Copper toxicity and sulfur metabolism in Chinese cabbage are affected by UV radiation

Biomass production, dry matter content, specific leaf area and pigment content of Chinese cabbage were all quite similar, when plants were grown in the absence or presence of UV-A + B (2.2 mW cm⁻²). Elevated Cu²⁺ concentrations (2–10 μM) in the root environment and UV radiation had negative synergistic effects for Chinese cabbage and resulted in a more rapid and stronger decrease in plant biomass production and pigment content. The quantum yield of photosystem II photochemistry (F_v/F_m) was only decreased at ≥5 μM Cu²⁺ in the presence of UV radiation, when leaf tissue started to become necrotic. The enhanced Cu toxicity in the presence of UV was largely due to a UV-induced enhanced accumulation of Cu in both roots and shoots. An enhanced Cu content strongly affected the uptake and assimilation of sulfur in plants. The total sulfur content of the root increased at ≥2 μM Cu²⁺ in presence of UV and at 10 μM Cu²⁺ in absence of UV and that of the shoot increased at ≥2 μM Cu²⁺ in presence of UV and at ≥5 μM Cu²⁺ in absence of UV. In the shoot it could be attributed mainly to an increase in sulfate content. Moreover, there was a strong increase in the water-soluble non-protein thiol content upon Cu²⁺ exposure in the root and, to a lesser extent in the shoot, both in the presence and absence of UV. The regulation of the uptake of sulfate responded to the occurrence of Cu toxicity directly, since it was more rapidly affected in the presence than in the absence of UV radiation. For instance, the expression and activity of the high affinity sulfate transporter, Sultr1;2, were enhanced at ≥2 μM in the presence of UV, and at ≥5 μM Cu²⁺ in the absence of UV. In the shoot, the expression of the vacuolar sulfate transporter, Sultr4;1, was upregulated at ≥5 μM Cu²⁺ in the presence and absence of UV whilst the expression of a second vacuolar sulfate transporter, Sultr4;2, was upregulated at 10 μM Cu²⁺ in the presence of UV. It is suggested that high Cu tissue levels may interfere/react with the signal compounds involved in the regulation of expression and activity of sulfate transporters. The expression of adenosine 5′-phosphosulfate reductase in the root was hardly affected and was slightly down-regulated at 2 μM in the presence of UV and at 10 μM in the absence of UV. The expression and activity of sulfate transporters were enhanced upon exposure at elevated Cu²⁺ concentrations; this may not be simply due to a greater sulfur demand at higher Cu levels, but more likely is the consequence of Cu toxicity, since it occurred more rapidly in the presence compared to the absence of UV.     

Copper,zinc superoxide dismutase of Curcuma aromatica is a kinetically stable protein

This study details on cloning and characterization of Cu,Zn superoxide dismutase (Ca–Cu,Zn SOD) from a medicinally important plant species Curcuma aromatica. Ca–Cu,Zn SOD was 692 bp with an open reading frame of 459 bp. Expression of the gene in Escherichia coli cells followed by purification yielded the enzyme with K_m of 0.047 ± 0.008 μM and V_max of 1250 ± 24 units/mg of protein. The enzyme functioned (i) across a temperature range of −10 to +80 °C with temperature optima at 20 °C; and (ii) at pH range of 6–9 with optimum activity at pH 7.8. Ca–Cu,Zn SOD retained 50% of the maximum activity after autoclaving, and was stable at a wide storage pH ranging from 3 to 10. The enzyme tolerated varying concentrations of denaturating agent, reductants, inhibitors, trypsin, was fairly resistant to inactivation at 80 °C for 180 min (k_d, 6.54 ± 0.17 × 10⁻³ min⁻¹; t_1/2, 106.07 ± 2.68 min), and had midpoint of thermal transition (T_m) of 70.45 °C. The results suggested Ca–Cu,Zn SOD to be a kinetically stable protein that could be used for various industrial applications.     

