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1.
Astroglia cells structurally and nutritionally support neurons in the central nervous system. They play an important role in guiding the construction of the nervous system and controlling the chemical and ionic environment of neurons. They also represent the major sites for accumulation and immobilisation of toxic metal ions most probably connected with metallothioneins. For this reason astroglia cells possess high cytosolic levels of metallothioneins I, II and III (MT-I,II,III). Our aim was to establish the inducibility and metal binding of MTs in two human astrocytoma cell lines, U87 MG (astrocytoma–glioblastoma, grade IV) and IPDDC-2A (astrocytoma, grade II), on exposure to cadmium chloride (1 μM). MTs were identified by molecular weight (size exclusion chromatography) and their metal content (Cd, Zn and Cu) to follow the interactions between metals. We showed that MTs are constitutively expressed in both human astrocytoma cell lines. In accordance with the higher malignancy grade of U87 MG, the amount of MTs was higher in U87 MG than in IPDDC-2A cells. After 24 hours of exposure to Cd their expression greatly increased in both cell lines and they were capable of immobilising almost all water soluble Cd. Induction of MTs in U87 MG cells was additionally followed up to 48 hours with exposure to different concentrations of CdCl2 (1, 10 μM). Induction was a time dependent process throughout the period. Isoform III (identified by chromatographic separation of isoform III from I/II) was present at all exposure times, but only in traces with respect to the prevailing amounts of MT-I/II isoforms. So induction can be attributed to isoform I/II only.  相似文献   

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Metallothioneins (MTs) are ubiquitous, low‐molecular weight, cysteine‐rich proteins. Despite a well‐established protective role in metal excess detoxification, there is little data about their putative physiological functions, commonly assumed to be metal homeostasis and redox equilibrium. Protein–protein interactions should have provided useful information to unveil unsuspected functions, but reports on MT interactions are scarce. This is probably due to the MT metal‐dependent 3D structure, a fact that has been seldom taken into account when performing proteomic interaction assays. In the present work, we have detected that the two major D. melanogaster isoforms (MtnA and MtnB) interact with the peroxiredoxin (Prx) encoded by the gene Jafrac1, both in a clear metal‐dependent pattern. The MT–Prx interaction is further confirmed in Saccharomyces cerevisiae by assaying both yeast MTs (Crs5p and Cup1p) versus Tsa1p and Tsa2p, the Jafrac1 homologous Prxs in this organism. Thus, a new methodological approach to detect MT‐interacting proteins in different proteomes is established on the basis of assaying MTs in the form of different metal complexes. Furthermore, new perspectives to investigate the often hypothesized contribution of MTs to the redox physiological networks are open.  相似文献   

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Cryptococcus neoformans metallothioneins (MTs), CnMT1 and CnMT2, have been identified as essential infectivity and virulence factors of this pathogen. Both MTs are unusually long Cu‐thioneins, exhibiting protein architecture and metal‐binding abilities compatible with the hypothesis of resulting from three and five tandem repetitions of 7‐Cys motives, respectively, each of them folding into Cu5‐clusters. Through the study of the Zn(II)‐ and Cu(I)‐binding capabilities of several CnMT1 truncated mutants, we show that a 7‐Cys segment of CnMT1 folds into Cu5‐species, of additive capacity when joined in tandem. All the obtained Cu‐complexes share practically similar architectural features, if judging by their almost equivalent CD fingerprints, and they also share their capacity to restore copper tolerance in MT‐devoid yeast cells. Besides the analysis of the modular composition of these long fungal MTs, we evaluate the features of the Cys‐rich stretch spacer and flanking sequences that allow the construction of stable metal clusters by adjacent union of binding modules. Overall, our data support a mechanism by which some microbial MTs may have evolved to enlarge their original metal co‐ordination capacity under the specific selective pressure of counteracting the Cu‐based immunity mechanisms evolved by the infected hosts.  相似文献   

