Effects of substrata on the polarization of bovine endometrial epithelial cells in vitro

Summary Epithelial-cell function requires cellular polarity in which apical membrane surfaces have unique characteristics and cellular organelles are stratified. Physiological investigations of endometrial, epithelial cells would be enhanced greatly by the ability of a method to polarize cells in culture. This study investigates the effects of different substrata on polarization of cultured bovine endometrial epithelial cells. Fetal bovine endometrial epithelial-cell lines were developed from explant outgrowth. Epithelial monolayers were subcultured onto amniotic membranes, Millicell-HA membranes, or Millicell-CM membranes coated with rat-tail collagen, Matrigel, laminin, Vitrogen,or fibronectin. Cultures on these substrata were maintained at the air/liquid interface. Cells grown on either collagen-coated or uncoated Milli-cell membranes also were maintained submerged in medium. Excellent polarized morphology was attained in cultures grown at the air/liquid interface on amniotic membranes and rat-tail collagen-coated membranes. Lectin-binding patterns, to apical membranes of polarized epithelial cell cultures paralleled patterns of binding to bovine endometrial surfaces in vivo. Cultures on rat-tail collagen were maintained for several weeks. These methods provide a valuable system for studying the endometrium in vitro.     

Effects of endogenous and exogenous cholesterol on the ultrastructure and steroid secretion of undifferentiated rat adrenocortical cells in primary culture

Summary The present study was undertaken to define the effects of lipoprotein-derived cholesterol and endogenous, de novo synthesized cholesterol on the ultrastructure and function of undifferentiated rat adrenocortical cells [lipoprotein (HDL₃ and LDL) receptor-negative, zona glomerulosa-like adrenocortical cells] in primary culture. For this purpose human plasma high density lipoprotein (HDL₃) or low density lipoprotein (LDL) was added to culture medium devoid of cholesterol. Steroid secretion remained at the low basal level even after addition of lipoproteins, and the amount of intracellular lipid droplets did not increase. When mevinolin (0.96 µg/ml), an inhibitor of cholesterol synthesis, was added to the culture medium, a low secretion of corticosterone was measured both in serum-free and serum-containing media. Ultrastructurally, lipid droplets disappeared after treatment with mevinolin in both media used. At this concentration of mevinolin cell proliferation was similar to that in the controls, but at higher concentrations (4.8 or 9.6 µg/ml) proliferation was inhibited to 42% and 26% in serum-free medium, and 20% and 12% in serum-supplemented medium, respectively. This study demonstrates that cell proliferation and synthesis of corticosterone by undifferentiated rat adrenocortical cells is identical in the absence or presence of exogenous lipoprotein cholesterol. Inhibition of de novo cholesterol synthesis by mevinolin over a period of 7 days does not inhibit corticosterone secretion or proliferation of cells but decreases the amount of intracellular lipid droplets, thus suggesting utilization of intracellular cholesterol esters. However, higher concentrations of mevinolin inhibit proliferation of cells both in serum-free and serum-containing media.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

δ and κ Opiate receptors in primary astroglial cultures from rat cerebral cortex

The effects of , , and receptor-agonists on forskolin stimulated cyclic adenosine-3, 5-monophosphate (cAMP) formation were examined in astroglial enriched primary cultures from the cerebral cortex of newborn rats. Intracellular cAMP accumulation was quantified by radioimmunoassay. Morphine was used as a -receptor agonist, D-Ala-D-Leu-Enkephalin (DADLE) as a -receptor agonist and dynorphine 1–13 (Dyn) as a -receptor agonist. Basal cAMP levels were unaffected by either the opiate agonists or the antagonists used. In the presence of the cAMP stimulator forskolin, morphine had no significant effect on the cytoplasmic cAMP levels. DADLE caused a dose related inhibition of the forskolin stimulated cAMP accumulation. The effects of this receptor stimulation was blocked with the selective antagonist ICI 174.864. In the presence of Dyn, the forskolin stimulated cAMP accumulation was inhibited in a dose related manner. This receptor stimulation was blocked with the selective antagonist MR 2266. Co-administration of DADLE and Dyn resulted in a non additive inhibition of the forskolin stimulated accumulation of cAMP. These findings indicate that astroglial enriched cultures from the cerebral cortex of rats express and -receptors co-localized ont he same population of cells, and that these receptors are inhibitory coupled to adenylate cyclase.     

