pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+

Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5-6.75 (Kd approximately 4 nM), whereas a Kd approximately 20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 microM total Cu2+) on maximal activity regenerated was negligible at pH 6.25-8.0. However, this level of Cu2+ increased the inter-chain affinity by approximately 5-fold at pH 7.25. This effect resulted from an approximately 1.5-fold increased association rate constant (k(on)) and an approximately 3-fold reduced dissociation rate constant (k(off)). High affinity (Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.     

Influence of Ca, humic acid and pH on lead accumulation and toxicity in the fathead minnow during prolonged water-borne lead exposure

The present study examines the influence of Ca2+ as (CaSO4), dissolved organic carbon (DOC) and pH on chronic water-borne lead (Pb) toxicity to the larval fathead minnow (Pimephales promelas) under flow-through conditions. The 30 day LC50 for low hardness basic test water (19 mg CaCO3 L(-1)) was 39 (range: 27-51) microg dissolved Pb L(-1) and was greatly increased by increasing concentrations of CaSO4 and DOC to as much as 1903 (range: 1812-1992) mug dissolved Pb L(-1). Both reduced and increased pH (6.7 and 8.1, respectively) compared to control pH of 7.4 appeared to increase Pb toxicity substantially. Whole body Pb accumulation did not reflect water chemistry and thus exhibited no correlation with Pb induced mortality. One possible explanation for this lack of correlation is that mortality occurred predominantly during the first 4-6 days of exposure, whereas Pb accumulation was determined in surviving fish at the end of 30 days of exposure. Chronic Pb exposure resulted in a general iono-regulatory disturbance affecting K+, Na+ and Ca2+ homeostasis. However, recovery of Na+ and K+ levels and reversal of effects on Ca2+ homeostasis during continued exposure strongly suggest fathead minnow can acclimate to Pb. The gills accumulate the highest Pb concentrations during chronic exposure but the skeleton contains the largest mass of Pb by contributing up to approximately 80% of whole body Pb. In conclusion, water chemistry characteristics like Ca2+ and DOC should be considered for chronic water quality criteria.     

Toxic Effects of Three Heavy Metallic Ions on Rana zhenhaiensis Tadpoles

Heavy metal pollution is widespread in some areas of China and results in contamination of land, water, and air with which all living organisms interact. In this study, we used three heavy metallic ions(Cu2+, Pb2+ and Zn2+) to assess their toxicity effects on mortality, blood biomarker and growth traits(body length and body mass) of Rana zhenhaiensis tadpoles. The results showed that the toxicity levels of the three metallic ions were different when conducted with different experiment designs. For acute toxicity tests, Cu2+ was the most toxic with the highest tadpole mortality. The mortalities of tadpoles showed significant differences among the treatments at the same exposure time endpoints(24, 48, 72 and 96h). Results from repeated measures ANOVA indicated that metallic ion concentration, exposure time and their interactions significantly affected the mortalities of R. zhenhaiensis tadpoles. Also, the toxicity effects of all binary combinations of the three metallic ion treatments showed synergism. The half lethal concentrations(LC50) decreased with increasing exposure time during the experimental period, and the safe concentration(SC) values of Cu2+, Pb2+ and Zn2+ were different from each other. Combined and compared LC50 values with previous data reported, it is suggestes that the toxicity levels of metal pollution to anuran tadpoles should be species-and age-related. For blood biomarker tests, Zn2+ was the most toxic with the highest total frequencies of abnormal erythrocytic nucleus. All three metallic ions caused higher abnormal erythrocytic nucleus compared with control groups. In a chronic toxicity test, Pb2+ was the most toxic with lowest growth traits. Survival rate(except for 18 days), total body length and body mass showed significant differences among the treatments. These findings indicated that tadpoles of R. zhenhaiensis should be as a bioindicator of heavy metals pollution.     

Effects of water hardness and Cu and Zn on LC50 in Gambusia holbrooki

Abstract

The aim of this study was to determine by static bioassay whether water hardness affects the toxicity of Zn and Cu to a fish, Gambusia holbrooki Girard, 1859. The acute toxicity of selected heavy metals to G. holbrooki was determined in soft, hard and very hard water (25, 125 and 350 mg L^?1 as CaCO₃). Results showed that water hardness had a significant effect on Cu and on Zn toxicity on fish. Toxicity of Cu and of Zn increased with decreasing water hardness. The 96 hours LC₅₀ values for G. holbrooki were higher in the hard and very hard water compared with soft water. Water hardness had a much smaller effect upon the acute toxicity of Cu than that of Zn. It was observed that the 96 hours LC50 for Cu at the soft, hard and very hard water was found to be 0.017, 0.17 and 0.65 mg L^?1, respectively, while the 96 hours LC₅₀ for Zn at the soft, hard and very hard water was found to be 0.46, 48.1 and 121.6 mg L^?1, respectively.     

