The isatin-Schiff base copper(II) complex Cu(isaepy)(2) acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis

We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl) pyridine-N, N`] copper(II) [Cu(isaepy)(2)] was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)(2) to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)(2) increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to adenosine diphosphate. These data indicate that Cu(isaepy)(2) behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: (i) an early loss of mitochondrial transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (ATP) decrement. The causative energetic impairment mediated by Cu(isaepy)(2) in apoptosis is confirmed by experiments carried out with rho(0) cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity toward Cu(isaepy)(2). Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate: ATP ratio, is deeply involved in the apoptotic process because the overexpression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation, AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)(2)-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)(2) behaves as delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.

Spectroscopic evidences of the presence of hydrogenated species on the surface of copper during CO(2) electroreduction at low cathodic potentials

Carbon dioxide electroreduction on copper electrode was studied by surface enhanced Raman scattering (SERS) in K(2)SO(4) aqueous solutions with different pH values. CO(2) was bubbled into the solution at 0 V vs. Ag/AgCl, i.e., on an oxidized copper surface. In acidic solutions (pH around 2.5), at -0.2 V, bands indicative of the presence of ethylene on the electrode surface were detected. Although ethylene is knowledgably a product of CO(2) electroreduction on copper, it was not experimentally identified on the electrode`s surface at such a low cathodic potential in prior works. In solutions with pH around 2.5, CO bands were not observed, suggesting that hydrocarbons could be formed by a pathway that does not occur via adsorbed CO. In solutions with higher pHs, a complex spectral pattern, between 800 and 1700 cm(-1), was observed at approximately -0.4 V. The observed spectrum closely resembles those reported in the literature for adsorption of monocarboxylic acids with small chains. The spectral features indicate the presence of a structure containing a double C=C bond. a carboxyl group, and C-H bonds on the electrode`s surface. SERS spectra obtained in CO-saturated solution are also presented. However, in this case, no SERS bands were observed in the region between 800 and 1700 cm(-1) at low cathodic potentials. (c) 2009 Elsevier B.V. All rights reserved.

Peat as a natural solid-phase for copper preconcentration and determination in a multicommuted flow system coupled to flame atomic absorption spectrometry

The physical and chemical characteristics of peat were assessed through measurement of pH, percentage of organic matter, cationic exchange capacity (CEC), elemental analysis, infrared spectroscopy and quantitative analysis of metals by ICP OES. Despite the material showed to be very acid in view of the percentage of organic matter, its CEC was significant, showing potential for retention of metal ions. This characteristic was exploited by coupling a peat mini-column to a flow system based on the multicommutation approach for the in-line copper concentration prior to flame atomic absorption spectrometric determination. Cu(II) ions were adsorbed at pH 4.5 and eluted with 0.50 mol L(-1) HNO(3). The influence of chemical and hydrodynamic parameters, such as sample pH, buffer concentration, eluent type and concentration, sample flow-rate and preconcentration time were investigated. Under the optimized conditions, a linear response was observed between 16 and 100 mu g L(-1), with a detection limit estimated as 3 mu g L(-1) at the 99.7% confidence level and an enrichment factor of 16. The relative standard deviation was estimated as 3.3% (n = 20). The mini-column was used for at least 100 sampling cycles without significant variation in the analytical response. Recoveries from copper spiked to lake water or groundwater as well as concentrates used in hemodialysis were in the 97.3-111 % range. The results obtained for copper determination in these samples agreed with those achieved by graphite furnace atomic absorption spectrometry (GFAAS) at the 95% confidence level. (C) 2009 Elsevier B.V. All rights reserved.

