Urinary copper excretion before and after oral intake of D-penicillamine in parents of patients with Wilson's disease

Background: Urinary copper excretion higher than 100 mu g/24 h is useful for diagnosing Wilson's disease. D-Penicillamine challenge test may produce higher levels than 1400 mu g/24 h, allowing for better diagnostic accuracy. This study investigated whether heterozygotes reach this value and compared copper serum levels, ceruloplasmin, and urinary copper excretion before and after administering D-penicillamine to the parents of Wilson's disease patients. Methods: Fifty parents of adult patients were enrolled to obtain copper serum levels and ceruloplasmin along with 24-h urinary copper excretion before and after administering 1 g D-penicillamine. Results: Serum ceruloplasmin and copper levels were significantly lower in fathers than in mothers (mean 21.8 x 27.8 mg%; 71.4 x 88.0 mu g%; p <= 0.001). The mean of basal 24-h urinary copper excretion was higher in fathers (26.2 x 18.7 mu g/24 h, p = 0.01), but did not differ between the genders after D-penicillamine (521.7 x 525.3, range 31.6-1085.1 mu g/24 h, p = 0.8). Conclusions: The mean values of serum copper, ceruloplasmin, and basal urinary copper excretion were different between males and females. The current diagnostic threshold of 24-h urinary copper excretion after D-penicillamine was not reached by heterozygotes. The increased urinary copper excretion after D-penicillamine challenge was much higher than fivefold the upper limit of normal urinary copper excretion in the majority of heterozygotes and should not be taken into account when diagnosing Wilson's disease. (C) 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Design and synthesis of a new coumarin-based 'turn-on' fluorescent probe selective for Cu+2

The novel coumarin-based 'turn-on' fluorescent probe (E)-3-(2,5-dimethoxybenzylideneamino)-7-hydroxy-2H-chromen-2-one (MGM) was designed, synthesized, and characterized. This compound shows high selectivity for Cu+2, combined with a large fluorescence enhancement upon binding to Cu2+. Benesi-Hildebrand and Job plots demonstrate that the stoichiometry of the Cu+2 complex formed is 2:1. Preliminary studies employing epifluorescence microscopy demonstrated that Cu+2 could be imaged in human neuroblastoma SH-SY5Y cells treated with MGM. (c) 2012 Elsevier Ltd. All rights reserved.

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.

Organic and inorganic sources of zinc, copper and selenium in diets for dairy cows: intake, blood metabolic profile, milk yield and composition

The present study was carried out with the objective of evaluating the effects of feeding dairy cows with organic or inorganic sources of zinc (Zn), copper (Cu) and selenium (Se) on blood concentrations of these minerals, blood metabolic profiles, nutrient intake and milk yield and composition. Nineteen Holstein cows were selected and randomly assigned to two groups for receiving organic (n = 9) or inorganic (n = 10) sources of Zn, Cu and Se from 60 days before the expected date of calving to 80 days of lactation. Samples of feed, orts and milk were collected for analysis. Body condition score (BCS) was determined and blood samples were collected for analysis of Zn, Cu and Se concentrations, as well as for metabolic profile. Supplying organic or inorganic sources of Zn, Cu, and Se did not affect dry matter and nutrient intake, blood metabolic profile, milk yield and composition, plasma concentration of these minerals, and BCS or change the BCS in cows from 60 days before the expected date of calving to 80 days of lactation. An effect of time was observed on all feed intake variables, plasma concentrations of Zn and Se, milk yield, milk protein content, BCS and change in BCS.

Increasing the activity of copper(II) complexes against Leishmania through lipophilicity and pro-oxidant ability

Copper complexes with fluorinated beta-diketones were synthesized and characterized in terms of lipophilicity and peroxide-assisted oxidation of dihydrorhodamine as an indicator of redox activity. The biological activity of the complexes was tested against promastigotes of Leishmania amazonensis. Inhibition of trypanosomatid-specific trypanothione reductase was also tested. It was found that the highly lipophilic and redox-active bis(trifluoroacetylacetonate) derivative had increased toxicity towards promastigotes. These results indicate that it is possible to modulate the activity of metallodrugs based on redox-active metals through the appropriate choice of lipophilic chelators in order to design new antileishmanials. Further work will be necessary to improve selectivity of these compounds against the parasite.

Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na, K-ATPase activity by mammalian FXYD2 peptide

This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (gamma C-33) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K+, ATP and N-4(+)center dot K-0.5 for Na+ was unaffected. Exogenous pig FXYD2 increased the V-max for stimulation of gill Na,K-ATPase activity by Na+, K+ and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na, K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity. (C) 2012 Elsevier B.V. All rights reserved.

Dinuclear copper(II) complexes with valsartan. Synthesis, characterization and cytotoxicity

Two novel dinuclear complexes involving the antihypertensive drug valsartan and copper(II) ion have been prepared in water and DMSO. The complex compositions were determined as: [Cu(vals)(H(2)O)(3)](2)center dot 6H(2)O and [Cu(vals)(H(2)O)(2)DMSO](2)center dot 2H(2)O. They were thoroughly characterized by elemental and thermal analysis, spectrophotometric titrations and UV-visible, diffuse reflectance, FTIR, Raman and EPR spectroscopies. No effect of the ligand on two tested osteoblastic cell lines in culture (one normal MOT3E1 and one tumoral UMR106) was observed in concentrations up to 100 mu M. Higher concentrations of Valsartan are required to induce cytotoxicity in both cell lines. The antiproliferative effect of the tested complex ([Cu(vals) (H(2)O)(3)](2)center dot 6H(2)O) in a dose-response manner, was higher in the UMR106 osteoblastic cell line than that of the MC3T3E1 normal line at concentrations >= 100 mu M. Morphological alterations are in accordance with proliferative observations. (C) 2011 Elsevier Inc. All rights reserved.

Blood gas profile of copper-poisoned in sheep treated with ammonium tetrathiomolybdate

The aim of this study was to evaluate the blood gas profile of experimentally copper-poisoned sheep (in the pre-hemolytic, hemolytic and post-hemolytic phases) that have been treated or not treated with ammonium tetrathiomolybdate. Ten lambs of the Santa Ines breed were divided into two groups: control and ATTM (treated (ammonium tetrathiomolibydate). The animals were submitted to increasing doses of copper sulfate until macroscopic hemoglobinuria was detected. All of the control animals from died within four days of hemolytic crisis, and one sheep from ATTM died during the treatment. There was no difference in blood gas parameters between experimental groups. Higher values of pCO(2) were observed during the hemolytic crisis (HC) in both groups. The control group had higher mean values of hCO(3) in the times HC and 2 days after hemolytic crisis (dA) when compared with the time 15 before hemolytic crises (dB). The sheep that were treated with ATTM presented lower values of hCO(3) at 7dB and higher levels at the HC. The control and ATTM groups exhibited higher values of BE during the HC. Poisoning resulted in disorder in the acid-base equilibrium, characterized by metabolic alkalosis and respiratory acidosis. Treatment with ATTM was able to reverse the changes in acid-base balance in copper poisoning sheep.

Local electrochemical investigation of copper patina

The patination of copper is known for its complexity and heterogeneous formation. For a deeper investigation, a locally resolved surface analysis was considered. An exact determination of the accessed area and a potentiostatic control in a three-electrode configuration was reached with the use of the electrochemical microcell technique, which enables local electrochemical measurement such as local electrochemical impedance spectroscopy and cyclic voltammetry. Such a technique provides a unique way for performing the investigation of heterogeneities on electrode surfaces. The local electrochemical measurements on the artificially patinated surface have allowed distinguishing areas of different reactivity even when the analysis of the surface revealed a homogenous chemical composition of patina. Local measurements with the electrochemical microcell showed the presence of small defects on the patina layer that can be modelled by considering a hemispherical diffusion process at small active areas surrounded by larger less reactive domains.

On the optical properties of copper nanocubes as a function of the edge length as modeled by the discrete dipole approximation

This Letter reports an investigation on the optical properties of copper nanocubes as a function of size as modeled by the discrete dipole approximation. In the far-field, our results showed that the extinction resonances shifted from 595 to 670 nm as the size increased from 20 to 100 nm. Also, the highest optical efficiencies for absorption and scattering were obtained for nanocubes that were 60 and 100 nm in size, respectively. In the near-field, the electric-field amplitudes were investigated considering 514, 633 and 785 nm as the excitation wavelengths. The E-fields increased with size, being the highest at 633 nm. (c) 2012 Elsevier B.V. All rights reserved.

