exclusion size of macromolecules capable of diffusing across the cell wall and plasmalemma into the cytoplasm. Following an incubation period of 30 min, it was observed by conventional and confocal fluorescence microscopy that all derivatized macromolecules tested (20–140 kDa) could be incorporated into the cytoplasm, but not into the vacuole. This procedure did not appear to affect cell viability adversely. A normal doubling time was observed for these cells following the permeabilization procedure.Abbreviations FDA fluorescein diacetate - FITC-20 kDa, FITC-40 kDa, FITC-70 kDa dextrans fluorescein-derivatized 20-kDa, 40-kDa, and 70-kDa dextrans - IgG immunoglobulin G - kDa kilodalton Paramjit K. Gharyal wishes to thank the Nitrogen Availability Program at Michigan State University for financial support. We also thank Edwin de Feijter of Meridian Instruments for technical assistance in performing the confocal measurements. This work was supported by a grant from the U.S. — Israel Binational Agricultural Research and Development Fund (BARD project No. US-1384-87).     

Characteristics of aroma-active compounds in the pectin-elicited suspension culture of Zanthoxylum piperitum (prickly ash)

Zanthoxylum piperitum (prickly ash) was grown as a suspension culture in Schenk and Hildebrandt medium supplemented with 50 g sucrose l^–1 and 0.5 mg 2,4-dichlorophenoxyacetic acid l^–1 for 21 days with elicitation by pectin added at day 15. Volatile compounds were extracted from the culture and 3-hydroxy-2-butanone was identified by GC-MS as the most abundant compound, followed by -butyrolactone and 2,3-butanedione. 2,3-Butanedione, ethyl 3-methylbutyrate, and ethyl 2-methylpropanoate were identified as the most intense aroma-active compounds and represented the characteristic aroma of the culture.     

Detoxification of N-(phosphonoacetyl)-L-aspartate by carrot cells in suspension culture

Phototropic reversal of Phycomyces sporangiophores can be elicited by a change to darkness during steady-state phototropism. The reversal lasts 25–30 min under these conditions. Control experiments show that the reversal is not caused by gravitropism. Tropic reversal is also elicited by the removal of a barrier during an avoidance response, showing that the reversal occurs at the output of the sensory transduction chain.     

CHO工程细胞 (11G-S) 悬浮培养的无血清培养基的设计

以悬浮适应的表达重组尿激酶原 (Pro-urokinase,pro-UK) CHO工程细胞系11G-S为对象,采用Plackett-Burman实验设计及响应面分析法,设计支持CHO工程细胞 (11G-S) 悬浮生长的无血清培养基。以细胞密度为评价指标,在单因素实验的基础上采用Plackett-Burman实验设计对影响细胞生长的培养基添加成分进行考察,确定了3种对细胞生长明显促进作用的培养基添加成分：胰岛素、转铁蛋白及腐胺。继而利用响应面法分析了这3种添加成分的最佳水平范围,设计了一种适用于CHO工程细胞 (11G-S) 悬浮培养的无血清培养基SFM-CHO-S。11G-S细胞在SFM-CHO-S批次悬浮培养的细胞最大生长密度达到4.12×106 cells/mL,pro-UK的最大累积活性达到5 614 IU/mL,培养效果优于商品化的同类无血清培养基。     

皱皮木瓜开花结果习性观察初报

对长阳所产皱皮木瓜的开花结果习性作了形态上的描述。先开花后展叶,花序为聚伞花序,花果枝为特化的短缩叶丛枝,梨果发育分两个时期:幼果发育期和果实生长期。     

The effect of cadmium on cell cycle control in suspension culture cells of soybean

