Ultrastructural and Cytochemical Study on Mature Pollen of Pinus tabulaeformis

The pollen of Pinus tabulaeformis Cart. comprised two prothallial cells, a generative cell and a tube cell which degenerated at pollen maturation. The generative cell had its own cell wall, seperating from the intine of pollen, but with its side wall attached to the infine. Cytoplasmic channels were present on the side of the generative cell wall, which faced to the tube cell cytoplasm. The generative cell differed conspicuously from the tube cell. The main differences include: ( 1 ) The chromatin in the generative cell nucleus was condensed, but was dispersed and had numerous nueleare pores in the tube cell nucleus; (2)There was no microbody in the generative cell but many microbodies were present in the tube cell cytoplasm; (3)More inclusions were present in the tube cell than in the generative cell. Both the generative cell and the tube cells contained lipid bodies and amyloplasts in the cytoplasm, but there were more amyloplasts in the former. The tube cell also contained a few proteins which was absent in the generative cell. In addition, there were numerous mitochondria, polyribosomes, and a few endoplasmic reticulums and dictyosomes in the generative and tube cells. DAPI staining demonstrated numerous cytoplasmic DNA in both generative cell and tube cell. The mode of cytoplasmic inheritance, and the composition, structure and the nature of the pollen wall of P. tabulaefonnis are also discussed in this paper.     

改进的培养体系在小鼠体细胞核移植及重构胚ES细胞分离培养中的应用

取8周后的雌性昆明小鼠进行超排,取卵母细胞用作核受体,收集卵母细胞周围的卵丘细胞作核供体,进行体细胞核移植。核移植重构胚经SrCl2激活处理6h后,与改良的M16培养液和小鼠输卵管上皮细胞共培养;将发育到早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加含心肌细胞培养液的ES细胞培养液;把孵出的ICM进行消化接种培养,对孵出的ES细胞集落进行鉴定培养。结果显示,以小鼠卵丘细胞为核供体,体细胞核移植重构胚激活率为65．23％,囊胚发育率为11．69％;9个核移植重构囊胚中分离出ES细胞集落,分离率为2．77％;分离出的核移植ES细胞集落具有岛屿状团状隆起结构、碱性磷酸酶染色呈阳性,体外分化可形成类胚体,并能分化成上皮样或梭形细胞。ES细胞集落经常规冻存和复苏后,显示出同冻存前相似的集落形态,并具有较强的增殖能力。实验证实小鼠输卵管上皮细胞、改良的M16培养液及含心肌细胞培养液的ES细胞培养液可以更为成功地运用于小鼠的体细胞核移植及ES细胞的分离培养研究。     

成体心脏干细胞经静脉移植后在心梗小鼠体内的分布研究

目的：观察心肌梗死小鼠静脉移植成体心脏干细胞后,细胞在小鼠体内各器官的分布情况。明确心梗后静脉移植成体心脏干 细胞在小鼠体内的分布和归巢情况。方法：分离培养小鼠心脏成体干细胞,采用流式细胞仪鉴定细胞,通过亲脂性染料CM-DiI标 记细胞后行小鼠急性心肌梗死模型建立和细胞移植,分别在细胞移植后7、14、8 天取小鼠心脏、肝脏、脑、脊髓、肺脏,行冰冻切 片,在荧光显微镜下观察移植细胞在各组织器官存活和分布情况。结果：成体心脏干细胞分离培养后呈贴壁生长,流式细胞仪检 测显示细胞纯度>80%。CM-DiI标记后荧光显微镜下观察可见标记的细胞胞浆胞核均被染成呈明亮的红色。心肌梗死后经静脉 移植成体心脏干细胞,细胞在各组织中分布呈变化过程,7 天时,在肺脏和肝脏分布较多,至14 天和28 天时,肺脏和肝脏分布减 少,心脏分布逐渐增多,表现出向心脏的" 归巢" 现象,而脑和脊髓在28 天的观察时间内分布较少。结论：采用CM-DiI标记心 脏成体干细胞,操作简单,标记效果好,可用于短期的细胞体内追踪。小鼠心肌梗死后行经静脉成体心脏干细胞移植,28 天后细胞 在心脏的分布逐渐增多,表现出向心脏的" 归巢" 现象。     

Physico-chemical characteristics of cell walls from Arabidopsis thaliana microcalli showing different adhesion strengths

Changes in the composition and structure of cell walls and extracellular polysaccharides (ECP) were studied during the growth of suspension-cultured Arabidopsis thaliana microcalli. Three growth phases, namely the cell division phase, the cell expansion phase, and the stationary phase, were distinguished and associated with a decreasing cell cluster adhesion strength. Degradation of the homogalacturonan pectic backbone and of linear pectic side chains (1,4)-beta-D-galactan were observed concomitantly with the cell expansion and stationary phases and the decrease in cell adhesion. Also, in the stationary phase, branched (1,5)-alpha-L-arabinans were linearized. The AGP content of the culture medium increased while it decreased in the cell wall during cell growth and as cell adhesion decreased. These data suggest that, in addition to homogalacturonan, pectic side chains and AGP are involved in plant cell development and particularly in cell-cell attachment.     

