Histochemical Method for Detecting Nitric Oxide Synthase Activity in Cell Cultures

NADPH diaphorase histochemistry has been used extensively for detecting nitric oxide synthase (NOS) activity in various cell types including neuronal cell bodies, vascular endothelium, cells of the immune system and epithelial cells. The use of the diaphorase technique in cell cultures to study the induction of NOS has not been investigated. In this paper we report the use of diaphorase histochemistry as a good marker for the detection of NOS activity in cultured cells. This technique can be used in conjunction with other established techniques to determine the presence and activity of NOS in cultured cells.     

Nitric Oxide Is Involved in Cadmium-Induced Programmed Cell Death in Arabidopsis Suspension Cultures

Exposure to cadmium (Cd²⁺) can result in cell death, but the molecular mechanisms of Cd²⁺ cytotoxicity in plants are not fully understood. Here, we show that Arabidopsis (Arabidopsis thaliana) cell suspension cultures underwent a process of programmed cell death when exposed to 100 and 150 μm CdCl₂ and that this process resembled an accelerated senescence, as suggested by the expression of the marker senescence-associated gene12 (SAG12). CdCl₂ treatment was accompanied by a rapid increase in nitric oxide (NO) and phytochelatin synthesis, which continued to be high as long as cells remained viable. Hydrogen peroxide production was a later event and preceded the rise of cell death by about 24 h. Inhibition of NO synthesis by N^G-monomethyl-arginine monoacetate resulted in partial prevention of hydrogen peroxide increase, SAG12 expression, and mortality, indicating that NO is actually required for Cd²⁺-induced cell death. NO also modulated the extent of phytochelatin content, and possibly their function, by S-nitrosylation. These results shed light on the signaling events controlling Cd²⁺ cytotoxicity in plants.Cadmium (Cd²⁺) is a heavy metal with a long biological half-life, and its presence as a pollutant in agricultural soil is due mainly to anthropogenic activities. It is rapidly taken up by roots and enters the food chain, resulting in toxicity for both plants and animals (for review, see Sanità di Toppi and Gabbrielli, 1999). Cd²⁺ inhibits seed germination, decreases plant growth and photosynthesis, and impairs the distribution of nutrients. Overall, the symptoms of chronic exposure to sublethal amounts of Cd²⁺ mimic premature senescence (Rascio et al., 1993; McCarthy et al., 2001; Sandalio et al., 2001; Rodriguez-Serrano et al., 2006). Depending on the concentration, Cd²⁺ treatment of tobacco (Nicotiana tabacum) cell cultures and onion (Allium cepa) roots eventually triggers either necrosis or programmed cell death (PCD; Fojtovà and Kovařik, 2000; Behboodi and Samadi, 2004).Although Cd²⁺ is an environmental threat, the mechanisms by which it exerts its toxic effects in plants are not fully understood. In plant cells, Cd²⁺ is believed to enter through Fe²⁺, Ca²⁺, and Zn²⁺ transporters/channels (Clemens, 2006). Once in the cytosol, Cd²⁺ stimulates the production of phytochelatins (PCs), a glutathione-derived class of peptides containing repeated units of Glu and Cys, which bind the metal ions and transport them into the vacuole (Sanità di Toppi and Gabbrielli, 1999). Strong evidence exists that high (millimolar) concentrations of Cd²⁺ induce reactive oxygen species (ROS) bursts in plants, which might have a role in signaling and/or degenerative steps leading to cell death (Piqueras et al., 1999; Olmos et al., 2003; Cho and Seo, 2005; Garnier et al., 2006). Treatment with a lower, nontoxic Cd²⁺ concentration also caused increase in ROS production in pea (Pisum sativum) leaves and roots (Sandalio et al., 2001; Romero-Puertas et al., 2004; Rodriguez-Serrano et al., 2006) and Arabidopsis (Arabidopsis thaliana) cell cultures (Horemans et al., 2007).Nitric oxide (NO) is a gaseous reactive molecule with a pivotal signaling role in many developmental and response processes (for review, see Neill et al., 2003; Besson-Bard et al., 2008). In plants, it can be synthesized via several routes, either enzymatically or by chemical reduction of nitrite. Nitrate reductase and a root-specific plasma membrane nitrite-NO reductase also utilize nitrite as substrate. In animals, nitric oxide synthase (NOS) converts l-Arg into NO and l-citrulline. Although no plant NOS has been unambiguously identified yet, activity assays and pharmacological evidence suggests the existence of a NOS-like counterpart in plants. Depending on its concentration and possibly on the timing and localization of its production, NO can either act as an antioxidant or promote PCD, often in concert with ROS (Delledonne et al., 2001; Beligni et al., 2002; de Pinto et al., 2006). Extensive research has shown that NO plays a fundamental role in the hypersensitive response, but its involvement in other types of PCD, such as that resulting from mechanical stress and natural and cytokinin-induced senescence of cell cultures, has also been demonstrated (Garcês et al., 2001; Carimi et al., 2005). Because of its participation in numerous biotic and abiotic responses, NO has been proposed as a general stress molecule (Gould et al., 2003). However, the mechanisms by which NO determines its effects are far from being completely elucidated, and a number of downstream signaling pathways, involving Ca²⁺, cyclic GMP, and cyclic ADP-Rib, are involved (Neill et al., 2003; Besson-Bard et al., 2008). NO can also modulate biological responses by direct modification of proteins, reacting with Cys residues (S-nitrosylation), Tyr residues (nitration), or iron and zinc in metalloproteins (metal nitrosylation; Besson-Bard et al., 2008).The aim of this work is to study the plant responses to various concentrations of Cd²⁺ and, in particular, the role of ROS and NO in the signaling events leading to cell death. Cell cultures of the model plant Arabidopsis were chosen as an experimental system because the homogeneity and undifferentiated state of the cells, combined with the uniform delivery of the treatments, allow a clear and reproducible response. The results point to NO as a master regulator of Cd²⁺-induced cell death. Possible mechanisms that explain this evidence will be discussed.     

