Oxidative stress by <Emphasis Type="Italic">Helicobacter pylori</Emphasis> causes apoptosis through mitochondrial pathway in gastric epithelial cells

Helicobacter pylori is a gram negative bacterium that infects the human stomach of approximately half of the world’s population. It produces oxidative stress, and mitochondria are one of the possible targets and the major intracellular source of free radicals. The present study was aimed at determining mitochondrial alterations in H. pylori-infected gastric epithelial cells and its relationship with oxidative stress, one of the recognized causes of apoptotic processes. Cells were treated with a strain of H. pylori for 24 h. Cellular oxidative burst, antioxidant defense analysis, mitochondrial alterations and apoptosis-related processes were measured. Our data provide evidence on how superoxide acts on mitochondria to initiate apoptotic pathways, with these changes occurring in the presence of mitochondrial depolarization and other morphological and functional changes. Treatment of infected cells with Vitamin E prevented increases in intracellular ROS and mitochondrial damage consistent with H. pylori inducing a mitochondrial ROS mediated programmed cell death pathway.     

Abscisic-acid-induced turion formation in Spirodela polyrrhiza L. II. Ultrastructure of the turion; a stereological analysis

Abstract The ultrastructural features of the abscisic-acid-induced turion of Spirodela polyrrhiza are briefly described and a comparison between turion and vegetative frond tissue was made by stereological analysis. The turion is characterized by its small size, reniform shape, and dark-brown coloration; the mesophyll is undifferentiated and totally lacking the substantial acrenchyma development found in the vegetative frond. The turion cells have a smaller vacuole and a denser cytoplasm than the cells of the vegetative frond. Stereological analysis showed that the tissues differed quantitatively only in three main respects: air space formation, vacuolation, and starch and cell wall material accumulation. During development, it is suggested that the cells of the turion, while reaching the same final size as the vegetative frond cells, accumulate numerous starch grains, thick cell walls, and large deposits of tannins and anthocyanin pigment at the expense of the vacuolar expansion characteristic of the normal maturity programme. Certain features of the turion ultrastructure indicate a differential cell sensitivity to ABA.     

Mitochondrial function plasticity in <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> during growth in batch culture

The alterations in mitochondrial bioenergetics during growth in a batch culture of Acanthamoeba castellanii were studied. The capacity of cytochrome pathway-dependent respiration measured in vitro decreased from the intermediary phase, when cell division slowed down. The pattern of the cytochrome pathway capacity changes was paralleled from the intermediary phase by alterations in the amount of total (and reducible) membranous ubiquinone. These changes were accompanied by a decrease in mitochondrial reactive oxygen species production in vitro (when no energy-dissipating system was active), and almost no change in superoxide dismutase activity and protein level, thus indicating an equivalent need for this enzyme in oxidative stress defence in A. castellanii culture. On the other hand, a decrease in the activity and protein level of alternative oxidase and uncoupling protein was observed in vitro, when cells shifted from the exponential growth phase to the stationary phase. It turned out that the contribution of both energy-dissipating systems in the prevention of mitochondrial reactive oxygen species generation in vivo could lead to its constant level throughout the growth cycle of A. castellanii batch culture. Hence, the observed functional plasticity insures survival of high quality cysts of A. castellanii cells.     

Haemocyte apoptosis as a general cellular immune response of the snail, <Emphasis Type="BoldItalic">Lymnaea stagnalis</Emphasis>, to a toxicant

The effects of a xenobiotic on the circulating haemocytes of Lymnaea stagnalis were investigated after short-term (24 h, 96 h) and long-term (504 h) exposure of snails to environmental concentrations. Fomesafen, a pro-oxidant generator led to the activation of the haemocyte apoptotic program by promoting reactive oxygen species (ROS). Cells entering apoptosis underwent a series of events, both on the plasma membrane and in the mitochondria; these events were quantified by flow cytofluorometry. The data showed a loss of mitochondrial transmembrane potential (Δψm), which was dose-dependent and time-dependent and related to an increased release of superoxide anions. The phosphatidylserine that was exposed at the outer plasma membrane was not related to the disruption of either ROS or Δψm but was strongly correlated with the haemocyte concentration (total haemocyte count). This cascade of apoptotic processes occurred in a dose-independent manner and was not strengthened over time. The increase of circulating haemocytes depended upon the life span of the cells and might have reflected either facilitated cell turn-over or the accompanying presence of haemocytes phagocytosing apoptotic cells.     