Concerted involvement of cooperative proton–electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu_A and heme a₃/Cu_B are cooperatively linked to proton transfer at acid/base groups in the enzyme. H⁺/e⁻ cooperative linkage at Fe_a3/Cu_B is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H⁺/e⁻ linkage at heme a (and Cu_A). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H₂O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu_A and heme a₃/Cu_B in the soluble oxidase; (iii) H⁺ release in the external phase (i.e. H⁺ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H⁺/e⁻ linkage at heme a/Cu_A and heme a₃/Cu_B with acid/base clusters, C₁ and C₂ respectively, and protonmotive steps of the reduction of O₂ to water are involved in proton pumping.     

Effect of copper sources and levels on serum lipid profiles in Black Bengal (Capra hircus) kids

Twenty eight 2–3 month old castrated male Black Bengal kids (Capra hircus) were used to determine the effects of dietary Cu concentration on lipid metabolism. These kids were randomly assigned to one of seven treatments in a ((2 × 3) + 1) factorial arrangement. Factors were two sources of Cu (CuSO₄ versus Cu proteinate) fed at three dietary levels (10, 20 or 30 mg/kg) and the control group, where neither CuSO₄ nor Cu proteinate were supplemented. Kids were fed a basal diet containing maize (19.5%), soybean (17.0%), deoiled rice bran (56.5%), molasses (4.0%), di-calcium phosphate and salt (1.0% each), and mineral and vitamin mixture (0.5% each) supplements, at 3.5% of BW to meet NRC requirements for protein, energy, macro minerals and micro minerals, excluding Cu. The basal diet (DM basis) contained 5.7 mg Cu/kg, 122.5 mg Fe/kg, 110 mg Zn/kg, 0.26 mg Mo/kg and 0.32% S. CuSO₄ or Cu proteinate (Cu-P) was added to the basal diet at the rate of 10, 20 and 30 mg/kg. Kids were housed in a well-ventilated shed with facilities for individual feeding in aluminum plated metabolic cages in an open-sided barn. Blood samples were collected on Days 0, 30, 60 and 90 to determine serum cholesterol, high density lipoprotein (HDL), total lipid and phospholipids. Kids were slaughtered after metabolism trial and liver tissues were collected to determine the copper and zinc concentrations. Kids receiving Cu-P showed higher (P < 0.05) HDL, total lipid and phospholipid concentrations. Increase in dietary level of Cu significantly decreased (P < 0.05) serum cholesterol and increased serum HDL, total lipid and phospholipid concentrations. There was an increasing (P < 0.05) trend in liver Cu with the increased dietary level of Cu supplementation irrespective of source, but the increasing rate was greater with CuSO₄ than Cu-P supplementation. Kids’ diet containing 30 mg/kg CuSO₄ had 26% more liver Cu than those fed iso-amounts of Cu-P. Fecal Cu excretion was increased with the increasing dietary level of Cu, and excretion was reduced by the use of Cu-P in the diet. In conclusion, dietary supplementation of organic Cu in the form of copper proteinate had significant effects on lipid metabolism in goat kids. There was an increase in accumulation of Cu in the liver and excretion of Cu in feces with the increase of dietary level of Cu in the diet of Black Bengal kids.     

Trace mineral status and liver and blood parameters in sheep without mineral supply compared to local roe deer (Capreolus capreolus) populations

General health, clinical-chemical blood analysis and copper (Cu), zinc (Zn), selenium (Se) and vitamin E concentrations in plasma and liver tissue (wet weight, ww) of two extensive grazing sheep flocks without mineral supply were compared to the status of local roe deer (Capreolus capreolus) populations (liver samples). Both sheep flocks were classified as healthy except for a remarkable variation in body weight and a slight foot rot infection in one flock. Hematology of sheep was normal, and total protein and creatinine as well as activities of creatin kinase, aspartat-amino-transferase, alkaline phosphatase and gamma-glutamyl-transferase in plasma were within reference levels. The mean of glutamate dehydrogenase (13.8 U/l) was slightly elevated in one flock. Mean liver concentrations of Zn (38.9 and 43.5 mg/kg ww) and Cu (111 and 87.5 mg/kg ww) in sheep flocks were higher compared to the respective roe deer populations (27.5 and 36.3 mg Zn/kg ww; 18.3 and 28.6 mg Cu/kg ww). This is supposed to be caused by differences in Cu and Zn metabolism in sheep and roe deer. Selenium deficiency was diagnosed in liver samples of both sheep flocks (0.21 and 0.23 mg/kg ww). There were neither significant differences compared to roe deer (0.21 and 0.27 mg Se/kg ww) nor differences depending on location. Correlations between plasma and liver concentrations of Cu, Zn and Se were not significant in sheep. Means of vitamin E in liver samples (30.6 and 41.8 mg/kg ww) were higher in roe deer populations. This may be caused by the opportunity of selective browsing for wild ruminants, which allows access to younger plants which are higher in vitamin E.     

