Inhibition of NADPH oxidase subunits translocation by tea catechin EGCG in mast cell

The inhibitory mechanism of tea catechins for allergy remains undefined. We studied the effect of catechins, mainly EGCG, on the activation of mast cell line canine cutaneous mastocytoma cells (CM-MC). Compound 48/80 induced the degranulation in CM-MC dose dependently, whereas its release of beta-hexosaminidase was inhibited by EGCG and O-methylated EGCG (EGCG-Me). Both catechins were found to inhibit intracellular ROS generation dose dependently together with DPI. Intracellular ROS generation in human polymorphonuclear leukocytes was also inhibited by EGCG. Neither L-NAME, ebeselen nor NAC inhibited ROS generation. From the Western blot analysis of the subunits components of NADPH oxidase, we detected cytosolic subunits; p47(phox), p67(phox), p40(phox), rac2 and membrane subunits; gp91(phox), p22(phox) in CM-MC. Cytosolic subunits were translocated from cytosol to membrane time dependently after stimulation with compound 48/80. EGCG and DPI inhibited cytosolic subunits from translocating into membrane. These data suggest that EGCG inhibits the activation of NADPH oxidase in CM-MC.     

Sphingosine 1-phosphate receptors modulate intracellular Ca2+ homeostasis

Ligation of sphingosine 1-phosphate (S1P) to a set of specific receptors named S1P receptors (S1PRs) regulates important biological processes. Although the ability of S1P to increase cytosolic Ca2+ in various cell types is well known, the role of the individual S1PRs has not been fully characterized. Here, we provide a complete analysis of S1P-dependent intracellular Ca2+ homeostasis in HeLa cells. Overexpression of S1P2, or S1P3, but not S1P1, leads to a significant increase in cytosolic and mitochondrial [Ca2+] in response to S1P challenge. Moreover, cells ectopically expressing S1P2, or S1P3 exhibited an appreciable decrease of the free Ca2+ concentration in the endoplasmic reticulum, dependent on stimulation of receptors by S1P endogenously present in the culture medium which was accompanied by a reduced susceptibility to C2-ceramide-induced cell death. These results demonstrate a differential contribution of individual S1PRs to Ca2+ homeostasis and its possible implication in the regulation of cell survival.     

模拟失重增强大鼠脑动脉血管平滑肌细胞大电导钙激活钾通道功能

本工作旨在探讨短期模拟失重大鼠脑动脉血管平滑肌细胞（vascular smooth muscle cells,VSMCs）大电导钙激活钾通道（large conductance calcium-activated potassium channels,BKCa channels）功能的改变。以尾部悬吊大鼠模型模拟失重对脑血管的影响。应用激光扫描共聚焦显微镜测定VSMCs胞内游离钙浓度（[Ca^2＋]i）;采用细胞贴附模式,记录BKCa通道的单通道活动。结果表明,模拟失重1周后,大鼠脑动脉VSMCs的[Ca^2＋]i比对照组显著升高（P〈0．05）：BKCa通道的开放概率（Po）与平均开放时间（To）显著增加（P〈0．05）,而单通道电导与平均关闭时间（Tc）则无显著变化。总之,1周模拟失重可引起脑动脉VSMCs的BKCa通道功能显著增强,且与细胞[Ca^2＋]i的升高同步出现。结果提示,脑动脉VSMCs的离子通道机制可能参与介导模拟失重引起的脑血管适应性变化。     

Immunoregulatory effect of mesenchymal stem cell via mitochondria signaling pathways in allergic asthma

