Apoptosis In Animal Cells Research Articles

Fluorescent macrocycle-dye-anchor conjugates for sensing phospholipids in biomembranes

Negatively charged phospholipids can serve as biomarkers for a number of biological processes allowing detection of apoptosis in animal cells, drug-induced phospholipidosis as well as detection of bacteria. Selective detection of phospholipids thus represents an important but challenging goal. The development of fluorescent probes capable of selective recognition of specific phospholipids can be a promising solution to the problem. Along this line, we have developed the macrocycle-dye-anchor conjugates, in which the macrocycle recognizes the phosphate residue, hydrocarbon anchors ensure hydrophobic interaction with the membrane, and environment-sensitive fluorescent dye functions as a reporter unit. To the best of our knowledge, this is the first report of synthetic fluorescent probes for specific phospholipids using the macrocycle-dye-anchor concept. We have studied the effect of the structure of the anchor and the macrocycle on the resulting fluorescent response in the presence of two types of lipid vesicles: phosphatidylcholine as a model of healthy cells and phosphatidylcholine and phosphatidylserine (1:1), mimicking apoptotic cells. The properties of the obtained compounds were analyzed in various model media and in HeLa cells. The response of fluorescent probes has been found to be strongly dependent on the macrocycle structure, length of the anchor, nature of the buffer, and pH. The probes showed significant fluorescence enhancement in apoptotic cells, thus providing the proof of concept for the macrocycle-dye-anchor design.

Selenium Alleviates Chromium(VI)-Induced Ileum Damage and Cecal Microbial Disturbances in Mice.

Hexavalent chromium [Cr(VI)] is one of the most common environmental contaminants caused by its broad industrial applications. Importantly, exposure to Cr(VI) induces oxidative damage and apoptosis in animal cells. Studies have shown that selenium (Se) can alleviate the toxic effects of Cr(VI) by functioning as an antioxidant and/or by chelating Cr(VI) into biologically inert complexes, but the underlying mechanism remains unknown. Here, we evaluated whether Se can ameliorate ileum damage and cecal microbial disturbances induced by Cr(VI) in vivo. Mice administered Cr(VI) for 30days presented histopathological damage, reduced responses to oxidative stress, and increased expression of apoptosis-related genes in the ileum compared with those in the control (non-exposed) group. Se alleviated the histopathological damage and decreased the oxidative stress and apoptosis induced by Cr(VI) in the ileum. In addition, Cr(VI) disturbed cecal microflora, and it was partially reversed by Se treatment. These findings demonstrate that the damaging and potentially pathological effects of Cr(VI) on the ileum and cecal microflora can be effectively alleviated with Se treatment.

ProxyPhos sensors for the detection of negatively charged membranes.

Membrane-embedded negatively charged phospholipids (MENCP) can be used as biomarkers for a range of biological processes, including early detection of apoptosis in animal cells, drug-induced phospholipidosis, and selective detection of bacterial over animal cells. Currently, several technologies for the detection of apoptosis and bacterial cells are based on the recognition of MENCPs, including the AnnexinV stain and PSVue™ probes. As probes, these technologies have limitations, the most significant of which is the need for washing the unbound probe away to achieve optimal signal. In contrast, a turn-on chemosensor selective for MENCP would address this shortcoming, and allow for a more rapid protocol for the detection of apoptosis, bacteria and for other relevant applications. In this work, the aim was to explore whether ProxyPhos chemosensors, previously reported by our group for the detection of proximally phosphorylated peptides and proteins, could be re-purposed for the detection of MENCPs. Six lead ProxyPhos sensors were screened against synthetic vesicles containing biologically relevant negatively charged phospholipids including phosphatidic acid (PA), phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS). Through these screens, ProxyPhos sensors exhibiting high selectivity for the detection of MENCPs over zwitterionic lipids were identified. Particular selectivity was observed for PA and CL. Sensitivity of the lead sensors for MENCPs was suitable for the detection of apoptosis: ProxyPhos detected vesicles containing as little as 2.5% PS and detected camptothecin-induced apoptosis in mammalian cells in flow cytometry experiments. The results suggest that ProxyPhos sensors can be used for the detection of MENCPs in synthetic vesicles and live mammalian cells.

Dark-induced senescence of barley leaves involves activation of plastid transglutaminases.

