全反式维甲酸协同DAPT抑制胶质瘤干细胞的自我更新

前期研究脑表明,脑胶质瘤干细胞（glioma stem cells,GSCs）是胶质瘤发生和发展的重要因素,探索靶向干预GSCs生长有可能成为脑胶质瘤治疗的有效途径之一。该研究旨在阐明两种药物ATRA和Y-分泌酶抑制剂DAPT协同抑制GSCs自我更新的生物学效应。通过用台盼蓝排染法、克隆球形成试验和免疫印迹分析了两种药物的单独使用或联用对GSC样细胞PGCl和PGC2生长、成球能力和自我更新以及干细胞标志物表达的影响。结果发现,单独使用ATRA对PGCl生长有一定的抑制作用,而对PGC2生长几乎没有影响;DAPT对PGCs的生长抑制作用明显强于ATRA。高浓度ATRA（80μmol/L）能诱导PGCs的分化,降低PGCs成球大小,且成球效率降至5％～8％,而正常对照组为32％～35％;同样,DAPT（40μmol／L）也能降低PGCs成球大小,且成球效率降至2％～3％;低浓度ATRA（20μmol／L）和DAPT（5gmol／L）对PGCs自我更新能力和干性没有明显影响,而联合使用后其明显降低PGCs的成球大小,且成球效率降至3％～5％,促进细胞凋亡,并且明显抑制了干细胞标志物Nestin、CDl33、Sox2、Oct4的表达,提高了分化标志物GFAP的表达。该研究证明了低浓度的ATRA和DAPT能协同抑制脑胶质瘤干细胞PGCs的自我更新。研究结果将为脑胶质瘤的临床研究提供实验依据。     

Kinetics of manganese transport and gene expressions of manganese transport carriers in Caco-2 cell monolayers

Two experiments were conducted to investigate the kinetics of manganese (Mn) transport in Caco-2 cell monolayers and the gene expressions of Mn transport carriers in apical (AP) and basolateral (BL) membranes. In experiment 1, the cells were treated with the medium containing 146 μmol/L of Mn (MnSO₄·H₂O). Both the uptake and transport of Mn from AP–BL or from BL–AP at different time-points were assessed to determine the optimal time for kinetics of Mn transport. The transport of Mn increased linearly with higher efficiency values in AP–BL than in BL–AP direction, however, the uptake of Mn revealed an asymptotic pattern within 120 min. In experiment 2, the kinetics of Mn transport in AP–BL was determined with media containing Mn concentrations from 0 to 2,500 μmol/L at 40 and 120 min, respectively, and mRNA levels of divalent metal transporter 1 (DMT1) and ferroportin (FPN1) were determined in Caco-2 cells treated with the medium containing 0 or 800 μmol/L of Mn for 120 min. The kinetics of Mn transport showed a carrier-mediated process when Mn concentrations were lower than 1,000 μmol/L and a linear increment when Mn concentrations exceeded 1,000 μmol/L at either 40 or 120 min. Mn treatment decreased (P < 0.01) DMT1 mRNA level and increased (P < 0.01) FPN1 mRNA level. The results from the present study suggested that Mn transport in AP–BL fit both carrier-mediated saturable and non-saturable diffusion processes, and Mn transport carriers DMT1 and FPN1 mediate the apical uptake and basolateral exit of Mn in Caco-2 cells.     

