Effects of cholesterol and lipoproteins on endocytosis by a monocyte-like cell line

The human monocyte/macrophage-like cell line U937 is a cholesterol auxotroph. Incubation of these cells in the growth medium in which delipidated fetal calf serum has been substituted for fetal calf serum depletes cellular cholesterol and inhibits growth. The cholesterol requirement of these cells for growth can be satisfied by human low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL), but not by high-density lipoprotein (HDL). U937 cells can bind and degrade LDL via a high-affinity site and this recognition is altered by acetylation of LDL. This indicates that these cells express relatively high LDL receptor activity and low levels of the acetyl-LDL receptor. The cells were used to study the role of cholesterol in lectin-mediated and fluid-phase endocytosis. Growth of the cells in the medium containing delipidated fetal calf serum results in impairment of both concanavalin A-mediated endocytosis of horseradish peroxidase and concanavalin A-independent endocytosis of Lucifer Yellow. Supplementation of the medium with cholesterol prevents cellular cholesterol depletion, supports growth and stimulates Lucifer Yellow endocytosis but fails to restore horseradish peroxidase endocytosis. However, if the cells are incubated in the presence of no less than 40 μg LDL protein/ml to maintain normal cell cholesterol levels, concanavalin A-mediated endocytosis of horseradish peroxidase is activated. The effect of LDL is specific since neither VLDL nor HDL₃ at the same protein concentration activates horseradish peroxidase uptake by the cells. Furthermore, the activation of endocytosis by LDL is not inhibited by the inclusion of heparin or acetylation of the LDL indicating that binding of LDL to the LDL receptor is not required for these effects. The mediation of activation of horseradish peroxidase endocytosis by the lectin is presumed to involve binding of LDL to concanavalin A associated with the cell surface which in turn stimulates horseradish peroxidase binding and uptake by adsorptive endocytosis. The rate of fluid endocytosis and endosome formation seems to depend on cellular cholesterol content presumably because cholesterol is involved in maintaining the appropriate plasma membrane structure and fluidity.     

Cysteine protects freshly isolated cardiomyocytes against oxidative stress by stimulating glutathione peroxidase

Cysteine has been implicated in myocardial protection, although this is controversial and constrained by limited knowledge about the effects of cysteine at the cellular level. This study tested the hypothesis that a physiologically relevant dose of l-cysteine could be safely loaded into isolated cardiomyocytes leading to improved protection against oxidative stress. Freshly isolated adult rat ventricular cardiomyocytes were incubated for 2 h at 37°C with (cysteine incubated) or without (control) 0.5 mM cysteine prior to washing and suspension in fresh cysteine-free media. Cysteine incubated cells had higher intracellular cysteine levels compared to controls (9.6 ± 0.78 vs. 6.5 ± 0.65 nmol/mg protein, P < 0.02, n = 6 ± SE). Cell homeostasis indicators were similar in the two groups. Cysteine incubated cells had significantly higher glutathione peroxidase (GPx) activity (1.11 ± 0.23 vs. 0.54 ± 0.1 U/mg protein, P < 0.05, n = 5 ± SE) and significantly greater expression of GPx-1 (5.01 ± 0.48 vs. 3.01 ± 0.25 OD units/mm², P < 0.05, n = 4 ± SE) compared to controls. Upon exposure to H₂O₂, cysteine incubated cells generated fewer reactive oxygen species and took longer to show contractile changes and undergo hypercontracture. However, when cells were exposed to H₂O₂ in the presence of 0.05 mM of the GPx inhibitor mercaptosuccinic acid, this increased the control cells’ susceptibility to H₂O₂ and completely abolished the cysteine mediated protection. These results suggest a new role for cysteine in myocardial protection involving stimulation of glutathione peroxidase.     

