Nitrate reductase is inhibited in leaves of Triticum aestivum treated with high levels of copper

Copper is a potent sulfhydryl reagent which can also catalyse the generation of active oxygen. Since nitrate reductase (EC 1.6.6.1) is an SH-enzyme sensitive to oxidative environments, the relations among copper, active oxygen species and nitrate reductase (NR) activity are of interest. Foliar segments of wheat ( Triticum aestivum cv. Oasis) were floated on CuSO₄ solutions (up to 250 μ M ) for 24 h under continuous light. Copper decreased NR activity before affecting active oxygen generation as estimated by changes in oxidative parameters, including malondialdehyde, K⁺ leakage and chlorophyll degradation. Cysteine and Na-benzoate counteracted this decrease, suggesting an oxidative damage of the enzyme in leaves exposed to high copper levels. Copper-induced NR inactivation was further studied in the partially purified enzyme. Preincubation with CuSO₄ inhibited NR. Copper inhibition was reversed by subsequent incubation with EDTA, indicating that the metal bonded to key -SH groups of the enzyme. In addition, an ^˙OH-generating system (composed of CuSO₄, ascorbate and H₂O₂) irreversibly decreased the activity of purified NR to a greater extent than copper alone. Our results show that copper affects nitrogen metabolism by diminishing NR activity, involving a direct effect on key SH-groups and an indirect effect via attack by active oxygen species induced by the metal.     

Calcium-Activated Neutral Protease Activity Is Decreased in PC12 Cells After Ethanol Exposure

Abstract: Calcium-activated neutral protease activity was determined in PC12 cells exposed to ethanol for 96 h using a fluorescence-based assay with N -succinyl-Leu-Tyr 7-amido-4-methylcoumarin as the substrate. Stimulated activity was measured at high (1,400 µ M ) or low (140 µ M ) Ca²⁺ concentrations in the presence of 20 µ M ionomycin. Kinetic parameters were derived by fitting a model relating fluorescence intensity to time: F_t = F _final*(1 − e ^{− k obs t} ). Cell extracts were subjected to nondenaturing gel electrophoresis and casein zymography with quantification of the activity of the two calpain isoforms. Exposure to ethanol significantly decreased whole cell calpain activity measured by k _obs beginning at 20 m M , to 27.8% of control at 1,400 µ M Ca²⁺ and 29.2% of control at 140 µ M Ca²⁺ in the presence of 20 µ M ionomycin. No changes in μ-calpain or m-calpain activities were found in cell extracts from cells exposed to 20 m M ethanol, whereas at 40 and 80 m M ethanol, significant decreases in both μ-calpain and m-calpain activities were discovered.     

Cell wall modifications of bean (Phaseolus vulgaris) cell suspensions during habituation and dehabituation to dichlobenil

Bean ( Phaseolus vulgaris L.) cell suspensions were adapted for growth in 12 µ M dichlobenil (2,6-dichlorobenzonitrile or DCB) by a stepwise increase in the concentration of the inhibitor in each subculture. Non-tolerant suspensions (I ₅₀  = 0.3 µ M ) gave rise to single cells or small clusters while tolerant cell suspensions (I ₅₀  = 30 µ M ) grown in DCB formed large clusters. The cells in these clusters were surrounded by a thick and irregular cell wall with a lamellate structure and lacking a differentiated middle lamella. Analysis of habituated cell walls by Fourier transform infrared spectroscopy and cell wall fractionation revealed: (1) a reduced amount of cellulose and hemicelluloses, mainly xyloglucan (2) qualitative and quantitative differences in pectin levels, and (3) a non-crystalline and soluble β-1,4-glucan. When tolerant cells were returned to medium lacking DCB, the size of the cell clusters was reduced; the middle lamella was only partly formed, and the composition of the cell wall gradually reverted to that obtained with non-tolerant cells. However, dehabituated cells (I ₅₀  = 12 µ M ) were 40-fold more tolerant to DCB than non-tolerant cells and were only 2.5-fold more sensitive than tolerant cells.     

