BCL-2-Related Protein Expression in Apoptosis: Oxidative Stress Versus Serum Deprivation in PC12 Cells

Abstract: Expression of the BCL-2 protein family members, BAX, BAK, BAD, BCL-xL, BCL-xS, and BCL-2, was measured (by western blotting using specific antibodies) in PC12 cells before and during apoptosis induced by either H₂O₂ treatment or by serum deprivation and during rescue from apoptosis by nerve growth factor (NGF). H₂O₂-induced apoptosis, as measured by DNA fragmentation, caused: (a) a dose-dependent increase in BAX, (b) a dose-independent increase in BAK, and (c) a dose-dependent inhibition of BAD expression. By comparison, apoptosis induced by serum deprivation resulted in a time-dependent decrease in both BAX and BAK, along with a dramatic and sudden decrease in BAD expression. However, when PC12 cells were incubated in an apoptosis-sparing medium (i.e., NGF-supplemented serum-free medium), both BAX and BAK were increased significantly, whereas BAD expression remained inhibited. BCL-xL expression was increased by H₂O₂ but unaffected by serum deprivation or long-term NGF treatment. Neither BCL-2 nor BCL-xS expression could be detected in PC12 cells under the experimental conditions tested. Our results show that the expression of BAX, BAK, BAD, and BCL-xL is altered in a stimulus-dependent manner but cannot be used to define whether a cell will undergo or survive apoptosis. The similarity between changes in expression of BCL-2-related proteins induced by H₂O₂ exposure and NGF rescue could reflect activation in part of a common antioxidant pathway.     

Roles for Protein Kinase C and Mitogen-Activated Protein Kinase in Nicotine-Induced Secretion from Bovine Adrenal Chromaffin Cells

Abstract: Both the Ca²⁺/phospholipid-dependent protein kinases (protein kinases C, PKCs) and mitogen-activated protein kinases (MAPKs) have been implicated as participants in the secretory response of bovine adrenomedullary chromaffin cells. To investigate a possible role for these kinases in exocytosis and the relationship of these kinases to one another, intact chromaffin cells were treated with agents that inhibited each of the kinases and analyzed for catecholamine release and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)/MAPK activation after stimulation with secretagogues of differential efficacy. Of the three secretagogues tested, inactivation of PKCs by long-term phorbol 12-myristate 13-acetate (PMA) treatment or incubation with GF109203X had the greatest inhibitory effect on nicotine-induced catecholamine release and MEK/MAPK activation, a moderate effect on KCl-induced events, and little, if any, effect on Ca²⁺ ionophore-elicited exocytosis and MEK/MAPK activation. These results indicate that PKC plays a significant role in events induced by the optimal secretagogue nicotine and a lesser role in exocytosis elicited by the suboptimal secretagogues KCl and Ca²⁺ ionophore. Treatment of cells with the MEK-activation inhibitor PD098059 completely inhibited MEK/MAPK activation (IC₅₀ 1–5 µM) and partially inhibited catecholamine release induced by all secretagogues. However, PD098059 was more effective at inhibiting exocytosis induced by suboptimal secretagogues (IC₅₀～10 µM) than that induced by nicotine (IC₅₀～30 µM). These results suggest a more prominent role for MEK/MAPK in basic secretory events activated by suboptimal secretagogues than in those activated by the optimal secretagogue nicotine. However, PD098059 also partially blocked secretion potentiated by short-term PMA treatment, suggesting that PKC can function in part by signaling through MEK/MAPK to enhance secretion. Taken together, these results provide evidence for the preferential involvement of MEK/MAPK in basic secretory events activated by the suboptimal secretagogues KCl and Ca²⁺ ionophore and the participation of both PKC and MEK/MAPK in optimal secretion induced by nicotine.     

Enhanced antitumor effects of tumor antigen-pulsed dendritic cells by their transfection with GM-CSF gene

To investigate the biological characterization and antitumor activitites of GM-CSF gene-transfected dendritic cells, the splenic dendritic cells were infected with GM-CSF recombinant replication-deficient adenoviruses in vitro . Their enhanced expression of B7 was demonstrated by FACS analysis, and more potent stimulatory activity was confirmed by allogeneic MLR. Immunization of dendritic cells pulsed with irradiated B16 melanoma cells induced sig-nificant CTL and enabled host to resist the challenge of wild-type B16 cells. When they were transfected with GM-CSF gene subsequently, the induced CTL activity was higher, and the produced protection against B16 cell challenge and therapeutic effect on the mice with preestablished pulmonary melastases more effective. These data suggest that the dendritic cells pulsed with tumor antigen then transfected with GM-CSF gene can be used as an effective vaccine in tumor immunotherapy.     