Transglutaminases (E.C. 2.3.2.13) catalyze the post-translational modification of proteins by establishing ε-(γ-glutamyl) lysine isopeptide bonds and by the covalent conjugation of polyamines to endo-glutamyl residues of proteins. In light of the confirmed role of transglutaminases in animal cell apoptosis and only limited information on the role of these enzymes in plant senescence, we decided to investigate the activity of chloroplast transglutaminases (ChlTGases) and the fate of chloroplast-associated polyamines in Hordeum vulgare L. ‘Nagrad’ leaves, where the senescence process was induced by darkness (day 0) and continued until chloroplast degradation (day 12). Using an anti-TGase antibody, we detected on a subcellular level, the ChlTGases that were associated with destacked/degraded thylakoid membranes, and beginning on day 5, were also found in the stroma. Colorimetric and radiometric assays revealed during senescence an increase in ChlTGases enzymatic activity. The MS/MS identification of plastid proteins conjugated with exogenous polyamines had shown that the ChlTGases are engaged in the post-translational modification of proteins involved in photosystem organization, stress response, and oxidation processes. We also computationally identified the cDNA of Hv-Png1-like, a barley homologue of the Arabidopsis AtPng1 gene. Its mRNA level was raised from days 3 to 10, indicating that transcriptional regulation controls the activity of barley ChlTGases. Together, the presented results deepen our knowledge of the mechanisms of the events happened in dark-induced senescence of barley leaves that might be activation of plastid transglutaminases.Electronic supplementary materialThe online version of this article (doi:10.1007/s00726-014-1912-y) contains supplementary material, which is available to authorized users.

The expression of p53 and hsp70 proteins after treatment with Annona muricata Linn leaf for activating apoptotic and lead to homeostasis program of Raji cells

Purpose: Organic extracts of plant Annonaceae enhances apoptosis in animal cells and get the drives to reach a new homeostasis. The incidence rate of nasopharyngeal cancer in Indonesia is quite high. Protein 53Kd (p53) play a role in apoptosis process, being heat shock protein 70 (hsp70) play a role in homeostasis. The aim of this research is to identify the apoptotic effects of Annona muricata Linn leaf toward Raji cells by observing the p53 and hsp70 expression. Methods : Apoptotic assay was performed in 24 wells micro-culture plate. Raji cells were prepared as 2 × 10 4 cells in 100 ml RPMI media per well. Roswell Park Memorial Institute (RPMI) medium was created and solvent was controlled with Dimethyl Sulfoxide (DMSO) solvent 0.25. Apoptotic test was performed by calculating trypan-blue-dye exclution. The cells were then grown in micro-culture plate with media plus extract non-lethal concentration of partition and fractionation of Annona muricata Linn leaf. The sampling was performed for 24 hours. The number of living cells was calculated in each of these well and incubation time were determined. Immunohistochemical staining was done to identify the expression of p53 and hsp70. Results: The results showed that Raji cells treated with partition of Annona muricata Linn leaf in ethyl acetate solvent 133.00 % resulted in higher apoptosis. Another results showed that Raji cells treated with fractionation Annona muricata Linn leaf in ethyl acetate solvent 103.20 % resulted in higher apoptosis. The expression of p53 after treatment with fractionation Annona muricata Linn leaf was higher than before while hsp70 expression after treatment with fractionation Annona muricata Linn leaf was lower. Conclusion : The conclusion is the higher the dose of Annona muricata Linn the higher the p53 expression thereby activates apoptosis process The higher dose of Anonna muricata Linn also leads to lower hsp70 expression indicating stable homeostasis of Raji cells. ---------------------------------------------- Cite this article as: Astirin OP, Prayitno A, Artanti AN, Fitria MS, Witianingsih DA, Pranatami DA, Putra ST. The expression of p53 and hsp70 proteins after treatment with Annona muricata Linn leaf for activating apoptotic and lead to homeostasis program of Raji cells. Int J Cancer Ther Oncol 2014; 2(2):02028. DOI: 10.14319/ijcto.0202.8

Senescence and programmed cell death in plants: polyamine action mediated by transglutaminase.