Heavy metals inhibit plant growth. This proces may be directly or indirectly connected with mechanisms regulating cell division. We analyzed the effect of Cd²⁺ on cell cycle progression in partially synchronized soybean (Glycine max) cell suspension culture and followed the expression of cell cycle genes (cyclin B1 and cyclin-dependent kinase A - CDK-A). We have checked the hypothesis that Cd²⁺-induced impairment of cell division is connected with DNA damage. The [³H]-thymidine incorporation in cell cultures synchronized either with hydroxyurea (HU) or phosphate starvation have shown, that Cd²⁺ strongly affects the S phase of soybean cell cycle, by causing the earlier entry of cells into S phase and by decreasing the rate of DNA synthesis. RT-PCR analysis indicated that Cd²⁺ decreases the level of cyclin B1 mRNA and has no effect on CDK-A mRNA. The result of comet assay indicated the damaging effect of Cd²⁺ on DNA of soybean cells. We suggest that Cd²⁺ affects plant cell cycle at two major checkpoints: the G1/S — by damaging of DNA, and G2/M - by decreasing the level of cyclin B1 mRNA     

Fine structure of chemosensitive cells (glomus caroticum) in tissue culture

Summary Cells of the carotid body of both embryonic and 1 to 2-d-old rabbits were cultured in monolayer in primary tissue culture. The cells grew in their original association of type I and type II cells. The fine structure of both cell types was similar to that in vivo. Even after one week in culture their morphological characteristics, such as the dense-cored vesicles, were preserved. The prolonged synthesis of catecholamines in culture and the formation of new granular vesicles is discussed.The results were reported in part at 17. Tagung für Elektronenmikroskopie held on September 21–26, 1975 in BerlinThe authors want to thank Prof. Dr. D.W. Lubbers for his advice and helpful suggestions     

l-Phenylalanine ammonia-lyase in suspension culture cells of spruce (Picea abies)

The activity of l-phenylalanine ammonia-lyase (PAL) (EC 4.3.1.5) was determined in seedlings, callus cells, cell suspension cultures and in young needles of spruce (Picea abies) (L.) (Karst). PAL activity increased up to 10 fold in response to transferring suspension cultured cells into new cultivation medium. PAL was also induced about 10 fold when callus cells were transferrd into liquid medium. The increase was transient and it required the presence of a carbohydrate.In cell suspension cultures, grown in the dark (white cells), but not in light-grown cultures (green cells), PAL activity was induced up to 30 fold by UV-light.With a cell wall preparation of Rhizosphaera kalkhoffii, a forest pathogenic fungus, used as elicitor, the activity of PAL could be induced more than 10 fold. The degree of induction depended on the elicitor concentration. Induction was prevented by cycloheximide but not by actinomycin D.     

Partitioning of recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) from plant cell suspension culture in PEG/sodium phosphate aqueous two-phase systems

Partitioning of human granulocyte-macrophage colony stimulating factor (hGM-CSF) was achieved in the aqueous two-phase systems (ATPSs) using a crude extract of transgenic tobacco cell suspension culture. This study examined the effects of polyethylene glycol (PEG) molecular weight and concentration and the effects of sodium phosphate concentration in different PEG/sodium phosphate systems on the partition coefficient,K. The best ATPS system was 5% PEG 8,000/1.6 M sodium phosphate after 2 h of incubation at room temperature. In this system, hGM-CSF was partitioned in the PEG-rich phase with a yield of 57.99% andK _hGM-CSF of 8.12. In another system, 3% PEG 10,000/1.6 M sodium phosphate, hGM-CSF was also partitioned primarily in the top phase with a yield of 45.66% andK _hGM-CSF of 7.64 after 2 h of incubation at room temperature.     

利用微载体悬浮培养人脐带间充质干细胞

随着干细胞研究的不断深入,干细胞功能分化研究和临床应用转化的需求日益提升。人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUCMSCs)来源广泛,不仅自我更新能力强、能够分化成多种类型的成体细胞,而且其自身具有免疫调节能力,不易引发免疫排斥反应,在干细胞功能分化研究和临床应用中具有巨大应用前景和应用潜力。目前,传统的细胞培养方式培养效率低、细胞活性较差,不能满足日益增长的研究和应用需求。本研究利用微载体结合旋转瓶的悬浮培养方法,通过优化细胞接种量及转速等影响因素,快速获得大量高质量的人脐带间充质干细胞。经悬浮培养总细胞量可高达到7×10 ⁸个细胞/L,而且细胞活性较高,MSC 特异性标记物表达良好,在恢复平面培养后仍能维持MSC的正常细胞形态和增殖能力。高效脐带间充质干细胞悬浮培养体系的初步建立,为未来的干细胞功能分化研究和临床应用奠定了基础。     

