Anatomy of the vegetative organs and secretory structures of Rhaponticum carthamoides (Asteraceae)

Roots, stems, rhizomes and leaves of Rhaponticum carthamoides (Willd.) Iljin (a Siberian adaptogenic plant, originating from the Altai and Saian Mountains) of different ages were investigated by means of light and electron microscopy. Schizogenous secretory reservoirs occurred in every organ, and were located within the secondary xylem (adventitious roots and rhizome of young plants), at the interface of endodermis/cortical parenchyma (roots and hypocotyl), along phloem and primary xylem (older rhizome), around the vascular bundles (inflorescence stem, petiole and leaf midrib veins) and along phloem (cotyledonary and leaf veins). At the interface of endodermis/inner parenchyma, secretion accumulated in the intercellular spaces prior to the formation of proper epithelial cells. The secretion as observed by transmission electron microscopy comprised three components: soluble (i.e. absent from sections; probably phenolic), insoluble and strongly osmiophilic (probably phenolic) and insoluble, moderately osmiophilic (probably lipidic). Numerous osmiophilic oil droplets, similar to the lipidic secretion inside the reservoirs, local proliferation of rough endoplasmic reticulum and numerous multivesicular bodies characterized epithelial cells in all organs. Leucoplasts (in subterranean organs) with osmiophilic inclusions and peroxisomes with crystalloid inclusions were specific for parenchyma cells. Peltate glandular hairs were formed on leaf blades.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 144 , 207–233.     

Enhancement of berberine production by spermidine in Thalictrum minus cell suspension cultures

Berberine production in cell suspension cultures of Thalictrum minus L. var. hypoleucum Miq. was significantly enhanced by administration of spermidine, whereas other polyamines such as cadaverine, putrescine and spermine were ineffective. The results of experiments indicated that spermidine causes an increase of ethylene generation which is closely associated with activation of berberine biosynthesis.     

Stimulation of berberine secretion and growth in cell cultures of Thalictrum minus

Summary An endo-pectate lyase (PL; EC 4.2.2.2), originally cloned fiom the phytopathogenic bacterium Erwinia chrysanthemi EC16, was expressed in recA ^– E. coli strain DK1, purified to a single band by isoelectric focusing and used to induce berberine production in established plant suspension cultures of Thalictrum minus L. subsp. saxatile. Addition of 10^–9M pectate lyase c (PLc) stimulated berberine production and enhanced secretion of the alkaloid into the medium. A lower concentration of PLc, 10^–11M, stimulated a transient two-fold increase in cell growth rate relative to untreated cultures. Parallel changes in L-phenylalanine ammonia lyase (PAL; EC 4.3.1.5) activity with the rate of berberine synthesis and the inverse relationship between cell growth and berberine synthesis imply that berberine synthesis is stress-related in this cell line.     

The Effect of Amino Acids as Components of Nutrient Medium on the Accumulation of Protoberberine Alkaloids in the Cell Culture of <Emphasis Type="Italic">Thalictrum minus</Emphasis>

We studied the effects of amino acids on the biosynthesis of protoberberine alkaloids. When 5 mM tyrosine was added to the nutrient medium, the content of alkaloids was reduced by 23% and dry weight was only 77% of the control. On the medium with 1 mM L-tryptophan, the content of alkaloids was somewhat increased (by 20%). Other amino acids (sulfur-containing L-cysteine and L-methionine, and also L-proline and L-arginine) did not affect substantially the content of alkaloids. The addition of 1 and 5 mM L-phenylalanine, which is not a primary precursor to alkaloids, induced the accumulation of alkaloids by the 17th day of the growth cycle by 40 and 140%, respectively, as compared to control treatment. The comparison of various phenylalanine concentrations showed that 7 mM phenylalanine added on the 7– 8th day induced the highest accumulation of alkaloids in the culture medium (above 1 g/l). The content of alkaloids and soluble phenolic compounds increased threefold in both the medium and cells. None of the amino acid tested enhanced biomass accumulation.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 438–442.Original Russian Text Copyright © 2005 by Urmantseva, Gaevskaya, Karyagina, Bairamashvili.     

