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Claudia R da Silva, Gabriella S Almeida, Adriano Caldeira-de-Araújo, Alvaro C Leitão, Marcelo de Pádula
In Saccharomyces cerevisiae, disruption of genes by deletion allowed elucidation of the molecular mechanisms of a series of human diseases, such as in Wilson disease (WD). WD is a disorder of copper metabolism, due to inherited mutations in human copper-transporting ATPase (ATP7B). An orthologous gene is present in S. cerevisiae, CCC2 gene. Copper is required as a cofactor for a number of enzymes. In excess, however, it is toxic, potentially carcinogenic, leading to many pathological conditions via oxidatively generated DNA damage...
January 2016: Mutagenesis
C A Alesso, K F Discola, G Monteiro
In the yeast Saccharomyces cerevisiae, many genes are involved in the uptake, transport, storage and detoxification of copper. Large scale studies have noted that deletion of the gene ICS3 increases sensitivity to copper, Sortin 2 and acid exposure. Here, we report a study on the Δics3 strain, in which ICS3 is related to copper homeostasis, affecting the intracellular accumulation of this metal. This strain is sensitive to hydrogen peroxide and copper exposure, but not to other tested transition metals. At pH 6...
September 2015: Fungal Genetics and Biology: FG & B
Ozlenen Simsek Papur, Orhan Terzioglu, Ahmet Koc
The Wilson disease gene, a copper transporting ATPase (Atp7b), is responsible for the sequestration of Cu into secretory vesicles, and this function is exhibited by the orthologous Ccc2p in the yeast. In this study, we aimed to characterize clinically relevant new mutations of human ATP7B (p.T788I, p.V1036I and p.R1038G-fsX83) in yeast lacking the CCC2 gene. Expression of human wild type ATP7B gene in ccc2Δ mutant yeast restored the growth deficiency and copper transport activity; however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth...
2015: Journal of Trace Elements in Medicine and Biology
Yuchi Zhang, Chunming Liu, Yan Pan, Yanjuan Qi, Yuchun Li, Sainan Li
A novel procedure comprising ultrasonic-assisted dynamic extraction (UADE) coupled with two countercurrent-chromatography systems (parallel countercurrent chromatography, PCCC) was developed. This technique offers the possibility for simultaneous extraction, solvent configuration, purification, and isolation of target compounds. This new approach was applied to the fractionation and purification of isoflavones from red clover. The two-phase solvent system was prepared by automating the equipment. The lower aqueous phase of the two-phase solvent system was used as the UADE solution and as the mobile phase for PCCC...
June 2015: Analytical and Bioanalytical Chemistry
Hung-Mo Lin, John M Williamson
Some studies are designed to assess the agreement between different raters and/or different instruments in the medical sciences and pharmaceutical research. In practice, the same sample will be used to compare the agreement of two or more assessment methods for simplicity and to take advantage of the positive correlation of the ratings. The concordance correlation coefficient (CCC) is often used as a measure of agreement when the rating is a continuous variable. We present an approach for calculating the sample size required for testing the equality of two CCCs, H0: CCC1 = CCC2 vs...
2015: Journal of Biopharmaceutical Statistics
Anna S Degtyarenko, Marcel Handke, Karl W Krämer, Shi-Xia Liu, Silvio Decurtins, Eduard B Rusanov, Laurence K Thompson, Harald Krautscheid, Konstantin V Domasevitch
Rational combination of pyridazine, hydroxo and carboxylate bridging ligands led to the assembly of three types of mixed-ligand polynuclear Cu(II) clusters (A: [Cu2(μ-OH)(μ-pdz)(μ-COO)]; B: [Cu4(μ3-OH)2(μ-pdz)2]; C: [Cu5(μ-OH)2(μ-pdz)4(μ-COO)2(μ-H2O)2]) and their integration into 3D framework structures. Mixed-ligand complexes [Cu2(μ-OH){TMA}(L)(H2O)] (1), [Cu4(μ3-OH)2{ATC}2(L)2(H2O)2]·H2O (2) [Cu4(μ3-OH)2{TDC}3(L)2(H2O)2]·7H2O (3) (L = 1,3-bis(pyridazin-4-yl)adamantane; TMA(3-) = benzene-1,3,5-tricarboxylate, ATC(3-) = adamantane-1,3,5-tricarboxylate, TDC(2-) = 2,5-thiophenedicarboxylate) and [Cu5(μ-OH)2{X}4(L)2(H2O)2]·nH2O (X = benzene-1,3-dicarboxylate, BDC(2-), n = 5 (4) and 5-hydroxybenzene-1,3-dicarboxylate, HO-BDC(2-), n = 6 (5)) are prepared under hydrothermal conditions...
