No phytochelatin (PC2 and PC3) detected in Salix viminalis

Phytochelatins (PCs) have been detected in a large range of plant species, but their role in heavy-metal tolerance is unclear. Various clones of Salix viminalis are differently tolerant to heavy metals, and the aim of this work was to investigate whether PCs were differently expressed in tolerant compared with sensitive clones. In a long-term study, five clones with high or low metal tolerance were cultivated 21 days without or with Cd (1 or 10 µ M ), Cu (0.3 or 7 µ M ), Ni (15 µ M ), Pb (7 µ M ) or Zn (10 or 100 µ M ). Controls were further used in a short-term study where Cd (1 µ M ) was added and samples were collected 0, 15 and 30 min, 1, 3 and 24 h after start of treatment. PCs were analysed on high performance liquid chromatography (HPLC) using two different methods: post-column derivatization using Ellmans reagent and pre-derivatization with monobromobimane. Thlaspi caerulescens treated with Cd was used as internal PC standard. No PCs could be detected in Salix with either of the two methods in any of the treatments: different clones, metals, concentrations, plant parts or treatment time. The 16 thiol peaks shown were the same in both control and treated plants. Both HPLC methods showed PC peaks when Thlaspi was used but these peaks could not be associated with any of the 16 peaks. The amino acid composition of the 16 peaks was not the expected composition of that of PCs. Thus, Salix viminalis have no detectable levels of PCs, which in turn are not involved in heavy metal tolerance in Salix .     

Validation of an in vitro cytotoxicity test for four heavy metals using cell lines derived from a green sea turtle (<Emphasis Type="Italic">Chelonia mydas</Emphasis>)

Ten cell lines established from juvenile green sea turtles were tested and evaluated for their cytotoxic responses to four heavy metals: cadmium (Cd), chromium (Cr), zinc (Zn), and copper (Cu). Following a 24-h exposure to these metal salts at selected concentrations, test cells were comparatively characterized by morphology, viability, and proliferation. Experimental results indicated that all these metal salts were cytotoxic to these turtle cell lines at varied concentrations. Calculated 10% and 50% inhibitory concentration (IC₁₀ and IC₅₀) values revealed that the cytotoxicities of Cd and Cr were significantly more potent than the other two metal salts (p < 0.01). Comparative analysis of IC₁₀ values in these ten cell lines showed that turtle lung cells (GT-LG) are the most sensitive cell line to Cd, Cr, Zn, and Cu. Among these turtle cell lines, turtle liver cells (GT-LV) are more tolerant than other cells to Cd, Cr, and Zn, while GT-EYE cells are more tolerant to Cu, as determined by IC₅₀ values. Overall, GT-LG represents the most sensitive cells to heavy metal contamination and may be used for initial environmental monitoring, while the highly tolerant nature of GT-LV and GT-EYE cells to the tested heavy metals suggest their potential use as an emergency last-resort indicator of potential metal-related adverse effect on human health.     

Heavy Metal Resistance and Accumulation Characteristics in Willows

The resistance of Salix to Cu, Cd, Ni, and Zn was investigated in hydroponic culture, with phytoextraction potential evaluated for Cu. Root elongation (indicative of resistance level) was significantly affected, with considerable variation between and within individual clones. Resistance appeared to be clone- or hybrid-specific, rather than species-specific. S. caprea clones (and hybrids) were among the most resistant, but a secondary S. caprea clone from a different provenance was much less tolerant. S. viminalis and S. triandra clones were the most sensitive. Highest resistance was found in response to Cd, while Cu and Ni were extremely toxic. A resistant S. caprea ecotype originating from a metalliferous mine spoil was identified using this technique. Copper concentration reached a maximum of 2000, 400, and 82 μg g^-1 (d.wt) in roots, wood, and foliage, respectively, after 1 month in hydroponic culture. The level of variation in the response of Salix to metals may cause difficulties in phytoremediation screening programs, but may be essential in providing genetic variation for selection of metal resistance traits, where the contaminant profile is heterogeneous, mixed, or subject to change. Clone selection for metal phytoextraction is feasible, but a longer field-scale study on metal-contaminated soils is needed before their role in phytoremediation can be confirmed.     

