Bacterial killing by dry metallic copper surfaces

Metallic copper surfaces rapidly and efficiently kill bacteria. Cells exposed to copper surfaces accumulated large amounts of copper ions, and this copper uptake was faster from dry copper than from moist copper. Cells suffered extensive membrane damage within minutes of exposure to dry copper. Further, cells removed from copper showed loss of cell integrity. Acute contact with metallic copper surfaces did not result in increased mutation rates or DNA lesions. These findings are important first steps for revealing the molecular sensitive targets in cells lethally challenged by exposure to copper surfaces and provide a scientific explanation for the use of copper surfaces as antimicrobial agents for supporting public hygiene.     

Handling of Copper and Copper Oxide Nanoparticles by Astrocytes

Copper is an essential trace element for many important cellular functions. However, excess of copper can impair cellular functions by copper-induced oxidative stress. In brain, astrocytes are considered to play a prominent role in the copper homeostasis. In this short review we summarise the current knowledge on the molecular mechanisms which are involved in the handling of copper by astrocytes. Cultured astrocytes efficiently take up copper ions predominantly by the copper transporter Ctr1 and the divalent metal transporter DMT1. In addition, copper oxide nanoparticles are rapidly accumulated by astrocytes via endocytosis. Cultured astrocytes tolerate moderate increases in intracellular copper contents very well. However, if a given threshold of cellular copper content is exceeded after exposure to copper, accelerated production of reactive oxygen species and compromised cell viability are observed. Upon exposure to sub-toxic concentrations of copper ions or copper oxide nanoparticles, astrocytes increase their copper storage capacity by upregulating the cellular contents of glutathione and metallothioneins. In addition, cultured astrocytes have the capacity to export copper ions which is likely to involve the copper ATPase 7A. The ability of astrocytes to efficiently accumulate, store and export copper ions suggests that astrocytes have a key role in the distribution of copper in brain. Impairment of this astrocytic function may be involved in diseases which are connected with disturbances in brain copper metabolism.     

The significance of copper chelators in clinical and experimental application

The essentiality and redox-activity of copper make it indispensable in the mammalian system. However, a comprehensive understanding of copper metabolism and function has not been achieved. Copper chelators have been used as an approach to provide insights into copper acquisition, distribution, and disposition at both the cellular and organism level. Unfortunately, the understanding of effective copper chelators is predominantly based upon their chemical structures and their reactions with copper. The understanding of the efficacy of copper chelators in the biological system has been equivocal, thereby leading to under- or misleading-utilization of these agents in clinical and experimental approaches. Current use of copper chelators in vivo almost exclusively either limits the availability or focuses on the removal of copper in mammalian organ system. There are at least two aspects of copper chelators that are yet to be explored. First, copper chelators preferentially bind either cuprous or cupric. As a result, they potentially modulate copper redox-activity without removing copper from the system. Second, copper chelators are characterized as either membrane-permeable or -impermeable, thus would serve as an organ-selective copper delivery or deprivation system to manipulate the biological function of copper. Here we review clinically relevant copper chelators that have been experimentally or clinically studied for their role in manipulation of copper metabolism and function, paying critical attention to potentially more valuable usage of these agents.     

Effects of promoter mutations on the in vivo regulation of the cop operon of Enterococcus hirae by copper(I) and copper(II).

The cop operon of Enterococcus hirae encodes a repressor, CopY, a copper chaperone, CopZ, and two copper ATPases, CopA and CopB. Regulation of the cop operon is bi-phasic, with copper addition as well as copper chelation leading to induction. Using a plasmid-borne system with a reporter gene, induction of wild-type and mutant cop promoters by high and low copper conditions was investigated. Only mutations that impaired the interaction of CopY with both DNA binding sites had a marked effect on regulation, leading to hyperinduction by copper(I) or copper(II). Chelation of copper(II), but not copper(I), also induced the operon, but induction by copper chelation was not significantly affected by the mutations. E. hirae mutants with reduced extracellular copper reductase activity exhibited the same induction kinetics as wild-type cells. These results show that copper addition and copper chelation induce the cop operon by different routes.     

