Expression of gangliosides as receptors at the cell surface controls infection of NCTC 2071 cells by Sendai virus

The involvement of gangliosides as receptors for Sendai virus was established previously using experimentally produced receptor-deficient cells. In the search for a naturally occurring counterpart, NCTC 2071 cells emerged as a likely candidate. These cells in their native state were not agglutinated nor infected by Sendai virus, but were infected by the virus when the gangliosides GD1a, GT1b, or GQ1b were supplied in the culturing medium. Preliminary analysis indicated that NCTC 2071 cells contained an unusually high ratio of sialoglycoproteins to gangliosides. A brief treatment of the cell surface with the protease trypsin made greater than 99% of the native monolayer susceptible to infection by the wild-type virus which contains the viral attachment protein HN. (Incubation of the trypsin-treated cells with a temperature-sensitive mutant missing HN produced no detectable infection.) The increased binding of cholera toxin, a ganglioside-specific probe, after incubation of the cells with trypsin and sialidase, was consistent with the hypothesis that gangliosides more complex than GM1 are on the surface of NCTC 2071 cells and that trypsin treatment increases their accessibility. The presence of receptor gangliosides in lipid extracts of NCTC 2071 cells was confirmed by thin-layer chromatography of the ganglioside fraction and by the binding of cholera toxin. These results demonstrate that cells containing receptor gangliosides may still be resistant to infection because these are not expressed properly at the cell surface as receptors for interaction with the HN protein of Sendai virus.     

Effect of serum on the antiviral and anticellular activities of mouse interferon

Summary Fetal bovine serum markedly decreased the ability of mouse L-929 interferon preparations to inhibit the formation of L-929 clones, but did not affect their ability to inhibit vesicular stomatitis virus (VSV) plaque formation in these cells. This dissociation of effects by interferon preparations indicates that: 1. the mechanism of action of interferon for its anticlonal and antiviral activities is different; or 2. the molecule responsible for the anticlonal activity is a separate growth inhibitory factor.With 2 Figures     

A new assay system for quality control testing of a chemically defined medium

Summary Mouse NCTC clone 929 L (L-929) cells were propagated continuously over 2 yr in protein-free Eagles's minimal essential medium (EMEM). The cells designated L-929-WS were used for quality-control testing of protein-free EMEM as a model for more general medium quality tests. The parameter for the suggested quality control assay was the growth-promoting activity of the medium for L-929-WS cells. As an example of quality tests we assayed the growth promoting activity of EMEM exposed to various conditions of storage time and temperature. We established the sensitivity of the new assay by comparing it to the original cells L-929 grown in EMEM supplemented with 10% fetal bovine serum (FBS). The assay system using L-929-WS cells propagated continuously in protein-free medium proved to be much more sensitive. The sensitivity, however, was abolished by the addition of FBS in a concentration as low as 1% to a culture medium.     

Interferon-induced alterations in sialic acid and glycoconjugates of L-929 cells

Changes in Sialic acid and glycosphingolipid (GSL) metabolism were demonstrated in interferon (IFN)-treated L-929 cells. IFN induced an increase in total cell Sialic acid, sialoglycoproteins, and gangliosides, as shown by calorimetric and radiolabeling techniques. Expression of cell surface (neuraminidase-releasable) Sialic acid on IFN-treated cells was markedly elevated, particularly in the GSL fractions. The incorporation of [¹⁴C]galactose into glycoproteins, most neutral GSL homologs, and most ganglioside homologs also was elevated, with the more striking effects (two- to threefold) in the lipid fractions. An increase in the concentration of a ganglioside with the migration of GM₂, as measured by chemical staining of chromatograms, was also shown. The observed effects were IFN dose dependent at ranges from 10 to 10,000 U/ml. As shown previously, IFN-treated L-929 cells became resistant to lysis by virus-induced IFN-activated natural killer cells. Correlations between high levels of surface Sialic acid, resistance to NK cell-mediated lysis, and tumor invasiveness have been shown in other systems.     

Interferonogenicity of the bacterial lysates in human and murine cell cultures

The antibacterial JRS vaccine was found to be capable of inducing interferon production in human peripheral blood leukocytes and mouse bone marrow cells. The vaccine induced no interferon in L-929 cells or human diploid M-19 cells. Interferon appeared in the culture fluid within 4-6 hours after induction and reached the maximum levels in 18-24 hours. One-hour contact of the vaccine with leukocytes was sufficient for interferon induction. The interferon generated in human blood leukocytes was partially stable at pH 2.2; heating at 56 degrees C for 30 min reduced its activity 4-fold; antiserum to alpha-interferon inhibited it.     

Mouse fibroblast interferon modifies Salmonella typhimurium infection in infant mice.

