Selective,quantitative detection of cadmium and/or zinc by use of BTAN (2-aminobenzothiazolylazo-beta-naphthol)

Summary BTAN (Sumi Y et al. (1982) Histochemistry 73:481) was investigated as a histochemical Cd/Zn chelator. Cd-BTAN exhibits a main peak about 635 nm, while Zn-BTAN exhibits a main peak about 644 nm. The isobestic wavelength for Cd-BTAN and Zn-BTAN is 638 nm. The microscopical detection limit for Cd is about 25 amol/m², and for Zn about 5 amol/m². The relation between metal and bound chelator is fairly linear at a BTAN concentration more than 10-fold the metal concentration. Histochemical localization was fair to good, with a crystal size of up to 0.2–0.3 m. The chelate was unaffected by hydrophilic and largely also by hydrophobic mounting media. The original staining procedure proved erratic and was modified. Posttreatment with oxine to selectively demonstrate Cd in the presence of Zn (Sumi Y et al. 1982) seriously reduced the staining intensity. Post-treatment for 8–15 min with HCl, 0.5 mol/l, in 50% ethanol removed Cd-BTAN completely with little reduction of Zn staining intensity, even from sites with 5× as much Cd as Zn. It is concluded that BTAN permits direct quantitative detection of (Zn+Cd). Provided certain precautions are taken quantitative detection of Zn and quantitation of Cd in mixed Zn/Cd sites is possible by microphotometry of the stained section before and after differentiation for 8–15 min with the HCl/50% ethanol medium.     

Allosteric negative regulation of smt O/P binding of the zinc sensor,SmtB, by metal ions: a coupled equilibrium analysis

The Synechococcus PCC 7942 smt operon is responsible for cellular resistance to excess zinc and consists of two divergently transcribed genes, smtB and smtA. SmtB is the Zn(II)-sensing metal-regulated repressor of the system and binds to a 12-2-12 imperfect inverted repeat in the smtA O/P region. Using fluorescence anisotropy to monitor SmtB-smt O/P multiple equilibria, we show that four SmtB homodimers bind to a 40 bp oligonucleotide containing a single 12-2-12 inverted repeat. The binding affinities of the first two dimers are very tight (K(int) = 2.9 x 10(9) M(-1)) with the affinities of the third and fourth dimers lower by approximately 10- and approximately 30-fold, respectively. A single monomer equivalent of Zn(II), Cd(II), or Co(II) promotes disassembly of the oligomeric complex to a mixture of (P(2)).D and (P(2))(2).D SmtB dimer-DNA complexes with the intrinsic affinity of all SmtB homodimers for DNA greatly reduced by approximately 500-2000-fold. Substitution or derivatization of cysteines which comprise the alpha3N metal binding site (Cys14 and Cys61) [VanZile, M. L., et al. (2002) Biochemistry 41, 9765-9775] has no effect on allosteric negative regulation by Zn(II); in contrast, H106Q SmtB, harboring a single zinc-liganding substitution in the alpha5 metal binding site, is refractory to zinc-induced disassembly of SmtB-DNA complexes. The alpha5 metal binding sites are therefore regulatory for Zn(II) sensing in vitro and in vivo, while the high-affinity alpha3N sites play some other role. This finding for SmtB is the opposite of that previously determined for Staphylococcus aureus pI258 CadC, a Pb(II)/Cd(II)/Bi(III) sensor [Busenlehner, L. S., et al. (2002) J. Mol. Biol. 319, 685-701], thus providing insight into the origin of functional metal ion selectivity in this family of metal sensor proteins.     

2,2-dipyridyl binding to metal substituted horse liver alcohol dehydrogenase

The binding of 2,2-dipyridyl to metal substituted horse liver alcohol dehydrogenase was measured by spectrophotometric titrations. Large changes in the visible absorption spectra were seen for the Co2+, Cu2+ and Ni2+ hybrids upon coordination of 2,2-dipyridyl, due to a change in coordination number. The formation constants for binding to the Co2+ and Cd2+ hybrids are of the order 10(6) M-1, which means that these hybrids have a 500-fold higher affinity for 2,2-dipyridyl than the native Zn2+ enzyme. 2,2-dipyridyl has a 100-fold higher affinity for enzyme bound Cd2+ than for aqueous Cd2+ ions, while for Cu2+ and Zn2+ the opposite is the case. None of the substituted metal ions were removed from the active site during titration with the chelator 2,2-dipyridyl.     