PHYTOALEXIN DEFICIENT 4 affects reactive oxygen species metabolism,cell wall and wood properties in hybrid aspen (Populus tremula L. × tremuloides)

The PHYTOALEXIN DEFICIENT 4 (PAD4) gene in Arabidopsis thaliana (AtPAD4) is involved in the regulation of plant – pathogen interactions. The role of PAD4 in woody plants is not known; therefore, we characterized its function in hybrid aspen and its role in reactive oxygen species (ROS)‐dependent signalling and wood development. Three independent transgenic lines with different suppression levels of poplar PAD expression were generated. All these lines displayed deregulated ROS metabolism, which was manifested by an increased H₂O₂ level in the leaves and shoots, and higher activities of manganese superoxide dismutase (MnSOD) and catalase (CAT) in the leaves in comparison to the wild‐type plants. However, no changes in non‐photochemical quenching (NPQ) between the transgenic lines and wild type were observed in the leaves. Moreover, changes in the ROS metabolism in the pad4 transgenic lines positively correlated with wood formation. A higher rate of cell division, decreased tracheid average size and numbers, and increased cell wall thickness were observed. The results presented here suggest that the Populus tremula × tremuloides PAD gene might be involved in the regulation of cellular ROS homeostasis and in the cell division – cell death balance that is associated with wood development.     

Metabolic responses of Lemna minor to lead ions II. Induction of antioxidant enzymes in roots

Duckweed Lemna minor L. was grown on Wang culture medium supplemented with lead ions for 24 hours. Metal was tested at 1.5, 3 and 6 mg·dm⁻³ concentrations. The response of antioxidant enzymes, such as superoxide dismutase, catalase and peroxidase in lead-treated roots of duckweed was investigated. Lead ions had no effect on the spectrum of catalase and peroxidase isoenzymes while a new isoform of superoxide dismutase appeared on the Pb treated roots. A lead-depended increase in activities of superoxide dismutase and peroxidase was observed, whereas catalase activity was maintained at relatively constant values at lower lead concentrations and then decreased markedly below control level.     

Frond transformation system mediated by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> for <Emphasis Type="Italic">Lemna minor</Emphasis>

The Lemnaceae, known as duckweed, the smallest flowering aquatic plant, shows promise as a plant bioreactor. For applying this potential plant bioreactor, establishing a stable and efficient genetic transformation system is necessary. The currently favored callus-based method for duckweed transformation is time consuming and genotype limited, as it requires callus culture and regeneration, which is inapplicable to many elite duckweed strains suitable for bioreactor exploitation. In this study, we attempted to establish a simple frond transformation system mediated by Agrobacterium tumefaciens for Lemna minor, one of the most widespread duckweed species in the world. To evaluate the feasibility of the new transformation system, the gene CYP710A11 was overexpressed to improve the yield of stigmasterol, which has multiple medicinal purposes. Three L. minor strains, ZH0055, D0158 and M0165, were transformed by both a conventional callus transformation system (CTS) and the simple frond transformation system (FTS). GUS staining, PCR, quantitative PCR and stigmasterol content detection showed that FTS can produce stable transgenic lines as well as CTS. Moreover, compared to CTS, FTS can avoid the genotype constraints of callus induction, thus saving at least half of the required processing time (CTS took 8–9 months while FTS took approximately 3 months in this study). Therefore, this transformation system is feasible in producing stable transgenic lines for a wide range of L. minor genotypes.     

