THE EFFECTS OF HALOTHANE ON CULTURED MOUSE NEUROBLASTOMA CELLS : I. Inhibition of Morphological Differentiation

Mouse neuroblastoma cells (clone NB2a) were cultured in the presence of 0.3–2.1% halothane in the gas phase for up to 72 h. Halothane inhibited neurite extension dose dependently and virtually abolished microspike formation even at the lowest concentration tested. These effects were completely reversible. Electron microscopy demonstrated that microfilaments measuring 40–80 Å in diameter are the only fibrous organelles visible within microspikes. When the cells were exposed to halothane, no microfilamentous complexes could be identified in any cells and the subcortical regions of neurites often appeared devoid of individual microfilaments. Microtubules were still present in neurites after exposure to halothane concentrations at which microfilaments disappeared. However, at concentrations above 1.0%, microtubules gradually appeared to decrease in number. Short-term experiments showed that existing neurites and microspikes rapidly retracted when suddenly exposed to culture medium equilibrated with 1.0% halothane and quickly reformed when the halothane was removed. The inhibition of neuroblastoma cell differentiation by halothane appears to be mediated by disruption of 40–80 Å diameter microfilaments.     

STIMULATORY EFFECTS OF SUBSTANCE P ON NEURITE EXTENSION AND CYCLIC AMP LEVELS IN CULTURED NEUROBLASTOMA CELLS

Abstract— Synthetic substance P initially increased cyclic AMP levels and subsequently induced neurite extension in cultured neuroblastoma N 18 cells. The magnitude of these effects depended on the concentration of fetal calf serum (FCS) in the culture medium, being more evident in the presence of a lower (0.1%) concentration of FCS.
In Eagle's medium containing 0.1% FCS, low concentrations of substance P (10⁻⁷-10⁻⁵ M) increased cyclic AMP levels and stimulated neurite extension.
In Eagle's medium containing 5%FCS, both substance P at concentrations of 10⁻⁵-10⁻³M and dibutyryl cyclic AMP at concentrations of 10⁻⁴-10⁻²M increased cyclic AMP levels and stimulated neurite extension. The activities of acetylcholinesterase, (Na⁺+ K⁺)-, HCO₃⁻ and Mg²⁺ -stimulated-ATPase were also increased. Cell growth was inhibited.
Substance P at concentrations of 10-⁷-10⁻⁵M also stimulated the adenylate cyclase activity of a particulate fraction of N 18 in a concentration-dependent manner.     

多糖对滇紫草培养细胞的影响

黑节草多糖、火棘果果胶和琼脂加入到培养基中,均能促进滇紫草细胞合成紫草素。黑节草多糖促进紫草素合成的最适浓度为0.05—0.1％,大于最适浓度紫草素合成逐渐受到抑制。三种黑节草多糖单体中以多糖Ⅱ最为有效,紫草素产量为90.07mg/l。加入火棘果果胶和琼脂均对紫草素合成呈现促进作用。最后对多糖的作用机理以及多糖与寡糖的关系进行了讨论。     

UTILIZATION OF POLYUNSATURATED FATTY ACID SUPPLEMENTS BY CULTURED NEUROBLASTOMA CELLS1

Abstract— Brain phosphoglycerides are known to contain large amounts of polyunsaturated fatty acids (PUFA). However, neuroblastoma cells contain very low amounts of PUFA. Since the serum used for cell culture has been shown to be deficient in PUFA, a supplementation of this serum with various PUFA was undertaken. Linoleic, arachidonic and docosahexaenoic acids were incorporated in cell phosphoglycerides, mainly at the expense of oieic acid, while linolenic acid was only poorly incorporated. Linoleic. linolenic and arachidonic acids were transformed to their elongation and desaturation products; but the last step of transformation, which involves the action of a Δ 4 desaturase, was never observed. The levels of incorporation and transformation of exogenous PUFA could vary strikingly in different lines of neuroblastoma cells. The simultaneous addition of two PUFA (linoleic and linolenic acids) was followed by a reduction in the amount of their respective derivatives in cell phosphoglycerides. compared to that obtained when only one PUFA was given to the cells, suggesting a competitive inhibition of the desaturation of each PUFA. Such alterations in membrane lipids may provide a useful model for the study of membrane structure-function relationships.     