QTL analysis of cadmium and zinc accumulation in the heavy metal hyperaccumulator Thlaspi caerulescens

Thlaspi caerulescens (Tc; 2n = 14) is a natural Zn, Cd and Ni hyperaccumulator species belonging to the Brassicaceae family. It shares 88% DNA identity in the coding regions with Arabidopsis thaliana (At) (Rigola et al. 2006). Although the physiology of heavy metal (hyper)accumulation has been intensively studied, the molecular genetics are still largely unexplored. We address this topic by constructing a genetic map based on AFLP markers and expressed sequence tags (ESTs). To establish a genetic map, an F(2) population of 129 individuals was generated from a cross between a plant from a Pb/Cd/Zn-contaminated site near La Calamine, Belgium, and a plant from a comparable site near Ganges (GA), France. These two accessions show different degrees of Zn and, particularly, Cd accumulation. We analyzed 181 AFLP markers (of which 4 co-dominant) and 13 co-dominant EST sequences-based markers and mapped them to seven linkage groups (LGs), presumably corresponding to the seven chromosomes of T. caerulescens. The total length of the genetic map is 496 cM with an average density of one marker every 2.5 cM. This map was used for Quantitative Trait Locus (QTL) mapping in the F(2). For Zn as well as Cd concentration in root we mapped two QTLs. Three QTLs and one QTL were mapped for Zn and Cd concentration in shoot, respectively. These QTLs explain 23.8-60.4% of the total variance of the traits measured. We found only one common locus (LG6) for Zn and Cd (concentration in root) and one common locus for shoot and root concentrations of Zn (LG1) and of Cd (LG3). For all QTLs, the GA allele increased the trait value except for two QTLs for Zn accumulation in shoot (LG1 and LG4) and one for Zn concentration in root (LG1).     

Products of metal exchange reactions of metallothionein

Hepatic metallothionein (MT) isolated from Cd-exposed animals always contains Zn (2-3 mol/mol of protein) in addition to Cd (4-5 mol/mol of protein), and the two metals are distributed in a nonuniform, but reproducible, manner among the seven binding sites of the protein's two metal-thiolate clusters. Different methodologies of preparing rabbit liver Cd, Zn-MT in vitro were investigated to provide insight into why such a distinct mixture of mixed-metal clusters is produced in vivo and by what mechanism they form. 113Cd NMR spectra of the products of stepwise displacement of Zn2+ from Zn7-MT by 113Cd2+ show that Cd binding to the clusters is not cooperative (i.e., clusters containing exclusively Cd are not formed in preference to mixed-metal Cd, Zn clusters), there is no selective occupancy of one cluster before the other, and many clusters are produced with a nonnative metal distribution indicating that this pathway is probably not followed in vivo. In contrast, the surprising discovery was made that the native cluster compositions and their relative concentrations could be reproduced exactly by simply mixing together the appropriate amounts of Cd7-MT and Zn7-MT and allowing intermolecular metal exchange to occur. This heretofore unknown metal interchange reaction occurs readily, and the driving force appears to be the relative thermodynamic instability of three-metal clusters containing Cd. With this new insight into how Cd,Zn-MT is likely to be formed in vivo we are able for the first time to postulate rational explanations for previous observations regarding the response of hepatic Zn and metallothionein levels to Cd administration.     

Displacement of zinc and copper from copper-induced metallothionein by cadmium and by mercury: in vivo and ex vivo studies

The in vitro affinity of metals for metallothionein (MT) is Zn less than Cd less than Cu less than Hg. In a previous study Cd(II) and Hg(II) displaced Zn(II) from rat hepatic Zn7-MT in vivo and ex vivo (Day et al., 1984, Chem. Biol. Interact. 50, 159-174). The ability of Cd(II) or Hg(II) to displace Zn(II) and/or Cu(II) from metallothionein in copper-preinduced rat liver (Zn, Cu-MT) was assessed. Cd(II) and Hg(II) can displace zinc from (Zn, Cu)-MT both in vivo and ex vivo. The in vitro displacement of copper from MT by Hg(II) was not confirmed in vivo and ex vivo. Cd(II) treatment did not alter copper levels in (Zn, Cu)-MT, as expected. Hg(II) treatment, however, did not decrease copper levels in MT, but rather increased them. The sum of the copper increase and mercury incorporation into MT matched the zinc decrease under in vivo conditions and actually exceeded the zinc decrease under ex vivo conditions. Short-term exposure of rat liver to exogenous metals can result in incorporation of these metals into MT by displacement of zinc from pre-existing MT. Displacement of copper from pre-existing MT by mercury, as predicted by in vitro experiments, was not confirmed under the conditions of our in vivo and ex vivo experiments. This result is explainable based on the differing affinities and/or preferences of the two metal clusters in MT.     