全反式维甲酸下调肝癌细胞HepG2内AFP结合蛋白的表达

当成人肝细胞发生癌变,甲胎蛋白(alpha-fetoprotein,AFP)在血清中的含量会急剧增加.AFP可与细胞表面AFP结合蛋白(AFP binding protein,ABP)结合促使细胞增殖分化.全反式维甲酸(al1-trans retinoic acid,ATRA)通过与特异性维甲酸受体(retinoic acid receptor,RAR)结合发挥抑制肿瘤生长的作用.Western印迹检测肝癌细胞HepG2和HLE中ABP的表达.结果显示,ABP在HepG2细胞中高表达,在HLE细胞中无明显表达.这一结果与2种细胞AFP的表达情况一致.激光扫描共聚焦显微镜定位分析显示,ABP存在于HepG2细胞胞膜和胞浆,用80μmol/L ATRA处理HepG2细胞4 h,可导致RAR入核增加.用不同浓度(20～160μmol/L)ATRA处理HepG2细胞后培养36 h.Western印迹结果表明,细胞ABP的表达随着ATRA浓度的增高而越少,ATRA浓度达80μmol/L时,HepG2细胞的ABP表达减少,ATRA浓度为160μmol/L时,ABP几乎无表达;加入80μmol/L ATRA后,随着作用时间延长,HepG2细胞ABP表达逐渐减少,当作用时间为12 h时,ABP表达明显减少.结果表明,ABP的表达对ATRA的反应呈剂量和时间依赖性.免疫共沉淀结果表明,AFP、ABP及RAR这3种蛋白质具有互相结合的作用.这些结果为进一步深入研究AFP在肝癌发生过程中的作用机制提供了依据.     

全反式维甲酸抑制猪脂肪间充质干细胞的成脂分化

分离培养猪脂肪间充质干细胞（adipose mesenchymal stem cells, AMSCs）,流式细胞仪鉴定其表面标记.利用MTT比色检测不同浓度的全反式维甲酸（all trans retinoic acid, ATRA）对猪AMSCs增殖的影响;光学显微镜下观察猪AMSCs向脂肪细胞分化的形态学变化;油红O染色提取法分析不同浓度ATRA对猪AMSCs成脂分化的影响;RT PCR检测脂肪细胞分化标志基因LPL和aP2 mRNA的变化.MTT比色结果显示,生理浓度（1×10^－9～1×10^－8 mol/L）和药理浓度（1×10^－7～1×10^－5 mol/L）ATRA对猪AMSCs增殖均没有影响.油红O染色提取法结果表明,除1×10^－7　mol/L ATRA对猪AMSCs成脂分化没有影响外,生理浓度（1×10^－9～1×10^－8 mol/L）和其它药理浓度（1×10^－6～1×10^－5 mol/L）ATRA均显著抑制猪AMSCs成脂分化（P<0.05）.RT-PCR检测显示,ATRA显著抑制脂肪细胞分化标志基因LPL和aP2 mRNA表达（P<0.05）.     

Somatic embryogenesis and plant regeneration from cell suspension culture of niger (Guizotia abyssinica Cass.)

Somatic embryogenesis was achieved from cell suspension cultures of niger (Guizotia abyssinica Cass.). Initially, friable embryogenic calluses were induced from cotyledonary leaves of niger on Murashige and Skoog (MS) agar medium containing 5 μM 2,4-Dichlorophenoxyacetic acid (2,4-D) and 0.5 μM kinetin (KIN). Cell suspension cultures were established by using embryogenic calluses in MS liquid medium containing 5 μM 2,4-D and 0.5 μM KIN. Initiation of somatic embryogenesis and development up to globular stage from embryogenic cell clumps occurred in the liquid medium itself. Thereafter embryogenic cell aggregates were transferred to MS agar medium supplemented with 3 μM KIN for embryo differentiation, whereas maturation of somatic embryos occurred in MS agar medium containing 10 μM abscisic acid.     