Enzyme-bound copper of dopamine β-monooxygenase.: Activation of the holoenzyme by added copper and uncoupling of electron transfer from hydroxylation by copper salicylate

Preparations of dopamine β-monooxygenase containing a full complement of copper (4.2 copper atoms per tetramer) show increased ascorbate-supported catalytic activities after addition of an excess of copper ions. The significance ot this observation on the question of the number of copper atoms per active site is discussed.Low molecular weight copper complexes such as copper salicylate cause uncoupling of electron transport from hydroxylation. This uncoupling is probably the reason for the well-known inhibition of this enzyme observed at high copper concentration.The onset of inhibition by the copper chelator bathocuproine disulfonate occurs on a faster time scale than the removal of enzyme-bound copper. Nevertheless, the copper removal is sufficiently rapid to require that it be considered in interpretation of inhibition experiments with chelators.     

Isolation of 2′-Hydroxy-4′-methoxy propiophenone from Tribolium castaneum and T confusum

The copper binding properties were influenced by growth phase of cells, pH and concentration of copper in reaction mixtures. The efficiency of copper absorption increased with growth time and was largest at the mid-logarithmic growth phase. The time course of copper absorption was biphasic, that copper rapidly bound to cell surface for initial few minutes after addition of copper and then the copper was slowly transported into cells. The copper binding to the cell surface depended on the molecular form of copper complex in the reaction mixture and the ligand residue to copper on the cell surface. Double reciprocal plots of absorption velocity of copper vs. copper concentration gave straight lines at low concentration between 0.01 to 0.1 mm. The apparent affinity of copper to the cells of stationary growth phase was the same as that of logarithmic growth phase, that is, the Km values were about 0.01 mm. On the other hand, at high concentration of copper between 0.1 to 5.0 mm the apparent affinity decreased but the absorption velocity of copper remarkably increased. Zinc sulfate most strongly inhibited the copper absorption in this test. It was assumed that zinc competitively bound to the copper binding sites of cell surface.     

Nickel tolerance and copper - nickel co - tolerance in Mimulus guttatus from copper mine and serpentine habitats

Previous work on M. guttatus suggested that nickel tolerance in copper mine populations may also be given by the genes for copper tolerance. It has been shown that copper tolerance in M. guttatus is controlled by a single major gene, plus a number of minor genes (or modifiers) which elevate copper tolerance. Crosses between nickel tolerant individuals from three families and non - tolerants showed that nickel tolerance in M. guttatus is heritable. In order to study the effects of the major copper tolerance gene on copper - nickel co - tolerance in M. guttatus, homozygous copper tolerant and non - tolerant lines were screened against nickel. Significant differences occur between these lines for copper, but were not found when analysed for nickel, indicating that copper - nickel tolerance is not governed by the major gene for copper tolerance. To test whether the minor genes for copper have a pleiotropic effect on nickel tolerance, five selection lines derived from three copper mines (Copperopolis, Penn and Quail) in Calaveras county, California, which vary in degree of tolerance to copper, by the presence or absence of minor copper genes, were also screened against nickel. Two out of three of the lines from Copperopolis showed elevated tolerance to nickel, but two further selection lines derived from Penn and Quail copper mines gave no indication of increased nickel tolerance. These results suggest that the minor genes for copper do not give tolerance to nickel. This was confirmed by the screening of modifier lines, in which modifiers for differing degrees of copper tolerance were inserted into a non - tolerant background. Genotypes possessing fewer copper modifiers yielded higher nickel tolerance than those genotypes which have a greater number of modifiers. Thus nickel tolerance in this species is heritable and under the control of different genes to those producing copper tolerance.     

"Pulling the plug" on cellular copper: the role of mitochondria in copper export

Mitochondria contain two enzymes, Cu,Zn superoxide dismutase (Sod1) and cytochrome c oxidase (CcO), that require copper as a cofactor for their biological activity. The copper used for their metallation originates from a conserved, bioactive pool contained within the mitochondrial matrix, the size of which changes in response to either genetic or pharmacological manipulation of cellular copper status. Its dynamic nature implies molecular mechanisms exist that functionally couple mitochondrial copper handling with other, extramitochondrial copper trafficking pathways. The recent finding that mitochondrial proteins with established roles in CcO assembly can also effect changes in cellular copper levels by modulating copper efflux from the cell supports a mechanistic link between organellar and cellular copper metabolism. However, the proteins and molecular mechanisms that link trafficking of copper to and from the organelle with other cellular copper trafficking pathways are unknown. This review documents our current understanding of copper trafficking to, and within, the mitochondrion for metallation of CcO and Sod1; the pathways by which the two copper centers in CcO are formed; and, the interconnections between mitochondrial function and the regulation of cellular copper homeostasis.     