NMR determination of the global structure of the 113Cd derivative of desulforedoxin: investigation of the hydrogen bonding pattern at the metal center.

Desulforedoxin (Dx) is a simple homodimeric protein isolated from Desulfovibrio gigas (Dg) containing a distorted rubredoxin-like center with one iron coordinated by four cysteinyl residues (7.9 kDa with 36 amino acids per monomer). In order to probe the geometry and the H-bonding at the active site of Dx, the protein was reconstituted with 113Cd and the solution structure determined using 2D NMR methods. The structure of this derivative was initially compared with the NMR solution structure of the Zn form (Goodfellow BJ et al., 1996, J Biol Inorg Chem 1:341-353). Backbone amide protons for G4, D5, G13, L11 NH, and the Q14 NH side-chain protons, H-bonded in the X-ray structure, were readily exchanged with solvent. Chemical shift differences observed for amide protons near the metal center confirm the H-bonding pattern seen in the X-ray model (Archer M et al., 1995, J Mol Biol 251:690-702) and also suggest that H-bond lengths may vary between the Fe, Zn, and 113Cd forms. The H-bonding pattern was further probed using a heteronuclear spin echo difference (HSED) experiment; the results confirm the presence of NH-S H-bonds inferred from D2O exchange data and observed in the NMR family of structures. The presence of "H-bond mediated" coupling in Dx indicates that the NH-S H-bonds at the metal center have significant covalent character. The HSED experiment also identified an intermonomer "through space" coupling for one of the L26 methyl groups, indicating its proximity to the 113Cd center in the opposing monomer. This is the first example of an intermonomer "through space" coupling. Initial structure calculations produced subsets of NMR families with the S of C28 pointing away from or toward the L26 methyl: only the subset with the C28 sulfur pointing toward the L26 methyl could result in a "through space" coupling. The HSED result was therefore included in the structure calculations. Comparison of the Fe, Zn, and 113Cd forms of Dx suggests that the geometry of the metal center and the global fold of the protein does not vary to any great extent, although the H-bond network varies slightly when Cd is introduced. The similarity between the H-bonding pattern seen at the metal center in Dx, Rd (including H-bonded and through space-mediated coupling), and many zinc-finger proteins suggests that these H-bonds are structurally vital for stabilization of the metal centers in these proteins.     

Compartmentalization of Ca2+ in sickle cells

Control (AA) and sickle cell anemia (SS) erythrocytes were loaded with Ca-chelator (Quin2 or Benz2) to increase the cellular exchangeable Ca2+ pool and to measure the Ca2+ exchange fluxes and the cytosolic ionized Ca2+ ([Ca]i) (Lew et al., 1982, Nature, 298, 478). The chelator incorporation induced a decrease in the ATP content which was smaller in SS than in AA cells and partially reversible upon reincubation in a chelator-free medium. The amount of trapped chelator was determined by two methods: 45Ca binding to the chelator in Ca-ionophore treated cells in Ca-EGTA buffers and [3H]Quin2 incorporation. A slight over-estimation of the chelator content was found with the second method but incorporation was the same in both types of cells. The kinetics of 45Ca equilibration and 45Ca release were used to measure Ca2+ fluxes and [Ca]i in oxygenated chelator-loaded cells. SS cells, as compared to AA cells, exhibited a moderate increase in Ca2+ fluxes (30-75%) but [Ca]i remained in the same range (about 20 nM). Thus the excess of Ca2+ found in SS cells is not available for the Ca2+ pump or the K+ channel a conclusion in agreement with that of Bookchin et al. (1984, Cell Calcium, 5, 277). Analysis of the 45Ca kinetics showed that in AA cells, exchangeable Ca2+ behaved as one compartment. In SS cells, the existence of a second slowly-exchangeable Ca2+ compartment was demonstrated. This latter (3-5 mumol/l cells) was independent of the concentration of the chelator and thus could represent exchangeable Ca2+ enclosed within the intracellular inside-out vesicles recently observed in SS cells (Williamson et al., 1984, J. Cell. Biol., 99, 430a). Alternatively, these two kinetic pools could reflect heterogeneity of the SS cell population.     