人参总皂甙应用及改善细胞冻存的研究进展

人参作为一种名贵的药材,具有多方面的作用,近年来关于人参的成分之一人参总皂甙的研究也越来越多。大量实验研究表明,人参总皂甙对改善细胞冻存方面也有很大的作用。组织细胞经深低温冷冻技术处理后其活性能获得有效的保存,在液氮(-196℃)温度下,细胞、组织内各种酶的活力及代谢很低,几乎为零,生命处于所谓的"停滞"状态,在该状态下细胞的活性能得到最大限度的保存。目前常用的冷冻剂有海藻糖、丙二醇、丙三醇以及二甲基亚砜等。但这些冷冻剂均属于化学药剂,在一定程度上会对纤维细胞造成损伤,从而影响细胞的活性。人参总皂甙是从人参中提取出来的有效成分,其具有增强人体表面细胞活性,延缓细胞衰老的作用,为此本文将对人参总皂甙用于细胞冻存的效果进行综述,旨在为细胞的冻存技术提供参考依据。     

人乳头瘤病毒特异性T细胞系细胞冻存后细胞的存活率及功能

研究人乳头瘤病毒特异性T细胞系细胞冻存后细胞的存活率及功能。应用包含10%二甲基亚砜、90%小牛血清的冻存液冻存6个T细胞系(5个CD4 T细胞系,1个CD8 T细胞系)细胞,液氮中冻存32~54个月后复苏,台盼蓝染色法检测复苏后T细胞系细胞的存活率,用酶联免疫斑点法(enzyme-linked immunospot assay,ELISPOT)检测复苏后T细胞系细胞的功能。结果显示,6个T细胞系细胞液氮冻存解冻后细胞的存活率为24.7%~93.5%,过夜培养后细胞的存活率为2.5%~72.2%。CD8 T细胞系细胞的存活率高于CD4 T细胞系细胞。6个复苏后的T细胞系细胞在PHA诱导后均能分泌IFN-γ。人乳头瘤病毒特异性T细胞系细胞冻存复苏后能够保持较好的存活率和功能。     

脱水速率和降温速率对葡萄柚种子超低温保存的影响

以新鲜葡萄柚(Citrus paradise)种子为材料,使用3种脱水速率处理至约20%含水量,然后用饱和盐溶液平衡至10种不同的含水量,再进行简化的二步法超低温保存;同时,以经过15 ℃、50%相对湿度(RH)条件脱水至含水量约12%的葡萄柚种子为材料,使用程序降温仪进行5个降温速率和3个预冷温度的传统二步法超低温保存。结果表明,葡萄柚种子对脱水敏感,种子含水量越低,成苗率越低。而且,脱水速率越快,对种子的损伤越大,成苗率呈现75% RH>50% RH>15% RH;冷冻处理对种子有进一步的伤害,含水量6%～8%的种子超低温保存效果最佳,但脱水速率对超低温保存的种子成苗率的影响不明显。用传统二步法超低温保存葡萄柚种子,与预冷对照相比,经液氮冷冻的种子成苗率都有明显下降。在预冷对照组中,-2.5 ℃·min^-1降温速率的种子成苗率最高,在冷冻保存中-0.5 ℃·min^-1和-0.25 ℃·min^-1的降温速率最有利于超低温保存。预冷温度从-40 ℃降至-60 ℃,未超低温处理种子的发芽率降低,但超低温处理种子的发芽率提高。     

玻璃化冷藏条件对连香树定芽细胞存活率及微结构的影响

选择6种保护剂、2种玻璃化方式和2种低温处理方式对连香树定芽的冷藏条件进行筛选,用TTC还原法测定冷藏定芽的细胞存活率,并用电子显微镜观察定芽细胞冷冻时的受损情况,以优选连香树定芽的冷藏技术条件。结果表明:(1)保护剂、玻璃化方式、冷藏温度均对莲香树定芽的细胞存活率具有显著影响,不同冷藏处理的定芽组织细胞内冰晶形状、数量不同,细胞破裂数也不同。(2)连香树定芽的最佳冷藏条件为:用含甘油35.0mL、乙烯乙二醇20.0mL、DMSO 15.0mL、甘氨酸1.0mg和3%蔗糖脱氨MS液体培养基组成的100mL保护剂于真空下浸泡30min,再经-20℃预冻处理6h后,于液氮中玻璃化和冷藏60d,其定芽细胞的存活率最高,相对存活率达95%。(3)细胞微结构观察显示,在该优选条件下明显抑制了连香树定芽细胞内冰晶体的形成,消除了冰晶对细胞的涨破和刺伤,其细胞结构完整无损伤。     