Effect of copper deficiency on photosynthetic electron transport in wheat plants

Copper deficiency in wheat ( Triticum aestivum L. cv. Nazareno Stramppeli) markedly affects photosynthetic activity. Flag leaves of copper-deficient plants showed a 50% reduction of the photosynthetic rate expressed as mg CO₂ dm⁻²h⁻¹. The activities of PSI and PSII, determined for isolated chloroplasts, as well as fluorescence measurements on intact leaves of copper-deficient plants, indicated a low activity of photosynthetic electron transport. Ribulose bisphosphate carboxylase/oxygenase (Rubisco) activity was not affected by copper deficiency but copper deficiency affected the chloroplast ultrastructure, especially at the level of grana, where a disorganization of thylakoids is evident.     

The photosynthetic potential of canola embryos

Canola embryos are green during development, but the role of embryo chloroplasts is not known. The objective of this study was to characterize the structure and function of embryo chloroplasts. Observations obtained by SDS-PAGE and transmission electron microscopy showed that thylakoids from embryo chloroplasts contained the same chlorophyll-protein complexes, but exhibited a greater proportion of granal stacking, compared with thylakoids from leaf chloroplasts. When assayed using an oxygen electrode, photosynthetic electron transport and respiration were enhanced in canola embryos bathed in concentrations of sucrose below 0.47 M. Photosynthesis, measured as the rate of incorporation of ¹⁴CO₂, was much lower in embryos than leaves even though significant electron transport was detected. These results indicate that the primary role of chloroplasts in embryos is not to photoassimilate CO₂. Instead, canola embryos are photoheterotrophic and may use the light reactions to generate the ATP and NADPH required to fuel the conversion of maternally supplied sucrose to the fatty acids used in oil synthesis and storage. These results led us to propose that the current model of plastid differentiation be modified to include the development of photoheterotrophic chloroplasts in sink tissues. This pattern of plastid differentiation can then be used to explain patterns of embryo development.     

Gas exchange in intact isolated chloroplasts from Chlamydomonas reinhardtii during starch degradation in the dark

During starch degradation in intact isolated chloroplasts from Chlamydomonas reinhardtii gas exchange was studied with a mass spectrometer. Oxygen uptake by intact chloroplasts in the dark never exceeded 1.5% of the starch degradation rate [maximum 15 nmol O₂ (mg Chl)⁻¹ h⁻¹ consumed. 1 000 nmol glucose (mg Chl)⁻¹h⁻¹ degraded]. Evolution of CO₂ under aerobic conditions [9.8–28 nmol (mg Chl)⁻¹ h⁻¹] was stimulated by addition of 0.1–0.5 m M oxaloacetate [393–425 nmol CO₂ (mg Chl)⁻¹ h⁻¹]. Pyridoxal phosphate (5 m M ) inhibited starch degradation by more than 80%, but had no effect on O₂ uptake. Starch degradation rates and CO₂ evolution did not differ under acrobic and anaerobic conditions. Increasing P_i in the reaction medium from 0.5 m M to 5.0 m M stimulated starch degradation by 230 and 260% under aerobic and anaerobic conditions, respectively. A rapid autooxidation of reduced ferredoxin was observed in a reconstituted system consisting of purified Chlamydomonas ferredoxin, purified Chlamydomonas NADP-ferredoxin oxidoreductase (EC 1.6.7.1) and NADPH. Addition of isolated thylakoids from C. reinhardtii did not affect the rate of O₂ uptake. Our results clearly indicate the absence of any oxygen requirement during starch degradation in isolated chloroplasts.     

