Science.gov

Sample records for individual arabidopsis metallothioneins

  1. Overexpression of Nelumbo nucifera metallothioneins 2a and 3 enhances seed germination vigor in Arabidopsis.

    PubMed

    Zhou, Yuliang; Chu, Pu; Chen, Huhui; Li, Yin; Liu, Jun; Ding, Yu; Tsang, Edward W T; Jiang, Liwen; Wu, Keqiang; Huang, Shangzhi

    2012-03-01

    Metallothioneins (MTs) are small, cysteine-rich and metal-binding proteins which are involved in metal homeostasis and scavenging of reactive oxygen species. Although plant MTs have been intensively studied, their roles in seeds remain to be clearly established. Here, we report the isolation and characterization of NnMT2a, NnMT2b and NnMT3 from sacred lotus (Nelumbo nucifera Gaertn.) and their roles in seed germination vigor. The transcripts of NnMT2a, NnMT2b and NnMT3 were highly expressed in developing and germinating sacred lotus seeds, and were dramatically up-regulated in response to high salinity, oxidative stresses and heavy metals. Analysis of transformed Arabidopsis protoplasts showed that NnMT2a-YFP and NnMT3-YFP were localized in cytoplasm and nucleoplasm. Transgenic Arabidopsis seeds overexpressing NnMT2a and NnMT3 displayed improved resistance to accelerated aging (AA) treatment, indicating their significant roles in seed germination vigor. These transgenic seeds also exhibited higher superoxide dismutase activity compared to wild-type seeds after AA treatment. In addition, we showed that NnMT2a and NnMT3 conferred improved germination ability to NaCl and methyl viologen on transgenic Arabidopsis seeds. Taken together, these data demonstrate that overexpression of NnMT2a and NnMT3 in Arabidopsis significantly enhances seed germination vigor after AA treatment and under abiotic stresses.

  2. Purification and immunological identification of metallothioneins 1 and 2 from Arabidopsis thaliana.

    PubMed Central

    Murphy, A; Zhou, J; Goldsbrough, P B; Taiz, L

    1997-01-01

    Gene families encoding two types of metallothioneins (MTs) MT1 and MT2, have been identified in Arabidopsis thaliana, and their respective mRNAs have been shown to be regulated by copper in a tissue-specific manner (J. Zhou and P.B. Goldsbrough [1994] Plant Cell 6: 875-884; J. Zhou and P.B. Goldsbrough [1995] Mol Gen Genet 248: 318-328; A.S. Murphy and L. Taiz [1995] Plant Physiol 109: 1-10). However, to date the protein products have not been identified. To purify MT proteins from Arabidopsis, we isolated low-molecular-mass, copper-binding, thiol-rich proteins using selective precipitation followed by size-exclusion, copper-affinity, and thiol-affinity chromatographies. Polyclonal antibodies raised against Arabidopsis MT-glutathione-S-transferase fusion proteins cross-reacted with the 4.5- and 8-kD bands in immunoblots of low-molecular-mass, copper-binding proteins purified from seedling, mature leaf, and mature root tissues. The identity of the proteins was subsequently confirmed by amino acid sequencing. MT1 expression was constitutive in roots and inducible by copper in mature leaves; the reverse pattern was observed for MT2. MT2 expression was also concentrated in the growing tip of the root. The accumulation of the MT1- and MT2-encoded proteins thus parallels the regulation of their respective mRNAs with regard to tissue specificity and induction by copper. In addition, a new type of MT, designated MT3, was derived from the database, detected by reverse transcription-polymerase chain reaction, and tentatively identified at the protein level by amino acid sequencing of a 7-kD cysteine-rich polypeptide. PMID:9112777

  3. Expression of the rgMT gene, encoding for a rice metallothionein-like protein in Saccharomyces cerevisiae and Arabidopsis thaliana.

    PubMed

    Jin, Shumei; Sun, Dan; Wang, Ji; Li, Ying; Wang, Xinwang; Liu, Shenkui

    2014-12-01

    Metallothioneins (MTs) are cysteine-rich proteins of low molecular weight with many attributed functions, such as providing protection against metal toxicity, being involved in regulation of metal ions uptake that can impact plant physiology and providing protection against oxidative stress. However, the precise function of the metallothionein-like proteins such as the one coded for rgMT gene isolated from rice (Oryza sativa L.) is not completely understood. The whole genome analysis of rice (O. sativa) showed that the rgMT gene is homologue to the Os11g47809 on chromosome 11 of O. sativa sp. japonica genome. This study used the rgMT coding sequence to create transgenic lines to investigate the subcellular localization of the protein, as well as the impact of gene expression in yeast (Saccharomyces cerevisiae) and Arabidopsis thaliana under heavy metal ion, salt and oxidative stresses. The results indicate that the rgMT gene was expressed in the cytoplasm of transgenic cells. Yeast cells transgenic for rgMT showed vigorous growth compared to the nontransgenic controls when exposed to 7 mM CuCl2, 10 mM FeCl2, 1 M NaCl, 24 mM NaHCO3 and 3.2 mM H2O2, but there was no significant difference for other stresses tested. Similarly, Arabidopsis transgenic for rgMT displayed significantly improved seed germination rates over that of the control when the seeds were stressed with 100 μM CuCl2 or 1 mM H2O2. Increased biomass was observed in the presence of 100 μM CuCl2, 220 μM FeCl2, 3 mM Na2CO3, 5 mM NaHCO3 or 1 mM H2O2. These results indicate that the expression of the rice rgMT gene in transgenic yeast and Arabidopsis is implicated in improving their tolerance for certain salt and peroxide stressors.

  4. Metallothionein protection of cadmium toxicity

    SciTech Connect

    Klaassen, Curtis D. Liu, Jie; Diwan, Bhalchandra A.

    2009-08-01

    The discovery of the cadmium (Cd)-binding protein from horse kidney in 1957 marked the birth of research on this low-molecular weight, cysteine-rich protein called metallothionein (MT) in Cd toxicology. MT plays minimal roles in the gastrointestinal absorption of Cd, but MT plays important roles in Cd retention in tissues and dramatically decreases biliary excretion of Cd. Cd-bound to MT is responsible for Cd accumulation in tissues and the long biological half-life of Cd in the body. Induction of MT protects against acute Cd-induced lethality, as well as acute toxicity to the liver and lung. Intracellular MT also plays important roles in ameliorating Cd toxicity following prolonged exposures, particularly chronic Cd-induced nephrotoxicity, osteotoxicity, and toxicity to the lung, liver, and immune system. There is an association between human and rodent Cd exposure and prostate cancers, especially in the portions where MT is poorly expressed. MT expression in Cd-induced tumors varies depending on the type and the stage of tumor development. For instance, high levels of MT are detected in Cd-induced sarcomas at the injection site, whereas the sarcoma metastases are devoid of MT. The use of MT-transgenic and MT-null mice has greatly helped define the role of MT in Cd toxicology, with the MT-null mice being hypersensitive and MT-transgenic mice resistant to Cd toxicity. Thus, MT is critical for protecting human health from Cd toxicity. There are large individual variations in MT expression, which might in turn predispose some people to Cd toxicity.

  5. Yeast metallothionein function in metal ion detoxification.

    PubMed

    Ecker, D J; Butt, T R; Sternberg, E J; Neeper, M P; Debouck, C; Gorman, J A; Crooke, S T

    1986-12-25

    A genetic approach was taken to test the function of yeast metallothionein in metal ion detoxification. A yeast strain was constructed in which the metallothionein locus was deleted (cup1 delta). The cup1 delta strain was complemented with normal or mutant metallothionein genes under normal or constitutive regulatory control on high copy episomal plasmids. Metal resistance of the cup1 delta strain with and without the metallothionein-expressing vectors was analyzed. The normally regulated metallothionein gene conferred resistance only to copper (1000-fold); constitutively expressed metallothionein conferred resistance to both copper (500-fold) and cadmium (1000-fold), but not to mercury, zinc, silver, cobalt, nickel, gold, platinum, lanthanum, uranium, or tin. Two mutant versions of the metallothionein gene were constructed and tested for their ability to confer metal resistance in the cup1 delta background. The first had a deletion of a highly conserved amino acid sequence (Lys-Lys-Ser-Cys-Cys-Ser). The second was a hybrid gene consisting of the sequences coding for the first 20 amino acids of the yeast protein fused to the monkey metallothionein gene. Expression of these genes under the CUP1 promoter provided significant protection from copper, but none of the other metals tested. These results demonstrate that there is significant flexibility in the structural requirements for metallothionein to function in copper detoxification and that yeast metallothionein is also capable of detoxifying cadmium under conditions of constitutive expression.

  6. Multiscale digital Arabidopsis predicts individual organ and whole-organism growth

    PubMed Central

    Chew, Yin Hoon; Wenden, Bénédicte; Flis, Anna; Mengin, Virginie; Taylor, Jasper; Davey, Christopher L.; Tindal, Christopher; Thomas, Howard; Ougham, Helen J.; de Reffye, Philippe; Stitt, Mark; Williams, Mathew; Muetzelfeldt, Robert; Halliday, Karen J.; Millar, Andrew J.

    2014-01-01

    Understanding how dynamic molecular networks affect whole-organism physiology, analogous to mapping genotype to phenotype, remains a key challenge in biology. Quantitative models that represent processes at multiple scales and link understanding from several research domains can help to tackle this problem. Such integrated models are more common in crop science and ecophysiology than in the research communities that elucidate molecular networks. Several laboratories have modeled particular aspects of growth in Arabidopsis thaliana, but it was unclear whether these existing models could productively be combined. We test this approach by constructing a multiscale model of Arabidopsis rosette growth. Four existing models were integrated with minimal parameter modification (leaf water content and one flowering parameter used measured data). The resulting framework model links genetic regulation and biochemical dynamics to events at the organ and whole-plant levels, helping to understand the combined effects of endogenous and environmental regulators on Arabidopsis growth. The framework model was validated and tested with metabolic, physiological, and biomass data from two laboratories, for five photoperiods, three accessions, and a transgenic line, highlighting the plasticity of plant growth strategies. The model was extended to include stochastic development. Model simulations gave insight into the developmental control of leaf production and provided a quantitative explanation for the pleiotropic developmental phenotype caused by overexpression of miR156, which was an open question. Modular, multiscale models, assembling knowledge from systems biology to ecophysiology, will help to understand and to engineer plant behavior from the genome to the field. PMID:25197087

  7. Individual and joint activity of terpenoids, isolated from Calamintha nepeta extract, on Arabidopsis thaliana.

    PubMed

    Araniti, Fabrizio; Graña, Elisa; Reigosa, Manuel J; Sánchez-Moreiras, Adela M; Abenavoli, Maria Rosa

    2013-01-01

    Four terpenoids, camphor, pulegone, trans-caryophyllene and farnesene, previously found in Calamintha nepeta (L.) Savi methanolic extract and essential oils were assayed on germination and root growth of Arabidopsis thaliana (L.) Heynh. None of the terpenes, singularly or in combination, was able to inhibit the germination process. Farnesene and trans-caryophyllene caused a strong inhibitory effect on root growth, and pulegone, at the highest concentrations, reduced lateral root formation. Although the mixture of camphor-trans-caryophyllene with or without farnesene did not cause any effect on root growth, the addition of pulegone induced a marked synergistic activity. Moreover, the addition, at low concentration, of farnesene to pulegone-camphor-trans-caryophyllene mixture further increased the inhibitory effect on root elongation. These results suggested that the inhibitory effects caused by C. nepeta methanolic extract may depend on the combined action of different molecules.

  8. Voltammetric Methods in Metallothionein Research

    PubMed Central

    Navrátil, Tomáš

    2005-01-01

    The application of voltammetric methods using different rates of polarisation on HMDE reveal inert or labile behaviour of Cd- or Zn- complexes in the presence of excessive cadmium or zinc ions in solution. This phenomenon was demonstrated first on the simplest phytochelatin – complex of peptide (γ-Glu-Cys)2 Gly with cadmium, later on rabbit liver metallothioneins – Cd7 MT in the presence of cadmium and Cd5 Zn2 MT in the presence of zinc. Voltammetric methods can distinguish between labile and inert complexes present simultaneously and therefore could elucidate their role in reactions of metal ion transfer. Another method using different rates of polarisation – elimination voltammetry with linear scan – proved that S-tetracoordinated complexes of Cd(II) or Zn(II) in the above-mentioned metallothioneins on HMDE are reduced in the adsorbed state. This implies the possibility of increasing the sensitivity of identification or determination of the above complexes. On carbon composite electrode, similar behaviour of Cd-complexes as on HMDE was observed using differential pulse voltammetry. PMID:18365088

  9. Arsenic binding to Fucus vesiculosus metallothionein.

    PubMed

    Merrifield, Maureen E; Ngu, Thanh; Stillman, Martin J

    2004-11-05

    The seaweed Fucus vesiculosus is a member of the brown algae family. Kille and co-workers [Biochem. J. 338 (1999) 553] reported that this species contains the gene for metallothionein. Metallothionein is a metalloprotein having low molecular weight, and high cysteine content, which binds a range of metals. F. vesiculosus bioaccumulates arsenic from the aquatic environment [Mar. Chem. 18 (1986) 321]. In this paper we describe arsenic binding to F. vesiculosus metallothionein, characterized by electrospray ionization mass spectrometry. Five arsenic-MT species were detected with increasing As to protein ratios. These results provide important information about the metal-chelation behaviour of this novel algal metallothionein which is a putative model for arsenic binding to F. vesiculosus in vivo.

  10. Monoclonal antibodies against metallothioneins and metalloproteins.

    PubMed

    Talbot, B G; Bilodeau, G; Thirion, J P

    1986-10-01

    Monoclonal antibodies against rabbit metallothioneins (MT) were prepared by in vitro immunization of mouse lymphocytes with a mixture of the two forms of metallothionein MT1 and MT2. Six IgM antibodies (TN1,3,4,5,6,7) which bind to metallothionein were characterized. Antibody TN3 is specific for rabbit MT1 and does not react with any other MT's tested. TN5 is specific for both rabbit MT1 and MT2. TN7 is specific for rabbit MT2 but not MT1 and cross-reacts also with Chinese hamster, mouse and rat metallothioneins. The antibodies TN1, TN4 and TN6 bind not only to rabbit MT1 and MT2 but also to other metal binding proteins like alcohol dehydrogenase and carbonic anhydrase.

  11. Dietary cadmium and benzo(a)pyrene increased intestinal metallothionein expression in the fish Fundulus heteroclitus

    SciTech Connect

    Roesijadi, Guritno; Rezvankhah, Saeid; Perez-Matus, Alejandro; Mitelberg, A.; Torruellas, K.; Van Veld, P. A.

    2008-10-17

    To test the effect of dietary exposure to cadmium and benzo(a)pyrene on induction of metallothionein mRNA in the Fundulus heteroclitus, fish were individually fed a pelletized gel food containing cadmium, benzo(a)pyrene, or a combination of the two over a period of seven days, then analyzed for relative levels of metallothionein mRNA in the intestine, liver, and gill using real-time RT-qPCR. An initial experiment with only cadmium exposure showed an apparent 10-fold induction in the intestine, but no induction in liver or gill. Ingestion of contaminated pellets varied in individual fish, and because it was possible to monitor individual ingestion rates with our method, individual cadmium doses were estimated from the amount of ingested cadmium. When the levels of metallothionein mRNA were related to the dose to each fish, a linear dose-response relationship was observed for the intestine, but not the other organs, which showed no induction. In a second experiment, dose was controlled by placing the entire daily cadmium dose into a single contaminated pellet that was fed first (thereby, effectively controlling the effect of variable ingestion rates), and the interaction between cadmium and benzo(a)pyrene was also investigated. The intestine was again the primary organ for metallothionein induction by cadmium, with a 20-fold increase in metallothionein mRNA over control levels. When benzo(a)pyrene was administered together with cadmium, induction of metallothionein was potentiated by the presence of benzo(a)pyrene, with the main effect seen in the intestine, where already high levels of induction by cadmium alone increased by 1.74-fold when benzo(a)pyrene was present.

  12. Null Mutants of Individual RABA Genes Impact the Proportion of Different Cell Wall Components in Stem Tissue of Arabidopsis thaliana

    PubMed Central

    Lunn, Daniel; Gaddipati, Sanyasi R.; Tucker, Gregory A.; Lycett, Grantley W.

    2013-01-01

    In Arabidopsis, and other plants, the RABA GTPases (orthologous to the Rab11a of mammals) have expanded in number and diversity and have been shown to belong to eight sub clades, some of which have been implicated in controlling vesicles that traffic cell wall polymers and enzymes that synthesise or modify them to the cell wall. In order to investigate this, we have investigated whether T-DNA insertion knockouts of individual RABA genes belonging to different sub clades, impact on the composition of the plant cell wall. Single gene knockouts of the RABA1, RABA2 and RABA4 sub clades primarily affected the percentage composition of pectin, cellulose and hemicellulose within the cell wall, respectively, despite having no obvious phenotype in the whole plant. We hypothesise that vesicles carrying specific types of cargoes from the Golgi to the cell surface may be regulated by particular sub types of RABA proteins, a finding that could have wider implications for how trafficking systems work and could be a useful tool in cell wall research and other fields of plant biology. PMID:24124508

  13. Arsenic Induction of Metallothionein and Metallothionein Induction Against Arsenic Cytotoxicity.

    PubMed

    Rahman, Mohammad Tariqur; De Ley, Marc

    Human exposure to arsenic (As) can lead to oxidative stress that can become evident in organs such as the skin, liver, kidneys and lungs. Several intracellular antioxidant defense mechanisms including glutathione (GSH) and metallothionein (MT) have been shown to minimize As cytotoxicity. The current review summarizes the involvement of MT as an intracellular defense mechanism against As cytotoxicity, mostly in blood. Zinc (Zn) and selenium (Se) supplements are also proposed as a possible remediation of As cytotoxicity. In vivo and in vitro studies on As toxicity were reviewed to summarize cytotoxic mechanisms of As. Intracellular antioxidant defense mechanisms of MT are linked in relation to As cytotoxicity. Arsenic uses a different route, compared to major metal MT inducers such as Zn, to enter/exit blood cells. A number of in vivo and in vitro studies showed that upregulated MT biosynthesis in blood components are related to toxic levels of As. Despite the cysteine residues in MT that aid to bind As, MT is not the preferred binding protein for As. Nonetheless, intracellular oxidative stress due to As toxicity can be minimized, if not eliminated, by MT. Thus MT induction by essential metals such as Zn and Se supplementation could be beneficial to fight against As toxicity.

  14. Metallothioneins: potential therapeutic aids for wound healing in the skin.

    PubMed

    Lansdown, Alan B G

    2002-01-01

    Metallothioneins are a family of low molecular weight proteins containing approximately 30 % cysteine. Expression of the metallothionein gene is up-regulated in the skin following topical application of zinc and copper, and in wound margins particularly in regions of high mitotic activity. This induction of metallothionein in the wound margin may reflect its role in promoting cell proliferation and reepithelialiation. The action of metallothioneins in these processes may result from the large number of Zinc-dependent and copper-dependent enzymes required for cell proliferation and matrix remodeling. In addition, selected growth factors may modulate metallothionein gene expression and hence the ability of cells to proliferate. Recent studies suggest that metallothioneins may be an attractive target for development as vulnerary agents.

  15. Arsenic metalation of seaweed Fucus vesiculosus metallothionein: the importance of the interdomain linker in metallothionein.

    PubMed

    Ngu, Thanh T; Lee, Janice A; Rushton, Moira K; Stillman, Martin J

    2009-09-22

    The presence of metallothionein in seaweed Fucus vesiculosus has been suggested as the protecting agent against toxic metals in the contaminated waters it can grow in. We report the first kinetic pathway data for A3+ binding to an algal metallothionein, F. vesiculosus metallothionein (rfMT). The time and temperature dependence of the relative concentrations of apo-rfMT and the five As-containing species have been determined following mixing of As3+ and apo-rfMT using electrospray ionization mass spectrometry (ESI MS). Kinetic analysis of the detailed time-resolved mass spectral data for As3+ metalation allows the simulation of the metalation reactions showing the consumption of apo-rfMT, the formation and consumption of As1- to As4-rfMT, and subsequent, final formation of As5-rfMT. The kinetic model proposed here provides a stepwise analysis of the metalation reaction showing time-resolved occupancy of the Cys7 and the Cys9 domain. The rate constants (M(-1) s(-1)) calculated from the fits for the 7-cysteine gamma domain are k1gamma, 19.8, and k2gamma, 1.4, and for the 9-cysteine beta domain are k1beta, 16.3, k2beta, 9.1, and k3beta, 2.2. The activation energies and Arrhenius factors for each of the reaction steps are also reported. rfMT has a long 14 residue linker, which as we show from analysis of the ESI MS data, allows each of its two domains to bind As3+ independently of each other. The analysis provides for the first time an explanation of the differing metal-binding properties of two-domain MTs with linkers of varying lengths, suggesting further comparison between plant (with long linkers) and mammalian (with short linkers) metallothioneins will shed light on the role of the interdomain linker.

  16. Isolation and characterization of a hepatic metallothionein from mice.

    PubMed

    Nordberg, M; Nordberg, G F; Piscator, M

    1975-01-01

    Mice were given cadmium chloride orally for 18 weeks, after which they were given two subcutaneous injections of cadmium chloride. Cadmium-and zinc-containing proteins were obtained from livers by ultracentrifugation, Sephadex chromatography and isoelectric focusing. Metallothionein with apI of 4.2 at 8 degrees C was separated from other proteins (possibly including other forms of metallothionein) by the isoelectric procedure. The metallothionein with pI = 4.2 had a high E250, reflecting abundant Cd-SH bonds, in accord with amino acid analysis, which gave a cysteine content of 34.6 residues % and showed an absence of aromatic amino acids. These results indicated a very high purity of the form of metallothionein isolated after isoelectric focusing.

