钙和钙调素对机械刺激诱导的烟草悬浮培养细胞耐热性的调控

机械刺激（mechanical stimulation,MS）可提高烟草悬浮培养细胞的钙调素（calmodulin,CaM）活性,诱导烟草悬浮培养细胞耐热性的形成,外源Ca2＋可加强,而Ca2＋螯合剂EGTA、质膜Ca2＋通道阻塞剂La3＋和胞内Ca2＋通道阻断剂钌红（RR）,以及CaM拮抗剂氯丙嗪（CPZ）和三氟拉嗪（TFP）则削弱这种耐热性的形成。这些暗示Ca2＋和CaM参与MS诱导的烟草悬浮培养细胞耐热性形成的调控。     

钙信使系统对机械刺激诱导的烟草悬浮培养细胞中H_2O_2爆发的调控

机械刺激可诱导烟草悬浮培养细胞H2O2的爆发,外源Ca2＋有强化作用,而Ca2＋螯合剂EGTA、质膜Ca2＋通道阻塞剂La3＋、胞内Ca2＋通道阻断剂钌红,以及钙调素拮抗剂氯丙嗪和三氟拉嗪则削弱机械刺激诱导的氧化爆发。这暗示以钙和钙调素为核心的钙信使系统对机械刺激诱发的烟草悬浮培养细胞中H2O2的爆发有调控作用。     

细胞壁中的过氧化物酶参与机械刺激诱导的烟草悬浮培养细胞的氧化爆发

细胞壁中的过氧化物酶（CWPOD）是植物细胞中产生H2O2的酶源之一。机械刺激可提高烟草悬浮培养细胞中CWPOD的活性,促进烟草悬浮培养细胞中H2O2的积累,增加悬浮细胞培养介质的pH值。用CWPOD的抑制剂KCN或水杨苷异羟肟酸（SHAM）预处理烟草悬浮细胞后,机械刺激诱发的H2O2爆发和介质pH值的增加都不同程度地受到削弱。这些结果暗示CWPOD有可能参与了机械刺激诱发的烟草悬浮细胞中H2O2爆发的形成。     

机械刺激诱导的烟草悬浮培养细胞耐热性及其与过氧化氢的关系

机械刺激可诱发烟草悬浮培养细胞中H2O2的积累,提高烟草悬浮培养细胞在高温胁迫下的存活率和再生能力,缓解高温胁迫下的细胞活力丧失和膜伤害,外源H2O2预处理也可提高烟草悬浮细胞的耐热性。这些暗示H2O2作为信号分子可触发机械刺激诱导的烟草悬浮细胞耐热性形成。     

细胞外钙调素对烟草悬浮培养细胞蛋白质磷酸化作用的影响

用液体闪烁计数法研究了细胞外钙调素对烟草悬浮培养细胞质蛋白质磷酸化的作用。结果表明烟草细胞细胞质蛋白质磷酸化活性在细胞培养过程中逐渐增加,达到最高峰后又开始下降。在细胞质蛋白质磷酸化强度高峰时,加入抗CaM血清后,细胞质蛋白质磷酸化活性受到了部分抑制。加抗CaM血清后再补加CaM能够部分解除抗CaM血清对细胞质部分与细胞核部分蛋白质磷酸化的抑制作用。外加纯化钙调素可以引起烟草悬浮培养细胞细胞质蛋白质磷酸化的活性增强,并且这种增强作用具有时间（高峰为70min）与剂量（最适为CaM10-7mmol/L）依赖性。CaM引起的细胞质蛋白质磷酸化变化与红光所引起的细胞质蛋白质磷酸化变化在时间进程上是不相同的。     

