Heat Shock Induces Programmed Cell Death in Wheat Leaves

Programmed cell death (PCD) was triggered in wheat leaves by a heat treatment (42 °C). This treatment caused DNA fragmentation as shown in DNA laddering analysis and in terminal deoxynucleotidyl transferase-mediated deoxyuridine-5-triphosphate (dUTP) nick end labeling (TUNEL) analysis. Methanol and acetone treatment of leaves significantly blocked PCD. Western analysis indicated that a 65 kDa poly(ADP-ribose) polymerase-like protein was degraded during the treatment. However, high temperature (80 °C) treatment caused necrosis but not PCD.     

渗透胁迫诱导的小麦叶片细胞程序性死亡

用20%PEG6000（-0.63MPa）溶液对小麦（Triticumaestivum）根系进行渗透胁迫,在DNA琼脂糖凝胶电泳图谱上观察到明显的梯状DNA条带,表明PEG处理诱发了DNA核小体间的断裂,从而表现出典型的细胞程序性死亡的生化特征;末端脱氧核糖核酸转移酶介导的3′-OH末端标记法（terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end     

Programmed cell death remodels lace plant leaf shape during development

Programmed cell death (PCD) functions in the developmental remodeling of leaf shape in higher plants, a process analogous to digit formation in the vertebrate limb. In this study, we provide a cytological characterization of the time course of events as PCD remodels young expanding leaves of the lace plant. Tonoplast rupture is the first PCD event in this system, indicated by alterations in cytoplasmic streaming, loss of anthocyanin color, and ultrastructural appearance. Nuclei become terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive soon afterward but do not become morphologically altered until late stages of PCD. Genomic DNA is fragmented, but not into internucleosomal units. Other cytoplasmic changes, such as shrinkage and degradation of organelles, occur later. This form of PCD resembles tracheary element differentiation in cytological execution but requires unique developmental regulation so that discrete panels of tissue located equidistantly between veins undergo PCD while surrounding cells do not.     

Evidence of ceased programmed cell death in metaphloem sieve elements in the developing caryopsis of Triticum aestivum L.

Transmission electron microscopy (TEM) and fluorescence microscopy studies revealed that the metaphloem sieve elements (MSEs) in the ventral vascular bundle of the caryopses of developing wheat (Triticum aestivum L.) undergo a unique type of programmed cell death (PCD). Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive nuclei were observed at 3 and 4 days after flowering (DAF). Transmission electron microscopy studies of differentiating MSEs revealed increased vacuolation, nuclear degeneration, chromatin condensation and localization to the periphery of the nucleus, and partly dilated perinuclear spaces, all typical characteristics of PCD in plant cells. In addition, vacuoles were disrupted at the last stages of differentiation. These results demonstrate that MSE differentiation is a unique type of PCD with highly selective autophagic processes, in which PCD ceases just prior to death. During this cessation of PCD, vacuoles and the endoplasmic reticulum appear to be associated with selective organelle digestion.     

Sophorolipid produced from the new yeast strain Wickerhamiella domercqiae induces apoptosis in H7402 human liver cancer cells

The effects of sophorolipid on the growth and apoptosis of H7402 human liver cancer cells were investigated. By treatment with sophorolipid, a dose- and time-dependent inhibition of cell proliferation was observed. The cells developed many features of apoptosis, including condensation of chromatin, nuclear fragmentation, and appearance of apoptotic bodies, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling positive cells were stained dark brown. Sophorolipid treatment induced apoptosis in H7402 cells by blocking cell cycle at G1 phase and partly at S phase, activating caspase-3, and increasing Ca2+ concentration in cytoplasm. These findings may suggest a potential use of sophorolipid for liver cancer treatment.     

