The role of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion

The biochemical and biophysical roles of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion were studied. (1) Various kinds of cell, such as Ehrlich ascites tumor cells, mouse melanoma cells (B16-CW1 cells) and human epidermoid carcinoma cells (KB cells), could fuse in Ca2+-free medium containing a cheletor, glycoletherdiaminetetraacetic acid, in the same way as in Ca2+-containing medium. (2) The ATP content in Ehrlich ascites tumor cells decreased rapidly when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (3) Intracellular adenine nucleotides leaked out into the reaction medium when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (4) On addition of the virus, O2 consumption of Ehrlich ascites tumor cells decreased in Ca2+-free medium, but not in Ca2+-containing medium. (5) HVJ (Sendai virus) did not affect production of lactate by Ehrlich ascites tumor cells in both Ca2+-free medium and Ca2+-containing medium. These observations suggest that the role of extracellular Ca2+ in virus-induced cell fusion is to maintain the ATP and other intracellular metabolite contents at normal levels instead of triggering the fusion reaction itself.     

Subject index volume 38 (1985)

Reproducibly high yields of protoplasts were obtained from the unicellular asexual green alga Chlorella saccharophila (Krüger) Nadson 211-1a by treating the cells with polysaccharide degrading enzymes. A maximum of 32% of the protoplasts were able to regenerate cell walls and formed colonies when plated on a regeneration medium. Chlorophyll deficient mutants were isolated after X-ray irradiation of the green wild-type cells. These mutants differed in growth requirements and light sensitivity from each other and from the wild-type cells. Intraspecific protoplast fusion of a yellow with a white pigment mutant strain was accomplished by polyethylene glycol (PEG) and Ca²⁺ and green hybrid cells were obtained using selective conditions. Ultrastructural and morphological investigations carried out so far demonstrate differences between hybrid cells and the parental mutant cells as well as between hybrid cells and the green wild-type cells.     

狂犬疫苗单克隆抗体制备过程中细胞融合条件的探索

在充分了解SP2/0细胞系的生长、增殖和遗传特性后,以聚乙二醇(PEG)诱导动物细胞融合为契机,意在探索不同分子量、不同浓度的PEG作融合剂对诱导SP2/0细胞与脾细胞融合的最适条件,在HAT选择培养基下通过细胞融合率的变化进行比较。结果表明,分子量为4000,浓度为50%的PEG诱导的细胞融合率最高,为下一步制备狂犬病毒疫苗单克隆抗体奠定基础。     

Somatic hybridization of amino acid analogue-resistant cell lines of potato (Solanum tuberosum L.) by electrofusion

Summary Intraspecific somatic hybridization between amino acid analogue-resistant cell lines of potato (Solanum tuberosum L.) has been carried out following electrofusion of protoplasts. In initial analytical electrofusion experiments (1 mm electrode separation) optimal fusion conditions were determined by changing the fusion medium (addition of Ca and/or spermine) and the electrical parameters. Subsequently, in large scale experiments, cell suspension protoplasts of aec-1, a variant resistant to AEC, were fused with the same type of protoplasts of 5mt-26 or 5mt-27, both variants resistant to 5MT and cross-resistant to 3 FT. After an extensive selection procedure only somatic hybrid lines of aec-1 + 5mt-26 were obtained. The resistance traits of aec-1 and 5mt-26 were expressed fully, indicating that the variant characters involved are transmitted dominantly. Quantitative examination of the free amino acid content revealed characteristics of both the parental cell lines in most of the somatic hybrids. However, initially selected double resistant colonies from fusions of aec-1 + 5mt-27 lines appeared not to be somatic hybrids.Abbreviations AEC S-aminoethylcysteine - 3FT 3-fluorotyrosine - 5MT 5-methyltryptophan     