The role of phytochelatins in constitutive and adaptive heavy metal tolerances in hyperaccumulator and non-hyperaccumulator metallophytes

Using the gamma-glutamylcysteine synthetase inhibitor, L-buthionine-[S,R]-sulphoximine (BSO), the role for phytochelatins (PCs) was evaluated in Cu, Cd, Zn, As, Ni, and Co tolerance in non-metallicolous and metallicolous, hypertolerant populations of Silene vulgaris (Moench) Garcke, Thlaspi caerulescens J.&C. Presl., Holcus lanatus L., and Agrostis castellana Boiss. et Reuter. Based on plant-internal PC-thiol to metal molar ratios, the metals' tendency to induce PC accumulation decreased in the order As/Cd/Cu > Zn > Ni/Co, and was consistently higher in non-metallicolous plants than in hypertolerant ones, except for the case of As. The sensitivities to Cu, Zn, Ni, and Co were consistently unaffected by BSO treatment, both in non-metallicolous and hypertolerant plants, suggesting that PC-based sequestration is not essential for constitutive tolerance or hypertolerance to these metals. Cd sensitivity was considerably increased by BSO, though exclusively in plants lacking Cd hypertolerance, suggesting that adaptive cadmium hypertolerance is not dependent on PC-mediated sequestration. BSO dramatically increased As sensitivity, both in non-adapted and As-hypertolerant plants, showing that PC-based sequestration is essential for both normal constitutive tolerance and adaptive hypertolerance to this metalloid. The primary function of PC synthase in plants and algae remains elusive.     

Metal(loid)s and radionuclides cytotoxicity in Saccharomyces cerevisiae. Role of YCF1, glutathione and effect of buthionine sulfoximine

The presence of heavy metal(loid)s in soils and waters is an important issue with regards to human health. Taking into account speciation problems, in the first part of this report, we investigated under identical growth conditions, yeast tolerance to a set of 15 cytotoxic metal(loid)s and radionuclides. The yeast cadmium factor 1 (YCF1) is an ATP-Binding Cassette transporter mediating the glutathione detoxification of heavy metals. In the second part, metal(loid)s that could be handled by YCF1 and a possible re-localisation of the transporter after heavy metal exposure were evaluated. YCF1 and a C-terminal GFP fusion, YCF1-GFP, were overexpressed in wild-type and Deltaycf1 strains. Both forms were functional, conferring a tolerance to Cd, Sb, As, Pb, Hg but not to Ni, Zn, Cu, Ag, Se, Te, Cr, Sr, Tc, U. Confocal experiments demonstrated that during exposure to cytotoxic metals, the localisation of YCF1-GFP was restricted to the yeast vacuolar membrane. In the last part, the role of glutathione in this resistance mechanism to metal(loid)s was studied. In the presence of heavy metals, application of buthionine sulfoximine (BSO), a well-known inhibitor of gamma-glutamylcysteine synthetase, led to a decrease in the cytosolic pool of GSH and to a limitation of yeast growth. Surprisingly, BSO was able to phenocopy the deletion of gamma-glutamylcysteine synthetase after exposure to Cd but not to Sb or As. In the genetic context of gsh1 and gsh2 yeast mutants, the critical role of GSH for Cd, As, Sb and Hg tolerance was compared to that of wild-type and Deltaycf1.     