Research on polyamines (PAs) in plants laps a long way of about 50 years and many roles have been discovered for these aliphatic cations. PAs regulate cell division, differentiation, organogenesis, reproduction, dormancy-break and senescence, homeostatic adjustments in response to external stimuli and stresses. Nevertheless, the molecular mechanisms of their multiple activities are still matter of research. PAs are present in free and bound forms and interact with several important cell molecules; some of these interactions may occur by covalent linkages catalyzed by transglutaminase (TGase), giving rise to “cationization” or cross-links among specific proteins. Senescence and programmed cell death (PCD) can be delayed by PAs; in order to re-interpret some of these effects and to obtain new insights into their molecular mechanisms, their conjugation has been revised here. The TGase-mediated interactions between proteins and PAs are the main target of this review. After an introduction on the characteristics of this enzyme, on its catalysis and role in PCD in animals, the plant senescence and PCD models in which TGase has been studied, are presented: the corolla of naturally senescing or excised flowers, the leaves senescing, either excised or not, the pollen during self-incompatible pollination, the hypersensitive response and the tuber storage parenchyma during dormancy release. In all the models examined, TGase appears to be involved by a similar molecular mechanism as described during apoptosis in animal cells, even though several substrates are different. Its effect is probably related to the type of PCD, but mostly to the substrate to be modified in order to achieve the specific PCD program. As a cross-linker of PAs and proteins, TGase is an important factor involved in multiple, sometimes controversial, roles of PAs during senescence and PCD.

The Fusarium Mycotoxin Deoxynivalenol Can Inhibit Plant Apoptosis-Like Programmed Cell Death

The Fusarium genus of fungi is responsible for commercially devastating crop diseases and the contamination of cereals with harmful mycotoxins. Fusarium mycotoxins aid infection, establishment, and spread of the fungus within the host plant. We investigated the effects of the Fusarium mycotoxin deoxynivalenol (DON) on the viability of Arabidopsis cells. Although it is known to trigger apoptosis in animal cells, DON treatment at low concentrations surprisingly did not kill these cells. On the contrary, we found that DON inhibited apoptosis-like programmed cell death (PCD) in Arabidopsis cells subjected to abiotic stress treatment in a manner independent of mitochondrial cytochrome c release. This suggested that Fusarium may utilise mycotoxins to suppress plant apoptosis-like PCD. To test this, we infected Arabidopsis cells with a wild type and a DON-minus mutant strain of F. graminearum and found that only the DON producing strain could inhibit death induced by heat treatment. These results indicate that mycotoxins may be capable of disarming plant apoptosis-like PCD and thereby suggest a novel way that some fungi can influence plant cell fate.

BAX inhibitor-1 silencing suppresses white spot syndrome virus replication in red swamp crayfish, Procambarus clarkii

BAX inhibitor-1 (BI-1) was originally described as an anti-apoptotic protein in both animal and plant cells. BI-1 overexpression suppresses ER stress–induced apoptosis in animal cells. Inhibition of BI-1 activity could induce the cell death in mammals and plants. However, the function of BI-1 in crustacean immunity was unclear. In this paper, the full-length cDNA of a BI-1 protein in red swamp crayfish, Procambarus clarkii (PcBI-1) was cloned and its expression profiles in normal and infected crayfish were analyzed. The results showed that PcBI-1 was expressed in hemocytes, heart, hepatopancreas, gills, stomach, and intestines of the crayfish and was upregulated after challenged with Vibrio anguillarum and with white spot syndrome virus (WSSV). To determine the function of PcBI-1 in the innate immunity of the crayfish, the RNA interference against PcBI-1 was performed and the results indicated the hemocyte programmed cell death rate was increased significantly and WSSV replication was declined after PcBI-1 knocked down. Altogether, PcBI-1 plays an anti-apoptotic role, wherein high PcBI-1 expression suppresses programmed cell death, which is beneficial for WSSW replication in crayfish.

Antioxidant effect of protein-free silkworm hemolymph extract in mitochondrial membrane potential

Silkworm was used in the sericulture industry and also processed for food sources in some of Asian countries. In previous studies, it was reported that the 30K protein in silkworm, Bombyx mori hemolymph inhibited animal cell apoptosis. In this work, the antioxidative function of silkworm hemolymph was investigated using its protein-free extract. H2O2-induced ROS and O2·− were efficiently scavenged in Chinese hamster ovary cells when the culture medium was supplemented with silkworm hemolymph. The hemolymph extract prepared by deproteinization through ethanol precipitation completely scavenged DPPH radicals (IC50=0.025 mg/mL). When the extract was supplemented with protein-free medium, oxidative damage to mitochondria was efficiently diminished with the maintenance of the mitochondrial membrane potential. Lutein was identified as the major antioxidative constituents in the extract (52.06 μg/mg).