Antioxidative responses of Ocimum basilicum to sodium chloride or sodium sulphate salinization

Soils and ground water in nature are dominated by chloride and sulphate salts. There have been several studies concerning NaCl salinity, however, little is known about the Na₂SO₄ one. The effects on antioxidative activities of chloride or sodium sulphate in terms of the same Na⁺ equivalents (25 mM Na₂SO₄ and 50 mM NaCl) were studied on 30 day-old plants of Ocimum basilicum L., variety Genovese subjected to 15 and 30 days of treatment. Growth, thiobarbituric acid reactive substances (TBARS), relative ion leakage ratio (RLR), hydrogen peroxide (H₂O₂), ascorbate and glutathione contents as well as the activities of ascorbate peroxidase (APX, EC 1.11.1.11); glutathione reductase (GR, EC 1.6.4.2) and peroxidases (POD, EC 1.11.1.7) were determined. In leaves, growth was more depressed by 25 mM Na₂SO₄ than 50 mM NaCl. The higher sensitivity of basil to Na₂SO₄ was associated with an enhanced accumulation of H₂O₂, an inhibition of APX, GR and POD activities (with the exception of POD under the 30-day-treatment) and a lower regeneration of reduced ascorbate (AsA) and reduced glutathione (GSH). However, the changes in the antioxidant metabolism were enough to limit oxidative damage, explaining the fact that RLR and TBARS levels were unchanged under both Na₂SO₄ and NaCl treatment. Moreover, for both salts the 30-day-treatment reduced H₂O₂ accumulation, unchanged RLR and TBARS levels, and enhanced the levels of antioxidants and antioxidative enzymes, thus achieving an adaptation mechanism against reactive oxygen species.     

Sulfur stress-induced antioxidative responses in leaves of Triticum aestivum L.

Antioxidative responses were investigated in leaves of wheat (Triticum aestivum L.) grown at varying S levels ranging from deficiency to excess (1, 2, 4, 6 and 8 mM S). Optimum yield was observed in plants supplied with 4 mM S. Wheat responded to S deficiency and excess supply by decreasing growth of root and shoot. Chlorosis in young leaves was observed after 15 days of deficient S supply. The biomass and concentration of photoassimilatory pigments decreased in plants grown at 1, 2, 6 and 8 mM S supply. The concentration of thiobarbituric acid reactive substances (TBARS), cysteine, nonprotein thiol and hydrogen peroxide (H₂O₂) increased in plants grown under S stress. Accumulation of TBARS and H₂O₂ in leaves indicated oxidative damage in S-deficient and S-excess plants. Deficient and excess levels of S showed an increase in the activities of antioxidative enzymes superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), peroxidase (EC 1.11.1.7), ascorbate peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2).     

Oxidative stress and antioxidant responses of mulberry (Morus alba) plants subjected to deficiency and excess of manganese

The aim of the study was to relate the effects of deficiency and excess of Mn with the generation of reactive oxygen species (ROS) and altered cellular redox environment in mulberry (Morus alba L.) cv. Kanva-2 plants. Mn deficiency symptom appeared as mild interveinal chlorosis in middle leaves. Mn-excess did not produce any specific symptom. Leaf water potential (Ψ) was increased in Mn-deficient and Mn-excess mulberry plants. Mn-deficient leaves contained less Mn, less chloroplastic pigments and high tissue Fe, Zn and Cu concentrations. Starch content was increased with increasing Mn supply. While reducing sugar content increased in Mn-deficient and Mn-excess plants as well, non-reducing sugars remained unaffected in Mn-deficient plants and decreased in Mn-excess plants. Moreover, study of antioxidative responses, oxidative stress (H₂O₂ and lipid peroxidation) and cellular redox environment [dehydroascorbate (DHA)/ascorbic acid (AsA) ratio] in Mn-stressed mulberry plants was also undertaken. Both hydrogen peroxide and lipid peroxidation were enhanced in the leaves of Mn-deficient plants. Increased H₂O₂ concentration in Mn-excess leaves did not induce oxidative damage as indicated by no change in lipid peroxidation. The ratio of the redox couple (DHA/AsA) was increased both in Mn-deficient or Mn-excess plants. The activities of superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) increased in Mn-deficient plants. The activity of ascorbate peroxidase (EC 1.11.1.11) increased with increasing Mn supply. The results suggest that deficiency or excess of Mn induces oxidative stress through enhanced ROS generation and disturbed redox couple in mulberry plants.     