Binding of flunitrazepam to differentiating neurons cultured in a chemically defined,hormone-supplemented medium

[³H]Flunitrazepam (FNZ) binding to cortical neurons from fetal rat brain was investigated in vitro. The use of a synthetic medium specific for neurons made it possible to plot a developmental curve of³H-FNZ binding in an almost pure neuronal culture. Detectable specific binding was present in vitro at time 0 (that is, the 16th gestational day). A progressive increase of binding, due to an increment in the number of recognition sites, was observed on the subsequent days. The affinity of the specific binding sites to³H-FNZ was enhanced by the addition of exogenous GABA, whereas the density was not affected.     

在原代培养的大鼠肝细胞观察消炎痛的细胞保护作用

我们曾观察到消炎痛预处理能明显减轻四氯化碳和半乳糖胺对大鼠的肝损伤作用,本工作采用原代培养的大鼠肝细胞进一步观察了这一现象。结果表明,经消炎痛整体处理后分离的大鼠肝细胞,在原代培养的条件下,对四氯化碳的损伤仍然具有明显的抵抗作用,表现为细胞内酶的漏出少于对照组。正常大鼠离体肝细胞在原代培养条件下用消炎痛处理,在相当大的剂量和相当长的时间范围内未出现明显的抗损伤作用。结果提示:消炎痛整体处理可使肝细胞本身获得抗损伤的能力,而这种抗损伤能力的产生可能有赖于肝细胞外其它因素的参与。     

重组红细胞生成素对小鼠巨核细胞后期成熟及血小板生成的影响

本文应用血小板生成液体培养体系,检测了重组人红细胞生成素(r-EPO)对巨核细胞成熟及血小板生成的影响。r-EPO 能在1U 至6~U/ml 浓度范围内增加体系血小板数,r-EPO剂量与血小板数之间呈线性关系。r-EPO 还能促进巨核细胞 DNA 合成,并使 Ⅱ、Ⅳ 期巨核细胞比例增加,Ⅰ、Ⅱ 期巨核细胞比例减少。结果表明:r-EPO 可以促进巨核细胞成熟,并作为一种主要刺激因子,以增加血小板数的方式促进血小板生成。     

原生质体来源的大白菜悬浮细胞系的超低温保存

原生质体来源的大白菜 Brasstca campessris var.pekinsis 悬浮细胞系在二甲亚砜的保护下,能在液氮中(-196℃)长期冻存。加入山梨醇能增强保护作用;而加入甘露糖则降低保护作用。培养基对冻存也有明显的影响。在液氮中存放的时间长短对细胞存活率没有多大影响。冻后相对活性最高可达75.4％,恢复生长快,化冻后重新悬浮培养6天,生长量可达300-500％。遮光比不遮光对恢复更有利。冻存后恢复生长的悬浮细胞,能与未经冰冻的对照一样进行原生质体分离和培养。     

红豆草组织培养物的超低温保存及其超微结构的观察

红豆草(Onobrychis viciaefolia Scop.)组织培养物在5%DMSO+10%甘油+8%蔗糖的冰冻保护剂及以1℃/分钟的速度降温到-35—-40℃,停留2小时后,投入液氮,40℃水浴快速化冻等条件下,存活率达60—70%,并保持了高的分化能力。电子显微镜的观察结果表明,快速冰冻和1℃/分钟慢速冰冻至-35℃—40℃不停留,对细胞结构造成严重的致死性破坏;-35℃停留30分钟对细胞结构的损伤是可逆性的;停留2小时的其超微结构基本上与对照材料无明显差别。     

High density culture of hybridoma cells using a perfusion culture vessel with an external centrifuge

The influence of centrifugal force on the growth of cells was examined by exposing the cells of the mouse-human hybridoma X87 line to centrifugal force (100–500 G) for ten minutes twice a day and comparing the static culture with that of unexposed cells. In this experiment, both cell proliferation and specific antibody productivity were independent of the centrifugal effect, and gave the same results as in the case of no exposure to centrifugal force. High density cultivation of the mouse-human hybridoma X87 line was obtained by a perfusion system where the cells were separated from the culture medium by continuous centrifugation. In the serum-free culture, the maximum viable cell density exceeded 10⁷ cells/ml, and monoclonal antibody was stably produced for 37 days. The results in this culture were equivalent to those obtained by intermittent centrifugal cell separation from the culture medium, and separation by gravitational settlement.