Enhancing phytoremediative ability of Pisum sativum by EDTA application

The aim of our research was to demonstrate how the presence of EDTA affects resistance of pea plants to Pb and Pb-EDTA presence, and to show the effectivity of lead ions accumulation and translocation. It was determined that EDTA not only increased the amount of Pb taken up by plants but also Pb ion transport through the xylem and metal translocation from roots to stems and leaves. It can be seen in the presented research results that addition of the chelator with Pb limited metal phytotoxicity. We also demonstrated a significant effect of EDTA not only on Pb accumulation and metal transport to the aboveground parts but also on the profile and amount of thiol compounds: glutathione (GSH), homoglutathione (hGSH) or phytochelatins (PCs), synthesized by the plants. We observed a significant effect of the synthetic chelator on increasing the level of Pb accumulation in roots of plants treated with Pb including EDTA (0.5 and 1 mM). Pisum sativum plants treated only with 1 mM Pb(NO3)2 accumulated over 50 mg Pb x g(-1) dry wt during 4 days of cultivation. Whereas in roots of pea plants exposed to Pb+0.5 mM EDTA 35% more Pb was observed. When 1 mM EDTA was applied roots of pea accumulated over 67% more metal. The presence of EDTA also increased metal uptake and transport to the aboveground parts. In pea plants treated only with 1 mM lead nitrate less than 3 mg Pb x g(-1) dry wt was transported, whereas in P. sativum treated with Pb-EDTA doubled amount of Pb was observed in stems and leaves.     

Role of antioxidant defences in the species-specific response of isolated atria to menadione

In previous works we demonstrated that 2-methyl-1,4-naphthoquinone (menadione) causes a marked increase in the force of contraction of guinea pig and rat isolated atria. This inotropic effect was significantly higher in the guinea pig than in the rat and was strictly related to the amount of superoxide anion (O(2)(*-)), generated as a consequence of cardiac menadione metabolism through mitochondrial NADH-ubiquinone oxidoreductase. The present study was designed to further elucidate the basis of these quantitatively different positive inotropic responses. To this purpose, we measured O(2)(*-) and hydrogen peroxide (H(2)O(2)) produced by mitochondria isolated from guinea pig and rat hearts in the presence of 20 microM menadione. Moreover, we evaluated the menadione detoxification activity (DT-diaphorase) and the antioxidant defences of guinea pig and rat hearts, namely their GSH/GSSG content, Cu/Zn- and Mn-dependent superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx) activities. Our results indicate that DT-diaphorase activity and glutathione levels were similar in both animal species. By contrast, guinea pig mitochondria produced greater amounts of O(2)(*-) and H(2)O(2) than those of rat heart. This is probably due to both the higher Mn-SOD activity (2.93 +/- 0.02 vs. 1.95 +/- 0.06 units/mg protein; P < 0.05) and to the lower Gpx activity (10.09 +/- 0.30 vs. 32.67 +/- 1.02 units/mg protein; P < 0.001) of guinea pig mitochondria. A lower CAT activity was also observed in guinea pig mitochondria (2.40 +/- 0.80 vs. 6.13 +/- 0.20 units/mg protein; P < 0.01). Taken together, these data provide a rational explanation for the greater susceptibility of guinea pig heart to the toxic effect of menadione: because of the greater amount of O(2)(*-) generated by the quinone and the higher mitochondrial Mn-SOD activity, guinea pig heart is exposed to more elevated concentrations of H(2)O(2) that is less efficiently detoxified, because of lower Gpx and CAT levels of mitochondria.     