Dysregulation of intracellular copper trafficking pathway in a mouse model of mutant copper/zinc superoxide dismutase-linked familial amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are responsible for 20% of familial amyotrophic lateral sclerosis through a gain-of-toxic function. We have recently shown that ammonium tetrathiomolybdate, an intracellular copper-chelating reagent, has an excellent therapeutic benefit in a mouse model for amyotrophic lateral sclerosis. This finding suggests that mutant SOD1 might disrupt intracellular copper homeostasis. In this study, we investigated the effects of mutant SOD1 on the components of the copper trafficking pathway, which regulate intracellular copper homeostasis. We found that mutant, but not wild-type, SOD1 shifts intracellular copper homeostasis toward copper accumulation in the spinal cord during disease progression: copper influx increases, copper chaperones are up-regulated, and copper efflux decreases. This dysregulation was observed within spinal motor neurons and was proportionally associated with an age-dependent increase in spinal copper ion levels. We also found that a subset of the copper trafficking pathway constituents co-aggregated with mutant SOD1. These results indicate that the nature of mutant SOD1 toxicity might involve the dysregulation of the copper trafficking pathway, resulting in the disruption of intracellular copper homeostasis.     

Copper export from cultured astrocytes

Copper is an essential trace metal that is required as a catalytic co-factor or a structural component of several important enzymes. However, since excess of copper can also harm cells due to its potential to catalyse the generation of toxic reactive oxygen species, transport of copper and the cellular copper content are tightly regulated. Astrocytes are known to efficiently take up copper ions, but it was not known whether these cells are also able to export copper. Treatment of astrocyte-rich primary cultures for 24 h with copper chloride caused a concentration-dependent increase in the specific cellular copper content. During further 24 h incubation in the absence of copper chloride, the copper-loaded astrocytes remained viable and released up to 45% of the accumulated copper. The rate of copper export was proportional to the amount of cellular copper, was almost completely prevented by lowering the incubation temperature to 4 °C and was partly prevented by the endocytosis inhibitor amiloride. Copper export is most likely mediated by the copper ATPase ATP7A, since this transporter is expressed in astrocyte cultures and its cellular location is strongly affected by the absence or the presence of extracellular copper. The potential of cultured astrocytes to export copper suggests that astrocytes provide neighbouring cells in brain with this essential trace element.     

Regulation of copper uptake and transport in intestinal cell monolayers by acute and chronic copper exposure

Adaptation to high and low copper intake in mammals depends on the cellular control of influx, efflux and storage mechanisms of cellular copper concentrations. In the present study, we used an intestinal cell line (Caco-2), grown in bicameral chambers to study the effect of equilibrium loading with copper. We analyzed (64)Cu uptake from the apical surface, intracellular metal (Cu, Zn, Fe) content, (64)Cu transport into the basal chamber, and total copper, zinc and iron in the basal chamber. We found that the (64)Cu uptake is saturable, shows a linear response phase up to 1.5 microM reaching a plateau at 4-6 microM extracellular Cu. Intracellular copper increased 21.6-fold, from 1.5 to 32.4 mM (at 0.2-20.2 microM extracellular copper respectively). The time course for (64)Cu uptake and transport was linear when the cells were incubated with different copper concentrations. Uptake increased 10-fold when intracellular copper concentration was raised. Fluxes were lowest at 1.5 mM and highest at 32.4 mM Cu intracellular copper (2.03 and 20. 98 pmole (64)Cu insert(-1) h(-1), respectively). The apical-to-basolateral copper transfer rate was lower at 32.4 mM as compared to 1.5 mM intracellular copper (0.55-1.95 pmole (64)Cu insert(-1) h(-1), respectively). The total copper in the basal chamber increased 4.2-fold (from 3.04 to 12.85 pmole Cu insert(-1) h(-1)) when the intracellular copper concentration was raised. If cells are preincubated in a low copper medium most of the newly incorporated copper (64%) is transferred to the basolateral compartment. In contrast, under preloading with high copper concentration, only 4% of the fresh copper is transferred to the basal chamber; however, the intracellular copper contribution to this chamber increases by 4.2-fold. Thus, the process results in an increase in both storage and intracellular-to-basolateral flux of copper. In summary, our results indicate that copper fluxes from apical-to-cell and apical-to-basolateral domains are affected by intracellular copper concentration suggesting that mechanisms of copper transport involved in cellular adaptation to low and high copper exposure are different.     

