Biotransformation of Gastrodin by Cell Suspension Cultures of Catharanthus roseus

应用长春花 (Catharanthusroseus (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8d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。

Isolation of Protoplast and Ion Channel Recording in Plasma Membrane of Suspension Cells of Populus euphratica

The authors used suspension cells of Populus euphratica to isolate protoplast in the present study. Protoplasts were successfully obtained after 4 hours incubation in enzyme solution containing 1 0% cellulase “onozuka” R\|10, 0\^01% pectolyase Y\|23,0\^15% macerozyme R\|10 and 0\^1% hemicellulase at 25℃. Outward and inward single channels in plasma membrane were observed using cell\|attached recording of patch\|clamp technique. In this study, single channel records showed that more than one species of channel were obtained. These attempts in protoplast isolation and ion channel recording offers the opportunity to characterize cellular mechanisms of salt tolerance in tree species.

Productions of Taxol and Related Taxanes by Cell Suspension Cultures of Taxus yunnanensis

A high taxol yield cell line of Taxus yunnanensis Cheng et L. K. Fu keeps a high taxol_producing level after successive subcultures for more than eight years. In this study, eight taxanes were isolated from the suspension cell cultures of this cell line. Based on NMR and MS analyses, and comparison with literature data and standards, their structures were determined to be 2α,5α,10β_triacetoxy_14β_propionyloxy_4(20),11_taxadiene (1), 2α,5α,10β_triacetoxy_14β_(2′_methyl)_butyryloxy_4(20),11_taxadiene (2), 2α,5α,10β_14β_tetra_acetoxy_4 (20),11_taxadiene (3, taxuyunnanine C), 2α,5α,10β_triacetoxy_14β_(2′_methyl_3′_hydroxy)_butyryloxy_4(20),11_taxadiene (4, yunnanxane) and its 3′_epimer (5), baccatin Ⅳ (6), baccatin Ⅲ (7) and taxol (8), respectively. Among those compounds, 3, 5, 6 and 7 were reported to be isolated from the suspension cell cultures of T. yunnanensis for the first time. TLC and HPLC analyses indicated that the chemical constituents of the culture solution were similar to those of cultured cells. Moreover, the highest taxol content of this cell line reached 0.3% and the cell line could be applied for a large_scale culture.

Chemical Constituents of the Suspension Cell Cultures of Maytenus hookeri

Suspension cell cultures of Maytenus hookeri Loos. (Celastraceae) in SH media were established from the calli induced from the leaves and young steins of M. hookeri on MS media with the supplement of 2 mg/L 2,4-D and 0.1 mg/L KIN (kinetin). Ethyl acetate extract of the cultures showed inhibitory activities against Penicillium avellaneum UC-4376 which was sensitive to maytansinoids. Exhaustive isolation of natural products from a large scale of suspension cell cultures did not yield maytansine instead of affording nine compounds including one novel triterpenoid, named 2, 3-diacetoxyl maytenusone (1), and eight known ones including squalene (2), beta-sitosterol (3), 2', 3', 4-triacetyl-sitoindoside I (4), salaspermic acid (5), maytenonic acid (6), 2alpha-hydroxy-maytenonic acid (7), 6, 11,12-trihydroxy-8, 11, 13-abietrien-7-one (8) and 11, 12-dihydroxy-8, 11, 13-abietatrien-7-one (9) elucidated on the basis of 1D and 2D NMR data. The H-1-NMR and C-13-NMR assignments were made for 1, 5, 6 and 7, while the C-13-NMR assignments for 5 and 6 were revised. The chemical results suggested that the suspension cell cultures of M. hookeri did not produce maytansinoids under the reported experiment conditions.

Effects of Salt Stress on Epidermal Cell Expansion in Leaves of Arabidopsis thaliana

[Objective] The aim of this study was to investigate the effects of salt stress on cell expansion in Arabidopsis thaliana rosette leaves.[Method] Arabidopsis seedlings were treated by sodium chloride at the concentration of 0,100 or 150 mmol/L. At the 7th and 14th d of treatment,with nail enamel printing mark method and computer software,the leaf blades area and abaxial epidermal pavement cells area was measured and compared using statistical analysis in Excel. [Result] The growth of Arabidopsis rosette leaves was inhibited under salt stress. Leaves treated for 7 or 14 d expanded less compared with controls. The salt-mediated decrease in leaf expansion is associated with a decrease in abaxial pavement cell expansion. [Conclusion] The decreased leaf and epidermal cell expansion under salt stress is the most important characteristic of plant physiological response to salt stress.