Patterns of asexual reproduction in <Emphasis Type="Italic">Nannochloris bacillaris</Emphasis> and <Emphasis Type="Italic">Marvania geminata</Emphasis> (Chlorophyta,Trebouxiophyceae)

Non-flagellated vegetative green algae of the Trebouxiophyceae propagate mainly by autosporulation. In this manner, the mother cell wall is shed following division of the protoplast in each round of cell division. Binary fission type Nannochloris and budding type Marvania are also included in the Trebouxiophyceae. Phylogenetic trees based on the actin sequences of Trebouxiophyceae members revealed that the binary fission type Nannochloris bacillaris and the budding type Marvania geminata are closely related in a distal monophyletic group. Our results suggest that autosporulation is the ancestral mode of cell division in Trebouxiophyceae. To elucidate how non-autosporulative mechanisms such as binary fission and budding evolved, we focused on the cleavage of the mother cell wall. Cell wall development was analyzed using a cell wall-specific fluorescent dye, Fluostain I. Exfoliation of the mother cell wall was not observed in either N. bacillaris or M. geminata. We then compared the two algae by transmission electron microscopy with rapid freeze fixation and freeze substitution; in both algae, the mother cell wall was cleaved at the site of cell division, but remained adhered to the daughter cell wall. In N. bacillaris, the cleaved mother cell wall gradually degenerated and was not observed in the next cell cycle. In contrast, M. geminata daughter cells entered the growth phase of the next cell cycle bearing the mother and grandmother cell walls, causing the uncovered portion of the plane of division to bulge outward. Such a delay in the degeneration and shedding of the mother cell wall probably led to the development of binary fission and budding.     

Carbon dioxide assimilation from air and water by duckweed Spirodela polyrrhiza (L.) Schleid

Carbon dioxide assimilation by duckweed, S. polyrrhiza, was measured using a glass assimilation box and ¹⁴C-NaHCO₃, under different pH conditions of water. S. polyrrhiza assimilates carbon dioxide from both air and water. The carbon assimilation from air is comparable to the assimilation from water under normal pH conditions.     

Development of a novel, multi-analyte biosensor system for assaying cell division: identification of cell proliferation/death precursor events

A novel, miniaturized biosensor system was created by combining the electrophysiological response of immobilized cells with superoxide-sensing technology, optical and fluorescence microscopy. Vero cells were immobilized in a calcium alginate matrix (at a density of 1.7 x 10(6) cells ml(-1)). A 0.5 cm x 0.5 cm piece of cell-containing gel matrix was aseptically adhered on a glass microscope slide with a microfabricated gold electrode array, sealed with a cover slip and provided with Dulbecco's medium +10% (v/v) fetal calf serum every day by means of a capillary feeding tube. During a culture period of 7 days, the membrane potential of immobilized cells was continuously monitored, while cell division was assayed with an optical microscope. In addition, daily measurements of immobilized cell membrane potential, viability, RNA and calcium concentration, radical oxygen species (ROS) and glutathione accumulation, were conducted by fluorescence microscopy after provision of an appropriate dye. Superoxide accumulation was assayed by covering the electrodes with superoxide dismutase (SOD). Maximum cell membrane potential values and superoxide production were observed upon initiation of cell division. Using the novel biosensor, we were able to correlate seven different cell physiological parameters to each other and formulate a model for ROS-mediated signaling function on cell division and death. In addition, we were able to predict cell proliferation or death by comparing the relative response of the electrophysiological and superoxide sensor during the culture period.     