The effect of mouse fibroblast interferon on Salmonella typhimurium infection in infant mice was examined. The lethality to mice that had been given S. typhimurium intragastrically was significantly reduced in a dose-dependent manner when the mice were pretreated with fibroblast interferon. Lower doses of interferon delayed the development of disease. Interferon neutralized with anti-interferon globulin did not influence the lethality of S. typhimurium to mice. In mice treated with interferon there was also a reduced invasiveness of S. typhimurium in intestinal epithelial cells in vivo. It was further demonstrated in an in vitro system that interferon pretreatment of mouse L-929 cells inhibited the invasiveness of the bacteria in a dose-dependent manner. The in vitro inhibition was neutralized with anti-interferon globulin. The results indicate that interferon inhibits Salmonella bacteria from invading cells and establishing an intracellular state of infection. This may represent an important factor in the pathogenesis of disease.     

Interferon action: role of membrane gangliosides.

The antiviral activity of human (fibroblast + leukocyte) and mouse fibroblast interferon was neutralized by preincubation with ganglioside before application to cells. Ganglioside mixtures were as effective as pure gangliosides, with no particular ganglioside specificity observed for neutralization of human interferon. Ganglioside-agarose derivatives removed human interferon from solution; interferon was eluted with buffers containing urea, sodium dodecyl sulfate, and mercaptoethanol. Ganglioside-deficient transformed mouse cell lines were relatively insensitive to interferon action. Treatment of such cells with ganglioside led to an increase in membrane ganglioside content and in two of three cell lines to an increase in cell sensitivity to mouse interferon; in mouse SVS AL/N and TAL/N cells, GM₂, GT₁, and a mixture of crude gangliosides were effective whereas GM₁ and GD_1a were without effect. The interferon sensitivity of untransformed cells, which failed to take up appreciable ganglioside, was not increased by treatment with exogenous ganglioside; the sensitivity of one GM₁-deficient transformed cell line ($\text{K-Balb}\text{C-3T3}$) was not increased by pretreatment with ganglioside. These results indicate an interaction between gangliosides and several types of interferon and suggest a role for membrane gangliosides in the antiviral action of interferon.     

High-affinity binding of 125I-labeled mouse interferon to a specific cell surface receptor. IV. Mouse gamma interferon and cholera toxin do not compete for the common receptor site of alpha / beta interferon

The initial step in interferon action consists of the binding to a specific high-affinity cell surface receptor. Mouse α and β interferon have been shown to share a common receptor. We now present evidence that mouse γ interferon does not compete for this receptor on mouse L 1210 and L 929 cells. Cholera toxin which binds specifically to the membrane monosialoganglioside G_M1, and has been shown to inhibit interferon action, does not inhibit the specific binding of labeled mouse α/β interferon to L 1210 or L 929 cells. Conversely, mouse α/β interferon does not inhibit the specific binding of radioactive cholera toxin to L 929 cells. L 1210S cells which have a specific receptor for α/β interferon do not have a specific binding site for cholera toxin. We conclude from these studies that there is no evidence to indicate that cholera toxin and mouse α/β interferon share a common receptor.     

Author index for volume 132

It has previously been shown that a strain of thymidine kinase (tk)-deficient mouse L-929 cells was unable to respond to murine β-interferon by induction of an anti-viral state and synthesis of double-stranded, RNA-dependent enzymes. Sensitivity to interferon can be restored by introducing into the cells a segment of Herpes simplex virus DNA containing the viral tk gene. It is shown here that not all Ltk(?) cell strains are resistant to interferon, suggesting that expression of a tk gene is not a prerequisite for response to interferon. Introduction of various genes into the resistant Ltk(?) strain, either alone or together with DNA containing the Herpes virus tk gene, leads to restoration of interferon sensitivity only when tk-containing DNA is inserted, showing that the activation of interferon responsiveness is not an artifact of the gene transfer, selection, and cloning procedures. The results imply that a component of the Herpes virus DNA introduced into the cells is able to activate interferon sensitivity.     

Interferon enhances the fragility of lysosomes in L-929 mouse fibroblasts.

Infection of interferon-treated L-929 mouse fibroblasts with vaccinia WR virus is followed by severe cytolysis within 3 to 4 h. It is shown that this cytolysis cannot be caused by enzymes released from lysosomes into the cytosol. However, there is evidence that homologous interferon has a noxious effect on lysosomes. This phenomenon appears to be another aspect of the anticellular functions of interferon.     

p67K kinase in different tissues and plasma of control and interferon-treated mice