Hydrogen peroxide leads to cell damage and apoptosis in the gill of the freshwater crab Sinopotamon henanense (Crustacea,Decapoda)

Hydrogen peroxide (H₂O₂), a major reactive oxygen species, has been shown to be a critical mediator of apoptosis induced by several toxic metals such as cadmium. In this study, we used the freshwater crab Sinopotamon henanense to study whether H₂O₂ can cause apoptosis in gill cells. The crabs were incubated in H₂O₂ and the DNA fragmentation, ultrastructural changes and caspase-3/8/9 activities were measured. The results showed that in freshwater crab, H₂O₂ was found to induce apoptosis, as confirmed by DNA fragmentation analysis and morphological observation of transmission electron microscopy. This apoptosis occurs in a concentration-dependent pattern. During the apoptotic process, caspase-3, caspase-8 and caspase-9 were activated by H₂O₂. In addition, multiple physiological and pathological changes of gill cells were discovered after 24 h exposure to 5 mM H₂O₂, including aggregation and condensation of nuclear chromatin, appearance of extremely irregular nuclei with finger-like buds, disappearance of the organelles around the nuclei, swollen and dissolved cristae of mitochondria. We propose that H₂O₂-induced stress leads to mitochondria lesions oxidative injury and triggers apoptotic response through the caspase pathway in freshwater crab.     

Trimetazidine protects low-density lipoproteins from oxidation and cultured cells exposed to H2O2 from DNA damage

Trimetazidine is a well-established anti-ischemic drug, which has been used for long time in the treatment of pathological conditions related with the generation of reactive oxygen species. However, although extensively studied, its molecular mode of action remains largely unknown. In the present study, the ability of trimetazidine to protect low-density lipoproteins (LDL) from oxidation and cultured cells from H₂O₂-induced DNA damage was investigated. Trimetazidine, tested at concentrations 0.02 to 2.20 mM, was shown to offer significant protection to LDL exposed to three different oxidizing systems, namely copper, Fe/ascorbate, and met-myoglobin/H₂O₂. The oxidizability of LDL was estimated by measuring, (i) the lag period, (ii) the maximal rate of conjugated diene formation, (iii) the total amount of conjugated dienes formed, (iv) the electrophoretic migration of LDL protein in agarose gels (REM), and (v) the inactivation of the enzyme PAF-acetylhydrolase present in LDL. In addition, the presence of trimetazidine decreased considerably the DNA damage in H₂O₂-exposed Jurkat cells in culture. H₂O₂ was continuously generated by the action of glucose oxidase at a rate of 11.8 ± 1.5 μM per min (60 ng enzyme per 100 μl), and DNA damage was assessed by the single cell gel electrophoresis assay (also called comet assay). The protection offered by trimetazidine in this system (about 30% at best) was transient, indicating modification of this agent during its action. These results indicate that trimetazidine can modulate the action of oxidizing agents in different systems. Although its mode of action is not clarified, the possibility that it acts as a lipid barrier permeable transition metal chelator is considered.     

Nuclear morphology and c‐Jun N‐terminal kinase 1 expression differentiate serum‐starved oxidative stress signalling from hydrogen peroxide‐induced apoptosis in retinal neuronal cell line

Oxidative stress induced by serum starvation and H₂O₂ exposure, both triggers apoptosis in retinal neuronal cell line RGC‐5 (retinal ganglion cell‐5). We have examined whether, despite excess generation of ROS (reactive oxygen species) and apoptosis induction, there is any dissimilarity in nuclear morphology and apoptotic signalling pathway in RGC‐5 under these conditions. Sub‐confluent cells were treated either with H₂O₂ or maintained in SFM (serum‐free medium). ROS level was detected along with nuclear morphology and ultrastructural analysis. Generation of excess intracellular ROS, nuclear localization of Bax and caspase 3 activation along with decrease of cellular viability, confirmed apoptosis induction in RGC‐5 by 72 h serum starvation and 500 M H₂O₂ exposure for 1 h. Nuclear swelling as supported by nuclear cytoplasmic ratio and conspicuous black spots with nuclear remodelling were observed only upon SFM, but not with H₂O₂ treatment. Serum starvation did not alter JNK1 (c‐Jun N‐terminal kinase 1) expression, although nuclear translocation and higher level of pJNK (phospho‐JNK) was evident. Conversely, H₂O₂ exposure blocked the expression and activation of JNK1 to phospho‐JNK as a negligible level of pJNK was present in the cytoplasm. Despite similar ROS generation in both the conditions, difference in nuclear morphology and JNK1 expression leads to the hypothesis that RGC‐5 cells may follow different signalling pathways when challenged with serum starvation and H₂O₂.     