Photosynthetic Activity during the Life Cycle of Synchronous Skeletonema Cells

A culture of Skeletonema costatum grown at a light intensity of 3 klux and at 20°C was synchronized in diurnally intermittent illumination of 12 hour light and 12 hour dark. The culture was hardly fully synchronous as the cell division period lasted about 9 hours. The cell division started in the middle of the light period. The concentration of the pigments: chlorophyll a, chlorophyll 6 and fucoxanthin and the rate of light-saturated photosynthesis were followed every hour during the 24 hour period. Both the concentration of pigments and the photosynthetic activity showed a rhythmical variation. The concentration per cell of all three pigments examined increased during the development of the cells and decreased automatically during the period of cell division. An increase in the pigment concentration was found only in the light period. The rate of light-saturated photosynthesis calculated per unit of cell number increased during the cell development and decreased during the division period. The increase in the photosynthetic activity at light-saturation started about 4 hours after the end of cell division, which was 4 hours before the light was turned on while the increase in the concentration of chlorophyll a first started 1–2 hours after this moment. The variation in photosynthetic activity was compared with that found by other workers. The results found with Chlorella ellipsoidea by Japanese scientists (Nihci et al.) was explained as an inhibition phenomenon because the cells were not adapted to the experimental conditions.     

The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts

The present study investigated changes in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in isolated mesophyll protoplasts and cell cultures of the cucumber Cucumis sativus cv. Marketer. Although only a minor increase in the level of nitrogen oxide (NO) was observed during the first 7 days of culture following protoplast isolation, a substantial accumulation of ROS was detected. Compounds known to modulate endogenous ROS and RNS levels were employed to study their role in cucumber protoplast regeneration and growth. Supplementing the culture medium with the NO donors S-nitrosoglutathione and sodium nitroprusside and the ROS scavenger ascorbate significantly increased protoplast viability and cell density. In contrast, cell density was significantly decreased following the addition of catalase to the medium. Scavenging of ROS and RNS induced the formation of cucumber microcalli, thus suggesting a differential role of NO in the maintenance of cell viability and in the control of cell division. Our findings confirm the crucial role of controlled ROS and RNS production in both protoplast regeneration and cellular growth and differentiation.     

Oxidative stress by antimicrobial peptide pleurocidin triggers apoptosis in Candida albicans

Pleurocidin (GWGSFFKKAAHVGKHVGKAALTHYL-NH₂), found in skin mucous secretions of the winter flounder Pleuronectes americanus, is known to possess a high potency and broad-spectrum antimicrobial peptide without cytotoxicity. In this study, to investigate the impact of pleurocidin on apoptotic progress, we observed morphological and physiological changes in Candida albicans. In cells exposed to pleurocidin, intracellular reactive oxygen species (ROS) which is a major cause of apoptosis were increased, and hydroxyl radicals were especially a large part of ROS. The increase of ROS induced oxidative stress and mitochondrial membrane depolarization which causes release of pro-apoptotic factors. Using FITC-VAD-FMK staining, we confirmed activation of yeast metacaspases which lead to apoptosis and phosphatidylserine externalization at early stage apoptosis was observed using annexin V FITC. In addition, pleurocidin induced-apoptotic cells underwent apoptotic morphological changes, showing the reduced cell size (low FSC) and enhanced intracellular density (high SSC) in flow cytometry dot plots. Under the influence of oxidative stress, DNA and nuclei were fragmented and condensed in cells, and they were visualized by 4′,6-diamidino-2-phenylindole (DAPI) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. These apoptotic phenomena represent that oxidative stress by inducing pleurocidin must be an important factor of the apoptotic process in C. albicans.     

Role of hydrolytic enzymes and oxidative stress in autolysis and morphology of <Emphasis Type="Italic">Blakeslea trispora</Emphasis> during β-carotene production in submerged fermentation

The role of hydrolytic enzymes (proteases and chitinase) and oxidative stress in the autolysis and morphology of Blakeslea trispora during β-carotene production from a chemically defined medium in shake flask culture was investigated. The process of cellular autolysis was studied by measuring the changes in biomass dry weight, pH, concentration of β-carotene, specific activity of the hydrolytic enzymes and micromorphology of the fungus using a computerized image analysis system. In addition, the phenomenon of autolysis was associated with high concentrations of reactive oxygen species (ROS). The accumulation of ROS produced during fermentation causes oxidative stress in B. trispora. Oxidative stress was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). The profile of the specific activities of the above enzymes appeared to correlate with the oxidative stress of the fungus. The high activities of CAT and SOD showed that B. trispora is found under oxidative stress during β-carotene production. The culture began to show signs of autolysis nearly in the growth phase and autolysis increased significantly during the production phase. The morphological differentiation of the fungus was a result of the degradation of the cell membrane by hydrolytic enzymes and oxidative stress. Increased β-carotene production is correlated with intense autolysis of clumps, which has as a consequence the increase of the freely dispersed mycelia.     

