Establishment and Growth of Embryogenic Suspension Cultures of Ocotea catharinensis Mez. (Lauraceae)

The focus of this study was to test the effects of 2,4-D, sucrose, culture media and initial inocula on the development of embryogenic suspension cultures of Ocotea catharinensis Mez. (Lauraceae). Suspension cultures were established in half-strength MS medium supplemented with 2% (w/v) sucrose either in the absence or in the presence of 2.2 μM 2,4-D, when higher cell viability was achieved. Under this culture condition the maximum fresh weight increase occurred in the fourth week. The cultures were yellow and consisted of a mixture of highly cytoplasmic single cells and small cell aggregates (<0.25 mm). The best proportion of inoculum per volume of medium for suspension culture development was 5% (w/w). Suspension cultures consisting of somatic embryos at the globular and cotyledonary stages (structures ranging from 1 to 3 mm) were successfully established on half-strength MS supplemented with 2% (w/w) sucrose through repetitive embryogenesis from the desiccated mature somatic embryos used as initial inoculum. The failure to initiate liquid cultures from non-desiccated mature somatic embryos was overcome by pre-treatment with air desiccation and reduction of the water content to 6.1 g H₂O g⁻¹ dry weight.     

Cell Cultures of Ecdysteroid-Containing Ajuga reptansand Serratula coronata Plants

Cell suspension cultures of Ajuga reptans L. and Serratula coronata L. were derived from long-cultured calluses. Their growth patterns and morphophysiological characteristics indicated that these cells adapted well to grow in suspensions. During the growth cycle of cell suspension, the main ecdysteroid, among others present in plants, was 20-hydroxyecdyson (20E) for both cell lines. The content of 20E in cell suspension of A. reptans was 4–8 times higher than in the intact plant. After long culturing, the ecdysteroid profile in cell suspensions of S. coronata (20E, inokosterone, makisterone A, ecdyson, and a nonidentified metabolite) became similar to that of the intact plant. The ecdysteroids accumulated with a periodicity during subculturing cycle.     

寡聚糖诱导悬浮培养南方红豆杉细胞的凋亡(英)

在真菌 (Fusariumoxysporumf.vasinfectum (Atkinson)SnyderetHansen)寡聚糖诱导悬浮培养南方红豆杉(Taxuschinensis (Pilger)Rehd .var.mairei (LemeeetL啨ｖｌ.)ChengetL .K .Fu)细胞生产紫杉醇的体系中发现细胞出现凋亡 ,次生代谢增强。电镜观察到细胞核质和原生质出现凝集现象 ,液泡内出现大量的高电子致密体。核DNA经琼脂糖凝胶电泳 ,呈 2 0 0bp的整数倍的梯状条带 (ladders) ;而对照组细胞核DNA完整 ,呈大片段 ,细胞完整 ,细胞器发达 ,但紫杉醇合成速率很低。加入寡聚糖后 ,细胞防御系统开启 ,细胞生长停止 ,次生代谢物酚类物质大量积累且次生壁加厚 ,多酚氧化酶活性迅速提高 ,苯丙烷类代谢途径的关键酶苯丙氨酸解氨酶的活性在 1h后急速提高 ,目的产物紫杉醇在诱导后 72h达到峰值 ,比对照组提高了 6倍 ,且细胞凋亡的出现与紫杉醇合成的峰值具有时间上的一致性。     

寡聚糖诱导悬浮培养南方红豆杉细胞的凋亡

在真菌(Fusarium oxysporum f.vasinfectum (Atkinson) Snyder et Hansen)寡聚糖诱导悬浮培养南方红豆杉(Taxus chinensis (Pilger) Rehd.var.mairei (Lemee et Lévl.) Cheng et L.K.Fu)细胞生产紫杉醇的体系中发现细胞出现凋亡,次生代谢增强.电镜观察到细胞核质和原生质出现凝集现象,液泡内出现大量的高电子致密体.核DNA经琼脂糖凝胶电泳,呈200 bp的整数倍的梯状条带(ladders);而对照组细胞核DNA完整,呈大片段,细胞完整,细胞器发达,但紫杉醇合成速率很低.加入寡聚糖后,细胞防御系统开启,细胞生长停止,次生代谢物酚类物质大量积累且次生壁加厚,多酚氧化酶活性迅速提高,苯丙烷类代谢途径的关键酶苯丙氨酸解氨酶的活性在1 h后急速提高,目的产物紫杉醇在诱导后72 h达到峰值,比对照组提高了6倍,且细胞凋亡的出现与紫杉醇合成的峰值具有时间上的一致性.     

