Lead tolerance and accumulation in the gametophytes of the fern Athyrium yokoscense

The fern Athyrium yokoscense is known to be highly tolerant to lead toxicity, and is a lead hyperaccumulator that can accumulate over 1,000 g g^–1 of lead in its dry matter. In this work, we examined whether the gametophytic generation of A. yokoscense also resists lead toxicity like the sporophytic generation. Spore germination in A. yokoscense was more tolerant to Pb²⁺, compared to that in other fern species, such as Pteridium aquilinum, Lygodium japonicum and Pteris vittata. In addition, the early gametophyte development of A. yokoscense was not much affected by 10 M Pb²⁺, as evaluated from the prothallial growth and rhizoid development. We also showed that Athyrium gametophytes could accumulate more than 10,000 g g^–1 of lead, and that the lead was localized in the cytosol and vacuole of rhizoidal cells, as determined by a transmission electron micrograph. These results indicate that Athyrium gametophytes have the ability to accumulate lead in the rhizoids. Furthermore, the gametophytes were found to include a large amount of proanthocyanidins (condensed tannins). Because proanthocyanidins have a latent ability to complex with lead ions, the possible roles of proanthocyanidins in the lead tolerance and accumulation of Athyrium gametophytes are discussed.     

Recent evolution of sedimentary heavy metals in a coastal lagoon contaminated by industrial wastewaters (Pialassa Baiona,Ravenna, Italy)

From 2000 to 2002, sediment contamination by Cd, Cu, Hg, Pb and Zn was analysed in the Pialassa Baiona salt marsh, which receives petrochemical wastewaters from the industrial district of Ravenna (Italy). The recent contamination levels were compared with data of previous studies carried out in 1982, in order to assess whether environmental policies and remedial measures reduced sediment pollution. Sedimentary profiles of Cu and Pb were homogeneous along the uppermost 0–10 cm horizon, which corresponded to the sedimentation in the last 30 years. Concentrations of Zn attained a peak (up to 800 mg kg⁻¹ dry weight) in the 0–4 cm sediment horizon, which was assumed to correspond to the last 10–15 years. A wide-spread contamination by Hg was detected in the salt marsh as well as in the main channel with peaks up to 20–40 mg kg⁻¹ dry weight. Nonetheless, recent sediments resulted less contaminated, since Hg discharge from industrial plants ceased about 20 years ago. Contamination levels by Hg values were two orders of magnitude higher than the international sediment quality standards. Cadmium, which was analysed for the first time in 2000–2002, attained a peak in the surface layers (1–2.5 mg kg⁻¹ d.w.), with a progressive decline along the sediment column. Through comparison with pre-industrial values detected in the deeper sediment horizons (before 1920), Hg showed the highest enrichment factor, up to 300 times. Cd and Zn concentrations in recent sediments were from 2 to 10 times higher than background values. In terms of possible adverse effects, Hg posed the highest risk, and Cd and Zn were frequently above the recommended thresholds.     

Speciation, stability constants and structures of complexes of copper(II), nickel(II), silver(I) and mercury(II) with PAMAM dendrimer and related tetraamide ligands