Periodicity in cell division and physiological behavior of ditylum brightwellii,a marine planktonic diatom,during growth in light-dark cycles

Summary Cells of Ditylum brightwellii, a large marine centric diatom, were partially synchronized by employing an appropriate light-dark cycle. At 20°C this consisted of 8 hrs of illumination at an intensity of 0.05 cal/cm² min. A single 2.8 l culture was studied over a 20 day period by diluting the culture daily to a standard cell concentration. The sequence of events in cell development was as follows: daughter cells were formed late in the light period, in the dark they elongated and the numerous chromatophores began dividing. A minimum cell buoyancy was observed in the dark concurrent with cell elongation. Increase in cell phosphorus took place in the dark period. The photosynthetic rate of cells removed during the dark period decreased to a minimum. In the following light period photosynthetic rate increased to a maximum, photosynthetic pigments, cell carbon, nitrogen, and carbohydrate increased and cell division again took place. Cell silica content increased concomitant with cell division. Details of cell morphology during cell division, based upon light microscopy, are reported.Contribution of the Scripps Institution of Oceanography.     

亚硝酸盐胁迫对罗氏沼虾血细胞及其抗氧化酶活力的影响

【背景】亚硝酸盐是虾类集约化养殖过程中最常见的毒性污染物之一,研究亚硝酸盐胁迫对罗氏沼虾血细胞的毒性以及抗氧化酶在抗胁迫防御中的作用,能够为罗氏沼虾养殖过程中的亚硝酸盐中毒防治提供理论参考。【方法】以不同浓度(0、1、5和10 mg·L~(-1))的亚硝态氮(NO_2~--N)对罗氏沼虾进行胁迫,于胁迫后的0、6、12、24和48 h取样,应用流式细胞术检测血细胞活性氧(ROS)含量和细胞凋亡率,同时测定血细胞总数(THC)和胞内抗氧化酶活力。【结果】1 mg·L~(-1)NO_2~--N在48 h内对血细胞ROS含量、凋亡率和THC均无显著影响。5 mg·L~(-1)NO_2~--N胁迫24 h,血细胞ROS含量显著上升,THC显著下降,胁迫48 h凋亡率显著提高。10 mg·L~(-1)NO_2~--N胁迫6 h,血细胞ROS含量和凋亡率均显著上升,胁迫12 h THC显著下降。血细胞的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和谷胱甘肽过氧化物酶(GPx)的活力均不同程度地被NO_2~--N胁迫所诱导,CAT活力主要在胁迫前期提高,而GPx活力在胁迫后期提高。【结论与意义】亚硝酸盐存在浓度和时间毒性效应,一定浓度的亚硝酸盐会诱导虾血细胞产生ROS,这些ROS的过量产生诱导了血细胞发生凋亡,继而导致THC下降,这一氧化胁迫过程可能是亚硝酸盐对罗氏沼虾产生细胞毒性的重要机制之一。抗氧化酶活力的诱导表明抗氧化酶在亚硝酸盐胁迫过程中发挥防御作用。     

Apoptotic cell death in suspension cultures of Taxus chinensis var. mairei

Apoptotic cell death was observed in suspension cultures of Taxus chinensis var. mairei under normal cultivation conditions by using microscopy, total DNA agarose gel electrophoresis and in situ end-labeling of fragmented DNA. The morphological and biochemical changes of cells occurred mainly in the non-dividing cell clusters, indicating that the T. chinensis cells died mainly by apoptosis. There exists a close relationship between cell apoptosis and Taxol formation. Taxol concentration increased with the increase in content of apoptotic cells and reached a maximum (14.2 mg l^–1) after 23 days of culture, corresponding to a maximum ratio of apoptotic to total cells of about 13%.     