Cell Apoptosis Induced by Oligosaccharide in Suspension Cultures of Taxus chinenesis

Taxol production of Taxus chinensis（Pilger） Rehd. var.mairei (Lemeeet Lévl.) Cheng et L. K. Fu induced by oligosaccharide from Fusarium oxysporum f.vasinfectum (Atkinson) Snyder et Hansen was studied in suspension cultures, and it was found that oligosaccharide triggered cell apoptosis. Under transmission electron microscope the following morphological changes were observed: cell shrinkage, condensation of cytoplasm, nuclear fragmentation, and the increase of high electron density bodies in vacuole in great quantity. In oligosaccharide treated cells, agarose gel electrophoresis revealed that DNA was digested into oligonucleosomal fragments that were times of 200 bp appearing as DNA ladders. Control cells were in normal physiological state, they were intact, abundant in organelle and with integral nucleus DNA, and the rate of taxol biosynthesis in these cells was very low. After the oligosaccharide to the culture system, the defense system of cells was elicited and the secondary metabolism was strengthened, i.e. phenolics were accumulated in the medium, the activity of polyphenol oxidase (PPO) was increased quickly and secondary wall of cells was thickened. The activity of L phenylalanine ammonia lyase (PAL), the critical enzyme of the phenylpropanoid pathway, was increased promptly 1 h after elicitation. The rate of taxol production was improved sharply and the maximal taxol concentration at 72 h was six times that of control. Appearance of cell apoptosis was accompanied with the highest concentration of taxol in suspension cultures.     

Effects of Host Cell Density on Cell Infection Level in Antheraea eucalypti (Lepidoptera: Saturniidae) Cell Cultures Persistently Infected with Nosema bombycis (Microsporida: Nosematidae)

ABSTRACT. Spores of Nosema bombycis Y9101, isolated from the beet armyworm, Spodoptera exigua , were primed with an alkaline solution and inoculated into Antheraea eucalypti cell cultures. Infected cells were subcultured every five days at three cell densities (2.5 × 10³, 5.0 × 10³, and 1.0 × 10⁴ cells/cm²). A difference was observed in the spread of N. bombycis Y9101 infection between low-density and higher-density cultures of host cells. The host cell density did not affect the productivity of secondary infective forms of the parasite. The principal factor determining the rate of microsporidian infection in vitro was the number of host cells existing within the reach of extruded short-coiled polar tubes from spores germinated intracellularly.     

蛇根木细胞悬浮培养进行紫杉醇的生物转化

通过理化及光谱学方法,从蛇根木(Rautwolfia serpentina(L.)Benth.et Kurz.)悬浮细胞内分离到3个紫杉醇同系物,高分辨1H-NMR和MS结构分析表明,它们分别为10-脱乙酰紫杉醇、baccatinⅢ和10-deacetylbaccatinⅢ.本实验未检测出紫杉醇的苷化或羟基化衍生物.     

Ontogenesis secretion and senescence of Tocoyena bullata (Vell.) Mart. (Rubiacaeae) colleters

Colleters are secretory structure present on many families including Rubiaceae. Particular characteristics have been described about colleters secretory cells, however senescence process are still under debate. Tocoyena bullata (Vell.) Mart. (Rubiaceae) shoot apex were collected at Jardim Botânico do Rio de Janeiro, RJ/Brazil. Stipules were separated and fragments were fixed in 2.5% glutaraldehyde and 4.0% formaldehyde in 0.05 m sodium cacodylate buffer, pH 7.2, post fixed in 1.0% osmium tetroxide in the same buffer, dehydrated in acetone, critical‐point‐drying, sputtered coated and observed. For light microscopy fragments were fixed and dehydrated, infiltrated with historesin and stained with 1% toluidine blue. For transmission electron microscopy, the samples were infiltrated with Epoxi resin. Colleters are present on stipule adaxial surface. On the beginning of development, these structures are recognized as small projections. Later on, colleters differentiated and secrete by cuticle rupture. The colleters senescence occurs in a concomitant and indissoluble way of programmed cell death. Ultrastructural analyses during the process strongly suggest the senescence is based on a non‐autolitic programmed cell death. T. bullata colleters, present at stipule abaxial surface are cylindrical secretory structures. Colleters secretory cells originated as stipule projections; differentiate; secrete and senesce by programmed cell death. The secretion and the cell dead occurs in a concomitantly and indissoluble way.     