  17. Mammalian Metallothionein-2A and Oxidative Stress

    PubMed Central

    Ling, Xue-Bin; Wei, Hong-Wei; Wang, Jun; Kong, Yue-Qiong; Wu, Yu-You; Guo, Jun-Li; Li, Tian-Fa; Li, Ji-Ke

    2016-01-01

    Mammalian metallothionein-2A (MT2A) has received considerable attention in recent years due to its crucial pathophysiological role in anti-oxidant, anti-apoptosis, detoxification and anti-inflammation. For many years, most studies evaluating the effects of MT2A have focused on reactive oxygen species (ROS), as second messengers that lead to oxidative stress injury of cells and tissues. Recent studies have highlighted that oxidative stress could activate mitogen-activated protein kinases (MAPKs), and MT2A, as a mediator of MAPKs, to regulate the pathogenesis of various diseases. However, the molecule mechanism of MT2A remains elusive. A deeper understanding of the functional, biochemical and molecular characteristics of MT2A would be identified, in order to bring new opportunities for oxidative stress therapy. PMID:27608012

  18. Different redox states of metallothionein/thionein in biological tissue

    PubMed Central

    Krężel, Artur; Maret, Wolfgang

    2006-01-01

    Mammalian metallothioneins are redox-active metalloproteins. In the case of zinc metallothioneins, the redox activity resides in the cysteine sulfur ligands of zinc. Oxidation releases zinc, whereas reduction re-generates zinc-binding capacity. Attempts to demonstrate the presence of the apoprotein (thionein) and the oxidized protein (thionin) in tissues posed tremendous analytical challenges. One emerging strategy is differential chemical modification of cysteine residues in the protein. Chemical modification distinguishes three states of the cysteine ligands (reduced, oxidized and metal-bound) based on (i) quenched reactivity of the thiolates when bound to metal ions and restoration of thiol reactivity in the presence of metal-ion-chelating agents, and (ii) modification of free thiols with alkylating agents and subsequent reduction of disulfides to yield reactive thiols. Under normal physiological conditions, metallothionein exists in three states in rat liver and in cell lines. Ras-mediated oncogenic transformation of normal HOSE (human ovarian surface epithelial) cells induces oxidative stress and increases the amount of thionin and the availability of cellular zinc. These experiments support the notion that metallothionein is a dynamic protein in terms of its redox state and metal content and functions at a juncture of redox and zinc metabolism. Thus redox control of zinc availability from this protein establishes multiple methods of zinc-dependent cellular regulation, while the presence of both oxidized and reduced states of the apoprotein suggest that they serve as a redox couple, the generation of which is controlled by metal ion release from metallothionein. PMID:17134375

  19. Drosophila melanogaster metallothionein genes: Selection for duplications

    SciTech Connect

    Lange, B.W.

    1989-01-01

    The metallothionein genes of Drosophila melanogaster, Mtn and Mto, may play an important role in heavy-metal detoxification. In order to investigate the possibility of increased selection for duplications of these genes in natural populations exposed to high levels of heavy metals, I compared the frequencies of such duplications among flies collected from metal-contaminated and non-contaminated orchards in Pennsylvania, Tennessee, and Georgia. Contaminated of collection sites and of local flies was confirmed by atomic absorption spectrosphotometry. Six-nucleotide-recognizing restriction enzyme analysis was used to screen 1666 wild third chromosomes for Mtn duplications. A subset (327) of these lines was screened for Mto duplications: none were found. Cadmium tolerance test performed on F{sub 2} progeny of wild females failed to detect a difference in tolerance levels between flies from contaminated orchards and flies from control orchards. Estimates of sequence diversity among a subsample (92) of the chromosomes used in the duplication survey, including all 27 Mtn duplication chromosomes, were obtained using four-nucleotide-recognizing restriction enzyme analysis.

  20. Metallothionein in rabbit kidneys preserved for transplantation.

    PubMed

    Elinder, C G; Lundgren, G; Nordberg, M; Palm, B; Piscator, M

    1984-03-01

    Thirteen rabbits were given repeated cadmium injections to achieve cadmium concentrations in kidney cortex ranging from 0.05 to 1 mmole Cd/kg wet weight. Another four animals served as controls. One kidney from each animal was frozen directly to -70 degrees C whereas the other kidney was kept for 24 hr at +4 degrees C in a preservative (Sachs' solution) to simulate conditions for preservation of human donor kidneys before transplantation. Protein binding of cadmium, zinc and copper in kidney homogenates and the concentration of metallothionein (MT) were measured in the kidney that was frozen directly and in the kidney that had been preserved. No gross differences in either the protein binding of cadmium, zinc and copper or in the MT content were seen between the directly frozen and preserved kidneys from the same animal. This indicates that MT is not rapidly broken down in rabbit kidneys which have been preserved similarly to human donor kidneys for 24 hr in a standard preservative solution prior to a transplantation.

  1. Expression of mouse metallothionein genes in tobacco

    SciTech Connect

    Maiti, I.B.; Yeargan, R.; Wagner, G.J.; Hunt, A.G. )

    1990-05-01

    We have expressed a mouse metallothionein (NT) gene in tobacco under control of the cauliflower mosaic virus (CaMV) 35S promoter and a pea ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) gene promoter. Seedlings in which MT gene expression is driven by the 35S promoter are resistant to toxic levels of cadmium. Mature plants carrying the 35S-MT gene accumulate less Cd in their leaves when exposed to low levels of Cd in laboratory growth conditions. Plants with the rbcS-MT construction express this gene in a light-regulated and tissue-specific manner, as expected. Moreover, the MT levels in leaves in these plants are about 20% of those seen in 35S-MT plants. These plants are currently being tested for Cd resistance. In addition, a small field evaluation of 35S-MT lines for Cd levels is being evaluated. These experiments will address the possibility of using MTs to alter Cd levels in crop species.

  2. Metallothionein immunolocalization in actinic skin nonmelanoma carcinomas.

    PubMed

    Borges Júnior, Paulo C; Ribeiro, Rosy I M A; Cardoso, Sérgio V; Berbet, Alceu L C; Rocha, Ademir; Espindola, Foued S; Loyola, Adriano M

    2007-06-01

    Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most frequent skin cancer. Its pathogeny is linked to genotoxic effects of actinic radiation exposure, especially to ultraviolet wavelength. Metallothionein (MT) is a low-molecular weight protein with high affinity for heavy metal. Its intracellular function has been related to heavy metals and free-radical detoxification, although many studies linked MT to protective action against actinic mutagenesis. In other way, overexpression in malignant tumors has been related to worse prognosis. We aimed to evaluate MT immunohistochemical expression in skin cancer associated to actinic radiation. Twenty-six BCC cases, 20 SCC, and 6 normal skin fragments were investigated. Immunohistochemical assay were performed by streptavidin-biotin-peroxidase technique with standard monoclonal antibody (E9). In normal skin, immunostaining was observed in basal layer of the epithelium. In the epithelium adjacent to tumors, suprabasal layer was also intensely labeled. Mean MT immunostaining indices were 18.5+21.2% for BCC and 69.1+14.4% for SCC. This difference was statistically significant. Higher MT expression in SCC as compared with BCC suggests association with tumoral aggressiveness.

  3. Metallothionein dimers studied by nano-spray mass spectrometry.

    PubMed

    Hathout, Yetrib; Reynolds, Kristy J; Szilagyi, Zoltan; Fenselau, Catherine

    2002-01-15

    Both transient and stable dimers of metallothionein have been characterized, based on earlier studies using NMR, circular dichroism and size-exclusion chromatography. Here additional characterization is provided by nanospray mass spectrometry. Rapid redistribution of metal ions between monomeric Cd7- and Zn7-metallothionein 2a is monitored by nanospray. An experiment in which theses two forms of the monomeric protein are separated by a dialysis membrane, which will pass metal ions but not proteins, confirms that a transient dimer must form for metal ions to be redistributed. On the other hand, size-exclusion chromatography of reconstituted Zn7- or Cd7-metallothionein revealed the presence of monomeric and dimeric species. These dimers do not equilibrate readily to form monomers and they are shown to be covalent.

  4. X-Ray Absorption Spectroscopy of Cuprous-Thiolate Clusters in Saccharomyces Cerevisiae Metallothionein

    SciTech Connect

    Zhang, L.; Pickering, I.J.; Winge, D.R.; George, G.N.

    2009-05-28

    Copper (Cu) metallothioneins are cuprous-thiolate proteins that contain multimetallic clusters, and are thought to have dual functions of Cu storage and Cu detoxification. We have used a combination of X-ray absorption spectroscopy (XAS) and density-functional theory (DFT) to investigate the nature of Cu binding to Saccharomyces cerevisiae metallothionein. We found that the XAS of metallothionein prepared, containing a full complement of Cu, was quantitatively consistent with the crystal structure, and that reconstitution of the apo-metallothionein with stoichiometric Cu results in the formation of a tetracopper cluster, indicating cooperative binding of the Cu ions by the metallothionein.

  5. Critical exposure level of cadmium for elevated urinary metallothionein-An occupational population study in China

    SciTech Connect

    Chen Liang; Jin Taiyi . E-mail: tyjin@shmu.edu.cn; Huang, Bo; Nordberg, Gunnar; Nordberg, Monica

    2006-08-15

    Cadmium is a well-known nephrotoxic agent with extremely long biological half-time of 15-30 years in humans. To prevent nephrotoxicity induced by cadmium, it is necessary to identify specific and sensitive biomarkers of cadmium exposure and renal damage, and to define critical exposure levels related to minimal nephrotoxicity in humans. In this study, urinary cadmium (UCd) and blood cadmium (BCd) were used as cadmium exposure indicators, urinary {beta}{sub 2}-microglobulin (UB2M), N-acetyl-{beta}-D-glucosaminidase (UNAG) and albumin (UALB) were applied as the effect biomarkers of tubular and glomerular dysfunction. The relationship between urinary metallothionein (UMT) and cadmium exposure biomarkers as well as effect biomarkers was examined. Significant correlations were found between the UMT and BCd, and UCd. At the same time, UB2M, UALB and UNAG showed positive correlation with UMT as well. According to this result, cadmium-exposed individuals with renal dysfunction excreted more metallothionein than those without. Dose-response relationships between UCd and urinary indicators of renal dysfunction were studied. The critical concentration of UCd was quantitatively estimated by the benchmark dose (BMD) method. The lower confidence limit of the BMD-10 (BMDL) of UCd (3.1 {mu}g/g Cr) related to increased excretion of urinary metallothionein was slightly higher than that for UNAG (2.7 {mu}g/g Cr), but lower than those of UB2M (3.4 {mu}g/g Cr) and UALB (4.2 {mu}g/g Cr). The results demonstrate that UMT may be used as a sensitive biomarker of renal tubular dysfunction in cadmium-exposed populations.

  6. Metallothionein function and genetic regulation in yeast

    SciTech Connect

    Ecker, D.J.; Butt, T.R.; Crooke, S.T.

    1986-05-01

    Copper resistance in yeast is mediated by the CUP1 locus which codes for yeast metallothionein (MT). A genetic approach was taken to study yeast MT gene regulation and to test the function of MT in the detoxification of metal ions other than copper. A yeast strain was constructed (cup1/sup ..delta../) in which the MT structural and regulatory sequences were deleted. The deleted gene was then replaced with the following genetically modified forms of MT on high copy episomal plasmid (YE/sup p/ 13): 1) the intact yeast gene with normal structural and regulatory sequences; 2) a constitutively expressed yeast promoter (TDH) running the yeast MT structural gene. Metal resistance in the cup1/sup ..delta../ strain and the cup1/sup ..delta../ strain transformed with the MT plasmid constructions was compared on metal-supplemented agar plates. Both of the high copy MT plasmids conferred in excess of 500-fold greater copper resistance to the cup1/sup ..delta../ strain. Increased cadmium resistance was not observed in any of the strains that had MT under normal regulatory control. However, the strain with constitutively expressed MT was in excess of 1000-fold more resistant to cadmium. Neither of the MT constructions conferred resistance to Hg,Zn,Co,Ni,Ag,Au,Pt,La,U or Sn. MT gene induction measured by the analysis of MT mRNA on northern blots showed that the yeast MT promoter is not induced by Cd, Zn, Au, Hg, Ag, superoxide, hydrogen peroxide, steroid hormones or heat shock.

  7. Label-free bead-based metallothionein electrochemical immunosensor.

    PubMed

    Nejdl, Lukas; Nguyen, Hoai Viet; Richtera, Lukas; Krizkova, Sona; Guran, Roman; Masarik, Michal; Hynek, David; Heger, Zbynek; Lundberg, Karin; Erikson, Kristofer; Adam, Vojtech; Kizek, Rene

    2015-08-01

    A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 μL/min). In stationary arrangement it is possible to detect MT concentrations up to 30 ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5 ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 μg/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed.

  8. Cadmium and copper metallothioneins in the American lobster, Homarus americanus

    SciTech Connect

    Engel, D.W.; Brouwer, M.

    1986-03-01

    Lobsters were fed cadmium-rich oysters for 28 days, and the induction of cadmium metallothionein and its relation to concentrations of cadmium, copper, and zinc in the digestive gland and gills was determined. A portion of the tissues also was retained for determining the cytosolic distribution of these metals by gel filtration and ion-exchange chromatography. The digestive gland contained a majority of the cadmium, copper, and zinc, and both cadmium and zinc were actively accumulated from the oysters. Gel chromatography of the digestive gland cytosol showed that initially only copper was bound to a protein with a molecular weight in the range of metallothionein (i.e., 10,000-7000). However, after feeding on cadmium-laden oysters for 28 days, both cadmium and copper were bound to the metallothioneinlike protein. Further purification of the cadmium/copper protein by ion-exchange chromatography showed that a large portion of the copper and all of the cadmium did not bind to DEAE-Sephacel. The induction of cadmium metallothionein in the digestive gland is correlated with tissue cadmium concentration. Coincident with the induction of the cadmium metallothionein was a cytosolic redistribution of copper. The distribution of zinc was not affected.

  9. Increased metallothionein content in rat liver induced by x irradiation and exposure to high oxygen tension

    SciTech Connect

    Shiraishi, N.; Aono, K.; Utsumi, K.

    1983-08-01

    X irradiation and exposure to high oxygen tension are known to induce lipid peroxidation. The effects of these stresses on hepatic content of metallothionein, which may be involved in the regulation of zinc and copper metabolism, have been studied. The amount of metallothionein in rat liver was increased 11-fold by a high dose of X irradiation (1000 R). Increased metallothionein content (about 15 times) was also observed in liver of rats exposed to high oxygen tension for 3 days.

  10. Immunochemical Detection of Metallothionein in Specific Epithelial Cells of Rat Organs

    NASA Astrophysics Data System (ADS)

    Danielson, Keith G.; Ohi, Seigo; Huang, P. C.

    1982-04-01

    The distribution of a heavy metal binding protein, metallothionein, was studied immunocytochemically by using antimetallothionein antibody and the immunoperoxidase staining technique on histological sections of liver, kidney, intestine, lung, and testis from cadmium-treated rats. These tissues either accumulate heavy metals (e.g., liver, kidney, and testis) or are exposed to metal by ingestion or inhalation (intestine and lung). Staining for metallothionein was observed intracellularly in epithelial parenchymal cells of the liver and kidney; all hepatocytes and most renal tubular cells stained for the protein. Accumulation of metallothionein was not seen in connective tissue cells surrounding either blood vessels or renal tubules. Extracellular localization of metallothionein was also observed in the liver sinusoids and within the lumina of the renal tubules, suggesting a metal transport or excretory function for this protein. Surface columnar epithelial cells of the intestinal villi indicated the presence of metallothionein but connective tissue cells of the lamina propria were negative for the protein. The granular secretory Paneth cells of the small intestine also stained strongly for metallothionein as did respiratory epithelial cells of the lung. In the testis, metallothionein was detected in the Sertoli cells and interstitial cells but not within the spermatogonia. Sertoli cells are closely associated with the developing spermatogonia and appear to serve a nutritive role in spermatogenesis. Because of the secretory, absorptive, or nutritive function of the metallothionein-localizing cells in the organs studied, we suggest that metallothionein may be involved in metal storage or transport in addition to its commonly proposed detoxification role.

  11. Sheep have an unusual variant of the brain-specific metallothionein, metallothionein-III.

    PubMed Central

    Chung, Roger S; Holloway, Adele F; Eckhardt, Bedrich L; Harris, Julie A; Vickers, James C; Chuah, Meng Inn; West, Adrian K

    2002-01-01

    Sheep metallothionein-III (MT-III) cDNA was isolated from a brain cDNA library and characterized. In contrast with MT-III from other species, sheep MT-III cDNA is predicted to encode a protein with significantly different metal-binding properties, owing to the loss of three of its cysteine residues. RT-PCR from other sheep confirmed that this aberrant structure is ubiquitous in this species. MT-III was successfully isolated from sheep brain, demonstrating that the cDNA does give rise to a protein product of the predicted structure. Sheep MT-III is similar to other mammalian MT-IIIs in that it retains the Cys-Pro-Cys-Pro motif which is thought to encode growth-inhibitory activity, and we show that it is likewise able to inhibit neuron survival in vitro. This is the first naturally occurring variant of MT-III (or any other major mammalian MT gene) which gives rise to a protein product. These findings are discussed in light of proposed roles of MT in the mammalian brain. PMID:11931634

  12. Inheritance of a functional mouse metallothionein gene in tobacco

    SciTech Connect

    Maiti, I.B.; Wagner, G.J.; Yeargan, R.; Hunt, A.G. )

    1989-04-01

    Morphologically normal plants were obtained from progeny (Ro, R1 and R2) originating from tobacco leaf tissue transformed with Agrobacterium tumefaciens containing a chimeric gene for kanamycin resistance an the mouse metallothionein cDNA gene directed by the constitutive promote 35S from CaMV. Integration and expression in R1 progeny was demonstrated by Southern, Northern blot analysis and metallothionein assay. Kanamycin resistance analysis of R1 and R2 progeny showed inheritance to be as a dominant Mendelian trait. Seedlings obtained from self-fertilized transgenic tobacco are more tolerant to cadmium stress than nontransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration was about 20% lower than that in nontransformed controls.

  13. Metallothionein – Immunohistochemical Cancer Biomarker: A Meta-Analysis

    PubMed Central

    Gumulec, Jaromir; Raudenska, Martina; Adam, Vojtech; Kizek, Rene; Masarik, Michal

    2014-01-01

    Metallothionein (MT) has been extensively investigated as a molecular marker of various types of cancer. In spite of the fact that numerous reviews have been published in this field, no meta-analytical approach has been performed. Therefore, results of to-date immunohistochemistry-based studies were summarized using meta-analysis in this review. Web of science, PubMed, Embase and CENTRAL databases were searched (up to April 30, 2013) and the eligibility of individual studies and heterogeneity among the studies was assessed. Random and fixed effects model meta-analysis was employed depending on the heterogeneity, and publication bias was evaluated using funnel plots and Egger's tests. A total of 77 studies were included with 8,015 tissue samples (4,631 cases and 3,384 controls). A significantly positive association between MT staining and tumors (vs. healthy tissues) was observed in head and neck (odds ratio, OR 9.95; 95% CI 5.82–17.03) and ovarian tumors (OR 7.83; 1.09–56.29), and a negative association was ascertained in liver tumors (OR 0.10; 0.03–0.30). No significant associations were identified in breast, colorectal, prostate, thyroid, stomach, bladder, kidney, gallbladder, and uterine cancers and in melanoma. While no associations were identified between MT and tumor staging, a positive association was identified with the tumor grade (OR 1.58; 1.08–2.30). In particular, strong associations were observed in breast, ovarian, uterine and prostate cancers. Borderline significant association of metastatic status and MT staining were determined (OR 1.59; 1.03–2.46), particularly in esophageal cancer. Additionally, a significant association between the patient prognosis and MT staining was also demonstrated (hazard ratio 2.04; 1.47–2.81). However, a high degree of inconsistence was observed in several tumor types, including colorectal, kidney and prostate cancer. Despite the ambiguity in some tumor types, conclusive results are provided in the tumors of

  14. A microscaled mercury saturation assay for metallothionein in fish.

    PubMed

    Shaw-Allen, Patricia; Elliott, Muriel; Jagoe, Charles H

    2003-09-01

    A mercury (Hg) saturation assay for measuring metallothionein (MT) in fish liver was modified by optimizing binding conditions to minimize the mercury and tissue consumed. The revised method uses stable Hg at low concentrations instead of 203Hg. At the reduced Hg concentrations used, MT concentrations in livers homogenized in saline appeared to increase systematically with dilution in both bluegill sunfish (Lepomis macrochirus) and largemouth bass (Micropterus salmoides). This error suggested a binding limitation due to sulfhydryl oxidation or competition for and removal of mercury by non-MT proteins. Homogenizing tissues in trichloroacetic acid (TCA) eliminated the interference. To further evaluate the method, the protocol was tested in the laboratory and field. Metallothionein in bluegill injected with 0.6 mg/kg zinc chloride increased at a rate of 0.03 nmole MT/g liver/h (r2 = 0.53, p = 0.001). Linearity improved when data were corrected for protein content (r2 = 0.74, p < 0.0001). Metallothionein levels in bluegill from a coal ash-contaminated environment were significantly increased over that of hatchery-reared sunfish (F = 20.17, p = 0.0003). The microscaled procedure minimizes concerns related to radioisotope use and waste generation while retaining the high sensitivity of the 203Hg assay.

  15. Quantifying protein synthesis and degradation in Arabidopsis by dynamic 13CO2 labeling and analysis of enrichment in individual amino acids in their free pools and in protein.

    PubMed

    Ishihara, Hirofumi; Obata, Toshihiro; Sulpice, Ronan; Fernie, Alisdair R; Stitt, Mark

    2015-05-01

    Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied (13)CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%-4% d(-1)), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark.

  16. Evidence of an altered protective effect of metallothioneins after cadmium exposure in the digenean parasite-infected cockle (Cerastoderma edule).

    PubMed

    Baudrimont, M; de Montaudouin, X

    2007-02-01

    The aim of the present study was to analyse the relation between parasitism and subsequent metallothioneins (MT) in the case of metal contamination. Experimental exposure of parasitized and unparasitized cockles (Cerastoderma edule) to cadmium (Cd) was performed, with the cockle as first or second intermediate host of 2 digenean species. After 7 days of Cd exposure in microcosms, cockles infected as first intermediate host by Labratrema minimus exhibited metal concentrations in tissues double that in uninfected cockles. Jointly, MT concentrations of parasitized cockles were not modified in comparison with uninfected individuals in which concentrations were increased 4.3-fold compared with controls. In cockles experimentally infected as the second intermediate host by Himasthla elongata, cadmium concentrations significantly increased again in parasitized cockles compared with uninfected individuals in contaminated conditions. Simultaneously, MT concentrations in healthy cockles increased, whereas they significantly decreased in parasitized individuals. Therefore, the presence of digenean parasites in Cd-exposed cockles leads to a maintenance or a decrease in MT concentrations compared with healthy individuals, whereas Cd accumulation in tissues is significantly increased. These experiments indicate a significant alteration of the protective effect of metallothioneins towards metals which could consequently enhance cockle vulnerability. Moreover, these results highlight the limit of the use of MT as a biomarker of metal pollution in field monitoring if parasitism is not taken into account.

  17. Assessment of metallothionein and antibodies to metallothionein in normal and autistic children having exposure to vaccine-derived thimerosal.