Helper factors derived from autologous mixed lymphocyte cultures

Human tonsillar lymphocytes infected with Epstein-Barr virus (EBV) were cultured at a cell concentration of 2 × 10⁶/ml in a 0.01-ml volume in microtest plates and cell lines composed of relatively small numbers of clones (oligoclonal) were established. Culture supernatants of 1020 cell lines thus established were screened for anti-phosphorylcholine (PC) antibody production by passive hemagglutination (PHA) assay and 10 cell lines with high anti-PC PHA titers were obtained. Plaque-forming cell (PFC) assay of these cell lines revealed that in seven cell lines more than 1% of the total cells and especially in two cell lines more than 10% of the total cells were anti-PC antibody-producing cells. Free PC concentrations required to inhibit PHA (8 PHA titer) and PFC (50% inhibition) correlated well in the same cell lines, but among the different cell lines considerable differences (10- to 1000-fold) of the PC concentration were obtained, indicating that anti-PC antibody-producing cells with different binding affinities to PC were transformed in these cell lines. PHA inhibition by monospecific antisera against immunoglobulin heavy-chain classes revealed that anti-PC antibodies produced in these cell lines were of the IgM class. Cloning experiments were performed and one clone stably producing anti-PC antibodies was obtained.     

Relationship between the rearrangement of immunoglobulin genes, the appearance of a B lymphocyte antigen, and immunoglobulin synthesis in murine pre-B cell lines

Eighteen Abelson virus-transformed immature B cell lines were established and immunoglobulin biosynthesis, expression of a B lymphocyte antigen detected by a monoclonal antibody, and rearrangement of immunoglobulin genes in these cell lines were studied. Only one cell line (A1) synthesized micro-chains but no light chains, and the other cell lines synthesized no detectable immunoglobulins. None of the cell lines established had detectable membrane-associated IgM. Fifteen cell lines expressed a B lymphocyte antigen on their cell surfaces. In three cell lines, however, the majority (greater than 99%) of cells did not express this antigen. Heavy chain genes were rearranged on both chromosomes in all the cell lines, although one heavy chain gene was deleted in three cell lines. In 12 of 18 cell lines, one or both kappa-chain genes were rearranged. In six cell lines, however, both kappa-chain genes remained in embryonic form; lambda-chain genes were in embryonic form in all the cell lines. These results suggested the hierarchy of Ig gene rearrangements, beginning with mu and proceeding to kappa and then to lambda. JH rearrangement was also shown to precede the appearance of a B lymphocyte antigen. In three cell lines (A1-A3), which were considered subclones derived from a single common precursor, it was suggested that one rearranged JH gene was functional, and the other was nonfunctional, indicating that allelic exclusion already operated in pre-B cells.     

Butanol-extractable and detergent-solubilized cell surface components from murine large cell lymphoma cells associated with adhesion to organ microvessel endothelial cells.

Metastatic variant cell lines of the murine RAW117 large cell lymphoma were used to study the cell surface components involved in syngeneic tumor cell/microvessel endothelial cell interactions. Poorly liver-metastatic parental RAW117-P cell line adhered to murine hepatic sinusoidal endothelial cell monolayers at significantly lower rates than the liver-selected, highly liver-metastatic RAW117-H10 line and both cell lines were poorly adherent to lung microvessel and bovine aorta endothelial cells. Viable, 2% 1-butanol-treated RAW117-H10 tumor cells formed fewer liver tumor nodules in experimental metastasis assays than untreated H10 cells and were significantly less adherent to murine hepatic sinusoidal endothelial cell monolayers. When 2% 1-butanol extracts of metabolically labeled or CHAPS detergent lysates of cell surface-labeled tumor cells were analyzed for their ability to bind to fixed microvessel endothelial cell monolayers, quantitative differences were found in the extractable tumor cell surface components that bound to the different organ-derived microvessel endothelial cells. Cell surface components (1-butanol extractable), of Mr approximately 85,000-90,000 and approximately 37,000-40,000 bound to hepatic sinusoidal endothelial cell monolayers to a greater extent than to murine lung microvessel endothelial or bovine aortic endothelial cell monolayers, whereas tumor cell surface components of Mr approximately 45,000, approximately 33,000, and approximately 25,000 bound similarly to endothelial cells regardless of origin. The results suggest but do not prove that tumor cell/endothelial cell adhesion involves multiple tumor cell surface components, some of which commonly bind to various endothelial cells and others for which binding may be dictated by the tissue origin and type of endothelial cell. Particular RAW117 butanol-extractable cell membrane components were associated with tumor cell-endothelial cell adhesion, and these components could be responsible, in part, for the preferential adhesion of RAW117 cells to liver sinusoidal endothelial cells and metastasis to liver.     