Is there a role for copper in neurodegenerative diseases?

Copper is an essential metal in living organisms; thus, the maintenance of adequate copper levels is of vital importance and is highly regulated. Dysfunction of copper metabolism leading to its excess or deficiency results in severe ailments. Two examples of illnesses related to alterations in copper metabolism are Menkes and Wilson diseases. Several proteins are involved in the maintenance of copper homeostasis, including copper transporters and metal chaperones. In the last several years, the beta-amyloid-precursor protein (beta-APP) and the prion protein (PrP(C)), which are related to the neurodegenerative disorders Alzheimer and prion diseases respectively, have been associated with copper metabolism. Both proteins bind copper through copper-binding domains that also have been shown to reduce copper in vitro. Moreover, this ability to reduce copper is associated with a neuroprotective effect exerted by the copper-binding domain of both proteins against copper in vivo. In addition to a functional link between copper and beta-APP or PrP(C), evidence suggests that copper has a role in Alzheimer and prion diseases. Here, we review the evidence that supports both, the role of beta-APP and PrP(C), in copper metabolism and the putative role of copper in neurodegenerative diseases.     

Response of Gram-positive bacteria to copper stress

The Gram-positive bacteria Enterococcus hirae, Lactococcus lactis, and Bacillus subtilis have received wide attention in the study of copper homeostasis. Consequently, copper extrusion by ATPases, gene regulation by copper, and intracellular copper chaperoning are understood in some detail. This has provided profound insight into basic principles of how organisms handle copper. It also emerged that many bacterial species may not require copper for life, making copper homeostatic systems pure defense mechanisms. Structural work on copper homeostatic proteins has given insight into copper coordination and bonding and has started to give molecular insight into copper handling in biological systems. Finally, recent biochemical work has shed new light on the mechanism of copper toxicity, which may not primarily be mediated by reactive oxygen radicals.     

“Pulling the plug” on cellular copper: The role of mitochondria in copper export

Mitochondria contain two enzymes, Cu,Zn superoxide dismutase (Sod1) and cytochrome c oxidase (CcO), that require copper as a cofactor for their biological activity. The copper used for their metallation originates from a conserved, bioactive pool contained within the mitochondrial matrix, the size of which changes in response to either genetic or pharmacological manipulation of cellular copper status. Its dynamic nature implies molecular mechanisms exist that functionally couple mitochondrial copper handling with other, extramitochondrial copper trafficking pathways. The recent finding that mitochondrial proteins with established roles in CcO assembly can also effect changes in cellular copper levels by modulating copper efflux from the cell supports a mechanistic link between organellar and cellular copper metabolism. However, the proteins and molecular mechanisms that link trafficking of copper to and from the organelle with other cellular copper trafficking pathways are unknown. This review documents our current understanding of copper trafficking to, and within, the mitochondrion for metallation of CcO and Sod1; the pathways by which the two copper centers in CcO are formed; and, the interconnections between mitochondrial function and the regulation of cellular copper homeostasis.     

Copper transport and Alzheimer’s disease

This brief review discusses copper transport in humans, with an emphasis on knowledge learned from one of the simplest model organisms, yeast. There is a further focus on copper transport in Alzheimer’s Disease (AD). Copper homeostasis is essential for the well-being of all organisms, from bacteria to yeast to humans: survival depends on maintaining the required supply of copper for the many enzymes, dependent on copper for activity, while ensuring that there is no excess free copper, which would cause toxicity. A virtual orchestra of proteins are required to achieve copper homeostasis. For copper uptake, Cu(II) is first reduced to Cu(I) via a membrane-bound reductase. The reduced copper can then be internalised by a copper transporter where it is transferred to copper chaperones for transport and specific delivery to various organelles. Of significance are internal copper transporters, ATP7A and ATP7B, notable for their role in disorders of copper deficiency and toxicity, Menkes and Wilson’s disease, respectively. Metallothioneins and Cu/Zn superoxide dismutase can protect against excess copper in cells. It is clear too, increasing age, environmental and lifestyle factors impact on brain copper. Studies on AD suggest an important role for copper in the brain, with some AD therapies focusing on mobilising copper in AD brains. The transport of copper into the brain is complex and involves numerous players, including amyloid precursor protein, Aβ peptide and cholesterol.     