Phytoextraction of Risk Elements by Willow and Poplar Trees

To characterize the phytoextraction efficiency of two clones of willow trees (Salix x smithiana Willd., Salix rubens) and two clones of poplar trees (Populus nigra x maximowiczii, Populus nigra Wolterson) were planted in contaminated soil (0.4–2.0 mg Cd.kg^?1, 78–313 mg Zn.kg^?1, 21.3–118 mg Cu.kg^?1). Field experiment was carried out in Czech Republic. The study investigated their ability to accumulate heavy metals (Cd, Zn, and Cu) in harvestable plant parts. The poplars produced higher amount of biomass than willows. Both Salix clones accumulated higher amount of Cd, Zn and Cu in their biomass (maximum 6.8 mg Cd.kg^?1, 909 mg Zn.kg^?1, and 17.7 mg Cu.kg^?1) compared to Populus clones (maximum 2.06 mg Cd.kg^?1, 463 mg Zn.kg^?1, and 11.8 mg Cu.kg^?1). There were no significant differences between clones of individual species. BCs for Cd and Zn were greater than 1 (the highest in willow leaves). BCs values of Cu were very low. These results indicate that Salix is more suitable plant for phytoextraction of Cd and Zn than Populus. The Cu phytoextraction potential of Salix and Populus trees was not confirmed in this experiment due to low soil availability of this element.     

Zn,Cd, S and trace metal bioaccumulation in willow (Salix spp.) cultivars grown hydroponically

Willows (Salix spp.) can be used to phytoremediate soils contaminated by Zn and Cd under certain conditions. In this study, the ability of 14 Salix cultivars to concentrate Cd, Zn and S in leaves was measured in hydroponic culture with 10 and 200 µM Cd and Zn, respectively, in the nutrient medium. The cultivars showed a wide range of biomass yields, tolerance to metals, and foliar concentrations of Zn and Cd, with some cultivars accumulating up to 1000 mg kg^?1 Zn, 70 mg kg^?1 Cd and 10,000 mg kg^?1 S with only mild phytotoxicity symptoms attributable to excess Zn. Cultivars with higher foliar Zn concentrations tended to have higher foliar Cd concentrations as well, and competition between Zn and Cd for uptake was observed. Exposure of Salix cultivars to Cd and Zn did not affect foliar concentrations of secondary metabolites such as polyphenols, but trace metal concentrations in leaves were significantly reduced (Fe and Cu) or increased (Mn) by exposure to excess Zn and Cd. Sulfur-XANES spectroscopy showed foliar S to be predominantly in highly oxidized (sulfate plus sulfonate) and reduced (thiol) forms, with oxidized S more prevalent in willows with the highest total S content.     

Strategies of cadmium and zinc resistance in willow by regulation of net accumulation

This work was performed to find out if metal resistant clones of Salix viminalis L. are capable to achieve high resistance to the metals by regulating their net accumulation. Salix clones with low or high resistance in combination with low or high accumulation capacity of either Zn or Cd were cultivated from cuttings in nutrient solution. The investigation included leakage and uptake experiments using ⁶⁵Zn or ¹⁰⁹Cd and analysis of root cation exchange capacity (CEC). Some plants were pre-treated with unlabeled 0.5 μM Cd or 2.5 μM Zn 24 h prior to the experiments to induce possible tolerance mechanisms. To find out if the regulation was a metabolic process, experiments were also performed with 2,4-dinitrophenol (DNP). Clones with high resistance and low Cd accumulation had higher efflux of Cd compared to the other clones, in both untreated and Cd pre-treated plants. This indicates a constitutive property to lower Cd accumulation by high Cd leakage. Pre-treatment with 0.5 μM Cd diminished the Cd net uptake to a level near zero in all clones, likely to be due to decreased the Cd uptake. In contrast, resistant clones with high Cd accumulation had the highest root CEC, which may be used to bind up Cd in the free space. No clear regulation of Zn net uptake was found in Zn-resistant clones. Pre-treatment with Zn decreased the uptake of Zn into the free space in Zn-resistant clones. The resistant high-accumulating clones, however, showed the highest leakage of Zn in both untreated and pre-treated plants, a constitutive process not related to high accumulation. Neither the influx nor the efflux of Cd or Zn was affected by DNP indicating passive transport across the plasma membrane.     

