The soluble proteome of tobacco Bright Yellow-2 cells undergoing H₂O₂-induced programmed cell death

Plant programmed cell death (PCD) is a genetically controlled process that plays an important role in development and stress responses. Reactive oxygen species (ROS) are key inducers of PCD. The addition of 50 mM H?O? to tobacco Bright Yellow-2 (TBY-2) cell cultures induces PCD. A comparative proteomic analysis of TBY-2 cells treated with 50 mM H?O? for 30 min and 3 h was performed. The results showed early down-regulation of several elements in the cellular redox hub and inhibition of the protein repair-degradation system. The expression patterns of proteins involved in the homeostatic response, in particular those associated with metabolism, were consistently altered. The changes in abundance of several cytoskeleton proteins confirmed the active role of the cytoskeleton in PCD signalling. Cells undergoing H?O?-induced PCD fail to cope with oxidative stress. The antioxidant defence system and the anti-PCD signalling cascades are inhibited. This promotes a genetically programmed cell suicide pathway. Fifteen differentially expressed proteins showed an expression pattern similar to that previously observed in TBY-2 cells undergoing heat shock-induced PCD. The possibility that these proteins are part of a core complex required for PCD induction is discussed.     

Hydrogen peroxide, nitric oxide and cytosolic ascorbate peroxidase at the crossroad between defence and cell death

An increase in the production of reactive oxygen species (ROS) is a typical event occurring during different stress conditions and activating conflicting responses in plants. In order to investigate the relevance of different timing and amounts of ROS production, tobacco (Nicotiana tabacum) Bright Yellow-2 (TBY-2) cells were incubated with different amounts of glucose plus glucose oxidase, for generating H(2)O(2) during time, or directly with known amounts of H(2)O(2). Data presented here indicate that, in TBY-2 cells, a difference in H(2)O(2) level is a critical point for shifting metabolic responses towards strengthening of antioxidant defences, or their depletion with consequent cell death. Timing of ROS production is also critical because it can determine programmed cell death (PCD) or necrosis. Depending on the different kinds of activated cell death, ascorbate (ASC) and glutathione (GSH) pools are altered differently. Moreover, an H(2)O(2)-dependent activation of nitric oxide synthesis is triggered only in the conditions inducing PCD. Ascorbate peroxidase (APX) has been analysed under different conditions of H(2)O(2) generation. Under a threshold value of H(2)O(2) overproduction, a transient increase in APX occurs, whereas under conditions inducing cell necrosis, the activity of APX decreases in proportion to cell death without any evident alteration in APX gene expression. Under conditions triggering PCD, the suppression of APX involves both gene expression and alteration of the kinetic characteristics of the enzyme. The changes in ASC, GSH and APX are involved in the signalling pathway leading to PCD, probably contributing to guaranteeing the cellular redox conditions required for successful PCD.     

Oxidative stress and mitochondrial dysfunctions are early events in narciclasine-induced programmed cell death in tobacco Bright Yellow-2 cells

Narciclasine (NCS) is a plant growth inhibitor isolated from the secreted mucilage of Narcissus tazetta bulbs. It is a commonly used anticancer agent in animal systems. In this study, we provide evidence to show that NCS also acts as an agent in inducing programmed cell death (PCD) in tobacco Bright Yellow-2 (TBY-2) cell cultures. NCS treatment induces typical PCD-associated morphological and biochemical changes, namely cell shrinkage, chromatin condensation and nuclear DNA degradation. To investigate possible signaling events, we analyzed the production of reactive oxygen species (ROS) and the function of mitochondria during PCD induced by NCS. A biphasic behavior burst of hydrogen peroxide (H(2)O(2)) was detected in TBY-2 cells treated with NCS, and mitochondrial transmembrane potential (MTP) loss occurred after a slight increase. Pre-incubation with antioxidant catalase (CAT) and N-acetyl-L-cysteine (NAC) not only significantly decreased the H(2)O(2) production but also effectively retarded the decrease of MTP and reduced the percentage of cells undergoing PCD after NCS treatment. In conclusion, our results suggest that NCS induces PCD in plant cells; the oxidative stress (accumulation of H(2)O(2)) and the MTP loss play important roles during NCS-induced PCD.     