The competition between methyl viologen and monodehydroascorbate radical as electron acceptors in spinach thylakoids and intact chloroplasts

In spinach thylakoids prepared from intact chloroplasts by shocking in the presence of ascorbate to preserve the operation of ascorbate peroxidase, the rate of oxygen uptake with methyl viologen as acceptor decreased in response to the addition of H₂O₂. Such a decrease was not observed in the presence of KCN or when the thylakoids lost ascorbate peroxidase activity. Illumination of intact chloroplasts in the presence of H₂O₂ and methyl viologen showed an initial rate of oxygen exchange, which is intermediate between the initial rate of oxygen evolution in the presence of H₂O₂ alone and steady-state oxygen uptake in the presence of methyl viologen. The data showed that monodehydroascorbate radical generated in ascorbate peroxidase reaction could compete with methyl viologen for electrons supplied by the electron transport chain in both thylakoids and intact chloroplasts. During the illumination of intact chloroplasts the rate of oxygen uptake increased. The presence of nigericin swiftly led to steady-state oxygen uptake, and to a clear-cut 1:1 relationship between the electron transport rate estimated from fluorescence assay and the electron transport rate determined from oxygen uptake, taking the stoichiometry 1O₂:4e. The increase in oxygen uptake was attributed to the cessation of monodehydroascorbate radical generation brought about by consumption of intrachloroplast ascorbate in the peroxidase reactions, and the effects of nigericin were explained by acceleration of such consumption. The competition between methyl viologen and monodehydroascorbate radical in the intact chloroplasts was estimated under various conditions.     

Chloroplast ultrastructure in two Crassulacean species and an F1 hybrid with differing biomass δ13C values

Abstract. The chloroplasts of two species of the Crassulaceae and their F₁ hybrid were compared by electron microscopy. The two species had contrasting leaf tissue δ¹³C values of −25°/_∞ ( Sedum greggii ) and −13°/_∞ ( Cremnophila linguifolia ), and the F₁ hybrid had a value of − 18°/_∞. S. greggii had a mean of 8.9 thylakoids per granum in contrast to C. linguifolia which had a mean of only 3.8 thylakoids per granum. The F₁ hybrid had a mean of 6.4 thylakoids per granum. Crystaloids were observed in S. greggii and the hybrid but not in C. linguifolia     

Dopamine Neurotoxicity: Inhibition of Mitochondrial Respiration

Abstract: Dopamine, due to metabolism by monoamine oxidase or autoxidation, can generate toxic products such as hydrogen peroxide, oxygen-derived radicals, semiquinones, and quinones and thus exert its neurotoxic effects. Intracerebroventricular injection of dopamine into rats pretreated with the monoamine oxidase nonselective inhibitor pargyline caused mortality in a dose-dependent manner with LD₅₀ = 90 µg. Norepinephrine was less effective with LD₅₀ = 141 µg. The iron chelator desferrioxamine completely protected against dopamine-induced mortality. In the absence of pargyline more rats survived, indicating that the products of dopamine enzymatic metabolism are not the main contributors to dopamine-induced toxicity. Biochemical analysis of frontal cortex and striatum from rats that received a lethal dose of dopamine did not show any difference from control rats in lipid and protein peroxidation and glutathione reductase and peroxidase activities. Moreover, dopamine significantly reduced the formation of iron-induced malondialdehyde in vitro, thus suggesting that earlier events in cell damage are involved in dopamine toxicity. Indeed, dopamine inhibited mitochondrial NADH dehydrogenase activity with IC₅₀ = 8 µ M , and that of norepinephrine was twice as much (IC₅₀ = 15 µ M ). Dopamine-induced inhibition of NADH dehydrogenase activity was only partially reversed by desferrioxamine, which had no effect on norepinephrine-induced inhibition. These results suggest that catecholamines can cause toxicity not only by inducing an oxidative stress state but also possibly through direct interaction with the mitochondrial electron transport system. The latter was further supported by the ability of ADP to reverse dopamine-induced inhibition of NADH dehydrogenase activity in a dose-dependent manner.     