细胞外钙调素对烟草悬浮培养细胞蛋白质磷酸化作用的影响

用液体闪烁计数法研究了细胞外钙调素对烟草悬浮培养细胞质蛋白质磷酸化的作用。结果表明：烟草细胞细胞质蛋白质磷酸化活性在细胞培养过程中逐渐增加,达到最高峰后又开始下降。在细胞质蛋白质磷酸化强度高峰时,加入抗CaM血清后,细胞质蛋白质磷酸化活性受到了部分抑制。加抗CaM血清后再补加CaM能够部分解除抗CaM血清对细胞质部分与细胞核部分蛋白质磷酸化的抑制作用。外加纯化钙调素可以引起烟草悬浮培养细胞细胞质蛋白质磷酸化的活性增强,并且这种增强作用具有时间（高峰为70min）与剂量（最适为CaM10^-7mmol/L）依赖性。CaM引起的细胞质蛋白质磷酸化变化与红光所引起的细胞质蛋白质磷酸化变化在时间进程上是不相同的。     

烟草培养细胞中钙离子,钙调素与细胞质流的关系

烟草愈伤组织的培养细胞中,当钙离子载体将Ca~(2 )导入细胞时,细胞质流停止.CaM拮抗剂试验表明,高钙使细胞质流停止的效应可能与CaM无关,除W7外的多种CaM拮抗剂都明显而且可逆地抑制细胞质流。酶联免疫吸附分析(ELISA)检出培养细胞中存在有CaM。间接酶标免疫组织化学分析进一步证明CaM存在于胞质条纹中。     

17β-雌二醇诱导血管内皮细胞一氧化氮释放及其与细胞内钙的关系

利用小牛胸主动脉内皮细胞（ＢＡＥＣｓ）作为模型,观察１７β－雌二醇（Ｅ２）ＢＡＥＣｓ一氧化氮（ＮＯ）释放、一氧化氮合酶（ｅＮＯＳ）ｍＲＮＡ表达和细胞内钙（〔Ｃａ＾２＋〕ｉ）的影响,以及雌激素受体（ＥＲ）拮抗剂ｔａｍｏｘｉｆｅｎ和ＮＯＳ抑制剂（Ｌ－ＮＡＭＥ）的作用。结果显示,Ｅ２（１０＾－１２￣１０＾－８ｍｏｌ／Ｌ）呈尝试依赖性促进ＢＡＥＣｓ中ＮＯ的释放,以１０＾－８ｍｏｌ／Ｌ浓度Ｅ２处理ＢＡＥＣｓ     

外源钙调素与拟南芥悬浮培养细胞结合位点的观测及其胞外效应

以拟南芥悬浮培养细胞为实验体系,借助外源荧光及同位素标记钙调素,研究结果表明外源钙调素不能被主动内吞入细胞内,而是主要以完整分子形式结合在细胞外表面;外源纯化钙调素可促进正向型质膜囊泡中的鸟苷酸三磷酸水解酶活性升高,也可引起拟南芥悬浮细胞质游离钙离子浓度的特异升高,表明外源钙调素可能通过细胞表面位点跨膜信号转换为细胞内信号,从而调节生物学活性。     

Ca~(2+)参与NO对蚕豆气孔运动的调控

观察了Ca2 + 、Ca2 + 的螯合剂和Ca2 + 通道抑制剂对NO调控的蚕豆气孔运动的影响。结果表明 ,NO的供体 1～ 10 0 μmol/LSNP (sodiumnitroprusside ,硝普纳 )可诱导气孔关闭 ;除去表皮条缓冲液中的Ca2 + 后 ,NO不再影响气孔的运动 ;Ca2 + 的螯合剂EGTA和BAPTA几乎可以完全抑制NO诱导的气孔关闭作用 ;胞内钙通道抑制剂钌红 (rutheniumred)和L型Ca2 + 通道阻断剂硝苯吡啶 (nifedipine)能够减弱SNP诱导气孔运动的关闭趋势 ;加入Ca2 + 通道抑制剂LaCl3 ,则外源NO失去其诱导气孔关闭的作用。说明在NO调控的气孔运动中 ,在NO信号途径的下游可能涉及来自胞内和胞外Ca2 + 的参与 ,并且胞外Ca2 + 更为重要。     