    PubMed

    Singh, Vijendra K; Hanson, Jeff

    2006-06-01

    Allergic autoimmune reaction after exposure to heavy metals such as mercury may play a causal role in autism, a developmental disorder of the central nervous system. As metallothionein (MT) is the primary metal-detoxifying protein in the body, we conducted a study of the MT protein and antibodies to metallothionein (anti-MT) in normal and autistic children whose exposure to mercury was only from thimerosal-containing vaccines. Laboratory analysis by immunoassays revealed that the serum level of MT did not significantly differ between normal and autistic children. Furthermore, autistic children harboured normal levels of anti-MT, including antibodies to isoform MT-I (anti-MT-I) and MT-II (anti-MT-II), without any significant difference between normal and autistic children. Our findings indicate that because autistic children have a normal profile of MT and anti-MT, the mercury-induced autoimmunity to MT may not be implicated in the pathogenesis of autism.

  18. Age-related changes of metallothionein 1/2 and metallothionein 3 expression in rat brain.

    PubMed

    Scudiero, Rosaria; Cigliano, Luisa; Verderame, Mariailaria

    2017-01-01

    Neurodegeneration is one of the main physiological consequences of aging on brain. Metallothioneins (MTs), low molecular weight, cysteine-rich proteins that bind heavy-metal ions and oxygen-free radicals, are commonly expressed in various tissues of mammals. MTs are involved in the regulation of cell proliferation and protection, and may be engaged in aging. Expression of the ubiquitous MTs (1 and 2) and the brain specific MT3 have been studied in many neurodegenerative disorders. The research results indicate that MTs may play important, although not yet fully known, roles in brain diseases; in addition, data lack the ability to identify the MT isoforms functionally involved. The aim of this study was to analyse the level of gene expression of selected MT isoforms during brain aging. By using real-time PCR analysis, we determined the MT1/2 and MT3 expression profiles in cerebral cortex and hippocampus of adolescent (2months), adult (4 and 8months), and middle-aged (16months) rats. We show that the relative abundance of all types of MT transcripts changes during aging in both hippocampus and cortex; the first effect is a generalized decrease in the content of MTs transcripts from 2- to 8-months-old rats. After passing middle age, at 16months, we observe a huge increase in MT3 transcripts in both cortical and hippocampal areas, while the MT1/2 mRNA content increases slightly, returning to the levels measured in adolescent rats. These findings demonstrate an age-related expression of the MT3 gene. A possible link between the increasing amount of MT3 in brain aging and its different metal-binding behaviour is discussed.

  19. Storage of Hg in the ileum of Blatella germanica: biochemical characterization of metallothionein.

    PubMed

    Bouquegneau, J M; Ballan-Dufrancais, C; Jeantet, A Y

    1985-01-01

    Cockroach ileum has a high capability to concentrate mercury compared with other tissues. Part of the mercury contained in the soluble phase of this organ is bound to metallothionein. It is suggested that mercury of the insoluble phase is stored in lysosomes under a polymerized metallothionein form.

  20. METALLOTHIONEIN GENE TRANSCRIPTION AS AN INDICATOR OF METAL EXPOSURE IN FATHEAD MINNOWS

    EPA Science Inventory

    Metallothionein is a cysteine rich, low molecular weight, metal binding protein. Basal levels of endogenous metallothioneins (MT) have been reported in all eucaryotes. MT has been shown to play an essential role in regulating physiological requirements of essential metals such a...

  1. One-step purification of metallothionein extracted from two different sources.

    PubMed

    Honda, Rubens T; Araújo, Roziete Mendes; Horta, Bruno Brasil; Val, Adalberto L; Demasi, Marilene

    2005-06-25

    We describe a one-step purification of hepatic metallothionein from the Amazon fish Colossoma macropomum injected with cadmium and from the copper-loaded metallothionein from the yeast Saccharomyces cerevisiae, performed by affinity chromatography through metal-chelating columns. Yeast metallothionein was purified from Cu2+-loaded resin and eluted by a continuous EDTA gradient whereas hepatic metallothionein extracted from fishes was purified by Ni2+-loaded resin and eluted by a continuous imidazol gradient. Purified metallothioneins were evaluated by SDS-PAGE and characterized by UV spectra of the apo- and Cd2+-loaded protein. This method allowed high purity and yield as well as rapid one-step extraction of both metal-loaded and apoprotein.

  2. Blockade of Metallothioneins 1 and 2 Increases Skeletal Muscle Mass and Strength.

    PubMed

    Summermatter, Serge; Bouzan, Anais; Pierrel, Eliane; Melly, Stefan; Stauffer, Daniela; Gutzwiller, Sabine; Nolin, Erin; Dornelas, Christina; Fryer, Christy; Leighton-Davies, Juliet; Glass, David J; Fournier, Brigitte

    2017-03-01

    Metallothioneins are proteins that are involved in intracellular zinc storage and transport. Their expression levels have been reported to be elevated in several settings of skeletal muscle atrophy. We therefore investigated the effect of metallothionein blockade on skeletal muscle anabolism in vitro and in vivo We found that concomitant abrogation of metallothioneins 1 and 2 results in activation of the Akt pathway and increases in myotube size, in type IIb fiber hypertrophy, and ultimately in muscle strength. Importantly, the beneficial effects of metallothionein blockade on muscle mass and function was also observed in the setting of glucocorticoid addition, which is a strong atrophy-inducing stimulus. Given the blockade of atrophy and the preservation of strength in atrophy-inducing settings, these results suggest that blockade of metallothioneins 1 and 2 constitutes a promising approach for the treatment of conditions which result in muscle atrophy.

  3. Characterization of a novel rice metallothionein gene promoter: its tissue specificity and heavy metal responsiveness.

    PubMed

    Dong, Chun-Juan; Wang, Yun; Yu, Shi-Shi; Liu, Jin-Yuan

    2010-10-01

    The rice (Oryza sativa L.) metallothionein gene OsMT-I-4b has previously been identified as a type I MT gene. To elucidate the regulatory mechanism involved in its tissue specificity and abiotic induction, we isolated a 1 730 bp fragment of the OsMT-I-4b promoter region. Histochemical β-glucuronidase (GUS) staining indicated a precise spacial and temporal expression pattern in transgenic Arabidopsis. Higher GUS activity was detected in the roots and the buds of flower stigmas, and relatively lower GUS staining in the shoots was restricted to the trichomes and hydathodes of leaves. No activity was observed in the stems and seeds. Additionally, in the root of transgenic plants, the promoter activity was highly upregulated by various environmental signals, such as abscisic acid, drought, dark, and heavy metals including Cu²(+) , Zn²(+) , Pb²(+) and Al³(+) . Slight induction was observed in transgenic seedlings under salinity stress, or when treated with Co²(+) and Cd²(+) . Promoter analysis of 5'-deletions revealed that the region -583/-1 was sufficient to drive strong GUS expression in the roots but not in the shoots. Furthermore, deletion analysis indicated important promoter regions containing different metal-responsive cis-elements that were responsible for responding to different heavy metals. Collectively, these findings provided important insight into the transcriptional regulation mechanisms of the OsMT-I-4b promoter, and the results also gave us some implications for the potential application of this promoter in plant genetic engineering.

  4. Shaping mechanisms of metal specificity in a family of metazoan metallothioneins: evolutionary differentiation of mollusc metallothioneins

    PubMed Central

    2011-01-01

    Background The degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu+ or Cd2+. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion. Results HpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd2+, Zn2+ or Cu2+ and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd6-HpCdMT and Cu12-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by

  5. Effect of metallothionein core promoter region polymorphism on cadmium, zinc and copper levels in autopsy kidney tissues from a Turkish population

    SciTech Connect

    Kayaalti, Zeliha; Mergen, Goerkem; Soeylemezoglu, Tuelin

    2010-06-01

    Metallothioneins (MTs) are metal-binding, low molecular weight proteins and are involved in pathophysiological processes like metabolism of essential metals, metal ion homeostasis and detoxification of heavy metals. Metallothionein expression is induced by various heavy metals especially cadmium, mercury and zinc; MTs suppress toxicity of heavy metals by binding themselves to these metals. The aim of this study was to investigate the association between the - 5 A/G metallothionein 2A (MT2A) single nucleotide polymorphism (SNP) and Cd, Zn and Cu levels in the renal cortex from autopsy cases. MT2A core promoter region - 5 A/G SNP was analyzed by PCR-RFLP method using 114 autopsy kidney tissues and the genotype frequencies of this polymorphism were found as 87.7% homozygote typical (AA), 11.4% heterozygote (AG) and 0.9% homozygote atypical (GG). In order to assess the Cd, Zn and Cu levels in the same autopsy kidney tissues, a dual atomic absorption spectrophotometer system was used and the average levels of Cd, Zn and Cu were measured as 95.54 {+-} 65.58 {mu}g/g, 181.20 {+-} 87.72 {mu}g/g and 17.14 {+-} 16.28 {mu}g/g, respectively. As a result, no statistical association was found between the - 5 A/G SNP in the MT2A gene and the Zn and Cu levels in the renal cortex (p > 0.05), but considerably high accumulation of Cd was monitored for individuals having AG (151.24 {+-} 60.21 {mu}g/g) and GG genotypes (153.09 {mu}g/g) compared with individuals having AA genotype (87.72 {+-} 62.98 {mu}g/g) (p < 0.05). These results show that the core promoter region polymorphism of metallothionein 2A increases the accumulation of Cd in human renal cortex.

  6. Metallothionein-like protein in lobsters (Homarus americanus)

    SciTech Connect

    Ray, J.; White, M.

    1981-12-01

    A metallothionein-like protein (MLP) was isolated from naturally cadmium-contaminated lobster hepatopancreas, gills, and green glands. Between 76-99% of the total cadmium was associated with this protein (molecular weight 9,500 daltons) while the remainder was associated with both high (>68,000 daltons) and low (<6,000 daltons) molecular weight proteins. MLP was not present in uncontaminated lobster hepatopancreas and only 1% of the total cadmium was associated with the gel filtration fraction corresponding to the protein.

  7. Inheritance and expression of the mouse metallothionein gene in tobacco

    SciTech Connect

    Maiti, I.B.; Wagner, G.J.; Yeargan, R.; Hunt, A.G. )

    1989-11-01

    Genetically engineered seedlings obtained from self-fertilized transgenic tobacco (Nicotiana tabacum) contained and expressed the mouse metallothionein and kanamycin resistance marker genes and were more tolerant to cadmium stress than untransformed controls. Cadmium accumulation in leaves of transgenic seedlings exposed to a low, field-like Cd concentration (0.02 micromolar) was about 20% lower than that in untransformed controls. Genetic analysis of R1 and R2 progeny showed inheritance of the marker gene to be as a dominant Mendelian trait. These results suggest the possibility of developing transgenic plants with modified tolerance to heavy metal stress and food crops having lower Cd content.

  8. Promiscuity and preferences of metallothioneins: the cell rules

    PubMed Central

    2011-01-01

    Metalloproteins are essential for many cellular functions, but it has not been clear how they distinguish between the different metals to bind the correct ones. A report in BMC Biology finds that preferences of two metallothionein isoforms for two different cations are due to inherent properties of these usually less discriminating proteins. Here these observations are discussed in the context of the cellular mechanisms that regulate metal binding to proteins. See research article: http://www.biomedcentral.com/1741-7007/9/4 PMID:21527046

  9. Metallothionein metabolism in the streptozotocin-diabetic rat

    SciTech Connect

    Chen, M.L.; Failla, M.L.

    1986-03-05

    Earlier reports from their laboratory showed the induction of the insulin-deficient diabetic state in adult rats was associated with an accumulation of zinc, copper, and a metallothionein-like zinc and copper binding protein in the soluble fraction of liver and kidney. Based upon chromatographic and electrophoretic properties, -SH to metal ratio and amino acid composition, they now report that elevated concentrations of metallothioneins (MT)-I and -II are indeed present in diabetic rat liver and kidney cytosol. The relative rates of MT synthesis in tissues from diabetic and control rats were measured by comparing incorporation of /sup 35/S-cysteine into MT vs. total cytoplasmic proteins at 5 h after injection of the precursor. The relative rates of MT synthesis in livers from rats diabetic for 10 d and fed either chow or purified diet containing 13 or 35 ppm copper were 1.4, 2.3 and 2.8 times greater, respectively, than control rats fed the same diets. Higher relative rates of MT synthesis were also observed in kidneys from diabetic rats fed purified diets compared to controls. Maximal relative rates of MT synthesis in diabetic liver and kidney were observed at 4 and 10 d, respectively, after onset of diabetes. The half-lives of cytoplasmic MT in liver and kidney from diabetic (10 d) rats were 1.3 and 2.6 days, respectively; half-lives of MT in control liver and kidney were 5.0 and 2.1 days, respectively.

  10. Metallothionein expression in the rat brain following KA and PTZ treatment.

    PubMed

    Juárez-Rebollar, Daniel; Manjarrez, Joaquín; Nava-Ruíz, Concepción; Zaga-Clavellina, Verónica; Flores-Espinosa, Pilar; Heras-Romero, Yesica; Díaz-Ruíz, Araceli; Méndez-Armenta, Marisela

    2015-09-01

    Epilepsy is a neurological disorder that has been associated with oxidative stress therefore epilepsy models have been develop such as kainic acid and pentylenetetrazol are usually used to understanding of the molecular mechanisms of this disease. We examined the metallothionein expression in rat brains of treated with kainic acid and pentylenetetrazol. Increase in metallothionein and nitrotirosyne immunoreactivity of both seizures epilepsy models was observed. Moreover, we show a significant increase on levels of MT expression. These results suggest that the increase of metallothionein expression is related with kainic acid and pentylenetetrazol treatments as response to damage mediated by oxidative stress.

  11. Bivalve vulnerability is enhanced by parasites through the deficit of metallothionein synthesis: A field monitoring on cockles (Cerastoderma edule)

    NASA Astrophysics Data System (ADS)

    Baudrimont, M.; de Montaudouin, X.; Palvadeau, A.

    2003-05-01

    The effect of the parasite Labratrema minimus (digenean trematode) on metallothioneins (MTs) biosynthesis by the cockle Cerastoderma edule (bivalve) was investigated. An in situ monitoring allowed to describe and compare monthly variations of MTs concentrations in cockles with and without L. minimus in a site free of metai pollution (Banc d'Arguin, Arcachon Bay, France). These concentrations are correlated with the reproductive cycle of the bivalve: (I) in spent cockles, MTs concentrations are higher in infected cockles. probably due to host tissue lysis: (2) during maturation, MTs biosynthesis increases in all cockles. However, concentrations are lower in parasitized individuals in relation with castration by parasites. Therefore, parasite infection in cockles, which can affect 100% of individuals, may contribute to a higher vulnerability of organisms to metal contamination.

  12. Impact of digenean parasite infection on metallothionein synthesis by the cockle (Cerastoderma edule): a multivariate field monitoring.

    PubMed

    Baudrimont, Magalie; de Montaudouin, Xavier; Palvadeau, Audrey

    2006-05-01

    Metallothioneins (MT) are proteins that play an important role in metabolism of essential metals and detoxification of trace metals from living organisms. Their synthesis is induced by metal pollution but can also be exacerbated by other factors such as reproduction processes. In this context, we monitored MT concentrations in a cockle Cerastoderma edule (marine bivalve) population and highlighted the effect of a castrating digenean parasite, Labratrema minimus. In spent cockles, MT levels were low (ca. 5 nmol sites g(-1), fresh weight) but slightly higher in parasitized individuals. During gametogenesis, MT synthesis increased in all cockles, but concentrations were lower in parasitized individuals (18 against 27 nmol sites g(-1), fw in unparasitized cockles) in relation with gonad damage by parasites. Therefore, it is suggested that parasite infection in cockles can modulate MT synthesis that could consequently interfere with the response of these protective proteins in case of metal contamination.

  13. Differential Hepatic Metal and Metallothionein Levels in Three Feral Fish Species along a Metal Pollution Gradient

    PubMed Central

    Bervoets, Lieven; Knapen, Dries; De Jonge, Maarten; Van Campenhout, Karen; Blust, Ronny

    2013-01-01

    The accumulation of cadmium, copper and zinc and the induction of metallothioneins (MT) in liver of three freshwater fish species was studied. Gudgeon (Gobio gobio), roach (Rutilus rutilus) and perch (Perca fluviatilis) were captured at 6 sampling sites along a cadmium and zinc gradient and one reference site in a tributary of the Scheldt River in Flanders (Belgium). At each site up to 10 individuals per species were collected and analyzed on their general condition factor (K), hepatosomatic index (HSI) and gonadosomatic index (GSI). From each individual fish the liver was dissected and analyzed on Cd, Cu and Zn and MT-content. Although not all species were present at each site, hepatic Cd and Zn levels generally followed the pollution gradient and highest levels were measured in perch, followed by roach and gudgeon. Nevertheless also an effect of site was observed on this order. MT-levels appeared to be the highest in gudgeon although differences with the other species were not very pronounced and depended on the site. Significant relationships were found between hepatic zinc accumulation and MT levels. For each species the ratio MTtheoretical/ MTmeasured was calculated, which gives an indication of the relative capacity to induce MTs and thus immobilize the metals. Perch had the lowest capacity in inducing MTs (highest ratio). Relationships between hepatic metal levels and fish condition indices were absent or very weak. PMID:23556004

  14. Isolation and characterization of Mytilus edulis metallothionein genes.

    PubMed

    Leignel, Vincent; Laulier, Marc

    2006-01-01

    Metallothioneins (MTs) are crucial proteins in all organisms for the regulation of essential metals and the detoxification of heavy metals. Many studies have estimated MT levels in mussel tissues to detect marine metal pollution. In this study, we investigated the MT gene structures of the forms present in Mytilus edulis (blue mussel). One MT-10 (2413 bp) gene and one MT-20 (1906 bp) gene were obtained. These MT genes contain three exons and two long introns. The splicing signals for MT-10 and MT-20 were GTA(T/A)GT-(C/T)AG. The structural organization (length of intron, splicing signals, AT content) of MT-10 and MT-20 is compared with other MT genes.

  15. Separation of two forms of rabbit metallothionein by isoelectric focusing.

    PubMed

    Nordberg, G F; Nordberg, M; Piscator, M; Vesterberg, O

    1972-02-01

    Rabbits were given repeated injections of cadmium chloride. Cadmium- and zinc-containing protein fractions were obtained from the livers of these animals by precipitation procedures and Sephadex G-75 chromatography. The protein thus obtained showed several characteristics similar to those of the earlier described protein metallothionein. Further separation by isoelectric focusing showed two main protein peaks with isoelectric points at 3.9 and 4.5 respectively. Amino acid analysis of these two forms showed similar content of most amino acids [residues per cent.: cysteine (28%), aspartate (8%), threonine (5-6%), serine (12%), glycine (7%), alanine (13%), methionine (2%), isoleucine (2%)] but with a small difference in content of lysine (12 and 13% respectively), proline (9 and 5% respectively) and glutamate (2 and 4% respectively). The two forms of the protein both contained cadmium, but only the one with pI4.5 contained also significant amounts of zinc.

  16. Metallothionein genes in Drosophila melanogaster constitute a dual system.

    PubMed Central

    Mokdad, R; Debec, A; Wegnez, M

    1987-01-01

    We have selected a metallothionein (MT) cDNA clone from a cadmium-resistant Drosophila melanogaster cell line. This clone includes an open reading frame coding for a 43-amino acid protein whose characteristics are a high cysteine content (12 cysteines, 28% of all residues) and a lack of aromatic amino acids. This protein differs markedly from the Drosophila MT (Mtn gene) previously reported [Lastowski-Perry, D., Otto, E. & Maroni, G. (1985) J. Biol. Chem. 260, 1527-1530). The MT system of Drosophila thus consists of at least two distantly related genes, in sharp contrast with vertebrate MT systems, in which the different members of MT gene families display high similarity. The gene corresponding to our MT cDNA (Mto) is inducible in Drosophila cell lines and in both larval and adult flies. Images PMID:3106973

  17. Identification of a Copper-Binding Metallothionein in Pathogenic Mycobacteria

    PubMed Central

    Gold, Ben; Deng, Haiteng; Bryk, Ruslana; Vargas, Diana; Eliezer, David; Roberts, Julia; Jiang, Xiuju; Nathan, Carl

    2009-01-01

    A screen of a genomic library from Mycobacterium tuberculosis (Mtb) identified a small, unannotated open reading frame (MT0196) that encodes a 4.9-kDa, cysteine-rich protein. Despite extensive nucleotide divergence, the amino acid sequence is highly conserved among mycobacteria that are pathogenic in vertebrate hosts. We synthesized the protein and found that it preferentially bound up to 6 Cu(I) ions in a solvent-shielded core. Copper, cadmium and compounds that generate nitric oxide or superoxide induced the gene’s expression in Mtb up to a thousand-fold. The native protein bound copper within Mtb and partially protected Mtb from copper toxicity. We propose that the product of the MT0196 gene be named mycobacterial metallothionien (MymT). To our knowledge, MymT is the first metallothionein of a Gram-positive bacterium with a demonstrated function. PMID:18724363

  18. Covalent sequestration of the nitrogen mustard mechlorethamine by metallothionein.

    PubMed

    Antoine, M; Fabris, D; Fenselau, C

    1998-09-01

    The research reported here demonstrates covalent binding to the metal-binding protein metallothionein (MT) by the therapeutic nitrogen mustard mechlorethamine. The most surprising aspect of this interaction is the selectivity of the alkylating agent for specific residues of MT. A combination of MS and proteolytic and enzymatic methods was used to deduce specific locations of mechlorethamine alkylation. These experiments indicated that alkylation occurs predominantly in the carboxyl domain of MT, with one molecule of mechlorethamine covalently cross-linking two cysteine residues. Electrospray MS revealed the retention of all seven metal ions in the cross-linked MT/mechlorethamine adducts, highlighting the uniqueness of this protein. Computerized docking experiments supported the hypothesis that selective binding precedes selective alkylation, and the structure of the drug indicates the minimal structural requirements for this binding. These results support the idea that MT overexpressed in tumor cells contributes to the inactivation of anticancer drugs.

  19. Metallothionein gene activation in the earthworm (Lumbricus rubellus).

    PubMed

    Höckner, M; Dallinger, R; Stürzenbaum, S R

    2015-05-08

    In order to cope with changing environmental conditions, organisms require highly responsive stress mechanisms. Heavy metal stress is handled by metallothioneins (MTs), the regulation of which is evolutionary conserved in insects and vertebrates and involves the binding of metal transcription factor 1 (MTF-1) to metal responsive elements (MREs) positioned in the promoter of MT genes. However, in most invertebrate phyla, the transcriptional activation of MTs is different and the exact mechanism is still unknown. Interestingly, although MREs are typically present also in invertebrate MT gene promoters, MTF-1 is notably absent. Here we use Lumbricus rubellus, the red earthworm, to study the elusive mechanism of wMT-2 activation in control and Cd-exposed conditions. EMSA and DNase I footprinting approaches were used to pinpoint functional binding sites within the wMT-2 promoter region, which revealed that the cAMP responsive element (CRE) is a promising candidate which may act as a transcriptional activator of invertebrate MTs.