The binding of cupric ions to 1-carboxymethylhistidine-119-ribonuclease

Binding of Cu(2+) by 1-carboxymethylhistidine-119-ribonuclease was investigated by using diligand metal ion buffers. A single Cu(2+)-binding site was found over the Cu(2+) concentration range studied. The binding constants for this site were 8.33x10(5) (+/-2%)m(-1) and 1.57x10(4) (+/-6%)m(-1) at pH7.0 and 6.1 respectively. An estimate of the pH-independent Cu(2+)-binding constant suggests that the most avid Cu(2+)-binding site has disappeared after carboxymethylation. This is consistent with an earlier report that binding of Cu(2+) at the most avid site is associated with the loss of enzymic activity.     

Effects of water hardness and alkalinity on the toxicity of uranium to a tropical freshwater hydra (Hydra viridissima)

In tropical Australian freshwaters, uranium (U) is of potential ecotoxicological concern, largely as a consequence of mining activities. Although the toxicity of uranium to Australian freshwater biota is comprehensive, by world standards, few data are available on the effects of physicochemical variables, such as hardness, alkalinity, pH and organic matter, on uranium speciation and bioavailability. This study determined the individual effects of water hardness (6.6, 165 and 330 mg l^-1 as CaCO₃) and alkalinity (4.0 and 102 mg l^-1 as CaCO₃), at a constant pH (6.0), on the toxicity (96 h population growth) of uranium to Hydra viridissima (green hydra). A 50-fold increase in hardness (Ca and Mg concentration) resulted in a 92% (two-fold) decrease in the toxicity of uranium to H. viridissima [i.e . an increase in the EC₅₀ value and 95% confidence interval from 114 (107-121) to 219 (192-246) µg l^-1]. Conversely, at a constant hardness (165 mg l-1 as CaCO₃), the toxicity of uranium to H. viridissima was not significantly (P > 0.05) affected by a 25-fold increase in alkalinity (carbonate concentration) [i.e. EC₅₀ values of 177 (166-188) and 171 (150-192) µg l^-1 at 4.0 and 102 mg l^-1 as CaCO₃, respectively]. A knowledge of the relationship between water chemistry variables, including hardness and alkalinity, and uranium toxicity is useful for predicting the potential ecological detriment in aquatic systems, and can be used to relax national water quality guidelines on a site-specific basis.     

Studies of the Ca2+ transport mechanism of human erythrocyte inside-out plasma membrane vesicles. I. Regulation of the Ca2+ pump by calmodulin

Calcium accumulation by human erythrocyte inside-out vesicles was linear for at least 30 min in the presence of ATP. In untreated inside-out vesicles, 3.76 +/- 1.44 nmol of calcium/min/unit of acetylcholinesterase were transported, compared with 10.57 +/- 2.05 (+/- S.D.; n = 11) in those treated with calmodulin. The amount of calmodulin necessary for 50% activation of Ca2+ accumulation was 60 +/- 22 ng/ml (+/- S.D.; n = 4). The Km (Ca2+) for calmodulin-stimulated accumulation was 0.8 +/- 0.05 microM (+/- S.D.; n = 5) using Ca2+ /ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) buffers, or 25 microM with direct addition of unbuffered calcium. In the absence of calmodulin, these values were 0.4 and 60 microM, respectively, Km (ATP) values of 90 and 60 microM in the presence and absence of calmodulin, respectively, were measured at constant magnesium concentration (3 mM). In the presence of calmodulin, a broad pH profile is exhibited from pH 6.6 to 8.2. Maximal calcium accumulation occurs at pH 7.8. In the absence of calmodulin, the pH profile exhibits a linear upward increase from pH 7.0 to 8.2. The (Ca2+-Mg2+)-ATPase activity, measured under identical conditions, was 2.40 +/- 0.72 nmol of Pi/min/unit of acetylcholinesterase in the untreated vesicles and 11.29 +/- 2.87 nmol of Pi/min/unit of acetylcholinesterase (+/- S.D.; n = 4) in calmodulin-treated vesicles. A stoichiometry of 1.6 Ca2+/ATP hydrolyzed was determined in the absence of calmodulin; in the presence of calmodulin, this ratio was decreased to 0.94 Ca2+/ATP hydrolyzed.     