Asthma is a complicated lung disease, which has increased morbidity and mortality rates in worldwide. There is an overlap between asthma pathophysiology and mitochondrial dysfunction and MSCs may have regulatory effect on mitochondrial dysfunction and treats asthma. Therefore, immune-modulatory effect of MSCs and mitochondrial signaling pathways in asthma was studied.After culturing of MSCs and producing asthma animal model, the mice were treated with MSCs via IV via IT. BALf's eosinophil Counting, The levels of IL-4, −5, −13, −25, –33, INF-γ, Cys-LT, LTB4, LTC4, mitochondria genes expression of COX-1, COX-2, ND1, Nrf2, Cytb were measured and lung histopathological study were done.BALf's eosinophils, the levels of IL-4, −5, −13, −25, –33, LTB4, LTC4, Cys-LT, the mitochondria genes expression (COX-1, COX-2, Cytb and ND-1), perivascular and peribronchial inflammation, mucus hyper-production and hyperplasia of the goblet cell in pathological study were significantly decreased in MSCs-treated asthma mice and reverse trend was found about Nrf-2 gene expression, IFN-γ level and ratio of the INF-γ/IL-4.MSC therapy can control inflammation, immune-inflammatory factors in asthma and mitochondrial related genes, and prevent asthma immune-pathology.     

MPK9 and MPK12 function in SA-induced stomatal closure in Arabidopsis thaliana

Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.     

细胞浆中PrP蛋白的表达及其细胞毒性作用的研究

为了探讨在胞浆中表达的PrP的理化特征以及对细胞活性的影响,我们构建了胞浆型PrP(CytoPrP)真核表达质粒,瞬时转染人神经母细胞瘤SH-SY5Y细胞,通过蛋白酶敏感性实验检测CytoPrP及其蛋白酶抗性,利用MTT和Trypan Blue细胞计数检测CytoPrP的细胞毒性作用.结果显示,CytoPrP在胞浆内的存在受蛋白酶抑制剂的控制;与野生型PrP相比,CytoPrP具有相对较强的蛋白酶K(PK)抗性.MTT和细胞计数实验均显示,Cyto-PrP的存在可诱导明显的细胞毒性效应,而CytoPrP的细胞毒性作用受蛋白酶抑制剂含量的影响,呈剂量依赖关系.上述结果为研究CytoPrP在朊病毒病的发病机制中的意义提供了一定的科学数据.     

Ca2+参与水杨酸诱导蚕豆气孔运动时的信号转导

在一定条件下,外源水杨酸(SA)可以诱导蚕豆(Vicia faba L.)气孔关闭,阻止气孔张开。以Fluo-3—AM作为Ca^2 的荧光探针,利用激光共聚焦扫描显微技术,对水杨酸调控气孔运动中保卫细胞胞质Ca^2 的变化趋势及Ca^2 的来源进行了研究。结果表明,水杨酸可引起胞质Ca^2 增加,这种变化发生在气孔开度改变之前。Ca^2 螯合剂BAPTA（1,2-bis(2-amino phenox-y)ethane-N,N,N′,N′-tetraacetic acid,1mmol/L)几乎可以完全抑制水杨酸诱导气孔开度减小的作用;胞外Ca^2 螯合剂EGTA(2mmol/L)、质膜Ca^2 通道抑制剂尼群地平(nifedipine,NIF,1μmol/L)和LaCl3(1mmol／L)可不同程度地减弱水杨酸诱导气孔关闭的效应。BAPTA(1mmol／L)预处理后,水杨酸不再引起胞质Ca^2 含量改变;尼群地平能够降低水杨酸引起的胞质Ca^2 增加的幅度。说明Ca^2 可能参与水杨酸诱导气孔运动的信号转导。水杨酸引起胞内升高的Ca^2 可能既来自胞外又来自胞内,胞内Ca^2 库可能是其主要来源。     

Lipid bodies in oxidized LDL-induced foam cells are leukotriene-synthesizing organelles: a MCP-1/CCL2 regulated phenomenon