Hydrogen sulfide delays nicotinamide-induced premature senescence via upregulation of SIRT1 in human umbilical vein endothelial cells

The present study was designed to investigate the effect of hydrogen sulfide on cellular senescence of human umbilical vascular endothelial cells (HUVECs CC-2517) and its underlying mechanism. The premature senescence-like phenotype HUVECs (the fourth passage) was induced by treatment with nicotinamide (NAM, an inhibitor of SIRT1, 5 mmol/L, 12 h). Cells were cultured with sodium hydrosulfide (NaHS, 12.5, 25, 50 and 100 μmol/L) for 48 h in premature senescence-like phenotype HUVECs. The fourth passage of HUVECs was considered as young group. Senescence-associated (SA)-β-galactosidase activities were detected to evaluate cell senescence, and the expression of SA heterochromatin foci (SAHF) was visualized by DAPI DNA staining. The mRNA and protein levels of SIRT1 were detected using RT-PCR and western blotting analysis, respectively. The results showed that β-galactosidase positive cells and the formation of SAHF were markedly increased after treatment with NAM (5 mmol/L) for 12 h. We also found that NaHS (12.5 μmol/L) had no effect on the percentage of SA β-gal positive cells and the expression of SAHF, and the hallmarks decreased at the concentration of 25 and 50 μmol/L, reaching the minimum at 50 μmol/L, while the percentage of SA β-gal positive cells and the expression of SAHF increased at the concentration of 100 μmol/L. Furthermore, we found that both on protein and mRNA levels of SIRT1 in the Y+N+S50 group was significantly increased compared with that in Y+N group. In conclusion, NaHS delays senescence of HUVECs induced by NAM via upregulation of SIRT1 expression.     

Effects of Homocysteine on ERK Signaling and Cell Proliferation in Fetal Neural Stem Cells In Vitro

The aim of the present study was to determine if the excitatory amino acid homocysteine (Hcy) alters ERK signaling and cell proliferation in fetal neural stem cells (NSCs) in vitro. NSCs were isolated from fetal rats and grown in serum-free suspension medium. The cells were identified as NSCs by their expression of immunoreactive Sox2. NSCs were assigned to one of four treatment groups: vehicle control, low-dose Hcy group (Hcy-L, medium contained 30 μmol/L Hcy), middle-dose Hcy group (Hcy-M, 100 μmol/L Hcy) and high-dose Hcy group (Hcy-H, 300 μmol/L Hcy). Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Protein expression levels of ERK1/2 and phosphorylated ERK1/2 were detected by Western blot. The effects of Hcy on NSC death, including apoptosis, were assessed by using flow cytometry and trypan blue exclusion. The results showed that NSCs grew as neurospheres in the serum-free medium. Hcy decreased ERK1/2 protein phosphorylation and NSC proliferation, but it did not induce cell death or apoptosis within the concentration from 30 to 300 μmol/L. The above results are consistent with the hypothesis that Hcy decreases fetal NSC proliferation by inhibiting ERK signaling.     

Low expression of ERK signaling pathway affecting proliferation,cell cycle arrest and apoptosis of human gastric HGC-27 cells line

This study was carried out for the first time to examine the potential role and the underlying mechanisms of Lycopene in the gastric cancer HGC-27 cells. HGC-27 cells were seeded onto heat-sterilized coverslips in six-well plates and exposed to Lycopene (5, 10, 20, 30 and 40 μmol/L) for periods of 72 h at 37 °C. Results showed that Lycopene (5, 10, 20, 30 and 40 μmol/L) dose-dependently increased NBT positive rate and decreased lactate dehydrogenase activity in HGC-27 cells. In addition, Lycopene (5, 10, 20, 30 and 40 μmol/L) inhibited proliferation and induced G0–G1 phase cell cycle arrest in HGC-27 cells. Western blot and FQRT-PCR analysis showed that Lycopene decreased pERK and extracellular signal-regulated kinase (ERK) protein and mRNA expression in a dose-dependent manner. These findings demonstrate that Lycopene inhibited gastric cancer HGC-27 cells growth and stimulated its apoptosis via the suppressing ERK signaling pathway.     

