Cu(GlyGlyHis) effects on MCF7 cells: Copper uptake, reactive oxygen species generation and membrane topography changes

Elevated levels of copper have been detected in various types of human cancer cells, such as breast cancer cells, and a number of mechanisms have been proposed to explain the action and influence of copper on tumor progress. In this work, we found that stimulating the proliferation of mammary epithelial MCF7 cells with the high-redox-potential copper complex Cu (GlyGlyHis) is associated with the copper-induced intracellular generation of reactive oxygen species (ROS) that induces lipid peroxidation and causes increased roughness of external cell membranes, which leads to the formation of larger cell domes. The results presented herein provide new insights into the molecular link between copper and the proliferation of breast cancer cells and, consequently, into the mechanism by which changes in redox balance and ROS accumulation regulates cell membrane roughness. (C) 2012 Elsevier Inc. All rights reserved.

hCTR1: A human gene for copper uptake identified by complementation in yeast

The molecular mechanisms responsible for the cellular uptake of copper in mammalian cells are unknown. We describe isolation of a human gene involved in this process by complementation of the yeast high-affinity copper uptake mutant, ctr1. Besides complementing ctr1 growth defect on nonfermentable media, the human gene also rescues iron transport and SOD1 defects in ctr1 yeast. Overexpression of the gene in yeast leads to vulnerability to the toxicity of copper overload. In addition, its expression in ctr1 yeast significantly increases the level of cellular copper, as demonstrated by atomic absorption. We propose this gene as a candidate for high-affinity copper uptake in humans and by analogy have named it hCTR1. The hCTR1 and yeast CTR1 predicted transmembrane proteins are 29% identical, but the human protein is substantially smaller in both the extracellular metal-binding and intracellular domains. An additional human gene similar to hCTR1, here named hCTR2, was identified in a database search. Both hCTR1 and hCTR2 are expressed in all human tissues examined, and both genes are located in 9q31/32. These studies, together with the previously recognized functional and sequence similarity between the Menkes/Wilson copper export proteins and CCC2 in yeast, demonstrate that similar copper homeostatic mechanisms are used in these evolutionarily divergent organisms.

The Menkes/Wilson disease gene homologue in yeast provides copper to a ceruloplasmin-like oxidase required for iron uptake.

The CCC2 gene of the yeast Saccharomyces cerevisiae is homologous to the human genes defective in Wilson disease and Menkes disease. A biochemical hallmark of these diseases is a deficiency of copper in ceruloplasmin and other copper proteins found in extracytosolic compartments. Here we demonstrate that disruption of the yeast CCC2 gene results in defects in respiration and iron uptake. These defects could be reversed by supplementing cells with copper, suggesting that CCC2 mutant cells were copper deficient. However, cytosolic copper levels and copper uptake were normal. Instead, CCC2 mutant cells lacked a copper-dependent oxidase activity associated with the extracytosolic domain of the FET3-encoded protein, a ceruloplasmin homologue previously shown to be necessary for high-affinity iron uptake in yeast. Copper restored oxidase activity both in vitro and in vivo, paralleling the ability of copper to restore respiration and iron uptake. These results suggest that the CCC2-encoded protein is required for the export of copper from the cytosol into an extracytosolic compartment, supporting the proposal that intracellular copper transport is impaired in Wilson disease and Menkes disease.

Bacterial stimulation of copper phytoaccumulation by bioaugmentation with rhizosphere bacteria

Copper contaminated areas pose environmental health risk to living organisms. Remediation processes are thus required for both crop production and industrial activities. This study employed bioaugmentation with copper resistant bacteria to improve phytoremediation of vineyard soils and copper mining waste contaminated with high copper concentrations. Oatmeal plant (Avena sativa L) was used for copper phytoextraction. Three copper resistant bacterial isolates from oatmeal rhizosphere (Pseudomonas putida A1 Stenotrophomonas maltophilia A2 and Acinetobacter calcoaceticus A6) were used for the stimulation of copper phytoextraction. Two long-term copper contaminated vineyard soils (Mollisol and Inceptisol) and copper mining waste from Southern Brazil were evaluated. Oatmeal plants substantially extracted copper from vineyard soils and copper mining waste. As much as 1549 mg of Cu kg(-1) dry mass was extracted from plants grown in Inceptisol soil. The vineyard Mollisol copper uptake (55 mg Cu kg(-1) of dry mass) in the shoots was significantly improved upon inoculation of oatmeal plants with isolate A2 (128 mg of Cu kg(-1) of shoot dry mass). Overall oatmeal plant biomass displayed higher potential of copper phytoextraction with inoculation of rhizosphere bacteria in vineyard soil to the extent that 404 and 327 g ha(-1) of copper removal were respectively observed in vineyard Mollisol bioaugmented with isolate A2 (S. maltophilia) and isolate A6 (A. calcoaceticus). Results suggest potential application of bacterial stimulation of phytoaccumulation of copper for biological removal of copper from contaminated areas. (C) 2010 Elsevier Ltd. All rights reserved.