Copper hexacyanoferrate nanoparticles modified electrodes: A versatile tool for biosensors

Copper hexacyanoferrate nanoparticles of about 30 nm in size have been prepared by the sonochemical irradiation of a mixture of aqueous potassium ferricyanide and copper chloride solutions. The nanoparticles were immobilized onto fluorine doped tin oxide (FTO) electrodes by using the electrostatic deposition layer-by-layer technique (LbL), obtaining electroactive films with electrocatalytic properties towards H2O2 reduction, providing higher currents than those observed for electrodeposited bulk material, even in electrolytes containing NH4+, Na+ and K+. The nanoparticles assembly was used as mediator in a glucose biosensor by immobilizing glucose oxidase enzyme by both, cross-linking and LbL. techniques. Sensitivities obtained were dependent on the immobilization method ranging from 1.23 mu A mmol(-1) L cm(-2) for crosslinking to 0.47 mu A mmol(-1) L cm(-2) for LbL; these values being of the same order than those obtained with electrodes where the amount of enzyme used is much higher. Moreover, the linear concentration range where the biosensors can operate was 10 times higher for electrodes prepared with the LbL immobilization method than with the conventional crosslinking one. (C) 2008 Elsevier B.V. All rights reserved.

The characterisation of the protective film formed by benzotriazole on the 90/10 copper-nickel alloy surface in H2SO4 media

The nature of the protective film formed by benzotriazole (BTAH) on the surface of the 90/10 CuNi alloy in deaerated 0.5 mol L-1 H2SO4 solution containing Fe(III) ions as oxidant was investigated by weight-loss, calorimetric measurements, and by surface-enhanced Raman spectroscopy (SERS). The SERS measurements show that the protective film is composed by the [Cu(I)BTA](n), polymeric complex and that the BTAH molecules are also adsorbed on the electrode surface. A modification of the BET isotherm for adsorption of gases ill solids is proposed to describe the experimental results obtained from weight-loss experiments that suggest an adsorption in multilayers. Electrochemical studies of copper and nickel in 0.5 mol L-1 H2SO4 in presence and absence of BTAH have also been made as an aid to interpret the results. The calculated adsorption free energy of the cuprous benzotriazolate on the surface of the alloy is in accordance with the value for pure copper. (C) 2007 Elsevier Ltd. All rights reserved.

Use of copper and gold electrodes as sensitive elements for fabrication of an electronic tongue: Discrimination of wines and whiskies

A low-cost method is proposed to classify wine and whisky samples using a disposable voltammetric electronic tongue that was fabricated using gold and copper substrates and a pattern recognition technique (Principal Component Analysis). The proposed device was successfully used to discriminate between expensive and cheap whisky samples and to detect adulteration processes using only a copper electrode. For wines, the electronic tongue was composed of copper and gold working electrodes and was able to classify three different brands of wine and to make distinctions regarding the wine type, i.e., dry red, soft red, dry white and soft white brands. Crown Copyright (C) 2011 Published by Elsevier B.V. All rights reserved.

Cadmium, copper, mercury, and zinc concentrations in tissues of the King Crab (Pseudocarcinus gigas) from southeast Australian waters

The concentrations of cadmium, copper, mercury, and zinc were determined in muscle (body, claw, and leg), hepatopancreas, and gill tissues of Pseudocarcinus gigas, an exceptionally large, long-lived, and deep-dwelling crab species. The accumulation patterns observed are discussed in terms of both intra- and interspecies variations, with particular attention to the possible consequences of the extreme size and depth range of P. gigas. Metal concentrations did not depend significantly on sex of the crab. Significant differences between tissues were detected for all metals, and the distribution of metal between the tissues was different for each metal. Significant correlations were found between metal concentrations in the various tissues and crab size, and these are discussed and rationalised. The concentrations of mercury and zinc in muscle tissue increased with crab size and were high compared to other crab species. The concentrations of cadmium and copper present in edible tissues were not especially high compared to other crab species, but the concentration of cadmium in the hepatopancreas is of dietary concern.