Use of Bromodeoxyuridine For Cell Kinetic Studies In Intact Animals

Abstract. A method is described for the use of BUdR for tracing cell proliferation patterns in the intestinal mucosa of intact mice.
The method has several distinct advantages over existing methods.
Bromodeoxyuridine (BUdR) is a well-established alternative to tritiated thymidine ([³H]TdR) as a tracer for studying DNA replication. However, its use in cytological as opposed to biochemical studies has been largely confined to examination of metaphase spreads, particularly analysis of sister chromatid exchange (Block, 1982). For this, BUdR incorporation into DNA has been demonstrated using the fluorescent dye Hoechst 33258, together with fluorescence microscopy (Latt, 1973), or Giemsa staining (Perry & Wolf, 1974). Recently, introduction of a monoclonal antibody which recognizes BUdR in single-stranded DNA (Gratzner, 1982) has enabled BUdR to be used for studying cell cycle kinetics in a manner exactly analogous to the use of [³H]TdR. This has been reported for whole cells in suspension and in monolayer (Dolbeare et al. , 1983; Dean et al. , 1984; Raza et al. , 1984). BUdR included in tissue culture medium is taken up and incorporated into newly synthesized DNA via the same pyrimidine salvage pathway as [³H]TdR (thymidine kinase). A concentration of as little as 10 μm—well below cytotoxic levels (Cerni, 1984)—is sufficient to give readily detectable labelling by immunocytochemistry with a pulse of less than 15 min. the validity of BUdR labelling for cell kinetic studies has been well established in comparisons with other methods by Dolbeare et al. (1983), Dean et al. (1984), and Raza et al. (1984).
We describe here the use of BUdR together with an immunocytochemical detection system applied to sections of wax-embedded tissues, which provides a convenient method of cell cycle analysis in intact animals.     

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.     

镉锌超积累植物伴矿景天焚烧飞灰的重金属浸出特性及安全处置

为探究超积累植物伴矿景天焚烧灰浸出回收有价金属的可行性,以800℃流化床焚烧旋风灰为材料,研究盐酸、硝酸、氯化铵以及不同浓度、液固比和浸出时间对飞灰中重金属浸出规律的影响,同时评价浸出残渣的安全性.结果 表明:浸提剂种类、浓度及液固比是影响浸出的重要参数,浸出时间的影响较小,酸性浸提剂浸出能力明显强于氯化铵盐溶液浸出;...     

The potential of Thlaspi caerulescens for phytoremediation of contaminated soils

Uptake of Cd, Zn, Pb and Mn by the hyperaccumulator Thlaspi caerulescens was studied by pot trials in plant growth units and in populations of wild plants growing over Pb/Zn base-metal mine wastes at Les Malines in the south of France. The pot trials utilised metal-contaminated soils from Auby in the Lille area. Zinc and Cd concentrations in wild plants averaged 1.16% and 0.16% (dry weight) respectively. The unfertilised biomass of the plants was 2.6 t/ha. A single fertilised crop with the above metal content could remove 60 kg of Zn and 8.4 kg Cd per hectare. Experiments with pot-grown and wild plants showed that metal concentrations (dry weight basis) were up to 1% Zn (4% Zn in the soil) and just over 0.1% Cd (0.02% Cd in the soil). The metal content of the plants was correlated strongly with the plant-available fraction in the soils as measured by extraction with ammonium acetate and was inversely correlated with pH. Bioaccumulation coefficients (plant/soil metal concentration quotients) were in general higher for Cd than for Zn except at low metal concentrations in the soil. There was a tendency for these coefficients to increase with decreasing metal concentrations in the soil. It is proposed that phytoremediation using Thlaspi caerulescens would be entirely feasible for low levels of Cd where only a single crop would be needed to halve a Cd content of 10 g/g in the soil. It will never be possible to remediate elevated Zn concentrations within an economic time frame (<10 yr) because of the lower bioaccumulation coefficient for this element coupled with the much higher Zn content of the soils.     

Effects of land irrigation with partially-treated wastewater on Frankia survival and infectivity

Phytoextraction of Cd by some populations of Thlaspi caerulescens which have the ability to co-hyperaccumulate Cd and Zn requires information about the distribution of both metals within the plant at the organ-level. This work was conducted to determine whether the distribution and solubility of Cd and Zn in Thlaspi caerulescens are affected by the age of plant and organ, and whether Cd and Zn have a common distribution in the plant in soils contaminated by both metals. A series of pot experiments were conducted where a Cd- and Zn-hyperaccumulating population was grown on soils contaminated by Cd and Zn. Temporal changes in metal concentration of roots and of shoots was recorded, along with the water and CaCl₂ solubility of metals in the plant organs. Also, leaves were grouped according to their age and their respective content of Cd and Zn was measured. Both metals were present at higher concentrations in leaves than in roots. The whole-plant content of Zn decreased with time while that of Cd increased or remained unchanged. At harvest, young leaves exhibited higher Cd concentration than older, but the reverse was true for Zn. Both metals were more soluble in dry leaves and senescent leaves than in fresh material, and Zn was more water-soluble than Cd. In conclusion, the distribution of Cd and Zn in the hyperaccumulator T. caerulescensvaried according to the organ and plant age, and Cd and Zn were shown to have a different distribution within the plant.     