  20. Characterization of calf liver Cu,Zn-metallothionein: naturally variable Cu and Zn stoichiometries.

    PubMed

    Chen, P; Onana, P; Shaw, C F; Petering, D H

    1996-07-15

    Cu,Zn-metallothioneins were purified from bovine calf liver in order to examine the stoichiometry of metal binding to the protein. Copper and zinc analyses were carried out by atomic absorption spectrophotometry. Consistent quantitative thiolate analyses were obtained spectrophotometrically with Ellman's reagent and amperometrically with phenylmercuric acetate. These were used to define protein concentration. A complementary method to assess the sum of the thiol and Cu(I) content of metallothionein involved titration of the reducing equivalents of the protein with ferricyanide. The stoichiometry of reaction was consistent with the oxidation of all the sulphydryl groups to disulphides and all of the bound Cu from the cuprous to the cupric oxidation state. Accordingly to these methods, total numbers of zinc plus copper ions bound to metallothionein isolated from a number of calf livers centred on about 7, 10-12, or 15 g-atoms of metal per mol of protein. The reactivity of ferricyanide and 4,7-phenylsulphonyl-2,9-dimethyl-1, 10-phenanthroline (BCS) with Cu,Zn-metallothioneins of various metal ratios was assessed. Zinc metallothionein reacted almost entirely in two slow steps with ferricyanide. As the Cu content of the protein increased, the fraction of reaction occurring in the time of mixing increased in parallel. BCS was able to remove 70-80% of metallothionein-bound Cu as Cu(I). The rest was resistant to reaction.

  1. Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability.

    PubMed

    Ruiz, Oscar N; Alvarez, Derry; Torres, Cesar; Roman, Laura; Daniell, Henry

    2011-06-01

    Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) and accumulate mercury in high concentrations within plant cells. Real-time PCR analysis showed that up to 1284 copies of the mt1 gene were found per cell when compared with 1326 copies of the 16S rrn gene, thereby attaining homoplasmy. Past studies in chloroplast transformation used qualitative Southern blots to evaluate indirectly transgene copy number, whereas we used real-time PCR for the first time to establish homoplasmy and estimate transgene copy number and transcript levels. The mt1 transcript levels were very high with 183,000 copies per ng of RNA or 41% the abundance of the 16S rrn transcripts. The transplastomic lines were resistant up to 20 μm mercury and maintained high chlorophyll content and biomass. Although the transgenic plants accumulated high concentrations of mercury in all tissues, leaves accumulated up to 106 ng, indicating active phytoremediation and translocation of mercury. Such accumulation of mercury in plant tissues facilitates proper disposal or recycling. This study reports, for the first time, the use of metallothioneins in plants for mercury phytoremediation. Chloroplast genetic engineering approach is useful to express metal-scavenging proteins for phytoremediation.

  2. Metallothionein induction as a measure of response to metal exposure in aquatic animals.

    PubMed Central

    Roesijadi, G

    1994-01-01

    Metallothioneins (MTs) are considered central in the intracellular regulation of metals such as copper, zinc, and cadmium. Increased MT synthesis is associated with increased capacity for binding these metals and protection against metal toxicity. Recent advances in the biochemistry and molecular biology of MTs have facilitated research on MTs in aquatic species. For the bivalve mollusc Crassostrea virginica, a species frequently used in studies on the toxicology and environmental monitoring of metals, the primary structure for MT has been deduced from analysis of the proteins and cDNA. Procedures for analysis of MT synthesis and MT gene expression have been applied in studies of response to metal exposure. Induction of specific MT forms by Cd is concentration- and time-dependent. The levels of MT-bound metals exhibit a strong relationship with the cytosolic metal concentrations in a metal-exposed natural population of oysters. Ribonuclease protection assays using sequence-specific antisense RNA probes have shown that the MT mRNA structure in this natural population exhibits considerable individual variability in the 3'-untranslated region. Although yet to be substantiated, the possibility exists that the distribution of this variability may be related to the level of environmental metal contamination. One probe derived from the coding region is suitable for use in quantitative RPAs for oyster MT mRNAs. PMID:7713043

  3. Metallothionein-1 and nitric oxide expression are inversely correlated in a murine model of Chagas disease

    PubMed Central

    Gonzalez-Mejia, Martha Elba; Torres-Rasgado, Enrique; Porchia, Leonardo M; Salgado, Hilda Rosas; Totolhua, José-Luis; Ortega, Arturo; Hernández-Kelly, Luisa Clara Regina; Ruiz-Vivanco, Guadalupe; Báez-Duarte, Blanca G; Pérez-Fuentes, Ricardo

    2014-01-01

    Chagas disease, caused by Trypanosoma cruzi, represents an endemic among Latin America countries. The participation of free radicals, especially nitric oxide (NO), has been demonstrated in the pathophysiology of seropositive individuals with T. cruzi. In Chagas disease, increased NO contributes to the development of cardiomyopathy and megacolon. Metallothioneins (MTs) are efficient free radicals scavengers of NO in vitro and in vivo. Here, we developed a murine model of the chronic phase of Chagas disease using endemic T. cruzi RyCH1 in BALB/c mice, which were divided into four groups: infected non-treated (Inf), infected N-monomethyl-L-arginine treated (Inf L-NAME), non-infected L-NAME treated and non-infected vehicle-treated. We determined blood parasitaemia and NO levels, the extent of parasite nests in tissues and liver MT-I expression levels. It was observed that NO levels were increasing in Inf mice in a time-dependent manner. Inf L-NAME mice had fewer T. cruzi nests in cardiac and skeletal muscle with decreased blood NO levels at day 135 post infection. This affect was negatively correlated with an increase of MT-I expression (r = -0.8462, p < 0.0001). In conclusion, we determined that in Chagas disease, an unknown inhibitory mechanism reduces MT-I expression, allowing augmented NO levels. PMID:24676665

  4. Polymorphism of Metallothionein 2A Modifies Lead Body Burden in Workers Chronically Exposed to the Metal.

    PubMed

    Fernandes, Kelly Christine Marques; Martins, Airton Cunha; Oliveira, Andréia Ávila Soares de; Antunes, Lusânia Maria Greggi; Cólus, Ilce Mara de Syllos; Barbosa, Fernando; Barcelos, Gustavo Rafael Mazzaron

    2016-01-01

    Lead (Pb) is a metal that accumulates in the human body, inducing several adverse health effects. One of the proteins responsible for the distribution of metal in the body is metallothionein (MT), which is expressed by different genes, and it is supposed that genetic variation in the genes that encode MTs may affect the Pb body burden. The present study aimed to evaluate the genetic effects of the polymorphism of MT2A (single nucleotide polymorphism rs10636; Cx2192;G) on blood Pb levels (BLL) of workers from car battery factories who are chronically exposed to the metal. In total, 221 men participated in the study; genomic DNA from whole blood was extracted, and genotyping of MT2A was performed by TaqMan assays; BLL were quantified by inductively coupled plasma mass spectrometry (ICP-MS). BLL were 25 ± 14 µg/dl (range 1.9-68); BLL were positively correlated with duration of work and smoking status. Individuals who carried at least one C allele had higher BLL than those with the GG genotype (β = -0.45; p = 0.025, multivariable linear regression analyses). Taken together, our data support the hypothesis that polymorphisms in genes related to the transport of Pb, such as MTs, may modulate the concentrations of the metal in the body and, consequently, adverse health effects induced by Pb exposure.

  5. Characterisation and genetic polymorphism of metallothionein gene CgMT4 in experimental families of Pacific oyster Crassostrea gigas displaying summer mortality.

    PubMed

    David, Elise; Tanguy, Arnaud; Moraga, Dario

    2012-02-01

    Summer mortality events have been observed in Pacific oyster Crassostrea gigas for several decades. This paper examines the selective pressure exerted by summer mortality on the polymorphism of a newly identified oyster metallothionein gene. CgMT4 cDNA and genomic sequences were obtained. CgMT4 was studied in two generations of oysters reared in three sites on the French Atlantic coast, using single strand conformation polymorphism analysis. Four alleles were detected. Individuals carrying genotype MT4-CD seem to have higher susceptibility to summer risk conditions. The MT4 gene could be a potential new genetic marker for susceptibility; further validation studies are recommended.

  6. Arabidopsis hybrid speciation processes

    PubMed Central

    Schmickl, Roswitha; Koch, Marcus A.

    2011-01-01

    The genus Arabidopsis provides a unique opportunity to study fundamental biological questions in plant sciences using the diploid model species Arabidopsis thaliana and Arabidopsis lyrata. However, only a few studies have focused on introgression and hybrid speciation in Arabidopsis, although polyploidy is a common phenomenon within this genus. More recently, there is growing evidence of significant gene flow between the various Arabidopsis species. So far, we know Arabidopsis suecica and Arabidopsis kamchatica as fully stabilized allopolyploid species. Both species evolved during Pleistocene glaciation and deglaciation cycles in Fennoscandinavia and the amphi-Beringian region, respectively. These hybrid studies were conducted either on a phylogeographic scale or reconstructed experimentally in the laboratory. In our study we focus at a regional and population level. Our research area is located in the foothills of the eastern Austrian Alps, where two Arabidopsis species, Arabidopsis arenosa and A. lyrata ssp. petraea, are sympatrically distributed. Our hypothesis of genetic introgression, migration, and adaptation to the changing environment during the Pleistocene has been confirmed: We observed significant, mainly unidirectional gene flow between the two species, which has given rise to the tetraploid A. lyrata. This cytotype was able to escape from the narrow ecological niche occupied by diploid A. lyrata ssp. petraea on limestone outcrops by migrating northward into siliceous areas, leaving behind a trail of genetic differentiation. PMID:21825128

  7. THE EMBRYOLETHALITY OF LIPOPOLYSACCHARIDE IN CD-1 AND METALLOTHIONEIN I-II NULL MICE: LACK OF A ROLE FOR INDUCED ZINC DEFICIENCY OR METALLOTHIONEIN INDUCTION

    EPA Science Inventory

    ABSTRACT

    Lipopolysaccharide (LPS) is embryolethal in CD-1 mice. LPS induces metallothionein (MT) via cytokines, including TNF-, IL-1 and IL-6, which initiate and maintain the acute phase response. Maternal hepatic MT induction in pregnant rats, by diverse toxicants, can ...

  8. A highly sensitive fluorescence probe for metallothioneins based on tiron-copper complex

    NASA Astrophysics Data System (ADS)

    Xiao, Xilin; Xue, Jinhua; Liao, Lifu; Huang, Mingyang; Zhou, Bin; He, Bo

    2015-06-01

    The fabrication of tiron-copper complex as a novel fluorescence probe for the sensitive directly detection of metallothioneins at nanomolar levels was demonstrated. In Britton-Robinson (B-R) buffer (pH 7.50), the interaction of bis(tiron)copper(II) complex cation [Cu(tiron)2]2+ and metallothioneins enhanced the fluorescence intensity of the system. The fluorescence enhancement at 347 nm was proportional to the concentration of metallothioneins. The mechanism was studied and discussed in terms of the fluorescence spectra. Under the optimal experimental conditions, at 347 nm, there was a linear relationship between the fluorescence intensity and the concentration of the metallothioneins in the range of 8.80 × 10-9-7.70 × 10-7 mol L-1, with a correlation coefficient of r = 0.995 and detection limit 2.60 × 10-9 mol L-1. The relative standard deviation was 0.77% (n = 11), and the average recovery 94.4%. The method proposed was successfully reliable, selective and sensitive in determining of trace metallothioneins in fish visceral organ samples with the results in good agreement with those obtained by HPLC.

  9. Yeast and Mammalian Metallothioneins Functionally Substitute for Yeast Copper-Zinc Superoxide Dismutase

    NASA Astrophysics Data System (ADS)

    Tamai, Katherine T.; Gralla, Edith B.; Ellerby, Lisa M.; Valentine, Joan S.; Thiele, Dennis J.

    1993-09-01

    Copper-zinc superoxide dismutase catalyzes the disproportionation of superoxide anion to hydrogen peroxide and dioxygen and is thought to play an important role in protecting cells from oxygen toxicity. Saccharomyces cerevisiae strains lacking copper-zinc superoxide dismutase, which is encoded by the SOD1 gene, are sensitive to oxidative stress and exhibit a variety of growth defects including hypersensitivity to dioxygen and to superoxide-generating drugs such as paraquat. We have found that in addition to these known phenotypes, SOD1-deletion strains fail to grow on agar containing the respiratory carbon source lactate. We demonstrate here that expression of the yeast or monkey metallothionein proteins in the presence of copper suppresses the lactate growth defect and some other phenotypes associated with SOD1-deletion strains, indicating that copper metallothioneins substitute for copper-zinc superoxide dismutase in vivo to protect cells from oxygen toxicity. Consistent with these results, we show that yeast metallothionein mRNA levels are dramatically elevated under conditions of oxidative stress. Furthermore, in vitro assays demonstrate that yeast metallothionein, purified or from whole-cell extracts, exhibits copper-dependent antioxidant activity. Taken together, these data suggest that both yeast and mammalian metallothioneins may play a direct role in the cellular defense against oxidative stress by functioning as antioxidants.

  10. Regulation of metallothionein gene expression and cellular zinc accumulation in a rat small intestinal cell line

    SciTech Connect

    Carlson, J.M.; Cousins, R.J. )

    1991-03-15

    The effects of extracellular zinc concentration on metallothionein gene expression and cellular zinc accumulation were studied in IRD-98 cells. This is a non-transformed clonal line established by Negrel, et al. from fetal rat small intestine which possess characteristics of small bowel epithelial cells. Cells were maintained in DMEM and grown to confluent monolayers. The response to media zinc concentrations over the range of 5-150 {mu}mol/L was assessed. After 24 h in culture, cell zinc and metallothionein protein concentrations were significantly increased in cells provided higher levels of media zinc. Subsequent time course experiments showed that cells exposed to higher zinc levels had significant elevations in both metallothionein mRNA, peaking at 24 h, and metallothionein protein increasing through 48 h. Furthermore, cell zinc concentrations were significantly increased. At 48 h of culture, greater than 50% of the additional cellular zinc accumulated could be attributed to elevated metallothionein protein levels. These cells represent a zinc-responsive model to examine the mechanism of zinc uptake and transcellular transport by intestinal cells and the regulatory factors involved.

  11. Immunohistochemical expression of metallothionein in pleomorphic adenoma of minor salivary glands: a role in the control of apoptosis?

    PubMed

    Miranda Viana, Alessandra de Castro; Ribeiro, Daniela Cotta; Florêncio, Taynara Nunes Guedes; Santos, Vanessa Torres; Sousa, Alexandre Andrade; Aguiar, Maria Cássia Ferreira

    2013-07-01

    Pleomorphic adenoma is the most common benign neoplasm of both the major and minor salivary glands. The histological features are diverse and are characterized by the involvement of epithelial-myoepithelial structures. Metallothionein is a cysteine-rich protein present in myoepithelial cells of several benign and malignant neoplasms. The function of metallothionein is associated with DNA protection, oxidative stress and apoptosis. The purpose of this study was to evaluate the expression of metallothionein in pleomorphic adenoma of the minor salivary glands. Additionally, we investigated the association of the clinicopathological features of the lesions with metallothionein, specifically its association with Bcl-2, in an attempt to evaluate the role of metallothionein in the control of apoptosis. Thirty-five cases of pleomorphic adenoma were selected and immunohistochemistry was performed for metallothionein and Bcl-2 proteins. The proteins were quantified by the Quickscore method. The samples showed epidemiological characteristics similar to those described in the literature. We did not find an association between the clinicopathological characteristics of pleomorphic adenomas and the proteins studied, but an association between metallothionein and Bcl-2 was demonstrated. The results suggest that metallothionein may have a role in the control of apoptosis in pleomorphic adenoma.

  12. A mercury saturation assay for measuring metallothionein in fish

    SciTech Connect

    Dutton, M.D. . Dept. of Zoology); Stephenson, M. . Environmental Science Branch); Klaverkamp, J.F. )

    1993-07-01

    An accurate, rapid, sensitive, and simple method using mercury saturation for quantifying metallothionein (MT) is described. A complex solution of enzymatic and nonenzymatic thiols, including rabbit liver MT-2, and supernatants from homogenized samples of rainbow trout liver were incubated in the presence of [sup 203]Hg in 10% trichloroacetic acid. Excess Hg was bound to an removed by chicken egg albumin, which denatured on contact with the acidic assay medium. After centrifugation, MT labeled with [sup 203]Hg remained in the TCA supernatant and was estimated using known stoichiometry for Hg-MT binding. A dilution series was used to establish that nonspecific metal binding, a common problem with other metal saturation assays, is negligible. Analysis of hepatic MT with high Cu content from rainbow trout demonstrated virtually complete displacement of Cu, Cd, and Zn by Hg. When compared to other metal-saturation assays developed for vertebrates, this method requires the least number of technical steps, and one-third or less of total preparatory and analytical time.

  13. Production of metallothionein polyclonal antibodies using chickens as model.

    PubMed

    Ortiz-Bueno, Angélica María; León-Chávez, Bertha Alicia; Ruiz-Tagle, Alejandro; Lozano-Zarain, Patricia; Castro-Caballero, Leopoldo; Achanzar, William E; Brambila, Eduardo

    2009-09-01

    The production of polyclonal antibodies (pAbs) against metallothioneins (MT) has been done in mammals. In this work, we describe a model where pAbs against rat liver MT were produced in chickens. Liver MT-1 and MT-2 isoforms isolated from rats were used as immunogens. MT was purified by exclusion chromatography and MT isoforms isolated by ionic exchange chromatography. Chickens were immunized with each isoform emulsified with Freund adjuvant over 6 weeks. MT-pAbs obtained from egg yolk were purified by ammonium sulfate precipitation followed by thiophilic interaction chromatography. MT-pAbs were characterized by ELISA, SDS-PAGE electrophoresis, and Western blot assays. Results showed significant titers (1:1,000) of MT-1 and MT-2 IgY in the eggs collected 30 days after the first immunization as determined by a direct ELISA assay; results also show a cross-reaction between MT-1 and MT-2 isoforms: however, the Abs obtained did not react with other non-MT proteins in hepatic homogenates. Sensitivity assays showed that MT-pAbs detected MT-1 and MT-2 at nanogram levels. These data suggest that chickens are an alternative model for producing pAbs against mammal high-homology proteins such as MT.

  14. Amino acid sequence and comparative antigenicity of chicken metallothionein.

    PubMed Central

    McCormick, C C; Fullmer, C S; Garvey, J S

    1988-01-01

    The complete amino acid sequence of metallothionein (MT) from chicken liver is reported. The primary structure was determined by automated sequence analysis of peptides produced by limited acid hydrolysis and by trypsin digestion. The comparative antigenicity of chicken MT was determined by radioimmunoassay using rabbit anti-rat MT polyclonal antibody. Chicken MT consists of 63 amino acids as compared to 61 found in MTs from mammals. One insertion (and two substitutions) occurs in the amino-terminal region, a region considered invariant among mammalian MTs. Eighteen of the 20 cysteines in chicken MT were aligned with cysteines from other mammalian sequences. Two cysteines near the carboxyl terminus are shifted by one residue due to the insertion of proline in that region. Overall, the chicken protein showed approximately equal to 68% sequence identity in a comparison with various mammalian MTs. The affinity of the polyclonal antibody for chicken MT was decreased by 2 orders of magnitude in comparison to that of a mammalian MT (rat MT isoforms). This reduced affinity is attributed to major substitutions in chicken MT in the regions of the principal determinants of mammalian MTs. Theoretical analysis of the primary structure predicted the secondary structure to consist of reverse turns and random coils with no stable beta or helix conformations. There is no evidence that chicken MT differs functionally from mammalian MTs. PMID:2448773

  15. Prognostic impact of metallothionein on oral squamous cell carcinoma.

    PubMed

    Cardoso, Sérgio V; Barbosa, Hugo M; Candellori, Ignez M; Loyola, Adriano M; Aguiar, Maria Cássia F

    2002-08-01

    Metallothionein (MT), a low-molecular-weight protein with high cysteine content, seems to be related to neoplastic resistance to oncologic treatment and therefore has been studied as a prognostic factor for a variety of human malignant tumors. MT overexpression in neoplasms of ectodermal origin is usually associated with a poor prognosis. MT expression was evaluated in 60 samples of oral squamous cell carcinoma by immunohistochemistry to study its prognostic influence on oral cancer. Possible associations of MT immunoexpression were also investigated with respect to clinical stage (TNM), histological grading, and proliferation index (Ki-67) of the lesions. No significant statistical correlation was observed among these variables. The impact on overall survival was assessed by uni and multivariate statistical tests. Mean MT labeling index was 60%. High MT labeling indexes (over 76%) predicted shorter survival in univariate statistical analysis. In multivariate analysis, MT labeling index and clinical stage were independent prognostic factors. MT overexpression in oral squamous cell carcinoma seems to be related to a worse prognosis for patients.

  16. Correlation of metallothionein expression with apoptosis in nasopharyngeal carcinoma

    PubMed Central

    Jayasurya, A; Bay, B H; Yap, W M; Tan, N G

    2000-01-01

    The expression of metallothionein (MT), an intracellular ubiquitous low molecular weight protein thiol with antioxidant properties, was studied in nasopharyngeal cancer (NPC) and correlated with the apoptotic index. Immunohistochemical staining of randomly selected, formalin-fixed and paraffin-embedded normal and malignant nasopharyngeal tissues were analysed for the expression of MT using the commercially available E9 antibody directed against MT I and MT II isoforms. The corresponding apoptosis labelling indices were evaluated by the TUNEL method. Localization of MT at the ultrastructural level was studied by immunogold labelling. All the tumour sections (17 specimens) showed MT-immunopositivity. A direct correlation between the percentage of MT-positive cells and the staining intensity was noted (P< 0.001; Pearson's r = 0.95). There was absence of cytoplasmic staining and only nuclear staining (with localization in the nucleoplasm) was demonstrated in the tumour cells. In normal epithelium of the nasopharynx, the basal layer was stained. An inverse relationship was observed between the level of MT expression and the apoptotic index in the NPC tissues (P = 0.0059; Pearson's r = –0.6380). The results suggest that overexpression of MT in NPC may protect the tumour cells from entering into the apoptotic process and thereby contribute to tumour expansion. Preferential localization of MT in the nuclei of NPC cells may possibly enhance radioresistance since radiotherapy is known to eradicate tumour cells by free radical-induced apoptosis. © 2000 Cancer Research Campaign PMID:10735506

  17. Metallothionein-I induction by stress in specific brain areas.

    PubMed

    Hidalgo, J; Campmany, L; Martí, O; Armario, A

    1991-10-01

    The distribution of metallothionein-I (MT) in several areas of the brain and its induction by immobilization stress has been studied in the rat. MT content was highest in hippocampus and midbrain and lowest in frontal cortex and pons plus medulla oblongata. Immobilization stress for 18 hours (which was accompanied by food and water deprivation) significantly increased MT levels in the frontal cortex, pons plus medulla oblongata and hypothalamus, but not in midbrain and hippocampus. The effect of stress on MT levels was specific as food and water deprivation along had no significant effect on MT levels in any of the brain areas studied. The effect of stress on MT levels was independent of changes in cytosolic Zn content; this was generally unaffected by stress or food and water deprivation but decreased in pons plus medulla oblongata from stressed rats. The results suggest that MT is induced more significantly in the brain areas that are usually involved in the response of animals to stress.