Cadmium exposure modifies lactotrophs activity associated to genomic and morphological changes in rat pituitary anterior lobe

Cadmium (Cd) is widely used in industrial applications and is an important contaminant of agricultural products. As an endocrine disruptor, Cd modifies the hormone release of pituitary anterior lobe (PAL). This work was undertaken to evaluate a possible association between phospholipase D (PLD) and prolactin mRNA expressions and the activity of lactotrophs and folliculostellate cells (FSC) in PAL of Cd exposed adult male Wistar rats (Cd, 0.133 mM per liter for 2 months). The PALs were submitted to immunohistochemical and morphometric analysis to determine the percentage of lactotrophs (PRL-ir) and FSC (S-100-ir). Cultured PAL cells were stained with Hoechst 33258 to determine the presence of alterations in nuclear morphology consistent with apoptosis. The expressions of PLD and prolactin mRNA were assessed by RT-PCR. Cd treated rats showed a decrease of PLD mRNA levels that can be associated to both high number of apoptotic cells and increase of S-100 protein expression in FSC. Cd decreased prolactin mRNA expression, number of lactotrophs and percentage of PRL-ir suggesting a low availability of prolactin to be secreted from PAL. Cd modifies the lactotrophs activity of pituitary gland through biochemical, genomic and morphological changes and contributes directly or indirectly to the levels of serum prolactin.     

镉通过引发氧化应激和线粒体损伤诱导PK-15细胞凋亡

镉（cadmium,Cd）是一种生物累积性的有毒重金属元素,能够在肾组织大量蓄积并引起肾发生病变和功能损伤。前期研究证实,Cd处理能够引起猪肾PK-15细胞的活性氧（reactive oxygen species,ROS）水平升高和细胞死亡,但详细机制有待进一步研究。本研究以PK-15细胞为研究对象,通过CCK-8检测、透射电镜观察、DCFH-DA标记、JC-1染色、彗星实验和流式细胞术等研究手段,分别检测Cd处理后的细胞活性、形态变化、ROS生成、线粒体膜电位Δψm、DNA损伤及细胞凋亡情况。CCK-8实验结果显示,CdCl2处理后PK-15细胞活性下降,且呈时间和剂量依赖性;形态学观察发现,CdCl2处理引起PK-15细胞皱缩、变圆,细胞核固缩、染色质凝聚,线粒体肿胀、线粒体嵴减少或消失;荧光染色和流式细胞术检测结果显示,CdCl2处理引起PK-15细胞内ROS水平升高、线粒体膜电位Δψm下降和DNA损伤,最终导致细胞凋亡。Western印迹结果显示,CdCl2处理组中促凋亡蛋白质Bax表达量上调,抑凋亡蛋白质Bcl-2表达量下调,并且CdCl2处理组检测到了活化状态的裂解胱天蛋白酶3(cleaved caspase 3)。此外,ROS清除剂N-乙酰基-L-半胱氨酸（N-acetyl-L-cysteine,NAC）缓解了CdCl2引起的线粒体损伤、DNA损伤和细胞凋亡。综上所述,Cd通过引发氧化应激和线粒体损伤诱导PK-15细胞凋亡。     

Cadmium Induces Apoptosis in Pancreatic β-Cells through a Mitochondria-Dependent Pathway: The Role of Oxidative Stress-Mediated c-Jun N-Terminal Kinase Activation

Cadmium (Cd), one of well-known highly toxic environmental and industrial pollutants, causes a number of adverse health effects and diseases in humans. The growing epidemiological studies have suggested a possible link between Cd exposure and diabetes mellitus (DM). However, the toxicological effects and underlying mechanisms of Cd-induced pancreatic β-cell injury are still unknown. In this study, we found that Cd significantly decreased cell viability, and increased sub-G1 hypodiploid cells and annexin V-Cy3 binding in pancreatic β-cell-derived RIN-m5F cells. Cd also increased intracellular reactive oxygen species (ROS) generation and malondialdehyde (MDA) production and induced mitochondrial dysfunction (the loss of mitochondrial membrane potential (MMP) and the increase of cytosolic cytochrome c release), the decreased Bcl-2 expression, increased p53 expression, poly (ADP-ribose) polymerase (PARP) cleavage, and caspase cascades, which accompanied with intracellular Cd accumulation. Pretreatment with the antioxidant N-acetylcysteine (NAC) effectively reversed these Cd-induced events. Furthermore, exposure to Cd induced the phosphorylations of c-jun N-terminal kinases (JNK), extracellular signal-regulated kinases (ERK)1/2, and p38-mitogen-activated protein kinase (MAPK), which was prevented by NAC. Additionally, the specific JNK inhibitor SP600125 or JNK-specific small interference RNA (si-RNA) transfection suppressed Cd-induced β-cell apoptosis and related signals, but not ERK1/2 and p38-MAPK inhibitors (PD98059 and SB203580) did not. However, the JNK inhibitor or JNK-specific si-RNA did not suppress ROS generation in Cd-treated cells. These results indicate that Cd induces pancreatic β-cell death via an oxidative stress downstream-mediated JNK activation-triggered mitochondria-regulated apoptotic pathway.     