应用两种方法诱导SH-SY5Y细胞分化的研究

目的：观察全反式维甲酸（ATRA）处理和ATRA与十四烷酰佛波醇乙酸酯（TPA）序贯处理（ATRA/TPA）对人类神经母细胞瘤细胞系SH-SY5Y细胞增殖抑制和形态分化的影响。方法：应用10μM ATRA处理6天和10μM ATRA处理3天继以80 nMTPA处理3天这两种方法使SH-SY5Y细胞分化;用倒置光学显微镜动态观察SH-SY5Y细胞形态学变化;并用MTT比色法比较两种分化方法对SH-SY5Y细胞的体外抗增殖作用。结果：ATRA处理和ATRA与TPA序贯处理对SH-SY5Y细胞都有抗增值和诱导细胞分化作用,细胞形态发生明显的变化,分化成神经元表型,前者主要表现为两端带有长突起的纺锤体样细胞形态,而后者主要是由细胞体延伸出多个突起的多边形的细胞。ATRA分化6天的细胞的存活率下降为78.7%±2.0%。当去除ATRA后,继续培养1天的细胞存活率上升为89%±0.2%,而继续培养2天的细胞存活率为86.3%±1.4%;ATRA与TPA序贯分化6天细胞存活率下降为75.9±0.4%。当去除TPA后,继续培养一天的细胞存活率为75.5±0.7%,继续培养2天的细胞存活率为74.9±1.0%。结论：维甲酸（ATRA）处理和ATRA与十四烷酰佛波醇乙酸酯（TPA）序贯处理（ATRA/TPA）均能明显诱导SH-SY5Y细胞分化。这两种分化细胞为神经科学的研究提供了优良的体外培养模型细胞,尤其是ATRA与TPA序贯处理能获得分化完全而稳定的神经元样细胞。     

Micropropagation of Semecarpus anacardium L. from mature tree-derived nodal explants

A protocol was established for micropropagation of Semecarpus anacardium L. from mature tree-derived twigs. Sixty percent of aseptic cultures were obtained by surface sterilization with Bavistin, liquid detergent, and cefotaxime. Elongated twigs collected before flowering were optimum for in vitro culture initiation. Meristematic activity was triggered at all concentrations of thidiazuron (TDZ) incorporated into Woody Plant Medium. TDZ suppressed elongation of axillary buds, resulting into swollen meristems and upon its elimination multiple shoot primordia formation and differentiation were noted. Differentiation and shoot elongation were slower in explants pre-cultured with higher concentrations of TDZ. Swollen axillary meristems pre-cultured on TDZ (9.08 and 13.62 μM) failed to differentiate, whereas TDZ at 2.27 μM was optimal for shoot differentiation and elongation. Multiple bud induction was favored by 4.45 μM of TDZ. Differentiation of multiple shoot primordia by repeated subculturing on growth regulator-free medium and rooting was 100% in filter-paper supported half-strength liquid medium containing 7.38 μM IBA. Rooting was 90% in shoots placed directly in half-strength liquid medium with 2.46 μM IBA. Rooted plantlets hardened in soil:sand mixture (1:1) were transferred to green house. Genetic uniformity of in vitro raised clones with mother plant was confirmed by Inter-Simple Sequence Repeat markers.     

Optimized shoot regeneration for Indian soybean: the influence of exogenous polyamines

A simple and efficient regeneration protocol was established for soybean [Glycine max (L.) Merrill]. Cotyledonary node explants from 7-day-old in vitro seedlings were used as explants. The effect of different plant growth regulators [N ⁶ –benzyladenine (BA), kinetin (KT), thidiazuron (TDZ), gibberellic acid (GA₃), zeatin riboside (ZTR), indole-3-acetic acid (IAA), and indole-3-butyric acid (IBA)] along with polyamines (Spermidine, spermine, and putrescine) were investigated at different stages of regeneration using direct organogenesis system. Exogenous spermidine (137.69 μM) in shoot induction medium containing optimal BA concentration (2.22 μM) induced maximum number of shoots (39.02 shoots/explant) compared to BA (2.22 μM) alone. Regenerated shoots elongated well in shoot elongation medium containing GA₃ (1.45 μM) and spermine (74.13 μM), and developed profuse roots in root induction medium containing putrescine (62.08 μM). Rooted plantlets were successfully hardened and acclimatized with a survival rate of 92 %. The amenability of the standardized protocol using cultivar PK 416 was tested on four more Indian soybean cultivars JS 90–41, Hara soy, Co1, and Co2 of which PK 416 was found to be the best responding cultivar, with a maximum of 96.94 % shoot induction.     