  18. Zinc and dexamethasone induce metallothionein accumulation by endothelial cells

    SciTech Connect

    Briske-Anderson, M.; Bobilya, D.J.; Reeves, P.G. )

    1991-03-11

    Several tissues increase their metallothionein (MT) concentration when exposed to elevated amounts of plasma Zn. Endothelial cells form the blood vessels that supply all tissues and constitute a barrier between cells of tissues and the blood. This study examined the ability of endothelial cells to synthesize MT and accumulate Zn in response to high amounts of Zn and dexamethasone. Bovine pulmonary endothelial cells were grown to confluence in Minimum Essential Medium with Earle's salts and 10% fetal calf serum. The monolayer was maintained for 2 d prior to use in medium containing EDTA-dialyzed serum. This low Zn medium was replaced with one containing 1, 6, 25, 50, 100, 150, or 200 {mu}M Zn and incubated for 24 hr before harvesting the cells. MT was quantified by the cadmium binding assay. Cellular Zn concentrations were analyzed by atomic absorption after a nitric acid digestion. The MT concentration was elevated in response to Zn concentrations of 100 {mu}M or more. Cellular Zn concentration was elevated when media Zn was 25 {mu}M or more. MT and cellular Zn concentrations were positively correlated. In another study, inclusion of 0.1 {mu}M dexamethasone in the media increased concentration at all Zn concentrations studied. However, the inclusion of 0.3 {mu}M cis-platinum had no effect. In conclusion, endothelial cells in culture respond to elevated amounts of Zn and dexamethasone in the media by accumulating Zn and MT.

  19. Influence of metallothionein-1 localization on its function.

    PubMed Central

    Levadoux-Martin, M; Hesketh, J E; Beattie, J H; Wallace, H M

    2001-01-01

    Metallothioneins (MTs) have a major role to play in metal metabolism, and may also protect DNA against oxidative damage. MT protein has been found localized in the nucleus during S-phase. The mRNA encoding the MT-1 isoform has a perinuclear localization, and is associated with the cytoskeleton; this targeting, due to signals within the 3'-untranslated region (3'-UTR), facilitates nuclear localization of MT-1 during S-phase [Levadoux, Mahon, Beattie, Wallace and Hesketh (1999) J. Biol. Chem. 274, 34961-34966]. Using cells transfected with MT gene constructs differing in their 3'-UTRs, the role of MT protein in the nucleus has been studied. Chinese hamster ovary cells were transfected with either the full MT gene (MTMT cells) or with the MT 5'-UTR and coding region linked to the 3'-UTR of glutathione peroxidase (MTGSH cells). Cell survival following exposure to oxidative stress and chemical agents was higher in cells expressing the native MT gene than in cells where MT localization was disrupted, or in untransfected cells. Also, MTMT cells showed less DNA damage than MTGSH cells in response to either hydrogen peroxide or mutagen. After exposure to UV light or mutagen, MTMT cells showed less apoptosis than MTGSH cells, as assessed by DNA fragmentation and flow cytometry. The data indicate that the perinuclear localization of MT mRNA is important for the function of MT in a protective role against DNA damage and apoptosis induced by external stress. PMID:11284736

  20. Role of metallothionein-III following central nervous system damage.

    PubMed

    Carrasco, Javier; Penkowa, Milena; Giralt, Mercedes; Camats, Jordi; Molinero, Amalia; Campbell, Iain L; Palmiter, Richard D; Hidalgo, Juan

    2003-06-01

    We evaluated the physiological relevance of metallothionein-III (MT-III) in the central nervous system following damage caused by a focal cryolesion onto the cortex by studying Mt3-null mice. In normal mice, dramatic astrogliosis and microgliosis and T-cell infiltration were observed in the area surrounding the lesioned tissue, along with signs of increased oxidative stress and apoptosis. There was also significant upregulation of cytokines/growth factors such as tumor necrosis factor-alpha, interleukin (IL)-1 alpha/beta, and IL-6 as measured by ribonuclease protection assay. Mt3-null mice did not differ from control mice in these responses, in sharp contrast to results obtained in Mt1- Mt2-null mice. In contrast, Mt3-null mice showed increased expression of several neurotrophins as well as of the neuronal sprouting factor GAP-43. Thus, unlike MT-I and MT-II, MT-III does not affect the inflammatory response elicited in the central nervous system by a cryoinjury, nor does it serve an important antioxidant role, but it may influence neuronal regeneration during the recovery process.

  1. Purification of human brain metallothionein by organic and reversed-phase high-performance liquid chromatography under acidic conditions.

    PubMed

    Cartel, N J

    1996-02-09

    A simplified high-performance liquid chromatographic method for the detection of metallothioneins, notably metallothionein-III, has been developed. In order to purify metallothionein, differential acetone precipitation at 50% (v/v) and at 80% (v/v) was employed on a 20% normal human brain homogenate. The reconstituted pellet was injected into a C18 microbore reversed-phase HPLC column, equilibrated with 0.1% trifluoroacetic acid, and developed at a flow-rate of 800 microliter/min with a linear gradient from 0% to 60% acetonitrile in 0.094% trifluoroacetic acid for 60 min. Western blots indicated that metallothioneins-I and II eluted at 16% acetonitrile and metallothionein-III eluted at 37% acetonitrile.

  2. The Arabidopsis Circadian System

    PubMed Central

    McClung, C. Robertson; Salomé, Patrice A.; Michael, Todd P.

    2002-01-01

    Rhythms with periods of approximately 24 hr are widespread in nature. Those that persist in constant conditions are termed circadian rhythms and reflect the activity of an endogenous biological clock. Plants, including Arabidopsis, are richly rhythmic. Expression analysis, most recently on a genomic scale, indicates that the Arabidopsis circadian clock regulates a number of key metabolic pathways and stress responses. A number of sensitive and high-throughput assays have been developed to monitor the Arabidopsis clock. These assays have facilitated the identification of components of plant circadian systems through genetic and molecular biological studies. Although much remains to be learned, the framework of the Arabidopsis circadian system is coming into focus. Dedication This review is dedicated to the memory of DeLill Nasser, a wonderful mentor and an unwavering advocate of both Arabidopsis and circadian rhythms research. PMID:22303209

  3. Red blood cell metallothionein as an indicator of zinc status during pregnancy RBC metallothionein, zinc status and pregnancy

    PubMed Central

    Caulfield, Laura E.; Donangelo, Carmen M.; Chen, Ping; Junco, Jorge; Merialdi, Mario; Zavaleta, Nelly

    2008-01-01

    Objective to describe the levels and patterns of change in red blood cell (RBC) metallothionein (MT) during pregnancy and the neonate, and relate RBCMT to other indicators of zinc and iron status. Research Methods & Procedures As part of a double-masked controlled trial of prenatal zinc supplementation among 242 Peruvian pregnant women, we determined RBCMT at enrollment (10–16 wk), 28 and 36 wk gestation, and in the cord blood at delivery in 158 women (86 who received daily supplements containing 60 mg iron and 250 ug folic acid, and 72 whose supplements also contained 25 mg zinc). In addition we measured plasma and urinary zinc concentrations, and hemoglobin and serum ferritin, and on a limited sample, we measured RBC zinc and placental MT. Results RBCMT increased during pregnancy, and levels in the cord blood approximated maternal values at 36 wk. Only RBC zinc at 36 wk differed by supplement type (P <0.05). Increases in RBCMT over pregnancy were however, related to early pregnancy RBC zinc and inversely with the decline in plasma zinc from baseline to 36 weeks gestation. Conclusion Changes in RBCMT throughout pregnancy were consistent with the hypothesized role of MT in regulating zinc homeostasis. RBCMT appears to not be responsive during pregnancy to changes in zinc status achieved with supplements. PMID:18602250

  4. Cardiac-specific overexpression of metallothionein rescues nicotine-induced cardiac contractile dysfunction and interstitial fibrosis.

    PubMed

    Hu, Nan; Guo, Rui; Han, Xuefeng; Zhu, Baocheng; Ren, Jun

    2011-04-10

    Cigarette smoking is a devastating risk factor for cardiovascular diseases and nicotine is believed the main toxin component responsible for the toxic myocardial effects of smoking. Nonetheless, neither the precise mechanism of nicotine-induced cardiac dysfunction nor effective treatment is elucidated. The aim of this study was to evaluate the impact of cardiac-specific overexpression of heavy metal scavenger metallothionein on myocardial geometry and mechanical function following nicotine exposure. Adult male friend virus B (FVB) wild-type and metallothionein mice were injected with nicotine (2 mg/kg/d) intraperitoneally for 10 days. Mechanical and intracellular Ca²+ properties were examined. Myocardial histology (cross-sectional area and fibrosis) was evaluated by hematoxylin and eosin (H&E) and Masson trichrome staining, respectively. Oxidative stress and apoptosis were measured by fluoroprobe 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H₂DCFDA) fluorescence and caspase-3 activity, respectively. Nicotine exposure failed to affect the protein abundance of metallothionein. Our data revealed reduced echocardiographic contractile capacity (fractional shortening), altered cardiomyocyte contractile and intracellular Ca²+ properties including depressed peak shortening amplitude, maximal velocity of shortening/relengthening, resting and electrically-stimulated rise in intracellular Ca²+, as well as prolonged duration of relengthening and intracellular Ca²+ clearance in hearts from nicotine-treated FVB mice, the effect of which was ameliorated by metallothionein. Biochemical and histological findings depicted overt accumulation of reactive oxygen species (ROS), apoptosis and myocardial fibrosis without any change in myocardial cross-sectional area following nicotine treatment, which was mitigated by metallothionein. Taken together, our findings suggest the antioxidant metallothionein may reconcile short-term nicotine exposure

  5. Metal release in metallothioneins induced by nitric oxide: X-ray absorption spectroscopy study.

    PubMed

    Casero, Elena; Martín-Gago, José A; Pariente, Félix; Lorenzo, Encarnación

    2004-12-01

    Metallothioneins (MTs) are low molecular weight proteins that include metal ions in thiolate clusters. The capability of metallothioneins to bind different metals has suggested their use as biosensors for different elements. We study here the interaction of nitric oxide with rat liver MTs by using in situ X-ray absorption spectroscopy techniques. We univocally show that the presence of NO induces the release of Zn atoms from the MT structure to the solution. Zn ions transform in the presence of NO from a tetrahedral four-fold coordinated environment in the MT into a regular octahedral six-fold coordinated state, with interatomic distances compatible with those of Zn solvated in water.

  6. Mechanism of protection by metallothionein against acetaminophen hepatotoxicity

    SciTech Connect

    Saito, Chieko; Yan, H.-M.; Artigues, Antonio; Villar, Maria T.; Farhood, Anwar; Jaeschke, Hartmut

    2010-01-15

    Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the US. Metallothionein (MT) expression attenuates APAP-induced liver injury. However, the mechanism of this protection remains incompletely understood. To address this issue, C57BL/6 mice were treated with 100 mumol/kg ZnCl{sub 2} for 3 days to induce MT. Twenty-four hours after the last dose of zinc, the animals received 300 mg/kg APAP. Liver injury (plasma ALT activities, area of necrosis), DNA fragmentation, peroxynitrite formation (nitrotyrosine staining), MT expression, hepatic glutathione (GSH), and glutathione disulfide (GSSG) levels were determined after 6 h. APAP alone caused severe liver injury with oxidant stress (increased GSSG levels), peroxynitrite formation, and DNA fragmentation, all of which were attenuated by zinc-induced MT expression. In contrast, MT knockout mice were not protected by zinc. Hydrogen peroxide-induced cell injury in primary hepatocytes was dependent only on the intracellular GSH levels but not on MT expression. Thus, the protective effect of MT in vivo was not due to the direct scavenging of reactive oxygen species. Zinc treatment had no effect on the early GSH depletion kinetics after APAP administration, which is an indicator of the metabolic activation of APAP to its reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI). However, MT was able to effectively trap NAPQI by covalent binding. We conclude that MT scavenges some of the excess NAPQI after GSH depletion and prevents covalent binding to cellular proteins, which is the trigger for the propagation of the cell injury mechanisms through mitochondrial dysfunction and nuclear DNA damage.

  7. Prognostic significance of metallothionein expression in renal cell carcinoma

    PubMed Central

    Mitropoulos, Dionisios; Kyroudi-Voulgari, Aspasia; Theocharis, Stamatis; Serafetinides, Efraim; Moraitis, Epaminondas; Zervas, Anastasios; Kittas, Christos

    2005-01-01

    Background Metallothionein (MT) protein expression deficiency has been implicated in carcinogenesis while MT over expression in tumors is indicative of tumor resistance to anti-cancer treatment. The purpose of the study was to examine the expression of MT expression in human renal cell carcinoma (RCC) and to correlate MT positivity, the pattern and extent of MT expression with tumor histologic cell type and nuclear grade, pathologic stage and patients' survival. Patients and methods The immunohistochemical expression of MT was determined in 43 formalin-fixed and paraffin-embedded RCC specimens, using a mouse monoclonal antibody that reacts with both human MT-I and MT-II. Correlation was sought between immunohistochemical (MT positivity, intensity and extension of staining) and clinico-pathological data (histological cell type, tumor nuclear grade, pathologic stage and patients' survival). Results Positive MT staining was present in 21 cases (49%), being mild/moderate and intense in 8 and 13 cases, respectively. The pattern was cytoplasmic in 7 cases and was both cytoplasmic and nuclear in 14 cases. MT expression in a percentage of up to 25% of tumor cells (negative MT staining included) was observed in 31 cases, in a percentage 25–50% of tumor cells in 7 cases, and in a percentage of 50–75% of tumor cells in 5 cases. There was no significant correlation of MT intensity of staining to histological type, stage and patients' survival, while it was inversely correlated to higher tumor nuclear grade. MT extent of staining did not correlate with histological type, nuclear grade, and pathologic stage while a statistically significant association was found with patients' survival. Conclusions The inverse correlation between MT staining intensity and tumor nuclear grade in RCC suggests a role of MT in tumor differentiation process. Since extent of MT expression is inversely correlated with survival it may be possibly used as a clinical prognostic parameter. PMID

  8. Cadmium modulates adipocyte functions in metallothionein-null mice

    SciTech Connect

    Kawakami, Takashige; Nishiyama, Kaori; Kadota, Yoshito; Sato, Masao; Inoue, Masahisa; Suzuki, Shinya

    2013-11-01

    Our previous study has demonstrated that exposure to cadmium (Cd), a toxic heavy metal, causes a reduction of adipocyte size and the modulation of adipokine expression. To further investigate the significance of the Cd action, we studied the effect of Cd on the white adipose tissue (WAT) of metallothionein null (MT{sup −/−}) mice, which cannot form atoxic Cd–MT complexes and are used for evaluating Cd as free ions, and wild type (MT{sup +/+}) mice. Cd administration more significantly reduced the adipocyte size of MT{sup −/−} mice than that of MT{sup +/+} mice. Cd exposure also induced macrophage recruitment to WAT with an increase in the expression level of Ccl2 (MCP-1) in the MT{sup −/−} mice. The in vitro exposure of Cd to adipocytes induce triglyceride release into culture medium, decrease in the expression levels of genes involved in fatty acid synthesis and lipid hydrolysis at 24 h, and at 48 h increase in phosphorylation of the lipid-droplet-associated protein perilipin, which facilitates the degradation of stored lipids in adipocytes. Therefore, the reduction in adipocyte size by Cd may arise from an imbalance between lipid synthesis and lipolysis. In addition, the expression levels of leptin, adiponectin and resistin decreased in adipocytes. Taken together, exposure to Cd may induce unusually small adipocytes and modulate the expression of adipokines differently from the case of physiologically small adipocytes, and may accelerate the risk of developing insulin resistance and type 2 diabetes. - Highlights: • Cd causes a marked reduction in adipocyte size in MT-null mice. • Cd enhances macrophage migration into adipose tissue and disrupt adipokine secretion. • MT gene alleviates Cd-induced adipocyte dysfunctions. • Cd enhances the degradation of stored lipids in adipocytes, mediated by perilipin. • Cd induces unusually small adipocytes and the abnormal expression of adipokines.

  9. Metallothionein-induced zinc partitioning exacerbates hyperoxic acute lung injury

    PubMed Central

    Lee, Sang-Min; McLaughlin, Joseph N.; Frederick, Daniel R.; Zhu, Lin; Thambiayya, Kalidasan; Wasserloos, Karla J.; Kaminski, Iris; Pearce, Linda L.; Peterson, Jim; Li, Jin; Latoche, Joseph D.; Peck Palmer, Octavia M.; Stolz, Donna Beer; Fattman, Cheryl L.; Alcorn, John F.; Oury, Tim D.; Angus, Derek C.; Pitt, Bruce R.

    2013-01-01

    Hypozincemia, with hepatic zinc accumulation at the expense of other organs, occurs in infection, inflammation, and aseptic lung injury. Mechanisms underlying zinc partitioning or its impact on extrahepatic organs are unclear. Here we show that the major zinc-binding protein, metallothionein (MT), is critical for zinc transmigration from lung to liver during hyperoxia and preservation of intrapulmonary zinc during hyperoxia is associated with an injury-resistant phenotype in MT-null mice. Particularly, lung-to-liver zinc ratios decreased in wild-type (WT) and increased significantly in MT-null mice breathing 95% oxygen for 72 h. Compared with female adult WT mice, MT-null mice were significantly protected against hyperoxic lung injury indicated by reduced inflammation and interstitial edema, fewer necrotic changes to distal airway epithelium, and sustained lung function at 72 h hyperoxia. Lungs of MT-null mice showed decreased levels of immunoreactive LC3, an autophagy marker, compared with WT mice. Analysis of superoxide dismutase (SOD) activity in the lungs revealed similar levels of manganese-SOD activity between strains under normoxia and hyperoxia. Lung extracellular SOD activity decreased significantly in both strains at 72 h of hyperoxia, although there was no difference between strains. Copper-zinc-SOD activity was ∼4× higher under normoxic conditions in MT-null compared with WT mice but was not affected in either group by hyperoxia. Collectively the data suggest that genetic deletion of MT-I/II in mice is associated with compensatory increase in copper-zinc-SOD activity, prevention of hyperoxia-induced zinc transmigration from lung to liver, and hyperoxia-resistant phenotype strongly associated with differences in zinc homeostasis during hyperoxic acute lung injury. PMID:23275622

  10. SPINK1 promotes colorectal cancer progression by downregulating Metallothioneins expression

    PubMed Central

    Tiwari, R; Pandey, S K; Goel, S; Bhatia, V; Shukla, S; Jing, X; Dhanasekaran, S M; Ateeq, B

    2015-01-01

    Colorectal cancer (CRC) is the third most common cancer in the world, and second leading cause of cancer deaths in the US. Although, anti-EGFR therapy is commonly prescribed for CRC, patients harboring mutations in KRAS or BRAF show poor treatment response, indicating an ardent demand for new therapeutic targets discovery. SPINK1 (serine peptidase inhibitor, Kazal type 1) overexpression has been identified in many cancers including the colon, lung, breast and prostate. Our study demonstrates the functional significance of SPINK1 in CRC progression and metastases. Stable knockdown of SPINK1 significantly decreases cell proliferation, invasion and soft agar colony formation in the colon adenocarcinoma WiDr cells. Conversely, an increase in these oncogenic phenotypes was observed on stimulation with SPINK1-enriched conditioned media (CM) in multiple benign models such as murine colonic epithelial cell lines, MSIE and YAMC (SPINK3-negative). Mechanistically, SPINK1 promotes tumorigenic phenotype by activating phosphatidylinositol 3-kinase (PI3K/AKT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways, and the SPINK1-positive WiDr cells are sensitive to AKT and MEK inhibitors. Importantly, SPINK1 silencing mediated upregulation of various Metallothionein isoforms, considered as tumor suppressors in CRC, confer sensitivity to doxorubicin, which strengthens the rationale for using the combinatorial treatment approach for the SPINK1-positive CRC patients. Furthermore, in vivo studies using chicken chorioallantoic membrane assay, murine xenograft studies and metastasis models further suggest a pivotal role of SPINK1 in CRC progression and metastasis. Taken together, our study demonstrates an important role for the overexpressed SPINK1 in CRC disease progression, a phenomenon that needs careful evaluation towards effective therapeutic target development. PMID:26258891

  11. Role of Metallothionein in Post-Burn Inflammation.

    PubMed

    Zhang, Wei; Xie, Yongjun; Liu, Weihua; Xu, Xuefeng; Chen, Xuelian; Liu, Hairong; Liu, Yueming

    2016-04-01

    Metallothioneins (MTs) are a family of low molecular-weight and cysteine-rich metalloproteins that regulate metal metabolism and protect cells from oxygen free radicals. Recent studies suggested that MTs have some anti-inflammatory effects. However, the role of MTs in post-burn inflammation remains unclear. This study is designed to investigate the role of MTs in post-burn inflammation in a mouse burn model. MT-I/II null (-/-) and C57BL/6 wild-type (WT) mice were randomly divided into sham burn, burn, Zn treated, and Zn-MT-2 treated groups. The inflammatory cytokines levels were measured by enzyme-linked immunosorbent assay (ELISA). Myeloperoxidase (MPO) activity was determined by spectrophotometry. In in vitro study, exogenous MT-2 was added to macrophages that were stimulated with burn serum in the presence or absence of a p38 MAPK inhibitor SB203580. The IL-6 and TNF-α messenger RNA (mRNA) expression were detected by quantitative real-time polymerase chain reaction. The levels of p38 expression were determined by Western blot. Burn induced increased inflammatory cytokines such as interleukin (IL)-1β, IL-6, tumor necrosis factors-α, and macrophage chemoattractant protein-1 production in burn wound and serum. The MPO activities in the lung and heart were also increased after burn. These effects were significantly more prominent in MT (-/-) mice than in WT mice. Furthermore, these effects were inhibited by administration of exogenous MT-2 to both WT and MT (-/-) mice. Exogenous MT-2 inhibited the p38 expression and abrogated the increase of IL-6 and TNF-α mRNA expression from macrophages that were stimulated with burn serum. The effect of MT-2 was not further strengthened in the presence of SB203580. MTs may have a protective role against post-burn inflammation and inflammatory organ damage, at least partly through inhibiting the p38 MAPK signaling.