Heavy metal stress and sulfate uptake in maize roots

ZmST1;1, a putative high-affinity sulfate transporter gene expressed in maize (Zea mays) roots, was functionally characterized and its expression patterns were analyzed in roots of plants exposed to different heavy metals (Cd, Zn, and Cu) interfering with thiol metabolism. The ZmST1;1 cDNA was expressed in the yeast (Saccharomyces cerevisiae) sulfate transporter mutant CP154-7A. Kinetic analysis of sulfate uptake isotherm, determined on complemented yeast cells, revealed that ZmST1;1 has a high affinity for sulfate (Km value of 14.6 +/- 0.4 microm). Cd, Zn, and Cu exposure increased both ZmST1;1 expression and root sulfate uptake capacity. The metal-induced sulfate uptakes were accompanied by deep alterations in both thiol metabolism and levels of compounds such as reduced glutathione (GSH), probably involved as signals in sulfate uptake modulation. Cd and Zn exposure strongly increased the level of nonprotein thiols of the roots, indicating the induction of additional sinks for reduced sulfur, but differently affected root GSH contents that decreased or increased following Cd or Zn stress, respectively. Moreover, during Cd stress a clear relation between the ZmST1;1 mRNA abundance increment and the entity of the GSH decrement was impossible to evince. Conversely, Cu stress did not affect nonprotein thiol levels, but resulted in a deep contraction of GSH pools. Our data suggest that during heavy metal stress sulfate uptake by roots may be controlled by both GSH-dependent or -independent signaling pathways. Finally, some evidence suggesting that root sulfate availability in Cd-stressed plants may limit GSH biosynthesis and thus Cd tolerance are discussed.     

Differential antioxidant enzyme and thiol responses of tolerant and non-tolerant clones of <Emphasis Type="Italic">Chloris barbata</Emphasis> to cadmium-stress

Tolerant and non-tolerant clones of Chloris barbata Sw. obtained, respectively, from an erstwhile mercury contaminated solid waste dump site near a chloralkali plant and a non-contaminated (control) site were subjected to cadmium-stress by growing the rooted cuttings in water containing CdSO₄, 13 and 130 μM. Differences between the two clones in their response to cadmium-stress were noted in root growth, and also with respect to certain biochemical parameters. Whereas catalase activity decreased and non protein-thiol levels increased in the non-tolerant clone, the level of protein-thiol alone increased significantly in the tolerant clone in response to cadmium-stress. No remarkable differences between the clones, however, were noted with respect to total soluble protein, peroxidase activity and lipid peroxidation. Remarkably the two clones responded differently to buthionine sulfoximine, an inhibitor of glutathione and/or phytochelatin synthesis, which inhibited root growth significantly in non-tolerant clone but not in the tolerant clone. Buthionine sulfoximine, nonetheless, could potentate cadmium toxicity in either of the clones, but more effectively in the tolerant clone. The high sensitivity of tolerant-clone to the combined treatment of BSO and Cd in the present study could, therefore, be attributed to the cumulative oxidative stress generated synergistically by BSO and Cd.     

Metal accumulation and response of antioxidant enzymes in seedlings and adult sunflower mutants with improved metal removal traits on a metal-contaminated soil

Sunflower mutant lines with an enhanced tolerance and metal accumulation capacity obtained by mutation breeding have been proposed for Zn, Cd and Cu removal from metal-contaminated soils in previous studies. However, soils contaminated with trace elements induce various biochemical alterations in plants leading to oxidative stress. There is a lack of knowledge concerning the metal accumulation and antioxidant responses during the growth and development of sunflowers. This study, therefore, aimed to characterise metal accumulation and possible metal detoxification mechanisms in young seedlings and adult sunflowers. Beside the inbred line, two mutant lines with an improved growth and enhanced metal uptake capacity on a metal contaminated soil were investigated in more detail.Sunflowers cultivated on a metal-contaminated soil in the greenhouse showed a decrease in shoot biomass and chlorophyll concentration in two different developmental stages. Adult sunflowers showed a lower sensitivity to metal toxicity than young seedlings, whereas mutant lines were more tolerant to metal stress than the control. Mutant lines also produced a higher amount of carotenoids on a metal-contaminated soil than on the control soil, indicating a possible protective mechanism of sunflower mutants against oxidative stress caused by Cd and excess Zn.Heavy metals primarily increased the activity of antioxidant enzymes involved in the ascorbate–glutathione cycle in sunflower leaves. Activity of dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR) and glutathione reductase (GR) was strongly increased in young seedlings exposed to heavy metals. The enzyme activities were even more pronounced in mutant lines. A significantly increased ascorbate peroxidase (APOX) activity in adult sunflowers exposed to heavy metals indicated an elevated use of ascorbate after a longer exposure to metal stress.An increased antioxidant level corresponded to a high Cd and Zn accumulation in young and adult sunflowers. Metal distribution, zinc translocation in particular, from the root into the shoot tissue obviously increased during sunflower growth and ripening. Altogether, these results suggest that sunflower plants, primarily the mutant lines, possess an efficient defence mechanism against oxidative stress caused by metal toxicity. A good tolerance of sunflowers toward heavy metals coupled with an increased metal accumulation capacity might contribute to an efficient removal of heavy metals from a polluted area.     