The homeostasis of iron, copper, and zinc in paracoccidioides brasiliensis, cryptococcus neoformans var. Grubii, and cryptococcus gattii: a comparative analysis

Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensis Pb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways.

Expression and cellular localization of Copper transporter 2 (Ctr2) in Mus musculus

The Ctr family is an essential part of the copper homeostasis machinery and its members share sequence homology and structural and functional features. Higher eukaryotes express two members of this family Ctr1 and Ctr2. Numerous structural and functional studies are available for Ctr1, the only high affinity Cu(I) transporter thus far identified. Ctr1 holigotrimers mediate cellular copper uptake and this protein was demonstrated to be essential for embryonic development and to play a crucial role in dietary copper acquisition. Instead very little is known about Ctr2, it bears structural homology to the yeast vacuolar copper transporter, which mediates mobilization of vacuolar copper stores. Recent studies using over-expressed epitope-tagged forms of human Ctr2 suggested a function as a low affinity copper transporter that can mediate either copper uptake from the extracellular environment or mobilization of lysosomal copper stores. Using an antibody that recognizes endogenous mouse Ctr2, we studied the expression and localization of endogenous mouse Ctr2 in cell culture and in mouse models to understand its regulation and function in copper homeostasis. By immunoblot we observed a regulation of mCtr2 protein levels in a copper and Ctr1 dependent way. Our observations in cells and transgenic mice suggest that lack of Ctr1 induces a strong downregulation of Ctr2 probably by a post-translational mechanism. By indirect immunofluorescence we observed an exclusive intracellular localization in a perinuclear compartment and no co-localization with lysosomal markers. Immunofluorescence experiments in Ctr1 null cells, supported by sequence analysis, suggest that lysosomes may play a role in mCtr2 biology not as resident compartment, but as a degradation site. In appendix a LC-mass method for analysis of algal biotoxins belonging to the family of PsP (paralytic shellfish poisoning) is described.

The copper transporter CTR1 provides an essential function in mammalian embryonic development

Copper serves as an essential cofactor for a variety of proteins in all living organisms. Previously, we described a human gene (CTR1;SLC31A1) that encodes a high-affinity copper-uptake protein and hypothesized that this protein is required for copper delivery to mammalian cells. Here, we test this hypothesis by inactivating the Ctr1 gene in mice by targeted mutagenesis. We observe early embryonic lethality in homozygous mutant embryos and a deficiency in copper uptake in the brains of heterozygous animals. Ctr1−/− embryos can be recovered at E8.5 but are severely developmentally retarded and morphologically abnormal. Histological analysis reveals discontinuities and variable thickness in the basement membrane of the embryonic region and an imperfect Reichert's membrane, features that are likely due to lack of activity in the collagen cross-linking cupro-enzyme lysyl oxidase. A collapsed embryonic cavity, the absence of an allantois, retarded mesodermal migration, and increased cell death are also apparent. In the brains of heterozygous adult mice, which at 16 months are phenotypically normal, copper is reduced to approximately half compared with control littermates, implicating CTR1 as the required port for copper entry into at least this organ. A study of the spatial and temporal expression pattern of Ctr1 during mouse development and adulthood further shows that CTR1 is ubiquitously transcribed with highest expression observed in the specialized epithelia of the choroid plexus and renal tubules and in connective tissues of the eye, ovary, and testes. We conclude that CTR1 is the primary avenue for copper uptake in mammalian cells.

Isolation of plant growth promoting bacteria from torch lake sediments and their role in phytoremediation of metal contaminated soil