Involvement of mitochondria in oxidative stress-induced cell death in mouse zygotes

Accumulation of reactive oxygen species during aging leads to programmed cell death (PCD) in many cell types but has not been explored in mammalian fertilized eggs, in which mitochondria are "immature," in contrast to "mature" mitochondria in somatic cells. We characterized PCD in mouse zygotes induced by either intensive (1 mM for 1.5 h) or mild (200 microM for 15 min) hydrogen peroxide (H(2)O(2)) treatment. Shortly after intensive treatment, zygotes displayed PCD, typified by cell shrinkage, cytochrome c release from mitochondria, and caspase activation, then terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in condensed pronuclei. On the other hand, after mild treatment, zygotes arrested developmentally and showed neither cytochrome c release nor caspase activation over 48 h; until 72 h, 46% zygotes exhibited TUNEL staining, and 88% of zygotes lost plasma membrane integrity. Interestingly, mild oxidative treatment induced a decline in mitochondrial membrane potential and disruption of the mitochondrial matrix. Taken together, these results suggest that oxidative stress caused by H(2)O(2) induces PCD in mouse zygotes and that mitochondria are involved in the early phase of oxidative stress-induced PCD. Furthermore, mitochondrial malfunction also may contribute to cell cycle arrest, followed by cell death, triggered by mild oxidative stress.     

Phenylephrine protects cardiomyocytes from starvation-induced apoptosis by increasing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is known to be a "housekeeping" protein; studies in non-cardiomyocytic cells have shown that GAPDH plays pro-apoptotic role by translocating from cytoplasm to the nucleus or to the mitochondria. However, the cardiovascular roles of GAPDH are unknown. We observed that phenylephrine (PE) (100 μM) protected against serum and glucose starvation -induced apoptosis in neonatal rat cardiac myocytes as assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and mitochondrial membrane potential depolarization. GAPDH glycolysis activity was positively correlated with the antiapoptotic action of PE. GAPDH activity inhibition blunted PE-induced protection of the mitochondrial membrane potential and cardiomyocytes. PE-induced Bcl-2 protein increase, Bax mitochondrial decrease and inhibition of cytochrome C release and Caspase 3 activation, as well as ROS production were blunted by GAPDH activity inhibition. Moreover, GAPDH overexpression provided protection against starvation-induced cardiomyocyte apoptosis in vitro and ischemia-induced cardiac infarction in vivo. Inhibition of Akt prevented PE-induced GAPDH activity increase and cardiomyocytes protection. In conclusion, the present study provides the first direct evidence of an antiapoptotic role of GAPDH in PE-induced cardiomyocytes protection; GAPDH activity elevation mainly affects the mitochondria-induced apoptosis.     

The scutellum of germinated wheat grains undergoes programmed cell death: identification of an acidic nuclease involved in nucleus dismantling

Programmed cell death (PCD) is a crucial phenomenon in the life cycle of cereal grains. In germinating grains, the scutellum allows the transport of nutrients from the starchy endosperm to the growing embryo, and therefore it may be the last grain tissue to undergo PCD. Thus, the aim of this work was to analyse whether the scutellum of wheat grains undergoes PCD and to perform a morphological and biochemical analysis of this process. Scutellum cells of grains following germination showed a progressive increase of DNA fragmentation, and the TUNEL assay showed that PCD extended in an apical-to-basal gradient along the scutellum affecting epidermal and parenchymal cells. Electron-transmission microscopy revealed high cytoplasm vacuolation, altered mitochondria, and the presence of double-membrane structures, which might constitute symptoms of vacuolar cell death, whereas the nucleus appeared lobed and had an increased heterochromatin content as the most distinctive features. An acid- and Zn(2+)-dependent nucleolytic activity was identified in nuclear extracts of scutellum cells undergoing PCD. This nuclease was not detected in grains imbibed in the presence of abscisic acid, which inhibited germination. This nucleolytic activity promoted DNA fragmentation in vitro on nuclei isolated from healthy cells, thus suggesting a main role in nucleus dismantling during PCD.     