Effect of cadmium on antioxidant enzyme activities and lipid peroxidation in the gills of the clam Ruditapes decussatus

Metals are known to influence the oxidative status of marine organisms, and antioxidant enzymes have been often proposed as biomarkers of effect. The clam Ruditapes decussatus is a well-known metal bioindicator. In this species cadmium (Cd) induces metallothionein (MT) synthesis only after 7 days of exposure. Before MT synthesis is induced, the other mechanisms capable of handling the excess of Cd are unknown. In order to identify some of these mechanisms, variations in antioxidant systems (superoxide dismutase, catalase, selenium-dependent glutathione peroxidase and non-selenium-dependent glutathione peroxidase), malondialdehyde (MDA) and MT were studied in the gills of R. decussatus exposed to different Cd concentrations (4, 40 and 100 gl^-1) for 28 days. These parameters, together with total proteins and Cd concentrations, were measured in the gills of the clams over different periods of exposure. Results indicate that Cd accumulation increased linearly in the gills of R. decussatus with the increase in Cd concentration. This increase induces an imbalance in the oxygen metabolism during the first days of Cd exposure. An increase in cytosolic superoxide dismutase (SOD) activity and a decrease in mitochondrial SOD activity was observed at the same time as or after a decrease in cytosolic and mitochondrial catalase activity and of selenium-dependent and non-selenium-dependent glutathione peroxidase activity. After 14 days of exposure, Cd no longer affect these enzymes but there was elevation of other cellular activities, such as MDA and MT production. MT bound excess Cd present in the cell. These variations in these parameters suggest their potential use as biomarkers of effects such as oxidative stress resulting from Cd contamination in molluscs.     

含硒类球红细菌的研究

为了确定类球红细菌转硒培养的最佳条件 ,研究了无机硒的加入浓度、时间以及分批补料培养对菌体生长和转硒效率的影响。实验表明 ,无机硒的浓度低于 1× 10 -5mol/L时 ,对类球红细菌的生长基本没有影响 ,并能将6 3.9%的无机硒转化为有机硒。转硒的最佳时间是在接种后 12h左右 ,此时转硒效率最高。实验还表明 ,分批补料培养可以提高菌体浓度 ,可使转硒效率和绝对量增加。体内试验表明 ,用 5mL/kgbw和 10mL/kgbw剂量的含硒类球红细菌灌养小鼠 ,可以使其全血GSH Px酶活性提高 2 0 .9%和 2 5 .5 % ,使其血清丙二醛 (MDA)含量降低2 1.0 %和 2 3.2 %。     

Glutaredoxins catalyze the reduction of glutathione by dihydrolipoamide with high efficiency

Glutaredoxins (Grx) are small (approximately 12kDa) proteins which catalyze thiol disulfide oxidoreductions involving glutathione (GSH) and disulfides in proteins or small molecules. Here, we present data which demonstrate the ability of glutaredoxins to catalyze the reduction of oxidized glutathione (GSSG) by dihydrolipoamide (DHL), an important biological redox catalyst and synthetic antioxidant. We have designed a new assay method to quantify the rate of reduction of GSSG and other disulfides by reduced lipoamide and have tested a set of eight recombinant Grx from human, rat, yeast, and E. coli. Lipoamide dependent activity is highest with the large atypical E. coli Grx2 (k(cat)=3.235 min(-1)) and lowest for human mitochondrial Grx2a (k(cat)=96 min(-1)) covering a wider range than k(cat) for the standard reduction of hydroxyethyldisulfide (HED) by GSH (290-2.851 min(-1)). The lipoamide/HED activity ratio was highest for yeast Grx2 (1.25) and E. coli Grx2 and lowest for E. coli Grx1 (0.13). These results suggest a new role for Grxs as ancillary proteins that could shunt reducing equivalents from main catabolic pathways to recycling of GSSG via a lipoyl group, thus serving biochemical functions which involve GSH but without NAD(P)H consumption.     

Modulation of the levels of NMDA receptor subunit mRNA and the bindings of [3H]MK-801 in rat brain by chronic infusion of subtoxic dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 l/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [³H]MK-801 binding are involved in the MK-801-infused adult rats.     