Putative P1B-type ATPase from the bacterium Achromobacter xylosoxidans A8 alters Pb2 +/Zn2 +/Cd2 +-resistance and accumulation in Saccharomyces cerevisiae

PbtA, a putative P_1B-type ATPase from the Gram-negative soil bacterium Achromobacter xylosoxidans A8 responsible for Pb^2 +/Zn^2 +/Cd^2 +-resistance in Escherichia coli, was heterologously expressed in Saccharomyces cerevisiae. When present in Zn^2 +- and Pb^2 +/Cd^2 +-hypersensitive S. cerevisiae strains CM137 and DTY168, respectively, PbtA was able to restore Zn^2 +- and Pb^2 +-resistant phenotype. At the same time, the increase of Pb, Zn, and Cd accumulation in yeast was observed. However, Cd^2 +-tolerance of the pbtA-bearing yeasts dramatically decreased. The PbtA-eGFP fusion protein was localized primarily in the tonoplast and also in the plasma membrane and the perinuclear region corresponding to the endoplasmic reticulum at later growth stages. This indicates that PbtA protein is successfully incorporated into membranes in yeasts. Since PbtA caused a substantial increase of Pb^2 +/Zn^2 +-resistance and accumulation in baker's yeast, we propose its further use for the genetic modification of suitable plant species in order to obtain an effective tool for the phytoremediation of sites polluted by toxic transition metals.     

Interaction of dietary zinc and growth implants on weight gain,carcass traits and zinc in tissues of growing beef steers and heifers

In two separate studies, 60 beef heifers (379 kg BW) and 60 beef steers (348 kg BW) were randomly assigned to six treatments in 2×3 factorial arrangements. The treatments were with or without Synovex^® implants combined with either a control diet or diets supplemented with 200 ppm Zn from ZnSO₄ or zinc methionine (Zn-Met). Near the mid-point of the feeding periods, cattle were vaccinated with a modified live virus and subsequent titers and concentrations of immunoglobulin G (IgG) were measured. Liver and blood samples were obtained 1 week prior to the start of the experiments and at intervals during the experiments. In experiment 1, average daily gains of beef heifers were (P<0.05) affected by the interaction of implant and source of dietary Zn. Compared to control and ZnSO₄ treatments, supplementation with Zn-Met increased (P<0.05) the concentration of Zn in serum. Antibody titers and concentrations of IgG in serum were highest (P<0.05) in heifers fed ZnSO₄ compared to heifers fed the control or Zn-Met supplemented diets. The Synovex-H^® implant reduced the concentrations of Zn and Cu in liver. In experiment 2, Synovex-S^® implants improved (P<0.05) weight gains of steers supplemented with 200 ppm dietary Zn from ZnSO₄ compared to non-implanted steers. However, the implant had no effect when Zn-Met was the dietary Zn source. The implant increased (P<0.05) concentrations of Zn in liver of steers supplemented with 200 ppm dietary Zn and reduced Zn in liver of steers fed the control diet. Implanted steers had higher (P<0.05) Cu status and IgG concentrations in serum than non-implanted steers. Steers supplemented with either ZnSO₄ or Zn-Met had greater (P<0.05) concentrations of Zn in liver and plasma than steers fed the control diet. These results indicate both the level and source of Zn supplementation in diets of feedlot cattle affect their response to growth implants.     