In this study, we isolated eight copper-resistant bacteria from Torch Lake sediment contaminated by copper mine tailings (stamp sand). Sequence analysis of gyrB and rpoD genes revealed that these organisms are closer to various Pseudomonas species. These eight bacterial isolates were also resistant to zinc, cesium, lead, arsenate and mercury. Further characterization showed that all the strains produced plant growth promoting indole-3-acetic acid (IAA), iron chelating siderophore and solubilized mineral phosphate and metals. The effect of bacterial inoculation on plant growth and copper uptake by maize (Zea mays) and sunflower (Helianthus annuus) was investigated using one of the isolates (Pseudomonas sp. TLC 6-6.5-4) with higher IAA production and phosphate and metal soubilization, which resulted in a significant increase in copper accumulation in maize and sunflower, and an increase in the total biomass of maize. Genes involved in copper resistance of Pseudomonas sp. TLC 6-6.5-4 was analyzed by transposon mutational analysis. Two copper sensitive mutants with significant reduction in copper resistance were identified: CSM1, a mutant disrupted in trp A gene (tryptophan synthase alpha subunit); CSM2, a mutant disrupted in clpA gene (ATP-dependent Clp protease). Proteomic and metabolomic analysis were performed to identify biochemical and molecular mechanisms involved in copper resistance using CSM2 due to its lower minimum inhibitory concentration compared with CSM1 and the wild type. The effect of different bacterial inoculation methods on plant growth, copper uptake and soil enzyme activities was investigated. Four different delivery methods were used including soil inoculation (before or after plant emergence), seed coating and root dipping. Soil inoculation before sowing seeds and coating seeds with PGPB led to better growth of maize, higher copper uptake and an increase in soil invertase and dehydrogenase activities. Proteomic and metabolomic analyses were performed to investigate the effect of bacterial inoculation on maize grown in normal soil and stamp sand. Our results revealed that bacterial inoculation led to environment-dependent effects on maize proteome and metabolome.

Complexation of copper by sewage sludge-derived dissolved organic matter: Effects on soil sorption behaviour and plant uptake

The complexation of Cu by sewage sludge-derived dissolved organic matter (SSDOM) is a process by which the environmental significance of the element may become enhanced due to reduced soil sorption and, hence, increased mobility. The work described in this paper used an ion selective electrode procedure to show that SSDOM complexation of Cu was greatest at intermediate pH values because competition between hydrogen ions and Cu for SSDOM binding sites, and between hydroxyl ions and SSDOM as Cu ligands, was lowest at such values. Batch sorption experiments further showed that the process of Cu complexation by SSDOM provided an explanation for enhanced desorption of Cu from the solid phase of a contaminated, organic matter-rich, clay loam soil, and reduced adsorption of Cu onto the solid phase of a sandy loam soil. Complexation of Cu by SSDOM did not affect uptake of Cu by spring barley plants, when compared to free ionic Cu, in a sand-culture pot experiment. However, it did appear to lead to greater biomass yields of the plant; perhaps indicating that the Cu-SSDOM complex had a lower toxicity towards the plant than the free Cu ion.

Ferric iron uptake genes are differentially expressed in the presence of copper sulfides in Acidithiobacillus ferrooxidans strain LR

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Biosynthesis and uptake of copper nanoparticles by dead biomass of Hypocrea lixii isolated from the metal mine in the Brazilian Amazon Region

A biological system for the biosynthesis of nanoparticles (NPs) and uptake of copper from wastewater, using dead biomass of Hypocrea lixii was analyzed and described for the first time. The equilibrium and kinetics investigation of the biosorption of copper onto dead, dried and live biomass of fungus were performed as a function of initial metal concentration, pH, temperature, agitation and inoculum volume. The high biosorption capacity was observed for dead biomass, completed within 60 min of contact, at pH 5.0, temperature of 40 °C and agitation speed of 150 rpm with a maximum copper biosorption of 19.0 mg g(-1). The equilibrium data were better described using the Langmuir isotherm and kinetic analysis indicated that copper biosorption follows a pseudo-second-order model. The average size, morphology and location of NPs biosynthesized by the fungus were determined by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and transmission electron microscopy (TEM). NPs were mainly spherical, with an average size of 24.5 nm, and were synthesized extracellularly. The X-ray diffraction (XRD) analysis confirms the presence of metallic copper particles. Infrared spectroscopy (FTIR) study revealed that the amide groups interact with the particles, which was accountable for the stability of NPs. This method further confirmed the presence of proteins as stabilizing and capping agents surrounding the copper NPs. These studies demonstrate that dead biomass of Hypocrea lixii provides an economic and technically feasible option for bioremediation of wastewater and is a potential candidate for industrial-scale production of copper NPs.