Sorting out the role of nitric oxide in cadmium-induced Arabidopsis thaliana programmed cell death

As a vital cell-signaling molecule, nitric oxide (NO) has been reported to regulate toxic metal responses in plants. Our recent report has suggested that caspase-3-like protease activation was detected in Arabidopsis (Arabidopsis thaliana) after Cd2+ treatment. NO contributed caspase-3-like protease activation in Cd2+ induced Arabidopsis thaliana programmed cell death (PCD), which was mediated by MPK6. It was first shown that NO promotes Cd2+-induced Arabidopsis PCD by promoting MPK6-mediated caspase-3-like activation. Our study contributed to the understanding of NO signaling pathway in Cd2+-induced Arabidopsis thaliana PCD. Although several studies have revealed that NO regulates plant PCD, compared with the study of signaling pathways involved in animal cell apoptosis, the mechanism of NO function still remains elusive and the molecular mechanisms of MAPK are far from clear in Cd2+-induced PCD. By using the fluorescence techniques and the Arabidopsis seedlings as the reference model, the subsequent researches have been performed to obtain comprehensive understanding of Cd2+-induced plant PCD.

Sphingolipid long chain base phosphates can regulate apoptotic-like programmed cell death in plants

Sphingolipids are ubiquitous components of eukaryotic cells and sphingolipid metabolites, such as the long chain base phosphate (LCB-P), sphingosine 1 phosphate (S1P) and ceramide (Cer) are important regulators of apoptosis in animal cells. This study evaluated the role of LCB-Ps in regulating apoptotic-like programmed cell death (AL-PCD) in plant cells using commercially available S1P as a tool. Arabidopsis cell cultures were exposed to a diverse array of cell death-inducing treatments (including Cer) in the presence of S1P. Rates of AL-PCD and cell survival were recorded using vital stains and morphological markers of AL-PCD. Internal LCB-P levels were altered in suspension cultured cells using inhibitors of sphingosine kinase and changes in rates of death in response to heat stress were evaluated. S1P reduced AL-PCD and promoted cell survival in cells subjected to a range of stresses. Treatments with inhibitors of sphingosine kinase lowered the temperature which induced maximal AL-PCD in cell cultures. The data supports the existence of a sphingolipid rheostat involved in controlling cell fate in Arabidopsis cells and that sphingolipid regulation of cell death may be a shared feature of both animal apoptosis and plant AL-PCD.

Attenuation of host NO production by MAMPs potentiates development of the host in the squid-vibrio symbiosis

Bacterial pathogens typically upregulate the host's production of nitric oxide synthase (NOS) and nitric oxide (NO) as antimicrobial agents, a response that is often mediated by microbe-associated molecular patterns (MAMPs) of the pathogen. In contrast, previous studies of the beneficial Euprymna scolopes/Vibrio fischeri symbiosis demonstrated that symbiont colonization results in attenuation of host NOS/NO, which occurs in high levels in hatchling light organs. Here, we sought to determine whether V. fischeri MAMPs, specifically lipopolysaccharide (LPS) and the peptidoglycan derivative tracheal cytotoxin (TCT), attenuate NOS/NO, and whether this activity mediates the MAMPs-induced light organ morphogenesis. Using confocal microscopy, we characterized levels of NOS with immunocytochemistry and NO with a NO-specific fluorochrome. When added exogenously to seawater containing hatchling animals, V. fischeri LPS and TCT together, but not individually, induced normal NOS/NO attenuation. Further, V. fischeri mutants defective in TCT release did not. Experiments with NOS inhibitors and NO donors provided evidence that NO mediates apoptosis and morphogenesis associated with symbiont colonization. Attenuation of NOS/NO by LPS and TCT in the squid-vibrio symbiosis provides another example of how the host's response to MAMPs depends on the context. These data also provide a mechanism by which symbiont MAMPs regulate host development.

Mitochondrial pathway leading to programmed cell death induced by aluminum phytotoxicity in Arabidopsis

Recent studies have reported some apoptosis-like characters under Al treatment including nucleus morphology changes and appearance of nucleus fragmentation in plant cells. Our recent report has suggested that mitochondrial oxidative burst, mitochondrial swelling and mitochondrial transmembrane potential (MTP) disrupt play crucial roles in Al-induced caspase-3-like activation and programmed cell death (PCD). And Complex I and III might be the sources of Al-induced mitochondrial reactive oxygen species (ROS) through interaction between Al and iron-sulfur (Fe-S) protein. Our study contributed to the understanding of mitochondria-dependent cellular signaling cascade of plant biological responses in Al-induced PCD. However, the mitochondria-dependent mechanism in Al-induced PCD needs further improvement, and the roles of mitochondria functional proteins are still poorly understood compared with the study of signaling pathways involved in animal cell apoptosis. By using the fluorescence techniques and the Arabidopsis mesophyll protoplasts as a reference model, the subsequent researches have been carried out to obtain comprehensive understanding of Al-induced plant PCD.