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[Ni(C11H9N2O5)2(H2O)2]?3(C3H7NO) ( 1 ) and [Co(C11H9N2O5)2(H2O)2]?3(C3H7NO) ( 2 ) are synthesized and characterized by elemental analysis, FT‐IR spectra, magnetic susceptibility, and thermal analysis. In addition, the crystal structure of Ni(II) complex is presented. Both complexes show distorted octahedral geometry. In 1 and 2, metal ions are coordinated by two oxygen atoms of salicylic residue and two nitrogen atoms of maleic amide residue from two ligands, and two oxygen atoms from two water molecules. In this paper, both compounds showed excellent inhibitory effects against human carbonic anhydrase (hCA) isoforms I, and II, α‐glycosidase, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE). Compounds 1 and 2 had Ki values of 18.36 ± 4.38 and 26.61 ± 7.54 nM against hCA I and 13.81 ± 3.02 and 29.56 ± 6.52 nM against hCA II, respectively. On the other hand, their Ki values were found to be 487.45 ± 54.18 and 453.81 ± 118.61 nM against AChE and 199.21 ± 50.35 and 409.41 ± 6.86 nM against BChE, respectively.  相似文献   

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To get insights into the functions of metallothionein (MT) in plant response to multiple stresses, expressions of 10 rice MT genes (OsMTs) and 7 Arabidopsis MT genes (AtMTs) were comprehensively analyzed under combined heavy metal and salt stress. OsMT1a, OsMT1b, OsMT1c, OsMT1g, and OsMT2a were increased by different heavy metals. Notably, ABA remarkably increased OsMT4 up to 80-fold. Combined salt and heavy metals (Cd, Pb, Cu) synergistically increased OsMT1a, OsMT1c, and OsMT1g, whereas combined salt and H2O2 or ABA synergistically increased OsMT1a and OsMT4. Heavy metals decreased AtMT1c, AtMT2b, and AtMT3 but cold or ABA increased AtMT1a, AtMT1c, and AtMT2a. AtMT4a was markedly increased by salt stress. Combined salt and other stresses (Pb, Cd, H2O2) synergistically increased AtMT4a. Taken together, these findings suggest that MTs in monocot and dicot respond differently to combined stresses, which provides a valuable basis to further determine the roles of MTs in broad stress tolerance.  相似文献   

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Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that bind to heavy metals. Type-1 MTs function under various abiotic stresses, including exposure to the cadmium ion. We have now isolated theBrassica rapa type-1 metallothioneirt gene (BrMT1)using yeast systems, and have found that it confers resistance to Cd in otherwise Cd-sensitive yeast. Using a constitutive CaMV35S promoter and an RbsS transit peptide, we successfully targeted BrMT1 to the chloroplastsof Arabidopsis. Overexpression in either the chloroplasts or the cytosol effectively detoxified cadmium and H2O2 stresses in transgenicArabidopsis. in particular, the chloropfast-targeted BrMTl was associated with a significant reduction in paraquat-induced chlorosis and the accumulation of H2O2. This is the first report regarding the effects of type-1 MT1 targeted to chloroplasts. Our results suggest that this may be applicable to the development of plants with enhanced tolerance against environmental stresses.  相似文献   

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Several populations with different metal tolerance, uptake and root-to-shoot transport are known for the metal hyperaccumulator plant Thlaspi caerulescens. In this study, genes differentially expressed under various Zn exposures were identified from the shoots of two T. caerulescens accessions (calaminous and non-calaminous) using fluorescent differential display RT-PCR. cDNA fragments from 16 Zn-responsive genes, including those encoding metallothionein (MT) type 2 and type 3, MRP-like transporter, pectin methylesterase (PME) and Ole e 1-like gene as well as several unknown genes, were eventually isolated. The full-length MT2 and MT3 sequences differ from those previously isolated from other Thlaspi accessions, possibly representing new alleles or isoforms. Besides the differential expression in Zn exposures, the gene expression was dependent on the accession. Thlaspi homologues of ClpP protease and MRP transporter were induced at high Zn concentrations. MT2 and PME were expressed at higher levels in the calaminous accession. The MTs and MRP transporter expressed in transgenic yeasts were capable of conferring Cu and Cd tolerance, whereas the Ole e 1-like gene enhanced toxicity to these metals. The MTs increased yeast intracellular Cd content. As no significant differences were found between Arabidopsis and Thlaspi MTs, they apparently do not differ in their capacity to bind metals. However, the higher levels of MT2 in the calaminous accession may contribute to the Zn-adapted phenotype.  相似文献   