Copper-dependent iron assimilation pathway in the model photosynthetic eukaryote chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a valuable model for studying metal metabolism in a photosynthetic background. A search of the Chlamydomonas expressed sequence tag database led to the identification of several components that form a copper-dependent iron assimilation pathway related to the high-affinity iron uptake pathway defined originally for Saccharomyces cerevisiae. They include a multicopper ferroxidase (encoded by Fox1), an iron permease (encoded by Ftr1), a copper chaperone (encoded by Atx1), and a copper-transporting ATPase. A cDNA, Fer1, encoding ferritin for iron storage also was identified. Expression analysis demonstrated that Fox1 and Ftr1 were coordinately induced by iron deficiency, as were Atx1 and Fer1, although to lesser extents. In addition, Fox1 abundance was regulated at the posttranscriptional level by copper availability. Each component exhibited sequence relationship with its yeast, mammalian, or plant counterparts to various degrees; Atx1 of C. reinhardtii is also functionally related with respect to copper chaperone and antioxidant activities. Fox1 is most highly related to the mammalian homologues hephaestin and ceruloplasmin; its occurrence and pattern of expression in Chlamydomonas indicate, for the first time, a role for copper in iron assimilation in a photosynthetic species. Nevertheless, growth of C. reinhardtii under copper- and iron-limiting conditions showed that, unlike the situation in yeast and mammals, where copper deficiency results in a secondary iron deficiency, copper-deficient Chlamydomonas cells do not exhibit symptoms of iron deficiency. We propose the existence of a copper-independent iron assimilation pathway in this organism.

Creatine supplementation and riluzole treatment provide similar beneficial effects in copper, zinc superoxide dismutase (G93A) transgenic mice

This study investigated the effects of riluzole (Ril), creatine (Cr) and a combination of these treatments on the onset and progression of clinical signs and neuropathology in an animal model of familial amyotrophic lateral sclerosis, the G93A transgenic mouse (n=13–17 per group). The onset of clinical signs was delayed (P<0.05) by about 12 days in all treatment groups compared with control; however, no differences occurred between treatments. All animals were killed at 199 days of age. At the end of the experimental period the severity of clinical signs was less (P<0.05) with all treatments compared with control. Again no differences between treatments were observed. The treatments had no effect on the number of neurons in ventral horns of the lumbar region of the spinal cord. Transgenic mice ingesting Cr displayed elevated (P<0.05) total Cr levels in cerebral hemispheres (5%) and spinal cord (8%), but not skeletal muscles. These data demonstrate that treatment with Ril and Cr were both effective in delaying disease onset and clinical disability. To the age of killing, no additional benefit was conferred by co-administration of Ril and Cr.

Purification and membrane reconstitution of catalytically active Menkes copper-transporting P-type ATPase (MNK; ATP7A)

The MNK (Menkes disease protein; ATP7A) is a major copper- transporting P-type ATPase involved in the delivery of copper to cuproenzymes in the secretory pathway and the efflux of excess copper from extrahepatic tissues. Mutations in the MNK (ATP7A) gene result in Menkes disease, a fatal neurodegenerative copper deficiency disorder. Currently, detailed biochemical and biophysical analyses of MNK to better understand its mechanisms of copper transport are not possible due to the lack of purified MNK in an active form. To address this issue, we expressed human MNK with an N-terminal Glu-Glu tag in Sf9 [Spodoptera frugiperda (fall armyworm) 9] insect cells and purified it by antibody affinity chromatography followed by size-exclusion chromatography in the presence of the non-ionic detergent DDM (n-dodecyl b-D-maltopyranoside). Formation of the classical vanadate-sensitive phosphoenzyme by purified MNK was activated by Cu(I) [EC50=0.7 µM; h (Hill coefficient) was 4.6]. Furthermore, we report the first measurement of Cu(I)-dependent ATPase activity of MNK (K0.5=0.6 µM; h=5.0). The purified MNK demonstrated active ATP-dependent vectorial 64Cu transport when reconstituted into soya-bean asolectin liposomes. Together, these data demonstrated that Cu(I) interacts with MNK in a co-operative manner and with high affinity in the sub-micromolar range. The present study provides the first biochemical characterization of a purified full-length mammalian copper-transporting P-type ATPase associated with a human disease.

Functional studies on the Wilson copper P-Type ATPase and toxic milk mouse mutant

The Wilson protein (WND; ATP7B) is an essential component of copper homeostasis. Mutations in the ATP7B gene result in Wilson disease, which is characterised by hepatotoxicity and neurological disturbances. In this paper, we provide the first direct biochemical evidence that the WND protein functions as a copper-translocating P-type ATPase in mammalian cells. Importantly, we have shown that the mutation of the conserved Met1386 to Val, in the Atp7B for the mouse model of Wilson disease, toxic milk (tx), caused a loss of Cu-translocating activity. These investigations provide strong evidence that the toxic milk mouse is a valid model for Wilson disease and demonstrate a link between the loss of catalytic function of WND and the Wilson disease phenotype.