Variation in the uptake of Arsenic, Cadmium, Lead, and Zinc by different species of willows Salix spp. grown in contaminated soils

The experiment assessed the variability of in seven clones of willow plants of high biomass production (Salix smithiana S-218, Salix smithiana S-150, Salix viminalis S-519, Salix alba S-464, Salix ’Pyramidalis’ S-141, Salix dasyclados S-406, Salix rubens S-391). They were planted in a pots for three vegetation periods in three soils differing in the total content of risk elements. Comparing the calculated relative decrease of total metal contents in soils, the phytoextraction potential of willows was obtained for cadmium (Cd) and zinc (Zn), moderately contaminated Cambisol and uncontaminated Chernozem, where aboveground biomass removed about 30% Cd and 5% Zn of the total element content, respectively. The clones showed variability in removing Cd and Zn, depending on soil type and contamination level: S. smithiana (S-150) and S. rubens (S-391) demonstrated the highest phytoextraction effect for Cd and Zn. For lead (Pb) and arsenic (As), the ability to accumulate the aboveground biomass of willows was found to be negligible in both soils. The results confirmed that willow plants show promising results for several elements, mainly for mobile ones like cadmium and zinc in moderate levels of contamination. The differences in accumulation among the clones seemed to be affected more by the properties of clones, not by the soil element concentrations or soil properties. However, confirmation and verification of the results in field conditions as well as more detailed investigation of the mechanisms of cadmium uptake in rhizosphere of willow plants will be determined by further research.     

Zinc and copper effects on metal-tolerant and non-tolerant clones ofAgrostis tenuis (Poaceae)

The toxicity of various Cu and Zn concentrations, as well as their mutual influence upon tolerant and non-tolerant clones ofAgrostis tenuis originating from soils of different metal content has been studied. Specific resistance has been clearly established. Co-existence of toxic metals causes an increase in toxicity. Uptake of metals (Cu and Zn) obviously occurs rather independently, and the toxic activity of the one is not affected competitively by the presence of the other. Above normal concentrations of Cu and Zn negatively affect cell division and growth in roots of non-tolerant genotypes. There is less ability to store metals than in tolerant genotypes.     

Effect of plants on the bioavailability of metals and other chemical properties of biosolids in a column study

The effects of metal-accumulating plants (Salix x reichardtii and Populus balsamifera) on the chemical properties and dynamics of metals in biosolids were investigated using different techniques including diffusive gradients in thin films (DGT), sequential extraction procedures and partitioning coefficient (K(d)). Plants could effectively extract Cd, Ni, and Zn and decreased dissolved organic carbon (DOC). The presence of plants increased the potential bioavailability of these metals, as assessed by an increase in the ratio of metal measured by DGT and metals in the solution. The plants affected the Cd, Ni, and Zn pools (soluble/exchangeable; Fe/Mn oxide and organic matter bound) characterised by sequential extraction and K(d) but did not reduce the total metals in either substrate. However, plants had no effect on Cu, presumably because of the effective buffering of available Cu by organic matter in both solution and solid phases. A high density of plant roots was associated with increased leaching of metals.     

Heavy metals in rat liver cadmium binding protein.