In vitro shoot growth, direct organogenesis and somatic embryogenesis promoted by silver nitrate in Coffea dewevrei

Direct differentiation of shoot buds in Coffea dewevrei was evident from the seedling shoots with collar region and also from collar region end of hypocotyl segments in presence of 40 μM AgNO₃, 8.88 μM of BA and 2.85 μM of IAA. Apart from this, shoot end of hypocotyl explants mainly supported yellow friable callus or somatic embryos. Subsequent transfer to the same medium induced secondary somatic embryogenesis. The collar region of the hypocotyl explants not only showed direct organogenesis by producing 1–3 shoots per explant and also able to produce globular somatic embryos and embryogenic yellow friable callus. Similarly direct somatic embryogenesis along with yellow friable embryogenic callus formation on 1/2 strength MS medium comprising 1.47 μM IAA, 2.22 μM BA and 40 μM AgNO₃ was noticed from cut portion of in vitro leaf and stalk of regenerated plants. The microshoots rooted well upon subculturing onto the same medium in 6 weeks and showed 60 % survival in green house and resumed growth upon hardening.     

P(HPMA-APMA)-ATRA的合成及其对HL-60细胞诱导分化能力的评估

以N-(2-羟丙基)甲基丙烯酰胺(HPMA),N-(3-氨基丙基)甲基丙烯酰胺(APMA)和全反式维甲酸(ATRA)为原料,采用自由基溶液聚合法设计合成P(HPMA-APMA)-ATRA,并用核磁共振氢谱对该化合物进行结构表征.相比于单体ATRA,聚合物的水溶性显著增加,同时可通过胞吞作用进入细胞.3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法评估聚合物和单体ATRA对人早幼粒白血病细胞HL-60生长的抑制作用,流式细胞术检测两者对HL-60细胞周期分布及细胞表面抗原CD11b表达的影响,进一步结合氯化硝基四氮唑蓝(NBT)还原法评估聚合物诱导HL-60细胞分化的能力.结果显示,聚合物比单体ATRA具有更强的细胞生长抑制活性,其IC50值分别为1.03和4.09μmol/L;聚合物还具有更高的G0/G1期细胞阻滞效应,1.2μmol/L时,聚合物比单体ATRA的G0/G1期细胞率高出17.7%;同样,0.4μmol/L聚合物与2.4μmol/L单体ATRA诱导HL-60的NBT还原能力相当,0.8μmol/L聚合物与2.4μmol/L单体ATRA诱导HL-60细胞表面抗原CD11b表达相当,表明聚合物比单体ATRA具有更强的诱导HL-60细胞向粒细胞分化的能力,其药效增强3~4倍.     

Factors affecting biohydrogen production by unicellular halotolerant cyanobacterium Aphanothece halophytica