Single-wall carbon nanotube interactions with copper-oxamato building block of molecule-based magnets probed by resonance Raman spectroscopy

The electronic interactions between the [Cu(opba)]2- anions (where opba is orthophenylenebis (oxamato)) and single-wall carbon nanotubes (SWCNTs) were investigated by resonance Raman spectroscopy. The opba can form molecular magnets, and the interactions of opba with SWCNTs can produce materials with very different magnetic/electronic properties. It is observed that the electronic interaction shows a dependence on the SWCNT diameter independent of whether they are metallic or semiconducting, although the interaction is stronger for metallic tubes. The interaction also is dependent on the amount of complex that is probably adsorbed on the carbon surface of the SWCNTs. Some charge transfer can be also occurring between the metallic complex and the SWCNTs. Copyright (c) 2012 John Wiley & Sons, Ltd.

The promoter of filamentation (POF1) protein from Saccharomyces cerevisiae is an ATPase involved in the protein quality control process

Background: The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene. Results: Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo. Conclusions: Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

Morphology, microstructure and electrocatalytic properties of activated copper surfaces

Morphologic changes on copper surfaces upon applying an established potential protocol were examined by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The results showed a good correlation between the time employed in the electrode activation and the resulting microstructure and electrochemical activity.

An alternative method for the simultaneous determination of copper and lead for quality control of sugar cane spirit using a nanotube-based sensor

The development of an electroanalytical method for simultaneous determination of copper and lead ions in sugar cane spirit (cachaça) using carbon paste electrode modified with ascorbic acid and carbon nanotubes (CPE-AaCNT) is described. Squarewave voltammetry (SWV) with anodic stripping was employed, and this technique was optimized with respect to the following parameters: frequency (50 Hz), amplitude (100 mV) and scan increment (9 mV). The analytical curves were linear in the range from 0.0900 to 7.00 mg L- 1 for lead and copper. The limits of detection were 48.5 and 23.9 µg L- 1 for lead and copper, respectively. The developed method was applied to the simultaneous determination of copper and lead in five commercial samples of sugar cane spirit. The results were in good agreement with those obtained by F AAS/GF AAS (flame atomic absorption spectrometry/graphite furnace atomic absorption spectrometry) and showed that CPE-AaCNT can be successfully employed in the simultaneous determination of these metals in real sugar cane spirit samples.

ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H+-ATPase subcellular vesicle trafficking

The effect of angiotensin II (ANG II) or arginine vasopressin (AVP) alone or plus atrial natriuretic peptide (ANP) on H+-ATPase subcellular vesicle trafficking was investigated in MDCK cells following intracellular pH (pHi) acidification by exposure to20 mMNH4Cl for 2 min in a Na+-free solution containing Schering 28080, conditions under which H+-AT-Pase is the only cell mechanism for pHi recovery. Using the acridine orange fluorescent probe (5mM) and confocal microscopy, the vesicle movement was quantified by determining, for each experimental group, the mean slope of the line indicating the changes in apical/basolateral fluorescence density ratio over time during the first 5.30 min of the pHi recovery period. Under the control conditions, the mean slope was 0.079 ± 0.0033 min-1 (14) and it increased significantly with ANG II [10-12 and 10-7 M, respectively to 0.322 ± 0.038 min-1 (13) and 0.578 ± 0.061 min-1 (12)] or AVP [10-12 and 10-6 M, respectively to 0.301 ± 0.018 min-1 (12) and 0.687 ± 0.049 min-1 (11)]. However, in presence of ANP (10-6 M, decreases cytosolic free calcium), dimethyl-BAPTA/AM (5 × 10-5 M, chelates intracellular calcium) or colchicine (10-5 M, 2-h preincubation; inhibits microtubule-dependent vesicular trafficking) alone or plus ANG II or AVP the mean slopes were similar to the control values, indicating that such agents blocked the stimulatory effect of ANG II or AVP on vesicle trafficking. The results suggest that the pathway responsible for the increase in cytosolic free calcium and the microtu-bule-dependent vesicular trafficking are involved in this hormonal stimulating effect. Whether cytosolic free calcium reduction represents an important direct mechanism for ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H+-ATPase subcellular vesicle trafficking, or is a side effect of other signaling pathways which will require additional studies.