体外培养和冷冻保存对微囊化细胞生长和内皮抑素表达的影响

微囊化基因工程细胞移植治疗肿瘤是一种新兴的肿瘤治疗方法,如果将此技术应用到临床研究,就需要制备大量的细胞活性良好、重组蛋白表达量高的生物微胶囊。体外培养和冷冻保存是生物微胶囊制备过程中两个重要的环节,因此需要考察体外培养和冷冻保存对微囊化重组基因细胞生长和蛋白表达的影响。以重组CHO细胞为模型,考察了体外培养时间和冷冻保存对微囊化细胞在动物体内生长和内皮抑素表达的影响及体外培养时间对微囊化细胞冷冻保存的影响。结果表明:体外培养时间对微囊化细胞在动物体内生长、内皮抑素表达和微囊稳定性具有较大的影响,体外不培养和培养4d的微囊化细胞在小鼠腹腔内生长良好、内皮抑素表达量高,并且微囊稳定性好,而体外培养8d的微囊化细胞在移植后的第26天破裂。体外培养时间对微囊化细胞冷冻保存也具有较大的影响,体外培养4d和8d的微囊化细胞在液氮中冷冻保存40d,复苏后细胞生长良好、内皮抑素表达量高,而冻存前未经过体外培养的微囊化细胞,复苏后细胞几乎全部死亡。综上所述,生物微胶囊在体外比较适宜的培养时间为4d。并且冷冻保存对微囊化细胞在动物体内生长、内皮抑素表达和微囊稳定性没有显著的影响。     

中国人肿瘤细胞中O~6-甲基鸟嘌呤DNA甲基转移酶活性与对嘧啶亚硝脲敏感性的关系

 分析了20株中国人肿瘤细胞的O~6-甲基鸟嘌呤DNA甲基转移酶(O~6-MT)活性及对嘧啶亚硝脲ACNU的敏感性,发现两者间有较好的线性关系,O~6-MT低,对ACNU敏感,提示O~6-MT可作为使用ACNU对肿瘤化疗时的一项予见性指标。     

Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures

We have recently described Arabidopsis cell suspension cultures that can be effectively synchronised. Here, we describe procedures that allow clonal-transformed cell suspension lines to be produced using Agrobacterium-mediated transformation, and an optimised and straightforward procedure for the cryopreservation and recovery of both parental and transformed lines. Frozen cultures show 90% viability and rapid re-growth after recovery. We show that the cryopreservation procedure is equally applicable to the frequently used tobacco bright yellow (BY)2 cell suspension culture, and that cell cycle synchronisation capacity of parental lines is maintained after both transformation and recovery from cryopreservation. The techniques require no specialised equipment, and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures and providing security against both contamination and cumulative somaclonal variation. Finally, the ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for high-throughput analysis.     

Antibiotic and Disinfectant Resistance in Tap Water Strains – Insight into the Resistance of Environmental Bacteria

Although antibiotic-resistant bacteria (ARB) have been isolated from tap water worldwide, the knowledge of their resistance patterns is still scarce. Both horizontal and vertical gene transfer has been suggested to contribute to the resistance spread among tap water bacteria. In this study, ARB were isolated from finished water collected at two independent water treatment plants (WTPs) and tap water collected at several point-of-use taps during summer and winter sampling campaigns. A total of 24 strains were identified to genus or species level and subjected to antibiotic and disinfectant susceptibility testing. The investigated tap water ARB belonged to phyla Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes. The majority of the isolates proved multidrug resistant and resistant to chemical disinfectant. Neither seasonal nor WTP-dependent variabilities in antibiotic or disinfectant resistance were found. Antibiotics most effective against the investigated isolates included imipenem, tetracyclines, erythromycin, and least effective – aztreonam, cefotaxime, amoxicillin, and ceftazidime. The most resistant strains originate from Afipia sp. and Methylobacterium sp. Comparing resistance patterns of isolated tap water ARB with literature reports concerning the same genera or species confirms intra-genus or even intra-specific variabilities of environmental bacteria. Neither species-specific nor acquired resistance can be excluded.     