Refinement and field validation of a biotic ligand model predicting acute copper toxicity to Daphnia magna

A previously developed biotic ligand model (BLM) was validated for its capacity to predict acute 48-h EC(50) values of copper to Daphnia magna in 25 reconstituted media with different pH values and concentrations of artificial dissolved organic carbon, Ca, Mg and Na. Before the BLM validation, fitting of measured (with a copper ion-selective electrode) and calculated (with the BLM) Cu(2+)-activity was performed by adjusting the WHAM model V (i.e. the metal-organic speciation part of the BLM) copper-proton exchange constant to pK(MHA)=1.9. Using this value, the 48-h EC(50) values observed agreed very well with BLM-predicted EC(50) values for tests performed at pH<8, but not at all for tests performed at pH>8. Additional experiments demonstrated that this was due to toxicity of the CuCO(3) complex, which is the most abundant inorganic copper species at pH>8. This was incorporated into the initial BLM by allowing the binding of CuCO(3) (next to Cu(2+) and CuOH(+)) to the biotic ligand of D. magna. The affinity of CuOH(+) and CuCO(3) for the biotic ligand was approximately five- and 10-fold lower than that of Cu(2+), respectively. With the refined BLM, 48-h EC(50) values could be accurately predicted within a factor of two not only in all 25 reconstituted media, but also in 19 natural waters. This validated and refined BLM could support efforts to improve the ecological relevance of risk assessment procedures applied at present.     

Interaction between pesticides and essential metal copper increases the accumulation of copper in the kidneys of rats

Male Sprague-Dawley rats, weighing 175-200 g, six per group were fed AIN 93M diet (CON) or diets containing 500 mg Ca (LCa), 7 mg Zn (LZn), 2 mg Cu (LCu), 60 mg Zn (HZn), or 12 mg Cu (HCu) per kilogram of diet in the following combinations: control (CON), LCa+LZn (LC+Z), LCa+LZn+LCu (LC+Z+C), or HZn+HCu (HZ+C) without or with a pesticides mixture (PM); Endosulfan, Thiram, and Acephate were added to the diets at 25% of LD50/kg. Rats were fed for 2 wk (small intestinal changes) or 4 wk (tissues changes). Plasma Zn was 47% lower than CON in the experimental groups. Plasma Cu and ceruloplasmin concomitantly decreased in the LC+Z+C group and increased with the addition of PM. Kidney Cu was 40% lower in LC+Z+C group, than CON and increased by 31% with PM; in the HZ+C+PM group, kidney Cu was 38% higher than the HZ+C group. Mucosal and small intestines Ca declined by 47% in all experimental groups; PM increased Zn in the LC+Z+C and HZ+C groups; PM further decreased intestinal and mucosal Cu retention in the LC+Z+C and HZ+C groups. Data suggest that low levels of PM in the diet can induce Cu accumulation in the kidney when dietary Zn and Cu are low or high.     

The interactions of water hardness and pH with the acute toxicity of zinc to the brown trout, Salmo trutta L.

Exposure of brown trout, Salmo trutta , to zinc under continuous flow conditions over 96 h showed that both water hardness and pH exert major influences on the toxicity of the metal. 96-h LC50 values for total zinc ranged from <0.14mg 1⁻¹ in alkaline soft water (pH 8; lOmg 1⁻¹ as CaCO₃) to 3.20 mg 1⁻¹ in acidic hard water (pH 5; 204 mg 1⁻¹ as CaCO₃). A variable reduction in zinc toxicity in hard water compared with soft water over the pH range 4–9 was attributed to high external calcium. Zinc toxicity was positively correlated with decreasing acidity over the pH range 5–7, the metal being most toxic at pH 8–9 where metal complexes predominate. Below pH 5 metal toxicity also increased, irrespective of hardness. Water hardness and pH interacted with zinc toxicity in a complex manner, apparently dependent on physical and chemical transformations of the metal, and as changes in uptake. detoxification and excretion by the fish.     