The acid-base properties and Cu(II), Ni(II), Ag(I) and Hg(II) binding abilities of PAMAM dendrimer, L, and of the simple model compounds, the tetraamides of EDTA and PDTA, L¹, were studied in solution by pH-metric methods and by ¹H NMR and UV-Vis spectroscopy. PAMAM is hexabasic and six pK_a values have been determined and assigned. PAMAM forms five identifiable complexes with copper(II), [CuLH₄]⁶⁺, [CuLH₂]⁴⁺, [CuLH]³⁺, [CuL]²⁺ and [CuLH_-1]⁺ in the pH range 2-11 and three with nickel(II), [NiLH]³⁺, [NiL]²⁺ and [NiLH_-1]⁺ in the pH range 7-11. The complex [CuLH₄]⁶⁺, which contains two tertiary nitrogen and three amide oxygen atoms coordinated to the metal ion, is less stable than the analogous EDTA and PDTA tetraamide complexes [CuL¹]²⁺, which contain two tertiary nitrogen and four amide oxygen atoms, due to ring size and charge effects. With increasing pH, [CuLH₄]⁶⁺ undergoes deprotonation of two coordinated amide groups to give [CuLH₂]⁴⁺ with a concomitant change from O-amide to N-amidate coordination. Surprisingly and in contrast to the tetraamide complexes [CuL¹]²⁺, these two deprotonation steps could not be separated. As expected the nickel(II) complexes are less stable than their copper(II) analogues. The tetra-N-methylamides of EDTA, L¹(b), and PDTA form mononuclear and binuclear complexes with Hg(II). In the case of L¹(b) these have stoichiometries HgL¹(b)Cl₂, [HgL¹(b)H₋₂Cl₂]²⁻, [Hg₂L¹(b)Cl₂]²⁺, Hg₂L¹(b)H₋₂Cl₂ and [Hg₂L¹(b)H₋₅Cl₂]³⁻. Based on ¹H NMR and pH-metric data the proposed structure for HgL¹(b)Cl₂, the main tetraamide ligand containing species in the pH range <3-6.5, contains L¹(b) coordinated to the metal ion through the two tertiary nitrogens and two amide oxygens while the structure of [HgL¹(b)H₋₂Cl₂]²⁻, the main tetraamide ligand species at pH 7.5-9.0, contains the ligand similarly coordinated but through two amidate nitrogen atoms instead of amide oxygens. The proposed structure of [Hg₂L¹(b)Cl₂]²⁺, a minor species at pH 3-6.5, also based on ¹H NMR and pH-metric data, contains each Hg(II) coordinated to a tertiary amino nitrogen, two amide oxygens and a chloride ligand while that of [Hg₂L¹(b)H₋₅Cl₂]³⁻, contains each Hg(II) coordinated to a tertiary amino nitrogen, two amidate nitrogens, a chloride and a hydroxo ligand in the case of one of the Hg(II) ions. The parent EDTA and PDTA amides only form mononuclear complexes. PAMAM also forms dinuclear as well as mononuclear complexes with mercury(II) and silver(I). In the pH range 3-11 six complexes with Hg(II) i.e. [HgLH₄Cl₂]⁴⁺, [HgLH₃Cl₂]³⁺, [Hg₂LCl₂]²⁺, [Hg₂LH₋₁Cl₂]⁺, [HgLH₋₁Cl₂]⁻ and [HgLH₋₂Cl₂]²⁻ were identified and only two with Ag(I), [AgLH₃]⁴⁺ and [Ag₂L]²⁺. Based on stoichiometries, stability constant comparisons and ¹H NMR data, structures are proposed for these species. Hence [HgLH₄Cl₂]⁴⁺ is proposed to have a similar structure to [CuLH₄]⁶⁺ while [Hg₂LCl₂]²⁺has a similar structure to [Hg₂L¹(b)H₋₅Cl₂]³⁻.     

Insertion reactions of organic anhydrides and the Cu(I) alkoxide [CuOBu]

The synthesis and structural characterization of the copper salts [Cu₈(benzoate)₈(THF)₆] (1), [Cu₂{(CO₂)₂C₆H₂(Boc)₂}dppm₂]₂ (2) and [Cu₂{(CO₂)₂C₁₀H₄(Boc)₂}dppm₂]₂ (3) [Boc = tert-butoxycarbonyl, dppm = 1,2-bis(diphenylphosphino)methane] prove that cyclic organic anhydrides and dianhydrides readily insert into the Cu-O bond of [CuO^tBu] forming carboxylate ligands with ester functionalities in the ligand periphery. [Mn₃(o-Boc-benzoate)₆(DME)₂] (4) (DME = 1,2-dimethoxyethane) was synthesized by the metathesis reaction of [Cu(I)(o-Boc-benzoate)] with MnCl₂.     