The effects of several physiological parameters on H₂ production rate in the unicellular halotolerant cyanobacterium Aphanothece halophytica were investigated. Under nitrogen deprivation, the growth of cells was inhibited, but H₂ production rate was enhanced approximately fourfold. Interestingly, cells grown under sulfur deprivation exhibited a decrease in cell growth, H₂ production rate, and bidirectional hydrogenase activity. Glucose was the preferred sugar source for H₂ production by A. halophytica, but H₂ production decreased at high glucose concentrations. H₂ production rate was optimum when cells were grown in the presence of 0.75 M?NaCl, or 0.4 μM?Fe³⁺, or 1 μM?Ni²⁺. The optimum light intensity and temperature for H₂ production were 30 μmol photons m^?2?s^?1 and 35 °C, respectively. A two-stage culture of A. halophytica was performed in order to overcome the reduction of cell growth in N-free medium. In the first stage, cells were grown in normal medium to accumulate biomass, and in the second stage, H₂ production by the obtained biomass was induced by growing cells in N-free medium supplemented with various chemicals for 24 h. A. halophytica grown in N-free medium containing various MgSO₄ concentrations had a high H₂ production rate between 11.432 and 12.767 μmol H₂ mg?chlorophyll a (chl a)^?1?h^?1, a 30-fold increase compared to cells grown in normal medium. The highest rate of 13.804 μmol H₂ mg?chl a ^?1?h^?1 was obtained when the N-free growth medium contained 0.4 μM Fe³⁺. These results suggested the possibility of using A. halophytica and some other halotolerant cyanobacteria thriving under extreme environmental conditions in the sea as potential sources for H₂ production in the future.     

S-Adenosylmethionine-induced adipogenesis is accompanied by suppression of Wnt/β-catenin and Hedgehog signaling pathways

S-Adenosylmethionine (SAM) plays a crucial role as a methyl donor in various biological processes and has been previously shown to be involved in adipogenesis in skeletal muscle. This study was conducted to explore the mechanism of SAM inducing adipogenesis in skeletal muscle. Adipose precursor cells, 3T3-L1, and C2C12 cells, were induced into adipogenic differentiation by addition of SAM in MDI-differentiation media (0.5 mmol/L isobutylmethylxanthine, 1 μm/L dexamethasone, and 10 μg/mL insulin) to explore the role of SAM in promoting adipogenesis. Subsequently, cells were cultured with a medium containing SAM alone at the beginning of differentiation to test the relationship between SAM-induced adipogenesis and Wnt/β-catenin, and Hedgehog signaling pathways that control the cell commitment to adipogenic- or myogenic-differentiation. We found SAM possessed an additive effect with MDI in promoting adipogenesis of 3T3-L1 and C2C12 cells at the beginning of adipogenic differentiation. SAM could also individually induce cell adipogenesis in a dose-dependent manner. Moreover, the expression of Wnt/β-catenin and Hedgehog signals and their targets were suppressed by SAM (P < 0.05). These results demonstrate that SAM, as an increasingly accepted nutritional supplement, can initiate adipogenesis of adipose precursor cells derived from adipose and muscle tissues, a function at least partly correlated with the suppression of Wnt/β-catenin and Hedgehog pathways.     

Shoot organogenesis from leaf explants of Dayaoshania cotinifolia W. T. Wang

Dayaoshania cotinifolia W. T. Wang is a rare and endangered member of the Gesneriaceae family which is endemic to China. To conserve this species, an efficient in vitro propagation and regeneration system via shoot organogenesis was established from young leaf explants. Adventitious shoot induction was possible within 50–60 d on basal Murashige and Skoog medium supplemented with 1–3 μM 6-benzyladenine, although 5 μM 6-benzyladenine induced hyperhydricity. Basal medium containing 1–5 μM thidiazuron induced fewer shoots, while 1–5 μM α-naphthaleneacetic acid induced numerous adventitious roots and a few adventitious shoots. However, when thidiazuron and α-naphthaleneacetic acid were combined, both the induction percentage and number of shoots increased. Leaf explants cultured on induction medium supplemented with 1–5 μM 2,4-dichlorophenoxyacetic acid become necrotic and died. Induction medium supplemented with 1 μM α-naphthaleneacetic acid and 1–3 μM 6-benzyladenine was optimal for inducing adventitious shoots as was the combination of 1–3 μM thidiazuron and 1 μM α-naphthaleneacetic acid. Induction medium containing 2.0 μM 6-benzyladenine and 0.5 μM indole-3-acetic acid was optimal for the multiplication of adventitious shoots. Rooting was achieved on half-strength MS medium supplemented with 3.0 μM indole-3-acetic acid or α-naphthaleneacetic acid and 0.1% activated charcoal. Plantlets were transplanted to a mixture of sand, vermiculite, and humus (1:1:1); 92% survived. This protocol is a unique and effective means to micropropagate this rare and important plant and could serve as a solution for in vitro and ex vitro conservation.     