  12. Tissue-specific metallothionein gene expression in liver and intestine by dexamethasone, interleukin-1. alpha. and elevated zinc status

    SciTech Connect

    Hempe, J.M.; Carlson, J.M.; Cousins, R.J. )

    1990-02-26

    Intestinal metallothionein has been implicated in the regulation of zinc absorption. Glucocorticoids and cytokines mediate hepatic metallothionein gene expression but the effects of these hormones in the small intestine are unclear. In this experiment, rats were injected ip with dexamethasone (DEX), recombinant human interleukin-1{alpha} (ILK-1), or ZnSO{sub 4}. Data collected 0. 3, 6,9, or 12 hour post-injection showed tissue specific regulation of metallothionein gene expression. Liver metallothionein mRNA (determined by hybridization analysis) were increased by DEX, IL-1 and ZnSO{sub 4}. In contrast, the intestine was completely refractory to IL-1. DEX did not affect intestinal metallothionein but did enhance mucosal accumulation of {sup 65}Zn by ligated duodenal loops. Absorption of {sup 65}Zn was not affected by IL-1 or DEX but was inversely related to elevated intestinal metallothionein protein induced in response to ZnSO. Plasma zinc was depressed by DEX and IL-1 and elevated in rats injected with ZnSO{sub 4} but was not related to {sup 54}Zn absorption. Tissue-specific induction of metallothionein may constitute a mechanism for independently regulating both tissue zinc distribution and zinc absorption.

  13. Effects of metallothionein-3 and metallothionein-1E gene transfection on proliferation, cell cycle, and apoptosis of esophageal cancer cells.

    PubMed

    Tian, Z Q; Xu, Y Z; Zhang, Y F; Ma, G F; He, M; Wang, G-Y

    2013-10-17

    Metallothionein (MT)-3 has cell growth inhibitory activity, and is the only currently known MT subtype with unique physiological functions. The expression levels of MT-1E, a subtype of MT-1, were positively correlated with the degree of esophageal cancer malignancy. The present study aimed to investigate the effects of MT-3 and MT-1E gene transfection on the proliferation, cell cycle, and apoptosis of esophageal cancer cells. The cationic liposome method was used to transfect the esophageal cancer strains Eca-109 and TE13. Reverse transcription-polymerase chain reaction was used to detect target gene expression, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction was applied to detect cell proliferation, and flow cytometry was used for cell cycle and apoptosis detection. Esophageal cancer cells with MT-3 and MT-1E gene transfection showed high expression of the foreign target gene and mRNA. Cells with MT-3 gene transfection showed markedly inhibited proliferation (P < 0.05), a significantly higher proportion of cells in the G0/G1 phase (P < 0.05), a significantly lower proportion of cells in the S phase (P < 0.05), and a significantly increased apoptosis rate (P < 0.05). Cells with MT-1E gene transfection did not show significant changes in proliferation, cell cycle, or apoptosis rate (P > 0.05). Therefore, the upregulation of MT-3 gene expression can inhibit esophageal cancer cell proliferation and induce apoptosis, which may be achieved by blocking the tumor cell growth cycle, whereas effects of the MT-1E gene on the proliferation of esophageal cancer cells were not evident.

  14. Expression, purification of metallothionein genes from freshwater crab (Sinopotamon yangtsekiense) and development of an anti-metallothionein ELISA

    PubMed Central

    Zhang, Hao; Zhou, Hui

    2017-01-01

    Using the phoA-fusion technology, the recombinant metallothionein (MT) from freshwater crab (Sinopotamon yangtsekiense) has been successfully produced in Escherichia coli. MT purified from the bacterial suspension showed one polypeptide with a molecular weight of 7 kDa by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). Western-blotting confirmed the polypeptides had a specific reactivity with mouse polyclonal MT anti-serum. Based on the purified MT and MT anti-serum, the reaction parameters for an enzyme-linked immunosorbent assay (ELISA) were developed. The direct coating ELISA showed a higher linear relationship compared to antibody sandwich coating ELISA. The optimal dilution rates of purified MT anti-serum and coating period were shown to be 1:160,000 and 12 hours at 4°C. At 37°C, the appropriate reaction duration of the first antibody and the second antibody were 2 hours and 1 hour, respectively. According to these optimal parameters, the standard linear equation, y = 0.0032x + 0.1769 (R2 = 0.9779, x, y representing MT concentration and OD450 value), was established for the determination of MT concentration with a valid range of 3.9–500 ng/ml. In verification experiments, the mean coefficients of variation of the intra-assay and inter-assay were 3.260% and 3.736%, respectively. According to the result of MT recovery, ELISA with an approaching 100% MT recovery was more reliable and sensitive than the Cd saturation assay. In conclusion, the newly developed ELISA of this study was precise, stable and repeatable, and could be used as a biomarker tool to monitor pollution by heavy metals. PMID:28350826

  15. The Cd(II)-binding abilities of recombinant Quercus suber metallothionein: bridging the gap between phytochelatins and metallothioneins.

    PubMed

    Domènech, Jordi; Orihuela, Rubén; Mir, Gisela; Molinas, Marisa; Atrian, Sílvia; Capdevila, Mercè

    2007-08-01

    In this work, we have analyzed both at stoichiometric and at conformational level the Cd(II)-binding features of a type 2 plant metallothionein (MT) (the cork oak, Quercus suber, QsMT). To this end four peptides, the wild-type QsMT and three constructs previously engineered to characterize its Zn(II)- and Cu(I)-binding behaviour, were heterologously produced in Escherichia coli cultures supplemented with Cd(II), and the corresponding complexes were purified up to homogeneity. The Cd(II)-binding ability of these recombinant peptides was determined through the chemical, spectroscopic and spectrometric characterization of the recovered clusters. Recombinant synthesis of the four QsMT peptides in cadmium-rich media rendered complexes with a higher metal content than those obtained from zinc-supplemented cultures and, consequently, the recovered Cd(II) species are nonisostructural to those of Zn(II). Also of interest is the fact that three out of the four peptides yielded recombinant preparations that included S(2-)-containing Cd(II) complexes as major species. Subsequently, the in vitro Zn(II)/Cd(II) replacement reactions were studied, as well as the in vitro acid denaturation and S(2-) renaturation reactions. Finally, the capacity of the four peptides for preventing cadmium deleterious effects in yeast cells was tested through complementation assays. Consideration of all the results enables us to suggest a hairpin folding model for this typical type 2 plant Cd(II)-MT complex, as well as a nonnegligible role of the spacer in the detoxification function of QsMT towards cadmium.

  16. Induction and characterization of hepatic metallothionein in the channel catfish following exposure to cadmium

    SciTech Connect

    Schlenk, D.; Zhang, Y.S.

    1994-12-31

    The channel catfish (Ictalurus punctatus) is a commercially important fish found throughout the United States. Thus, understanding how this organism responds to environmental contaminants is of critical concern. The stress protein, metallothionein allows organisms to cope with excessive exposures of heavy metals, such as cadmium. Following a 5 mg/kg intraperitoneal injection of cadmium chloride, two metal binding proteins of approximately 8 kilodaltons and two MRNA bands of approximately 400 base pairs were induced in liver homogenates of juvenile channel catfish. Spectrophotometric examination of the purified proteins indicated that each protein was an isoform of metallothionein. Static aqueous exposures of 7 day duration to varied doses of cadmium chloride increased expression of metallothionein mRNA in a dose dependent manner. Significant (p {<=} 0.5) expression of MRNA was observed following exposures to cadmium chloride of 50/{mu}g/L or higher, but expression was unaltered by exposure to 10 {mu}g/L. Future studies will examine the time course of hepatic metallothionein expression in channel catfish following cadmium chloride exposure.

  17. Protective effect of metallothionein on cadmium toxicity in isolated rat hepatocytes.

    PubMed Central

    Din, W S; Frazier, J M

    1985-01-01

    An isolated rat hepatocyte preparation was used to study the cellular toxicity of cadmium and the protective effects of metallothionein on cadmium-induced toxicity. Exposure of primary suspension cultures of isolated rat hepatocytes to Cd2+ (0-35.7 microM) for 15 min resulted in a dose-dependent reduction in the synthesis of cellular proteins during a subsequent 6 h incubation. Such inhibition could not be correlated with cellular lethality or gross membrane damage. Pre-induction of metallothionein in hepatocytes by zinc treatment in vivo of donor rats protected hepatocytes in vitro from cadmium-induced inhibition of protein synthesis. The protective effects in zinc-pre-induced hepatocytes are not due to alterations in the level of total cellular cadmium, but could be accounted for by the redistribution of intracellular cadmium in the presence of high levels of zinc-metallothionein. The data suggest that metallothionein exerts its protective effect by a kinetic detoxification mechanism, i.e. a decrease in reactive intracellular cadmium. PMID:4052053

  18. Unexpected Interactions of the Cyanobacterial Metallothionein SmtA with Uranium.

    PubMed

    Acharya, Celin; Blindauer, Claudia A

    2016-02-15

    Molecules for remediating or recovering uranium from contaminated environmental resources are of high current interest, with protein-based ligands coming into focus recently. Metallothioneins either bind or redox-silence a range of heavy metals, conferring protection against metal stress in many organisms. Here, we report that the cyanobacterial metallothionein SmtA competes with carbonate for uranyl binding, leading to formation of heterometallic (UO2)(n)Zn4SmtA species, without thiol oxidation, zinc loss, or compromising secondary or tertiary structure of SmtA. In turn, only metalated and folded SmtA species were found to be capable of uranyl binding. (1)H NMR studies and molecular modeling identified Glu34/Asp38 and Glu12/C-terminus as likely adventitious, but surprisingly strong, bidentate binding sites. While it is unlikely that these interactions correspond to an evolved biological function of this metallothionein, their occurrence may offer new possibilities for designing novel multipurpose bacterial metallothioneins with dual ability to sequester both soft metal ions including Cu(+), Zn(2+), Cd(2+), Hg(2+), and Pb(2+) and hard, high-oxidation state heavy metals such as U(VI). The concomitant protection from the chemical toxicity of uranium may be valuable for the development of bacterial strains for bio-remediation.

  19. GENETIC BACKGROUND BUT NOT METALLOTHIONEIN PHENOTYPE DICTATES SENSITIVITY TO CADMIUM-INDUCED TESTICULAR INJURY IN MICE

    EPA Science Inventory

    Genetic Background but not Metallothionein Phenotype Dictates Sensitivity to
    Cadmium-Induced Testicular Injury in Mice

    Jie Liu1,2, Chris Corton3, David J. Dix4, Yaping Liu1, Michael P. Waalkes2
    and Curtis D. Klaassen1

    ABSTRACT

    Parenteral administrati...

  20. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    SciTech Connect

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J.; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.

  1. Determination of the Cd/S cluster stoichiometry in Fucus vesiculosus metallothionein.

    PubMed

    Merrifield, Maureen E; Chaseley, Jennifer; Kille, Peter; Stillman, Martin J

    2006-03-01

    The seaweed Fucus vesiculosus is unusual when compared with other algal species, in that it can survive in toxic-metal-contaminated aquatic environments. The metallothionein gene has been identified in F. vesiculosus by Kille and co-workers (Morris, C. A., Nicolaus, B., Sampson, V., Harwood, J. L., and Kille, P. (1999) Biochem. J. 338, 553), which suggests a possible protective mechanism against toxic metals for this species. We report the first detailed study of the metal binding properties of F. vesiculosus metallothionein using UV absorption, circular dichroism (CD), and electrospray mass spectral techniques. The overall metal-to-sulfur ratios of this novel algal protein when bound to divalent cadmium and zinc were determined to be Cd(6)S(16) and Zn(6)S(16), respectively. Mixed Cd/Zn species were also formed when Cd(2+) was added to the Zn-containing Fucus metallothionein. Only one conformation was identified at low pH for the native protein. Analysis of the UV absorption, CD, and ESI-MS spectral data recorded during stepwise, acid-induced demetalation supports a two-domain structure for the protein, with two 3-metal binding sites. The data suggest that one of the domains is significantly less stable than the other, and we tentatively propose from the arrangement of cysteines in the sequence that the two domains are M(3)S(7) and M(3)S(9) (where M = Cd(2+) or Zn(2+)). While the M(3)S(9) cluster is known in the beta clusters of crab, lobster, and mammalian metallothioneins, the M(3)S(7) is a hitherto unknown cluster structure. Metallothionein in F. vesiculosus is thought to act as a protective mechanism against incoming toxic metals. The metal binding studies reported are a putative model for metal binding in vivo.

  2. Expression of the zinc transporters genes and metallothionein in obese women.

    PubMed

    dos Santos Rocha, Paula Beatriz Krebsky; de Castro Amorim, Amanda; de Sousa, Artemizia Francisca; do Monte, Semíramis Jamil Hadad; da Mata Sousa, Luiz Claudio Demes; do Nascimento Nogueira, Nadir; Neto, José Machado Moita; do Nascimento Marreiro, Dilina

    2011-11-01

    Research has investigated the participation of zinc transport proteins and metallothionein in the metabolism of this mineral. However, studies about the genetic expression of these proteins in obese patients are scarce. The study determined the expression of zinc transporter protein codifying genes (ZnT-1, Zip-1 and Zip-3) and of metallothionein in 55 obese women, aged between 20 and 56 years. The assessment of body composition was carried out using anthropometric measurements and bioelectrical impedance. Zinc intake was obtained by recording diet over a 3-day period, and the nutritional analysis was carried out using NutWin software version 1.5. The plasmatic and erythrocytary zinc were analyzed by atomic absorption spectrophotometry (λ=213. 9 nm). The determination of mRNA expression of the zinc transporter proteins and metallothionein was carried out using blood, using the RT-PCR method. The mean values of body mass index were 37.9±5.5 kg/m2. The average intake of zinc was 9.4±2.3 mg/day. The analysis of the zinc plasma concentrations showed values of 58.4±10.9 μg/dL. The mean values of zinc in the erythroytes were 38.7±9.1 μg/g Hb. The metallothionein gene had a higher expression in the blood, when compared to zinc transporters ZnT-1, Zip-1, and Zip-3 (p=0.01). The study shows that there are alterations in the biochemical parameters of zinc in obese patients assessed, as well as higher expression of the codifying gene metallothionein, when compared to the investigated zinc transporters.

  3. Metallothionein deficiency aggravates depleted uranium-induced nephrotoxicity

    SciTech Connect

    Hao, Yuhui; Huang, Jiawei; Gu, Ying; Liu, Cong; Li, Hong; Liu, Jing; Ren, Jiong; Yang, Zhangyou; Peng, Shuangqing; Wang, Weidong; Li, Rong

    2015-09-15

    Depleted uranium (DU) has been widely used in both civilian and military activities, and the kidney is the main target organ of DU during acute high-dose exposures. In this study, the nephrotoxicity caused by DU in metallothionein-1/2-null mice (MT −/−) and corresponding wild-type (MT +/+) mice was investigated to determine any associations with MT. Each MT −/− or MT +/+ mouse was pretreated with a single dose of DU (10 mg/kg, intraperitoneal injection) or an equivalent volume of saline. After 4 days of DU administration, kidney changes were assessed. After DU exposure, serum creatinine and serum urea nitrogen in MT −/− mice significantly increased than in MT +/+ mice, with more severe kidney pathological damage. Moreover, catalase and superoxide dismutase (SOD) decreased, and generation of reactive oxygen species and malondialdehyde increased in MT −/− mice. The apoptosis rate in MT −/− mice significantly increased, with a significant increase in both Bax and caspase 3 and a decrease in Bcl-2. Furthermore, sodium-glucose cotransporter (SGLT) and sodium-phosphate cotransporter (NaPi-II) were significantly reduced after DU exposure, and the change of SGLT was more evident in MT −/− mice. Finally, exogenous MT was used to evaluate the correlation between kidney changes induced by DU and MT doses in MT −/− mice. The results showed that, the pathological damage and cell apoptosis decreased, and SOD and SGLT levels increased with increasing dose of MT. In conclusion, MT deficiency aggravated DU-induced nephrotoxicity, and the molecular mechanisms appeared to be related to the increased oxidative stress and apoptosis, and decreased SGLT expression. - Highlights: • MT −/− and MT +/+ mice were used to evaluate nephrotoxicity of DU. • Renal damage was more evident in the MT −/− mice after exposure to DU. • Exogenous MT also protects against DU-induced nephrotoxicity. • MT deficiency induced more ROS and apoptosis after exposure to

  4. Clinical significance of metallothioneins in cell therapy and nanomedicine

    PubMed Central

    Sharma, Sushil; Rais, Afsha; Sandhu, Ranbir; Nel, Wynand; Ebadi, Manuchair

    2013-01-01

    Mammalian metallothioneins (MTs) are low molecular weight (6–7 kDa) cysteine-rich proteins that are specifically induced by metal nanoparticles (NPs). MT induction in cell therapy may provide better protection by serving as antioxidant, anti-inflammatory, antiapoptotic agents, and by augmenting zinc-mediated transcriptional regulation of genes involved in cell proliferation and differentiation. Liposome-encapsulated MT-1 promoter has been used extensively to induce growth hormone or other genes in culture and gene-manipulated animals. MTs are induced as a defensive mechanism in chronic inflammatory conditions including neurodegenerative diseases, cardiovascular diseases, cancer, and infections, hence can serve as early and sensitive biomarkers of environmental safety and effectiveness of newly developed NPs for clinical applications. Microarray analysis has indicated that MTs are significantly induced in drug resistant cancers and during radiation treatment. Nutritional stress and environmental toxins (eg, kainic acid and domoic acid) induce MTs and aggregation of multilamellar electron-dense membrane stacks (Charnoly body) due to mitochondrial degeneration. MTs enhance mitochondrial bioenergetics of reduced nicotinamide adenine dinucleotide–ubiquinone oxidoreductase (complex-1), a rate-limiting enzyme complex involved in the oxidative phosphorylation. Monoamine oxidase-B inhibitors (eg, selegiline) inhibit α-synuclein nitration, implicated in Lewy body formation, and inhibit 1-methyl 4-phenylpyridinium and 3-morpholinosydnonimine-induced apoptosis in cultured human dopaminergic neurons and mesencephalic fetal stem cells. MTs as free radical scavengers inhibit Charnoly body formation and neurodegenerative α-synucleinopathies, hence Charnoly body formation and α-synuclein index may be used as early and sensitive biomarkers to assess NP effectiveness and toxicity to discover better drug delivery and surgical interventions. Furthermore, pharmacological

  5. Identification of a Functional Homolog of the Yeast Copper Homeostasis Gene ATX1 from Arabidopsis1

    PubMed Central

    Himelblau, Edward; Mira, Helena; Lin, Su-Ju; Cizewski Culotta, Valeria; Peñarrubia, Lola; Amasino, Richard M.

    1998-01-01

    A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and inflorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO4, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis. PMID:9701579

  6. Identification of a functional homolog of the yeast copper homeostasis gene ATX1 from Arabidopsis

    SciTech Connect

    Himelblau, E.; Amasino, R.M.; Mira, H.; Penarrubia, L.; Lin, S.J.; Culotta, V.C.

    1998-08-01

    A cDNA clone encoding a homolog of the yeast (Saccharomyces cerevisiae) gene Anti-oxidant 1 (ATX1) has been identified from Arabidopsis. This gene, referred to as Copper CHaperone (CCH), encodes a protein that is 36% identical to the amino acid sequence of ATX1 and has a 48-amino acid extension at the C-terminal end, which is absent from ATX1 homologs identified in animals. ATX1-deficient yeast (atx1) displayed a loss of high-affinity iron uptake. Expression of CCH in the atx1 strain restored high-affinity iron uptake, demonstrating that CCH is a functional homolog of ATX1. When overexpressed in yeast lacking the superoxide dismutase gene SOD1, both ATX1 and CCH protected the cell from the reactive oxygen toxicity that results from superoxide dismutase deficiency. CCH was unable to rescue the sod1 phenotype in the absence of copper, indicating that CCH function is copper dependent. In Arabidopsis CCH mRNA is present in the root, leaf, and in fluorescence and is up-regulated 7-fold in leaves undergoing senescence. In plants treated with 800 nL/L ozone for 30 min, CCH mRNA levels increased by 30%. In excised leaves and whole plants treated with high levels of exogenous CuSO{sub 4}, CCH mRNA levels decreased, indicating that CCH is regulated differently than characterized metallothionein proteins in Arabidopsis.

  7. Transfer of copper and zinc from ionic and metallothionein-bound forms to Cu, Zn--superoxide dismutase.

    PubMed

    Suzuki, K T; Kuroda, T

    1995-03-01

    Reactivity in transfer of copper (Cu) and zinc (Zn) to their binding sites of superoxide dismutase (SOD) was examined in vitro by the HPLC/atomic absorption spectrophotometry. Ionic Cu (cuprous and cupric ions) were incorporated more efficiently than the metal bound to metallothionein. Cu binds not only to the Cu-binding site but also to the Zn-binding site. Although Zn in the reaction medium and the metal bound to the Zn-binding site of SOD affected little the reactivity in binding of ionic Cu, they disturbed the reactivity of Cu bound to metallothionein to the Cu-binding site. Both ionic and metallothionein-bound Zn were transferred at a comparable efficiency to the Zn-binding site but not to the Cu-binding site. Co-existing ionic Cu but not metallothionein-bound Cu in the medium inhibited the binding of Zn to SOD. The results indicate that ionic Cu can be transferred to both Cu- and Zn-binding sites of SOD more efficiently than metallothionein-bound Cu, while both ionic and metallothionein-bound Zn are transferred only to Zn-binding site at a comparable efficiency.

  8. Metallothionein-mediated neuroprotection in genetically engineered mouse models of Parkinson's disease

    PubMed Central

    Ebadi, Manuchair; Brown-Borg, Holly; Refaey, Hesham El; Singh, Brij B.; Garrett, Scott; Shavali, Shaik; Sharma, Sushil K.

