Xylogenesis in tissue culture II: Microtubules,cell shape and secondary wall patterns

Summary InZinnia suspension cultures, two general categories of tracheary element (TE) secondary wall patterns can be distinguished: bands and webs. Band patterns are found in elongated cells or regions of cells, web patterns in isodiametric cells or regions of cells. Interphase cortical microtubule arrays, organized before overt differentiation occurs, determine both the shape of the cell and whether band or web patterns will be deposited at the time of TE formation. By altering cell shape and consequently also altering the interphase microtubule array, it is possible to control the type of wall pattern which is deposited.These results provide support for the hypothesis which states that the organization of interphase cortical microtubule arrays (i.e., random or parallel), which laterally associate during tracheary element differentiation, determines the pattern in which secondary walls will be deposited.     

Immunofluorescent and calcofluor white staining of developing tracheary elements inZinnia elegans L. Suspension cultures

Summary Xylogenesis has been studied in primary suspension cultures ofZinnia elegans L.: The wall patterns produced in culture closely resemble those described for intact tissues (annular, spiral, reticulate, scalariform, pitted). Using fluorescence microscopy and immuno-cytochemical techniques we have followed both the changes in wall deposition and microtubule organization during xylogenesis. Calcofluor white has been used to detect secondary wall deposition before it can be observed using either phase contrast or polarization optics. The development of tracheary elements can be divided into three stages: 1. microtubules grouped into bands without secondary wall deposition evident; 2. groups of microtubules subtending wall material only visible using Calcofluor white; 3. a complex microtubule pattern reflected by well developed wall thickenings detected using Calcofluor, phase contrast and polarization optics.     

Xylogenesis in tissue culture III: Continuing wall deposition during tracheary element development

Summary DifferentiatingZinnia cultures have two bursts of tracheary element (TE) formation which resemble the production of proto- and metaxylem in higher plants. TEs in the first burst have annular, spiral or reticulate secondary wall patterns while those in the second burst have reticulate, scalariform or pitted walls. Continuing wall deposition in TEs results in the transformation of annular or spiral patterns into scalariform or pitted. Indirect immunofluorescent observation of TE microtubules (MTs) during continuing wall deposition indicates an annular/spiral pattern is deposited first followed by the introduction of new arrays of MTs which guide later, in-filling wall deposition.Abbreviations TE Tracheary element - MTs Microtubules     

Tobacco protoplasts differentiate into elongate cells without net microtubule depolymerization

Summary Microtubules (MTs) are important for plant cell morphogenesis because they influence the deposition of cell plate and wall components. It has been observed that tobacco protoplasts contain a disordered MT array in the cortex. Following several days in culture, these protoplasts become elongate cells with an orderly cortical MT array. The transformation of the MT array may occur by net depolymerization of the disordered MTs and repolymerization of MTs into an ordered array, or by movement of the array as an integral unit. To experimentally distinguish between these two possibilities, the drug taxol was used to stabilize MTs. Protoplasts derived from suspension cultured tobacco,Nicotiana tabacum, were grown in a medium containing the two plant hormones -naphthaleneacetic acid and benzyladenine, in the presence or absence of 10M taxol. Changes in cell size and shape were quantified using a video image analysis system. Cell elongation had begun within 48h of protoplast conversion, in both treatments, and continued for 7 days. Immunolocalization of tubulin showed that, in the majority of cells, MTs were disorganized immediately following protoplast conversion. After elongation, the MT arrays were observed to have reoriented to an ordered state. Taxol-treated protoplasts were found to elongate faster and to a greater extent than the non-treated controls. Additionally, the cortical array of taxol-treated protoplasts reorganized more quickly. These data indicate that the net depolymerization of disordered cortical MTs is not necessarily required for the differentiation of a protoplast into an elongate cell.Abbreviations APM amiprophosmethyl - BSA bovine serum albumin - DIC differential interference contrast - DTT dithiothreitol - EGTA ethylenegrycol-bis-(-aminoethyl ether)N,N,N,N-tetra-acetic acid - ELISA enzyme-linked immunosorbent assay - FMS Fukuda, Murashige, and Skoog - MS Murashige and Skoog - MT(s) microtubule(s) - PBS phosphate buffered saline - PIPES piperazine-N,N-bis (2-ethanesulfonic acid, 1.5 sodium) - PM plasma membrane - Tris Tris(hydroxymethyl)amino-methane     