A new rare distamycin a-inducible fragile site,fra(11)(p15.1), found in two acute nonlymphocytic leukemia (ANLL) patients with t(7;11)(p15-p13;p15)

Summary Fragile sites were analyzed in normal peripheral lymphocytes from two acute nonlymphocytic leukemia patients with t(7;11)(p15-p13;p15) leukemic cells. To induce expression of fragile sites, cultures were exposed to folate deprivation (M-F10), BrdU, distamycin A, or Hoechst 33258. Fragility at 11p15.1 was induced by distamycin A and Hoechst 33258 but was not seen in M-F10, BrdU, and control cultures. Fra(11)(p15.1) was found neither in healthy Japanese subjects (0 in 845) nor in patients with leukemia or other hematologic disorders without the t(7;11) (0 in 126). From these results, fra(11)(p15.1) can now be calssified as a rare distamycin A-inducible fragile site. Furthermore, this fra(11)(p15.1) coincided with one of the breakpoints of the t(7;11)(p15-p13;p15).     

Comparative Genomics of Phylogenetically Diverse Unicellular Eukaryotes Provide New Insights into the Genetic Basis for the Evolution of the Programmed Cell Death Machinery

Programmed cell death (PCD) represents a significant component of normal growth and development in multicellular organisms. Recently, PCD-like processes have been reported in single-celled eukaryotes, implying that some components of the PCD machinery existed early in eukaryotic evolution. This study provides a comparative analysis of PCD-related sequences across more than 50 unicellular genera from four eukaryotic supergroups: Unikonts, Excavata, Chromalveolata, and Plantae. A complex set of PCD-related sequences that correspond to domains or proteins associated with all main functional classes—from ligands and receptors to executors of PCD—was found in many unicellular lineages. Several PCD domains and proteins previously thought to be restricted to animals or land plants are also present in unicellular species. Noteworthy, the yeast, Saccharomyces cerevisiae—used as an experimental model system for PCD research, has a rather reduced set of PCD-related sequences relative to other unicellular species. The phylogenetic distribution of the PCD-related sequences identified in unicellular lineages suggests that the genetic basis for the evolution of the complex PCD machinery present in extant multicellular lineages has been established early in the evolution of eukaryotes. The shaping of the PCD machinery in multicellular lineages involved the duplication, co-option, recruitment, and shuffling of domains already present in their unicellular ancestors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fasudil Mesylate Protects PC12 Cells from Oxidative Stress Injury via the Bax-Mediated Pathway

We previously reported that fasudil mesylate (FM) improves neurological deficit and neuronal damage in rats with ischemia following middle cerebral artery occlusion and reperfusion in vivo. In this study, the properties of FM on hydrogen peroxide (H₂O₂)-induced oxidative stress insult in cultured PC12 cells as well as the underlying mechanisms were investigated in vitro. Pretreatment with FM (5, 10 μM) prior to H₂O₂ exposure significantly elevated cell viability, reduced cell apoptosis by MTT assay, LDH assay, Hoechst 33258 dye staining, and FM also decreased the accumulation of reactive oxygen species (ROS) by DCFH-DA staining and NBT test. Furthermore, FM also reversed the upregulation of Bax/Bcl-2 ratio, the downstream cascade following ROS. FM protected PC12 cells from oxidative stress insult via down-regulating the Bax/Bcl-2 ratio. These findings indicate that a direct effect of fasudil mesylate on PC12 cells may be partly responsible for its protective effect against oxidative stress injury.     