    2013-01-01

    Parkinson's disease is characterized by a progressive loss of dopaminergic neurons in the substantia nigra zona compacta, and in other sub-cortical nuclei associated with a widespread occurrence of Lewy bodies. The cause of cell death in Parkinson's disease is still poorly understood, but a defect in mitochondrial oxidative phosphorylation and enhanced oxidative and nitrative stresses have been proposed. We have studied controlwt (C57B1/6), metallothionein transgenic (MTtrans), metallothionein double gene knock (MTdko), α-synuclein knock out (α-synko), α-synuclein–metallothionein triple knock out (α-syn–MTtko), weaver mutant (wv/wv) mice, and Ames dwarf mice to examine the role of peroxynitrite in the etiopathogenesis of Parkinson's disease and aging. Although MTdko mice were genetically susceptible to 1, methyl, 4-phenyl, 1,2,3,6-tetrahydropyridine (MPTP) Parkinsonism, they did not exhibit any overt clinical symptoms of neurodegeneration and gross neuropathological changes as observed in wv/wv mice. Progressive neurodegenerative changes were associated with typical Parkinsonism in wv/wv mice. Neurodegenerative changes in wv/wv mice were observed primarily in the striatum, hippocampus and cerebellum. Various hallmarks of apoptosis including caspase-3, TNFα, NFκB, metallothioneins (MT-1, 2) and complex-1 nitration were increased; whereas glutathione, complex-1, ATP, and Ser(40)-phosphorylation of tyrosine hydroxylase, and striatal 18F-DOPA uptake were reduced in wv/wv mice as compared to other experimental genotypes. Striatal neurons of wv/wv mice exhibited age-dependent increase in dense cored intra-neuronal inclusions, cellular aggregation, proto-oncogenes (c-fos, c-jun, caspase-3, and GAPDH) induction, inter-nucleosomal DNA fragmentation, and neuro-apoptosis. MTtrans and α-Synko mice were genetically resistant to MPTP-Parkinsonism and Ames dwarf mice possessed significantly higher concentrations of striatal coenzyme Q10 and metallothioneins (MT 1, 2

  9. Increased radioresistance of tumor cells exposed to metallothionein-inducing agents

    SciTech Connect

    Renan, M.J.; Dowman, P.I. )

    1989-12-01

    In this study, we have determined the radiosensitivity parameters of cells exposed in vitro to metallothionein-inducing agents. Three well-characterized tumor cell lines were chosen for investigation: HeLa, B16, and WHFIB. We have shown that exposure of cells in vitro to a heavy metal (cadmium), followed by irradiation, enhances cell survival for two out of three cell lines studied. As measured by the mean inactivation dose, the radioresistance increases by a factor of 1.6 for HeLa cells, 1.4 for WHFIB, and a negligible factor for B16 cells. An additional effect was noted when different classes of metallothionein inducers (such as serum factors, cadmium, and dexamethasone) were allowed to act together. Also, we found that the increase in radioresistance exhibits a peak at exposure times of approximately 10 h; longer exposure to inducing agents results in a reduction in radioresistance.

  10. Metallothionein of the terrestrial mollusc Helix pomatia as a possible biomarker for environmental stressors

    SciTech Connect

    Berger, B.; Dallinger, R.; Moore, C.

    1995-12-31

    Metallothioneins (MTs) are metal-binding proteins believed to function in homeostasis and detoxification of metals. MT synthesis is induced by various metals and chemical and physical stressors. MTs can therefore be considered as part of the cellular stress response. To test the effects of stressors on MT levels in the Roman snail (Helix pomatia) the authors exposed 8 individuals each to cadmium, copper, low temperature and X-ray radiation. MT concentrations were measured by a Thiomolybdate-Cd-Chelex assay in midgut gland and mantle tissues. This method allows to determine both the Cu-containing and the Cd,Zn-containing MT pool. The basal levels of MT in midgut gland and mantle of controls were about 2 mg MT g{sup {minus}1} fresh weight. In both organs about 80% of MT belongs to the Cu-MT pool indicating their importance for the metabolism of essential metals in non-stressed snails. As expected, changes of MT concentrations were most pronounced after cadmium exposure, MT levels in midgut glands rising to 4.2 mg MT g{sup {minus}1} f.w. and the proportion of Cd,Zn-MT increasing from 20% (controls) to 55%. This is an indication for the switching of MT function from the homeostatic control of metals towards their detoxification, Cadmium exposure as well as most of the other stressors did not affect MT concentrations in the mantle. MT levels in this organ decreased due to X-ray radiation, showing the ability of MT to function as a radical scavenger. There exists a complex pattern of increase and decrease of MT concentrations depending on the kind of stressor and the type of organ. By means of statistical methods it was possible to distinguish between the control and stressed MT status of the tissue. Moreover, the results prove that MTs should be measured in metabolically different organs and that different MT pools (Cu-MT, Cd,Zn-MT) should be considered, if these proteins are used as biomarkers for environmental stress.

  11. Quantifying Protein Synthesis and Degradation in Arabidopsis by Dynamic 13CO2 Labeling and Analysis of Enrichment in Individual Amino Acids in Their Free Pools and in Protein1[OPEN

    PubMed Central

    Fernie, Alisdair R.; Stitt, Mark

    2015-01-01

    Protein synthesis and degradation represent substantial costs during plant growth. To obtain a quantitative measure of the rate of protein synthesis and degradation, we supplied 13CO2 to intact Arabidopsis (Arabidopsis thaliana) Columbia-0 plants and analyzed enrichment in free amino acids and in amino acid residues in protein during a 24-h pulse and 4-d chase. While many free amino acids labeled slowly and incompletely, alanine showed a rapid rise in enrichment in the pulse and a decrease in the chase. Enrichment in free alanine was used to correct enrichment in alanine residues in protein and calculate the rate of protein synthesis. The latter was compared with the relative growth rate to estimate the rate of protein degradation. The relative growth rate was estimated from sequential determination of fresh weight, sequential images of rosette area, and labeling of glucose in the cell wall. In an 8-h photoperiod, protein synthesis and cell wall synthesis were 3-fold faster in the day than at night, protein degradation was slow (3%–4% d−1), and flux to growth and degradation resulted in a protein half-life of 3.5 d. In the starchless phosphoglucomutase mutant at night, protein synthesis was further decreased and protein degradation increased, while cell wall synthesis was totally inhibited, quantitatively accounting for the inhibition of growth in this mutant. We also investigated the rates of protein synthesis and degradation during leaf development, during growth at high temperature, and compared synthesis rates of Rubisco large and small subunits of in the light and dark. PMID:25810096

  12. Protective effects of selenium on cadmium toxicity in rats: Role of altered toxicokinetics and metallothionein

    SciTech Connect

    Wahba, Z.Z.; Coogan, T.P.; Rhodes, S.W.; Waalkes, M.P. )

    1993-02-01

    Selenium prevents the toxicity of the carcinogenic metal cadmium through undefined mechanisms. In this study, the authors determined the effects of selenium on cadmium toxicokinetics and on the ability of cadmium to induce metallothionein, a metal-binding protein that is thought to confer tolerance to cadmium toxicity. To assess the acute protective effects of selenium, male Wistar (WF/NCr) rats were given selenium (as SeO[sub 2]; 10 [mu]mol/kg, sc) at [minus]24, 0, and +24 h relative to cadmium (as CdCl[sub 2]; 45 [mu]mol/kg, sc). Over a 14-d period this dose of cadmium killed 6 out of 10 rats, while 100% of the cadmium-treated rats given concurrent selenium treatments survived. The acute increases in testicular weight that were seen with cadmium, indicative of edematous damage, were also prevented by concurrent selenium treatments. Further studies assessed the distribution and excretion of cadmium and its ability to induce metallothionein in rats given 40 [mu]mol Cd/kg, sc, at time 0 and selenium (10 [mu]mol/kg, sc) at [minus]24 and 0 h. Selenium treatments enhanced cadmium accumulation at 24 h in the liver (23%), testes (145%), and epididymis (35%) but reduced renal accumulation by more than half. Urine samples, collected at 0-3, 3-6, and 6-24 h following cadmium administration, indicted a markedly reduced excretion of cadmium in selenium treated rats during all time periods. The synthesis of metallothionein was stimulated to a much lesser extent by cadmium in selenium-treated rat kidney (41% decrease) but was unaffected in liver. The levels of cadmium-binding proteins within the testes were markedly reduced by cadmium treatment, an effect unmodified by selenium treatments. These results suggest selenium prevents acute cadmium toxicity through a mechanism that does not involve induction of metallothionein and in spite of a markedly enhanced retention of cadmium. 50 refs., 1 fig., 4 tabs.

  13. Changes in Metallothionein Level in Rat Hepatic Tissue after Administration of Natural Mouldy Wheat

    PubMed Central

    Vasatkova, Anna; Krizova, Sarka; Adam, Vojtech; Zeman, Ladislav; Kizek, Rene

    2009-01-01

    Mycotoxins are secondary metabolites produced by microfungi that are capable of causing disease and death in humans and other animals. This work was aimed at investigation of influence of mouldy wheat contaminated by pathogenic fungi producing mycotoxins on metallothionein levels in hepatic tissue of rats. The rats were administrating feed mixtures with different contents of vitamins or naturally mouldy wheat for 28 days. It was found that the wheat contained deoxynivalenol (80 ± 5 μg per kg of mouldy wheat), zearalenone (56 ± 3 μg/kg), T2-toxin (20 ± 2 μg/kg) and aflatoxins as a sum of B1, B2, G1 and G2 (3.9 ± 0.2 μg/kg). Rats were fed diets containing 0, 33, 66 and 100% naturally moulded wheat. Control group 0, 33, 66 and 100% contained vitamins according to Nutrient Requirements of Rats (NRC). Other four groups (control group with vitamins, vit33, vit66 and vit100%) were fed on the same levels of mouldy wheat, also vitamins at levels 100% higher than the previous mixtures. We determined weight, feed conversion and performed dissection to observe pathological processes. Changes between control group and experimental groups exposed to influence of mouldy wheat and experimental groups supplemented by higher concentration of vitamins and mouldy wheat were not observed. Livers were sampled and did not demonstrate significant changes in morphology compared to control either. In the following experiments the levels of metallothionein as a marker of oxidative stress was determined. We observed a quite surprising trend in metallothionein levels in animals supplemented with increased concentration of vitamins. Its level enhanced with increasing content of mouldy wheat. It was possible to determine a statistically significant decline (p<0.05) between control group and groups of animals fed with 33, 66 and 100% mouldy wheat. It is likely that some mycotoxins presented in mouldy wheat are able to block the mechanism of metallothionein synthesis. PMID:19399242

  14. Metallothionein and heavy metals in brown trout (Salmo trutta) and European eel (Anguilla anguilla): a comparative study.

    PubMed

    Linde, A R; Sánchez-Galán, S; Klein, D; García-Vázquez, E; Summer, K H

    1999-10-01

    The levels and the cellular distribution of heavy metals, and the extent by which the metals binds to metallothionein (MT) in brown trout (Salmo trutta) and European eel (Anguilla anguilla), were analyzed in order to assess the natural conditions of MT and heavy metals in these two fish species. There were no differences in heavy metals and MT concentrations between males and females of brown trout in a nonreproductive status and between adult brown trout individuals. Brown trout presented higher Cu content than European eel. The cellular distribution of Cu was also different between the two fish species; while in brown trout most of the Cu was in the noncytosolic fraction, Cu was mainly located in the cytosol in European eel. However, the cellular distribution of Zn, Cd, and Pb was similar in the two fish species. There was also an important difference in the metal content of MT between both species. Whereas, in brown trout, Cu-binding MT represented 75% of total metal-binding MT, this value was 25% in European eel. The between-species differences found in this study are intrinsic characteristics not associated with environmental factors. These results establish the basis to use MT as a bioindicator.

  15. Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?

    PubMed Central

    Gil-Moreno, Selene; Jiménez-Martí, Elena; Palacios, Òscar; Zerbe, Oliver; Dallinger, Reinhard; Capdevila, Mercè; Atrian, Sílvia

    2015-01-01

    Snail metallothioneins (MTs) constitute an ideal model to study structure/function relationships in these metal-binding polypeptides. Helix pomatia harbours three MT isoforms: the highly specific CdMT and CuMT, and an unspecific Cd/CuMT, which represent paralogous proteins with extremely different metal binding preferences while sharing high sequence similarity. Preceding work allowed assessing that, although, the Cys residues are responsible for metal ion coordination, metal specificity or preference is achieved by diversification of the amino acids interspersed between them. The metal-specific MT polypeptides fold into unique, energetically-optimized complexes of defined metal content, when binding their cognate metal ions, while they produce a mixture of complexes, none of them representing a clear energy minimum, with non-cognate metal ions. Another critical, and so far mostly unexplored, region is the stretch linking the individual MT domains, each of which represents an independent metal cluster. In this work, we have designed and analyzed two HpCdMT constructs with substituted linker segments, and determined their coordination behavior when exposed to both cognate and non-cognate metal ions. Results unequivocally show that neither length nor composition of the inter-domain linker alter the features of the Zn(II)- and Cd(II)-complexes, but surprisingly that they influence their ability to bind Cu(I), the non-cognate metal ion. PMID:26703589

  16. Identification of metallothionein subisoforms in HPLC using accurate mass and online sequencing by electrospray hybrid linear ion trap-orbital ion trap mass spectrometry.

    PubMed

    Mounicou, Sandra; Ouerdane, Laurent; L'Azou, Béatrice; Passagne, Isabelle; Ohayon-Courtès, Céline; Szpunar, Joanna; Lobinski, Ryszard

    2010-08-15

    A comprehensive approach to the characterization of metallothionein (MT) isoforms based on microbore HPLC with multimodal detection was developed. MTs were separated as Cd(7) complexes, detected by ICP MS and tentatively identified by molecular mass measured with 1-2 ppm accuracy using Orbital ion trap mass spectrometry. The identification was validated by accurate mass of the corresponding apo-MTs after postcolumn acidification and by their sequences acquired online by higher-energy collision dissociation MS/MS. The detection limits down to 10 fmol and 45 fmol could be obtained by ESI MS for apo- and Cd(7)-isoforms, respectively, and were lower than those obtained by ICP MS (100 fmol). The individual MT isoforms could be sequenced at levels as low as 200 fmol with the sequence coverage exceeding 90%. The approach was successfully applied to the identification of MT isoforms induced in a pig kidney cell line (LLC-PK(1)) exposed to CdS nanoparticles.

  17. Experimental exposure to cadmium affects metallothionein-like protein levels but not survival and growth in wolf spiders from polluted and reference populations.

    PubMed

    Eraly, Debbie; Hendrickx, Frederik; Bervoets, Lieven; Lens, Luc

    2010-06-01

    Both local adaptation and acclimation in tolerance mechanisms may allow populations to persist under metal pollution. However, both mechanisms are presumed to incur (energetic) costs and to trade-off with other life-history traits. To test this hypothesis, we exposed Pardosa saltans (Lycosidae) spiderlings originating from metal-polluted and unpolluted sites to a controlled cadmium (Cd) treatment, and compared contents of metal-binding metallothionein-like proteins (MTLPs), internal metal concentrations, and individual survival and growth rates with a reference treatment. While increased MTLP concentrations in offspring originating from both polluted and unpolluted populations upon exposure indicates a plastic tolerance mechanism, survival and growth rates remain largely unaffected, independent of the population of origin. However, MTLP and Cd concentrations were not significantly correlated. We suggest that MTLP production may be an important mechanism enabling P. saltans populations to persist in ecosystems polluted with heavy metals above a certain level.

  18. Alterations in prey capture and induction of metallothioneins in grass shrimp fed cadmium-contaminated prey

    SciTech Connect

    Wallace, W.G.; Hoexum Brouwer, T.M.; Brouwer, M.; Lopez, G.R.

    2000-04-01

    The aquatic oligochaete Limnodrilus hoffmeisteri from a Cd-contaminated cove on the Hudson River, Foundry Cove, New York, USA, has evolved Cd resistance. Past studies have focused on how the mode of detoxification of Cd by these Cd-resistant worms influences Cd trophic transfer to the grass shrimp Palaemonetes pugio. In the present study, the authors investigate reductions in prey capture in grass shrimp fed Cd-contaminated prey. They also investigate the induction of metal-binding proteins, metallothioneins, in these Cd-exposed shrimp. Grass shrimp were fed field-exposed Cd-contaminated Foundry Cove oligochaetes or laboratory-exposed Cd-contaminated Artemia salina. Following these exposures, the ability of Cd- dosed and control shrimp to capture live A. salina was compared. Results show that shrimp fed laboratory-exposed Cd-contaminated A. salina for 2 weeks exhibit significant reductions in their ability to successfully capture prey (live A. salina). Reductions in prey capture were also apparent, though not as dramatic in shrimp fed for 1 week on field-exposed Cd-contained Foundry Cove oligochaetes. Shrimp were further investigated for their subcellular distribution of Cd to examine if alterations in prey capture could be linked to saturation of Cd-metallothionein. Cd-dosed shrimp produced a low molecular weight CD-binding metallothionein protein in a dose- and time-dependent manner. Most importantly, successful prey capture decreased with increased Cd body burdens and increased Cd concentration bound to high molecular weight proteins.

  19. Production of a bifunctional hybrid molecule B72.3/metallothionein-1 by protein engineering.

    PubMed Central

    Xiang, J; Koropatnick, J; Qi, Y; Luo, X; Moyana, T; Li, K; Chen, Y

    1993-01-01

    A hybrid anti-tumour B72.3 antibody/metallothionein protein B72.3MT-1 was produced by the construction of the expression vector mpSV2neo-EP1-B72.3MT-1. This vector contained the neo gene as a selection marker, the murine immunoglobulin promoter and enhancer, and the hybrid B72.3 heavy chain gene fragment with mouse metallothionein-1 cDNA gene ligated into its CH2 domain. The expression vector was transfected to the heavy chain loss mutant B72.3Mut(K) cell line. The hybrid protein B72.3MT-1 was purified from transfectant supernates using a Protein G column. We showed that the hybrid protein retained the binding reactivity for the TAG72 antigen as the original B72.3 antibody, and the metal-binding capacity of the native metallothionein molecule. Therefore, the bifunctional hybrid protein B72.3MT-1 may be very useful in cancer imaging when labelled with radionuclides such as 99mTc. Images Figure 3 Figure 6 Figure 7 PMID:8495976

  20. Behaviour of the oxidant scavenger metallothionein in hypoxia-induced neotropical fish.

    PubMed

    Hauser-Davis, Rachel Ann; Bastos, Frederico Freire; Dantas, Rafael Ferreira; Tobar, Santiago Alonso Leitão; da Cunha Bastos Neto, Jayme; da Cunha Bastos, Vera Lucia Freire; Ziolli, Roberta Lourenço; Arruda, Marco Aurélio Zezzi

    2014-05-01

    The pacu (Piaractus mesopotamicus) is a hypoxia-tolerant neotropical fish species. There is little or no information in this species regarding biochemical adaptations to waters with different oxygen concentrations, such as the production of reactive oxygen species and antioxidant scavengers, which might be of interest in the study of antioxidant defense mechanisms. Metallothioneins (MT) have been widely applied as biomarkers for metal exposure in fish liver, and, recently, in bile. These metalloproteins, however, have also been reported as free radical scavengers, although studies in this regard are scarce in fish. In this context, normoxic and hypoxic controlled experiments were conducted with pacu specimens and MT levels were quantified in both liver and bile. Reduced glutathione (GSH) indicative of oxidative stress, and thiobarbituric acid reactive substances (TBARS), indicative of lipid peroxidation, were also determined in liver. The results demonstrate that hypoxic fish present significantly lower metallothionein levels in liver and bile and lower reduced glutathione levels in liver, whereas lipid peroxidation was not significantly different between hypoxic and normoxic fish. The results of the present study seem to suggest that metallothioneins may actively participate in redox regulation in hypoxic fish in both bile and liver. MT levels in these organs may be temporarily suppressed, supporting the notion that down-regulation of oxidant scavengers during the oxidative burst is important in defense signaling in these adapted organisms.

  1. Starch Metabolism in Arabidopsis

    PubMed Central

    Streb, Sebastian; Zeeman, Samuel C.

    2012-01-01

    Starch is the major non-structural carbohydrate in plants. It serves as an important store of carbon that fuels plant metabolism and growth when they are unable to photosynthesise. This storage can be in leaves and other green tissues, where it is degraded during the night, or in heterotrophic tissues such as roots, seeds and tubers, where it is stored over longer time periods. Arabidopsis accumulates starch in many of its tissues, but mostly in its leaves during the day. It has proven to be a powerful genetic system for discovering how starch is synthesised and degraded, and new proteins and processes have been discovered. Such work has major significance for our starch crops, whose yield and quality could be improved by the application of this knowledge. Research into Arabidopsis starch metabolism has begun to reveal how its daily turnover is integrated into the rest of metabolism and adapted to the environmental conditions. Furthermore, Arabidopsis mutant lines deficient in starch metabolism have been employed as tools to study other biological processes ranging from sugar sensing to gravitropism and flowering time control. This review gives a detailed account of the use of Arabidopsis to study starch metabolism. It describes the major discoveries made and presents an overview of our understanding today, together with some as-yet unresolved questions. PMID:23393426

  2. Separation and quantitation of metallothioneins by high-performance liquid chromatography coupled with atomic absorption spectrophotometry

    SciTech Connect

    Lehman, L.D.; Klaassen, C.D.

    1986-03-01

    A rapid, reproducible, and sensitive high-performance liquid chromatography (HPLC) method for the determination of the concentrations of metallothionein-I (MT-I) and metallothionein-II (MT-II) in rat liver has been developed. Metallothioneins (MTs) were separated and quantitated by anion-exchange high-performance liquid chromatography coupled with atomic absorption spectrophotometry (AAS). Purified rat liver MT-I and MT-II, used as standards for developing the method, were easily resolved, eluting at 7.5 and 10.4 min, respectively. To establish standard curves, protein concentrations of solutions of the purified MTs were determined by the Kjeldahl method for the determination of nitrogen, after which the standards were saturated with Cd (final concentration of 50 ppm Cd). Rat liver cytosols obtained from untreated and Cd- or Zn-treated rats were prepared for HPLC-AAS analysis by saturation with Cd (50 ppm Cd) followed by heat denaturation (placing in a boiling water bath for 1 min). Based on the method of standard additions, recovery of MTs exceeded 95% and repeated injection of a sample yielded a coefficient of variance of approximately 2%. A detection limit of 5 ..mu..g MT/g liver was established for the method. Only MT-II was detected in untreated rats, whereas following exposure to Cd or Zn, both forms of MTs were detected. Concentrations of total MTs in liver of untreated and Cd- or Zn-treated rats were also determined by the Cd/hemoglobin radioassay (which fails to distinguish MT-I from MT-II) and indicated that results obtained with the HPLC-AAS method compared favorably to the Cd/hemoglobin radioassay.

  3. Cadium pathways during gestation and lactation in control vs. metallothionein 1,2-knockout mice.

    SciTech Connect

    Brako, E. E.; Wilson, A. K.; Jonah, M. M.; Blum, C. A.; Cerny, E. A.; Williams, K. L.; Bhattacharyya, M. H.; Winona State Univ.; Benedictine Univ.; Dominican Univ.