Myriophyllum aquaticum as a biomonitor of water heavy metal input related to agricultural activities in the Xanaes River (Córdoba,Argentina)

The aim of the present study was to assess the temporal variation of the heavy metal content (Co, Cu, Fe, Mn, Ni, Pb, and Zn) in surface water and sediments in relation to agricultural practices in the Xanaes River (Córdoba, Argentina). A second objective was to analyze possible relationships between the input of heavy metals on surface water and sediment, heavy metal accumulation and physiological changes in the aquatic plant Myriophyllum aquaticum. Samples were taken from the river at two contrasting sites (between April 2010 and August 2010): (1) a pristine area (mountain site), and (2) an area with intensive agricultural activity located at 60 km down river (agricultural site). The total concentration of heavy metals in surface water was higher in samples collected at the agricultural site but in sediments only the Mn concentration was higher than at the mountain site. The Fe and Mn concentrations in surface water at the agricultural site exceeded the recommended values for Argentinean Legislation of 300 μg L⁻¹ for Fe and 100 μg L⁻¹ for Mn. The accumulations of Zn and Mn in M. aquaticum were higher at the agricultural site and more elevated than the Zn and Mn concentrations in sediments at the same sites and sampling times. At the agricultural site, temporal variations of Cu, Fe and Zn were relatively similar for plants and water column, but the levels of the metals in plants were displaced over time. These results suggest that the levels of pollutants in the river came in pulses from the riverbank. These results show the potential use of M. aquaticum as a suitable accumulation biomonitor at the early stages of heavy metal pollution in rivers.     

Metal uptake by medicinal plant species grown in soils contaminated by a smelter

The hypothesis tested in this study was if medicinal plants could be grown as alternative crops in heavy metal polluted soils without contamination of the final marketable produce. Furthermore, medicinal crops may offer a phytoremediation option for mildly heavy metal polluted agricultural soils. The effect of metal-enriched soils was evaluated in five medicinal species (Bidens tripartita L., Leonurus cardiaca L., Marrubium vulgare L., Melissa officinalis L. and Origanum heracleoticum L.). Soils were sampled in the vicinities of the Non-Ferrous Metals Combine (Pb–Zn smelter) near Plovdiv, Bulgaria, from plots at 0.5 km (soil 1), 3 km (soil 2), 6 km (soil 3) and 9 km (control soil) from the smelter. Cadmium, Pb and Zn concentration in soil 1 were above the critical total (HNO₃-extractable) concentrations for these elements in soils. Generally, heavy metals in soil 1 decreased dry mater yields of the five species relative to the control. However, the essential oil content of M. vulgare, M. officinalis and O. heracleoticum was within the usual range for respective species and was not affected by the treatments. The overall metal uptake was in the order: B. tripartita > M. vulgare > O. heracleoticum > L. cardiaca > M. officinalis for Cd, L. cardiaca = M. vulgare > B. tripartita = M. officinalis = O. heracleoticum for Pb, L. cardiaca = M. vulgare > O. heracleoticum > B. tripartita = M. officinalis for Cu and B. tripartita > L. cardiaca = M. vulgare > M. officinalis = O. heracleoticum for Mn and Zn. Overall, metal concentration in plant parts was in the order: roots > leaves > flowers > stems for Cd, Pb and Cu, leaves > roots > flowers > stems for Mn and Zn. The concentration of Cd, Pb, Cu and Zn in plant tissue correlated to the exchangeable (EXCH) and the carbonate (CARB) bound fractions of metals in soil. Heavy metals caused disruptions of the plasma membrane of some root cortical cells and alterations in chloroplasts thylakoids in plants grown in soil 1. Metal content in teas prepared from the species was negligible, the essential oils were free of metals. Generally, the transfer factor (TF) was less than 1, indicating the tested species did not have a significant phytoextraction potential. This study demonstrated the three essential oil species M. vulgare, M. officinalis and O. heracleoticum can be grown as alternative high-value crops in metal polluted agricultural soils around the smelter and provide metal-free marketable produce.     