高产花色苷玫瑰茄细胞系的筛选

花色苷在植物中呈现粉红、红、紫红、紫等颜色,可以用作食品、药品及化妆品的着色剂,亦有药用价值。作为食品添加剂,颜色较合成色素自然,且安全无毒性。早在１９８７年,Ｍｉｚukami^[1]就建议用植物细胞培养物生产花色苷类代替合成色素。所有的植物培养细胞都是异源性的。各细胞之间产花色苷的能力相差很大^[2].因为产花色苷的细胞系带有颜色标记,所以容易识别并通过肉眼选择即可获得高产花色苷的细胞系。筛选的方法很多,如平板饲喂法^[3]、小细胞团法^[4]、细胞块法^[5]、肉眼观察直接挑选法及细胞分栋器法^[6]等。高产系花色苷的含量可增加几倍到几十倍,而且产量稳定。本文采用平板法及小细胞团法筛选高产花色苷的玫瑰茄(Hibiscus sabdariffa L.)细胞系。     

Biotransformation of Paclitaxel (Taxol) by the Cell Suspension Cultures of Rauwolfia serpentina

The biotransformation of paclitaxel (TaxolⓇ) by the cell suspension cultures of Rauwolfia serpentina (L.) Benth. et Kurz. were investigated. Three Paclitaxel-based intracellular metabolites were detected from the cell filter cake and were, by high field ¹H-NMR and MS data, identified as 10-deacetyltaxol, baccatin Ⅲ, and 10-deacetylbaccatin Ⅲ. No glucosidated or hydroxylated derivatives were checked out in this incubation experiment.     

大蒜化学成分的气-质联用分析

本文采用环己烷、乙酸乙酯和正丁醇对大蒜（Allium sativum L.）新鲜鳞茎的95%乙醇提取物进行萃取,并与水蒸气蒸馏法提取的挥发油成分进行比较,用GC-MS对其成分进行定性和定量分析.在环己烷萃取物中共检出112个成分,鉴定了38个化合物,占环己烷萃取物总量的80.08%;在乙酸乙酯萃取物中检出86个成分,鉴定了26个化合物,占乙酸乙酯萃取物总量的56.70%;在正丁醇萃取物中未检出挥发性成分.在水蒸气蒸馏法提取的挥发油中共检出109个成分,鉴定了29个化合物,占挥发油总量的83.58%.大蒜95%乙醇提取物的环己烷和乙酸乙酯萃取物及大蒜挥发油中皆以含硫化合物为主.在环己烷和乙酸乙酯萃取物中,阿霍烯的含量分别为生药的0.00395%和0.00145%,大蒜中阿霍烯含量达0.00540%.     

Inula viscosa (L.) Aiton Ethanolic Extract Inhibits the Growth of Human AGS and A549 Cancer Cell Lines

The present study shows the chemical profile and cytotoxic properties of the ethanolic extracts of Inula viscosa from Northeast Algeria. The extract was obtained by maceration using ethanol. Its phenolic profile was determined using ultra-high-performance liquid chromatography coupled with a diode array detector and an electrospray mass spectrometer (UHPLC-DAD-ESI/MS), which allowed the identification and quantification of 17 compounds, 1,5-O-caffeoylquinic acid being the most abundant. The cytotoxic activity was assessed against human gastric cancer (AGS) and human non-small-cell lung cancer (A549) cell lines, whereas ethanolic extract elicited nearly 60 % and 40 % viability loss toward AGS and A549 cancer cells, respectively. Results also showed that cell death is caspase-independent and confirmed the involvement of RIPK1 and the necroptosis pathway in the toxicity induced by the I. viscosa extract. In addition, the ethanolic extract would not provoke morphological traits in the cancer cells. These findings suggest that I. viscosa can be a source of new antiproliferative drugs or used in preparation plant-derived pharmaceuticals.     

Compact Cell Settlers for Perfusion Cultures of Microbial (and Mammalian) Cells

As microbial secretory expression systems have become well developed for microbial yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, it is advantageous to develop high cell density continuous perfusion cultures of microbial yeast cells to retain the live and productive yeast cells inside the perfusion bioreactor while removing the dead cells and cell debris along with the secreted product protein in the harvest stream. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel, patent‐pending compact cell settlers that can be used more efficiently with microbial perfusion bioreactors to achieve high cell densities and bioreactor productivities. Reproducible results from numerous month‐long perfusion culture experiments using these devices attached to the 5 L perfusion bioreactor demonstrate very high cell densities due to substantial sedimentation of the larger live yeast cells which are returned to the bioreactor, while the harvest stream from the top of these cell settlers is a significantly clarified liquid, containing less than 30% and more typically less than 10% of the bioreactor cell concentration. Size of cells in the harvest is smaller than that of the cells in the bioreactor. Accumulated protein collected from the harvest and rate of protein accumulation is significantly (> 6x) higher than the protein produced in repeated fed‐batch cultures over the same culture duration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:913–922, 2017     