The simultaneous determination of heavy metals in microsamples of chromatographically isolated cadmium-binding protein (Cd-BP) from rat liver was performed by neutron activation analysis. The results suggested that metals other than those already reported (Cd, Zn, Cu, and Hg) can bind the protein. These observations were confirmed by in vivo radiotracer experiments by injecting i.p. 21 labelled metal ions in cadmium-treated rats. Of the metals tested, 109Cd, 65Zn, 64Cu, 203Hg, 106Ag and 113Sn were found incorporated in the Cd-BP. The incorporation of 35S-cysteine, used as an indicator of Cd-BP biosynthesis, was increased in rats exposed to cadmium as compared to untreated animals. In order to establish the influence of other metal ions on the biosynthesis of Cd-BP and the incorporation of cadmium in the protein, in vivo experiments were carried out by i.p. injection of 109Cd and 35S-cysteine. In the presence of 42 metal ions no influence was observed on the incorporation of the two radioisotopes in the Cd-BP. These observations tend to support the hypothesis that cadmium can act as a highly specific inducer of Cd-BP and that this protein might be involved in the metabolism of several heavy metals.     

Root development and heavy metal phytoextraction efficiency: comparison of different plant species in the field

Heavy metal phytoextraction is a soil remediation technique which implies the optimal use of plants to remove contamination from soil. Plants must thus be tolerant to heavy metals, adapted to soil and climate characteristics and able to take up large amounts of heavy metals. Their roots must also fit the spatial distribution of pollution. Their different root systems allow plants to adapt to their environment and be more or less efficient in element uptake. To assess the impact of the root system on phytoextraction efficiency in the field, we have studied the uptake and root systems (root length and root size) of various high biomass plants (Brassica juncea, Nicotiana tabacum, Zea mays and Salix viminalis) and one hyperaccumulator (Thlaspi caerulescens) grown in a Zn, Cu and Cd contaminated soil and compared them with total heavy metal distribution in the soil. Changes from year to year have been studied for an annual (Zea mays) and a perennial plant (Salix viminalis) to assess the impact of the climate on root systems and the evolution of efficiency with time and growth. In spite of a small biomass, T. caerulescens was the most efficient plant for Cd and Zn removal because of very high concentrations in the shoots. The second most efficient were plants combining high metal concentrations and high biomass (willows for Cd and Zn and tobacco for Cu and Cd). A large cumulative root density/aboveground biomass ratio (L_A/B), together with a relative larger proportion of fine roots compared to other plants seemed to be additional favourable characteristics for increased heavy metal uptake by T. caerulescens. In general, for all plants correlations were found between L _A/B and heavy metal concentrations in shoots (r=0.758^***, r=0.594^***, r=0.798^*** (P<0.001) for Cd, Cu and Zn concentrations resp.). Differences between years were significant because of variations in climatic conditions for annual plants or because of growth for perennial plants. The plants exhibited also different root distributions along the soil profile: T. caerulescens had a shallow root system and was thus best suited for shallow contamination (0.2 m) whereas maize and willows were the most efficient in colonising the soil at depth and thus more applicable for deep contamination (0.7 m). In the field situation, no plant was able to fit the contamination properly due to heterogeneity in soil contamination. This points out to the importance and the difficulty of choosing plant species according to depth and heterogeneity of localisation of the pollution.     

Effects of dexamethasone on metallothionein induction by Zn, Cu, and Cd in Chang liver cells

Metallothioneins (MTs) were induced in Chang liver cells by the metals, Zn, Cu and Cd, and the glucocorticoid hormone, dexamethasone. When 116 microM Zn, 32 microM Cu and 18 microM Cd, and 10(-7) M dexamethasone, respectively, were administered for 9 h, MTs induced by each inducer in the cells reached maximum levels. The maximum accumulation of MT level induced by dexamethasone was the lowest of the four inducers investigated; the levels induced by Zn, Cu and Cd were 4.7, 1.2 and 1.5 times of that induced by dexamethasone. When dexamethasone was added to the cells together with the heavy metals (Zn, Cu and Cd), dexamethasone had an additive effect on the maximum MT accumulations induced by heavy metals as compared to when induction was conducted using one of heavy metals alone or by dexamethasone alone. However, dexamethasone did almost not effect the metal accumulations in the cells, although the maximum MT levels induced by heavy metal increased by dexamethasone. These results suggest that the process of MT induction by heavy metals and that by dexamethasone are independent of one another. When dexamethasone was added to the cells together with a high concentration of Cu (32 microM) induced the maximum MT accumulation, Cu transport into the cells decreased by 20-40% of that into non-treated cells, which was statistically significant.     