沙打旺与苜蓿属间体细胞杂交

以豆科牧草沙打旺为一亲本,碘乙酰胺处理的紫花苜蓿发根农杆菌A_4菌株转化系为另一亲本,通过PEG-高pH,高钙法诱导原生质体融合。在不加外源激素的DPD 培养基上有效地筛选了杂种细胞。经培养首次得到沙打旺( )紫花苜蓿的属间体细胞杂种。尽管双亲原生质体均已丧失分化植株的能力,但杂种细胞系R_1仍得到苗的分化。杂种R_1细胞的染色体数检查、冠瘿碱检测、同工酶和RAPD 分析结果,都证实了其杂种特性。     

沙打旺与苜蓿属间体细胞杂交

以豆科牧草沙打旺为一亲本,碘乙酰胺处理的紫花苜蓿发根农杆菌A4菌株转化系为另一亲本,通过PEG-高pH,高钙法诱导原生质体融合。在不加外源激素的DPD培养基上有效地筛选了杂种细胞。经培养首次得到沙打旺( )紫花苜蓿的属间体细胞杂种。尽管双亲原生质体均已丧失分化植株的能力,但杂种细胞系R1仍得到苗的分化。杂种R1细胞的染色体数检查、冠瘿碱检测、同工酶和RAPD分析结果,都证实了其杂种特性。     

分泌人源性抗链球菌溶血素O单克隆抗体人—人杂交瘤细胞株的建立

自从淋巴细胞杂交瘤1975年创立以来（Kohler et al.,1975）越来越显示其优越性,（Sikora et al.,1982）,从其发展看来,前景广阔,针对恶性肿瘤,白血病和组织移植等提出了崭新的疗法。由于鼠源性单克隆抗体在临床应用上往往引起过敏反应和形成免疫复合物的危险性,激励着科学家们去开拓人—人杂交瘤,尽管前进的道路上还存在着许多困难,但人们还是不屈不挠地前进。目前,世界上共有三个人—人杂交瘤系统。（1）0lsson和Kaplan等培育的SKO—007人骨髓瘤细胞系统。（2）Croce等培育的GM 1500细胞系统。（3）Clark等的RPMI8226细胞系统。并且都进行了人—人杂交瘤的研究,但是多数杂交瘤阳性率低和分泌抗体量少。国内北京、上海、西安和昆明等都正在开展本项研究。我们从1984年8月,开始人单抗的研究,也碰到许多困难,深深体会到建立一个具有我国特色的人—人杂交瘤系统是十分重要的,现将我们的工作简要报告如下。     

NaCl alleviates polyethylene glycol-induced water stress in the halophyte species Atriplex halimus L

Atriplex halimus L. is a C4 xero-halophyte species well adapted to salt and drought conditions. To collect information on the physiological impact of low salt levels on their water-stress resistance, seedlings were exposed for 6 d to nutrient solution containing either 0% or 15% polyethylene glycol 10,000 (PEG), in the presence or in the absence of 50 mM NaCl. Similar experiments were performed with one PEG-resistant and one PEG-sensitive selected cell line exposed for 50 d to 0% or 15% PEG on standard Linsmaier and Skoog (LS) medium, on LS medium supplemented with 50 mM NaCl, or on Na+-free medium. NaCl mitigated the deleterious impact of PEG on growth of both whole plants and PEG-sensitive cell lines and improved the ability of stressed tissues to perform osmotic adjustment (OA). Water stress reduced CO2 net assimilation rates quantified in the presence of high CO2 and low O2 levels (A), stomatal conductance and transpiration, but NaCl improved water use efficiency of PEG-treated plants through its positive effect on A values, especially in young leaves. PEG increased the internal Na+ concentration. The resistant cell line accumulated higher concentration of Na+ than the PEG-sensitive one. The complete absence of Na+ in the medium endangered the survival of both cell lines exposed to PEG. Although Na+ by itself contributed only for a small part to OA, NaCl induced an increase in proline concentration and stimulated the synthesis of glycinebetaine in response to PEG in photosynthetic tissues. Soluble sugars were the main contributors to OA and increased when tissues were simultaneously exposed to PEG and NaCl compared with PEG alone, suggesting that Na+ may influence sugar synthesis and/or translocation.     

