Insulin rapidly increases diacylglycerol by activating de novo phosphatidic acid synthesis

The mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes were examined. When [3H]arachidonate labeling of phospholipids was used as an indicator of phospholipase C activation, transient increases in [3H]diacylglycerol were observed between 0.5 and 10 minutes after the onset of insulin treatment. With [3H]glycerol labeling as an indicator of de novo phospholipid synthesis, [3H]diacylglycerol was increased maximally at 1 minute and remained elevated for 20 minutes. [3H]Glycerol-labeled diacylglycerol was largely derived directly from phosphatidic acid. Insulin increased de novo phosphatidic acid synthesis within 5 to 10 seconds; within 1 minute, this synthesis was 60 times greater than that of controls. Thus, the initial increase in diacylglycerol is due to both increased hydrolysis of phospholipids and a burst of de novo phosphatidic acid synthesis. After 5 to 10 minutes, de novo phosphatidic acid synthesis continues as a major source of diacylglycerol. Both phospholipid effects of insulin seem important for generating diacylglycerol and other phospholipid-derived intracellular signaling substances.     

Immune specific induction of interferon production in cultures of human blood lymphocytes

Human blood lymphocytes stimulated with nonviral antigens in vitro produce an antiviral substance with the biological and biochemical characteristics of interferon. The induced response was specific for cells obtained from immune donors. Cells from nonimmune donors did not produce interferon on exposure to these substances. The quantity of interferon produced by antigen stimulation was related to concentration of antigen over a relatively narrow range; with higher concentrations induction was decreased. Interferon production was maximum during days 4 to 7 in culture. In contrast, phytohemagglutinin-induced interferon was primarily produced during the first 4 days in culture.     

Endogenous regulation of macrophage proliferative expansion by colony-stimulating factor-induced interferon

Stimulation of cultures of murine bone-marrow cells with specific macrophage growth factor (colony-stimulating factor I) resulted in the production of type I interferon. Neutralization of this endogenous interferon by antiserum directed against interferons alpha and beta resulted in a significant enhancement of mononuclear phagocyte proliferation from committed marrow precursors. The effect of the antiserum was lost in cultures depleted of adherent cells, an indication that an adherent regulatory cell (or cells) in the marrow limits mononuclear phagocyte proliferation by producing antiproliferative interferon in response to high levels of specific growth factor.     

Diagnostic ultrasound and sister chromatid exchanges: failure to reproduce positive findings

Human lymphocytes were exposed in vitro to ultrasound from two clinical devices, one of which was previously reported to have increased the frequency of sister chromatid exchanges. The ultrasonic exposures had no significant effect on the frequency of sister chromatid exchanges from three blood donors. Exposure to ultrasound also had no effect on cell cycle progression. A concomitant positive control (mitomycin C) resulted in a significant increase in sister chromatid exchanges.     

Tunicamycin enhances the antiviral and anticellular activity of interferon

The inhibitory effects of interferon on virus multiplication and cell growth are significantly enhanced by treatment with tunicamycin. Potentiation of antiviral activity was found only with enveloped viruses and not with nonbudding viruses. Changes in the plasma membrane of treated cells may account for this effect, since enveloped viruses bud from the cell surface as a terminal step.     

Generation of calcium oscillations in fibroblasts by positive feedback between calcium and IP3

A wide variety of nonexcitable cells generate repetitive transient increases in cytosolic calcium ion concentration ([Ca2+]i) when stimulated with agonists that engage the phosphoinositide signalling pathway. Current theories regarding the mechanisms of oscillation disagree on whether Ca2+ inhibits or stimulates its own release from internal stores and whether inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) also undergo oscillations linked to the Ca2+ spikes. In this study, Ca2+ was found to stimulate its own release in REF52 fibroblasts primed by mitogens plus depolarization. However, unlike Ca2+ release in muscle and nerve cells, this amplification was insensitive to caffeine or ryanodine and required hormone receptor occupancy and functional IP3 receptors. Oscillations in [Ca2+]i were accompanied by oscillations in IP3 concentration but did not require functional protein kinase C. Therefore, the dominant feedback mechanism in this cell type appears to be Ca2+ stimulation of phospholipase C once this enzyme has been activated by hormone receptors.     

Interferon production and action in mouse, hamster and somatic hybrid mouse-hamster cells

A hybrid mouse-hamster cell line was developed from a mouse cell line which produces a high titer of interferon and is sensitive to its action, and a hamster cell line which produces little interferon and is relatively insensitive to its action. Parental cell lines demonstrated complete species specificity with respect to interferon production and action. The hybrid cells produced interferon (or interferons) effective when tested on the mouse cell line and primary hamster cells; the hybrids were sensitive to the action of both mouse and hamster interferons. Hybrid cells produced ten times more hamster interferon than the parent hamster cell line and were eight times more sensitive to hamster interferon than the parent hamster cell line.     