Step-fortifications of nutrients in mammalian cell culture

A series of high-density media for mammalian cell culture were developed by step-fortifications of most nutrient components in RPMI-1640 medium. Each medium constituting the series was constructed to meet in vitro cell growth limitations. Four different cell lines were cultivated in the media series, and their growth characteristics were observed. Maximum cell densities varied in the range of 0.4 to 1.3 x 10(7) cells/mL, depending on cell lines. Cell growth responses to each of the media series were analyzed in terms of cell density and cell mass. Step increases of cell mass in the range of 1.3 to 3.7 g/L were observed according to the step-fortifications of nutrients. Also, the characteristics of each cell line were compared in terms of metabolic yields and specific productions of lactic acid and ammonium ion. The effect of step-fortifications of nutrients on the production of monoclonal antibody was also examined. Apparent differences in metabolic characteristics among cell lines were observed. Experimental results suggested that the different cell sizes and metabolic characteristics of each cell line resulted in cell-line-specific responses to the step-fortifications. The significant influence of nutritional fortifications on high-density culture of mammalian cells was evaluated. (c) 1993 John Wiley & Sons, Inc.     

Correlation of cell counts and indices in testicular FNAC with histology in male infertility.

OBJECTIVE: To evaluate the value of percentage cell counts and cell indices in testicular fine needle aspiration cytology (FNAC) in male infertility and their correlation with histologic categories as seen in open testicular biopsies. STUDY DESIGN: Differential cell counts were performed, and cell indices, including spermatic index, Sertoli cell index and sperm-Sertoli cell index, were calculated in testicular fine needle aspiration (FNA) smears in 30 azoospermic males whose open testicular biopsies were classified as normal spermatogenesis in 10 cases, maturation arrest in 5, hypospermatogenesis in 6, Sertoli cell only syndrome in 5 and tubular/peritubular sclerosis in 4. RESULTS: In normal spermatogenesis, FNA smears showed up to 40% Sertoli cells, and spermatozoa were the predominant spermatogenetic cell type. There was a progressive increase in Sertoli cell percentage and Sertoli cell index and reduction in spermatozon percentage, spermatic index and sperm-Sertoli cell index with increasing severity of reduction in spermatogenesis in different histologic categories. The differences between mean counts and indices in normal spermatogenesis and other histologic categories were statistically significant (P < .01). CONCLUSION: The percent cell counts and cell indices in testicular FNAC correlate with histologic categories and are useful in evaluating male infertility.     

Virus–host cell interactions in vaccine production cell lines infected with different human influenza A virus variants: A proteomic approach

A proteomic approach was used to investigate the dynamic cellular host cell response induced by influenza virus infection in two different vaccine production cell lines, MDCK and Vero. For identification of proteins possibly involved in global host cell response mechanisms and virus–host cell interactions in these producer cell lines, quantitative 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were performed. In particular, host cell proteome alterations caused by infection with influenza virus variants showing differences in replication characteristics in MDCK cells were compared. Moreover, the host cell response to virus infection in Vero cells with respect to their deficiency in interferon (IFN) production and the need for virus adaptation to optimize productivity of this cell line were analyzed. Several proteins with differential abundance profiles were identified and Western blot analysis was performed for further confirmation of selected proteins. IFN-induced signal transduction, cytoskeleton remodeling, vesicle transport and proteolysis were the principal pathways that changed in infected MDCK cells. In Vero cells alterations of cell interactions, heat shock and oxidative stress response were detected. The findings will improve understanding of host cell response mechanisms during influenza vaccine production and viral strategies to evade these responses and to replicate efficiently in different cell lines.     