四种鱼类外周血红细胞细胞周期及DNA含量

通过流式细胞仪测量了兴国红鲤（Ｃｙｐｒｉｎｕｓ ｃａｒｐｉｏ Ｖａｒ． Ｓｉｎｇｕｏｎｅｎｓｉｓ）、奥利亚罗非鱼（Ｓａｒｏｔｈｅｒｏｄｏｎ ａｕｒｅａ）、 鳙（Ａｒｉｓｔｉｃｈｔｈｙｓ ｎｏｂｉｌｉｓ Ｒ．）和团头鲂（Ｍｅｇａｌｏｂｒａｍａ ａｍｂｌｙｃｅｐｈａｌａ Ｙｉｎ．）红细胞的ＤＮＡ含量,四种鱼与鸡红细胞ＤＮＡ含量的比值分别为１·６５、０．９６、０．９１、１·１５,以鸡红细胞ＤＮＡ含量２．３ｐｇ／Ｎ计算,四种鱼二倍体体细胞的 ＤＮＡ含量分别为３．８０ ｐｇ／Ｎ, ２． ２２ｐｇ／Ｎ、２． ０８ｐｇ／Ｎ、 ２． ６６ｐｇ／Ｎ。另外,四种鱼外周血红细胞分别有２６％、 ２６％、 ２３％、 ２４％的细胞处于 ＤＮＡ合成期（Ｓ）,ＤＮＡ合成后期（Ｇ２）和分裂期（Ｍ）,这表明这四种鱼类的外周血红细胞并非失去分裂能力的特化细胞群,它们表现了较强的细胞周期现象。这有可能是这几种硬骨鱼类外周血仍具有造血功能的缘故。     

Interactions between diurnal temperature fluctuations and salinity on expansion growth and water status of young tomato plants

Expansion growth is limited if the difference between day and night temperature (DIF) is negative. Growth is also limited high salinity. Expansion growth of tomato seedlings was studied under day/night temperatures of 16/24°C and 24/16°C, and nutrient solution salinities of 3 and 15 mS cm^-1 to ascertain whether interactions exist between the two stress forms. Water status was also studied in order to assess possible mechanisms of growth retardation. A significant interaction between DIF and salinity was found for all recorded growth variables. Hypocotyl length, plant height, leaf area and fresh and dry weight were lower at negative DIF than at positive, the reduction being greater at low salinity than at high. Increased salinity also reduced growth, more so at positive DIF than at negative. Growth reduction at negative DIF was accompanied increased shoot water and osmotic potentials. Pressure potential was unaffected DIF. Growth reduction at high salinity was accompanied reduced water and osmotic potentials. Pre-dawn pressure potential was increased at high salinity, whereas no effect of salinity on pressure potential at midday was found. The differences in effects on water status between the two stress forms may suggest differing mechanisms of growth retardation.     

Down-Regulation of T-Cell Proliferation in Response to Soluble Anti-CD3 Antibodies through Development of Redirected Cytolytic Activity Eliminating Costimulatory Cells

CD4⁺ T-depleted spleen cells (CD8⁺ T cells) activated by anti-CD3 antibodies (aCD3) suppressed proliferation of CD8⁺ T-depleted spleen cells (CD4⁺ T cells) and fresh normal T cells in response to aCD3. Antigen-nonspecific cytolytic activity was induced in splenic CD8⁺ T cells by stimulation with aCD3 and showed the peak level on day 3, whereas cytolytic activity induced in CD4⁺ T cells was weak. Intact Ig but not F(ab')₂ of aCD3 induced and mediated cytolytic activity. Correspondingly, the cytolytic activity induced by aCD3 was directed against target cells bearing Ig-binding Fc-receptor activity and cytolysis was inhibited by the addition of free Ig into the assay system. We showed that aCD3-activated T cells carried a high level of aCD3 on their surface at the time after the peak proliferation when they attained high cytolytic activity. This raised the possibility that the anti-CD3-induced aCD3-redirected cytolytic activity eliminated Fc-receptor-bearing costimulatory cells in the culture for down-regulation of the T-cell proliferation. This view was supported by partial restoration of anti-CD3-induced low responsiveness of CD8⁺ T cells by the addition of fresh costimulatory cells. These results suggested a new pathway of down-regulation of T-cell proliferation by aCD3-activated cytolytic CD8⁺ T cells.     