Inhibition of cell motility by interferon

Interferon derived from human leukocytes, human fibroblasts, and mouse fibroblasts was found to inhibit the motility of cultured cells. It inhibits the tumor-induced motility of capillary endothelial cells as well as the spontaneous migration of other cell types. The ability of a given preparation of interferon to inhibit the motility of a given cell type is proportional to its antiviral activity in that particular cell type. Antiserum to human leukocyte interferon neutralizes both the motility-inhibitory activity and the antiviral activity of this preparation.     

Cell-to-cell transfer of interferon-induced antiproliferative activity

Interferon-treated cells rapidly and efficiently transferred the antiproliferative activity of interferon to untreated cells. This phenomenon was not due to the carry-over of interferon by the interferon-treated cells. Thus, to evoke an antiproliferative state, interferon did not directly contact each cell in a population. The results suggest a novel mechanism by which interferon may indirectly regulate cell growth, and suggests that cells other than those of the immune system may play a role in controlling tumor growth in tissue where cell-to-cell contact occurs.     

Inhibition of development of exoerythrocytic forms of malaria parasites by gamma-interferon

A specific DNA probe was used to study the effect of recombinant rat, mouse, and human gamma-interferon (gamma-IFN) on the course of sporozoite-induced malaria infections. In mice and rats infected with sporozoites of Plasmodium berghei, mouse and rat gamma-IFN's strongly inhibited the development of the exoerythrocytic forms in the liver liver cells of the hosts, but not the development of the erythrocytic stages. The degree of inhibition of the exoerythrocytic forms was proportional to the dose of gamma-IFN administered, but was independent of the number of sporozoites used for challenge. A 30 percent reduction in the development of exoerythrocytic forms in rat liver was achieved when 150 units (about 15 nanograms of protein) of rat gamma-IFN were injected a few hours before sporozoite challenge; the reduction was 90 percent or more with higher doses of gamma-IFN. The effect was less pronounced if the gamma-IFN was administered 18 hours before or a few hours after challenge. Human gamma-IFN also diminished the parasitemia in chimpanzees infected with sporozoites of the human malaria parasite Plasmodium vivax. The target of gamma-IFN activity may be the infected hepatocytes themselves, as shown by in vitro experiments in which small doses of the human lymphokine inhibited the development of exoerythrocytic forms of Plasmodium berghei in a human hepatoma cell line. These results suggest that immunologically induced interferon may be involved in controlling malaria infection under natural conditions.     

Strain dependence of the antiproliferative action of interferon on murine erythroid precursors

Electrophoretically pure mouse interferon inhibits erythropoietin-dependent proliferation of committed erythroid precursors (CFU-E) obtained either from adult mouse bone marrow or from 14-day fetal mouse livers. The degree of inhibition is significantly influenced by the genotype of the cell donor; about ten times as much interferon is required to inhibit proliferation of CFU-E from C57BL/6 than is needed for comparable inhibition of CFU-E from BALB/c or Swiss mice. These strain-dependent results point to the existence of genes that influence the degree of the inhibitory effect of interferon on cell multiplication.     

Human chromosome 21 dosage: effect on the expression of the interferon induced antiviral state

Human primary skin fibroblasts trisomic for chromosome 13, 18, or 21 and diploid human skin fibroblasts were induced for an antiviral response with human interferon. The cells that were trisomnic for chromosome 21 were three to seven times more sensitive to protection by human interferon than the normal diploid or trisomic 18 or 13 fibroblasts. The differential response in trisomnic 21 cells is consistent with the known assignment of the human antiviral gene to chromosome 21.     

中华鳖胚胎肝成纤维细胞的原代培养及其聚肌苷酸胞苷酸(Poly I:C)刺激模型构建

为建立中华鳖胚胎肝成纤维细胞的体外分离培养体系,研究聚肌苷酸胞苷酸(Poly I:C)刺激对细胞干扰素生成通路相关因子的影响,从而最终构建基于该成纤维细胞的干扰素生成通路激活模型,以发育至23期的中华鳖胚胎为实验材料,采用胰蛋白酶消化法得到肝成纤维细胞,结合细胞形态学、生长曲线和PCR法进行鉴定,并分析其生物学特性.之...     