Binding of Hoechst 33258 to chromatin in situ

The binding of Hoechst 33258 to rat thymocytes, human lymphocytes, and NHIK 3025 tissue culture cells was studied by measuring the fluorescence and light scattering of the cells as functions of dye concentration using flow cytometry. The results indicated that there were two different modes of binding of Hoechst 33258 to chromatin in situ at physiological pH. Type 1 binding, which dominated at total dye/phosphate ratios below 0.1 (0.15, M), was characterized by a binding constant of the order 10(7) M-1 and fluorescence with high quantum yield. Further binding of the dye resulted in a reduced blue/green fluorescence ratio, indicating that secondary sites were occupied. Binding at secondary sites above a certain density (0.1 less than or equal to bound dye/phosphate less than or equal to 0.2) induced strong quenching of fluorescence and precipitation of chromatin. Precipitation was quantitated by measuring the large-angle (greater than or equal to 15 degrees) light scattering of the cells above 400 nm, i.e., outside the Hoechst 33258/DNA absorption spectrum, as a function of dye concentration. In contrast, the light scattering at 365 nm, i.e., within the absorption spectrum of Hoechst 33258/DNA, was independent of the total dye/phosphate ratio. The coefficient of variation of the light-scattering (greater than or equal to 400 nm) histograms decreased with Hoechst 33258 concentration. Type 2 binding to histone-depleted chromatin was cooperative (Hill-coefficient approximately 2) and the apparent binding constant was 2-3 X 10(5) M-1 as determined from quenching and precipitation data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative spatiotemporal analysis of root aerenchyma formation processes in maize due to sulphate, nitrate or phosphate deprivation

Nitrate (N), phosphate (P) or sulphate (S) deprivation causes aerenchyma formation in maize (Zea mays L.) nodal roots. The exact mechanisms that trigger the formation of aerenchyma under these circumstances are unclear. We have compared aerenchyma distribution across the nodal roots of first whorl (just emerging in 10-day-old seedlings), which were subject to S, N or P deprivation over a period of 10?days in connection with oxygen consumption, ATP concentration, cellulase and polygalacturonase activity in the whole root. The effect of deprivation on aerenchyma formation was examined using light and electron microscopy, along with in situ detection of calcium and of reactive oxygen species (ROS) by fluorescence microscopy. Aerenchyma was not found in the root base regardless of the deprivation. Programmed cell death (PCD) was observed near the root tip, either within the first two days (-N) or a few days later (-S, -P) of the treatment. Roots at day?6 under all three nutrient-deprived conditions showed signs of PCD 1?cm behind the cap, whereas only N-deprived root cells 0.5?cm behind the cap showed severe ultrastructural alterations, due to advanced PCD. The lower ATP concentration and the higher oxygen consumptions observed at day?2 in N-, P- and S-deprived roots compared to the control indicated that PCD may be triggered by perturbations in energy status of the root. The peaks of cellulase activity located between days?3 (-N) and 6 (-P), along with the respective alterations in polygalacturonase activity, indicated a coordination which preceded aerenchyma formation. ROS and calcium seemed to contribute to PCD initiation, with ROS possessing dual roles as signals and eliminators. All the examined parameters presented both common features and characteristic variations among the deprivations.     

The endoplasmic reticulum stress induced by highly expressed OsrAAT reduces seed size via pre-mature programmed cell death

The high accumulation of a recombinant protein in rice endosperm causes endoplasmic reticulum (ER) stress and in turn dramatically affects endogenous storage protein expression, protein body morphology and seed phenotype. To elucidate the molecular mechanisms underlying these changes in transgenic rice seeds, we analyzed the expression profiles of endogenous storage proteins, ER stress-related and programmed cell death (PCD)-related genes in transgenic lines with different levels of Oryza sativa recombinant alpha antitrypsin (OsrAAT) expression. The results indicated that OsrAAT expression induced the ER stress and that the strength of the ER stress was dependent on OsrAAT expression levels. It in turn induced upregulation of the expression of the ER stress response genes and downregulation of the expression of the endogenous storage protein genes in rice endosperm. Further experiments showed that the ER stress response upregulated the expression of PCD-related genes to disturb the rice endosperm development and induced pre-mature PCD. As consequence, it resulted in decrease of grain weight and size. The mechanisms for the detriment seed phenotype in transgenic lines with high accumulation of the recombinant protein were elucidated.     

信号分子介导藻类细胞程序性死亡的研究进展

藻类是水生态系统中的重要初级生产者, 在物质转换和能量迁移过程中发挥重要作用。细胞程序性死亡(PCD)作为一种细胞自我调控的死亡模式, 受到多种信号分子的控制。研究发现藻类细胞在遭受环境胁迫的情况下, 在形态和生理上均表现出类PCD的特征, 同时伴随着活性氧/一氧化氮/钙离子(ROS/NO/Ca2+)水平的变化。研究认为, ROS/NO/Ca2+作为信号分子介导藻细胞内的caspase-like酶活性变化, 从而触发藻细胞的类程序性死亡。然而, 对信号分子是如何在环境胁迫下的藻类细胞中引发类PCD仍知之甚少。文章综述了信号分子ROS/NO/Ca2+介导藻类类PCD的研究进展以及信号分子间的级联关系, 并对今后类PCD在该领域待开展的研究进行了展望。     