一氧化氮合酶（NOS）在膀胱移行细胞癌中的表达及其意义

目的检测膀胱移行细胞癌中一氧化氮合酶（nitric oxide synthase,NOS）的表达,并分析其表达与肿瘤病理特性的关系.方法采用免疫组织化学技术检测35例膀胱移行细胞癌标本、12例癌旁粘膜标本及8例正常膀胱粘膜标本中一氧化氮合酶三种亚型的表达情况.结果 35例肿瘤标本中nNOS、iNOS、eNOS阳性表达率分别为74.3%、85.7%、42.9%,膀胱移行细胞癌中iNOS表达较正常膀胱粘膜增高.但移行细胞癌、癌旁粘膜、正常粘膜三组间nNOS及eNOS表达无差别.nNOS、iNOS、eNOS表达与膀胱移行细胞癌分期分级可能无相关性.结论 iNOS在膀胱移行细胞癌中表达增高,可能参与膀胱移行细胞癌的发生发展.     

白桦BpPIN5基因启动子组织定位及外源激素应答分析

PIN家族蛋白作为IAA的极性输出载体,在植物胚胎发育、器官发育和向性生长,尤其是植物叶序、叶脉的形成及维管组织分化过程中起关键作用。为了明确白桦（Betula platyphylla）BpPIN5基因对外源激素的应答特性,实验以白桦全基因组DNA为参考,克隆获得BpPIN5基因的上游1 447 bp启动子序列,采用PLACE在线软件对该序列含有的顺式作用元件进行预测,结果表明,BpPIN5启动子序列含有生长素（IAA）、赤霉素（GA）、水杨酸（SA）、脱落酸（ABA）、茉莉酸甲酯（MeJA）、乙烯（ET）等不同类型的生长素响应元件。实验构建了pro-BpPIN5：：GUS载体进行白桦转基因,GUS组化染色分析显示,BpPIN5启动子在白桦叶裂顶端、细叶脉及根中有转录活性。分别用IAA、GA、MeJA、SA及ABA激素处理转基因白桦,结果显示,BpPIN5启动子对上述5种激素在白桦第1叶片的裂叶边缘、第2叶的叶柄及根组织部位均有应答,且响应变化基本一致。研究结果为揭示白桦BpPIN5基因功能提供参考。     