Cryopreservation of Digitalis lanata Ehrh. cell cultures: Preculture and freeze tolerance

Cryopreservation experiments were performed with Digitalis lanata cell cultures. The main stress was laid on the behaviour of the cells during the preculture period and the capacity of various preculture additives to induce freeze tolerance. The following compounds were used as preculture additives: trehalose, mannitol, sucrose, melibiose, proline, and sorbitol. They are listed in the order of their respective efficiency. Using trehalose, high post-thaw viability rates were achieved and the cells resumed growth after a short lag period. Melibiose was used as a preculture additive for the first time. Its suitability was in the range of that of sucrose. Proline and sorbitol were not able to induce freeze tolerance in Digitalis cells. Cell viability showed a considerable decrease at the beginning of the preculture period. This reduction was found to be transient in the presence of trehalose, mannitol, sucrose, and melibiose. The damaging effects of proline and sorbitol were too severe to be compensated for by the cells. The PAL activity increased markedly in the presence of proline, whereas the trehalose-treated and the control cells behaved nearly identical to one another.     

Primates: Models for red cell transfusion studies—Cryopreservation and survival of transfused red cells in primates

Primates are excellent models for study of blood transfusion in humans. Erythrocytes of chimpanzees, gibbons, baboons, and rhesus monkeys have a half life (T/2) of 14 to 16 days and a life span (T/10) of approximately 50 to 60 days, which is about half of that found in man. Red cells of primates were cryopreserved by freezing using either a droplet method or the low-glycerol rapid-freeze procedure. Thawed cells survive normally when transfused into the same species. Transfusion of incompatible isologous blood in alloimmunized baboons, in the presence of high titer antibodies, showed survival with small volumes to be virtually nil, but with large volumes, a short normal survival period was followed by a “collapse” phenomenon similar to that seen in humans.     

光强对砀山酥梨石细胞形成的影响及其与内源IAA、ZR和ABA含量的关系

研究光强对砀山酥梨果实发育过程中石细胞形成的影响,探讨了内源激素和梨石细胞形成的关系。结果表明:梨花后第1周~11周是石细胞形成期,石细胞含量为高光强<中光强<弱光强<极弱光强;内源IAA、ZR含量于花后第1周与第7周分别出现2次高峰;花后第1周内源ABA含量最高后迅速下降,IAA、ZR和ABA含量为高光强>中光强>弱光强>极弱光强。高光强促进砀山酥梨果实发育前期IAA、ZR和ABA合成,减少石细胞的积累。     

4个品系小鼠胚胎干细胞系的建立

探索高效的不同品系的小鼠胚胎干细胞的建系方法。B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠,孕马血清促性腺激素(pregnant mare serum gonadotrophin,PMSG) 人绒毛膜促性腺激素(human chorionic gonadotropin,HCG)促排,3.5天交配后(days post coitus,dpc)冲洗子宫取囊胚,或者2.5dpc冲洗输卵管,卵裂球体外培养获取囊胚。囊胚种植到小鼠成纤维细胞饲养层上干细胞培养液培养,4～5天内细胞团扩增后玻璃毛细管挑出,种植到新的饲养层上过夜再行胰蛋白酶消化,3～4天传代一次。对所建立的小鼠ES细胞系进行形态学、染色体核型、AKP染色、体内外分化能力,干细胞分子标记物荧光免疫染色等鉴定。获得10株小鼠胚胎干细胞,具有典型的胚胎干细胞生长特性,符合ES细胞的鉴定标准。结果表明成功的建立了来自B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠的10株ES细胞系。内细胞团挑出过夜增殖后消化的培养方法可能有助于提高ES细胞的建系率。     

亚硝基化在植物细胞死亡及防御反应中的作用

亚硝基化是近年来新发现的不依赖于环磷酸鸟苷的一氧化氮信号转导途径, 是一氧化氮分子通过共价结合修饰靶蛋白的半胱氨酸残基从而改变其功能的过程。该文重点综述了近年来亚硝基化在细胞死亡和抗病反应这两个紧密关联的生物学过程中的最新研究成果, 总结了亚硝基化通过修饰和调控靶蛋白从而促进或抑制细胞死亡和抗病反应, 并对现有研究结果中某些不一致之处提出自己的观点。最后根据动物学领域的最新研究进展对植物学领域未来亚硝基化的研究方向进行了展望。     