Metal uptake by medicinal plant species grown in soils contaminated by a smelter

The hypothesis tested in this study was if medicinal plants could be grown as alternative crops in heavy metal polluted soils without contamination of the final marketable produce. Furthermore, medicinal crops may offer a phytoremediation option for mildly heavy metal polluted agricultural soils. The effect of metal-enriched soils was evaluated in five medicinal species (Bidens tripartita L., Leonurus cardiaca L., Marrubium vulgare L., Melissa officinalis L. and Origanum heracleoticum L.). Soils were sampled in the vicinities of the Non-Ferrous Metals Combine (Pb–Zn smelter) near Plovdiv, Bulgaria, from plots at 0.5 km (soil 1), 3 km (soil 2), 6 km (soil 3) and 9 km (control soil) from the smelter. Cadmium, Pb and Zn concentration in soil 1 were above the critical total (HNO₃-extractable) concentrations for these elements in soils. Generally, heavy metals in soil 1 decreased dry mater yields of the five species relative to the control. However, the essential oil content of M. vulgare, M. officinalis and O. heracleoticum was within the usual range for respective species and was not affected by the treatments. The overall metal uptake was in the order: B. tripartita > M. vulgare > O. heracleoticum > L. cardiaca > M. officinalis for Cd, L. cardiaca = M. vulgare > B. tripartita = M. officinalis = O. heracleoticum for Pb, L. cardiaca = M. vulgare > O. heracleoticum > B. tripartita = M. officinalis for Cu and B. tripartita > L. cardiaca = M. vulgare > M. officinalis = O. heracleoticum for Mn and Zn. Overall, metal concentration in plant parts was in the order: roots > leaves > flowers > stems for Cd, Pb and Cu, leaves > roots > flowers > stems for Mn and Zn. The concentration of Cd, Pb, Cu and Zn in plant tissue correlated to the exchangeable (EXCH) and the carbonate (CARB) bound fractions of metals in soil. Heavy metals caused disruptions of the plasma membrane of some root cortical cells and alterations in chloroplasts thylakoids in plants grown in soil 1. Metal content in teas prepared from the species was negligible, the essential oils were free of metals. Generally, the transfer factor (TF) was less than 1, indicating the tested species did not have a significant phytoextraction potential. This study demonstrated the three essential oil species M. vulgare, M. officinalis and O. heracleoticum can be grown as alternative high-value crops in metal polluted agricultural soils around the smelter and provide metal-free marketable produce.     

Synthesis,structures and reactivity of N-donor-functionalised Cu(I) aryloxides

The preparation and characterisation of the Cu(I) aryloxides [Cu₁₆(3-pyO)₁₆(dppm)₈] (1), [{Cu₂(2-pyO)₂(dppm)}₂] (2) and [{Cu₃(μ₃-6-OQ)₂(dppm)₃}{(6-HOQ)₂(μ-6-OQ)}] (3) (dppm = 1,2-bis-diphenylphosphinomethane, 6-HOQ = 6-hydroxyquinoline, py = pyridine) are described. A first attempt to employ organic anhydrides in insertion reactions with Cu(I) aryloxides was made producing the one-dimensional coordination polymer 1/_∞[Cu₃(3-pyO)(CO₂C₂H₄Boc)(dppm⁻)(dppm)]_∞ (4) (Boc = tert-butoxycarbonyl).     

Heavy metal pollution in surface water and sediment: A preliminary assessment of an urban river in a developing country

The concentration and chemical fractionation of globally alarming six heavy metals (Cr, Ni, Cu, As, Cd and Pb) were measured in surface water and sediment of an urban river in Bangladesh. The decreasing trend of metals were observed in water as Cr > Cu > As > Ni > Pb > Cd and in sediment as Cr > Ni > Cu > Pb > As > Cd. The level of studied metals exceeded the safe limits of drinking water, indicated that water from this river is not safe for drinking and/or cooking purposes. However, the investigated metals showed low mobility except for Cd and Pb which could pose a severe threat to the aquatic environment. Contamination factor (CF) and geoaccumulation index (I_geo) demonstrated that most of the sediment samples were moderately to heavily contaminated by Cr, As, Cd and Pb. The pollution load index (PLI) values were above one (>1) indicates progressive deterioration of the sediment quality. The extent of pollution by heavy metals in the river Korotoa implies that the condition is much frightening to the biota and inhabitants in the vicinity of the river as well.     