Ionic mechanisms controlling behavioral responses of paramecium to mechanical stimulation

A mechanical stimulus applied to the anterior part of Paramecium causes a transient increase in membrane permeability to calcium. This permits a calcium current to flow into the cell, causing the membrane potential to approach the equilibrium level for calcium. The transient depolarization which results elicits a reversal in the direction of ciliary beat. When the organisms are free-swimming this is seen as the reversed locomotion of Jennings' "avoiding reaction." In contrast, a mechanical stimulus applied to the posterior part results in increased permeability to potassium ions, and hence an outward potassium current. The hyperpolarization which results causes an increase in the frequency of ciliary beat in the normal direction. In free-swimming specimens this is seen as an increase in the velocity of forward locomotion.     

Interferon binding: the first step in establishment of antiviral activity

Chick cells incubated at 1 degrees C with interferon fail to develop antiviral activity, but this activity appears subsequent to a 7-hour incubation at 37 degrees C after removal of interferon by repeated washings. Treatment with actinomycin D blocks the development of the latter activity. Cells incubated with interferon at 1 degrees C for up to 1 hour and then washed and incubated for 2 hours at 37 degrees C develop a degree of antiviral activity proportional to the concentration of interferon at initial incubation; at any concentration, the antiviral activity increased with the duration of initial incubation at 1 degrees C, but a maximal response was reached at 10 or 20 minutes. Treatment with trypsin after incubation with interferon at 1 degrees C inhibited development of antiviral activity. Interferon is rapidly bound to a superficial cell site, and this binding is necessary for development of antiviral activity in chick cells.     

Interferon-resistant cell line lacks fatty acid cyclooxygenase activity

A clone of L1210 mouse leukemia cells selected for resistance to both the antiviral and anticellular properties of mouse interferon were essentially devoid of fatty acid cyclooxygenase activity. Experiments in which broken cell preparations were mixed or the two cell types were cultivated together failed to indicate the presence of a diffusible enzyme inhibitor. Fatty acid lipoxygenase activity of similar magnitude was detectable in both cell types. A selective impairment of fatty acid cyclooxygenase in interferon-resistant cells is consistent with recently described data suggesting that this enzyme may play a crucial role in mediating the antiviral and anticellular effects of interferon.     

Cilia: activation coupled to mechanical stimulation by calcium influx

Ciliated epithelial cells in the oviduct of Necturus maculosus were stimulated mechanically by brief dimpling with a microstylus. This treatment produlced a transient depolarization of the membrane, and a transient increase in the frequency of ciliary beating. The increase in frequency of ciliary beating was related to the concentration of extracellular calcium ion, decreasing with reductiotn in calcium. Addition of lanthanum was followed by a decrease in spontaneous ciliary aictivity and a hyperpolarization of the membrane. In the presence of lanthanum, the transietnt depolarization in response to mechanical stimulation had a shorter timte course, and the concomitant increase in ciliary frequency was greatly reduced. It is concluded that calciuml ions enter the cell as a result of mechanical stimulationi of the membrane, and that calcium influx leads to an increase in the frequency of ciliary activity.     

Depression of circulating interferon response in Balb-c mice after urethan treatment

Urethan, when given to female Balb/c mice, impaired the capacity of these animals to produce circulating interferon. The effect appeared rapidly after a single injection of either 1 or 1.5 milligrams of urethan per gram of body weight and was of short duration. The possibility that this inhibition of the production of interferon plays a role in the enhancement of viral leukemia by urethan should now be considered.     

人肿瘤坏死因子相关凋亡诱导配体基因植物表达载体的构建及瞬时表达

人肿瘤坏死因子相关凋亡诱导配体具有特异性诱导多种肿瘤细胞凋亡而对正常细胞无毒性的特点,被认为是肿瘤凋亡疗法较有希望的候选药物。植物生物反应器在药用蛋白质生产方面具备较大优势。构建了可溶性人肿瘤坏死因子相关凋亡诱导配体基因的植物表达载体,进行本氏烟(Nicotiana benthamiana)瞬时表达,并对目的蛋白的体外活性进行检测。结果显示,本氏烟叶片中目的蛋白平均表达量为68.60 pg/mg TSP;在浓度为200 pg/m L时,对NCI-H460细胞株的抑制率为28.75%。研究表明,经密码子优化的编码基因能够在受体植物中表达出有活性的目的蛋白,为开展目的基因的稳定转化工作奠定了基础。     

Interferon inducers in vitro: difference in sensitivity to inhbitiros of RNA and protein synthesis

Interferon can be induced by diverse agents in a variety of mammalian cell cultures through apparently two mechanisms. One results in an early (2 to 10 hours) appearance of interferon and is relatively resistant to inhibition by actinomycin, puromycin, or fluorophenylalanine. A second mechanism results in a late (18 to 24 hours) appearance of interferon and is more sensitive to inhibition by these inhibitors. The molecular basis for each mechanism is unclear. Since each interferon inducer may have multiple effects on the cell, the differences observed may not necessarily reflect a fundamental difference in the mechanism of interferon stimulation.