Effects of water hardness and alkalinity on the toxicity of uranium to a tropical freshwater hydra (Hydra viridissima)

In tropical Australian freshwaters, uranium (U) is of potential ecotoxicological concern, largely as a consequence of mining activities. Although the toxicity of uranium to Australian freshwater biota is comprehensive, by world standards, few data are available on the effects of physicochemical variables, such as hardness, alkalinity, pH and organic matter, on uranium speciation and bioavailability. This study determined the individual effects of water hardness (6.6, 165 and 330 mg l^-1 as CaCO₃) and alkalinity (4.0 and 102 mg l^-1 as CaCO₃), at a constant pH (6.0), on the toxicity (96 h population growth) of uranium to Hydra viridissima (green hydra). A 50-fold increase in hardness (Ca and Mg concentration) resulted in a 92% (two-fold) decrease in the toxicity of uranium to H. viridissima [i.e. an increase in the EC₅₀ value and 95% confidence interval from 114 (107-121) to 219 (192-246) µg l^-1]. Conversely, at a constant hardness (165 mg l-1 as CaCO₃), the toxicity of uranium to H. viridissima was not significantly (P &gt; 0.05) affected by a 25-fold increase in alkalinity (carbonate concentration) [i.e. EC₅₀ values of 177 (166-188) and 171 (150-192) µg l^-1 at 4.0 and 102 mg l^-1 as CaCO₃, respectively]. A knowledge of the relationship between water chemistry variables, including hardness and alkalinity, and uranium toxicity is useful for predicting the potential ecological detriment in aquatic systems, and can be used to relax national water quality guidelines on a site-specific basis.     

Acute, sublethal and combination effects of azadirachtin and Bacillus thuringiensis toxins on Helicoverpa armigera (Lepidoptera: Noctuidae) larvae

The efficacy of neem (1500 ppm azadirachtin (AI)), Delfin WG, a biological insecticide based on selected strain of Bacillus thuringiensis Berliner (Bt) subspecies kurstaki, and Cry1Ac protein, either individually or in combination, were examined against first to fourth instar Helicoverpa armigera (Hübner) larvae. Using an oral administration method, various growth inhibitory concentrations (EC) and lethal concentrations (LC) were determined for each bioagent. Combinations of sublethal concentrations of Bt spray formulation with azadirachtin at EC50 or EC95 levels not only enhanced the toxicity, but also reduced the duration of action when used in a mixture. The LC20 and LC50 values for Cry1Ac toxin were 0.06 and 0.22 microg ml-1, respectively. Bt-azadirachtin combinations of LC50+EC20 and LC50+EC50 result in 100% mortality. The mortality also was significant in LC20+EC20 and LC20+EC50 mixtures. These studies imply that the combined action is not synergistic but complimentary, with azadirachtin particularly facilitating the action of Bt. The Bt spray-azadirachtin combination is more economical than combinations that involve isolating the toxic protein, as the Bt spray formulations can be combined in a spray mixture with neem. These combinations may be useful for controlling bollworm populations that have acquired resistance to Bt as they may not survive the effect of mixture. Azadirachtin may be useful as a means of reducing the endotoxin concentrations in a mixture, to promote increased economic savings and further reduce the probability of resistance development to either insect control agent.     

The toxicity and physiological effects of copper on the freshwater pulmonate snail, Lymnaea stagnalis