Lipid-laden foam macrophages are emerging as key players in early atherogenesis. Even though cytoplasmic lipid bodies (lipid droplets) are now recognized as organelles with cell functions beyond lipid storage, the mechanisms controlling lipid body biogenesis within macrophages and their additional functions in atherosclerosis are not completely elucidated. Here we studied oxLDL-elicited macrophage machinery involved in lipid body biogenesis as well as lipid body roles in leukotriene (LT) synthesis. Both in vivo and in vitro, oxLDL (but not native LDL) induced rapid assembly of cytoplasmic lipid bodies-bearing ADRP within mice macrophages. Such oxLDL-elicited foamy-like phenotype was a pertussis toxin-sensitive process that depended on a paracrine activity of endogenous MCP-1/CCL2 and activation of ERK. Pretreatment with neutralizing anti-MCP-1/CCL2 inhibited macrophage ADRP protein expression induced by oxLDL. By directly immuno-localizing leukotrienes at their sites of synthesis, we showed that oxLDL-induced newly formed lipid bodies function as active sites of LTB₄ and LTC₄ synthesis, since oxLDL-induced lipid bodies within foam macrophages compartmentalized the enzyme 5-lipoxygenase and five lipoxygenase-activating protein (FLAP) as well as newly formed LTB₄ and LTC₄. Consistent with MCP-1/CCL-2 role in ox-LDL-induced lipid body biogenesis, in CCR2 deficient mice both ox-LDL-induced lipid body assembly and LT release were reduced as compared to wild type mice. In conclusion, oxLDL-driven foam cells are enriched with leukotriene-synthesizing lipid bodies – specialized organelles whose biogenic process is mediated by MCP-1/CCL2-triggered CCR2 activation and ERK-dependent downstream signaling – that may amplify inflammatory mediator production in atherosclerosis.     

Modulation of calcium signalling by mitochondria

In this review we will attempt to summarise the complex and sometimes contradictory effects that mitochondria have on different forms of calcium signalling. Mitochondria can influence Ca²⁺ signalling indirectly by changing the concentration of ATP, NAD(P)H, pyruvate and reactive oxygen species — which in turn modulate components of the Ca²⁺ signalling machinery i.e. buffering, release from internal stores, influx from the extracellular solution, uptake into cellular organelles and extrusion by plasma membrane Ca²⁺ pumps. Mitochondria can directly influence the calcium concentration in the cytosol of the cell by importing Ca²⁺ via the mitochondrial Ca²⁺ uniporter or transporting Ca²⁺ from the interior of the organelle into the cytosol by means of Na⁺/Ca²⁺ or H⁺/Ca²⁺ exchangers. Considerable progress in understanding the relationship between Ca²⁺ signalling cascades and mitochondrial physiology has been accumulated over the last few years due to the development of more advanced optical techniques and electrophysiological approaches.     

Comparative Detection of Calcium Fluctuations in Single Female Sex Cells of Tobacco to Distinguish Calcium Signals Triggered by in vitro Fertilization

Double fertilization is a key process of sexual reproduction in higher plants. The role of calcium in the activation of female sex cells through fertilization has recently received a great deal of attention. The establishment of a Ca²⁺-imaging technique for living, single, female sex cells is a difficult but necessary prerequisite for evaluating the role of Ca²⁺ in the transduction of external stimuli, including the fusion with the sperm cell, to internal cellular processes. The present study describes the use of Fluo-3 for reporting the Ca²⁺ signal in isolated, single, female sex cells, egg cells and central cells, of tobacco plants. A suitable loading protocol was optimized by loading the cells at pH 5.6 with 2 μM Fluo-3 for 30 min at 30  °C. Under these conditions, several key factors related to in vitro fertilization were also investigated in order to test their possible effects on the [Ca²⁺]_cyt of the female sex cells. The results indicated that the bovine serum albumin-fusion system was superior to the polyethlene glycol-fusion system for detecting calcium fluctuations in female sex cells during fertilization. The central cell was fertilized with the sperm cell in bovine serum albumin; however, no evident calcium dynamic was detected, implying that a transient calcium rise might be a specific signal for egg cell fertilization.