Selenium and Zinc against Aβ<Subscript>25–35</Subscript>-Induced Cytotoxicity and Tau Phosphorylation in PC12 Cells and Inhibits γ-cleavage of APP

Amyloid beta (Aβ) is the main component of the amyloid plaques that accumulate in the brains of Alzheimer patients. The present study was conducted to investigate whether the combined treatment with selenium (Se) and zinc (Zn) offers more beneficial effects than that provided by either of them alone in reversing Aβ_25–35-induced neurotoxicity in PC12 cells. Cells were pretreated with 0.1 μmol/L of Se and Zn for 4 h, after treated with 10 mmol/L Aβ_25–35 for 24 h. Cells were divided into control and five treated groups, and received either 10 mmol/L Aβ_25–35,10 mmol/L Aβ_25–35 + 0.1 μmol/L Se, 10 mmol/L Aβ_25–35 + 0.1 μmol/L Zn, 10 mmol/LAβ_25–35 + 0.1 μmol/L Se + 0.1 μmol/L Zn, or 0.1 μmol/L Se + 0.1 μmol/L Zn. The result showed that cell viability was decreased in MTT metabolic rate; LDH release and MDA, H₂O₂, and NO levels were increased and the GSK-3β and phosphorylated tau protein level were increased in Aβ_25–35-treated group (P < 0.05 or P < 0.01), which whole changes were attenuated by Se and Zn and Se combined Zn. In order to evaluate whether the Se and Zn have an effect on processing pathway of amyloid precursor protein (APP), we examined the activity of γ-secretase in primary cultured cortical neuron cells. ELISA analysis showed that Se and Zn could inhibit the activity of γ-secretase. Then we also investigated the effect of Se and Zn on the Aβ_1–40 concentration and APP-N-terminal fragment expression from APP695 stably transfected Chinese hamster ovary (CHO) cells. APP695 stably transfected CHO cells were treated with 0.1 μmol/L Se and Zn; cells were divided into control and four treated groups, which received either 0.5 M DAPT, 0.1 μmol/L Se, 0.1 μmol/L Zn, or 0.1 μmol/L Se + 0.1 μmol/L Zn. Se and Zn could decrease Aβ_1–40 production and increase the APP-N-terminal fragment protein expression. These experiments indicate that Se and Zn have a protective effect on AD pathology that a possible mechanism is inhibiting the activity of γ-secretase to decreasing Aβ_1–40 production further influencing the APP processing. Altogether, our findings may provide a novel therapeutic target to treat AD sufferers.     

The positive effect of arabinogalactan on induction of somatic embryogenesis in Quercus bicolor followed by embryo maturation and plant regeneration

This is the first report on the successful induction of somatic embryogenesis in swamp white oak from leaf and shoot apex explants excised from in vitro shoot cultures derived from 6- to 7-year-old trees. We demonstrated that arabinogalactan from larch wood (2–4 mg/L) promoted embryogenesis in the three genotypes evaluated by increasing the frequency of somatic embryogenesis, the embryogenic sites per explant, and by speeding the onset of embryo initiation. The explants were cultured sequentially on three culture media consisting of Murashige and Skoog (MS) salts and vitamins supplemented with 500 mg/L casein hydrolysate and different concentrations of α-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA). Somatic embryogenesis induction frequencies of up to 12.4, 4.5, and 0.7 % were obtained for the three genotypes. Clonal embryogenic lines were maintained by repetitive embryogenesis following culture on MS medium containing 0.44 μM BA with or without 0.27 μM NAA. Before germination, cotyledonary-stage embryos were cultured for 4 weeks in maturation medium (MS medium with half-strength macronutrients) containing 6 % sorbitol. Germination response was significantly improved by applying a 2-month cold storage as a post-maturation treatment. The mineral formulation and plant growth regulator content of the germination medium influenced the frequency of plantlet conversion with the best results achieved on Gresshoff and Doy medium with BA (0.25–0.44 μM). This procedure resulted in over 50–60 % of germinating embryos exhibiting continuous root growth and either epicotyl elongation or shoot development.     