Study on the Browning in Cell Suspension Culture of Taxus cuspidata

[Objective] This study aimed to investigate the browning of T. cuspidata cells in suspension culture and provide the guidance for the cell suspension culture of T. cuspidata. [Method] T. cuspidata callus was used as experimental materials, to explore the effect of different medium, N/P ratio, pH, shaking speed, illumination time and light intensity and other factors on browning of T. cuspidata cells in suspension culture. [Result] Non-browning callus was transferred to 2MB5 medium （pH 7.0） for illumination culture at 22℃ under light intensity of 1 500 lx with shaking speed of 90 r/min for 24 h. Results showed that the cell browning was significantly inhibited. [Conclusion] This study laid the foundation for cell suspension culture of T. cuspidata and had important significance to the large-scale industrial production of paclitaxel.

Studies on Parameters Affecting Embryogenesis of Protoplasts Isolated from Suspension of Embryogenic Cells in Loblolly Pine

Protoplasts of embryogenic suspension cells of loblolly pine (Pinus taeda L).were isolated at exponential growth stage.Influences of various concentrations of basal medium,levels of BA,and concentrations of inositol on the differentiation of embryonal suspensor mass (ESM),early stage somatic embryos (ESE) ,and lae stage somatic embryos (LSE) were investigated .A study of the effect of various concentrations of LP basal medium sowed that the optimal basal medium concentration of ESM,ESE,and LSE differentiation was 1.25 LP medium.The effects of various levels of BA and inositol showed that the optimal concentrations of BA for the formation of ESM,ESE and LSE were 4 mg/L ,2mg/L and 1mg/L,respectively ,and the optimal concentrations of inositol for the ESM ,ESE and LSM formation were 400mg/L,800mg/L and 1,200mg/L,respectively.

Analytical model for power dissipation in cell membranes in suspensions exposed to electric fields

Due to interaction among cells, it is too complex to build an exactanalytical model for the power dissipation within the cell membrane in suspensions exposed toexternal fields. An approximate equivalence method is proposed to resolve this problem. Based on theeffective medium theory, the transmembrane voltage on cells in suspensions was investigated by theequivalence principle. Then the electric field in the cell membrane was determined. Finally,analytical solutions for the power dissipation within the cell membrane in suspensions exposed toexternal fields were derived according to the Joule principle. The equations show that theconductive power dissipation is predominant within the cell membrane in suspensions exposed todirect current or lower frequencies, and dielectric power dissipation prevails at high frequenciesexceeding the relaxation frequency of the exposed membrane.

MORPHOLOGICAL MODEL FOR RHEOLOGICAL PROPERTIES OF PLANT CELL SUSPENSION CULTURE SYSTEM

This paper puts forward a physical and mathematical model for the rheological properties of a plant cell suspension culture system.The model can explain why the system is pseudoplastic satisfactorily,thus the rheological properties of the system as the effect of the flow behavior index on plant cell concentration are interpreted correctly and the mechanism of the rheological properties of the system is further understood.Therefore the model can be applied in the technological design and optimum conditions of the system and the reformation,evaluation and scale up of reactors.

Selective 3-OH Isomerization of Resibufogenin by Cell Suspension Cultures of Ginkgo biloba

Aim To modify the structure of resibufogenin by using Ginkgo bilobasuspension. Methods Young leaves of Ginkgo biloba were differentiated into callus in MS medium withonly 2,4-D as plant growth regulator. The callus was then transferred aseptically to liquid MSmedium exoge-nously supplemented with appropriate concentration of 6-BA, NAA and 2,4-D to establishsuspension cell culture system. Resibufogenin was administered into the well-grown cell cultures andincubated for 4 d. The products dissolved in the liquid phase of the cultures were extracted andpurified by silica gel column chromatography gradiently eluted with petroleum ether and acetonesystem. Results One transformed product was obtained in 40% yield after 4 d incubation, which wasidentified as 3-epi-resibufogenin on the basis of FAB MS, ~1H NMR and ^(13)C NMR spectroscopicanalysis and corresponding data reported in literature. Conclusion G. biloba suspension cultures canbe used as an enzyme system to biotransform resibufogenin, an animal-originated bufadienolide, into3-epi-resibufogenin.