An Integrated Mitochondrial ROS Production and Scavenging Model: Implications for Heart Failure

It has been observed experimentally that cells from failing hearts exhibit elevated levels of reactive oxygen species (ROS) upon increases in energetic workload. One proposed mechanism for this behavior is mitochondrial Ca²⁺ mismanagement that leads to depletion of ROS scavengers. Here, we present a computational model to test this hypothesis. Previously published models of ROS production and scavenging were combined and reparameterized to describe ROS regulation in the cellular environment. Extramitochondrial Ca²⁺ pulses were applied to simulate frequency-dependent changes in cytosolic Ca²⁺. Model results show that decreased mitochondrial Ca²⁺uptake due to mitochondrial Ca²⁺ uniporter inhibition (simulating Ru360) or elevated cytosolic Na⁺, as in heart failure, leads to a decreased supply of NADH and NADPH upon increasing cellular workload. Oxidation of NADPH leads to oxidation of glutathione (GSH) and increased mitochondrial ROS levels, validating the Ca²⁺ mismanagement hypothesis. The model goes on to predict that the ratio of steady-state [H₂O₂]_m during 3Hz pacing to [H₂O₂]_m at rest is highly sensitive to the size of the GSH pool. The largest relative increase in [H₂O₂]_m in response to pacing is shown to occur when the total GSH and GSSG is close to 1 mM, whereas pool sizes below 0.9 mM result in high resting H₂O₂ levels, a quantitative prediction only possible with a computational model.     

Cerium Oxide Nanoparticles Protect Endothelial Cells from Apoptosis Induced by Oxidative Stress

Oxidative stress is well documented to cause injury to endothelial cells (ECs), which in turn trigger cardiovascular diseases. Previous studies revealed that cerium oxide nanoparticles (nanoceria) had antioxidant property, but the protective effect of nanoceria on ROS injury to ECs and cardiovascular diseases has not been reported. In the current study, we investigated the protective effect and underlying mechanisms of nanoceria on oxidative injury to ECs. The cell viability, lactate dehydrogenase release, cellular uptake, intracellular localization and reactive oxygen species (ROS) levels, endocytosis mechanism, cell apoptosis, and mitochondrial membrane potential were performed. The results indicated that nanoceria had no cytotoxicity on ECs but had the ability to prevent injury by H₂O₂. Nanoceria could be uptaken into ECs through caveolae- and clathrin-mediated endocytosis and distributed throughout the cytoplasma. The internalized nanoceria effectively attenuated ROS overproduction induced by H₂O₂. Apoptosis was also alleviated greatly by nanoceria pretreatment. These results may be helpful for more rational application of nanoceria in biomedical fields in the future.     

Lipoproteins from normolipidemic and dyslipidemic subjects modify the thromboxane A2 generation by platelets in clotting human blood

The study was performed to investigate the influence of lipoproteins (LP) on the thromboxane (TX) A₂ formation capacity of platelets in clotting whole blood in vitro. The different lipoprotein fractions VLDL, LDL, HDL₂ and HDL₃ were isolated from blood of normo- or dyslipidemic volunteers by ultracentrifugation. These lipoproteins were incubated in blood with different levels of serum total cholesterol (TC) taken from normolipidemics (TC < 200 mg/dl), moderate hypercholesterolemics (TC: 200–250 mg/dl) or subjects with high cholesterol level (TC > 250 mg/dl), respectively. The amount of serum TXA₂ formed within 60 min at 37°C was measured by enzyme immunoassay. The results obtained show that the efficacy of separate LP fractions to influence the TXA₂ production depends not only on the type of LP fraction but also on the source of plasma used for isolation of LP and on the cholesterol level in the blood for incubation: LDL taken from normolipidemics or moderate hyperlipidemics inhibited the TXA₂ formation in blood from normolipidemics (P < 0.02, respectively), but enhanced it in blood from persons with moderate hypercholesterolemia (P < 0.05). LDL from hyperlipidemics enhanced TXA₂ production in blood from hyperlipidemics (P < 0.05). The HDL₂ fractions inhibited the TXA₂ formation in blood from normo- and hypercholesterolemics (P < 0.02, resp.), but there was no effect of HDL₂ in clotting blood from persons with moderate hypercholesterolemia. All HDL₃ fractions tested inhibited the TXA₂ formation in all types of blood used for clotting (P < 0.02, resp.), probably due to their great cholesterol accepting capacity.     