Effect of the culture medium and biotic stimulation on taxane production in Taxus globosa Schltdl in vitro cultures

Taxus globosa is the only species of the Taxus genus that grows in Mexico. In this study, callus cultures from leaves and young shoots of T. globosa were established in Gamborg’s B5 medium supplemented with 2,4-dichlorophenoxiacetic acid (2 mg/L), kinetin (0.5 mg/L) and gibberellic acid (0.25 mg/L). Callus growth and taxane production were evaluated using two culture media: Woody Plant Medium and Gamborg’s B5 supplemented with picloram (2 mg/L), kinetin (0.1 mg/L) and gibberellic acid (0.5 mg/L). The effect of the inoculum size (50, 100 and 150 g FW/L) and culture media (Woody Plant Medium and Gamborg’s B5) with and without the presence of methyl jasmonate (100 μM) on T. globosa cell suspensions was assessed. Taxane analysis revealed that the calli in Gamborg’s B5 produced taxol (50 μg/g DW), baccatin III, 10-deacetyl baccatin III and 10-deacetyl taxol. Woody Plant Medium also induced the production of taxol, although to a lesser extent. The optimum inoculum size was 50 g FW/L. In cell suspension cultures, both media had a significant effect on taxane production when supplemented with methyl jasmonate. In Woody Plant Medium, at day 14, a total concentration of 197.999 μg/L of taxol, 160.622 μg/L of baccatin III, 633.724 μg/L of 10-deacetyl baccatin III and 229.611 μg/L 10-deacetyl taxol were obtained, with total excretion of baccatin III and 10-deacetyl taxol to the culture medium. In Gamborg’s B5, cephalomanine was obtained at a concentration of 91.428 μg/L without elicitation, and all taxanes were excreted to the medium to a variable extent.     

A system for the transformation and regeneration of the recretohalophyte Limonium bicolor

Limonium bicolor, a typical recretohalophyte, has a specialized salt-secreting structure in the epidermis called the salt gland and plays a significant role in improving saline land. Understanding the molecular mechanisms of salt secretion and salt gland development requires an efficient L. bicolor transformation system, which is described in this report. Leaf explants were incubated with Agrobacterium tumefaciens strain EHA105 harboring the plasmid pTCK303 containing the β-glucuronidase gene (GUS) as the transgene reporter and the hygromycin B resistance gene as a selectable marker. Up to 96.9% of leaves were induced to regenerate shoots on an Murashige and Skoog (MS) medium supplemented with 4.4 μM 6-benzyladenine and 1.1 μM α-naphthaleneacetic acid; roots were induced on the MS medium containing 2.5 μM indole-3-butyric acid. This tissue culture system was suitable for Agrobacterium-mediated transformation of L. bicolor. Pre-cultivated explants (2 d old) were incubated with Agrobacterium (0.6–0.7 at OD₆₀₀) in a shaking culture for 20 min; the explants and bacterium were co-cultivated for 4 d in the dark before the explants were transferred to a selection medium containing 8 mg/L hygromycin B and 600 mg/L piperacillin sodium (added to prevent continued Agrobacterium growth). Histochemical assays and PCR to detect the GUS gene showed that transformation frequency was 4.43%. Quantitative PCR and Northern blotting further verified the integration and presence of the GUS gene in L. bicolor. This is the first report of an Agrobacterium-based transformation system for L. bicolor. The system will facilitate a research on the identity and function of genes involved in salt gland development and salt secretion.