白桦HD-Zip基因家族生物信息学及应答盐胁迫分析

HD-Zip转录因子蛋白家族是植物特有的一类转录因子蛋白,在植物生长发育和抵抗逆境胁迫等过程中发挥着重要作用。利用白桦全基因组数据库,获得白桦35条HD-Zip蛋白序列,参考拟南芥中该家族的分类方法,将这些成员分成HD-ZipⅠ-Ⅳ四个亚家族,并对这些成员的蛋白保守结构域、氨基酸组成、染色体分布、和理化性质等进行了预测和分析。从高盐处理的白桦幼苗根组织的转录组数据,鉴定了7个差异表达的基因。本研究为进一步研究白桦HD-Zip家族基因调控白桦耐盐性的功能提供了理论支持。     

逆转录病毒载体介导诱导型NO合酶在神经细胞中表达

为了深入研究诱导型一氧化氮合酶基因表达产物在阿片耐受和依赖中作用,采用脂质体介导基因转染技术,将ｉＮＯＳ ｃＤＮＡ重组逆转录病毒载体导入ＮＧ１０８－１５神经细胞,获得Ｇ４１８抗性克隆,命名为ＮＧ－ＬＮＣＸｉＮＯＳ细胞。ＤＮＡ印迹杂交,ＰＣＲ扩增及ＲＴ－ＰＣＲ和蛋白质免疫印迹杂交分析,证实ＮＧ－ＬＮＣＸｉＮＯＳ细胞有外源ｉＮＯＳ基因整合,转录和表达;ＮＡＤＰＨ黄递酶（ＮＡＤＰＨ ｄｉａｐｈｏｒａｓｅ     

白桦BpJMJ18基因启动子克隆及表达分析

为探究BpJMJ18基因在植物生长发育过程中的功能，本研究利用PCR技术克隆白桦（Betula platyphylla）BpJMJ18基因的启动子，通过生物信息学分析发现，该启动子序列中除了包含TATA-box和CAAT-box等基本顺式作用元件外，还具有光响应元件和多种激素应答相关的元件；进而构建植物表达载体pBI101-BpJMJ18pro：：GUS，并用农杆菌介导的瞬时转化法侵染白桦，对转基因株系进行GUS染色分析，结果发现BpJMJ18基因启动子能够驱动GUS基因在白桦的主根、侧根、根尖、叶片的维管束和嫩茎中均检测到表达。上述结果说明白桦BpJMJ18启动子具有启动活性，可能影响植物的生长发育。     

TNF-α、IL-1β及LPS对血管平滑肌细胞一氧化氮合酶基因表达的影响及作用机制研究

通过ＲＮＡ印迹分析和亚硝酸盐含量测定检查ＴＮＦ－α、ＩＬ－１β和ＬＰＳ对大鼠血管平滑肌细胞（ＶＳＭＣ）诱导型一氧化氮合酶（ｉＮＯＳ）基因表达及ＮＯ生成的影响．结果表明,ＴＮＦ－α、ＩＬ－１β和ＬＰＳ均能显著诱导ＶＳＭＣｉＮＯＳ基因表达和促进ＮＯ生成,其作用强度与浓度和作用时间有关;双因素（ＴＮＦ－α＋ＬＰＳ,ＬＰＳ＋ＩＬ－１β）对诱导ｉＮＯＳ基因表达及ＮＯ生成产生协同作用．ＰｏｌｙｍｙｘｉｎＢ和地塞米松可部分抑制ＴＮＦ－α对ｉＮＯＳ基因表达的诱导作用及ＮＯ生成     

Hypo- and Hyperglycemia Impair Endothelial Cell Actin Alignment and Nitric Oxide Synthase Activation in Response to Shear Stress