Magnetostructural correlations and catecholase-like activities of μ-alkoxo-μ-carboxylato double bridged dinuclear and tetranuclear copper(II) complexes

Two μ-alkoxo-μ-carboxylato bridged dinuclear copper(II) complexes, [Cu₂(L¹)(μ-HCO₂)] (1) ((H₃L¹ = 1,3-bis(5-bromosalicylideneamino)-2-propanol)), [Cu₂(L²)(μ-HCO₂)] · dmf (2) (H₃L² = 1,3-bis(3,5-chlorosalicylideneamino-2-propanol)), and two μ-alkoxo-μ-dicarboxylato doubly bridged tetranuclear copper(II) complexes, [{Cu₂(L³)}₂(μ-O₂C–C(CH₃)₂–CO₂)] · 5H₂O · 3CH₃OH (3) ((H₃L³ = 1,3-bis(salicylid-deneamino)-2-propanol)) and [{Cu₂(L³)}₂(μ- O₂CCH₂–C₆H₄–CH₂CO₂)] · 2H₂O (4) have been prepared and characterized. The single crystal X-ray analysis shows that the structures of complexes 1 and 2 are dimeric with two adjacent copper(II) atoms bridged by μ-alkoxo-μ-carboxylato ligands with the Cu⋯Cu distances and Cu–O(alkoxo)–Cu angles are 3.511 Å and 132.85° for 1, 3.517 Å and 131.7° for 2, respectively. Complexes 3 and 4 consist of μ-alkoxo-μ-dicarboxylato doubly bridged tetranuclear Cu(II) complexes with mean Cu–Cu distances and Cu–O–Cu angles of 3.158 Å and 108.05° for 3 and 3.081 Å and 104.76° for 4, respectively. Magnetic measurements reveal that 1 and 2 are strong antiferromagnetically coupled with 2J = −156 and −152 cm⁻¹, respectively, while 3 and 4 exhibit ferromagnetic coupling with 2J = 86 and 155.2 cm⁻¹, respectively. The 2J values of 1–4 are linearly correlated to the Cu–O–Cu angles. Dependence of the pH at 25 °C on the reaction rate of oxidation of 3,4-di-tert-butylcatechol (3,5-dtbc) to the corresponding quinone catalyzed by 1–4 was studied. Complexes 1–4 exhibit high catecholase-like activity at pH 9.0 and 25 °C for oxidation of 3,5-di-tert-butylcatechol.     

Isostructural M(RL)2(hfac)2 complexes with RL = 5-(4-[N-tert-butyl-N-aminoxyl]phenyl)pyrimidine

5-(4-(N-tert-Butyl-N-aminoxylphenyl))pyrimidine (RL, 4PPN) forms crystallographically isostructural and isomorphic pseudo-octahedral M(RL)₂(hfac)₂ complexes with M(hfac)₂, M = Zn, Cu, Ni, Co, and Mn. Multiple close contacts occur between sites of significant spin density of the organic radical units. Magnetic behavior of the Zn, Cu, Ni, Co complexes appears to involve multiple exchange pathways, with multiple close crystallographic contacts between sites that EPR (of 4PPN) indicates to have observable spin density. Powder EPR spectra at room temperature and low temperature are reported for each complex. Near room temperature, the magnetic moments of the complexes are roughly equal to those expected by a sum of non-interacting moments (two radicals plus ion). As temperature decreases, AFM exchange interactions become evident in all of the complexes. The closest fits to the magnetic data were found for a 1-D Heisenberg AFM chain model in the Zn(II) complex (J/k = (?)7 K), and for three-spin RL—M—RL exchange in the other complexes (J/k = (?)26 K, (?)3 K, (?)6 K, for Cu(II), Ni(II), and Co(II) complexes, respectively).     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Evidence of century-scale environmental changes: Trace element in tree-ring from Fuling Mausoleum Shenyang,China