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Characterization of Cd-binding proteins has great analytical interest due to the high toxicity of Cd to living organisms. Metallothioneins (MTs), as Cd(II)-binding proteins are of increasing interest, since they form very stable Cd chelates and are involved in many detoxification processes. In this work, inductively coupled plasma octopole reaction cell mass spectrometry and nanospray ionization time-of-flight mass spectrometry were used in parallel and combined with two-dimensional chromatography: size exclusion followed by reversed-phase high performance liquid chromatography, to study metal complexes of MT isoforms produced in hepatic cytosols of Mus musculus during exposure experiments to Cd. Exposure experiments were carried out by subcutaneous injection of a growing dose of the toxic element ranging from 0.1 to 1.0 mg of Cd per kg of body weight per day during 10 days. A control group and three exposure groups at days 2, 6 and 10 of exposure were studied, and different cadmium, copper and zinc complexes with MTs isoforms were isolated and characterized from the two most exposed groups. The results allow gaining insight into the mechanisms involved in metal detoxification by MTs, showing the changes in the stoichiometry of metal complexes–MTs along cadmium exposure.  相似文献   

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Metallobiologists have, at large, neglected soil dwelling invertebrates; exceptions are the nematode (Caenorhabditis elegans) and snails (Helix pomatia and Cantareus aspersus). This review aims to compare and contrast the molecular, protein and cellular mechanisms of the multifunctional nematode and snail metallothioneins (MTs). The C. elegans genome contains two MT genes, mtl-1, which is constitutively expressed in the pharynx and likely to act as an essential and/or toxic metal sensor, and mtl-2, which plays a negligible role under normal physiological conditions but is strongly induced (as mtl-1) in intestinal cells upon metal exposure. It has been possible to follow the intricate phenotypic responses upon the knockdown/knockout of single and multiple MT isoforms and we have started to decipher the multifunctional role of C. elegans MTs. The snails have contributed to our understanding regarding MT evolution and diversity, structure and metal-specific functionality. The H. pomatia and C. aspersus genomes contain at least three MT isoform genes. CdMT is responsible for cadmium detoxification, CuMT is involved in copper homeostasis and Cd/CuMT is a putative ancestral MT possibly only of minor importance in metal metabolism. Further investigations of nematode, snail and other invertebrate MTs will allow the development of alternative biomarker approaches and lead to an improved understanding of metallobiology, protein evolution and toxicogenomics.  相似文献   

12.
Metallothioneins (MTs) are ubiquitous proteins with the capacity to bind heavy metal ions (mainly Cd, Zn or Cu), and they have been found in animals, plants, eukaryotic and prokaryotic micro‐organisms. We have carried out a comparative analysis of ciliate MTs (Tetrahymena species) to well‐known MTs from other organisms, discussing their exclusive features, such as the presence of aromatic amino acid residues and almost exclusive cysteine clusters (CCC) present in cadmium‐binding metallothioneins (CdMTs), higher heavy metal‐MT stoichiometry values, and a strictly conserved modular–submodular structure. Based on this last feature and an extensive gene duplication, we propose a possible model for the evolutionary history of T. thermophila MTs. We also suggest possible functions for these MTs from consideration of their differential gene expressions and discuss the potential use of these proteins and/or their gene promoters for designing molecular or whole‐cell biosensors for a fast detection of heavy metals in diverse polluted ecosystems.  相似文献   