铝对心肌细胞动作电位及其兴奋性的影响

目的 :观察氯化铝 (AlCl3)对豚鼠乳头状肌动作动位及其兴奋性的影响。方法 :利用细胞内微电极技术引导动作电位 (AP) ,经微机采集AP图形并测算跨膜电位各参数。结果 :①AlCl30 .5 ,1,2 ,4mmol/L使动作电位复极5 0 %时程 (APD50 )和复极 10 0 %时程 (APD10 0 )先延长后大幅度缩短 ,呈现双相效应 ,其中AlCl32mmol/L作用 5min时使APD50 从 (182± 2 7)ms延长至 (2 0 2± 3 2 )ms(P <0 0 5 ) ,作用 3 0min时使APD50 缩短至 (14 4± 17)ms(P <0 0 1) ;②AlCl32 ,4mmol/L使动作电位幅度 (APA)及 0期最大除极速度 (Vmax)减小 ,兴奋性下降。结论 :AlCl3可能对Na 内流有抑制作用 ,对Ca2 内流呈现先促进后抑制的双相效应。     

苯扎贝特对培养的牛主动脉血管平滑肌细胞增殖的影响

目的探讨苯扎贝特对体外培养的牛血管平滑肌细胞(vascular smooth muscle cells,VSMC)增殖的影响,以及可能的信号转导通路.方法采用改良的贴块法培养小牛胸主动脉VSMC,α-actin单克隆抗体免疫组织化学染色鉴定培养细胞.以MTT法反映VSMC增殖情况;用Westem Blot方法检测与细胞增殖有关的丝裂原激动蛋白激酶(MAPK)信号传导通路.结果苯扎贝特呈剂量依赖性抑制VSMC增殖,其抑制增殖的作用主要是通过MAPK传导途径.结论苯扎贝特通过抑制VSMC增殖可能参与延缓动脉粥样硬化及经皮冠状动脉腔内成行术(percutaneous transluminal coronaryangioplasty,PTCA)术后再狭窄的发生、发展.     

丁酸对体外培养的人鼻咽癌上皮细胞的影响

无论是自发的、病毒引起的或致癌物诱发的恶性转化的哺乳类细胞的体外培养,其形态多发生改变,总是变得近似圆形,边缘突起短而少,细胞致密和折光性强,同时失去生长接触抑制,降低细胞与细胞之间和细胞与生长底物之间的粘着性等特性。近年报道了关于短链脂肪酸如丁酸(或丁酸钠)对细胞能产生明显的影响,能抑制培养细胞的分裂,可诱发一些上皮性细胞产生形态的改变,可使转化的细胞     

亮氨酸脑啡肽抑制培养大鼠主动脉平滑肌细胞增殖

实验以培养的ＳＤ大鼠胸主动脉平滑肌细胞为模型,用四唑盐比色地及＾３Ｈ－ＴｄＲ掺入技术动态观察发现亮氨酸脑啡肽可显著抑制血管平滑肌细胞的增殖及ＤＮＡ的合成,阿片受体阻断剂纳洛酮对ＶＳＭＣｓ的增殖及ＤＮＡ的合成无影响,但ＮＡＬ可拮抗亮氨酸脑啡肽对ＶＳＭＣｓ增殖的抑制作用。     

丁酸钠对人胃癌和食道癌细胞的生物学效应

5mM丁酸钠可使人胃癌MGc803细胞形态发生改变,细胞变长,出现胞浆突起,趋向于成纤维细胞形态的变化,使食道癌ECa109细胞的核/质比下降。同时能抑制细胞增殖,使细胞生长率下降,丁酸钠对细胞增殖的抑制作用是随其浓度的增加而增强的,具有剂量依赖关系。丁酸钠可促进人胃癌MGc803细胞胞质中微管和食道癌ECa109细胞内中等纤维的组装,这种变化对丁酸钠处理后细胞形态的改变有关。丁酸钠可使细胞阻断于G_1期。??对3’,5’-cAMP—PDE(cAMP-磷酸二酯酶)活力具有强的抑制作用,并提高细胞内cAMP水平,从而抑制细胞增殖。这些结果表明丁酸钠对人胃癌MGc803和食道癌ECa109细胞具有一定的诱导分化作用。     