The effect of taxol onTriticum preprophase root cells: preprophase microtubule band organization seems to depend on new microtubule assembly

Summary To examine whether preprophase microtubule band (PPB) organization occurs by rearrangement of pre-existing, or by assembly of new microtubules (Mts), we treated root cells ofTriticum turgidum with taxol, which stabilizes pre-existing Mts by slowing their depolymerization. With taxol early preprophase cells failed to form a normal PPB and PPB narrowing was prevented in cells that had already formed a wide one. The PPB became persistent in prometaphase cells and the formation of multipolar prophase-prometaphase spindles was induced. These data favour the suggestion that PPB formation and narrowing, as well as prophase spindle development, are dynamic processes depending on continuous Mt assembly at the PPB site and in the perinuclear cytoplasm.Abbreviations Mt microtubule - MTOC microtubule organizing centre - PPB preprophase microtubule band - DMSO dimethyl sulfoxide     

Photoautotrophic growth of Marchantia polymorpha L. cells in suspension culture

A cell line of M. polymorpha was grown photoautotrophically in liquid suspension culture using 1% CO₂ in air as sole carbon source. The growth rate in terms of cell dry-weight during the exponential phase was 0.171 and the doubling time was 1.76 d. The rate of increase in chlorophyll was 1.6 times higher than the growth rate. The highest content of chlorophyll was 24 mg g^-1 dry weight, and the photosynthetic activity of the cells in the exponential phase, as calculated from the growth rate, was at least 60 mol mg^-1 chlorophyll h^-1.     

Effects of marine organisms extracts on microtubule integrity and cell cycle progression in cultured cells

Marine organisms have been reported to be a rich source of biologically active compounds, but when compared with plants, their use is much more restrict in popular medicine. Among marine animals, sponges and ascidians are two of the most prominent sources of new compounds with cytotoxic potential. In this study, we tested 40 extracts of marine sponges and ascidians from southeastern Brazilian coast aiming to evaluate their anti-proliferative effects on cultured cells. The extracts of Amorphinopsis sp., Arenosclera brasilensis, Cystodytes dellechiajei, Cliona aff. celata, Didemnum sp., Hadromerida and Scopalina ruetzleri showed an IC₅₀≤30 μg/mL and produce strong effects on microtubules organization and on the cell cycle progression.     

Time course and auxin sensitivity of cortical microtubule reorientation in maize roots

Summary The kinetics of MT reorientation in primary roots ofZea mays cv. Merit, were examined 15,30,45, and 60 min after horizontal positioning. Confocal microscopy of longitudinal tissue sections showed no change in MT orientation 15 and 30 min after horizontal placement. However, after 45 and 60 min, MTs of the outer 4–5 cortical cell layers along the lower side were reoriented. In order to test whether MT reorientation during graviresponse is caused by an auxin gradient, we examined the organization of MTs in roots that were incubated for 1 h in solutions containing 10^–9 to 10^–6M IAA. IAA treatment at 10^–8M or less showed no major or consistent changes but 10^–7 M IAA resulted in MT reorientation in the cortex. The auxin effect does not appear to be acid-induced since benzoic acid (10^–5M) did not cause MT reorientation. The region closest to the maturation zone was most sensitive to IAA. The data indicate that early stages of gravity induced curvature occur in the absence of MT reorientation but sustained curvature leads to reoriented MTs in the outer cortex. Growth inhibition along the lower side of graviresponding roots appears to result from asymmetric distribution of auxin following gravistimulation.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether) N,N,NN-tetraacetic acid - MTs cortical microtubules - QC quiescent center - MES/TRIS 2-(N-morpholino)ethanesulfonic acid/tris(hydroxymethyl)aminomethane - IAA indole-3-acetic acid - PBS phosphate buffered saline - PHEMD [60 mM Pipes (piperazine-diethanesulfonic acid), 25 mM Hepes (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid), 10 mM EGTA, 2mM MgCl₂ pH7.0 adjusted with NaOH] containing 5% dimethyl sulfoxide     

Taxol maintains organized microtubule patterns in protoplasts which lead to the resynthesis of organized cell wall microfibrils