Sorting out the role of reactive oxygen species during plant programmed cell death induced by ultraviolet-C overexposure

Previous studies have reported that light is required for activating Arabidopsis programmed cell death (PCD) induced by ultraviolet-C (UV-C) overexposure, and a caspase-like protease cleaving the caspase-3 substrate Asp-Glu-Val-Asp (DEVDase activity) is induced during this process. Our recent report has suggested that a quick burst of reactive oxygen species (ROS), which is mainly derived from mitochondria and chloroplasts, is induced in a light dependent manner during the early stages of UV-induced plant PCD. Concomitantly, the mitochondria undergo serious dysfunction including the MTP loss and the changes in distribution and mobility, which ultimately lead to apoptotic-cell death. Though some of signaling molecules have been elucidated in this type of plant cell death, the molecular mechanism about UV-induce Arabidopsis PCD is still poorly understood when comparing with the study of signaling pathways involved in animal cell apoptosis induced by UV. By using the Arabidopsis mesophyll protoplasts as a reference model, we have begun to shed light on the complexity of signaling pathway in UV-induced plant PCD. Recently we have tried to real-time detect the presence of caspase-like proteolytic activation, and to sort out the key role of ROS as well as to further assess the relationship between the ROS production and caspase-like activation in this type of plant apoptotic cell death.

Oligochitosan induces cell death and hydrogen peroxide accumulation in tobacco suspension cells

Oligochitosan has been shown to induce several plant defense responses. In the present work, the effect of oligochitosan on tobacco cell survival was investigated. The results showed that oligochitosan caused tobacco cell death in a dose-dependent manner. About 40.6 % tobacco cells died when cultured for 72 h after 500 μg ml −1 oligochitosan treatment. Certain aspects of this cell death process appeared to be similar to apoptosis in animal cells. These included shrinkage of cytoplasm and condensation of chromatin. Oligochitosan also induced H 2O 2 accumulation in tobacco cell suspension culture. The role of H 2O 2 in the signal transduction that leads to cell death was investigated. Co-treatment of tobacco cells with oligochitosan and catalase inhibited H 2O 2 accumulation but did not inhibit the induction of cell death. The results suggested that apoptosis-like cell death of tobacco cells induced by oligochitosan is independent of H 2O 2 signal pathway.

Temporal and spatial activation of caspase-like enzymes induced by self-incompatibility in Papaver pollen

Caspase-like proteases are universal mediators of programmed cell death (PCD). Because plants have no caspase homologs, establishing the nature of their caspase-like activities is of considerable importance to our understanding of PCD in plants. Caspase-3, displaying DEVD specificity, is a key executioner caspase in animal cells. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants by inhibiting incompatible pollen. In Papaver rhoeas, SI activates a caspase-3-like/DEVDase activity in incompatible pollen that plays a pivotal role in regulating PCD. Here we characterize the SI-induced caspase-like activities in detail; our work provides insights into the temporal and spatial activation of plant caspase-like enzymes. We show that SI also activates a VEIDase and a LEVDase and that the VEIDase plays a role in SI-induced PCD. The DEVDase and VEIDase are activated remarkably rapidly: detectable within 1-2 h after SI induction; the LEVDase activity peaks later. Importantly, we show live-cell imaging of a DEVDase activity in a higher plant cell; the SI-activated DEVDase has a cytosolic and nuclear localization. We also demonstrate that SI induces a rapid and substantial cytosolic acidification that matches the in vitro pH optima for the SI-induced caspase activities. Because both cytosolic acidification and nuclear caspase localization are observed during apoptosis in animal cells, our data provide striking parallels between SI-induced PCD and apoptosis in animal cells.

MicroRNAs: recently discovered key regulators of proliferation and apoptosis in animal cells : Identification of miRNAs regulating growth and survival.

The relatively recent discovery of miRNAs has added a completely new dimension to the study of the regulation of gene expression. The mechanism of action of miRNAs, the conservation between diverse species and the fact that each miRNA can regulate a number of targets and phenotypes clearly indicates the importance of these molecules. In this review the current state of knowledge relating to miRNA expression and gene regulation is presented, outlining the key morphological and biochemical features controlled by miRNAs with particular emphasis on the key phenotypes that impact on cell growth in bioreactors, namely proliferation and apoptosis.