Solute distribution between vacuole and cytosol of sugarcane suspension cells: Sucrose is not accumulated in the vacuole

The compartmentation of solutes in suspension cells of Saccharum sp. during different growth phases in batch culture was determined using CuCl₂ to permeabilize the plasma membrane of the cells. The efflux of cytosolic and vacuolar pools of sugars, cations and phosphate was monitored, and the efflux data for phosphate were compared and corrected using data from compartmentation analysis of phosphate as determined by ³¹P-nuclear magnetic resonance spectroscopy. The results show that sucrose is not accumulated in the vacuoles at any phase of the growth cycle. On the other hand, glucose and fructose are usually accumulated in the vacuole, except at the end of the cell-culture cycle when equal distribution of glucose and fructose between the cytosol and the vacuole is found. Both Na⁺ and Mg²⁺ are preferentially located in the vacuoles, but follow the same tendency as glucose and fructose with almost complete location in the vacuole in the early culture phases and increasing cytosolic concentration with increasing age of the cell culture. Potassium ions are always clearly accumulated in the cytosol at a concentration of about 80 mM; only about 20% of the cellular K⁺ is located inside the vacuole. Cytosolic phosphate is little changed during the cell cycle, whereas the vacuolar phosphate pool changes according to total cellular phosphate. In general there are two different modes of solute compartmentation in sugarcane cells. Some solutes, fructose, glucose, Mg²⁺ and Na⁺, show high vacuolar compartmentation when the total cellular content of the respective solute is low, whereas in the case of ample supply the cytosolic pools increase. For other solutes, phosphate and K⁺, the cytosolic concentration tends to be kept constant, and only excess solute is stored in the vacuole and remobilized under starvation conditions. The behaviour of sucrose is somewhat intermediate and it appears to equilibrate easily between cytosol and vacuole.Abbreviation NMR nuclear magnetic resonance The very cooperative help by Dr. J. Reiner with the ³¹P-NMR measurements and the technical assistance by D. Keis are gratefully acknowledged. This research was supported by the Deutsche Forschungsgemeinschaft and by Fonds der Chemischen Industrie.     

Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media

The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.     

Primary culture of normal pituitary cells of carp (cyprinus carpio) for the study of gonadotropin release

Summary A method for preparing enzymaticlaly dispersed pituitary cell cultures of carp (Cyprinus carpio) is described. The cultures have been used to assay a synthetic analog of gonadotropin releasing hormone (GnRH) and to determine the specificity of steroids able to affect gonadotropin (GtH) release in vitro. Time course secretion studies indicated that by 48 h incubation, in the presence of 500 pM GnRH, cumulative secretion of gonadotropin (719 ng±90/2.5×10⁵ cells) had exceeded that of controls (446 ng±106/2.5×10⁵ cells). Estradiol-17β, progesterone, testosterone, and 11-ketotestosterone showed different inhibitory effects on pituitary basal GtH release. Based on the results, it was concluded that carp pituitary cell cultures can be applied to investigations of several aspects of the hypothalamo-hypophysial-gonadal system. This investigation was supported by the Deutsche Forschungsgemeinschaft, Bonn, FRG.     

Purification of extensin from cell walls of tomato (hybrid of Lycopersicon esculentum and L. peruvianum) cells in suspension culture