Cell cycle-dependent regulation of telomerase activity by auxin, abscisic acid and protein phosphorylation in tobacco BY-2 suspension culture cells

Telomerase is a specialized RNA-directed DNA polymerase that adds telomeric repeats onto the ends of linear eukaryotic chromosomes. It was recently reported that the low, basal level of telomerase activity markedly increased at early S-phase of the cell cycle, and auxin further increased the S-phase-specific telomerase activity in tobacco BY-2 cells. In this study we show that abscisic acid (ABA), a phytohormone known to induce the cyclin-dependent protein kinase inhibitor, effectively abolished both the auxin- and S-phase-specific activation of telomerase in a concentration- and time-dependent fashion in synchronized tobacco BY-2 cells. These results suggest that there exists a hormonal cross-talk between auxin and ABA for the regulation of telomerase activity during the cell cycle of tobacco cells. Treatment of synchronized BY-2 cells with the protein kinase inhibitor staurosporine or H-7 effectively prevented the S-phase-specific activation of telomerase activity. By contrast, when okadaic acid or cantharidin, potent inhibitors of protein phosphatase 2A (PP2A), was applied to the cells, the S-phase-specific high level of telomerase activity was continuously maintained in the cell cycle for at least 14 h after release from M-phase arrest. Incubation of tobacco cell extracts with exogenous PP2A rapidly abrogated in vitro telomerase activity, while okadaic acid and cantharidin blocked the action of PP2A, effectively restoring in vitro telomerase activity. Taken together, these findings are discussed in the light of the suggestion that antagonistic functions of auxin and ABA, and reciprocal phosphorylation and dephosphorylation of telomerase complex, are necessarily involved in the cell cycle-dependent modulation of telomerase activity in tobacco cells.     

肺腺癌A549/DDP细胞周期变化及其多药耐药性

用Fura 2 /AM标记药物敏感的肺腺癌细胞A54 9和抗顺铂药物的肺腺癌细胞A54 9/DDP两种细胞胞内游离Ca2 ,用碘化丙锭 (PI)标记细胞DNA ,检测其胞内Ca2 的变化及两种细胞增殖能力和细胞周期 .实验结果表明 ,抗药性细胞株A54 9/DDP胞浆内游离Ca2 的浓度仅为药物敏感细胞株A54 9的 1/ 3左右 ,同时前者的细胞增殖能力较后者明显增强 ,而且细胞周期也明显缩短 .当用BAPTA AM和EGTA或A2 3187和Thapsigargin处理细胞以降低或升高其胞内自由Ca2 浓度时可改变细胞的生长周期 ,二者也呈现明显差别 .这些结果表明 ,对顺铂产生耐药性的人肺腺癌A54 9/DDP细胞胞内Ca2 浓度的降低 ,可能影响细胞的增殖 ,缩短细胞的生长周期 ,特别是影响起决定作用的G1期 ,从而有利于肿瘤细胞多药耐药特性的维持     

构建稳定表达RFP及嘌呤霉素抗性的K562细胞株

目的构建稳定表达红色荧光蛋白（red fluorescent protein,RFP）和嘌呤霉素（puromycin）抗性的K562．PM．RFP细胞株,便于慢性粒细胞性白血病研究中K562细胞的观察和筛选。方法采用PCR法获得RFP片段,将其插入到慢病毒pGC-FU-3FLAG-IRES—Puromycin载体中获得pGC—PM—RFP重组质粒,经脂质体转染到293T细胞中获得慢病毒LV—PM—RFP,有限稀释法检测慢病毒在293T细胞中的转染效率,用包装获得的慢病毒感染K562细胞,经嘌呤霉素筛选获得RFP阳性的K562-PM—RFP细胞株。结果PCR及测序结果证实目的基因RFP正确克隆至慢病毒质粒中,经慢病毒LV—PM-RFP感染的K562细胞能在嘌呤霉素抗性培养基中存活,并稳定表达RFP。结论成功构建了慢病毒重组质粒pGC—PM-RFP,并获得了携带RFP及嘌呤霉素抗性基因的K562-PM—RFP细胞株。