Complexes of the tripodal nitrilotrimethylenetrisphosphonic (H6L) and P,P',P''-triphenylnitrilotrimethylenetrisphosphinic (H3L) acids with the copper(II) ion. Synthesis and charaterization of [Hpy][Cu(H3L)(H2O)] and [Cu(HL)(py)]2.2Me2CO

The complexes [Hpy][Cu(H₃L)(H2O)] 1 (L⁶ = nitrilotrimethylenetrisphosphonate) and [Cu(HL°)(py)]₂·2Me₂CO 2 [(L°)³ = P,P,P" -triphenylnitrilotrismethylenetrisphosphinate)] have been isolated and characterized by X-ray crystallography, near IR-visible spectroscopy and magnetic measurements. The structure determination has shown the complexes to be constituted by monomeric and dimeric units respectively. In the monomer the metal atom is surrounded by the phosphonate ligand and a water molecule, with a geometry between a trigonal bipyramid and a square pyramid. The two copper atoms in the dimer are held together by an arm of the tripod ligand, with a pyridine molecule as additional ligand, and display octahedral geometry. The presence of monomeric and dimeric species in aqueous solutions of 1 and 2 has been shown by ESMS studies. The formation in water solution of the dimer [{Cu(H₃L)}₂]^2-, as a minor species, has been supported by potentiometric measurements, whereas only the monomeric anion [CuL°] has been ascertained to be present. In general the ligand H₃L° forms less stable copper(II) complexes than H₆L.

Endosomal trafficking of the Menkes copper ATPase ATP7A is mediated by vesicles containing the Rab7 and Rab5 GTPase proteins

The Cu-ATPase ATP7A (MNK) is localized in the trans-Golgi network (TGN) and relocalizes in the plasma membrane via vesicle-mediated traffic following exposure of the cells to high concentrations of copper. Rab proteins are organelle-specific GTPases, markers of different endosomal compartments; their role has been recently reviewed (Trends Cell Biol. 11(2001) 487). In this article we analyze the endosomal pathway of trafficking of the MNK protein in stably transfected clones of CHO cells, expressing chimeric Rab5-myc or Rab7-myc proteins, markers of early or late endosome compartments, respectively. We demonstrate by immunofluorescence and confocal and electron microscopy techniques that the increase in the concentration of copper in the medium (189 μM) rapidly induces a redistribution of the MNK protein from early sorting endosomes, positive for Rab5-myc protein, to late endosomes, containing the Rab7-myc protein. Cell fractionation experiments confirm these results; i.e., the MNK protein is recruited to the endosomal fraction on copper stimulation and colocalizes with Rab5 and Rab7 proteins. These findings allow the first characterization of the vesicles involved in the intracellular routing of the MNK protein from the TGN to the plasma membrane, a key mechanism allowing appropriate efflux of copper in cells grown in high concentrations of the metal.

Copper transport during lactation in transgenic mice expressing the human ATP7A protein

Both copper transporting ATPases, ATP7A and ATP7B, are expressed in mammary epithelial cells but their role in copper delivery to milk has not been clarified. We investigated the role of ATP7A in delivery of copper to milk using transgenic mice that over-express human ATP7A. In mammary gland of transgenic mice, human ATP7A protein was 10- to 20-fold higher than in control mice, and was localized to the basolateral membrane of mammary epithelial cells in lactating mice. The copper concentration in the mammary gland of transgenic dams and stomach contents of transgenic pups was significantly reduced compared to non-transgenic mice. The mRNA levels of endogenous Atp7a, Atp7b, and Ctr1 copper transporters in the mammary gland were not altered by the expression of the ATP7A transgene, and the protein levels of Atp7b and ceruloplasmin were similar in transgenic and non-transgenic mice. These data suggest that ATP7A plays a role in removing excess copper from the mammary epithelial cells rather than supplying copper to milk.