NO和茉莉酸甲酯对黄芩悬浮细胞生长及黄芩苷合成的影响

以硝普钠(sodium nitroprusside,SNP)为一氧化氮(nitric oxide,NO)的供体,向黄芩(Scutellaria baicalensis)悬浮培养细胞系中添加SNP和茉莉酸甲酯(methyl jasmonate,MJ),考察这两种诱导子在不同的添加时间、添加浓度及混合配比使用对黄芩悬浮细胞系生长和黄芩苷含量的影响。研究结果表明：低浓度的外源NO有利于细胞的生长,但对黄芩苷积累无作用,而MJ有利于黄芩苷的合成,但抑制细胞生长,且两者的适用浓度范围和添加时间存在差异。在细胞培养初期(0天)添加0.05 mmol·L~(-1)SNP,而在细胞生长对数中期(8天)添加10μmol·L~(-1)的MJ,细胞鲜重可达到对照的1.2倍,黄芩苷总量达到对照的2.96倍。     

缺氧缺糖对培养海马神经细胞中一氧化氮和钙离子的影响

在缺血性脑损伤中 ,NO起着重要作用。研究了原代培养的海马神经细胞中 ,缺氧缺糖对NO合成的影响。利用激光共聚焦显微镜和荧光指示剂 ,对胞内钙离子和NO的变化进行实时检测 ,并用HPLC检测了缺氧缺糖导致的谷氨酸释放。结果表明 ,缺氧缺糖引起胞内钙离子浓度升高和NO合成增加。经过 2 0min缺氧缺糖处理后 ,胞外谷氨酸的浓度比对照组高出约10 0 %。N 甲基 D 天冬氨酸 (N methyl D aspartate,NMDA)的拮抗剂MK 80 1对缺氧缺糖引起的细胞内钙离子和NO的升高有明显抑制作用。去除细胞外液的钙离子和加入钙调蛋白抑制剂三氟拉嗪都可以抑制缺氧缺糖引起的NO升高。以上结果提示 ,缺氧缺糖引起神经细胞NO合成增加 ,这种合成受谷氨酸释放 ,胞内钙离子浓度和钙调蛋白的调控。     

一种实时检测剪切力引起培养的大鼠动脉内皮细胞内一氧化氮含量的新方法

建立一个稳定和实时检测在不同剪切力作用下内皮细胞内一氧化氮含量的方法。利用流动小室建立内皮细胞剪切模型 ,在内皮细胞用DAF FM染色后 ,用Zeiss荧光共聚焦显微镜和ICCD摄象头检测细胞内的荧光强度。DAF FM的荧光强度可以反映一氧化氮的胞内含量。剪切力引起内皮细胞合成一氧化氮增加 ,并且这种作用是随着剪切力的增加而增加。剪切力的作用被一氧化氮合酶抑制剂L NAME全部抑制 ,被无Ca2 缓冲液部分抑制。这个方法可以实时反映一氧化氮含量的变化 ,可以用来研究剪切力引起一氧化氮变化的机制以及用来评价内皮细胞对剪切力的反应特性     

Direct and Continuous Electrochemical Measurement of Noradrenaline-Induced Nitric Oxide Production in C6 Glioma Cells

1. Nitric oxide (NO) production in C6 glioma cells was directly monitored in real time by electrochemical detection with a NO-specific biosensor.2. We present here the first direct evidence that noradrenaline elicits long-lasting NO production in C6 cells pretreated with lipopolysaccharide and interferon-, an effect blocked by N ^G-monomethyl-L-arginine, a NO synthase inhibitor.3. This direct electrochemical measurement of glia-derived NO should facilitate our understanding of the kinetics of glial signaling in glia-glia and glia-neuron networks in the brain.     