Summary We have investigated the effects of microtubule stabilizing conditions upon microtubule patterns in protoplasts and developed a new method for producing protoplasts which have non-random cortical microtubule arrays. Segments of elongating pea epicotyl tissue were treated with the microtubule stabilizing drug taxol for 1 h before enzymatic digestion of the cell walls in the presence of the drug. Anti-tubulin immunofluorescence showed that 40 M taxol preserved regions of ordered microtubules. The microtubules in these regions were arranged in parallel arrays, although the arrays did not always show the transverse orientation seen in the intact tissue. Protoplasts prepared without taxol had microtubules which were random in distribution. Addition of taxol to protoplasts with random microtubule arrangements did not result in organized microtubule arrays. Taxol-treated protoplasts were used to determine whether or not organized microtubule arrays would affect the organization of cell wall microfibrils as new walls were regenerated. We found that protoplasts from taxol-treated tissue which were allowed to regenerate cell walls produced organized arrays of microfibrils whose patterns matched those of the underlying microtubules. Protoplasts from untreated tissue synthesized microfibrils which were disordered. The synthesis of organized microfibrils by protoplasts with ordered microtubules arrays shows that microtubule arrangements in protoplasts influence the arrangement of newly synthesized microfibrils.Abbreviations DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - IgG immunoglobulin G - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - PBS phosphate buffered saline     

Alkaline phosphatase staining of pig and sheep epiblast cells in culture

To define better the characteristics of pig and sheep epiblast cells in culture, the cells were tested for the presence of alkaline phosphatase (AP), a biochemical marker characteristic of mouse embryonic stem cells. Pig and sheep epiblast cells were positive for AP staining both at isolation from the blastocyst and after primary in vitro culture. The innermost portion of the attendant endoderm surrounding the epiblast was also positive for AP staining during primary culture. AP staining was lost upon differentiation or senescence of the epiblast cells. Also, all differentiated epiblast-derived cell cultures were negative for AP staining, with the exception of neuron-like cultures. Epiblast-like cells were cultured from day 10 (pig) and day 13 (sheep) embryonic discs, and these cells were also AP positive until they differentiated. Trophectoderm-endoderm-like cells from embryonic discs were AP negative or weakly positive. AP is a convenient marker for undifferentiated pig and sheep epiblast cells in culture when used in conjunction with cell morphology analysis. © 1993 Wiley-Liss, Inc.     

Medium optimization for cell biomass yield and anthocyanin production in cell suspension culture of Melastoma malabathricum

Macronutrients influence the cell biomass yield and anthocyanin production in plant cell suspension cultures. However, different species respond differently for different nutrient concentrations. In this study, we tested the effect of different concentrations of macronutrients on the cell cultures of Melastoma malabathricum L. The results showed that the addition of NH₄NO₃ did not show any increment in the cell biomass but it enhanced the anthocyanin accumulation. Results indicated that CaCl₂.2H₂O and MgSO₄.7H₂O played a more important role in anthocyanin accumulation than the cell growth of M. malabathricum. However, the presence or absence of KNO₃ and KH₂PO₄ did not show any effect on either the cell biomass or the anthocyanin production.     

Regulation of induction of phenylalanine ammonia-lyase in suspension cultures of Phaseolus vulgaris

Total RNA was extracted from fast growing suspension cells of bean, the mRNA was translated and the products of protein synthesis analysed by gel electrophoresis. Actinomycin D (20 g ml^–1) added to the cultures 12 h before the induction of phenylalanine ammonia-lyase (PAL) activity by naphthylacetic acid (NAA) (1 mg/l) and kinetin (0.2 mg/l) failed to prevent the increased activity of the enzyme usually produced by this ratio of the plant growth hormones. PAL was isolated and purified from suspension cultured bean cells. The purified enzyme ran as a single band on polyacrylamide gel electrophoresis. The protein translated from RNA prepared from induced and non-induced cells was separated by gel electrophoresis and the bands of protein on the gels were compared. There was no evidence for an increase in the amount of PAL synthesised in vitro from the mRNA of induced cells even though these had 5 times the amount of activity of the enzyme compared with that of the non-induced cells. The results indicate that the induction of PAL activity is not immediately preceeded by an increase in the synthesis of PAL-mRNA by the cells. The control of the activity of the enzyme is discussed with respect to this finding.Abbreviations PAL phenylalanine ammonia-lyase - NAA 3naphthylacetic acid - DEAE Diethylamino ethyl - EDTA Ethylenediamine tetraacetate - SDS Sodium dodecyl sulphate     