Several recent studies have demonstrated that the freshwater pulmonate snail Lymnaea stagnalis is extremely sensitive to metals (Co, Ni, Pb) in chronic exposures. The objective of the current study was to evaluate the acute and chronic sensitivity of L. stagnalis to Cu and investigate the underlying mechanism(s) of toxic action. A 96-h LC50 of 31μg L(-1) Cu was estimated indicating L. stagnalis was moderately acutely sensitive to Cu relative to other aquatic organisms. However, in a 30-day chronic exposure using juvenile snails an EC20 of 1.8μg L(-1) Cu was estimated for snail growth making L. stagnalis the most sensitive organism tested to date for Cu. Hardness-based and BLM-based water quality criteria for Cu at the water quality conditions used in this study were 7.8 and 1.5μg L(-1), respectively, indicating L. stagnalis is significantly under-protected by hardness-based WQC. Investigations into the mechanism(s) of toxic action for Cu were conducted on young adult snails necessitating higher Cu exposures. Exposure to Cu at 12μg L(-1) resulted in no detectable effects on hemolymph osmolality, net Ca(2+) uptake, titratable acid excretion, or ammonia excretion. Exposure to 48μg L(-1) Cu was shown to significantly reduce (91%) net Ca(2+) uptake which is strongly correlated with shell deposition and corresponding snail growth. Snails exposed to 48μg L(-1) Cu also exhibited reduced ammonia excretion, a marked hemolymph acidosis, and a compensatory increase in titratable acid excretion. The reduction in net Ca(2+) uptake was hypothesized to be a secondary effect of Cu-induced inhibition of carbonic anhydrase, but no reduction in carbonic anhydrase activity was detected. Overall, it remains unclear whether inhibition of Ca(2+) uptake is a direct result of Cu exposure or, along with the other observed physiological effects, is secondary to an unidentified primary mode of toxic action. Given the hypersensitivity of L. stagnalis to Cu, further study into the mechanisms of action and effects of varying water chemistry on Cu toxicity is clearly warranted.     

Effects of copper on growth, reproduction, survival and haemoglobin in Daphnia magna

The effects of additions of CuSO4 X 5H2O to final concentrations between 0.0004 and 105 micrograms Cu l-1 on growth, reproduction, survival and haemoglobin content of Daphnia magna were studied in hard reconstituted water and compared to the response in the dilution water without addition of copper. Concentrations of copper are nominal values. The 48-hr EC50 (immobilization) for unfed neonates was 6.5 micrograms Cu l-1 and the 48-hr and 21-day LC50 for fed neonates were 18.5 and 1.4 microgram Cu l-1, respectively. Growth expressed as body length of juveniles after 7 days and adult females after 21 days was only reduced in survivors at the highest non-lethal concentration (6.6 micrograms Cu l-1). Reproduction was stimulated by low concentrations of copper. Optimal reproduction after 21 days was found between 0.001 and 0.1 microgram Cu l-1. Higher concentrations were partially inhibitory (0.4 microgram Cu l-1), stimulatory (0.8 and 1.6 microgram Cu l-1) or completely inhibitory (3.2 micrograms Cu l-1 and above). The stimulatory peak around 1 microgram Cu l-1 was accompanied by a reduced survival (above 0.4 microgram Cu l-1). The Zero Equivalent Point (ZEP) for reproduction at non-reduced survival was 0.23 microgram Cu l-1. This concentration should be "safe" for D. magna under prevailing conditions (reconstituted water with a hardness of 250 mg l-1 as CaCo3 and a synthetic diet based on fish food and baby gruel). The haemoglobin content was affected by copper in a complex pattern which was not related to growth, reproduction or survival.     

Derivation of a toxicity-based model to predict how water chemistry influences silver toxicity to invertebrates

The effect of altering water chemistry on acute silver toxicity to three invertebrate species, two Daphnids, Daphnia magna and Daphnia pulex, as well as an amphipod Gammarus pulex was assessed. In addition, the physiological basis of Ag(I) toxicity to G. pulex was examined. Daphnia magna and D. pulex were more sensitive than G. pulex and 48 h LC(50) values in synthetic ion poor water were 0.47, 0.65 and 2.1 microg Ag(I) l(-1), respectively. Increasing water [Cl(-)] reduced Ag(I) toxicity in all species, and increasing water [Ca(2+)] from 50 to 1,500 microM reduced Ag(I) toxicity in G. pulex. Whole body Na(+) content, but not K(+) or Ca(2+) was significantly reduced in G. pulex exposed to 6 microg Ag(I) l(-1) for 24 h, but there was no inhibition of whole body Na(+)/K(+)-ATPase activity. Both increasing water [Cl(-)] and [Ca(2+)] reduced this Ag(I)-induced Na(+) loss. For D. magna, the presence of 10 mg l(-1) humic acid or 0.5 microM 3-mercaptoproprionic acid (3-MPA) increased the 48 h LC(50) values by 5.9 and 58.5-fold, respectively, and for D. pulex the presence of 1 microM thiosulfate increased the 48 h LC(50) value by four-fold. The D. magna toxicity data generated from this study were used to derive a Daphnia biotic ligand model (BLM). Analysis of the measured LC(50) values vs. the predicted LC(50) values for toxicity data from the present and published results where water Cl(-), Ca(2+), Na(+) or humic acid were varied showed that 91% of the measured toxicity data fell within a factor of two of the predicted LC(50) values. However, the daphnid BLM could not accurately predict G. pulex toxicity. Additionally, the Daphnia BLM was under-protective in the presence of the organic thiols 3-MPA or thiosulphate and predicted an increase in the LC(50) value of 114- and 74-fold, respectively. The Daphnia toxicity based BLM derived from the present data set is successful in predicting Daphnia toxicity in laboratory data sets in the absence of sulfur containing compounds, but shows its limitations when applied to waters containing organic thiols or thiosulphate.     