Seasonal variation in antioxidant enzymes and the sprouting response of Gmelina arborea Roxb. nodal sectors cultured in vitro

Nodal sector explants of Gmelina arborea Roxb. showed seasonal variation in the sprouting of axillary buds in vitro. Explants from mature trees showed only 20% sprouting in summer, while those from seedlings and young trees showed over 85% sprouting in this season. In winter, there was a significant decrease in the sprouting response of explants from young and mature trees but not in that of seedling explants. We have attempted to correlate the sprouting response of the explants with their antioxidant status. Activities of three enzymes, namely ascorbate peroxidase, superoxide dismutase and guaiacol-dependent peroxidase which form a part of the antioxidant defense system of plants, were studied in the excised nodal sectors before and after placing them on culture medium. Prior to culture, higher activities of ascorbate peroxidase, superoxide dismutase and guaiacol-dependent peroxidase were observed during winter than during summer. During the culture period, ascorbate peroxidase, superoxide dismutase and guaiacol-dependent peroxidase showed variation depending on the season in which the explants were isolated, and the age of the donor. Axillary bud sprouting in vitro appeared to depend more on the physiological state of the donor, than on the oxidative stress generated during culture. This revised version was published online in June 2006 with corrections to the Cover Date.     

<Emphasis Type="Italic">FORMOSA</Emphasis> controls cell division and expansion during floral development in <Emphasis Type="Italic">Antirrhinum</Emphasis><Emphasis Type="Italic">majus</Emphasis>

Control of organ size is the product of coordinated cell division and expansion. In plants where one of these pathways is perturbed, organ size is often unaffected as compensation mechanisms are brought into play. The number of founder cells in organ primordia, dividing cells, and the period of cell proliferation determine cell number in lateral organs. We have identified the Antirrhinum FORMOSA (FO) gene as a specific regulator of floral size. Analysis of cell size and number in the fo mutant, which has increased flower size, indicates that FO is an organ-specific inhibitor of cell division and activator of cell expansion. Increased cell number in fo floral organs correlated with upregulation of genes involved in the cell cycle. In Arabidopsis the AINTEGUMENTA (ANT) gene promotes cell division. In the fo mutant increased cell number also correlates with upregulation of an Antirrhinum ANT-like gene (Am-ANT) in inflorescences that is very closely related to ANT and shares a similar expression pattern, suggesting that they may be functional equivalents. Increased cell proliferation is thought to be compensated for by reduced cell expansion to maintain organ size. In Arabidopsis petal cell expansion is inhibited by the BIGPETAL (BPE) gene, and in the fo mutant reduced cell size corresponded to upregulation of an Antirrhinum BPE-like gene (Am-BPE). Our data suggest that FO inhibits cell proliferation by negatively regulating Am-ANT, and acts upstream of Am-BPE to coordinate floral organ size. This demonstrates that organ size is modulated by the organ-specific control of both general and local gene networks. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Female reproductive development and pollen tube growth in diploid genotypes of<Emphasis Type="Italic"> Solanum cardiophyllum</Emphasis> Lindl.

We have characterized female gametophyte (megagametophyte) development and the kinetics of pollen tube growth in self-pollinated diploid genotypes (2n=2x=24) of Solanum cardiophyllum Lindl. that show normal seed formation. In this species megasporogenesis and megagametogenesis give rise to a female gametophyte of the Polygonum type composed of two synergids, an egg cell, a binucleated central cell and three antipodals; however, asynchronous abnormalities resembling mechanisms that prevail during the formation of second division restitution gametes were observed. In self-pollinated pistils at least 1–2% of germinating pollen tubes were able to reach the megagametophyte 60–84 hours after pollination (hap). Although the egg cell acquired a zygote-like morphology 60–84 hap, division of the primary endosperm nucleus was only observed 84 hap. The analysis of genetic variability in full-sib progeny confirmed that seeds are derived from sexual reproduction. These observations suggest that diploid genotypes of S. cardiophyllum can serve as an ideal system to genetically investigate true seed formation in a tuber-bearing Solanum species.