Induction of Nitric Oxide Synthase and Nitric Oxide-Mediated Apoptosis in Neuronal PC12 Cells After Stimulation with Tumor Necrosis FActor-α/Lipopolysaccharide

Abstract: Exposure of neuronal PC12 cells, differentiated by nerve growth factor, to tumor necrosis factor-α (TNF-α) and bacterial lipopolysaccharide (LPS) resulted in de novo synthesis of inducible nitric oxide synthase (iNOS) mRNA and protein with an increase up to 24 h. Brain NOS expression was unaffected. The induction of iNOS in differntiated PC12 cells was associated with cell death characterized by features of apoptosis, The NOS inhibitors N -monomethylarginine, aminoguanidine, and 2-amino-5,6-dihydro-6-methyl-4 H -1,3-thiazine HCl prevented TNF-α/LPS-induced cell death and DNA fragmentation, suggesting that the TNF-α/LPS-induced cell death is mediated by iNOS-derived NO. This hypothesis is supported by the finding that addition of l -arginine, which serves as a precursor and limiting factor of enzyme-derived NO production, potentiated TNF-α/LPS-induced loss of viability.     

Suppression of Cytokine Production in T Helper Type 2 Cells by Nitric Oxide in Comparison with T Helper Type 1 Cells

We examined the effect of nitric oxide (NO) on cytokine production in T helper (Th) cell subsets, using murine splenic CD4⁺ T cells and two types of Th clones. Interferon-gamma-treated murine peritoneal exudate cells (IFN-PEC) suppressed DNA synthesis to 60% of the control level in CD4⁺ T cells stimulated with the anti-CD3 monoclonal antibody. The production of IL-2 and IL-4 in the CD4⁺ T cells decreased to 63.2% and 9.2%, respectively, of the control value by co-culture with IFN-PEC. The addition of N^G-monomethyl-L -arginine (L-NMMA) partially recovered the suppression of DNA synthesis. In the presence of indomethacin, the suppression of DNA synthesis was partially inhibited and the reduction in the cytokine production caused by IFN-PEC was partially recovered. The simultaneous addition of N^G-monomethyl-L -arginine (L-NMMA) and indomethacin completely inhibited not only the suppression of DNA synthesis but also the reduction in the cytokine production caused by IFN-PEC. Moreover, DNA synthesis in the Th2 clone was suppressed to a greater extent than that in the Th1 clone by co-culture with IFN-PEC. This suppression in the Th1 clone was inhibited by the addition of L-NMMA, whereas the DNA synthesis in the Th2 clone was not recovered by L-NMMA. In addition, sodium nitroprusside (SNP) suppressed IL-4 production in the Th2 clone but had no effect on IL-2 production in the Th1 clone. In the experiment of the co-culture with IFN-PEC, the inhibitory-effect of NO on T cell activation was not clarified by the influence of prostaglandins. However, in conclusion, cytokine production in Th2 cells may be more susceptible to NO than that in Th1 cells.     

Synergistic Inhibition of Growth by Aluminum and Iron of Tobacco (Nicotiana tabacum L.) Cells in Suspension Culture

Growth of tobacco cells at the logarithmic phase was inhibitedafter treatment with both AlCl₃ and FeSO₄ in modified Murashige-Skoogmedium prepared without P_i and EDTA at pH 5.0 for up to 20 h,whereas cells treated with either AlCl₃ or FeSO₄ (at concentrationsup to 100 µM, respectively) grew normally. These resultssuggest the synergistic inhibition of growth by Al³⁺ and Fe²⁺ions. During the exposure to both salts, there was a lag timeof about 10 h before growth inhibition became apparent in culturesof cells upon treatment with the two salts together. Then theextent of inhibition increased and reached a maximum value afterexposure for 18 h. After the lag period, cells treated withboth salts, but not cells treated with either salt alone, exhibitedsignificant increases in the amounts of both aluminum and ironin the cells and could be stained with hematoxylin, with thenuclei staining especially strongly. Cells treated with bothsalts also exhibited a decrease in the number of cells thatcould be plasmolyzed in 1 M mannitol, a decrease in the numberof cells that could be stained with fluorescein diacetate, adecrease in the amount of potassium in cells, and an increasein the extent of lipid peroxidation. These results suggest thataluminum causes the peroxidation of lipids in the presence ofiron and that lipid peroxidation alters the permeability ofthe plasma membrane and leads to cell death. (Received April 18, 1994; Accepted November 11, 1994)