In order to detect the response of trees to environmental pollution in Shenyang during the last century, tree rings of two 100+ year old Chinese pine (Pinus tabulaeformis Carr.) were analyzed for nine trace element concentrations (K, Ca, Mg, Mn, Cu, Zn, Al, Pb and Cd) and compared to local environmental change. The concentrations of Cu, Zn and Al had significant (p < 0.01) increasing trends after 1940s and correlated significantly (p < 0.01), with increasing production of nonferrous metal from local industrial processing. Concentrations of nutrient element (K, Ca and Mg) showed significant (p < 0.01) increasing trend from heartwood to sapwood with declines in the last three decades. Significant correlation between climatic factors and element concentration in tree rings were also found, in which the temperature (November to May) and relative humidity in April and May showed significant positive correlation with Ca, Zn and Cu (p < 0.05) as well as Al (p < 0.01) concentrations in tree rings. April rainfall showed significant positive correlation with K and Zn (p < 0.05) as well as Al (p < 0.01) concentration in tree rings. The results suggest that tree rings from Chinese pine in Shenyang are sensitive to environmental change and have the potential to be used as a biomonitor for the environmental pollution. The results also demonstrate the feasibility of applying dendrochemical techniques to areas with limited samples.     

Copper/zinc and copper/selenium ratios,and oxidative stress as biochemical markers in recurrent aphthous stomatitis

ProjectRecurrent aphthous stomatitis (RAS) is a common oral mucosal disorder characterized by recurrent, painful oral aphthae, and oxidative stress presumably contributes to its pathogenesis. The aim of this study is to scrutinize the relationship between oxidative stress and serum trace elements (copper, Cu; zinc, Zn; selenium, Se), and to evaluate the ratios of Cu/Zn and Cu/Se in this disorder.ProcedurePatients with RAS (n = 33) and age- and sex-matched healthy control subjects (n = 30) were enrolled in this study. Malondialdehyde (MDA) concentrations in plasma and the activities of superoxide dismutase (SOD1; CuZnSOD), glutathione peroxidase (GPx) and catalase (CAT) in erythrocyte were determined as spectrophotometric. Also, the levels of Se, Zn and Cu in serum were determined on flame and furnace atomic absorption spectrophotometer using Zeeman background correction.Results and conclusionsOxidative stress was confirmed by the significant elevation in plasma MDA, and by the significant decrease in CAT, SOD1, and GPx (p < 0.05). When compared to controls, Zn and Se levels were significantly lower in patients, whereas Cu levels was higher in RAS patients than those in controls (p < 0.05). In addition, the correlation results of this study were firstly shown that there were significant and positive correlations between Se–CAT, Se–GPx, and Cu–MDA parameters, but negative correlations between Se–Cu, Se–MDA, Cu–CAT, Cu–SOD1 and Cu–GPx parameters in RAS patients. Furthermore, the ratios of Cu/Zn and Cu/Se were significantly higher in the patients than the control subjects (p < 0.05). Our results indicated that lipid peroxidation associated with the imbalance of the trace elements seems to play a crucial role in the pathogenesis of RAS. Furthermore, the serum Cu/Zn and Cu/Se ratios may be used as biochemical markers in these patients.     

Response of native grasses and Cicer arietinum to soil polluted with mining wastes: Implications for the management of land adjacent to mine sites