Radiobiology of alpha particles. III. Cell inactivation by alpha-particle traversals of the cell nucleus

Cell inactivation after exposure to collimated 3.5-MeV alpha particles in three hamster cell lines, V79, CHO-10B, and HS-23, one mouse cell line, C3H 10T1/2, and a human skin fibroblast cell line were studied. Several parameters were investigated for each cell line. Theoretical calculations were performed to find the distribution of energy deposited in the nuclear volume for each cell line. The mean number of alpha-particle traversals required to induce a lethal lesion varied between two for HS-23 cells and six for C3H 10T1/2 cells. The number of traversals per unit area and the total track length of alpha particles that inactivated a cell were found to be nearly constant for the hamster and mouse cell lines. These quantities were found to be lower for the human skin fibroblast cell line. The RBE values for all cell lines were found to be about 3.8 at 10% survival. Thus cell lines that are more sensitive to alpha radiation are also more sensitive to gamma radiation. The average number of alpha-particle traversals producing a single lethal lesion is greater than one. The passages of alpha particles through the cell nucleus that do not kill the cell may lead to carcinogenic effects.     

Flow microfluorometric studies of lectin binding to mammalian cells. II. Estimation of the surface density of receptor sites by multiparameter analysis

A flow-system multiparameter cell analyzer that simultaneously measures and processes fluorescence and cell volume signals from single cells was used to study the binding of fluorescein-conjugated Concanavalin A (Con A–F) to the cell surface. Cells reacted with Con A–F were passed through a flow chamber where sensors measured both cell volume and fluorescence of each individual cell. Sensor signals were electronically processed by first converting the cell volume signals to two-thirds power (proportional to surface area) and then forming the fluorescence-to-surface area ratio. These ratios, which were considered as estimates of the surface density of binding sites, were displayed as frequency distribution histograms using a multichannel pulse-height analyzer for various cell populations differing in cell size. Comparisons between cell lines showed characteristic differences in binding site density. Cell cycle dependent changes were not found for CHO cells synchronized by mitotic selection. An important benefit of this analysis method was the ability to quantitate very weak cell surface fluorescence.     

Protective effect of Bcl-2 in NSO myeloma cell culture is greater in more stressful environments

In the present study, the protective effects of Bcl-2 over-expression in a suspension culture (without any adaptation) and spent medium (low nutrient and high toxic metabolite conditions) were investigated. In the suspension culture without prior adaptation, the viability of the control cell line fall to 0% by day 7, whereas the Bcl-2 cell line had a viability of 65%. The difference in the viability and viable cell density between the Bcl-2 and control cell lines was more apparent in the suspension culture than the static culture, and became even more apparent on day 6. Fluorescence microscopic counting revealed that the major mechanism of cell death in the control cell line in both the static and suspension cultures was apoptosis. For the Bcl-2 cell lines, necrosis was the major mode of cell death in the static culture, but apoptosis became equally important in the suspension culture. When the NSO 6A1 cell line was cultured in spent medium taken from a 14 day batch culture, the control cell line almost completely lost its viability by day 5, whereas, the Bcl-2 still had a viability of 73%. The viable cell density and viability of the Bcl-2 cell line cultivated in fresh medium were 2.2 and 2.7 fold higher, respectively, than those of the control cultures. However, the viable cell density and viability of the Bcl-2 cultivated in the spent medium were 8.7 and 7.8 fold higher, respectively, than those of the control cultures. Most of the dead cells in the control cell line were apoptotic; whereas, the major cell death mechanisms in the Bcl-2 cell line were necrotic.     

太空环境改变生物工程细胞CHO(dhfr-

目的 :了解太空诱变对生物工程细胞CHO(dhfr^----     

Getting the right cells to the array: Gene expression microarray analysis of cell mixtures and sorted cells.

BACKGROUND: Most biological samples are cell mixtures. Some basic questions are still unanswered about analyzing these heterogeneous samples using gene expression microarray technology (MAT). How meaningful is a cell mixture's overall gene expression profile (GEP)? Is it necessary to purify the cells of interest before microarray analysis, and how much purity is needed? How much does the purification itself distort the GEP, and how well can the GEP of a small cell subset be recovered? METHODS: Model cell mixtures with different cell ratios were analyzed by both spotted and Affymetrix MAT. GEP distortion during cell purification and GEPs of purified cells were studied. CD34+ cord blood cells were purified and analyzed by MAT. RESULTS: GEPs for mixed cell populations were found to mirror the cell ratios in the mixture. Over 75% pure samples were indistinguishable from pure cells by their overall GEP. Cell purification preserved the GEP. The GEPs of small cell subsets could be accurately recovered by cell sorting both from model cell mixtures and from cord blood. CONCLUSIONS: Purification of small cell subsets from a mixture prior to MAT is necessary for meaningful results. Even completely hidden GEPs of small cell subpopulations can be recovered by cell sorting.     