Decavanadate is responsible for vanadate-induced two-dimensional crystals in sarcoplasmic reticulum

Two-dimensional protein crystals of the calcium pump protein of sarcoplasmic reticulum (SR) from fast skeletal muscle were induced using Na₃VO₃ as first described by Dux and Martonosi. These crystals exhibit repeat rows 11 nm apart which contain discrete units with 7 nm repeats. Four different methods of sample preparation for electron microscopy, i.e., negative staining, freezedrying, freeze-fracturing, and thin-sectioning electron microscopy, each give complimentary repeat units. The SR-membrane crystals exhibit surface structure by the freeze-drying technique and row-like structures on the normally smooth outer face of normal SR. The formation of the membrane crystals is dependent on the pH and concentration of the vanadate. Only conditions favoring the presence of decavanadate yield crystals. At low concentrations and neutral pH, decavanadate is unstable and with time converts to smaller oligomers and the monomer. The presence of membrane crystals was correlated with the life span of the decavanadate. Membrane crystals were obtained in the SR membrane from fast twitch muscle from light and heavy SR, referable to longitudinal and terminal cisternae as well as from reconstituted SR. Canine cardiac SR did not crystallize under these conditions.Abbreviations Tris (tris[hydroxymethyl])aminomethane - TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), 2-(2-hydroxy-1-bis[hydroxymethyl]ethyl)aminoethanesulfonic acid - SR sarcoplasmic reticulum - CPP calcium pump protein Dedicated to the memory of Prof. David E. Green, friend, mentor, and colleague.     

Adenine-nucleotide levels and metabolism-dependent membrane potential in cells of Nitellopsis obtusa Groves

The relationship between adenine-nucleotide levels and metabolism-dependent membrane potential was studied in cells of Nitellopsis obtusa. Effects of ADP and AMP in the presence of ATP on electrogenic pump activity were measured in the dark, using the continuous perfusion method. Both ADP and AMP acte as competitive inhibitors for ATP, the K_i value for either compound being about 0.4 mM. The role of ADP and AMP as regulating factors for the electrogenic pump was investigated under various metabolic conditions. Application of N₂ gas in the dark caused a significant membrane depolarization amounting to 90 mV, but cytoplasmic streaming and membrane excitability were not affected. Under anoxia, the ATP level decreased from 1.6 to 0.5 mM; ADP increased but only slightly, and AMP increased greatly. However, the time course of changes in the adenine nucleotides was not concurrent with that of the membrane-potential changes, thus, the adenine-nucleotide level changes cannot fully account for the N₂-elicited depolarization. Under light, although the membrane hyperpolarized, no significant changes in the adenine-nucleotide levels were observed. Therefore, the light-induced membrane hyperpolarization cannot be explained solely by changes in adenine-nucleotide levels.Abbreviations APW artificial pond water - E_m membrane potential - R_m membrane resistance     

Oxidative stress responses and shock proteins in the unicellular cyanobacterium Synechococcus R2 (PCC-7942)

Oxidative stress responses were tested in the unicellular cyanobacterium Synechococcus PCC 7942 (R2). Cells were exposed to hydrogen peroxide, cumene hydroperoxide and high light intensities. Activities of ascorbate peroxidase and catalase were correlated with the extent and time-course of oxidative stresses. Ascorbate peroxidase was found to be the major enzyme involved in the removal of hydrogen peroxide under the tested oxidative stresses. Catalase activity was inhibited in cells treated with high H₂O₂ concentrations, and was not induced under photo-oxidative stress. Regeneration of ascorbate in peroxide-treated cells was found to involve mainly monodehydroascorbate reductase and to a lesser extent dehydroascorbate reductase. The induction of the antioxidative enzymes was dependent on light and was inhibited by chloramphenicol. Peroxide treatment was found to induce the synthesis of eight proteins, four of which were also induced by heat shock.Abbreviations ASC ascorbate - DHA dehydroascorbate - MDA monodehydroascorbate - GSH reduced glutathione - GSSG oxidized glutathione - ASC Per ascorbate peroxidase - DHA red. dehydroascorbate reductase - MDA red. monodehydroascorbate reductase - GSSG red. glutathione reductase - HSP heat shock proteins - PSP peroxide shock proteins - C_m chloramphenicol