Reversal by light of deleterious effects of chilling on oxygen evolution, manganese and free fatty acid content in tomato thylakoids is not accompanied by restoration of the original membrane conformation

Free fatty acids (FFA) generated in thylakoids upon chilling of tomato leaves at 0°C for a few days result in release of functionally active Mn and inactivation of O₂ evolution. Chilling does not lead to a decrease in the extrinsic 16, 23 and 33 kDa polypeptides. Upon illumination of chilled leaves both Mn content and O₂ evolution in thylakoids are restored and FFA content is reduced to the level of the control. Photoactivation of O₂ evolution in chilled leaves does not change the ratio of unsaturated/saturated FFA. Constant Arrhenius activation energy (Ea) for O₂ evolution by thylakoids isolated from control leaves was found, whereas it increased at temperatures below 8.0 and 10.5°C in thylakoids from cold-treated and photoactivated leaves, respectively. This indicates that restoration of O₂ evolution as well as of FFA and Mn contents is not accompanied by a complete reversal of membrance conformation.     

Murine Glial Cells Regenerate NAD, After Peroxide-Induced Depletion, Using Either Nicotinic Acid, Nicotinamide, or Quinolinic Acid as Substrates

Abstract: The potential for regeneration of intracellular pyridine nucleotide levels from different precursors, after peroxide-induced NAD depletion, in cultured glial cells was investigated. Cultured murine glial cells showed a decrease in intracellular NAD levels of >40% after treatment with H₂O₂ (100 µ M ). Removal of the H₂O₂ followed by a 2-h incubation did not result in NAD recovery in the absence of precursors. However, NAD levels increased significantly in these cells after the following substrate additions, at minimum effective concentrations of 1 m M for quinolinic acid (QUIN), 500 µ M for nicotinamide, and 2 µ M for nicotinic acid. The regeneration of significant amounts of NAD from nicotinic acid at doses 250 and 500 times lower than either nicotinamide or QUIN indicates a preferred route for NAD biosynthesis in glial cells in vitro, probably via nicotinic acid phosphoribosylation.     

A Subtype of κ-Opioid Receptor Mediates Inhibition of High-Affinity GTPase Inherent in Gi1 in Guinea Pig Cerebellar Membranes

Abstract: The κ-opioid receptor agonists including U-50,488H and dynorphin A (1–17) in ranges of 0.1–100 n M inhibited the hydrolysis of GTP to GDP (P_i release) inherent in GTP-binding proteins (G proteins) in guinea pig cerebellar membranes. U-50,488H inhibited only high-affinity GTPase activity, not low-affinity activity. The action of this agonist was found to be biphasic, and there was no inhibition at concentrations >1 µ M . The inhibition was abolished by pretreatment with preactivated pertussis toxin (PTX) at concentrations >1 µg/ml but not with preactivated cholera toxin (30 µg/ml). Similar blockade of κ-receptor-mediated inhibition was also observed when membranes were pretreated with a low concentration (8 µ M ) of N -ethylmaleimide (NEM) at low temperature (4°C), which alkylates the cysteine residue to be ADP-ribosylated by PTX; but this treatment caused no significant change in κ-agonist binding. When purified G_i1, but not G_o, was reconstituted into membranes pretreated with NEM, the κ-receptor-mediated inhibition was recovered. These findings suggest that a subtype of κ-opioid receptor is coupled to inhibition of intrinsic activity of G_i1.     

Ultrastructural and Morphological Characteristics of Cultivated Wheat Growing on Copper-Polluted Fields

A cultivated Greek variety of wheat (Triticum aestivum L. cv. Vergina) growing in fields naturally polluted by outcrops of copper ores was investigated. Wheat plants show a negative response to increasing quantities of soil copper, including reduced growth and chlorosis. Copper toxicity was demonstrated in the laboratory by a rooting test; the frequency of mitoses declines sharply with increasing copper concentration in the nutrient solution. The mesophyll cells of polluted plants display a circular shape (in transverse sections) with a few chloroplasts parietally distributed, in contrast with the elongate or pleomorphic shape of control leaves that contain numerous chloroplasts crowded at the cell periphery. Ultrastructurally, the chloroplasts of polluted plants contain a poorly developed internal membrane system consisting of thylakoids arranged parallel to each other with only a few, rudimentary grana. In addition, a number of statistically significant differences were found, including the number of starch grains and plastoglobuli, chloroplast surface area, volume fraction of starch grains and, most important, the volume fraction of the internal membrane system. All ultrastructural changes are attributed to the toxic effect of high concentrations of soil copper.     