Effects of Metal Combinations on the Production of Phytochelatins and Glutathione by the Marine Diatom Phaeodactylum tricornutum

Copper, Cd and Zn can be found at elevated concentrations in contaminated estuarine and coastal waters and have potential toxic effects on phytoplankton species. In this study, the effects of these metals on the intracellular production of the polypeptides phytochelatin and glutathione by the marine diatom Phaeodactylum tricornutum were examined in laboratory cultures. Single additions of Cu and Cd (0.4 μM Cu² and 0.45 μM Cd²⁺) to the culture medium induced the production of short-chained phytochelatins ((γ-Glu-Cys)_n-Gly where n = 2–5), whereas a single addition of Zn (2.2 μM Zn²⁺) did not stimulate phytochelatin production. Combination of Zn with Cu resulted in a similar phytochelatin production compared with a single Cu addition. The simultaneous exposure to Zn and Cd led to an antagonistic effect on phytochelatin production, which was probably caused by metal competition for cellular binding sites. Glutathione concentrations were affected only upon exposure to Cd (85% increase) or the combination of Cd with Zn (65% decrease), relative to the control experiment. Ratios of phytochelatins to glutathione indicated a pronounced metal stress in response to exposures to Cu or Cd combined with Zn. This study indicates that variabilities in phytochelatin and glutathione production in the field can be explained in part by metal competition for cellular binding sites.     

A proposal for the metal geometry in yeast superoxide dismutase based on results from EXAFS spectroscopy

Extended x-ray absorption fine structure (EXAFS) spectra have been recorded at the Cu edge and Zn edge in native yeast superoxide dismutase and at the Cu edge and Cd edge in the yeast superoxide dismutase derivative, where Zn has been substituted with Cd. Two different metal ligand distances in the range 1.9-2.0 A and 2.3-2.4 are determined for the Cu and Zn metals. For Cd in the Zn site two different metal ligand distances about 2.2 A and 2.6 A, respectively, were found. The striking feature is the similarity between the amplitude and radii determined for both the Cu and Zn sites. The increased distances for Cd can be explained by the increased ionic radius of Cd relative to Cu and Zn. Based on these EXAFS results and other relevant knowledge about the metal geometries, we propose that histidine 61 (63) positioned between the Cu and Zn metals are in one subunit bound to Zn and in the other to Cu. This model explains the recently observed difference between the two metal sites in each subunit.     

Metal ion-catalysed hydrolysis of ampicillin in microbiological growth media

Anodic stripping voltammetry of bacterial growth medium containing copper(II) and ampicillin shows that Cu(II) is complexed by the antibiotic and that this complex decomposes to give Cu(II) complexes with ligands derived from ampicillin. At pH 7, substantial decomposition of ampicillin occurs over a few minutes, and even the very low levels of Cu(II) in Chelex-extracted medium are able effectively to catalyse the decomposition. The significance of this observation was shown during the screening of an Escherichia coli cosmid library for clones exhibiting increased resistance to Zn(II), Co(II) or Cd(II); the unexpected growth of the ampicillin-sensitive host E. coli strain on Luria-Bertani plates containing ampicillin and any of these metals was attributed to metal ion-catalysed decomposition of ampicillin. The instability of ampicillin (and other beta-lactam antibiotics) to metal ion-catalysed hydrolysis means that great care must be taken to ensure that such reactions do not occur in growth media. Furthermore, it is clear that double selection for resistance to ampicillin and metals such as Cu(II), Zn(II), Co(II) and Cd(II) is impossible.