Electron paramagnetic resonance evidence for binding of Cu(2+) to the C-terminal domain of the murine prion protein.

Transmissible spongiform encephalopathies in mammals are believed to be caused by scrapie form of prion protein (PrP(Sc)), an abnormal, oligomeric isoform of the monomeric cellular prion protein (PrP(C)). One of the proposed functions of PrP(C) in vivo is a Cu(II) binding activity. Previous studies revealed that Cu(2+) binds to the unstructured N-terminal PrP(C) segment (residues 23-120) through conserved histidine residues. Here we analyzed the Cu(II) binding properties of full-length murine PrP(C) (mPrP), of its isolated C-terminal domain mPrP(121-231) and of the N-terminal fragment mPrP(58-91) in the range of pH 3-8 with electron paramagnetic resonance spectroscopy. We find that the C-terminal domain, both in its isolated form and in the context of the full-length protein, is capable of interacting with Cu(2+). Three Cu(II) coordination types are observed for the C-terminal domain. The N-terminal segment mPrP(58-91) binds Cu(2+) only at pH values above 5.0, whereas both mPrP(121-231) and mPrP(23-231) already show identical Cu(II) coordination in the pH range 3-5. As the Cu(2+)-binding N-terminal segment 58-91 is not required for prion propagation, our results open the possibility that Cu(2+) ions bound to the C-terminal domain are involved in the replication of prions, and provide the basis for further analytical studies on the specificity of Cu(II) binding by PrP.     

Acute toxicity,critical body residues,Michaelis–Menten analysis of bioaccumulation,and ionoregulatory disturbance in response to waterborne nickel in four invertebrates: Chironomus riparius, Lymnaea stagnalis, Lumbriculus variegatus and Daphnia pulex

We investigated the bioaccumulation and acute toxicity (48 h or 96 h) of Ni in four freshwater invertebrate species in two waters with hardness of 40 (soft water) and 140 mg L^− 1 as CaCO₃ (hard water). Sensitivity order (most to least) was Lymnaea stagnalis > Daphnia pulex > Lumbriculus variegatus > Chironomus riparius. In all cases water hardness was protective against acute Ni toxicity with LC50 values 3–3.5 × higher in the hard water vs. soft water. In addition, higher water hardness significantly reduced Ni bioaccumulation in these organisms suggesting that competition by Ca and Mg for uptake at the biotic ligand may contribute to higher metal resistance. CBR50 values (Critical Body Residues) were less dependent on water chemistry (i.e. more consistent) than LC50 values within and across species by ~ 2 fold. These data support one of the main advantages of the Tissue Residue Approach (TRA) where tissue concentrations are generally less variable than exposure concentrations with respect to toxicity. Whole body Ni bioaccumulation followed Michaelis–Menten kinetics in all organisms, with greater hardness tending to decrease B_max with no consistent effect on K_d. Across species, acute Ni LC50 values tended to increase with both K_d and B_max values — i.e. more sensitive species exhibited higher binding affinity and lower binding capacity for Ni, but there was no correlation with body size. With respect to biotic ligand modeling, log K_NiBL values derived from Ni bioaccumulation correlated well with log K_NiBL values derived from toxicity testing. Both whole body Na and Mg levels were disturbed, suggesting that disruption of ionoregulatory homeostasis is a mechanism of acute Ni toxicity. In L. stagnalis, Na depletion was a more sensitive endpoint than mortality, however, the opposite was true for the other organisms. This is the first study to show the relationship between Na and Ni.     