An Integrated Mitochondrial ROS Production and Scavenging Model: Implications for Heart Failure

It has been observed experimentally that cells from failing hearts exhibit elevated levels of reactive oxygen species (ROS) upon increases in energetic workload. One proposed mechanism for this behavior is mitochondrial Ca²⁺ mismanagement that leads to depletion of ROS scavengers. Here, we present a computational model to test this hypothesis. Previously published models of ROS production and scavenging were combined and reparameterized to describe ROS regulation in the cellular environment. Extramitochondrial Ca²⁺ pulses were applied to simulate frequency-dependent changes in cytosolic Ca²⁺. Model results show that decreased mitochondrial Ca²⁺uptake due to mitochondrial Ca²⁺ uniporter inhibition (simulating Ru360) or elevated cytosolic Na⁺, as in heart failure, leads to a decreased supply of NADH and NADPH upon increasing cellular workload. Oxidation of NADPH leads to oxidation of glutathione (GSH) and increased mitochondrial ROS levels, validating the Ca²⁺ mismanagement hypothesis. The model goes on to predict that the ratio of steady-state [H₂O₂]_m during 3Hz pacing to [H₂O₂]_m at rest is highly sensitive to the size of the GSH pool. The largest relative increase in [H₂O₂]_m in response to pacing is shown to occur when the total GSH and GSSG is close to 1 mM, whereas pool sizes below 0.9 mM result in high resting H₂O₂ levels, a quantitative prediction only possible with a computational model.     

Activation of an oxidative burst is a general feature of sensitive plants exposed to the air pollutant ozone

Ozone exposure stimulates an oxidative burst in leaves of sensitive plants, resulting in the generation and accumulation of hydrogen peroxide (H₂O₂) in tobacco and tomato, and superoxide (O₂^–?) together with H₂O₂ in Arabidopsis accessions. Accumulation of these reactive oxygen species (ROS) preceded the induction of cell death, and both responses co‐occurred spatially in the periveinal regions of the leaves. Re‐current ozone exposure of the sensitive tobacco cv. Bel W3 in closed chambers or in the field led to an enlargement of existing lesions by priming the border cells for H₂O₂ accumulation. Open top chamber experiments with native herbaceous plants in the field showed that Malva sylvestris L. accumulates O₂^–? at those sites that later exhibit plant cell death. Blocking of ROS accumulation markedly reduced ozone‐induced cell death in tomato, Arabidopsis and M. sylvestris. It is concluded that ozone triggers an in planta generation and accumulation of H₂O₂ and/or O₂^–? depending on the species, accession and cultivar, and that both these reactive oxygen species are involved in the induction of cell death in sensitive crop and native plants.     

Inhibitory and enhancing effects of NO on H2O2 toxicity: Dependence on the concentrations of NO and H2O2

Nitric oxide (NO) has been shown to both enhance hydrogen peroxide (H₂O₂) toxicity and protect cells against H₂O₂ toxicity. In order to resolve this apparent contradiction, we here studied the effects of NO on H₂O₂ toxicity in cultured liver endothelial cells over a wide range of NO and H₂O₂ concentrations. NO was generated by spermine NONOate (SpNO, 0.001–1 mM), H₂O₂ was generated continuously by glucose/glucose oxidase (GOD, 20–300 U/l), or added as a bolus (200 μM). SpNO concentrations between 0.01 and 0.1 mM provided protection against H₂O₂-induced cell death. SpNO concentrations >0.1 mM were injurious with low H₂O₂ concentrations, but protective at high H₂O₂ concentrations. Protection appeared to be mainly due to inhibition of lipid peroxidation, for which SpNO concentrations as low as 0.01 mM were sufficient. SpNO in high concentration (1 mM) consistently raised H₂O₂ steady-state levels in line with inhibition of H₂O₂ degradation. Thus, the overall effect of NO on H₂O₂ toxicity can be switched within the same cellular model, with protection being predominant at low NO and high H₂O₂ levels and enhancement being predominant with high NO and low H₂O₂ levels.     

