Somatic embryogenesis and plant regeneration from undeveloped ovules and stigma/style explants of sweet orange navel group [ Citrus sinensis (L.) Osb.]

Somatic embryogenesis was induced and plant regeneration was obtained in 11 different genotypes of sweet orange navel group [Citrus sinensis (L.) Osb.] from cultures of stigma/style explants and undeveloped ovules. Explants were cultured on 3 different modifications of Murashige and Skoog medium: 500 mg l-1 malt extract; 500 mg l-1 malt extract and 4.6 μM kinetin; and 500 mg l-1 malt extract and 13.3 μM 6-benzylaminopurine. Sucrose (146 mM) was used as carbon source. Somatic embryogenesis occurred 1–3 months after culture initiation from undeveloped ovule and stigma/style cultures of all the genotypes tested. Somatic embryos developed into plantlets with a high frequency (74%) after transfer to Murashige and Skoog medium supplemented with 146 mM sucrose and 500 mg l-1 malt extract. Plants were successfully transferred to soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

Callus formation from protoplasys of Sorghum cell suspension cultures

Cell suspension cultures have been obtained from three cultivars of Sorghum bicolor L. Moench. Protoplasts readily obtained from these cultures underwent sustained cell division and callus formation.     

The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation

Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic and physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage.     

Simultaneous HPLC analysis of triamcinolone acetonide and budesonide in microdialysate and rat plasma: application to a pharmacokinetic study

A specific and reliable HPLC-PDA method for the quantitative determination of triamcinolone acetonide, budesonide and fluticasone propionate (as internal standards) in small volumes of microdialysate and rat plasma was developed. An efficient solid-phase extraction (SPE) procedure for plasma samples yielded extremely clean extracts with overall recovery of 104.3% and 95.7% for triamcinolone acetonide (TA) and fluticasone propionate, respectively. Plasma extracts obtained after SPE and microdialysis samples were directly injected on a C18 column to separation. The method has been validated with good linearity, sensitivity, specificity and high accuracy (RE -5.28% to 9.14%) and precision (CV 0.50% to 6.62%) on both matrices. In stability studies, TA and budesonide were stable during storage and assay procedures. The method was applied to a pharmacokinetic study in rodents using microdialysis to determine protein unbound TA concentrations in blood and muscle.     

Laminin-1 induces neurite outgrowth in human mesenchymal stem cells in serum/differentiation factors-free conditions through activation of FAK-MEK/ERK signaling pathways

Mesenchymal stem cells (MSCs) can be differentiated into cell types derived from all three germ layers by manipulating culture conditions in vitro. A multitude of growth and differentiation factors have been employed for driving MSCs towards a neuronal phenotype. In the present study, we investigated the potential of extracellular matrix (ECM) proteins—fibronectin, collagen-1, collagen-IV, laminin-1, and laminin-10/11, to induce a neuronal phenotype in bone marrow derived human MSCs in the absence of growth factors/differentiating agents. All of the ECM proteins tested were found to support adhesion of MSCs to different extents. However, direct interaction only with laminin-1 triggered sprouting of neurite-like processes. Cells plated on laminin-1 exhibited neurite out growth as early as 3 h, and by 24 h, the cells developed elaborate neurites with contracted cell bodies and neuronal-like morphology. Function-blocking antibodies directed against α6 and β1 integrin subunits inhibited neurite formation on laminin-1 which confirmed the involvement of integrin α6β1 in neurite outgrowth. Mechanistic studies revealed that cell adhesion to laminin-1 activated focal adhesion kinase (FAK), and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways. Abrogation of FAK phosphorylation by herbimycin-A inhibited neurite formation and also decreased activities of MEK and ERK. Pharmacological inhibitors of MEK (U0126) and ERK (PD98059) also blocked neurite outgrowth in cells plated on laminin-1. Our study demonstrates the involvement of integrin α6β1 and FAK-MEK/ERK signaling pathways in laminin-1-induced neurite outgrowth in MSCs in the absence of serum and differentiation factors.     

噻托溴铵及噻托溴铵联合布地奈德福莫特罗治疗COPD疗效分析

目的:观察噻托溴铵与布地奈德/福莫特罗联合吸入和噻托溴铵单独吸入对慢性阻塞性肺疾病(COPD)患者的疗效及安全性。方法:58例COPD患者随机分为2组:治疗组30例,给予噻托溴铵(18μg,1次/d)联合布地奈德/福莫特罗(160/4.5μg,2次/d)吸入治疗;对照组28例,给予噻托溴铵(18μg,1次/d)吸入治疗。观察患者治疗前、治疗8周和16周后肺功能及临床症状的变化。结果:治疗8周和16周后,2组FEV1和SGRQ症状评分均较治疗前明显改善(P<0.05),治疗组FEV1和SGRQ症状评分的改善显著高于对照组(P<0.05);治疗组白天使用沙丁胺醇次数更少(P<0.05)。各组均未出现明显不良反应。结论:噻托溴铵与布地奈德/福莫特罗联合吸入治疗COPD,疗效优于噻托溴铵单药治疗。     

Metabolism of diclofop-methyl (methyl-2-[4-(2',4'-dichlorophenoxy)phenoxy] propanoate) in cell suspensions of diploid wheat (Triticum monococcum)