Influence of long-term limitation of cell proliferation on the cell cycle duration

The hypothesis was tested that constant, long-term inhibition of cell proliferation is the primary cause of cellular senescence. Mouse L-929 fibroblasts were maintained in confluent cultures for periods of 6 and 12 months with cell viability maintained by regular replacement of medium and serum. The mitotic activity of the cell population under these conditions was one mitosis per 10⁴ cells. No changes in the duration of the cell cycle were observed when, following long-term quiescence, the cell were replated and grown at low cell density. The results do not support the hypothesis that prolonged suppression of cell proliferation induces cell senescence.     

大规模动物细胞培养技术研究进展

利用动物细胞大规模培养技术可生产多种生物制品,为提高细胞活力和表达水平及有利于表达产物的纯化,采用有多种添加成分的无血清培养基培养细胞,选择更有利于细胞生长又可提高培养细胞密度的微载体和条件温和、易操作、气体交换速度快的生物反应器,在线监控细胞生存环境和生理活动,减少培养过程培养基中的抑制因素,可创造更适合细胞生存的环境,提高表达水平,向细胞中导入抗凋亡基因,可提高细胞活性和蛋白产量。利用多也微载体以球转球方式大规模培养动物细胞有很好的发展前景。     

Cisplatin resistance induced by decreased apoptotic activity in non-small-cell lung cancer cell lines

We have investigated defective steps in apoptosis that might account for the development of resistance. For this purpose, A549 and Calu1 NSCLC (non-small-cell lung cancer) cell lines were treated with cisplatin to obtain resistant sub-lines. Gene expression profiles and the phosphorylation status of the BAD (Bcl-2/Bcl-XL-antagonist, causing cell death) protein were determined for each cell line. Cell death and cytochrome c release were analysed after treating cell lines with their appropriate cisplatin doses. Gene expression of BAD, Bid, caspases 4 and 6 were clearly decreased in the resistant cell lines, and the differential phosphorylation status of BAD also seemed to play a role in the development of cisplatin resistance. Since this is a new cisplatin-resistant Calu1 cell line, it is noteworthy that DNA fragmentation, apoptotic cell ratio and cytochrome c levels were most decreased in the CR-Calu1 cell line.     

Role of silicon in diatom metabolism. VII. Silicic acid-stimulated DNA synthesis in toluene-permeabilized cells of Cylindrotheca fusiformis.

Human diploid cell populations were fractionated on the basis of cell size by gravity sedimentation. This cell separation procedure yielded fractionated cell populations that were enriched for both large cell volumes and for slow and/or non-replicating cells (cells which did not have labeled nuclei after a 48 h incubation with [³H]TdR). It also yielded fractionated cell populations that were enriched for small cell volumes and for rapidly replicating cells (cells with labeled nuclei). However, upon reintroduction to tissue culture conditions, cell populations lost their fractionated properties and soon resembled unfractionated cell cultures at similar levels of in vitro passage.     

Epidermal growth factor induces cell cycle arrest and apoptosis of squamous carcinoma cells through reduction of cell adhesion

Most squamous epithelial cells are strictly anchorage-dependent cell types. We observed that epidermal growth factor (EGF) promoted the growth of A431 squamous carcinoma cells in suspension cultures but suppressed cell growth and induced apoptosis in monolayer cultures, suggesting that loss of adhesion is responsible for the effects observed in monolayer culture, before cell death. Consistent with this finding, we demonstrated that EGF reduced cell attachment, cell-cell interaction, and cell spreading. Treatment with EGF increased cell adhesion-regulated expression of p21 but suppressed expressions of cyclin A, D1, cdk2, and retinoblastoma protein (pRb), leading to cell cycle arrest and adhesion-regulated programmed cell death. To test directly whether promoting cell adhesion could reduce the effects of EGF, we grew cultures on plates coated with type II collagen. On these plates, cell adhesion was enhanced and EGF treatment had little effect on cell adhesion and apoptosis when cells were attached to the collagen. The collagen effects were dose dependent, and cell cycle and cell cycle-associated proteins were altered accordingly. Finally, when cultures were plated on bacterial Petri dishes, which completely disrupted cell attachment to substratum, the level of apoptosis was greatly higher and cell cycle was arrested as compared with monolayer cultures. Taken together, our results strongly suggest that the EGF-induced cell cycle arrest and apoptosis in monolayer cultures was the result of a decline in cell adhesion.