Soil washing of Pb, Zn and Cd using biodegradable chelator and permeable barriers and induced phytoextraction by Cannabis sativa

The feasibility of combined phytoextraction and in situ washing of soil contaminated with Pb (1750 mg kg^–1), Zn (1300 mg kg^–1), and Cd (7.2 mg kg^–1), induced by the addition of biodegradable chelator, [S,S] stereoisomere of ethylenediamine discuccinate ([S,S]-EDDS), was tested in soil columns with hemp (Cannabis sativa) as the phytoextracting plant. The addition of [S,S]-EDDS (10 mmol kg^–1 dry soil) yielded concentrations of 1026±442, 330.3±114.7 and 3.84±1.55 mg kg^–1 of Pb, Zn and Cd in the dry above-ground plant biomass, respectively. These concentrations were 1926, 7.5, and 11 times higher, respectively, compared to treatments with no chelator addition. Horizontal permeable barriers, composed of a 3 cm high layer of nutrient enriched sawdust and vermiculite mixture, and a 3 cm layer of soil, vermiculite and apatite mixture, were positioned 20, 30 and 40 cm deep in the soil. In chelator treatments, barriers placed 30 cm deep reduced leaching of Pb, Zn and Cd by 435, 4 and 53 times, respectively, compared to columns with no barrier, where 3.0, 4.3 and 3.3% of total initial Pb, Zn and Cd, respectively, was leached during 6-weeks water irrigation after chelator addition. Lower positioned barriers were almost equally effective in preventing leaching of Pb than barriers positioned closer to the soil surface, less effective for Cd, and did not prevent leaching of Zn. 2.53% of total initial Pb and 2.83% of Cd was washed from the contaminated soil and accumulated into the barrier. Combined method was less effective than simulated ex situ soil washing, where 14.2, 5.5 and 24.5% of Pb, Zn and Cd, respectively, were removed after 1-h extraction, but comparable effective to 48-h extraction. Abbreviations: BCF – bioconcentration factor; EDTA – ethylene diaminetetraacetate; HM – heavy metal; PP – phytoextraction potential; [S,S]-EDDS – [S,S]-ethylenediamine disuccinate.     

Metal ion requirement of bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase from rat liver

The metal ion requirement for both enzymatic activitiesof the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosaminekinase (E.C. 5.1.3.14/ 2.7.1.60), the key enzyme of N-acetylneuraminic acidbiosynthesis in ratliver, was investigated. UDP-N-acetylglucosamine 2-epimerase was active inimida-zole/HCl buffer in the complete absence of any metal ion. 200 mM Na + , K + , Rb + and Cs +activated enzymeactivity up to five-fold, whereas lower concentrations of thesemonovalent metal ions showed only a small effect on UDP-N-acetylglucosamine 2-epimeraseactivity. In sodium phosphate buffer the enzyme activitywas increased by 0.5 mM Mg , Sr , Ba and Mn , while in the presence of 200 mM NaCl UDP-N-acetyl-glucosamine2-epimerase activity showed astronger activation by these divalent metal ions. In imidazole/HClbuffer, UDP-N-acetylglucosamine2-epimerase activity was partially inhibited by 0.5 mM Be , Mg , Ba ,Mn , Sn and Fe , and completely inhibited by 0.5 mM Zn and Cd . Divalent metal ions were essen-tialforN-acetylmannosamine kinase activity, the most effective being Mg , followed byMn and Co .The optimal concentration of these metal ions was 3 mM. Less effective were Ni and Cd , whereas Ca ,Ba , Cu , Fe and Zn showed no effect on enzyme activity.     

Effective switch-on fluorescence sensing of zinc(II) ion by 8-aminoquinolino-beta-cyclodextrin/adamantaneacetic acid system in water

A water-soluble 8-aminoquinolino-beta-cyclodextrin/1-adamantaneacetic acid (1/ADA) system is prepared in situ and exhibits a unique switch-on fluorescence response to Zn(2+) over other common metal ions. Spectrophotometric studies demonstrate that this system can strongly coordinate Zn(2+) through a cyclodextrin/substrate/metal triple recognition mode, and the resulting 1/ADA/Zn(2+) ternary complex emits the blue-green fluorescence (lambda=490nm) that can be easily distinguished by eyes in aqueous solution. Significantly, the switch-on fluorescence response of 1/ADA to Zn(2+) is barely affected by various metal ions except Cu(2+). As a result, this system can behave as an efficient supramolecular fluorescence sensor for Zn(2+) in water.     

Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.