Accumulation of F-actin during cytokinesis inAllium. Correlation with microtubule distribution and the effects of drugs

Summary The distribution of F-actin in the phragmoplast/cell plate complex of formaldehyde-fixedAllium root cells was visualized with rhodaminephalloidin (RP). Increased RP fluorescence appears in late anaphase in a broad zone between separating chromosomes. The fluorescence is mostly amorphous in appearance and does not resemble the distinct actin fibers seen in interphase cells. The actin becomes more concentrated near the midplane by telophase and takes the form of a relatively bright layer of fluorescence adjacent to the forming cell plate. This distribution differs markedly from that of phragmoplast microtubules (MTs) which extend back from the plate toward the daughter nuclei. F-actin continues to accumulate in new parts of the expanding phragmoplast, while RP fluorescence gradually decreases near older portions of the plate. It disappears completely near the new wall in most interphase cells. Treatment of root tips with cytochalasin B or D before fixation markedly reduces RP fluorescence, but phragmoplast MTs remain. Colchicine or oryzalin treatment leads to the disappearance of both phragmoplast actin and MTs. The possible function of actin in the phragmoplast/cell plate complex is discussed.Abbreviations CB cytochalasin B - CD cytochalasin D - CIPC isopropyl N-(3-chlorophenyl-)carbamate - DIC differential interference contrast - MT microtubule - PBS phosphate buffered saline - PM plasmalemma - RP rhodamine-phalloidin     

紫杉醇抑制前列腺癌增殖的体内实验研究

目的探讨紫杉醇对人前列腺癌细胞PC-3增殖的体内抑制作用。方法建立体内绿色荧光蛋白（GFP）标记的人雄激素非依赖性前列腺癌细胞PC-3裸鼠原位移植瘤模型,观察紫杉醇对裸鼠前列腺癌原位移植瘤的体积、重量的影响。结果裸鼠模型体内实验显示,与对照组（100μL生理盐水）相比,紫杉醇处理组（0.5 mg/kg）在给药第18天后能显著抑制前列腺肿瘤的体积（P〈0.05）;紫杉醇处理组在抑制前列腺肿瘤重量方面与对照组相比亦有明显抑制作用（P〈0.05）。与对照组相比G31P处理组VEGF（P〈0.05）的表达差异具有统计学意义（免疫组化法）。结论紫杉醇在体内实验中能明显抑制人雄激素非依赖性前列腺癌细胞系PC-3的增殖。     

Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development

Two experiments were designed to assess the effectiveness of cryopreserving bovine MII oocytes using cryotops as the carrier system for vitrification. In the first experiment, we examined the developmental competence of oocytes after: (i) vitrification in open-pulled straws (OPS method); or (ii) vitrification in <0.1 μl medium droplet on the surface of a specially constructed fine polypropylene strip attached to a plastic handle (Cryotop method). In the second experiment, warmed oocytes that had been vitrified in OPS or cryotops were fixed to analyze spindle and chromosome configuration. In all experiments both cow and calf oocytes were used. Significantly different fertilization rates were observed between the vitrification groups: 31.5% and 20.2% for the cow and calf oocytes vitrified in OPS, respectively, versus 46.1% and 46.4% for the oocytes vitrified using cryotops. After in vitro fertilization, 3.8% of the calf oocytes and 5.3% of the cow oocytes developed to the blastocyst stage. All blastocysts from vitrified oocytes resulted from the Cryotop method. A significantly lower percentage of the OPS-vitrified calf oocytes showed a normal spindle configuration (37.8%) compared to control fresh oocytes (69.9%), while normal spindle and chromosome configurations were observed in a significantly higher proportion of the cryotop-vitrified calf oocytes (60.2%). For the cow oocytes, 60.6% in the OPS group and 60.3% in the Cryotop group exhibited a normal morphology after warming. These findings suggest the cryotop system is a more efficient carrier for vitrification than OPS for the cryopreservation of bovine oocytes.     