The effects of EGTA on vascular smooth muscle contractility in calcium-free medium

The effect of EGTA, commonly present in Ca2+-free physiological saline solution, on the contractile responses induced by Ca2+ and phenylephrine was studied in dog mesenteric arteries and aortas of rats and rabbits. EGTA substantially enhanced the contractile responses of these vascular strips or rings to added Ca2+ after a prolonged preincubation period in the Ca2+-free medium. The maximal level of the enhanced contractile responses was independent of EGTA concentration, but the rate of the maximal responses was faster at higher EGTA concentration, presumably as a result of faster removal of intracellular Ca2+. Such a Ca2+-induced response was sensitive to the Ca2+ antagonist, nifedipine. EGTA present at low concentrations (50 and 400 microM) in Ca2+-free medium also inhibited the phenylephrine-induced contractile response more prominently for the longer preincubation periods of the vascular tissues in Ca2+-free medium. Our results suggest that EGTA, even when added at low concentrations to the vascular smooth muscle for a sufficiently long period in Ca2+-free medium, may cause destabilization of the cell membranes leading to increased permeability to subsequently added Ca2+. EGTA may also remove the superficially bound Ca2+ and subsequently reduce the intracellular Ca2+ pool via extraction of the intracellular Ca2+ at the cell membrane surfaces.     

草木樨状黄芪和木本霸王的科间体细胞杂交

在成功培养原生质体的基础上, 用改进的PEG-高pH高钙法诱导草木樨状黄芪(Astragalus melilotoides)和木本霸王(Zygophyllum xanthoxylum)原生质体融合, 得到了科间体细胞杂种融合细胞。采用罗丹明-6G预处理草木樨状黄芪原生质体以及UV-B辐照霸王原生质体, 使双亲原生质体及其同源融合产物均不能持续分裂而死亡, 融合后的杂种细胞由于生理互补可恢复持续分裂能力而被筛选出来。融合产物经培养分裂获得了2个杂种细胞系, 其中1个分化出芽。染色体计数和分子鉴定证明了杂种的真实性。初步比较了杂种细胞系及亲本对盐分和水分胁迫的耐受性, 结果表明杂种细胞系对盐分和水分胁迫的耐受性介于两个亲本之间。     

Spectrofluorimetric and image recordings of spontaneous and lectin-induced cytosolic calcium oscillations in Jurkat T cells

Intracellular variations in Ca2+ concentrations have been measured in single Jurkat T lymphocyte variants (77 6.8 and E6.1) using Fura-2 as a probe. Under basal conditions, the cytosolic Ca2+ level is stable but some cells show spontaneous Ca2+ oscillations (frequency, 0.30 +/- 0.06 Hz). These oscillations are sensitive to the external concentration of Ca2+ since they can no longer be observed when the bathing solution is replaced (superfusion) with a Ca(2+)-free medium or when a Ca2+ chelator (EGTA) is added. Various changes in the cytosolic concentration of Ca2+ ([Ca2+]i) can be observed when the cells are exposed to the mitogenic lectin phytohemagglutinin (PHA, 80 nM). For instance, in the case of non-oscillating cells, the lectin induces either a rapid increase in [Ca2+]i that is followed by a sustained response (plateau) or it triggers Ca2+ spikes. In the case of experiments done in Ca(2+)-free medium, only the initial spike was observed. In the case of spontaneously oscillating cells, PHA induces a rapid increase in [Ca2+]i that is followed by a plateau where oscillations are absent. In every case, the PHA-dependent Ca2+ response is abrogated in a Ca(2+)-free medium. Computer simulations based on the model of Goldbeter et al. [27] show that the various Ca2+ responses of Jurkat cells are related to the cytosolic level of free Ca2+. Video imaging analyses show that the cellular Ca2+ responses are not homogeneous whether the observations are made in spontaneously oscillating Jurkat cells or when they are exposed to PHA.     