Biochemical basis of the high resistance to oxidative stress in<Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Aerobic organisms experience oxidative stress due to generation of reactive oxygen species during normal aerobic metabolism. In addition, several chemicals also generate reactive oxygen species which induce oxidative stress. Thus oxidative stress constitutes a major threat to organisms living in aerobic environments. Programmed cell death or apoptosis is a physiological mechanism of cell death, that probably evolved with multicellularity, and is indispensable for normal growth and development.Dictyostelium discoideum, an eukaryotic developmental model, shows both unicellular and multicellular forms in its life cycle and exhibits apparent caspase-independent programmed cell death, and also shows high resistance to oxidative stress. An attempt has been made to investigate the biochemical basis for high resistance ofD. discoideum cell death induced by different oxidants. Dose-dependent induction of cell death by exogenous addition of hydrogen peroxide (H₂O₂),in situ generation of H₂O₂ by hydroxylamine, and nitric oxide (NO) generation by sodium nitroprusside treatment inD. discoideum were studied. The AD₅₀ doses (concentration of the oxidants cusing 50% of the cells to die) after 24 h of treatment were found to be 0.45 mM, 4 mM and 1 mM, respectively. Studies on enzymatic antioxidant status ofD. discoideum when subjected to oxidative stress, NO and nutrient stress reveal that superoxide dismutase and catalase were unchanged; a significant induction of glutathione peroxidase was observed. Interestingly, oxidative stress-induced lipid membrane peroxidative damage could not be detected. The results shed light on the biochemical basis for the observed high resistance to oxidative stress inD. discoideum.     

Invitro culturedSpodoptera frugiperda insect cells: Model for oxidative stress-induced apoptosis

Cellular imbalance in the levels of antioxidants and reactive oxygen species (ROS) is directly associated with a number of pathological states and results in programmed cell death or apoptosis. We demonstrate the use ofin vitro culturedSpodoptera frugiperda (sf9) insect cells as a model to study oxidative stress induced programmed cell death. Apoptosis ofin vitro cultured sf9 cells was induced by the exogenous treatment of H₂O₂ to cells growing in culture. The AD₅₀ (concentration of H₂O₂ inducing about 50% apoptotic response) varied with the duration of treatment, batch to batch variation of H₂O₂ and the physiological state of cells. At 24 h post-treatment with H₂O₂ AD₅₀ was about 475 Μm. Apoptosis could also be induced byin situ generation of H₂O₂ by the inhibition of catalase activity upon hydroxylamine treatment. Hydroxylamine acted synergistically with H₂O₂ with an AD₅₀ of 2.2 mM. DMSO, a free radical scavenger, inhibited H₂O₂-induced apoptosis thereby confirming the involvement of reactive oxygen species. Exposure of cells to UV radiation (312 nm) resulted in a dose-dependent induction of apoptosis. These results provide evidence on the novel use of insect cells as a model for oxidative stress-induced apoptosis.     

Pycnogenol enhances endothelial cell antioxidant defenses

Summary

Reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂), superoxide anion (O₂^?), and hydroxyl radical (OH^?) have been implicated in mediating various pathological events such as cancer, atherosclerosis, diabetes, ischemia, inflammatory diseases, and the aging process. The glutathione (GSH) redox cycle and antioxidant enzymes—superoxide dismutase (SOD) and catalase (CAT)—play an important role in scavenging ROS and preventing cell injury. Pycnogenol has been shown to protect endothelial cells against oxidant-induced injury. The present study determined the effects of pycnogenol on cellular metabolism of H₂O₂ and O₂^? and on glutathione-dependent and -independent antioxidant enzymes in bovine pulmonary artery endothelial cells (PAEC). Confluent monolayers of PAEC were incubated with pycnogenol, and oxidative stress was triggered by hypoxanthine and xanthine oxidase or H₂O₂. Pycnogenol caused a concentration-dependent enhancement of H₂O₂ and O₂^? clearance. It increased the intracellular GSH content and the activities of GSH peroxidase and GSH disulfide reductase. It also increased the activities of SOD and CAT. The results suggest that pycnogenol promotes a protective antioxidant state by upregulating important enzymatic and nonenzymatic oxidant scavenging systems.     