13.
Oxidative stress is a major cause of cellular injury in a variety of human diseases including neurodegenerative disorders. Thus, removal of excessive reactive oxygen species (ROS) or suppression of ROS generation may be effective in preventing oxidative stress‐induced cell death. This study was designed to investigate the effect of icariside II (ICS II), a novel phosphodiesterase 5 inhibitor, on hydrogen peroxide (H2O2)‐induced death of highly differentiated rat neuronal PC12 cells, and to further examine the underlying mechanisms. We found that ICS II pre‐treatment significantly abrogated H2O2‐induced PC12 cell death as demonstrated by the increase of the number of metabolically active cells and decrease of intracellular lactate dehydrogenase (LDH) release. Furthermore, ICS II inhibited H2O2‐induced cell death through attenuating intracellular ROS production, mitochondrial impairment, and activating glycogen synthase kinase‐3β (GSK‐3β) as demonstrated by reduced intracellular and mitochondrial ROS levels, restored mitochondrial membrane potential (MMP), decreased p‐tyr216‐GSK‐3β level and increased p‐ser9‐GSK‐3β level respectively. The GSK‐3β inhibitor SB216763 abrogated H2O2‐induced cell death. Moreover, ICS II significantly inhibited H2O2‐induced autophagy by the reducing autophagosomes number and the LC3‐II/LC3‐I ratio, down‐regulating Beclin‐1 expression, and up‐regulating p62/SQSTM1 and HSP60 expression. The autophagy inhibitor 3‐methyl adenine (3‐MA) blocked H2O2‐induced cell death. Altogether, this study demonstrated that ICS II may alleviate oxidative stress‐induced autophagy in PC12 cells, and the underlying mechanisms are related to its antioxidant activity functioning via ROS/GSK‐3β/mitochondrial signalling pathways.  相似文献   

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Toxic effects of metals appear to be partly related to the production of reactive oxygen species (ROS), which can cause oxidative damage to cells. The ability of several redox active metals [Fe(III), Cu(II), Ag(I), Cr(III), Cr(VI)], nonredox active metals [Pb(II), Cd(II), Zn(II)], and the metalloid As(III) and As(V) to produce ROS at environmentally relevant metal concentrations was assessed. Cells of the freshwater alga Chlamydomonas reinhardtii P. A. Dang. were exposed to various metal concentrations for 2.5 h. Intracellular ROS accumulation was detected using an oxidation‐sensitive reporter dye, 5‐(and‐6)‐carboxy‐2′,7′‐dihydrodifluorofluorescein diacetate (H2DFFDA), and changes in the fluorescence signal were quantified by flow cytometry (FCM). In almost all cases, low concentrations of both redox and nonredox active metals enhanced intracellular ROS levels. The hierarchy of maximal ROS induction indicated by the increased number of stained cells compared to the control sample was as follows: Pb(II) > Fe(III) > Cd(II) > Ag(I) > Cu(II) > As(V) > Cr(VI) > Zn(II). As(III) and Cr(III) had no detectable effect. The effective free metal ion concentrations ranged from 10?6 to 10?9 M, except in the case of Fe(III), which was effective at 10?18 M. These metal concentrations did not affect algal photosynthesis. Therefore, a slightly enhanced ROS production is a general and early response to elevated, environmentally relevant metal concentrations.  相似文献   

16.
Lysophospholipids are intermediates of phospholipid metabolism resulting from stress and lysophospholipases detoxify lysophosphatidylcholine (lysoPC). Many lysophospholipases have been characterized in mammals and bacteria, but few have been reported from plants. Arabidopsis thaliana lysophospholipase 2 (lysoPL2) (At1g52760) was identified as a protein interactor of acyl‐CoA‐binding protein 2 (ACBP2) in yeast two‐hybrid analysis and co‐immunoprecipitation assays. BLASTP analysis indicated that lysoPL2 showed ~35% amino acid identity to the lysoPL1 family. Co‐localization of autofluorescence‐tagged lysoPL2 and ACBP2 by confocal microscopy in agroinfiltrated tobacco suggests the plasma membrane as a site for their subcellular interaction. LysoPL2 mRNA was induced by zinc (Zn) and hydrogen peroxide (H2O2), and lysoPL2 knockout mutants showed enhanced sensitivity to Zn and H2O2 in comparison to wild type. LysoPL2‐overexpressing Arabidopsis was more tolerant to H2O2 and cadmium (Cd) than wild type, suggesting involvement of lysoPL2 in phospholipid repair following lipid peroxidation arising from metal‐induced stress. Lipid hydroperoxide (LOOH) contents in ACBP2‐overexpressors and lysoPL2‐overexpressors after Cd‐treatment were lower than wild type, indicating that ACBP2 and lysoPL2 confer protection during oxidative stress. A role for lysoPL2 in lysoPC detoxification was demonstrated when recombinant lysoPL2 was observed to degrade lysoPC in vitro. Filter‐binding assays and Lipidex competition assays showed that (His)6‐ACBP2 binds lysoPC in vitro. Binding was disrupted in a (His)6‐ACBP2 derivative lacking the acyl‐CoA‐binding domain, confirming that this domain confers lysoPC binding. These results suggest that ACBP2 can bind both lysoPC and lysoPL2 to promote the degradation of lysoPC in response to Cd‐induced oxidative stress.  相似文献   