Dissemination and survival of non-indigenous bacterial genomes in pristine Antarctic environments

Continental Antarctic is perceived as a largely pristine environment, although certain localized regions (e.g., parts of the Ross Dependency Dry Valleys) are relatively heavy impacted by human activities. The procedures imposed on Antarctic field parties for the handling and disposal of both solid and liquid wastes are designed to minimise eutrofication and contamination (particularly by human enteric bacteria). However, little consideration has been given to the significance, if any, of less obvious forms of microbial contamination resulting from periodic human activities in Antarctica. The predominant commensal microorganism on human skin, Staphylococcus epidermidis, could be detected by PCR, in Dry Valley mineral soils collected from heavily impacted areas, but could not be detected in Dry Valley mineral soils collected from low impact and pristine areas. Cell viability of this non-enteric human commensal is rapidly lost in Dry Valley mineral soil. However, S. epidermidis can persist for long periods in Dry Valley mineral soil as non-viable cells and/or naked DNA.     

Tissue-specific cadmium accumulation and metallothionein-like protein levels during acclimation process in the Chinese crab Eriocheir sinensis

Aquatic organisms chronically exposed to cadmium can increase their resistance to a subsequent elevated exposure. In order to investigate mechanisms involved in acclimation process in the Chinese crab Eriocheir sinensis, we compared Cd level as well as metallothionein-like protein (MTLP) content in different tissues after direct acute exposure (i.e. 500 microg Cd L(-1) for 3 days), and after acute following chronic (i.e. 10 or 50 microg Cd L(-1) for 30 days) exposure. Cadmium accumulation occurred in the following order: anterior gill>hepatopancreas>posterior gill>carapace>hemolymph>muscle. As high concentrations as 188 microg Cd g(-1) w.w. were reported in anterior gills and seem to reach a saturation level. In these gills, the highest MTLP induction was observed after a direct acute exposure, for which a correlation with Cd content occurred. However, the Cd-binding potential by MTLPs was exceeded for any exposure condition. In hepatopancreas, the highest Cd level was reported for crabs acclimated during 30 days to 50 microg Cd L(-1) before challenging with an acute exposure. Moreover, we showed that MTLPs were induced during the acclimation process. In this organ, MTLPs are theoretically sufficient to bind all Cd. These results suggest that during a chronic exposure to 50 microg Cd L(-1), Chinese crabs acquire the capacity to hold more cadmium in hepatopancreas where it can be sequestrated by MTLPs. On the contrary, MTLP induction seems to be a rapid response to acute exposure in anterior gill, but is not sufficient to sequester all Cd. Other sequestration and/or detoxification mechanisms must take place in anterior gill to cope with high Cd levels.     

Theoretical model of the aqua-copper [Cu(H2O)5]+cation interactions with guanine

Pentaaqua complexes of Cu(I) with guanine were optimized at the DFT B3PW91/6-31G(d) level. For the most stable structures, vibration frequencies and NBO charges were computed followed by energy analyses. The order of individual conformers was very sensitive to the method and basis sets used for the calculation. Several conformers are practically degenerated in energy. The inclusion of an entropy term changes the order of the conformers stability. Water molecules associated at the N9 position of guanine are favored by the inclusion of the entropy correction. Bonding energies of Cu–O(aqua) interactions were estimated to be about 60 kcal mol^–1 and for Cu–N7 bonding in the range of 75–83 kcal mol^–1. The broad range in Cu–N interaction energies demonstrates the role of induction effects caused by water molecules associated at the various sites of guanine. The charge distribution of the guanine molecule is changed remarkably by the coordination of a Cu(I) cation, which can also change the base-pairing pattern of the guanine.     

The impact of blood contamination on the proteome of cerebrospinal fluid

Human cerebrospinal fluid (CSF) is in direct contact with the brain extracellular space. Beside the secretion of CSF by the choroid plexus the fluid also derives directly from the brain by the ependymal lining of the ventricular system and the glial membrane and from blood vessels in the arachnoid. Therefore, biochemical change in the brain may be reflected in the CSF. CSF is a potential source of protein molecular indices of central nervous system function and pathology. However, various amounts of blood contamination in CSF may arise during sample acquisition. The concentration of protein in the CSF is only 0.2 to 0.5% that of blood. Minor contamination of CSF with blood during collection of the fluid may dramatically alter the protein profile confounding the identification of potential biomarkers. We have analyzed CSF and CSF spiked with increasing amounts of whole blood using proteomic techniques. We detected at least four blood specific highly abundant proteins: hemoglobin, catalase, peroxiredoxin and carbonic anhydrase I. These proteins can be used as blood contamination markers for proteomic analysis of CSF. Proteins in blood contaminated CSF samples were less stable compared to neat CSF at 37 degrees C suggesting that blood borne protease may induce protein degradation in CSF during sample acquisition. This analysis was aimed at identification of proteins found primarily in CSF, those found primarily in blood and assessment of the impact of blood contamination on those proteins found in both fluids.     