Interferon-treated mouse cells show an enhanced level of protein kinase activity which is manifested by the phosphorylation of an endogenous 67,000-molecular weight protein (p67K kinase). This kinase activity can be assayed efficiently after its partial purification on poly(I) · poly(C)-Sepharose. We have previously shown that the p67K kinase is present in the liver, spleen and plasma (heparinized) of mice with high levels of circulating interferon. Here we confirm these results by treatment of mice with interferon and furthermore show that besides the liver and spleen, the level of p67K kinase is enhanced in several other tissues such as thymus, brain, pancreas, heart, and lung. The action of interferon in mice was further monitored by the assay of pppA(2′p5′A)n synthetase (2–5A synthetase) in different tissues. The level of 2-5A synthetase was enhanced several fold in the following tissues: heart, pancreas, thymus, liver, and spleen. The detection of 2–5A synthetase and p67K kinase activities in the different tissues of mice provides suitable markers for the response of each individual tissue toward treatment with interferon. The phosphorylated 67K protein (pp67K) from control and interferon-treated mouse L-929 cells and from the plasma and different tissues of control and interferontreated mice was characterized by two-dimensional gel electrophoresis. The isoelectric point (pl) of pp67K from the different tissues and L-929 cells was 8 to 8.5. On the other hand the pI of pp67K from the plasma had a range of 7.5 to 8. These results indicated that the presence of p67K kinase in the plasma of mice is not due to lysis of tissue cells.     

Production,purification and characterization of rat interferon

Summary Priming with heterologous mouse interferon, increased production of an antiviral substance induced in rat diploid fibroblasts by Newcastle disease virus. This substance was characterized as an acid stable interferon. This rat interferon exhibited marked cross-species antiviral activity when tested in mouse L929B cells, guinea pig embryo fibroblasts, human fibroblasts and bovine cells but was not active on chick embryo cells.     

三种贵金属烤瓷合金与高糖对小鼠成纤维细胞L-929增殖的影响

背景：糖尿病患者这一特殊群体口腔环境较为特殊,关于口腔修复材料在这类患者应用的安全性研究较少见报道。 目的：检测3种贵金属烤瓷合金与高糖对体外培养的小鼠成纤维细胞L-929增殖的影响。 方法：将89%金合金、74%金合金、银钯合金3种烤瓷材料浸泡在不同糖浓度(11.1,22.2,33.3 mmol/L)的培养液中浸提,将所得的浸提液与小鼠成纤维细胞L-929共培养。同期设立空白对照。 结果与结论：L-929在不同贵金属烤瓷合金和不同糖浓度的细胞培养液中的增殖活性不同,24,72 h两因素交互作用的P值均小于0.05。与阴性对照组相比,22.2 mmol/L糖浓度下89%金合金促进细胞的增殖(P=0.004),银钯合金则抑制细胞的增殖(P < 0.001),74%金合金在不同糖浓度的培养液中与阴性对照组相比其细胞的增殖活性差异无显著性意义。结果表明,上述3种材料中74%金合金在不同糖浓度下对细胞增殖的影响最小。 关键词：贵金属烤瓷合金;高糖;金合金;银钯合金;小鼠成纤维细胞;增殖 doi:10.3969/j.issn.1673-8225.2012.12.019     

贵金属烤瓷合金与高糖对细胞外液炎性因子的影响

背景：前期研究中发现高糖与烤瓷合金浸提液对小鼠成纤维细胞L-929的增殖存在交互效应。 目的：观察3种贵金属烤瓷合金与高糖对小鼠成纤维细胞L-929中白细胞介素6水平的影响。 方法：采用两因素析因设计实验研究不同糖浓度(11.1,22.2,33.3 mmol/L)下,89%金合金、74%金合金及银钯合金贵金属烤瓷合金浸提液,分别与小鼠成纤维细胞L-929体外共培养24h后,细胞外液中白细胞介素6水平的变化,并设未加入合金材料的空白对照为阴性对照组。 结果与结论：两因素析因设计分析结果表明,贵金属烤瓷合金浸提液的主效应有统计学意义(P=0.001),其中74%金合金与银钯合金白细胞介素6水平低于阴性对照组(P=0.004,0);糖浓度的主效应也有统计学意义(P=0.005), 33.3 mmol/L糖浓度组白细胞介素6水平低于11.1 mmol/L(正常)糖浓度组(P=0.001)。但贵金属烤瓷合金浸提液与糖浓度两因素对小鼠成纤维细胞L-929细胞外液中白细胞介素6水平的影响无交互作用(P=0.33)。     

Effect of different interferon preparations on the synthesis of cellular DNA

The relationship between the age of interferon producers and the capacity of interferon to inhibit DNA synthesis in L929 cells was demonstrated on interferon models produced by fibroblast cultures from newborn and adult mice in the presence of blood sera of hemologous ages. Inhibition of DNA synthesis by both types of interferon partially purified with the use of porous glass and having similar antiviral activity depended both on the dose of the preparation and on the time of cell incubation with it. Under equal conditions, the interferon produced by newborn mouse cells inhibited DNA synthesis 1.9-fold less effectively than interferon produced by adult mouse cells.     