Mine tailings are an environmental problem in Southern Spain because wind and water erosion of bare surfaces results in the dispersal of toxic metals over nearby urban or agricultural areas. Revegetation with tolerant native species may reduce this risk. We grew two grasses, Lygeum spartum and Piptatherum miliaceum, and the crop species Cicer arietinum (chickpea) under controlled conditions in pots containing a mine tailings mixed into non-polluted soil to give treatments of 0%, 25%, 50%, 75% and 100% mine tailings. We tested a neutral (pH 7.4) mine tailings which contained high concentrations of Cd, Cu, Pb and Zn. Water-extractable metal concentrations increased in proportion to the amount of tailings added. The biomass of the two grasses decreased in proportion to the rate of neutral mine-tailing addition, while the biomass of C. arietinum only decreased in relation to the control treatment. Neutron radiography revealed that root development of C. arietinum was perturbed in soil amended with the neutral tailings compared to those of the control treatment, despite a lack of toxicity symptoms in the shoots. In all treatments and for all metals, the plants accumulated higher concentrations in the roots than in shoots. The highest concentrations occurred in the roots of P. miliaceum (2500 mg kg^?1 Pb, 146 mg kg^?1 Cd, 185 mg kg^?1 Cu, 2700 mg kg^?1 Zn). C. arietinum seeds had normal concentrations of Zn (70–90 mg kg^?1) and Cu (6–9 mg kg^?1). However, the Cd concentration in this species was ～1 mg kg^?1 in the seeds and 14.5 mg kg^?1 in shoots. Consumption of these plant species by cattle and wild fauna may present a risk of toxic metals entering the food chain.     

Salt effects on β-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl⁻ salts) the effectiveness follows the series: Cu⁺², Fe⁺² > Zn⁺² > Li⁺ > Ca⁺² > Mg⁺² > Cs⁺ > NH₄⁺ > Rb⁺ > K⁺ > Na⁺ and for anions (Na⁺ salts) the series is: I⁻ > ClO₄⁻ > ⁻SCN > Br⁻ ≈ NO₃⁻ > Cl⁻ ≈ ⁻OAc > F⁻ ≈ SO₄^− 2. The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k_cat/K_m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pK_as and not to an effect on the pH-independent second-order rate constant, (k_cat/K_m)^lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pK_a1 increases (from 3.7 to 4.9) and the apparent pK_a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (k_cat/K_m)^lim (= 9 × 10⁴ M^− 1 s^− 1) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pK_as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pK_a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pK_a of 3.7 and dissociated from the group with a pK_a of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two pK_as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (k_cat/K_m)^lim, of the monoprotonated species. For other enzymes which may show such salt-induced pK_a shifts, this provides a convenient test for the role of reverse protonation.     

Effect of the dietary oregano (Origanum vulgare) on Cu and Zn balance in weaned piglets

A 4-week study conducted on 20 weaned piglets (average initial weight 15 kg) evaluated the effects of dietary oregano (Origanum vulgare) used in the presence/absence of phytase on the Cu and Zn balance, while reducing/eliminating their inclusion in the diet as inorganic salts. Oregano was harvested from the wild flora. The Cu and Zn concentrations that were taken into consideration (9.85 ppm and 53.31 pmm, respectively) were the consensus values obtained in an interlaboratory study. The piglets were assigned to 4 groups (C, E1, E2, E3), housed in individual metabolic cages and fed on corn–soybean meal-based diets. The diet of the control group (C) with addition of 1% inorganic mineral premix (MP), contained: 40.92 ppm Cu, 144.96 ppm Zn. The experimental diets differed from the C diet as follows: E1 – 3% oregano, 0% phytase (5000 PU/g), 0% MP; E2 – 3% oregano, 0.01% phytase, 0% MP; E3 – 3% oregano, 0% phytase, 0.5% MP, E4 – 3% oregano, 0.01% phytase, 0,5% premix. For groups E1, E2, E3 and E4, 0.5% Zn of the MP were included in the diet, because the dietary oregano amount did not meet the requirements (NRC) for piglets. The mineral balance was determined during 3 periods of 5 days each. The levels of Cu and Zn were measured by FAAS in the samples (weekly samples/piglet) of ingesta, faeces and urine. It was noticed that although the dietary Cu ingested by the groups without MP was 75% (10.08 ppm) lower than C, the absorption coefficients were only 47% (28.83) lower than for group C (54.22%), while in the groups with 0.5% MP, the absorption was just 10% (48.86%) lower than for group C. For Zn, where the amount ingested by the experimental groups was 33% (97.62 ppm) lower than for group C, the absorption coefficients were just 20% (46.3%) lower than for group C (57.64%). No significant differences were noticed for Cu and Zn in terms of apparent absorption, between the groups with/without phytase. The deposits of Cu and Zn in the main organs and serum (from slaughtered piglets) were also evaluated.