Uncontrolled blood glucose in people with diabetes correlates with endothelial cell dysfunction, which contributes to accelerated atherosclerosis and subsequent myocardial infarction, stroke, and peripheral vascular disease. In vitro, both low and high glucose induce endothelial cell dysfunction; however the effect of altered glucose on endothelial cell fluid flow response has not been studied. This is critical to understanding diabetic cardiovascular disease, since endothelial cell cytoskeletal alignment and nitric oxide release in response to shear stress from flowing blood are atheroprotective. In this study, porcine aortic endothelial cells were cultured in 1, 5.55, and 33 mM D-glucose medium (low, normal, and high glucose) and exposed to 20 dynes/cm² shear stress for up to 24 hours in a parallel plate flow chamber. Actin alignment and endothelial nitric oxide synthase phosphorylation increased with shear stress for cells in normal glucose, but not cells in low and high glucose. Both low and high glucose elevated protein kinase C (PKC) levels; however PKC blockade only restored actin alignment in high glucose cells. Cells in low glucose instead released vascular endothelial growth factor (VEGF), which translocated β-catenin away from the cell membrane and disabled the mechanosensory complex. Blocking VEGF in low glucose restored cell actin alignment in response to shear stress. These data suggest that low and high glucose alter endothelial cell alignment and nitric oxide production in response to shear stress through different mechanisms.     

白桦APETALA2基因启动子的克隆及其表达分析

APETALA2（AP2）转录因子亚家族普遍存在于植物中,参与植株的生长发育、胁迫应答和多种生理生化反应的信号传导。本研究从白桦（Betula platyphylla Suk.）基因组中克隆了AP2基因2 308 bp的启动子序列,生物信息学分析发现,该序列除具有TATA-box和CAAT box等高等植物普遍具有的保守元件外,还具有大量光响应元件和激素响应元件,如响应赤霉素、脱落酸、茉莉酸甲酯等的元件。将白桦AP2基因启动子克隆至pBI121-35S：：GUS植物表达载体中,命名为pBI121-proAP2：：GUS,用农杆菌介导法侵染白桦和拟南芥,并进行GUS染色分析,结果表明AP2基因启动子驱动下的GUS报告基因在整个拟南芥中都表达,在白桦的营养器官和雌花种翅及花柄中也有表达,说明其具有启动活性,可能参与该器官的发育。     

白桦CESA7基因启动子的克隆与表达分析

克隆得到了一个白桦纤维素合成酶基因（CESA7）GenBanK登录号（EU591531）启动子序列,通过序列分析发现该启动子含有多个不同功能的顺式作用元件,包括光响应元件、激素响应元件、叶片形态发育元件等,推测该启动子在白桦生长发育过程中具有关键作用。将BpCESA7启动子克隆至带有GUS报告基因的植物表达载体,命名为proBpCESA7-121-GUS,并利用农杆菌介导方法侵染白桦和拟南芥,然后通过GUS组织化学染色观察BpCESA7基因启动子的组织表达特性。结果在白桦的根、茎、叶和拟南芥的根,叶,萼片、雌蕊中检测到了GUS活性,说明BpCESA7基因启动子具有启动子活性,并且在白桦的根和叶中染色最深,表明BpCESA7基因在白桦根和叶中表达量较高,并且其存在组织表达特异性。     

悬浮培养南方红豆杉细胞的紫杉醇生物合成路径中诱导子的作用位点

作者研究开发出一种基于分析中间响应模式确定悬浮培养诱导子作用位点的新方法。研究结果表明,一个诱导子的作用位点存在于浓度变化方向相反的相邻两个中间代谢物之间;该方法的有效性在悬浮培养南方红豆杉(Taxus chinensis (Pilg.) Rehd. var. mairei (Lemee et Levl.) Cheng et L. K. Fu)生物合成紫杉醇过程中得以证实;经确定,甲基茉莉酮酸、硝酸根和柠檬酸铵的作用位点在baccatin Ⅲ至10-去乙酰基紫杉醇之间;水杉酸、花生四烯酸的作用位点存在3种可能性,即增强10-去乙酰基紫杉醇的合成、防止紫杉醇和cephalomannine的降解。该方法对指导诱导子配伍优化紫杉醇生产具有指导意义。     