Fendiline-Induced Ca2+ movement in A10 smooth muscle cells

The effect of fendiline, an anti-anginal drug, on cytosolic free Ca2+ levels ([Ca2+]i) in A10 smooth muscle cells was explored by using fura-2 as a Ca2+ indicator. Fendiline at concentrations between 10-50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 of 20 microM. External Ca2+ removal reduced the Ca2+ signal by 75%. Addition of 3 mM Ca2+ increased [Ca2+]i in cells pretreated with fendiline in Ca2+-free medium. The 50 microM fendiline-induced [Ca2+]i increase in Ca2+-containing medium was inhibited by 10 microM of La3+, nifedipine, or verapamil. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ store partly inhibited 50 microM fendiline-induced Ca2+ release; whereas pretreatment with 50 microM fendiline abolished 1 microM thapsigargin-induced Ca2+ release. Inhibition of phospholipase C activity with 2 microM U73122 did not alter 50 microM fendiline-induced Ca2+ release. Incubation with 50 microM fendiline for 10-30 min decreased cell viability by 10-20%. Together, the findings indicate that in smooth muscle cells fendiline induced [Ca2+]i increases. Fendiline acted by activating Ca2+ influx via L-type Ca2+ channels, and by releasing internal Ca2+ in a phospholipase C-independent manner. Prolonged exposure of cells to fendiline induced cell death.     

Calcium requirement for the inhibition by theophylline of histamine release from mast cells

When added to Ca2+-free Hanks' solution, Ca2+ (0.1-2.5 mM) had no significant effect on antigen-induced histamine release from rat mast cells, but Sr2+ (1.0-3.0 mM) dose-dependently increased the release. Ba2+ (1.0 and 2.0 mM) also enhanced the release. Ca2+ and Ba2+ inhibited compound 40/80-induced histamine release, in a dose-dependent manner. In ordinary Hanks' medium, theophylline and 3-isobutyl-1-methylxanthine (IBMX) dose-dependently inhibited the antigen-induced histamine release but these drugs were ineffective in Ca2+-free medium. Theophylline (1.0 mM) also inhibited compound 48/80-induced histamine release in the presence but not absence of Ca2+. There was an optimal Ca2+ concentration for the theophylline effect. Sr2+ but not Ba2+ could substitute for Ca2+ in supporting the theophylline effect. Theophylline (1.0 mM) and IBMX (1.0 mM) increased mast cell cyclic AMP levels both in the presence and absence of Ca2+. These results suggest that Ca2+ is required in the interaction of theophylline and specific sites on mast cells or in the mast cell response to theophylline which probably does not involve the cyclic AMP increase and is linked to the inhibition of histamine release.     

Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells

The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ [( Ca2+]i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37 degrees C with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15 degrees C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37 degrees C for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15 degrees C demonstrated in spotty cells a marked apparent blunting of Ca2+ transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]i was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]i measurements.     

The role of Ca2+ in dimethyl sulfoxide-induced differentiation of Friend erythroleukemia cells

Cultured Friend cells can be induced by dimethyl sulfoxide (Me2SO) and several other agents to mature along the erythroid pathway. Evidence has been presented that an increase in Ca2+ influx is an early and necessary prelude to the commitment to maturation by these cells (Levenson, R., Housman, D., and Cantley, L. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5948-5952). The simplest hypothesis supporting all the available data is that Me2SO and other inducers elevate the cytosolic Ca2+ concentration. We have now measured cytosolic Ca2+ using the fluorescent indicator quin-2, and find, contrary to expectation, a small decrease upon treatment of cells with Me2SO. Cytosolic Ca2+ was increased by raising the Ca2+ in the medium, but was not dramatically altered by addition of ouabain or monensin or by incubation in Na+-free medium. Measurement of total cell Ca2+ by a triple-labeling technique using 3H2O and 125I-albumin to determine cell water and extracellular space, respectively, revealed no significant change upon treatment with Me2SO for up to 40 h. A decrease in the initial rate of 45Ca2+ influx was observed in Me2SO-treated cells, when measured at 4 degrees C. These data do not support the hypothesis that an increase in cell Ca2+ is necessary for the induction of Friend cell differentiation or that Na+/Ca2+ exchange is a significant regulator of cytosolic Ca2+ in Friend cells.     