烟草花粉发育过程中的细胞凋亡及其与钙/钙调素依赖性蛋白激酶T1表达的关系(英文)

用原位末端标记及免疫组化显色方法,对烟草花药发育过程中的细胞凋亡进行了检测。结果表明绒毡层、环形细胞、药隔组织细胞在减数分裂后四分体时期开始凋亡(Plate I, C &D),而花药维管束细胞从小孢子母细胞时期就开始凋亡(Plate I, E)。这些特定的细胞在花药发育特定时期的凋亡担负着特定的作用。钙/钙调素依赖性蛋白激酶T1在这些凋亡细胞中大量特异表达(Plate II),表明钙信号途径参与了烟草花粉发育的细胞凋亡调控。     

Treadmill exercise suppresses ischemia-induced increment in apoptosis and cell proliferation in hippocampal dentate gyrus of gerbils

Cerebral ischemia resulting from transient or permanent occlusion of cerebral arteries leads to neuronal cell death and eventually causes neurological impairments. In the present study, the effects of treadmill exercise on apoptosis and cell proliferation in the hippocampal dentate gyrus following transient global ischemia in gerbils were investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunohistochemistry for caspase-3 and 5-bromo-2'-deoxyuridine (BrdU). It was shown that apoptotic cell death and cell proliferation in the hippocampal dentate gyrus were significantly increased following transient global ischemia in gerbils and that treadmill exercise suppressed the ischemia-induced increase in apoptosis and cell proliferation in the dentate gyrus. The present results suggest that treadmill exercise may protect cells from apoptotic death and aid in recovery from the central nervous system sequelae following stroke.     

Induction of metamorphosis in the marine gastropod Ilyanassa obsoleta: 5HT, NO and programmed cell death

The central nervous system (CNS) of a metamorphically competent larva of the caenogastropod Ilyanassa obsoleta contains a medial, unpaired apical ganglion (AG) of approximately 25 neurons that lies above the commissure connecting the paired cerebral ganglia. The AG, also known as the cephalic or apical sensory organ (ASO), contains numerous sensory neurons and innervates the ciliated velar lobes, the larval swimming and feeding structures. Before metamorphosis, the AG contains 5 serotonergic neurons and exogenous serotonin can induce metamorphosis in competent larvae. The AG appears to be a purely larval structure as it disappears within 3 days of metamorphic induction. In competent larvae, most neurons of the AG display nitric oxide synthase (NOS)-like immunoreactivity and inhibition of NOS activity can induce larval metamorphose. Because nitric oxide (NO) can prevent cells from undergoing apoptosis, a form of programmed cell death (PCD), we hypothesize that inhibition of NOS activity triggers the loss of the AG at the beginning of the metamorphic process. Within 24 hours of metamorphic induction, cellular changes that are typical of the early stages of PCD are visible in histological sections and results of a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in metamorphosing larvae show AG nuclei containing fragmented DNA, supporting our hypothesis.     

Ultrastructural identification of apoptotic nuclei using the TUNEL technique

Summary We describe an ultrastructural adaptation of the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) for the identification of DNA fragmentation. Thin sections of tissue embedded in hydrophilic resin were nick end labelled with biotinylated dUTP which was subsequently labelled with avidin conjugated to gold particles. The technique was validated by labelling the nuclei of L929-8 cells treated with tumour necrosis factor α. These cells are known to respond to treatment with the factor by undergoing apoptosis. The method was then used on tissue from the chick embryo which is known to be undergoing programmed cell death. This tissue was from the neural tube and the posterior necrotic zone of the limb bud, where cells can be identified as undergoing apoptosis based on the morphology of their nuclei. The method specifically labelled heterochromatin adjacent to the nuclear envelope as well as the associated with the nucleolus of cells from regions of the embryo where programmed cell death was expected. In addition to labelling the nuclei of cells that were clearly undergoing apoptosis, the method also identified nuclei of apparently normal cells. This method, used in conjunction with corroborating techniques, provides a means for the early detection of cells undergoing DNA fragmentation, before the onset of gross apoptotic morphology, and in cells that do not show classical apoptotic characteristics.     