Extensin, a hydroxyproline-rich glycoprotein comprising substantial amounts of -l-arabinose-hydroxyproline glycosidic linkages is believed to be insolubilized in the cell wall during host-pathogen interaction by a peroxidase/hydroperoxide-mediated cross-linking process. Both extensin precursor and extensin peroxidase were ionically eluted from intact water-washed tomato (hybrid) of Lycopersicon esculentum Mill. and L. peruvianum L. (Mill.) cells in suspension cultures and purified to homogeneity by a rapid and simple procedure under mild and non-destructive experimental conditions. The molecular weight of native extensin precursor was estimated to be greater than 240–300 kDa by Superose-12 gel-filtration chromatography. Extensin monomers have previously been designated a molecular weight of approximately 80 kDa. Our results indicate that salt-eluted extensin precursor is not monomeric. Agarose-gel electrophoresis, Superose-12-gel-filtration, extensin-peroxidase-catalysed cross-linking, Mono-S ion-exchange fast protein liquid chromatography (FPLC), and peptide-sequencing data confirmed the homogeneity of the extensin preparation. Evidence that the purified protein was extensin is attributed to the presence of the putative sequence motif — Ser (Hyp)₄ — within the N-terminal end of the protein. Treatment of extensin with trifluoroacetic acid demonstrated that arabinose was the principal carbohydrate. The amino-acid composition of the purified extensin was similar to those reported in the literature. The cross-linking of extensin in vitro upon incubation with extensin peroxidase and exogenous H₂O₂ was characteristic of other reported extensins. Furthermore, Mono-S ion-exchange FPLC of native extensin precursor resolved it into two isoforms, A (90%) and B (10%). The amino-acid compositions of extensin A and extensin B were found to be similar to each other and both extensins were cross-linked in vitro by extensin peroxidase.Abbreviations CM-cellulose carboxymethyl-cellulose - FPLC fast protein liquid chromatography - HF hydrogen fluoride - HRGP hydroxyproline-rich glycoprotein - Hyp hydroxyproline - V_c retention volume - TCA trichloroacetic acid - TFA tri-fluoroacetic acid This work was supported by a A.F.R.C. postdoctoral assistantship to Michael D. Brownleader. We thank Dr. Anthony K. Allen (Department of Biochemistry, Charing Cross and Westminster Hospital, London, UK) for performing the amino-acid analysis and Mrs. Margaret Pickering (Department of Biochemistry, Royal Holloway) for performing the peptide-sequence analysis of extensin. We also express our gratitide to Dr. A. Mort (Oklahoma State University) for performing the HF-deglycosylation of extensin.     

聚N-异丙基丙烯酰胺细胞培养平台的研究进展

聚N-异丙基丙烯酰胺(poly(N-isopropylacrylamide),PNIPAAm),温敏性聚合物,可利用其温敏特性替代酶类物质或细胞刮刀用于贴壁细胞的收获,从而有效避免酶解和机械损伤,可为生物医药领域提供品质优良的种子细胞。重点阐述促进细胞有效粘附和快速脱附的温敏性PNIPAAm二维平面的研发情况,包括选取特殊基材、引入亲水基团、调节反应物比率、控制聚合物厚度/密度、提供适宜外力等方式,从而有效改善细胞对温敏性平面的适应性并降低染菌风险以及减少低温处理对细胞的影响。同时介绍PNIPAAm微载体、支架和凝胶等温敏性三维培养介质的研究进展,此方式不仅增加细胞生长面积,更可以模拟体内微环境,从而保持细胞原始生理特征,同时实现大规模扩增和非酶解收获细胞以及组织器官修复和重构的目标。最后简单说明PNIPAAm培养平台的应用,PNIPAAm的研发为再生医学的发展提供了崭新思路。     

Incorporation of [214C]malonyl CoA into fatty acids by a cell-free extract of Catharanthus roseus suspension culture cells