Effects of NaCI and Ca^(2+) on Membrane Potential of Epidermal Cells of Maize Roots

The effects of salt-stress on plants involve not only the water stress caused by low osmotic pressure, but also the toxicity of excess Na^＋. A large amount of Na^＋ entering cells would reduce K^＋ uptake, which leads to an imbalance of K：Na ratio in cells. One of the reasons for the reduced K^＋-uptake is the closure of K^＋-channel which is controlled by membrane potential. Calcium is usually applied to improve the growth of plants on saline soils and shows positive influence in the integrality of cell membrane. This study applied glass microelectrode technique to monitoring the NaCl-induced changes of membrane potential of root epidermal cells of maize （Zea mays L., Denghai 11） seedlings at NaCl concentrations of 0, 8, 20, 50, 100, 200 mmol L^-1, respectively. The effect of Ca^2＋ on the changes of membrane potential caused by NaCl was also studied. The results showed that： NaCl caused cell membrane depolarization. The depolarization became greater and faster with increasing of NaCl concentration. Moreover, the extent of depolarization was positively correlated with NaCl concentration. The addition of calcium postponed the depolarization, and decreased the degree of depolarization caused by NaCl. High NaCl concentration leads to depolarization of maize root cell membrane, which can partly be counteracted by calcium.

Induction of SiHa Cells Apoptosis by Nanometer Realgar Suspension and Its Mechanism

The effects of nanometer realgar uterine cervix cancer cell line SiHa cells and suspension on proliferation and apoptosis of human oncogenic genes HPV16E6/E7 were investigated. A ＂micro-jet effiux＂ strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations （6.25, 12.5, 25 and 50 mg/L） for different durations （12, 24, 48 and 72 h）. The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MTT method. Special morphological changes of apoptosis were observed by transmission electron microscopy （TEM） and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry （FCM）. The expression of HPV 16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25--50 mg/L nanometer realgar suspension for 48 h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time- and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group （P〈0.01）, and G0/G1 phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50 mg/L for 48 h. RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.

Research on Electric Impedance Spectroscopy of Living Cell Suspensions by a Chip with Microelectrodes

A microfabricated electrical impedance spectroscopy (EIS) chip with microelectrodes was developed.The substrate and the electrodes of the chip were made of glass and gold,respectively.The experimental results demonstrated that the EIS-chip could distinguish different solutions (physiological saline,culture medium,living cell suspension etc.) by scanning from 10Hz to 45kHz.A 6-element circuit model was used for fitting the real part and the imaginary part admittance curves of the living cell suspension.An actual circuit was also built and tested to verify the 6-element circuit model proposed.The micro-EIS chip has several advantages including the use of small sample volumes,high resolution and ease of operation.It shows good application prospects in the areas of cellular electrophysioiogy,drug screening and bio-sensors etc.

Biomass accumulation of Panax vietnamensis in cell suspension cultures varies with addition of plant growth regulators and organic additives

Objective: To evaluate the impact of plant growth regulators including kinetin(KN),benzyl adenine and naphthalene acetic acid, yeast extract and casein hydrolyzate on biomass accumulation of Vietnamese ginseng Panax vietnamensis(P. vietnamensis) in cell suspension culture.Methods: Cell suspension cultures were established from friable calluses derived from leaves and petioles of 3-year-old in-vitro P. vietnamensis plants. The cell suspension cultures were grown in Murashige and Skoog basal media supplemented with various concentrations of KN, benzyl adenine, naphthalene acetic acid, and yeast extract and casein hydrolyzate.Results: All tested factors generated an increase in the cell biomass of P. vietnamensis in suspension culture, but the impact of each varies depended on the factor type, concentration, and incubation period. Addition of 2.0 mg/L KN resulted in the largest biomass increase after 24 d,(57.0 ± 0.9) and(3.1 ± 0.1) mg/m L fresh and dry weight, respectively,whereas addition of benzyl adenine or naphthalene acetic acid produced optimum levels of Panax cell biomass at 1.0 and 1.5 mg/L, respectively. Addition of the elicitor yeast extract led to a 1.4–2.4 fold increase in biomass of P. vietnamensis, while addition of casein hydrolyzate enhanced biomass accumulation 1.8–2.6 fold.Conclusions: The addition of each factor causes significant changes in biomass accumulation of P. vietnamensis. The largest biomass accumulation is from cultures grown in MS media containing 2.0 mg/L KN for 24 d. The outcome of the present study provides new insights into the optimal suspension culture conditions for studies on the in vitro cell biomass production of P. vietnamensis.