Increased polyamine biosynthesis enhances stress tolerance by preventing the accumulation of reactive oxygen species: T-DNA mutational analysis of Oryza sativa lysine decarboxylase-like protein 1

A highly oxidative stress-tolerant japonica rice line was isolated by T-DNA insertion mutation followed by screening in the presence of 50 mM H₂O₂. The T-DNA insertion was mapped to locus Os09g0547500, the gene product of which was annotated as lysine decarboxylase-like protein (GenBank accession No. AK062595). We termed this gene OsLDC-like 1, for Oryza sativa lysine decarboxylase-like 1. The insertion site was in the second exon and resulted in a 27 amino acid N-terminal deletion. Despite this defect in OsLDC-like 1, the mutant line exhibited enhanced accumulation of the polyamines (PAs) putrescine, spermidine, and spermine under conditions of oxidative stress. The generation of reactive oxygen species (ROS) in the mutant line was assessed by qRT-PCR analysis of NADPH oxidase (RbohD and RbohF), and by DCFH-DA staining. Cellular levels of ROS in osldc-like 1 leaves were significantly lower than those in the wild-type (WT) rice after exposure to oxidative, high salt and acid stresses. Exogenouslyapplied PAs such as spermidine and spermine significantly inhibited the stress-induced accumulation of ROS and cell damage in WT leaves. Additionally, the activities of ROS-detoxifying enzymes were increased in the homozygous mutant line in the presence or absence of H₂O₂. Thus, mutation of OsLDC-like 1 conferred an oxidative stress-tolerant phenotype. These results suggest that increased cellular PA levels have a physiological role in preventing stress-induced ROS and ethylene accumulation and the resultant cell damage.     

Index

Hydrogen peroxide (H₂O₂) can induce cell damage by generating reactive oxygen species (ROS), resulting in DNA damage and cell death. The aim of this study is to elucidate the protective effects of fisetin (3,7,3′,4′,-tetrahydroxy flavone) against H₂O₂-induced cell damage. Fisetin reduced the level of superoxide anion, hydroxyl radical in cell free system, and intracellular ROS generated by H₂O₂. Moreover, fisetin protected against H₂O₂-induced membrane lipid peroxidation, cellular DNA damage, and protein carbonylation, which are the primary cellular outcomes of H₂O₂ treatment. Furthermore, fisetin increased the level of reduced glutathione (GSH) and expression of glutamate-cysteine ligase catalytic subunit, which is decreased by H₂O₂. Conversely, a GSH inhibitor abolished the cytoprotective effect of fisetin against H₂O₂-induced cells damage. Taken together, our results suggest that fisetin protects against H₂O₂-induced cell damage by inhibiting ROS generation, thereby maintaining the protective role of the cellular GSH system.     

In vivo and in situ visualization of early physiological events induced by heavy metals in pea root meristem

Heavy metals (HMs) are toxic pollutants, which can negatively affect the physiological processes of plants; moreover, HMs can be present in the food chain endangering people’s health. The aim of this study was to investigate the early physiological events during HM exposure in the root tips of the food plant Pisum sativum L. Ten-day-old pea plants were treated with 100 μM CdCl₂ or CuSO₄, in nutrient solution for 48 h. We studied the rapid formation of different reactive oxygen species (hydrogen peroxide H₂O₂ and superoxide radical O₂^·−) and reactive nitrogen species (nitric oxide NO· and peroxynitrite ONOO⁻) together with membrane damage and cell death in the meristem cells of pea roots using in vivo and in situ microscopic methods. In our experimental system, copper and cadmium induced the formation of H₂O₂ and NO. Two hours of heavy metal treatments resulted in an increased O₂^·− formation; however, later the level of this reactive molecule dramatically decreased. We found that high levels of NO were needed for ONOO⁻ production under HM exposure. A fast loss of membrane integrity and decreased cell viability were detected in root tips of copper-treated plants. The effects of cadmium seemed to be slower compared to copper, but this non-essential metal also caused cell death. We concluded that viability decreased when NO and H₂O₂ levels were simultaneously high in the same tissues. Using the NO scavenger it was also evidenced that NO generation is essential for cell death induction under copper or cadmium stress.