Formation of archegonium chamber is associated with nucellar-cell programmed cell death in Ginkgo biloba

Summary. The archegonium chamber in Ginkgo biloba L. is a pathway for spermatozoids swimming towards the archegonium for fertilization. The objective of this study was to elucidate the mechanism of archegonium chamber formation. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay and DNA ladder demonstrated that the nucellar cell death, coordinated with the archegonium chamber formation, was a process of programmed cell death. Cytochemical localization of Ca²⁺ in these nucellar cells was determined by means of in situ precipitation with potassium pyroantimonate and electron microscopic visualization, in order to study the relation between Ca²⁺ and programmed cell death. The results showed an early uptake of the mitochondrial calcium particles in the nucellar cells undergoing programmed cell death. Together with other dynamic changes in Ca²⁺ subcellular distribution, this indicates that Ca²⁺ may play a role in the regulation of mitochondria-mediated programmed events in the nucellar cells. Correspondence and reprints: School of Pharmaceutical Sciences, Peking University, Beijing 100083, People’s Republic of China.     

Activation of monoamine oxidase isotypes by prolonged intake of aluminum in rat brain

Rats were fed 100 microM aluminum maltolate for one year in their drinking water. Brain aluminum contents have increased 4.2-fold in the aluminum-treated group, whereas no significant changes in the body weight, brain weight, and brain protein content were observed. Long-term aluminum feeding induced apoptosis as assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method and showed activatory effects on the catalytic efficiency (kcat/KM) of monoamine oxidase-A and monoamine oxidase-B up to 1.9- and 3.8-fold, respectively. The expression level of monoamine oxidase isotypes on the Western blot remained unchanged between the two groups, suggesting a change in post-translational regulation of the activities of monoamine oxidase isotypes by long-term aluminum feeding.     

Early generation of nitric oxide contributes to copper tolerance through reducing oxidative stress and cell death in hulless barley roots

The objective of this study was to investigate the specific role of nitric oxide (NO) in the early response of hulless barley roots to copper (Cu) stress. We used the fluorescent probe diaminofluorescein-FM diacetate to establish NO localization, and hydrogen peroxide (H₂O₂)-special labeling and histochemical procedures for the detection of reactive oxygen species (ROS) in the root apex. An early production of NO was observed in Cu-treated root tips of hulless barley, but the detection of NO levels was decreased by supplementation with a NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO). Application of sodium nitroprusside (a NO donor) relieved Cu-induced root inhibition, ROS accumulation and oxidative damage, while c-PTIO treatment had a synergistic effect with Cu and further enhanced ROS levels and oxidative stress. In addition, the Cu-dependent increase in activities of superoxide dismutase, peroxidase and ascorbate peroxidase were further enhanced by exogenous NO, but application of c-PTIO decreased the activities of catalase and ascorbate peroxidase in Cu-treated roots. Subsequently, cell death was observed in root tips and was identified as a type of programed cell death (PCD) by terminal deoxynucleotidyl transferase dUTP nick end labeling assay. The addition of NO prevented the increase of cell death in root tips, whereas inhibiting NO accumulation further increased the number of cells undergoing PCD. These results revealed that NO production is an early response of hulless barley roots to Cu stress and that NO contributes to Cu tolerance in hulless barley possibly by modulating antioxidant defense, subsequently reducing oxidative stress and PCD in root tips.     

Involvement of histone phosphorylation in apoptosis of human astrocytes after exposure to saline solution

We have previously found using inhibitors of protein phosphatase that phosphorylation of histones may be involved in thymocyte apoptosis. In this study, we examined whether histone modification occurs in astrocyte apoptosis induced by a pathological condition in the absence of drug. Incubation of cultured human astrocytes with growth medium for 24 h after exposure to saline solution for 30 min induced an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and nuclear condensation, biochemical and morphological hallmarks of apoptotic cell death. Acetic acid-urea-Triton X-100 (AUT) gel electrophoresis of the nuclear histone fraction and N-terminal peptide analysis showed that the treatment with saline solution caused rapid changes in phosphorylation of H2A subfamilies, but not in histone acetylation. The phosphorylation of the two subtypes increased markedly, whereas the phosphorylation of one subtype decreased. In contrast, exposure to ACF-95, an artificial cerebrospinal fluid (CSF), was associated with little induction of apoptotic cell death and induced less changes in histone phosphorylation. These results support the previous idea that chemical modification of histones is involved in the DNA fragmentation in astrocytes undergoing apoptosis.     