茉莉酸甲酯诱导下红豆杉细胞产生紫杉烷类物质群代谢轮廓分析

红豆杉细胞培养物经甲醇提取和固相萃取粗分离,进行反相高效液相色谱分析,获得了约含13个与紫杉醇极性相关的化合物色谱图,通过质谱联用和对照品参照,归属了这些化合物组成。进一步利用茉莉酸甲酯（MJ）诱导细胞,比较这些组分在诱导前后的相对色谱峰面积变化,结果表明,所有紫杉烷组分的浓度在诱导后都提高,其中以taxchinin M及其结构类似物提高最显著,紫杉醇,B-Ⅲ和B-Ⅵ等比C14位取代的taxuyunnanine C及其衍生物的浓度增加幅度要大,提示MJ对紫杉醇合成中非有效乙酰化旁路代谢有促进作用,而对C14位取代物生成的旁路促进作用不明显。本文为紫杉醇的生物合成研究提供了新的思路和方法。     

Evaluation of the Hydroxynitrile Lyase Activity in Cell Cultures of Capulin （Prunus serotina）

Enzymatic preparations obtained from young plants and cell cultures of capulin were screened for hydroxynitrile lyase activity. The threeweek old plants, grown under sterile conditions, were used to establish a solid cell culture. Crude preparations obtained from this plant material were evaluated for the transformation of benzaldehyde to the corresponding cyanohydrin (mandelonitrile). The results show that the crude material from roots, stalks, and leaves of young plants and calli of roots, stalks, internodes and petioles biocatalyzed the addition of hydrogen cyanide (HCN) to benzaldehyde with a modest to excellent enantioselectivity.     

狂犬病毒CTN—1株在Vero细胞上的适应传代研究

本文报导了用我国狂犬病毒固定毒人二倍体细胞适应株（ＣＴＮ－１）进行Ｖｅｒｏ细胞适应传代研究。通过连续传代培养,滴度可达８．０１ｏｇＬＤ５０／ｍｌ,达到了ＷＨＯ规定的不需浓缩的标准。病毒用０．０１ＭＯＩ感染细胞其产量与１Ｍｏｌ感染量相仿。病毒增殖高峰在４－５天,维持达１５天无明显下降,且可连续收获４－５次。因此,该毒种符合ＷＨＯ提出的疫苗生产毒种要求,可用于狂犬病疫苗生产。     

Typing of nitrogen-fixing Frankia strains by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry

Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) was evaluated as a technique to characterize strains of the nitrogen-fixing actinomycete Frankia. MALDI-TOF MS reliably distinguished 37 isolates within the genus Frankia and assigned them to their respective host infection groups, i.e., the Alnus/Casuarina and the Elaeagnus host infection groups. The assignment of individual strains to sub-groups within the respective host infection groups was consistent with classification based on comparative sequence analysis of nifH gene fragments, confirming the usefulness of MALDI-TOF MS as a rapid and reliable tool for the characterization of Frankia strains.     

Establishment of a New Human Glioblastoma Multiforme Cell Line (WJ1) and Its Partial Characterization

(1) A new human glioblastoma multiforme (GBM) cell line, WJ1, was established from the tissue derived from a 29-year-old patient diagnosed with a grade IV GBM. (2) The WJ1 cell line has been subcultured for more than 80 passages in standard culture media without feeder layer or collagen coatings. (3) GBM cells grow in vitro with distinct morphological appearance. Ultrastructural examination revealed large irregular nuclei and pseudo-inclusion bodies in nuclei. The cytoplasm contained numerous immature organelles and a few glia filaments. Growth kinetic studies demonstrated an approximate population doubling time of 60 h and a colony forming efficiency of 4.04%. The karyotype of the cells was hyperdiploid, with a large subpopulation of polyploid cells. Drug sensitivities of DDP, VP-16, tanshinone IIA of this cell line were assayed. They showed a dose- and time-dependent growth inhibition effect on the cells. (4) Orthotopic transplantation of GBM cells into athymic nude mice induced the formation of solid tumor masses about 6 weeks. The cells obtained from mouse tumor masses when cultivated in vitro had the same morphology and ultrastructure as those of the initial cultures. (5) This cell line may provide a useful model in vitro and in vivo in the cellular and molecular studies as well as in testing novel therapies for human glioblastoma multiforme.     

PFA: Program for the Quantitative Assessment of Cell Metabolism by Spectral Data Analysis

Assessment of mitochondrial oxidative metabolism has wide-ranging importance, from pharmacokinetic analysis to studies in cell viability and apoptosis. Here we present the Perfusion File Analyzer (PFA) application for the real-time analysis of spectral data to measure cytochrome c reduction, cytochrome a3 reduction, and other parameters important to cellular metabolism, which are collected during tissue perfusion experiments. Our current efforts are focused on quantitating changes in mitochondrial function by normalizing baseline drift of spectral data while addressing two major challenges: (1) a lack of real-time feedback from the system when aiming is compromised, and (2) an inability to adjust calculated data in the event of spectral shift. PFA has been developed to address these issues, and is currently used for quality assessment of human islets prior to clinical transplantation.