June 14, 2014: Dalton Transactions: An International Journal of Inorganic Chemistry
Allison R Rogala, Andrew P Morgan, Alexis M Christensen, Terry J Gooch, Timothy A Bell, Darla R Miller, Virginia L Godfrey, Fernando Pardo-Manuel de Villena
Inflammatory bowel disease (IBD) is an immune-mediated condition driven by improper responses to intestinal microflora in the context of environmental and genetic background. GWAS in humans have identified many loci associated with IBD, but animal models are valuable for dissecting the underlying molecular mechanisms, characterizing environmental and genetic contributions and developing treatments. Mouse models rely on interventions such as chemical treatment or introduction of an infectious agent to induce disease...
April 2014: Mammalian Genome: Official Journal of the International Mammalian Genome Society
V I Negruk, G I Eisner, T D Redichkina, N N Dumanskaya, D I Cherny, A A Alexandrov, M F Shemyakin, R G Butenko
A comparative analysis of the Vicia faba mitochondrial genome in whole plants and in longterm suspension culture has been conducted. Restriction fragment patterns of the mtDNA isolated from these two sources were notably different. Electronmicroscopic analysis also revealed significant differences. Large circular mtDNA patterns shifted from a 37-80 kb subpopulation, which was predominant in whole plants, to 18-34 kb subpopulations although in both classes notable quantities of circular molecules of 80 to 120 kb and more were also found...
July 1986: TAG. Theoretical and Applied Genetics. Theoretische und Angewandte Genetik
V I Negruk, N K Kaushik
A comparative analysis of the presence of minicircular DNA CCCIB in 16 different lines and cultivars of fertile Vicia faba L. plants was conducted. It was found that copy number of CCCIB ranged from several copies per mitochondrial genome to - probably - zero, depending on cultivar or line. Fertility of plants in these cases was not altered. We chose 10 cultivars and lines among 16 analysed. Mitochondria of five cultivars and lines contained about two CCCIB molecules per one CCCIA. The sixth cultivar contained CCCIB at copy number several times lower...
October 1988: TAG. Theoretical and Applied Genetics. Theoretische und Angewandte Genetik
Ayca Cankorur-Cetinkaya, Serpil Eraslan, Betul Kirdar
A high degree of conservation of the copper homeostasis pathway between yeast and humans makes yeast an ideal model organism for studying copper-related disorders. In this study, a system based integrative approach was used to investigate the genome-wide effects of the deletion of the yeast ortholog of Wilson and Menkes diseases encoding a Cu(2+)-transporting P-type ATPase (CCC2) in different copper containing media and to compare with the wild type. The experimental design applied in this study enabled the observation of the effect of CCC2 deletion, extracellular copper levels and interactive effects of both factors in S...
November 2013: Molecular BioSystems
Zsuzsanna Sasvari, Nikolay Kovalev, Peter D Nagy
Replication of plus-strand RNA viruses [(+)RNA viruses] is performed by viral replicases, whose function is affected by many cellular factors in infected cells. In this paper, we demonstrate a surprising role for Gef1p proton-chloride exchanger in replication of Tomato bushy stunt virus (TBSV) model (+)RNA virus. A genetic approach revealed that Gef1p, which is the only proton-chloride exchanger in Saccharomyces cerevisiae, is required for TBSV replication in the yeast model host. We also show that the in vitro activity of the purified tombusvirus replicase from gef1Δ yeast was low and that the in vitro assembly of the viral replicase in a cell extract was inhibited by the cytosolic fraction obtained from gef1Δ yeast...
February 2013: Journal of Virology
Dong-Hyuk Heo, In-Joon Baek, Hyun-Jun Kang, Ji-Hyun Kim, Miwha Chang, Chang-Min Kang, Cheol-Won Yun
The ATX1 deletion strain of Saccharomyces cerevisiae is more resistant to Cd(2+) than the wild-type. To investigate the function of Atx1 in Cd(2+) toxicity, we used a metal-binding assay to study the interaction between Atx1 and Cd(2+) in vitro. Using circular dichroism and two-hybrid analyses, we found that Atx1 can bind Cd(2+) specifically and that Cd(2+) binding to Atx1 affects the physical interaction between Atx1 and Ccc2. These results imply that Atx1 delivers Cd(2+) to Ccc2 and that this delivery is, at least in part, responsible for Cd(2+) toxicity in S...
February 2012: Biotechnology Letters
Rafael H F Valverde, Thiago Britto-Borges, Jennifer Lowe, Marcelo Einicker-Lamas, Elisabeth Mintz, Martine Cuillel, Adalberto Vieyra
Ccc2, the yeast copper-transporting ATPase, pumps copper from the cytosol to the Golgi lumen. During its catalytic cycle, Ccc2 undergoes auto-phosphorylation on Asp(627) and uses the energy gained to transport copper across the cell membrane. We previously demonstrated that cAMP-dependent protein kinase (PKA) controls the energy interconversion (Cu)E∼P → E-P + Cu when Ser(258) is phosphorylated. We now demonstrate that Ser(258) is essential in vivo for copper homeostasis in extremely low copper and iron concentrations...