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Salt marshes constitute major sinks for heavy metal accumulation but the precise impact of salinity on heavy metal toxicity for halophyte plant species remains largely unknown. Young seedlings of Kosteletzkya virginica were exposed during 3 weeks in nutrient solution to Cd 5 µM in the presence or absence of 50 mM NaCl. Cadmium (Cd) reduced growth and shoot water content and had major detrimental effect on maximum quantum efficiency (Fv/Fm), effective quantum yield of photosystem II (Y(II)) and electron transport rates (ETRs). Cd induced an oxidative stress in relation to an increase in O2?? and H2O2 concentration and lead to a decrease in endogenous glutathione (GSH) and α‐tocopherol in the leaves. Cd not only increased leaf zeatin and zeatin riboside concentration but also increased the senescing compounds 1‐aminocyclopropane‐1‐carboxylic acid (ACC) and abscisic acid (ABA). Salinity reduced Cd accumulation already after 1 week of stress but was unable to restore shoot growth and thus did not induce any dilution effect. Salinity delayed the Cd‐induced leaf senescence: NaCl reduced the deleterious impact of Cd on photosynthesis apparatus through an improvement of Fv/Fm, Y(II) and ETR. Salt reduced oxidative stress in Cd‐treated plants through an increase in GSH, α‐tocopherol and ascorbic acid synthesis and an increase in glutathione reductase (EC 1.6.4.2) activity. Additional salt reduced ACC and ABA accumulation in Cd+NaCl‐treated leaves comparing to Cd alone. It is concluded that salinity affords efficient protection against Cd to the halophyte species K. virginica, in relation to an improved management of oxidative stress and hormonal status.  相似文献   

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A determination method for Co(II), Fe(II) and Cr(III) ions by luminol‐H2O2 system using chelating reagents is presented. A metal ion‐chelating ligand complex with a Co(II) ion and a chelating reagent like ethylenediaminetetraacetic acid (EDTA) produced highly enhanced chemiluminescence (CL) intensity as well as longer lifetime in the luminol‐H2O2 system compared to metals that exist as free ions. Whereas free Cu(II) and Pb(II) ions had a strong catalytic effect on the luminol‐H2O2 system, significantly, the complexes of Cu(II) and Pb(II) with chelating reagents lost their catalytic activity due to the chelating reagents acting as masking agents. Based on the observed phenomenon, it was possible to determine Co(II), Fe(II) and Cr(III) ions with enhanced sensitivity and selectivity using the chelating reagents of the luminol‐H2O2 system. The effects of ligand, H2O2 concentration, pH, buffer solution and concentrations of chelating reagents on CL intensity of the luminol‐H2O2 system were investigated and optimized for the determination of Co(II), Fe(II) and Cr(III) ions. Under optimized conditions, the calibration curve of metal ions was linear over the range of 2.0 × 10‐8 to 2.0 × 10‐5 M for Co(II), 1.0 × 10‐7 to 2.0 × 10‐5 M for Fe (II) and 2.0 × 10‐7 to 1.0 × 10‐4 M for Cr(III). Limits of detection (3σ/s) were 1.2 × 10‐8, 4.0 × 10‐8 and 1.2 × 10‐7 M for Co(II), Fe(II) and Cr(III), respectively. Copyright © 2012 John Wiley & Sons, Ltd.  相似文献   

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