Mitochondria-Associated Hexokinases Play a Role in the Control of Programmed Cell Death inNicotiana benthamiana

Recent findings suggest a pivotal role for mitochondria-associated hexokinase in the regulation of apoptosis in animal cells. In this study, virus-induced gene silencing (VIGS) of a hexokinase-encoding Hxk1 caused necrotic lesions on leaves, abnormal leaf morphology, and retarded plant growth in Nicotiana benthamiana. Hxk1 was associated with the mitochondria, and this association required the N-terminal membrane anchor. VIGS of Hxk1 reduced the cellular glucose-phosphorylating activity to approximately 31% of control levels without changing the fructose-phosphorylating activity and did not alter hexose phosphate content severely. The affected cells showed programmed cell death (PCD) morphological markers, including nuclear condensation and DNA fragmentation. Similar to animal cell apoptosis, cytochrome c was released into the cytosol and caspase-9- and caspase-3-like proteolytic activities were strongly induced. Furthermore, based on flow cytometry, Arabidopsis thaliana plants overexpressing Arabidopsis HXK1 and HXK2, both of which are predominantly associated with mitochondria, exhibited enhanced resistance to H(2)O(2)- and alpha-picolinic acid-induced PCD. Finally, the addition of recombinant Hxk1 to mitochondria-enriched fractions prevented H(2)O(2)/clotrimazole-induced cytochrome c release and loss of mitochondrial membrane potential. Together, these results show that hexokinase critically regulates the execution of PCD in plant cells, suggesting a link between glucose metabolism and apoptosis.

Identification of a nuclear-localized nuclease from wheat cells undergoing programmed cell death that is able to trigger DNA fragmentation and apoptotic morphology on nuclei from human cells

PCD (programmed cell death) in plants presents important morphological and biochemical differences compared with apoptosis in animal cells. This raises the question of whether PCD arose independently or from a common ancestor in plants and animals. In the present study we describe a cell-free system, using wheat grain nucellar cells undergoing PCD, to analyse nucleus dismantling, the final stage of PCD. We have identified a Ca2+/Mg2+ nuclease and a serine protease localized to the nucleus of dying nucellar cells. Nuclear extracts from nucellar cells undergoing PCD triggered DNA fragmentation and other apoptotic morphology in nuclei from different plant tissues. Inhibition of the serine protease did not affect DNA laddering. Furthermore, we show that the nuclear extracts from plant cells triggered DNA fragmentation and apoptotic morphology in nuclei from human cells. The inhibition of the nucleolytic activity with Zn2+ or EDTA blocked the morphological changes of the nucleus. Moreover, nuclear extracts from apoptotic human cells triggered DNA fragmentation and apoptotic morphology in nuclei from plant cells. These results show that degradation of the nucleus is morphologically and biochemically similar in plant and animal cells. The implication of this finding on the origin of PCD in plants and animals is discussed.

Salt stress-induced programmed cell death in tobacco protoplasts is mediated by reactive oxygen species and mitochondrial permeability transition pore status

The status of mitochondrial permeability transition pore (PTP) and levels of reactive oxygen species (ROS) play key roles in regulating apoptosis in animal cells. To investigate if the PTP and cellular oxidation-reduction state are also involved in salt stress-induced programmed cell death (PCD) in tobacco (Nicotiana tabacum, cultivar BY-2) protoplasts, flow cytometry was used to simultaneously monitor ROS levels, PTP status and PCD. Increased ROS and decreased mitochondrial membrane potential (delta psi(m)) were observed before the appearance of PCD. Pre-treatment with an inhibitor of the PTP opening, cyclosporin A (CsA), effectively retarded the onset of PCD, the delta psi(m) decrease and the ROS content increase. Addition of ascorbic acid (AsA) during the salt stress significantly decreased the percentage of protoplasts undergoing PCD and ROS levels but increased delta psi(m). Hydrogen peroxide effectively induced the appearance of PCD and caused an increase in ROS and a decrease in delta psi(m). Pre-treatment of protoplasts with CsA weakened the effects of H2O2. All these results suggest that the open state of PTP and ROS are necessary elements for salt stress-induced PCD in tobacco protoplasts. The open states of PTP and ROS could promote each other suggesting that ROS could lead to a self-amplifying process. This positive feedback loop may act as an all-or-nothing switch, which is in good accordance with the hypothesis that PTP is an important coordinator and executioner of PCD in both animals and plants.