Xylogenesis and lignification in plant tissue cultures differently affected by 8-hydroxyquinoline sulphate

Summary An established callus tissue derived from mesophyll cells ofSedum telephium L. was used to assess the influence of 8-hydroxyquinoline sulphate (Quinosol) on the differentiation of tracheary cells.Electron microscopic study of cells from calli grown in the presence of Quinosol (20.55 and 41.10 M) showed no marked ultrastructural differences compared to controls at the same developmental stages. Growth of calli diminished with increasing concentrations of Quinosol, being drastically affected by the maximum dose used (82.20 M). On the other hand, xylogenesis, expressed as the number of tracheary elements to parenchyma cells, increased with increasing concentrations of the drug. However, lignin and phenolic content responded differently, increasing at a low Quinosol dose (20.55 M) but decreasing at higher concentrations of the drug (41.10 and 82.20 M).The impaired growth of the culture was due to decreased cell proliferation in spite of an increase in differentiation of xylem-like cells. However, the calli with numerous tracheary elements synthesized less lignin than calli with a lower percentage of such cells.     

Efficiently differentiating vascular endothelial cells from adipose tissue-derived mesenchymal stem cells in serum-free culture

Adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to be multipotent and to differentiate into various cell types, including osteocytes, adipocytes, chondrocytes, and neural cells. Recently, many authors have reported that ASCs are also able to differentiate into vascular endothelial cells (VECs) in vitro. However, these reports included the use of medium containing fetal bovine serum for endothelial differentiation. In the present study, we have developed a novel method for differentiating mouse ASCs into VECs under serum-free conditions. After the differentiation culture, over 80% of the cells expressed vascular endothelial-specific marker proteins and could take up low-density lipoprotein in vitro. This protocol should be helpful in clarifying the mechanisms of ASC differentiation into the VSC lineage.     

大鼠应激耐受多系分化细胞的分离、扩增改良技术

目的 探讨体外从大鼠骨髓间充质干细胞（BMSCs）中分离应激耐受多系分化细胞（Muse cells）及其扩增培养的技术方法.方法 分离、扩增、鉴定BMSCs,应用长时间应激方法酶消化分离Muse细胞,悬浮扩增培养4d后Western blot鉴定特异性抗原.结果 第3代生长良好的BMSCs中表达CD29、CD90而不表达CD45的占细胞总数的97.89%.长时间酶消化结合悬浮培养后,以酶消化8h组细胞表达CD105显著高于其余各组（P〈0.05）,符合Muse细胞特征.结论 Muse细胞可由BMSCs经消化分离扩增获得,可望为其进一步研究应用奠定基础.     

Cytoplasmic actomyosin fibrils in tissue culture cells

Summary A special cell line derived from a rat mammary adenocarcinoma (RMCD cells) displays a distinct pattern of actomyosin fibrils (AM fibrils) visible with phase contrast, Nomarski interference and polarized light optics. It was shown that the cytoplasmic AM fibrils are arranged as bundles of highly parallel F-actin filaments. The chemical nature of the filaments was identified by incubation with heavy meromyosin from rabbit skeletal muscle.These cytoplasmic actomyosin fibrils actively contract under isotonic conditions. This was shown by contraction experiments under polarized light optics, by cinematographic analysis and by direct proof of the contractility of AM fibrils isolated by laser micro-dissection. Thus, cytoplasmic AM fibrils can be assumed to represent structures essential for motive force generation in contraction processes in non-muscle cells.We thank Dr. W. Meier-Ruge and Mr. W. Bielser (Basic Medical Research Departments, Sandoz AG, Basle, Switzerland) for making the laser equipment available to us and for their kind cooperation during this investigation. Supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

红豆杉细胞周期时相与紫杉醇诱导合成的关系初探

利用植物细胞培养生产药用次生代谢产物 ,普遍存在着次生代谢物产量不稳定的问题。次生代谢物是一大类无明显生理功能或者非生长发育所必需的小分子有机化合物 ,其生物合成与积累一般发生在植物某些器官和组织发育的某个时期。因此 ,植物细胞的生长发育状态与次生代谢物产量有密切的关系。细胞周期时相在很大程度上反映细胞本身所处的状态 ,对细胞周期时相进行调控可能对次生代谢物产量具有重要影响 [1 ] 。诱导作用是提高植物次生代谢物产量的有效方法之一 [2 ,3]。我们以 2种经典同步化方法 ,建立红豆杉悬浮细胞初始周期时相模型 ,对不同…