Mechanisms of cytotoxicity of nickel ions based on gene expression profiles

This study investigated cytotoxic effects of Ni(II) to mouse fibroblast cells (L-929) on the level of gene expression profiles with cDNA microarray. The gene expression profiles of L-929 were detected after the cells were cultured in the medium with 200 microm Ni(II) for 24, 48 and 72 h, respectively, and the cytotoxicity of Ni(II) was evaluated with methylthiazoltetrazolium (MTT) assay. 20 up-regulated genes and 19 down-regulated genes were differentially expressed in all three-culture periods. Gene ontology analysis showed that the L-929 cells which responded to Ni(II) covered a broad range of functional gene groups including cellular biological process, molecular function, and cellular component. Ni(II) has extensive effects on cells by inhibiting cell proliferation and differentiation through inducing cell apoptosis, affecting cell development and influencing cholesterol metabolism.     

The influence of temperature on interferon in cell cultures of homoiothermic and heterothermic mammals

Subnormal temperature was found to depress the production of interferon by cultures of fibroblasts of homoiotherms and heterotherms after virus or poly I.poly C induction. However, in comparison to human (HEF) and mouse (MEF) fibroblasts (homoiotherms) induced with NDV-R or poly I.poly C, interferon production in fibroblasts of spotted sousliks (SL) (heterotherm) and in the aneuploid line of mouse origin (L929) exhibits a greater cold resistance. In contrast to HEF and MEF cells in SL and L929 cells interferon was produced even at 21 degrees C after induction with NDV-R and at 26 degrees C after induction with poly I.poly C. A comparison of alpha and gamma interferon production by mouse and spotted souslik leukocytes did not reveal such distinct differences. At a low temperature (26 degrees C) the production of NDV-R induced interferon was depressed, but it was higher after induction with LPS in both types of leukocytes. PHA and Con A induced interferon was produced in both types of leukocytes only at 37 degrees C. These data confirm that alpha, beta and gamma interferon induction is triggered by different mechanisms and suggest a resistance to cold of beta interferon production in heterothermic animals.     

Studies on the Mechanism of the Priming Effect of Interferon on Interferon Production by Cell Cultures Exposed to Poly(rI) · Poly(rC)

Interferon induction by poly(rI).poly(rC) in primary rabbit kidney and mouse L-929 cell cultures was markedly increased if the cells were previously treated with homologous interferon. This priming effect has been established with different times of exposure of the cells to poly(rI).poly(rC), and was most pronounced for short pulses of contact of the polynucleotide with the cells (10 s, 1 min). Treatment of the cells with pancreatic ribonuclease immediately after their exposure to poly(rI).poly(rC) brought about a relatively greater reduction of the interferon response in interferon-primed cells than it did in unprimed cell cultures. Priming of the cells with interferon did not increase cell-binding of poly(rI).poly(rC), whether this cell-binding was measured quantitatively (by radioactivity, upon exposure of the cells to radiolabeled polymer) or qualitatively (by antiviral activity, by assaying the cell extract for virus plaque reduction). Similarly, interferon priming did not alter the sensitivity of cell-associated poly(rI).poly(rC) to extraneous ribonuclease treatment. Finally, priming with interferon did not decrease the rate of degradation of cell-bound poly(rI).poly(rC) by cellular nucleases nor did it increase the anti-nuclease potency of the cells. The exact mechanism by which previous exposure of the cells to interferon enhances subsequent interferon production, induced by either synthetic polynucleotides or viruses, has not yet been resolved.     

Ultrastructural distribution of interferon receptor sites on mouse L fibroblasts grown in suspension: ganglioside blockade of ligand binding.

Murine beta-interferon (IFN) receptors on L929 cells grown in suspension culture were visualized by indirect immunoferritin electron microscopy. Ferritin label on these cells was associated primarily with the coated areas and coated pits of the membrane, in contrast to previous observations with L929 cells grown in a monolayer, which did not reveal such coated areas or pits but showed ferritin label distributed randomly on the cell membrane (Kushnaryov et al., Infect. Immun. 36:811-821, 1982). On about 15% of the cell sections from suspension-grown cells, the ferritin label was found outside coated membrane areas. These findings suggest that different cell populations exist with respect to the localization and possibly the affinity of IFN receptors. In the same experiment, exogenously added gangliosides blocked the binding to cell surfaces not only of 125I-labeled IFN but also of unlabeled IFN as revealed by an immunospecific ferritin labeling technique, providing direct evidence that gangliosides interfere with the binding of IFN to specific receptor sites on the surface of mouse L929 cells. These studies establish that the binding of IFN to cell membranes, depending on cell growth conditions, can involve coated areas and coated pits, to which certain hormones and toxins have been shown to bind.