脂多糖对血管平滑肌细胞一氧化氮合酶基因表达及细胞增殖的影响

应用ＲＮＡ印迹分析和亚硝酸盐含量测定检查脂多糖（ＬＰＳ）对大鼠血管平滑肌细胞（ＶＳＭＣ）一氧化氮合酶（ＮＯＳ）基因表达及ＮＯ合成的影响,用３Ｈ－ＴｄＲ参入实验观察ＬＰＳ对细胞ＤＮＡ合成的影响．结果表明,ＬＰＳ在诱导ＶＳＭＣｉＮＯＳｍＲＮＡ表达和促进ＮＯ合成的同时,抑制ＶＳＭＣＤＮＡ合成．证明ＬＰＳ的作用与其浓度和作用时间有关     

悬浮培养南方红豆杉细胞的紫杉醇生物合成路径中诱导子的作用位点

作者研究开发出一种基于分析中间响应模式确定悬浮培养诱导子作用位点的新方法.研究结果表明,一个诱导子的作用位点存在于浓度变化方向相反的相邻两个中间代谢物之间;该方法的有效性在悬浮培养南方红豆杉(Taxus chinensis(Pilg.)Rehd.var.mairei(Lemee et Levl.)Cheng et L. K.Fu)生物合成紫杉醇过程中得以证实;经确定,甲基茉莉酮酸、硝酸根和柠檬酸铵的作用位点在baccatinⅢ至10-去乙酰基紫杉醇之间;水杉酸、花生四烯酸的作用位点存在3种可能性,即增强10-去乙酰基紫杉醇的合成、防止紫杉醇和cephalomannine的降解.该方法对指导诱导子配伍优化紫杉醇生产具有指导意义.     

白桦开花位点Flowering Locus T（FT）基因的分离及其表达

FT及其同源基因在促进植物成花和发育阶段转变过程中起重要作用。应用RT-PCR和RACE技术分离了白桦FT基因的cDNA,全长为928 bp,其开放阅读框为525 bp,编码174个氨基酸。预测的蛋白质分子量为19.6 kDa,理论等电点为7.73。该预测蛋白序列含有保守的PEBP蛋白结构域,命名为BplFT,并在GenBank注册,登录号为JQ409561。该基因序列同其它16种植物的相似性为74%～93%,其中与无花果（Ficus carica）的相似度最高为93%,与拟南芥（Arabidopsis thaliana）的相似度最低为74%,并构建了该基因序列的进化树。通过qRT-PCR的方法检测BplFT基因在白桦不同时期不同组织中的转录表达,在营养器官的表达高于花器官,成熟组织要高于幼嫩的组织,在成熟茎中的表达量最高,推测BplFT基因在成熟的营养器官发育中起重要作用,并可能参与调控次生细胞壁的形成。另外,选择了白桦雄花序突变体进行该基因的转录表达分析,该基因在突变体雌花序、雄花序、幼叶及幼茎中均为上调表达,预示着BplFT基因不仅仅参与营养组织发育,在花器官发育中也具有一定的作用。     

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。     

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10 μmol.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。     

In Vivo Expression of Inducible Nitric Oxide Synthase in Cerebellar Neurons

Abstract: In the CNS, nitric oxide (NO) functions as both neuromodulator and neurotoxic agent. In vivo neuronal expression of NO synthase (NOS) has been attributed to constitutive NOS—both the neuronal and the endothelial types. The other class of NOS—the inducible NOS (iNOS)—is known to mediate toxic effects of NO in various tissues. In this study, we show for the first time that direct intracerebellar injection of endotoxin and cytokine (lipopolysaccharide and interferon-γ) induced in vivo neuronal expression of the iNOS gene, as demonstrated by fluorescent in situ hybridization and immunohistochemical staining analyzed by confocal laser-scanning microscopy. This raises the possibility that neuronal iNOS might contribute significantly to the vulnerability of the brain to various insults.