1α,25-dihydroxyvitamin D3 rapidly alters phospholipid metabolism in the nuclea envelope of osteoblasts

1α,25-Dihydroxyvitamin D₃ (1α, 25-(OH)₂D₃) has been shown to increase cytosolic calcium and inositol trophosphate levels in rat osteosarcoma cells (ROS 17/2.8) and to increase nuclear calcium in these cells. To determine the mechanism(s) of 1α, (OH)₂D₃-induced changes in the calcium, the effect of the hormone on phospholipid metabolism in isolated osteoblast nuclei wa assessed. 1α,25 (OH)₂D₃, 20 nM, increased inositol triphosphate levels in the nuclei after 5 min of treatment. The biologically inactive epimer, 1β,25-(OH)₂D₃, had no significant effect on inositol triphosphate levels. ATP, 1 mM, also increased inositol triphosphate levels in the isolated nuclei after 5 min. 1α,25-(OH)₂D₃, 20 nM, increased calcium in the isolated nuclei in the presence but not in the absence of extranuclear calcium with 5 min. Nuclear calcium was also increased within 5 min by ATP, 1 mM, and inositol triphosphate, 1 mM. The effects of ATP on nuclear calcium was not additive with 1α, 25-(OH)₂D₃, suggesting that these two agents increase nuclear calcium in these osteoblast-like cells by similar mechanisms. In summary, 1α,25-(OH)₂D₃ amd ATP rapidly increase inositol triphosphate levels in isolated from ROS 17/2.8 cells. The hormone, the nucleotide, and the inositol phospholipid nuclear calcium. Thus, the 1α,25-(OH)₂D₃ and ATP effects of nuclear calcium may be mediated by changes in phospholipid metabolism in the nuclei of these osteoblastlike cells. © Wiley-Liss, Inc.     

Prothrombin cleavage by human vascular smooth muscle cells: A potential alternative pathway to the coagulation cascade

Thrombin is a potent mitogen for human vascular smooth muscle cells (HVSMC) and its enzymatic activity is required for this function. The present study demonstrates that prothrombin is also mitogenic for HVSMC due to the generation of enzymatically active thrombin which occurs upon incubation of prothrombin with the cells. Analysis by SDS-PAGE, immunoblotting, and amino acid sequencing revealed that prothrombin incubated with HVSMC undergoes limited proteolysis. Prethrombin 1 was formed through cleavage at R¹⁵⁵-S¹⁵⁶. Cleavage at R²⁷¹-T²⁷² generated fragment 1.2 and prethrombin 2 whilst cleavage at R²⁸⁴-T²⁸⁵ yielded truncated prothrombin 2 (prethrombin 2′). However, cleavage at R³²⁰-I³²¹ which, during prothrombin activation produces two-chain α-thrombin, was not detectable. Studies on HVSMC-conditioned medium revealed that a similar pattern of prothrombin cleavage occurred by a cell-secreted factor(s). Amidolytic activity analysis indicated that 1–3% catalytically active thrombin-like activity was generated upon incubation of prothrombin with HVSMC-conditioned medium. By treating conditioned medium with various classes of proteinase inhibitors or hirudin, it was determined that prothrombin is cleaved by a cell-derived serine proteinase-like factor(s) at R²⁷¹-S²⁷² and by α-thrombin at R¹⁵⁵-S¹⁵⁶ and R²⁸⁴-T²⁸⁵. Antibodies neutralising the activity of either urokinase, tissue plasminogen activator, or factor Xa failed to alter the prothrombin cleaving activity of conditioned medium. This activity which may catalyse an alternative pathway for the generation of thrombin, was eluted from a gel filtration column as a single peak with apparent molecular mass of 30–40 kDa. © 1995 Wiley-Liss, Inc.     

T cell receptor-mediated Ca2+ signaling: Release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca²⁺ release from the intracellular stores followed by a sustained Ca²⁺ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca²⁺ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca²⁺ -ATPase inhibitor, stimulated Ca²⁺ entry into the cells by a mechanism other than emptying Ca²⁺ stores. In addition, Ca²⁺ entry into the Ca²⁺ -depleted cells was stimulated by low basal level of cytosolic Ca²⁺, not by the emptying of intracellular Ca²⁺ stores. Both the Ca²⁺ release and influx were dependent on high and low concentrations of extracellular Ca²⁺. At low concentrations, Mn²⁺ entered the cell through the Ca²⁺ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn²⁺ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca²⁺ release was inhibited by low concentrations of Ni²⁺, La³⁺, and EGTA, while Ca²⁺ influx was inhibited by high concentrations. Thus, in T cells Ca²⁺ influx occurs independently of IP3-dependent Ca²⁺ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca²⁺ influx. Extracellular Ca²⁺ influenced Ca²⁺ release and influx through the action of two plasma membrane Ca²⁺ entry pathways with different pharmacological and biochemical properties.