Distinct responses to copper stress in the halophyte Mesembryanthemum crystallinum

Selective gene expression allows the halophyte Mesembryanthemum crystallinum to survive a salt stress. To broaden our understanding of the environmental cues initiating diverse stress responses in this higher plant, unstressed and 0.4 M NaCl‐stressed plants were compared to plants treated with several concentrations of copper (CuSO₄), an increasingly relevant environmental heavy metal pollutant. Comparisons of control and copper‐stressed plants included germination, chlorophyll content, accumulation of proline, heat shock protein (HSP) 60 and a Crassulacean acid metabolism (CAM)‐specific marker enzyme, phospho enol pyruvate carboxylase (PEPCase). In germination and whole plant tests, M. crystallinum was significantly more tolerant to copper than Arabidopsis thaliana. Mature M. crystallinum plants stressed with 50 ppm CuSO₄ for 48 h became dehydrated. These plants produced a 4‐fold increase in proline concentration and accumulated both the CAM‐specific PEPCase and HSP 60 compared to controls. Higher levels of copper stress resulted in a 10‐fold increase in leaf proline content, 10‐fold HSP 60 accumulation but no detectable PEPCase protein compared to unstressed controls. HSP 60 did not accumulate under NaCl stress. Concurrent with copper‐induced genetic responses to stress, copper was accumulated and concentrated in leaves (3 500 ppm). Together, these results suggest that this halophyte copes with copper metal exposure through distinct genetic mechanisms.     

NaCl treatment markedly enhances H2O2-scavenging system in leaves of halophyte Suaeda salsa

The C₃ halophyte Suaeda salsa L. grown under the high concentration of NaCl (200 m M ) was used to investigate the role of the hydrogen peroxide (H₂O₂)-scavenging system [catalase, ascorbate peroxidase, glutathione reductase (GR), ascorbic acid, and glutathione (GSH)] in removal of reactive oxygen species. The activity of catalase (CAT, EC 1.11.1.6), ascorbate peroxidase (APX, EC 1.11.1.11), and GR (EC 1.6.4.2) increased significantly after 7 days of NaCl treatment. The isoform patterns of CAT and GR were not affected, but the staining intensities were significantly increased by NaCl treatment. Activities of both the thylakoid-bound APX or GR and stromal APX (S-APX) or GR in the chloroplasts were markedly enhanced under high salinity. Fifty percent of APX in the chloroplasts is thylakoid-bound APX. S-APX and GR activity represented about 74–78 and 64–71% of the total soluble leaf APX and GR activity, respectively. Salt treatment increased the contents of ascorbic acid and GSH. By contrast, a decreased content of H₂O₂ was found in the leaves of NaCl-treated S . salsa . The level of membrane lipid peroxidation decreased slightly after NaCl treatment. The plants grew well with high rate of net photosynthesis under high salinity. These data suggest that upregulation of the H₂O₂-scavenging system in plant cells, especially in the chloroplasts, is at least one component of the tolerance adaptations of halophytes to high salinity.     

Isolation and Characterization of a Cytosolic Phospholipase A2 from Bovine Adrenal Medulla

Abstract: We have recently demonstrated that bovine adrenal medulla contains a soluble phospholipase A₂ (PLA₂), which is localized in the cytosol. In the present study, this PLA₂ was purified 1,097-fold using sequential concanavalin A, hydrophobic interaction, anion exchange, gel filtration, and an additional anion exchange chromatography. The enzyme is activated over the range of 20–1,000 µ M Ca²⁺ and has a pH optimum near 8.0. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a molecular mass of 26 kDa and an isoelectric point of 4.6 as revealed by isoelectric focusing. The cytosolic PLA₂ is not inhibited by NaCl, and the enzymatic activity is stimulated at low concentrations of Triton X-100 (0.01%) and deoxycholate (1 m M ) but inhibited at higher concentrations (0.1% and 3 m M , respectively) of these detergents. Furthermore, heat treatment (57°C, 5 min) reduced the enzymatic activity by 80%, whereas glycerol (30%) increased the activity. p -Bromophenacylbromide, a frequently used irreversible inhibitor of type II PLA₂, has little effect until 100 µ M , and 2–10 m M dithiothreitol totally inactivated the enzyme. The purified PLA₂ displays a preference for phosphatidylcholine as a substrate but hydrolyzes phospholipid substrates with arachidonic acid or linoleic acid esterified at the sn -2 position to the same extent. It is concluded that the chromaffin cell cytosolic PLA₂, which was isolated and characterized in this study, represents a type of PLA₂ that has not been formerly reported in chromaffin cells. Additional research on the chromaffin cell cytosolic PLA₂ will help to reveal whether the enzyme is important for exocytosis.     