A cell-free extract containing the enzymes for de-novo synthesis, elongation and desaturation of fatty acids was prepared from cultured cells of Catharanthus roseus G. Don. ¹⁴C-Fatty acids synthesized by the extract from [2-¹⁴C]malonyl CoA substrate were palmitic (16:0), stearic (18:0) and oleic (18:1). Dialyzed extract was active and stable at room temperature and at 4° C, but was inactivated on boiling. There was an absolute requirement for NADPH for incorporation of [2-¹⁴C]malonyl CoA into total fatty acids. Escherichia coli acyl carrier protein stimulated total fatty-acid synthesis without affecting the relative ratio of individual fatty acids. Total fatty-acid synthesis at a rate of 45 nmol·mg^-1 protein·h^-1 occurred at a substrate level of 73 M malonyl CoA, cofactor levels of 500 M NADPH, 30 g·ml^-1 E. coli ACP, and 1.0 mg·ml^-1 extract protein. Total fatty acid synthesis was also sensitive to cerulenin and CoA levels. Variations in the relative abundance of individual ¹⁴C-fatty acids were regulated by concentrations of [¹⁴C]malonyl CoA. NADPH and ferredoxin, as well as by pH, temperature and length of incubation. Fatty-acid synthetase enzymes responsible for [¹⁴C]palmitic acid were rapidly saturated at a low substrate level (0.3 M malonyl CoA). Increasing the level of [2-¹⁴C]malonyl CoA permitted further synthesis of [¹⁴C]stearate and [¹⁴C]oleate. Desaturation of [¹⁴C]stearate to [¹⁴C]oleate was stimulated by increasing the levels of NADPH and ferredoxin. The desaturase and elongase enzymes were sensitive to acidic pH. The desaturase was also unstable at 41° C, although fatty acid synthetase and elongase were unaffected by this temperature.Abbreviation ACP Acyl carrier protein     

Conditioning of the culture medium by suspension cells and formation of somatic proembryo in Araucaria angustifolia (coniferae)

Summary Cell masses of Araucaria angustifolia cultured in LP liquid medium showed different uptake and utilization patterns for different sugars. Fructose was slowly utilized by cell growth during the lag phase. After this lag period, fructose concentration decreased quickly, corresponding to the start of linear growth. Glucose rapidly decreased following fructose depletion. The level of extracellular proteins in the culture medium increased for the first 5 d after cell inoculation, during the transition from the lag phase to the high-growth phase. The pH of the medium decreased through the first half of the culture period (4.94) and increased (5.6) thereafter. Proembryos originated from a single cell, without a cleavage process. Suspensor cells and proembryonal groups developed independently. Only embryogenic cells divided and formed a suspensor or a proembryo group, which differentiated further along the longitudinal axis. The pathway observed for proembryo formation in Araucaria differs from the model proposed for other conifers, which show cleavage polyembryony.     

Effect of culture pH on erythropoietin production by Chinese hamster ovary cells grown in suspension at 32.5 and 37.0 degrees C

To investigate the effect of culture pH in the range of 6.85-7.80 on cell growth and erythropoietin (EPO) production at 32.5 and 37.0 degrees C, serum-free suspension cultures of recombinant CHO cells (rCHO) were performed in a bioreactor with pH control. Lowering culture temperature from 37.0 to 32.5 degrees C suppressed cell growth, but cell viability remained high for a longer culture period. Regardless of culture temperature, the highest specific growth rate (mu) and maximum viable cell concentration were obtained at pH values of 7.00 and 7.20, respectively. Like mu, the specific consumption rates of glucose and glutamine decreased at 32.5 degrees C compared to 37.0 degrees C. In addition, they increased with increasing culture pH. Culture pH at 32.5 degrees C affected specific EPO productivity (q(EPO)) in a different fashion from that at 37 degrees C. At 37 degrees C, the q(EPO) was fairly constant in the pH range of 6.85-7.80, while at 32.5 degrees C, the q(EPO) was significantly influenced by culture pH. The highest q(EPO) was obtained at pH 7.00 and 32.5 degrees C, and its value was approximately 1.5-fold higher than that at pH 7.00 and 37.0 degrees C. The proportion of acidic EPO isoforms, which is a critical factor for high in vivo biological activity of EPO, was highest in the stationary phase of growth, regardless of culture temperature and pH. Although cell viability rapidly decreased in death phase at both 32.5 and 37.0 degrees C, the significant degradation of produced EPO, probably by the action of proteases released from lysed cells, was observed only at 37.0 degrees C. Taken together, through the optimization of culture temperature and pH, a 3-fold increase in maximum EPO concentration and a 1.4-fold increase in volumetric productivity were obtained at pH 7.00 and 32.5 degrees C when compared with those at 37.0 degrees C. These results demonstrate the importance of optimization of culture temperature and pH for enhancing EPO production in serum-free, suspension culture of rCHO cells.