Programmed cell death in the apical ganglion during larval metamorphosis of the marine mollusc Ilyanassa obsoleta

The apical ganglion (AG) of larval caenogastropods, such as Ilyanassa obsoleta, houses a sensory organ, contains five serotonergic neurons, innervates the muscular and ciliary components of the velum, and sends neurites into a neuropil that lies atop the cerebral commissure. During metamorphosis, the AG is lost. This loss had been postulated to occur through some form of programmed cell death (PCD), but it is possible for cells within the AG to be respecified or to migrate into adjacent ganglia. Evidence from histological sections is supported by results from a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which indicate that cells of the AG degenerate by PCD. PCD occurs after metamorphic induction by serotonin or by inhibition of nitric oxide synthase (NOS) activity. Cellular degeneration and nuclear condensation and loss were observed within 12 h of metamorphic induction by NOS inhibition and occur before loss of the velar lobes, the ciliated tissue used for larval swimming and feeding. Velar disintegration happens more rapidly after metamorphic induction by serotonin than by 7-nitroindazole, a NOS inhibitor. Loss of the AG was complete by 72 h after induction. Spontaneous loss of the AG in older competent larvae may arise from a natural decrease in endogenous NOS activity, giving rise to the tendency of aging larvae to display spontaneous metamorphosis in culture.     

Yariv reagent treatment induces programmed cell death in Arabidopsis cell cultures and implicates arabinogalactan protein involvement

Arabinogalactan proteins (AGPs) are a family of highly glycosylated, hydroxyproline-rich glycoproteins implicated in various aspects of plant growth and development. (beta-D-glucosyl)3 and (beta-D-galactosyl)3 Yariv phenylglycosides, commonly known as Yariv reagents, specifically bind AGPs in a non-covalent manner. Here (beta-D-galactosyl)3 Yariv reagent was added to Arabidopsis thaliana cell suspension cultures and determined to induce programmed cell death (PCD) by three criteria: (i) DNA fragmentation as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) of DNA 3'-OH groups; (ii) inter- nucleosomal DNA fragmentation as visualized by genomic Southern blotting; and (iii) structural changes characteristic of PCD including cytoplasmic shrinkage and condensation, chromatin condensation and nuclear membrane blebbing. These findings implicate AGP involvement in PCD in plants, presumably by perturbation of AGPs located at the plasma membrane-cell wall interface.     

Barley aleurone cell death is not apoptotic: characterization of nuclease activities and DNA degradation

Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevent PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmark of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. CA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.     

Endotoxin infusion in rats induces apoptotic and survival pathways in hearts

Inflammatory mediators of sepsis induce apoptosis in many cell lines. We tested the hypothesis that lipopolysaccharide (LPS) injection in vivo results in induction of early apoptotic and survival pathways as well as evidence of late-stage apoptosis in the heart. Hearts were collected from control rats and at 6, 12, and 24 h after LPS injection (4 mg/kg). Activation of an apoptotic pathway was identified by a 1,000-fold increase in caspase-3 activity at 24 h (P < 0.05). Confirmation of these results occurred when terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining identified myocardial cells undergoing DNA fragmentation with significant levels at 24 h post-LPS injection. LPS also caused early proapoptotic mRNA (Bax) to increase (16% at 24 h, P < 0.05), whereas the Bax protein initially decreased (35% at 6 h, P < 0.05) and then returned to baseline values by 24 h. Six hours after LPS injection, Bcl-2 (early prosurvival) mRNA levels increased, whereas its protein levels decreased (70%, P < 0.05) and then returned to baseline levels by 24 h. Mitochondrial cytochrome c levels decreased, suggestive of mitochondrial involvement. Thus involvement of proapoptotic and prosurvival pathways in the heart occurs during a septic inflammatory response.