The metabolism of the herbicide, diclofop-methyl (methyl-2-[4-(2′,4′-dichlorophenoxy)phenoxy]propanoate, in cell suspensions of resistant diploid wheat (Triticum monococcum L.) was determined 1, 8, and 24 h after treatment with ¹⁴C-diclofop-methyl. The ¹⁴C-labeled products were identified by thin layer chromatographic comparisons to appropriate standards. Eight hours after treatment with 5 μM diclofop-methyl in 0.8% acetone (neither of which were toxic to the cell suspensions) 87.2% (84.0% methanol soluble, 3.2% methanol insoluble) of the total ¹⁴C recovered (90.4%) was in the cells and 12.8% was in the medium. Major metabolites found in methanol extracts of the cells were diclofop (2-[4-(2′,4′-dichlorophenoxy)phenoxylpropionic acid), diclofop hydroxylated at an undetermined position on the 2,4-dichlorophenyl ring (ring-OH diclofop), and conjugates of ring-OH diclofop. Acid hydrolysis of the conjugated metabolite(s) yielded ring-OH diclofop and diclofop. Twenty-four hours after treatment 70–75% of the total ¹⁴C recovered was present as conjugated metabolites. With the exception of ring-OH diclofop, all metabolites present in the cells were also recovered from the medium. A metabolite found in low concentrations in the medium that yielded diclofop upon hydrolysis was identified as an ester conjugate. Toxic concentrations of diclofop-methyl (10 and 20μM) had no effect on the metabolism of the herbicide, although the rate of uptake was slower than for cells treated with 5 μM herbicide. The products of diclofop-methyl metabolism in cell suspensions of T. monococcum were compared to previous data from T. aestivum intact plant metabolism of diclofop-methyl.     

Formation of cyclotides and variations in cyclotide expression in <Emphasis Type="Italic">Oldenlandia affinis</Emphasis> suspension cultures

Cyclotides, a family of disulfide-rich mini-proteins, show a wide range of biological activities, making them interesting targets for pharmaceutical and agrochemical applications, but little is known about their natural function and the events that trigger their expression. An investigation of nutritional variations and irradiation during a batch process involving plant cell cultures has been performed, using the native African medical herb, Oldenlandia affinis, as a model plant. The results demonstrated the biosynthesis of kalata B1, the main cyclotide in O. affinis, in a combined growth/nongrowth-associated pattern. The highest concentration, 0.37 mg g⁻¹ dry weight, was accumulated in irradiated cells at 35 μmol m⁻² s⁻¹. Furthermore, 12 novel cyclotides were identified and the expression of various cyclotides compared in irradiated vs non-irradiated cultures. The results indicate that cyclotide expression varies greatly depending on physiological conditions and environmental stress. Kalata B1 is the most abundant cyclotide in plant suspension cultures, which underlies its importance as a natural defense molecule. The identification of novel cyclotides in suspension cultures, compared to whole plants, indicates that there may be more novel cyclotides to be discovered and that the genetic network regulating cyclotide expression is a very sensitive system, ready to adapt to the current environmental growth condition.     

四种前体对胀果甘草细胞悬浮培养生产甘草黄酮的调控效果评价

在胀果甘草细胞悬浮体系中加入苯丙氨酸、酪氨酸、肉桂酸和乙酸钠来研究前体对甘草细胞生产甘草黄酮的影响。结果显示,4种前体在合适的浓度下对细胞的生长没有明显的抑制作用,而且均能促进细胞内甘草黄酮的生物合成,但高浓度的肉桂酸对细胞的生长有一定的抑制作用。苯丙氨酸的最佳添加浓度为20 mg/L,酪氨酸、肉桂酸、乙酸钠的最佳添加浓度都是5 mg/L。此时,均可使培养体系的甘草黄酮产量高达100 mg/L以上,其中酪氨酸的添加使得产量高达对照的1.43倍。苯丙氨酸、肉桂酸和乙酸钠3种前体的添加时间均以第10天为宜,酪氨酸添加时间以第5天为最佳。而且,在添加苯丙氨酸和乙酸钠后的第3天收获细胞,此时细胞的生物量和甘草黄酮产量最大。此外,苯丙氨酸、乙酸钠的添加可以增加黄酮合成的关键酶之一——苯丙氨酸裂解酶的活性。酪氨酸对苯丙氨酸裂解酶影响不大,而肉桂酸的添加却导致其活性显著降低。     

Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4I g) fusion protein, a novel immunosuppressive agent, was expressed in transgenic rice cell suspension culture and its characteristics and in vitro activities were investigated. The expression vector pMYN409 was constructed to express hCTLA4I g under the control of rice alpha-amylase 3D (RAmy3D) promoter. Transgenic calli were prepared by particle bombardment mediated transformation and were screened for hCTLA4I g expression using ELISA. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4I g into the media up to 31.4 mg/L in flask culture. The rice-derived hCTLA4Ig (hCTLA4IgP) was purified from the culture media with affinity chromatography using protein A and compared with CHO-derived hCTLA4Ig (hCTLA4IgM). Recombinant hCTLA4IgP has molecular weight of approximately 50 kDa on SDS-PAGE under reducing condition, which is a little different from that of hCTLA4IgM probably due to the difference of carbohydrate chain structures. Purified hCTLA4IgP was biologically active and was confirmed to suppress T-cell proliferation.