Taxol对培养的人成纤维细胞内微管组装的影响

本实验用微管的PAP免疫酶细胞化学方法,研究了培养的小儿包皮成纤维细胞及其分离的中心体在taxol的作用下对微管组装的影响。实验结果表明taxol对低温(4℃)和微管解聚药物的处理具有拮抗作用,它阻止微管解聚,对微管具有稳定作用,并观察到taxol可降低中心体对微管组装所需的管蛋白临界浓度,增强中心体对微管的组装能力。Taxol对细胞内微管的影响,主要表现在促使微管呈束状浓集化,并随处理时间的延长,这种浓集化表现愈益明显,导致破坏胞质CMTC的正常分布。由于taxol能使微管浓集化,抑制其解聚,使得细胞从G_2期进入M期后,微管不解聚,从而不能形成正常的纺锤体,胞质不分裂,最后导致细胞微核化。用秋水仙酰胺处理后再加taxol时,我们观察到细胞CMTC与正常未经处理的细胞CMTC比较,呈相反的分布现象,这可能与秋水仙酰胺促使中心体与细胞核分离和taxol增强中心体对微管的组装有关。     

STUDIES ON THE BIOSYNTHESIS OF GLYCO-SPHINGOLIPIDS IN CULTURED MOUSE NEUROBLASTOMA CELLS: CHARACTERIZATION AND ACCEPTOR SPECIFICITIES OF N-ACETYLNEURAMINYL- AND N-ACETYLGALACTOSAMINYLTRANSFERASES

Abstract— The pathway of biosynthesis of N -acetylgalactosamine-containing gangliosides in mouse neuroblastoma has been studied using NB41A cells grown in monolayer tissue culture. Cell-free enzyme preparations catalyzed the transfer of NeuNAc from CMP-NeuNAc to lactosylceramide (GL-2a), to form G_M3. Asialo-G_M2 was neither an acceptor nor a competitive inhibitor of the sialyltransferase (CMP-NeuNAc: GL-2a N-acetylneuraminyltransferase, EC 2.4.99.-) under a variety of experimental conditions. Enzyme preparations also contained an N -acetylgalactosaminyltransferase (UDP-GalNAc. G_M3 N -acetylgalactosaminyltransferase, EC 2.4.1.-) which catalyzed the conversion of G_M3 to G_M2. No significant transfer of N -acetylgalactosamine to GL-2a could be demonstrated. The results of the glycosyltransferase assays support the concept that the first NeuNAc of brain gangliosides is introduced into GL-2a. The present data suggests that the occurrence of asialo-G_M2 in NB41A cells under some culture conditions is a consequence of the catabolism of higher gangliosides.     

REGULATION OF OXYGEN CONSUMPTION IN NEUROBLASTOMA CELLS: EFFECTS OF DIFFERENTIATION AND OF POTASSIUM

Abstract— The rate of oxygen consumption has been measured micromanometrically in fresh mouse neuroblastoma cells and in corresponding cells cultivated with and without 20% calf serum known to suppress differentiation. Fresh cells and cells cultivated in the serum depleted medium showed a relatively intense respiration (0.5–1.0 × 10⁻⁵μl/h/cell), whereas the proliferating, cultivated cells had a low rate of oxygen uptake (0.2 × 10^-5μl/h/cell), provided they did not differentiate morphologically during the 4 h of measurement in the serum-free diver medium. Both morphological and metabolic differentiation of such cells occurs rapidly but may be completely prevented by siliconization of the divers.
An increase of the potassium concentration in the medium to 50 mM had essentially no effect on cultivated, differentiated cells, but led to an abolishment of oxygen uptake by fresh cells. No stimulatory effect of potassium was observed.     