March 4, 2011: Journal of Biological Chemistry
Brad M Binder, Fernando I Rodríguez, Anthony B Bleecker
Plants utilize ethylene as a hormone to regulate multiple developmental processes and to coordinate responses to biotic and abiotic stress. In Arabidopsis thaliana, a small family of five receptor proteins typified by ETR1 mediates ethylene perception. Our previous work suggested that copper ions likely play a role in ethylene binding. An independent study indicated that the ran1 mutants, which display ethylene-like responses to the ethylene antagonist trans-cyclooctene, have mutations in the RAN1 copper-transporting P-type ATPase, once again linking copper ions to the ethylene-response pathway...
November 26, 2010: Journal of Biological Chemistry
Isabelle Morin, Simon Gudin, Elisabeth Mintz, Martine Cuillel
In yeast, copper delivery to the trans-Golgi network involves interactions between the metallo-chaperone Atx1 and the N-terminus of Ccc2, the P-type ATPase responsible for copper transport across trans-Golgi network membranes. Disruption of the Atx1-Ccc2 route leads to cell growth arrest in a copper-and-iron-limited medium, a phenotype allowing complementation studies. Coexpression of Atx1 and Ccc2 mutants in an atx1Delta ccc2Delta strain allowed us to study in vivo Atx1-Ccc2 and intra-Ccc2 domain-domain interactions, leading to active copper transfer into the trans-Golgi network...
August 2009: FEBS Journal
Chardeen Peter, Julie Laliberté, Jude Beaudoin, Simon Labbé
Copper amine oxidases (CAOs) have been proposed to be involved in the metabolism of xenobiotic and biogenic amines. The requirement for copper is absolute for their activity. In the fission yeast Schizosaccharomyces pombe, cao1(+) and cao2(+) genes are predicted to encode members of the CAO family. While both genes are expressed in wild-type cells, we determined that the expression of only cao1(+) but not cao2(+) results in the production of an active enzyme. Site-directed mutagenesis identified three histidine residues within the C-terminal region of Cao1 that are necessary for amine oxidase activity...
October 2008: Eukaryotic Cell
Yuriko Kobayashi, Keishi Kuroda, Keisuke Kimura, Jennafer L Southron-Francis, Aya Furuzawa, Kazuhiko Kimura, Satoshi Iuchi, Masatomo Kobayashi, Gregory J Taylor, Hiroyuki Koyama
Copper (Cu) is an essential element in plant nutrition, but it inhibits the growth of roots at low concentrations. Accessions of Arabidopsis (Arabidopsis thaliana) vary in their tolerance to Cu. To understand the molecular mechanism of Cu tolerance in Arabidopsis, we performed quantitative trait locus (QTL) analysis and accession studies. One major QTL on chromosome 1 (QTL1) explained 52% of the phenotypic variation in Cu tolerance in roots in a Landsberg erecta/Cape Verde Islands (Ler/Cvi) recombinant inbred population...
October 2008: Plant Physiology
Gloria Hsi, Lara M Cullen, Georgina Macintyre, Matthew M Chen, D Moira Glerum, Diane W Cox
ATP7B is a copper transporting P-type ATPase defective in the autosomal recessive copper storage disorder, Wilson disease (WND). Functional assessment of variants helps to distinguish normal from disease-causing variants and provides information on important amino acid residues. A total of 11 missense variants of ATP7B, originally identified in WND patients, were examined for their capacity to functionally complement a yeast mutant strain in which the yeast gene ortholog, CCC2, was disrupted. Solution structures of ATP7B domains were used to predict the effects of each variant on ATP7B structure...
April 2008: Human Mutation
Lucia Banci, Ivano Bertini, Christos T Chasapis, Antonio Rosato, Leonardo Tenori
Yeast Ccc2 is a P-type ATPase responsible for transport of copper(I) from the cytosol to the trans-Golgi network. It possesses a soluble cytosolic N-terminal region containing two copper(I)-binding domains. Homologous eukaryotic copper-transporting ATPases have from one to six domains. We have expressed a fragment encompassing residues 1-150 of Ccc2, which corresponds to the two domains, and found that the second domain was substantially less structured than the first. The first domain could bind copper(I) and interact with the partner protein Atx1 at variance with the second...
December 21, 2007: Biochemical and Biophysical Research Communications
Roger Miras, Isabelle Morin, Olivier Jacquin, Martine Cuillel, Florent Guillain, Elisabeth Mintz
Copper is both an essential element as a catalytic cofactor and a toxic element because of its redox properties. Once in the cell, Cu(I) binds to glutathione (GSH) and various thiol-rich proteins that sequester and/or exchange copper with other intracellular components. Among them, the Cu(I) chaperone Atx1 is known to deliver Cu(I) to Ccc2, the Golgi Cu-ATPase, in yeast. However, the mechanism for Cu(I) incorporation into Atx1 has not yet been unraveled. We investigated here a possible role of GSH in Cu(I) binding to Atx1...
February 2008: Journal of Biological Inorganic Chemistry: JBIC
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