镉胁迫对拟南芥的毒害作用及自噬现象的观测

镉离子(Cd2+)具有强植物毒性,可抑制植物生长,甚至导致植物死亡。为了研究重金属镉对拟南芥的毒害作用,采用叶绿素荧光技术、流式细胞技术、激光共聚焦技术及半定量RT-PCR技术,检测光合参数的变化、活性氧(reactive oxygen species,ROS)的累积、自噬的发生,以及病原相关蛋白(pathogenesis-related protein,PR)基因表达的变化。实验结果显示,随着50μmol/L CdCl2处理时间的延长,ROS和Cd2+在细胞中大量积累。而在镉胁迫的初期,会观察到自噬的发生及PR基因表达的变化。说明植物受到外界Cd2+作用的初期,会通过自噬及增强PR基因表达来抵抗外界胁迫。但随着处理时间的延长,植物细胞内累积了大量的ROS和Cd2+,当植物不足以通过自噬途径抵抗胁迫时,就会导致生长受阻,最终对光合系统造成损伤。     

Nitric oxide protects the mitochondria of anterior pituitary cells and prevents cadmium-induced cell death by reducing oxidative stress

Cadmium (Cd2+) is a highly toxic metal that affects the endocrine system. We have previously shown that Cd2+ induces caspase-3 activation and apoptosis of anterior pituitary cells and that endogenous nitric oxide (NO) protects these cells from Cd2+. Here we investigate the mechanisms by which NO exerts this protective role. Cd2+ (25 microM) reduced the mitochondrial membrane potential (MMP) as measured by flow cytometry. Cd2+-induced apoptosis was mitochondrial dependent since cyclosporin A protected the cells from this metal. Inhibition of NO synthesis with 0.5 mM L-NAME increased the effect of Cd2+ on MMP, whereas the NO donor DETANONOate (0.1 mM) reduced it. Cd2+ increased the production of reactive oxygen species (ROS) as measured by flow cytometry. This effect was electron-transfer-chain-dependent since it was inhibited by rotenone. In fact, rotenone reduced the cytotoxic effect of the metal. The action of Cd2+ on mitochondrial integrity was ROS dependent. Trolox, an antioxidant, inhibited the effect of the metal on the MMP. Cd2+-induced increase in ROS generation was reduced by DETANONOate. There are discrepancies concerning the role of NO in Cd2+ toxicity. Here we show that NO reduces Cd2+ toxicity by protecting the mitochondria from oxidative stress in a system where NO plays a regulatory role.     

Cadmium activates the mitogen-activated protein kinase (MAPK) pathway via induction of reactive oxygen species and inhibition of protein phosphatases 2A and 5

Cadmium (Cd), a highly toxic environmental pollutant, induces neurodegenerative diseases. Recently we have demonstrated that Cd may induce neuronal apoptosis in part through activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (Erk1/2) pathways. However, the underlying mechanism remains enigmatic. Here we show that Cd induced generation of reactive oxygen species (ROS), leading to apoptosis of PC12 and SH-SY5Y cells. Pretreatment with N-acetyl-L-cysteine (NAC) scavenged Cd-induced ROS, and prevented cell death, suggesting that Cd-induced apoptosis is attributed to its induction of ROS. Furthermore, we found that Cd-induced ROS inhibited serine/threonine protein phosphatases 2A (PP2A) and 5 (PP5), leading to activation of Erk1/2 and JNK, which was abrogated by NAC. Overexpression of PP2A or PP5 partially prevented Cd-induced activation of Erk1/2 and JNK, as well as cell death. Cd-induced ROS was also linked to the activation of caspase-3. Pretreatment with inhibitors of JNK (SP600125) and Erk1/2 (U0126) partially blocked Cd-induced cleavage of caspase-3 and prevented cell death. However, zVAD-fmk, a pan caspase inhibitor, only partially prevented Cd-induced apoptosis. The results indicate that Cd induction of ROS inhibits PP2A and PP5, leading to activation of JNK and Erk1/2 pathways, and consequently resulting in caspase-dependent and -independent apoptosis of neuronal cells. The findings strongly suggest that the inhibitors of JNK, Erk1/2, or antioxidants may be exploited for prevention of Cd-induced neurodegenerative diseases.