Establishment of cell suspension line of Populus tomentosa Carr

Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L^-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subcultured into a MS liquid medium supplemented with 1.5 mg·L^-1 2,4-D. The best subculture medium was MS ＋ 0.8 mg＇L-1 2,4-D ＋ 30 g·L^-1 sucrose with a subculture cycle of seven days.

Influences of Plant Growth Regulators， Basal Media and Carbohydrate Levels on Cell Suspension Culture of Panax ginseng

A cell suspension culture of Panax ginseng which may be continuously subcultured has been established. Embryogenic callus derived from clutured young leaves was used to initiate the culture.Plant growth regulators, basal medium formula and carbohydrate levels were examined to determine their various effects on suspension culture cell growth and development. The best selection of plant growth regulator, hasal medium and carbohydrate level is 2 mg / L 2,4-D: 0.5 mg / L KT,MS and 3% sucrose respectively.

Ex vivo differentiation of multipotent adult progenitor cells to skin epidermal cells

Objective： By establishing the indirect contact co-culture system, we studied the in vitro condition for MAPCs differentiating into epidermal cells and the transformation of MAPCs into epidermal cell phenotype. Methods： Cell culture insert membrane was used for substitute basal membrane and MAPCs, fibroblast cells （FCs） and mixture of MAPCs and epidermal cells and FCs were separately implanted into 2 sides of it. PKH26 was used to label cloned MAPCs; type IV collagen rapid adhering method was used to isolate and culture the skin epidermal cells from l-day-old SD rat. Results： Part of the MAPCs transformed into cells expressing keratin in the presence of peripheral epithelia and FCs. Type Ⅳ collagen rapid adhering method successfully selected rats＇ epidermal stem cells. The mixture of the 2 kinds of cells or indirect culture might promote the differentiation through mesenchymal factors secreted by dermis FC. Conclusion： We were the first to have established the in vitro model of MAPCs differentiation into epidermal cells, in which MAPCs were transformed into epithelium-like cells.

Ultrastructural and Extracellular Protein Changes in Cell Suspension Cultures of Populus euphratica Associated with Low Temperature-induced Cold Acclimation

Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A cell suspension culture was initiated from callus derived from plantlets of Populus euphratica. Cold acclimation was induced (LT50 of 17.5 ℃) in cell suspension at 45 ℃ in the dark for 30 days and the freezing tolerance increased from LT50 of 12.5 ℃ in nonacclimated cells to LT50 of 17.5 ℃ in cold-acclimated cells. Microvacuolation, cytoplasmic augmentation and accumulation of starch granules were observed in cells that were cold-acclimated by exposure to low temperatures. Several qualitative and quantitative changes in proteins were noted during cold acclimation. Antibodies to carrot extracellular (apoplastic) 36 kD antifreeze protein did not cross react on immunoelectroblots with extracellular proteins in cell suspension culture medium of Populus euphratica, indicating no common epitopes in the carrot 36 kD antifreeze protein and P. euphratica extracellular proteins. The relationship of these changes to cold acclimation in Populus euphratica cell cultures was discussed.

Ce^(4+)-Induced Apoptosis of Taxus cuspidata Cells in Suspension Culture

The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce 4+ were studied. The condensation and margination of chromatin were observed under the electron microscopy. DNA fragmentation ranged 'DNA ladder' on agarose gel electrophoresis. TdT mediated dUTP nick end labeling (TUNEL) analysis of the cells reveals that the nuclear DNA strand breaks can be identified by labeling free 3′ OH termini. These results suggest that Ce 4+ can induce apoptosis of Taxus cuspidata cells and also indicate that there is a certain relationship between apoptosis and secondary metabolite product Taxol.

Somatic Embryogenesis from Cell Suspension Cultures of Aspen Clone

Suspension cultures initiated from callus derived from petiole explants of aspen hybrid （Populus tremuloides×P. tremula） produced somatic embryos. Callus was induced on a MS medium supplemented with 5mg·L^-1 2,4-D and 0.05mg·L^-1 zeatin under light conditions. Embryogenic calli were obtained when a subsequent subculture of calli was suspended in the same basal medium with 10mg·L^-1 2,4-D. The highest number of globular embryos were induced from embryogenic calli by cell suspension culture in a MS liquid medium supplemented with 10mg·L^-1 2,4-D. Genotype and 2,4-D concentration were vital to the induction of embryogenic calli producing competent cells. Embryogenic calli for each genotype were heterogeneous. Green calli with gel-like consistency could yield more competent cells than light yellow embryogenic calli. However, some globular embryos broke into slices and some developed abnormally after one month of culture under the same or other hormonal conditions.