Cadmium accumulation in the chloroplast of Euglena gracilis

Intracellular distribution of Cd, cysteine, glutathione, and Cd-induced thiol peptides in Euglena gracilis cultured under photoheterotrophic conditions was studied. After 3 days of culture with 0.2 m M CdCl₂, 62% of the Cd accumulated by cells was equally distributed between the cytosolic and chloroplastic fractions. However, after 8 days, metal content increased in the crude chloroplastic fraction to 40% of total and decreased to 19% in the cytosol; in Percoll-purified chloroplasts the estimated content of Cd raised to 62%. Accumulation of Cd in chloroplasts could be mediated by a transporter of free Cd²⁺, since uptake of added CdCl₂ in isolated chloroplasts exhibited a hyperbolic type of kinetics with a K_m of 57 µ M and V_max of 3.7 nmol (mg protein)⁻¹ min⁻¹. The contents of cysteine and glutathione markedly increased in both chloroplasts (7–19 times) and cytosol (4–9 times) by exposure to Cd²⁺, although they were always higher in the cytosol. Thiol-containing peptides induced by Cd were mainly located in the cytosol after 3 days, and in the chloroplasts after 8 days of culture. The data suggested that Cd was compartmentalized into chloroplasts in a process that may involve the transport of free Cd and the participation of thiol-peptides.     

Effects of 1-Methyl-4-Phenylpyridinium on Isolated Rat Brain Mitochondria: Evidence for a Primary Involvement of Energy Depletion

Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP⁺) on the oxygen consumption, ATP production, H₂O₂ production, and mitochondrial NADH-CoQ₁ reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µ M MPP⁺ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP⁺ produced a concentration-dependent decrease in H₂O₂ production. Incubation of mitochondria with ADP for 60 min after loading with 100 µ M MPP⁺ caused no loss of complex I activity after washing of MPP⁺ from the mitochondrial membranes. These data are consistent with MPP⁺ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H₂O₂ by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H₂O₂ in the presence of Cu²⁺ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP⁺ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.     

Heterotrophic tobacco cell cultures during greening

Structural changes in cells and plastids are described that occur during the greening of an initially dark-grown cell suspension of tobacco ( Nicotiana tabacum L. cv. Xanthi). The pattern of cell growth during greening, expressed in dry weight or cell number, showed a classical sigmoid curve with a lag phase (T₀–T₂), an exponential phase (T₃–T₉) and a stationary phase (T₁₀–T₂₁). Achlorophyllous vacuolated cells (T₀), obtained after 3 culture cycles in the dark, contained amyloplasts devoid of lamellae. Exposure to light brought about an enrichment in cytoplasm and an amyloplast to proplastid transformation (starch loss) accompanied by chlorophyll synthesis. By T₃, many cells appeared meristematic and contained dividing proplastids with rudimentary single lamellae typical of those in intact meristematic leaf cells. As cell division occurred (T₃ to T₉), plastids replicated and their internal membrane system developed progressively into defined grana-intergrana thylakoids. By the stationary phase of cell growth (T₁₄), the lamellar system had reached a highly structured grana-intergrana network typical of higher plant chloroplasts. We have emphasized the analogies between the sequence of events (proplastid to chloroplast transition) during the greening of tobacco cells and that in developing intact leaves; in this respect the cell cultures provide a useful material for studies dealing with the biogenesis of structural or physiological events.