EFFECTS OF 50 Hz MAGNETIC FIELD EXPOSURE ON PROTEIN TYROSINE PHOSPHORYLATION IN CULTURED CELLS

Protein phosphorylation is an extremely important and widely used mechanism of cellular regulation. Here, the effects of 50 Hz magnetic fields (MFs) on tyrosine phosphorylation were studied. A Chinese hamster lung (CHL) cell line was exposed to 50 Hz magnetic fields at two intensities (0.4 mT and 0.8 mT) for different exposure durations, and western blot analysis was used to measure the degree of tyrosine phosphorylation of cellular proteins. Results showed that both 0.4 mT and 0.8 mT 50 Hz magnetic fields could affect the protein tyrosine phosphorylation in cultured cells. Both intensities could affect the tyrosine phosphorylation of 38 and 97.4 kDa proteins. In addition, 0.4 mT could affect tyrosine phosphorylation of 61.7, 105, and 112 kDa proteins, and 0.8 mT affected the tyrosine phosphorylation of 79 and 150 kDa proteins. Moreover, all the tyrosine phosphorylation changes of these proteins were time-dependent. The findings from this study demonstrated that under these experimental conditions, there was evidence that protein tyrosine phosphorylation was a possible process for ELF-EMF producing bioeffects.     

DIFFERENTIATION OF NEUROBLASTOMA CELLS IN CULTURE

1. An elevation of the intracellular level of cyclic AMP in neuroblastoma cells by prostaglandin E₁ by an inhibitor of cyclic AMP phosphodiesterase, or by analogues of cyclic AMP irreversibly induces many differentiated functions which are characteristic of mature neurones. These include formation of long neurites, increase in size of soma and nucleus associated with a rise in total RNA and protein contents, increase in activities of specific neural enzymes, loss of malignancy, increase in sensitivity of adenylate cyclase to catecholamines and blockade of cells in G₁-stage of the cell cycle. 2. Other agents, including serum-free medium, X-irradiation, 6-thioguanine, cytosine arabinoside, methotrexate, 5-bromodeoxyuridine, nerve growth factor, glial extract and hypertonic medium can induce some of the differentiated functions which are induced by high intracellular cyclic AMP. 3. Morphological differentiation and differentiated biochemical functions can each be expressed in the absence of the other. 4. Many of the responses of normal embryonic nerve cells to cyclic AMP are similar to those of neuroblastoma cells. 5. A working hypothesis for the malignancy of nerve cells has been proposed. This states that an abnormal regulation of cyclic AMP phosphodiesterase activity which allows the expression of high amounts of this enzyme in neuroblastoma cells, may be one of the early lesions during a malignant transformation of nerve cells. 6. A new experimental therapeutic model for the treatment of neuroblastoma is proposed. This involves the administration of sodium butyrate followed by the injection of l -dihydroxyphenylalanine (l-dopa) and prostaglandin E₁ in the presence of cyclic AMP phosphodiesterase inhibitor. 7. Recent studies have elucidated the control mechanisms of some differentiated functions in neuroblastoma cells. Cyclic AMP may become an important biological tool to probe the regulation and expression of many other differentiated functions in these cells. In addition to neuroblastoma cells, other neuronal culture systems are now available for investigating the problems of differentiation and maturation in nerve cells.     

肾上腺糖皮质激素对体外培养的人体肝癌细胞的影响电镜观察

本工作的目的系观察肾上腺糖皮质激素对离体培养的人体肝癌细胞(BEL- 7402)引起的形态学表型改变。肝癌细胞经浓度为7.6×10~(-6)的合成糖皮质激素——地塞米松处理48小时显示,体积较对照细胞显著较大,呈扁平多边形。电镜观察到:1)激素处理48小时、浓度为7.6×10~(-7)M及7.5×10~(-6)M的两个剂量组,线粒体偶有增大及融合现象;胞膜下外细胞质区域有短的微管出现。2)处理72小时的两个剂量组,肝癌细胞的微绒毛似有减少;经常见到体积明显增大的线粒体及成片的糖元;胞质内经常出现长的、纵横交错的微管,尤以7.5×10~(-6)M组明显,弥散分布的微丝和张力纤维出现的频率也远高于对照组。本文对肝癌细胞出现的表型变化的意义进行了讨论。