Biochemical Study on the Critical Period for Treatment of the Mottled Brindled Mouse

Hemizygous mottled brindled mice (Mobr/y mice) were treated by subcutaneous injection of copper and were decapitated on postnatal day 14. Cytochrome c oxidase (COX) activity of the brain mitochondria in the mice given 10 micrograms of copper/g on day 4 or 7 showed significant increases compared with that of untreated Mobr/y animals, and these mice had no neurological symptoms. Mice given 10 micrograms of copper/g on day 12 showed neither increases in COX activity nor clinical improvement. The brain levels of copper, noradrenaline, and dopamine in the mice treated on day 12 were the same as those in animals treated on day 4 or 7. The in vitro activities of dopamine-beta-hydroxylase of the brain were also the same among the treated mice, irrespective of the date of treatment. The results indicate that delays in copper treatment produce irreversible changes in COX activity of the brain and lead to clinical unresponsiveness to treatment.     

Atx1-like chaperones and their cognate P-type ATPases: copper-binding and transfer

Copper is an essential yet toxic metal ion. To satisfy cellular requirements, while, at the same time, minimizing toxicity, complex systems of copper trafficking have evolved in all cell types. The best conserved and most widely distributed of these involve Atx1-like chaperones and P_1B-type ATPase transporters. Here, we discuss current understanding of how these chaperones bind Cu(I) and transfer it to the Atx1-like N-terminal domains of their cognate transporter.     

Heterogenization of solvent-ligated copper(II) complexes on poly(4-vinylpyridine) for the catalytic cyclopropanation of olefins

[Cu(NCCH₃)₆][B(C₆F₅)₄]₂ and [Cu(NCCH₃)₆][B{C₆H₃(CF₃)₂}₄]₂ are immobilized on poly(4-vinylpyridine). Both resulting materials (Cu(II) complexes immobilized on polymer) are applicable as catalysts for the cyclopropanation of olefins at room temperature. The immobilized Cu(II) compounds are quite stable and recyclable for several catalytic runs, however with some decrease in the catalytic activity.     

A mixed-valence Cu(I)-Cu(II) and metal-metal bond containing coordination polymer obtained from an in situ oxidation reaction route

A new mixed-valence copper coordination polymer with copper-copper metal bonds in a two-dimensional network was generated from an in situ oxidation reaction route under hydrothermal conditions. The synthesis of this coordination polymer demonstrated that the novel compounds that may not be accessible using the known methods could be synthesized via an oxidation reaction route. The reaction conditions are mild enough to keep the building blocks intact during the oxidation and self-assembly process under hydrothermal conditions.     

Evaluation of decay and termite resistance of wood treated with copper in combination with boron and N′-N-(1, 8-naphthalyl) hydroxylamine (NHA-Na)

This study evaluated the relative ability of various combinations of copper sulfate with either boric acid or calcium-precipitating agent, N′-N-(1, 8-naphthalyl) hydroxylamine (NHA-Na), to inhibit fungal degradation and attack by Formosan subterranean termites (Coptotermes formosanus Shiraki). Wood specimens were treated with either 1%, 0.5%, or 0.1% concentrations of copper sulfate, boric acid, NHA-Na, copper sulfate + boric acid, or copper sulfate + NHA-Na mixtures. Treated specimens were subjected to laboratory decay-resistance tests by using petri dishes inoculated with the Basidiomycetes fungi Tyromyces palustris and Trametes versicolor for 12 weeks. Treated wood specimens were also subjected to termite-resistance tests under laboratory conditions. Increased efficacy of copper sulfate against the brown-rot fungus T. palustris was observed when either boric acid or NHA-Na was added. The most effective treatments against the fungi tested were NHA-Na only treatments at 1% and 0.5% concentration levels. Boric acid treatments were not able to protect wood against decay after leaching because of excessive leaching of boron. Similar results were obtained in termite-resistance tests in comparison with decay-resistance tests. These results indicate that the efficacy of the treatments in preventing fungal and termite attack is a function of the type of preservative.     

Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry

 Mammalian metallothioneins (MT) are known to maximally bind 12 copper ions in two six-Cu(I) ion clusters. Using electrospray ionization mass spectrometry of MT at pH 4.5, a four-Cu(I) ion cluster was observed intermediate to a fully formed six Cu(I) in a single domain or a fully formed Cu₁₂MT species. The four-Cu(I) cluster was observed in both MT1 and MT3 isoforms. Addition of increasing amounts of Cu(I) to MT at pH 4.5 resulted in prominent ions whoses masses were consistent with apo-MT, Cu₄MT, Cu₆MT, and Cu₁₂MT. The cooperativity of cluster formation was reduced at pH 2.5. Addition of Cu(I) to apo-MT at a reduced pH resulted in a series of ions consistent with Cu₄ to Cu₁₂MT species. However, formation of the tetracopper MT species remained cooperative at low pH, suggesting that this species is very stable. To determine whether the tetracopper cluster was formed in either the α or β domain, domain peptides of MT3 were used. Addition of Cu(I) to the apo β domain resulted in a peak consistent with the formation of a four-Cu(I) cluster. This is consistent with reports that Cu(I) ions bind preferentially to the β domain of MTs. Received: 2 June 1998 / Accepted: 21 August 1998     

Structure of Cu2(indomethacin)4 and the reaction with superoxide in aprotic systems

The copper complex of indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole acetate), a common anti-inflammatory drug, was prepared and characterized. Crystal structure determination revealed the dimeric form of the 1:2 complex, namely Cu₂(indomethacin)₄ · L₂, in the unit cell. Suprisingly, the copper-copper distance (263 pm) was very close to metallic copper (256 pm). The two coordination sites in the copper-copper axis can be readily replaced by superoxide. An intriguing similarity to Cu₂(acetate)₄ was seen.Due to the lipophilic nature of the indomethacin ligand, this copper complex reacted with superoxide in aprotic solvents. The superoxide dismutating activity was successfully demonstrated in Me₂SO/water and acetonitrile/water mixtures using the nitro-blue tetrazolium assay and pulse radiolysis. The second-order rate constant of 6 · 10⁹ M^?1 · s^?1 in strictly aqueous systems dropped only slightly to 1.1 · 10⁹ M^?1 · s^?1 when aprotic solvents were used. This is the fastest rate constant ever observed for a copper-dependent dismutation of superoxide. The KO₂-induced lipid peroxidation in both erythrocytes and liver microsomes was suppressed by 70% in the presence of 1 · 10^?10 mol · ml^?1 of Cu₂(indomethacin)₄. The inhibitory action dropped to 25% when Cu₂Zn₂superoxide dismutase was employed. The formation of copper · indomethacin in rat serum after administration of indomethacin was shown in vitro and in vivo.     

Identification of porphyrin present in apo-cytochrome c oxidase of copper-deficient yeast cells

Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells. Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells. The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a. When solubilized mitochondria from [³H]leucine and δ-amino[¹⁴C]levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed [³H]leucine associated with six bands and δ-amino[¹⁴C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with δ-amino[¹⁴C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thinlayer chromatography of the radioactivity found in 20% HCl showed an R_F value identical to that of purified porphyrin a. When δ-amino[³H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells wa converted into heme a, and this conversion was prevented by cycloheximidine.These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur. This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficieny.     

β-Carboline copper complex as a potential mitochondrial-targeted anticancer chemotherapeutic agent: Favorable attenuation of human breast cancer MCF7 cells via apoptosis

The development of preferentially selective cancer chemotherapeutics is a new trend in drug research. Thus, we designed and synthesized novel ternary complexes, [Cu(tryp)(Hnor)₂(DMSO)]NO₃ (1) and [Zn(tryp)(Hnor)₂(DMSO)]NO₃ (2) (tryp = DL-Tryptophane; Hnor = Norharmane, β-carboline; DMSO = Dimethyl sulfoxide), characterized with elemental analysis, FTIR, UV–vis, FL, NMR, ESI-MS, and molar conductivity. Furthermore, the TD-DFT studies with UV–vis and FTIR validated the proposed structures of 1 and 2. Moreover, we evaluated the HOMO-LUMO energy gap and found that 1 has a smaller energy gap than 2. Then, 1 and 2 were assessed for anticancer chemotherapeutic potential against cancer cell lines MCF7 (human breast cancer) and HepG2 (human liver hepatocellular carcinoma) as well as the non-tumorigenic HEK293 (human embryonic kidney) cells. The MTT assay illustrated the preferentially cytotoxic behavior of 1 when compared with that of 2 and cisplatin (standard drug) against MCF7 cells. Moreover, 1 was exposed to MCF7 cells, and the results indicated the arrest of the G2/M phases, which followed the apoptotic pathway predominantly. Generation of ROS, GSH depletion, and elevation in LPO validated the redox changes prompted by 1. These studies establish the great potential of 1 as a candidate for anticancer therapeutics.