High-Density Lipoprotein Inhibits the Uptake of Modified Low-Density Lipoprotein and the Expression of CD36 and Fc gamma RI

Aim: Modified low-density lipoprotein (mLDL), mainly upon oxidative and enzymatic modification, is the major atherogenic lipoprotein. Conversely, high-density lipoprotein (HDL) is considered anti-atherogenic because of its ability to remove cholesterol. The aim of this work was to analyze both the influence of HDL on the uptake of mLDL and the expression of CD36 and Fc gamma I receptors on monocytic cell lines during cell differentiation. Methods: Uptake of fluorescein isothiocyanate (FITC)-conjugated LDL and FITC-conjugated mLDL, i.e., copper-oxidized LDL (oxLDL) or trypsin enzyme modified LDL (enzLDL), was analyzed, as well as the expression of CD36 and Fc gamma RI in THP-1 and U937 cells, using flow cytometry. Results: HDL inhibited the uptake of mLDL, which varied in degree depending on the cell line or type of mLDL. Further, HDL rapidly decreased CD36 and Fc gamma RI involved in the uptake of mLDL. Conclusions: We demonstrate that modified LDL promotes specific LDL receptor-independent uptake by monocytic cell lines, and that the uptake of LDL and enzLDL is less than that of oxLDL. In this process, HDL diminishes the uptake of LDL or mLDL, which may involve the down-regulation of receptors (CD36 and Fc gamma I). This regulatory process represents another way by which HDL can be anti-atherogenic and it depends on the type of modification of LDL and the stage of differentiation of monocytes to macrophages.

Growth and nutrient uptake of coffee seedlings cultivated in nutrient solution with and without silicon addition

In recent years, the application of silicon (Si) in crops, including coffee, has become a common practice. The objective of this study was to assess the silicon uptake by coffee seedlings and its effects on plant growth, water and macro and micronutrient uptake. The research was conducted using nutrient solution in a greenhouse at the Departamento de Fitotecnia da Universidade Federal de Viçosa, in a completely randomized design with two treatments (with and without silicon) and three replications. Each plot consisted of three plants grown in a 800 mL vessel containing the treatment solutions. At every three days, water consumption, the concentration of OH - and the depletion of Si and K were assessed in the nutrient solutions. After 33 days, the plants were assessed with regard to their fresh and dry weight of leaves, roots and stem, shoot height and total length of the plant (shoot and root). Number of leaves and internodes, and the content and accumulation of silicon, macro, and micronutrients were also determined. The consumption of water, the amount of potassium uptake and, biomass accumulation were greater in plants grown in solution without silicon addition. However, the concentration of OH- in the solution and the amount of silicon uptake were greater in plants grown in solution with added silicon. Silicon accumulation was greater in leaves than in stem and roots. Silicon decreased coffee plant accumulation of phosphorus, potassium, calcium, zinc, copper and iron.

Copper and lead removal by peanut hulls: equilibrium and kinetic studies

This research work aims to study the use of peanut hulls, an agricultural and food industry waste, for copper and lead removal through equilibrium and kinetic parameters evaluation. Equilibrium batch studies were performed in a batch adsorber. The influence of initial pH was evaluated (3–5) and it was selected between 4.0 and 4.5. The maximum sorption capacities obtained for the Langmuir model were 0.21 ± 0.03 and 0.18 ± 0.02 mmol/g, respectively for copper and lead. In bi-component systems, competitive sorption of copper and lead was verified, the total amount adsorbed being around 0.21 mmol of metal per gram of material in both mono and bi-component systems. In the kinetic studies equilibrium was reached after 200 min contact time using a 400 rpm stirring rate, achieving 78% and 58% removal, in mono-component system, for copper and lead respectively. Their removal follows a pseudo-second-order kinetics. These studies show that most of the metals removal occurred in the first 20 min of contact, which shows a good uptake rate in all systems.

Secondary effects of catalytic diesel particulate filters: copper-induced formation of PCDD/Fs.

Potential risks of a secondary formation of polychlorinated dibenzodioxins/furans (PCDD/Fs) were assessed for two cordierite-based, wall-through diesel particulate filters (DPFs) for which soot combustion was either catalyzed with an iron- or a copper-based fuel additive. A heavy duty diesel engine was used as test platform, applying the eight-stage ISO 8178/4 C1 cycle. DPF applications neither affected the engine performance, nor did they increase NO, NO2, CO, and CO2 emissions. The latter is a metric for fuel consumption. THC emissions decreased by about 40% when deploying DPFs. PCDD/F emissions, with a focus on tetra- to octachlorinated congeners, were compared under standard and worst case conditions (enhanced chlorine uptake). The iron-catalyzed DPF neither increased PCDD/F emissions, nor did it change the congener pattern, even when traces of chlorine became available. In case of copper, PCDD/F emissions increased by up to 3 orders of magnitude from 22 to 200 to 12 700 pg I-TEQ/L with fuels of < 2, 14, and 110 microg/g chlorine, respectively. Mainly lower chlorinated DD/Fs were formed. Based on these substantial effects on PCDD/F emissions, the copper-catalyzed DPF system was not approved for workplace applications, whereas the iron system fulfilled all the specifications of the Swiss procedures for DPF approval (VERT).