Distinct metal dependence for catalytic and structural functions in the L-arabinose isomerases from the mesophilic Bacillus halodurans and the thermophilic Geobacillus stearothermophilus

L-Arabinose isomerase (AI) catalyzes the isomerization of L-arabinose to L-ribulose. It can also convert d-galactose to d-tagatose at elevated temperatures in the presence of divalent metal ions. The araA genes, encoding AI, from the mesophilic bacterium Bacillus halodurans and the thermophilic Geobacillus stearothermophilus were cloned and overexpressed in Escherichia coli, and the recombinant enzymes were purified to homogeneity. The purified enzymes are homotetramers with a molecular mass of 232 kDa and close amino acid sequence identity (67%). However, they exhibit quite different temperature dependence and metal requirements. B. halodurans AI has maximal activity at 50 degrees C under the assay conditions used and is not dependent on divalent metal ions. Its apparent K(m) values are 36 mM for L-arabinose and 167 mM for d-galactose, and the catalytic efficiencies (k(cat)/K(m)) of the enzyme were 51.4 mM(-1)min(-1) (L-arabinose) and 0.4 mM(-1)min(-1) (d-galactose). Unlike B. halodurans AI, G. stearothermophilus AI has maximal activity at 65-70 degrees C, and is strongly activated by Mn(2+). It also has a much higher catalytic efficiency of 4.3 mM(-1)min(-1) for d-galactose and 32.5 mM(-1)min(-1)for L-arabinose, with apparent K(m) values of 117 and 63 mM, respectively. Irreversible thermal denaturation experiments using circular dichroism (CD) spectroscopy showed that the apparent melting temperature of B. halodurans AI (T(m)=65-67 degrees C) was unaffected by the presence of metal ions, whereas EDTA-treated G. stearothermophilus AI had a lower T(m) (72 degrees C) than the holoenzyme (78 degrees C). CD studies of both enzymes demonstrated that metal-mediated significant conformational changes were found in holo G. stearothermophilus AI, and there is an active tertiary structure for G. stearothermophilus AI at elevated temperatures for its catalytic activity. This is in marked contrast to the mesophilic B. halodurans AI where cofactor coordination is not necessary for proper protein folding. The metal dependence of G. stearothermophilus AI seems to be correlated with their catalytic and structural functions. We therefore propose that the metal ion requirement of the thermophilic G. stearothermophilus AI reflects the need to adopt the correct substrate-binding conformation and the structural stability at elevated temperatures.     

Evidence for pH dependent Zn2+influx in K562 erythroleukemia cells: studies using ZnAF-2F fluorescence and 65Zn2+ uptake

Using both ZnAF-2F (a Zn2+ specific fluorophore) and 65Zn2+, we determined the rate of transporter mediated Zn2+ influx (presumably mediated by the SLC39A1 gene product, protein name hZIP1) under steady state conditions and studied the effects of extracellular acidification. When K562 erythroleukemia cells were placed in Zn2+ containing buffers (1-60 microM), the initial rate of 65Zn2+ accumulation mirrored the apparent rise in free intracellular Zn2+ concentrations sensed by ZnAF-2F. Therefore, newly transported Zn2+ equilibrated with the free intracellular Zn2+ pool sensed by ZnAF-2F. A new steady state with elevated free intracellular Zn2+ was established after about 30 min. An estimate of 11 microM for the Km and 0.203 nmol/mg/s for the Vmax were obtained for Zn2+ influx. 65Zn2+ uptake and ZnAF-2F fluorescent changes were inhibited by extracellular acidification (range tested: pH 8-6, IC50 = pH 6.34). The IC50 for proton effects was close to the pKa for histidine, suggesting conserved histidine residues present in SLC39A1 play a critical role in Zn2+ influx and are involved in the pH effect.