Synchronous fluorescence, UV-visible spectrophotometric, and voltammetric studies of the competitive interaction of bis(1,10-phenanthroline)copper(II) complex and neutral red with DNA

Constant wavelength synchronous fluorescence spectroscopy (CW-SFS), UV-visible absorption spectroscopy, and cyclic and differential pulse voltammetry were applied to investigate the competitive interaction of DNA with the bis(1,10-phenanthroline)copper(II) complex cation ([Cu(phen)(2)](2+)) and a fluorescence probe, neutral red dye (NR), in a tris-hydrogen chloride buffer (pH 7.4). The results show that both the [Cu(phen)(2)](2+)and the NR molecules can intercalate competitively into the DNA double-helix structure. The cyclic voltammetry method showed that both anodic and cathodic currents of [Cu(phen)(2)](2+) decreased on addition of the DNA and the intercalated [Cu(phen)(2)](2+)-DNA complex formed (beta = (4.14 +/- 0.24) x 10(3)). CW-SFS measurements were facilitated by the use of the three-way resolution of the CW-SFS for NR, [Cu(phen)(2)](2+), and NR-DNA. The important constant wavelength (CW) interval, Deltalambda, was shown to vary considerably when optimized (135, 58, and 98 nm for NR, NR-DNA, and [Cu(phen)(2)](2+), respectively). This approach clearly avoided the errors that otherwise would have arisen from the common assumption that Deltalambda is constant. Furthermore, a chemometrics approach, parallel factor analysis (PARAFAC), was applied to resolve the measured three-way CW-SFS data, and the results provided simultaneously the concentration information for the three reaction components, NR, [Cu(phen)(2)](2+), and NR-DNA, for the system at each equilibrium point. The PARAFAC analysis indicated that the intercalation of the [Cu(phen)(2)](2+) molecule into the DNA proceeds by exchanging with the NR probe and can be attributed to two parallel reactions. Comprehensive information was readily obtained; the replacement of the intercalated NR commenced immediately on introduction of [Cu(phen)(2)](2+), approximately 50% of NR was replaced by [Cu(phen)(2)](2+) at a concentration of 0.45 x 10(-5) mol L(-1), and nearly all of the NR was replaced at a [Cu(phen)(2)](2+) concentration of 2.50 x 10(-5) mol L(-1). This work has the potential to improve extraction of information from the fluorescence intercalator displacement (FID) assay.     

Effect of different concentrations of heavy metal ions on the normal physical and chemical characteristics of the hemolymph of Palanorbarius purpura (Mollusca: Bulinidae) in the norm and during a trematode infection

Short-term simultaneous effect of high concentrations (LC25, LC50, LC75) of heavy metal ions. (Cu2+, Zn2+, Cd2+ and Pb2+) and infection with trematode partenites Echinoparyphium aconiatum onto haemolymph of mollusk has been investigated. It was noted that low doses of toxicant (2.5 and 10 maximum admitted concentrations) have variable effect. In infected molluscs the concentration of haemoglobin decreases, while in intact ones it increases. In relation to this index, it was found, that the ion Cu2+ is highly toxic, Zn2+ and Cd(2+)--toxic, Pb(2+)--moderately toxic.     

Isolation and characteristics of scallop sarcoplasmic reticulum with calcium transport activity.

Sarcoplasmic reticulum with calcium transport activity has been isolated from the cross-striated adductor muscle of the scallop, which lives in cold (< or = 20 degrees C) sea water, by using pH 7.0 buffer solution both to homogenize the tissue and to sediment the membrane fraction. The yield of the preparation was 60-100 mg protein from 100 g of the scallop muscle. Ca(2+)-activated ATPase protein of about 100 kDa accounted for 40-50% of the protein preparation. The maximum activities of ATP-dependent, oxalate-facilitated calcium accumulation and Ca(2+)-ATPase were observed at a pH of about 7.0 and temperature of 20-30 degrees C, and their values were about 2 mumol Ca2+/mg of protein/min and about 3 mumol ATP hydrolysis/mg of protein/min, respectively. At 0 degree C, 10-20% of these activities was maintained, while at 37 degrees C, the activities were irreversibly lost. The Ca(2+)-ATPase activity was half-maximally activated at about 0.3 microM [Ca2+]. The ATPase activity exhibited non-Michaelian behavior with respect to ATP, with two different Km values of approximately 10 microM and 0.1-0.3 mM. GTP, CTP, and ITP were also hydrolyzed by the preparation at a rate of 10-30% of that of ATP. The preparation was stored at -80 degrees C with retention of function for about a year.