Inhibition of the heat shock response and synthesis of glucose-regulated proteins in Ca2+-deprived rat hepatoma cells

Rat hepatoma cells become refractory to the induction of heat shock proteins and highly resistant to severe hyperthermia when incubated in Ca2+-free medium. The Ca2+-depleted cells synthesize polypeptides identified as the glucose-regulated proteins, but these proteins do not appear to be directly involved in the inhibition of the heat shock response. The results suggest that a Ca2+-dependent metabolic process is involved in the generation of the heat shock signal and/or mediates a step in the subsequent cascade of events that leads to the induction of heat shock protein synthesis and cell death.     

Modulatory effect of HCO3- on rat mast cell exocytosis: cross-talks between bicarbonate and calcium.

HCO-3 modulation of histamine release and its relationship with the Ca2+ signal were studied in serosal rat mast cells. Histamine release was induced by Ca2+ mobilizing stimuli, namely compound 48/80, thapsigargin, Ca2+ chelators, ionophore A23187, and PMA and ionophore A23187 in a HCO-3-buffered medium or a HCO-3-free medium. The presence of HCO-3 reduced histamine release by 48/80, Ca2+ chelators, A23187, and PMA/A23187, but increased histamine release induced by thapsigargin. Histamine release by PMA was significantly higher in a HCO-3-free medium than in a HCO-3-free medium, as it was the PMA potentiation of histamine release by A23187. [Ca2+]i changes induced by these drugs were measured in fura-2-loaded mast cells. In thapsigargin and EGTA or BAPTA preincubated mast cells [Ca2+]i increase was higher in a HCO-3-buffered medium than in a HCO-3-free medium in the presence of Ca2+. On the contrary, in compound 48/80 and PMA/A23187 activated mast cells the [Ca2+]i increase is the same both in the presence and in the absence of HCO-3. The effect of HCO-3 on histamine release in serosal rat mast cells depends on the stimulus, but it is not related to the presence of Cl-. In thapsigargin-stimulated mast cells the effect of HCO-3 on histamine release may be related to the Ca2+ signal, but in compound 48/80, EGTA, and PMA/A23187-activated mast cells there is no relationship between intracellular Ca2+ and the inhibitory effect of HCO-3 on histamine release. Additionally, the PKC pathway is implicated in the inhibitory effect of HCO-3 on histamine release, the higher the chelation of calcium rendering the higher the enhancement of the response after adding calcium in the absence of HCO-3.     

Production of somatic hybrid tissues following chemical and electrical fusion of protoplasts from albino cell suspensions ofMedicago sativa andM. borealis

Protoplasts isolated from cell suspensions of albinoMedicago borealis andM. sativa were fused chemically, using two methods, and electrically. Although a small scale method of chemical fusion gave the highest fusion frequency, electrofusion was the superior technique on the basis of throughput of green somatic hybrid cell colonies. Chlorophyll-containing tissues were confirmed as being somatic hybrid by isoenzyme and cytological analyses. This is the first report of the application of albino complementation to produce somatic hybrid cells in forage legumes.Abbreviations AC alternating current - DC direct current - 2,4-D 2,4-dichlorophenoxyacetic acid - f.wt. fresh weight - PEG polyethylene glycol - K8P and K8 Kao (1977) protoplast and cell culture media - MS Murashige and Skoog (1962) medium - UM Uchimiya and Murashige (1974) medium