Clioquinol mediates copper uptake and counteracts copper efflux activities of the amyloid precursor protein of Alzheimer's disease

The key protein in Alzheimer's disease, the amyloid precursor protein (APP), is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Abeta amyloid peptide. Whereas overexpression of APP results in significantly reduced brain copper levels in three different lines of transgenic mice, knock-out animals revealed increased copper levels. A provoked rise in peripheral levels of copper reduced concentrations of soluble amyloid peptides and resulted in fewer pathogenic Abeta plaques. Contradictory evidence has been provided by the efficacy of copper chelation treatment with the drug clioquinol. Using a yeast model system, we show that adding clioquinol to the yeast culture medium drastically increased the intracellular copper concentration but there was no significant effect observed on zinc levels. This finding suggests that clioquinol can act therapeutically by changing the distribution of copper or facilitating copper uptake rather than by decreasing copper levels. The overexpression of the human APP or APLP2 extracellular domains but not the extracellular domain of APLP1 decreased intracellular copper levels. The expression of a mutant APP deficient for copper binding increased intracellular copper levels several-fold. These data uncover a novel biological function for APP and APLP2 in copper efflux and provide a new conceptual framework for the formerly diverging theories of copper supplementation and chelation in the treatment of Alzheimer's disease.     

Extrahepatic tissue copper concentrations in white perch with hepatic copper storage

Hepatic copper storage in man (Wilson's disease), Bedtington and West Highland white terriers, and white perch ( Morone americana ) is characterized by the progressive accumulation of copper in hepatic lysosomes bound to cytoprotective metallothionein. In man, saturation of the liver storage capacity results in the distribution of copper to extrahepatic tissues with multiple organ system dysfunction. To determine if extrahepatic tissue copper concentrations also increase in white perch, copper and zinc levels in liver, brain, heart, gills, serum, and bile were determined by atomic absorption spectrophotometry and compared to striped bass ( Morone saxatilis ). Results showed that brain copper concentrations in. white perch were elevated and significantly correlated with liver copper. Bile and serum copper also increased significantly with liver copper. Copper levels in heart and gill tissues were low. Liver zinc was increased in white perch but not to the same magnitude as copper, and was correlated significantly with liver copper; possibly a non-specific secondary increase related to an overall increase in hepatic metallothionein. Histochemical staining of liver with rubeimc acid for copper was proportional to copper concentrations, and clusters of positive mononuclear cells were also seen in brain and spleen. Foci of macrophages in spleen were also intensely positive with Perl's iron stain which may have been indicative of haemolysis. The patterns of copper distribution seen in white perch present a useful comparative model to study alterations in copper metabolism.     

The use of waste biomass of Sargassum sp. for the biosorption of copper from simulated semiconductor effluents

Seaweed Sargassum sp. biomass proved to be useful for the recovery of ionic copper from highly concentrated solutions simulating effluents from semiconductor production. In the case of solutions containing copper in the form of chloride, sulphate and nitrate salts, the best pH for the recovery of copper was 4.5. It was observed that copper biosorption from copper nitrate solutions was higher than the recovery of copper from copper chloride or sulphate solutions. The continuous system used was constituted of four column reactors filled with the biomass of Sargassum sp. and showed high operational stability. Biomass of Sargassum sp. in the reactors was gradually saturated from the bottom to the top of each column reactor. The biomass of Sargassum sp. in the first column was saturated first, followed by a gradual saturation of the remaining columns due to the pre-concentration caused by the biomass in the first column. The biomass of Sargassum in the bioreactors completely biosorbed the ionic copper contained in 63 L of copper sulphate solution, 72 L of copper chloride solution and 72 L of copper nitrate solution, all the solutions containing copper at 500 mg/L. Effluents produced after biosorption presented copper concentrations less than 0.5 mg/L.