    2003-01-01

    Effects of metallothionein (MT) on cadmium absorption and transfer pathways during gestation and lactation in mice were investigated. Female 129/SvJ metallothionein-knockout (MT1,2KO) and metallothionein-normal (MTN) mice received drinking water containing trace amounts of {sup 109}CdCl{sub 2} (0.15 ng Cd/ml; 0.074 {mu}Ci {sup 109}Cd/ml). {sup 109}Cd and MT in maternal, fetal, and pup tissues were measured on gestation days 7, 14, and 17 and lactation day 11. In dams, MT influenced both the amount of {sup 109}Cd transferred from intestine into body (two- to three-fold higher in MT1,2KO than MTN dams) and tissue-specific {sup 109}Cd distribution (higher liver/kidney ratio in MT1,2KO dams). Placental {sup 109}Cd concentrations in MT1,2KO dams were three- and seven-fold higher on gestation days 14 and 17, respectively, than in MTN dams. Fetal {sup 109}Cd levels were low in both mouse types, but at least 10-fold lower in MTN fetuses. MT had no effect on the amount of {sup 109}Cd transferred to pups via milk; furthermore, 85--90% of total pup {sup 109}Cd was recovered in gastrointestinal tracts of both types, despite high duodenal MT only in MTN pups. A relatively large percentage of milk-derived intestinal {sup 109}Cd was transferred to other pup tissues in both MT1,2KO and MTN pups (14 and 10%, respectively). These results demonstrate that specific sequestration of cadmium by both maternal and neonatal intestinal tract does not require MT. Although MT decreased oral cadmium transfer from intestine to body tissues at low cadmium exposure levels, MT did not play a major role in restricting transfer of cadmium from dam to fetus via placenta and to neonate via milk.

  4. Cucumber Metallothionein-Like 2 (CsMTL2) Exhibits Metal-Binding Properties

    PubMed Central

    Pan, Yu; Pan, Yanglu; Zhai, Junpeng; Xiong, Yan; Li, Jinhua; Du, Xiaobing; Su, Chenggang; Zhang, Xingguo

    2016-01-01

    We identified a novel member of the metallothionein (MT) family, Cucumis sativus metallothionein-like 2 (CsMTL2), by screening a young cucumber fruit complementary DNA (cDNA) library. The CsMTL2 encodes a putative 77-amino acid Class II MT protein that contains two cysteine (Cys)-rich domains separated by a Cys-free spacer region. We found that CsMTL2 expression was regulated by metal stress and was specifically induced by Cd2+ treatment. We investigated the metal-binding characteristics of CsMTL2 and its possible role in the homeostasis and/or detoxification of metals by heterologous overexpression in Escherichia coli cells. Furthermore, we produced a deletion mutant form of the protein, CsMTL2m, that contained the two Cys-rich clusters but lacked the spacer region, in E. coli. We compared the metal-binding properties of CsMTL2 with those of CsMTL2m, the β domain of human metallothionein-like protein 1 (HsMTXb), and phytochelatin-like (PCL) heterologously expressed in E. coli using metal-binding assays. We found that E. coli cells expressing CsMTL2 accumulated the highest levels of Zn2+ and Cd2+ of the four transformed cell types, with levels being significantly higher than those of control cells containing empty vector. E. coli cells expressing CsMTL2 had a higher tolerance for cadmium than for zinc ions. These findings show that CsMTL2 improves metal tolerance when heterologously expressed in E. coli. Future studies should examine whether CsMTL2 improves metal tolerance in planta. PMID:27916887

  5. A cadmium metallothionein gene of ridgetail white prawn Exopalaemon carinicauda (Holthuis, 1950) and its expression

    NASA Astrophysics Data System (ADS)

    Zhang, Jiquan; Wang, Jing; Xiang, Jianhai

    2013-11-01

    Metallothioneins (MTs) are a group of low molecular weight cysteine-rich proteins capable of binding heavy metal ions. A cadmium metallothionein ( EcMT — Cd) cDNA with a 189 bp open reading frame (ORF) that encoded a 62 amino acid protein was obtained from Exopalaemon carinicauda. Seventeen cysteines were in the deduced amino acid sequence, and the cysteine (Cys)-rich characteristic was revealed in different metallothioneins in other species. In addition, the deduced amino acid sequence did not contain any aromatic amino acid residues, such as tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe). EcMT—Cd mRNA was expressed in all tested tissues (the ovary, muscle, stomach, and hepatopancreas), and its expression profiles in the hepatopancreas were very different when shrimps were exposed to seawater containing either 50 μmol/L CuSO4 or 2.5 μmol/L CdCl 2. The expression of EcMT-Cd was significantly up-regulated in shrimp exposed to CuSO4 for 12 h and down-regulated in shrimps exposed to CdCl2 for 12 h. After 24 h exposure to both metals, its expression was down-regulated. By contrast, at 48 h the EcMT-Cd was up-regulated in test shrimps exposed to CdCl2. The transcript of EcMT-Cd was very low or even absent before the zoea stage, and the expression of EcMT-Cd was detected from mysis larvae-I, then its expression began to rise. In conclusion, a cadmium MT exists in E. carinicauda that is expressed in different tissues and during different developmental stages, and responds to the challenge with heavy metal ions, which provides a clue to understanding the function of cadmium MT.

  6. Metal accumulation and metallothionein concentrations in tree swallow nestlings near acidified lakes

    SciTech Connect

    St. Louis, V.; Breebaart, L. . Dept. of Zoology); Barlow, J.C. . Dept. of Zoology Royal Ontario Museum, Toronto, Ontario . Dept. of Ornithology); Klaverkamp, J.F. . Dept. of Fisheries and Oceans)

    1993-07-01

    The authors studied metal accumulation in hepatic and renal tissues of tree swallow (Tachycineta bicolor) nestlings at acidified and nonacid reference lakes in northwestern Ontario. Hepatic concentrations of metallothionein (metal-binding proteins, MT) in tree swallow nestlings were negatively correlated with pH of the nest-site lake. Combined concentrations of Cu and Zn in the liver were correlated with liver MT concentrations, but Cd was not. Although no overt signs of metal toxicity were observed in nestlings near acid lakes, the results clearly provided evidence that metals are transferred from acid lakes to birds and that these metals are correlated with increases in hepatic MT production.

  7. Highly selective and rapid arsenic removal by metabolically engineered Escherichia coli cells expressing Fucus vesiculosus metallothionein.

    PubMed

    Singh, Shailendra; Mulchandani, Ashok; Chen, Wilfred

    2008-05-01

    An arsenic-chelating metallothionein (fMT) from the arsenic-tolerant marine alga Fucus vesiculosus was expressed in Escherichia coli, resulting in 30- and 26-fold-higher As(III) and As(V) binding, respectively. Coexpression of the As(III)-specific transporter GlpF with fMT further improved arsenic accumulation and offered high selectivity toward As. Resting E. coli cells coexpressing fMT and GlpF completely removed trace amounts (35 ppb) of As(III) within 20 min, providing a promising technology for compliance with the As limit of 10 ppb newly recommended by the U.S. EPA.

  8. Metallothionein blocks oxidative DNA damage induced by acute inorganic arsenic exposure

    SciTech Connect

    Qu, Wei Waalkes, Michael P.

    2015-02-01

    We studied how protein metallothionein (MT) impacts arsenic-induced oxidative DNA damage (ODD) using cells that poorly express MT (MT-I/II double knockout embryonic cells; called MT-null cells) and wild-type (WT) MT competent cells. Arsenic (as NaAsO{sub 2}) was less cytolethal over 24 h in WT cells (LC{sub 50} = 11.0 ± 1.3 μM; mean ± SEM) than in MT-null cells (LC{sub 50} = 5.6 ± 1.2 μM). ODD was measured by the immuno-spin trapping method. Arsenic (1 or 5 μM; 24 h) induced much less ODD in WT cells (121% and 141% of control, respectively) than in MT-null cells (202% and 260%). In WT cells arsenic caused concentration-dependent increases in MT expression (transcript and protein), and in the metal-responsive transcription factor-1 (MTF-1), which is required to induce the MT gene. In contrast, basal MT levels were not detectable in MT-null cells and unaltered by arsenic exposure. Transfection of MT-I gene into the MT-null cells markedly reduced arsenic-induced ODD levels. The transport genes, Abcc1 and Abcc2 were increased by arsenic in WT cells but either showed no or very limited increases in MT-null cells. Arsenic caused increases in oxidant stress defense genes HO-1 and GSTα2 in both WT and MT-null cells, but to much higher levels in WT cells. WT cells appear more adept at activating metal transport systems and oxidant response genes, although the role of MT in these responses is unclear. Overall, MT protects against arsenic-induced ODD in MT competent cells by potential sequestration of scavenging oxidant radicals and/or arsenic. - Highlights: • Metallothionein blocks arsenic toxicity. • Metallothionein reduces arsenic-induced DNA damage. • Metallothionein may bind arsenic or radicals produced by arsenic.

  9. Metallothionein as bioindicator of freshwater metal pollution: European eel and brown trout.

    PubMed

    Linde, A R; Sánchez-Galán, S; Vallés-Mota, P; García-Vázquez, E

    2001-05-01

    The aim of this work was to evaluate the potential of metallothionein (MT) as a bioindicator of heavy metal pollution in brown trout and European eel in the field situation. River Ferrerias (North Spain) provided a good gradient of metal contamination: concentrations of heavy metals were elevated both in water and in sediments at the downstream (polluted) site and were low at the upstream (unpolluted) site. MT levels of brown trout exhibited statistically significant differences between sites. Although European eel at the polluted site had a higher MT content, differences were not significant. It is concluded that MT is a good bioindicator of heavy metal pollution in brown trout.

  10. The potential effect of metallothionein 2A - 5 A/G single nucleotide polymorphism on blood cadmium, lead, zinc and copper levels

    SciTech Connect

    Kayaalti, Zeliha Aliyev, Vugar; Soeylemezoglu, Tuelin

    2011-10-01

    Metallothioneins (MTs) are low molecular weight, cysteine-rich, metal-binding proteins. Because of their rich thiol groups, MTs bind to the biologically essential metals and perform these metals' homeostatic regulations; absorb the heavy metals and assist with their transportation and extraction. The aim of this study was to investigate the association between the metallothionein 2A (MT2A) core promoter region - 5 A/G single nucleotide polymorphism (SNP) and Cd, Pb, Zn and Cu levels in the blood samples. MT2A polymorphism was determined by the standard polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique using the 616 blood samples and the genotype frequencies were found as 86.6% homozygote typical (AA), 12.8% heterozygote (AG) and 0.6% homozygote atypical (GG). Metal levels were analyzed by dual atomic absorption spectrophotometer system and the average levels of Cd, Pb, Zn and Cu in the blood samples were 1.69 {+-} 1.57 ppb, 30.62 {+-} 14.13 ppb, 0.98 {+-} 0.49 ppm and 1.04 {+-} 0.45 ppm, respectively. As a result; highly statistically significant associations were detected between the - 5 A/G core promoter region SNP in the MT2A gene and Cd, Pb and Zn levels (p = 0.004, p = 0.012 and p = 0.002, respectively), but no association was found with Cu level (p = 0.595). Individuals with the GG genotype had statistically lower Zn level and higher Cd and Pb levels in the blood samples than individuals with AA and AG genotypes. This study suggests that having the GG genotype individuals may be more sensitive for the metal toxicity and they should be more careful about protecting their health against the toxic effects of the heavy metals. - Highlights: > MT2A -5A/G SNP has strong effect on the Cd, Pb and Zn levels in the blood. > MT2A GG individuals should be more careful for their health against metal toxicity. > This SNP might be considered as a biomarker for risk of disease related to metals.

  11. A Simple Metallothionein-Based Biosensor for Enhanced Detection of Arsenic and Mercury

    PubMed Central

    Irvine, Gordon W.; Tan, Swee Ngin; Stillman, Martin J.

    2017-01-01

    Metallothioneins (MTs) are a family of cysteine-rich proteins whose biological roles include the regulation of essential metal ions and protection against the harmful effects of toxic metals. Due to its high affinity for many toxic, soft metals, recombinant human MT isoform 1a was incorporated into an electrochemical-based biosensor for the detection of As3+ and Hg2+. A simple design was chosen to maximize its potential in environmental monitoring and MT was physically adsorbed onto paper discs placed on screen-printed carbon electrodes (SPCEs). This system was tested with concentrations of arsenic and mercury typical of contaminated water sources ranging from 5 to 1000 ppb. The analytical performance of the MT-adsorbed paper discs on SPCEs demonstrated a greater than three-fold signal enhancement and a lower detection limit compared to blank SPCEs, 13 ppb for As3+ and 45 ppb for Hg2+. While not being as low as some of the recommended drinking water limits, the sensitivity of the simple MT-biosensor would be potentially useful in monitoring of areas of concern with a known contamination problem. This paper describes the ability of the metal binding protein metallothionein to enhance the effectiveness of a simple, low-cost electrochemical sensor. PMID:28335390

  12. [Metallothionein-I/II in brain injury repair mechanism and its application in forensic medicine].

    PubMed

    Li, Dong; Li, Ru-bo; Lin, Ju-li

    2013-10-01

    Metallothionein (MT) is a kind of metal binding protein. As an important member in metallothionein family, MT-I/II regulates metabolism and detoxication of brain metal ion and scavenges free radicals. It is capable of anti-inflammatory response and anti-oxidative stress so as to protect the brain tissue. During the repair process of brain injury, the latest study showed that MT-I/II could stimulate brain anti-inflammatory factors, growth factors, neurotrophic factors and the expression of the receptor, and promote the extension of axon of neuron, which makes contribution to the regeneration of neuron and has important effect on the recovery of brain injury. Based on the findings, this article reviews the structure, expression, distribution, adjustion, function, mechanism in the repair of brain injury of MT-I/II and its application prospect in forensic medicine. It could provide a new approach for the design and manufacture of brain injury drugs as well as for age estimation of the brain injury.

  13. A Simple Metallothionein-Based Biosensor for Enhanced Detection of Arsenic and Mercury.

    PubMed

    Irvine, Gordon W; Tan, Swee Ngin; Stillman, Martin J

    2017-03-13

    Metallothioneins (MTs) are a family of cysteine-rich proteins whose biological roles include the regulation of essential metal ions and protection against the harmful effects of toxic metals. Due to its high affinity for many toxic, soft metals, recombinant human MT isoform 1a was incorporated into an electrochemical-based biosensor for the detection of As(3+) and Hg(2+). A simple design was chosen to maximize its potential in environmental monitoring and MT was physically adsorbed onto paper discs placed on screen-printed carbon electrodes (SPCEs). This system was tested with concentrations of arsenic and mercury typical of contaminated water sources ranging from 5 to 1000 ppb. The analytical performance of the MT-adsorbed paper discs on SPCEs demonstrated a greater than three-fold signal enhancement and a lower detection limit compared to blank SPCEs, 13 ppb for As(3+) and 45 ppb for Hg(2+). While not being as low as some of the recommended drinking water limits, the sensitivity of the simple MT-biosensor would be potentially useful in monitoring of areas of concern with a known contamination problem. This paper describes the ability of the metal binding protein metallothionein to enhance the effectiveness of a simple, low-cost electrochemical sensor.

  14. The metallothionein gene, TaMT3, from Tamarix androssowii confers Cd2+ tolerance in tobacco.

    PubMed

    Zhou, Boru; Yao, Wenjing; Wang, Shengji; Wang, Xinwang; Jiang, Tingbo

    2014-06-10

    Cadmium (Cd) is a nonessential microelement and low concentration Cd2+ has strong toxicity to plant growth. Plant metallothioneins, a class of low molecular, cystein(Cys)-rich and heavy-metal binding proteins, play an important role in both metal chaperoning and scavenging of reactive oxygen species (ROS) with their large number of cysteine residues and therefore, protect plants from oxidative damage. In this study, a metallothionein gene, TaMT3, isolated from Tamarix androssowii was transformed into tobacco (Nicotiana tobacum) through Agrobacterium-mediated leaf disc method, and correctly expressed under the control of 35S promoter. Under Cd2+ stress, the transgenic tobacco showed significant increases of superoxide dismutase (SOD) activity and chlorophyll concentration, but decreases of peroxidase (POD) activity and malondialdehyde (MDA) accumulation when compared to the non-transgenic tobacco. Vigorous growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weight were significantly larger than those of the non-transgenic tobacco under Cd2+ stress. These results demonstrated that the expression of the exogenous TaMT3 gene increased the ability of ROS cleaning-up, indicating a stronger tolerance to Cd2+ stress.

  15. Mercury accumulation and metallothionein expression from aquafeeds by Litopenaeus vannamei Boone, 1931 under intensive aquaculture conditions.

    PubMed

    Soares, T M; Coutinho, D A; Lacerda, L D; Moraes, M O; Rebelo, M F

    2011-02-01

    This study describes the accumulation of Hg and metallothionein gene expression in Litopenaeus vannamei Boone, 1931 with aquafeeds as the major source of Hg. Trials were conducted under controlled conditions in experimental tank facilities with high (indoor tanks) and low (outdoor tanks) Hg aquafeeds concentrations. Aquafeeds were the sole source of Hg for the shrimps and concentrations varied from 5.4 to 124 ng.g-1 d.w.. In the three animal fractions analysed; muscle (6,3 - 15,9 ng.g-1); hepatopancreas (5,1 - 22,0 ng.g-1) and exoskeleton (3,0 - 16,2 ng.g-1), Hg concentrations were significantly lower in the outdoor trials submitted to Hg-poor aquafeeds. Maximum shrimp muscle Hg concentrations were low (36.4 ng.g-1 w.w.) relative to maximum permissible concentrations for human consumption and Hg content in muscle and hepatopancreas were significantly correlated with Hg content in aquafeeds. Highest Hg concentrations in the exoskeleton of animals exposed to Hg-richer aquafeed, suggested that a detoxification mechanism is taking place. On the other hand the metallothionein suffered no variation in its relative expression in any of the experiments, meaning that the contact with feed containing the observed Hg concentrations were not sufficient to activate gene transcription. It was not possible, under the experimental design used, to infer Hg effects on the biological performance of the animals.

  16. Metallothionein immunoexpression in non-syndromic and syndromic keratocystic odontogenic tumour

    PubMed Central

    Johann, Aline-Cristina-Batista-Rodrigues; Caliari, Marcelo-Vidigal; Gomez, Ricardo-Santiago; Aguiar, Maria-Cássia-Ferreira; Mesquita, Ricardo-Alves

    2015-01-01

    Background To compare the metallothionein (MT) immunoexpression in non-syndromic and syndromic keratocystic odontogenic tumour (KOT), to correlate MT with cellular proliferation, and to evaluate the influence of inflammation in MT. Material and Methods Fourteen cases of KOT were submitted to immunohistochemistry for MT and Ki-67 analysis. The lesions were grouped according to their grade of inflammation, and statistical analysis was performed. Results MT was higher in non-syndromic KOT than in syndromic KOT (p<0.05). No statistical difference in Ki-67 could be identified; however, an inverse correlation was observed between MT and Ki-67 in both lesions. When analysing inflammation, non-syndromic KOT showed no differences in either MT or Ki-67. Conclusions The MT immunophenotype of syndromic KOT was different from non-syndromic KOT. MT might not be involved in the proliferation control of both KOT. MT and Ki-67 immunoexpressions proved to be unaffected by inflammation in non-syndromic KOT. Key words: Odontogenic tumours, basal cell nevus syndrome, metallothionein, Ki-67 Antigen, immunohistoche-mistry. PMID:25858080

  17. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A.

    PubMed

    Sato, Shoko; Shirakawa, Hitoshi; Tomita, Shuhei; Tohkin, Masahiro; Gonzalez, Frank J; Komai, Michio

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHR or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein-protein interactions with GR.

  18. The two distinctive metal ion binding domains of the wheat metallothionein Ec-1.

    PubMed

    Peroza, Estevão A; Kaabi, Ali Al; Meyer-Klaucke, Wolfram; Wellenreuther, Gerd; Freisinger, Eva

    2009-03-01

    Metallothioneins are small cysteine-rich proteins believed to play a role, among others, in the homeostasis of essential metal ions such as Zn(II) and Cu(I). Recently, we could show that wheat E(c)-1 is coordinating its six Zn(II) ions in form of metal-thiolate clusters analogously to the vertebrate metallothioneins. Specifically, two Zn(II) ions are bound in the N-terminal and four in the C-terminal domain. In the following, we will present evidence for the relative independence of the two domains from each other with respect to their metal ion binding abilities, and uncover three intriguing peculiarities of the protein. Firstly, one Zn(II) ion of the N-terminal domain is relative resistant to complete replacement with Cd(II) indicating the presence of a Zn(II)-binding site with increased stability. Secondly, the C-terminal domain is able to coordinate an additional fifth metal ion, though with reduced affinity, which went undetected so far. Finally, reconstitution of apoE(c)-1 with an excess of Zn(II) shows a certain amount of sub-stoichiometrically metal-loaded species. The possible relevance of these finding for the proposed biological functions of wheat E(c)-1 will be discussed. In addition, extended X-ray absorption fine structure (EXAFS) measurements on both, the full-length and the truncated protein, provide final evidence for His participation in metal ion binding.

  19. Identification and expression of an atypical isoform of metallothionein in the African clawed frog Xenopus laevis.

    PubMed

    Scudiero, Rosaria; Tussellino, Margherita; Carotenuto, Rosa

    2015-05-01

    Exploiting the annotation of the western clawed frog Silurana tropicalis genome, we identified a new metallothionein (MT) gene, exhibiting all the features to be considered an active gene, but with an atypical coding region, showing only 17 cysteine residues instead of the canonical 20 cysteines of vertebrate metallothioneins and two anomalous cysteine triplets. However, the presence of a gene in the genome does not ensure its effective expression. By using conventional and Real-Time PCR analyses, we demonstrated that this atypical MT is constitutively expressed throughout the life cycle of the African clawed frog Xenopus laevis; moreover, this gene is highly expressed in the adult liver, the major site of MT expression and synthesis in vertebrates. To our knowledge, the X. laevis MT described in this paper is the first sequence of a vertebrate MT showing only 17 cysteine residues, arranged in two Cys-Cys-Cys motifs. Phylogenetic analyses also demonstrated that the atypical X. laevis MT merges in the anuran clade, but is the most derived sequence among tetrapods MTs. Finally, Tajima's Relative Rate Test suggested a different evolutionary rate between the canonical X. laevis MT and this novel isoform.

  20. Involvement of metallothionein in cadmium accumulation and elimination in the clam ruditapes decussata

    SciTech Connect

    Bebianno, M.J.; Serafim, M.A.P.; Rita, M.F. )

    1994-11-01

    Cadmium is one of the most toxic pollutants in seawater because of its persistence, toxicity and potential for bioaccumulation. It is included on the [open quotes]black list[close quotes] of several international agreements established to regulate the input of pollutants into the marine environment. The deleterious effects of cadmium contamination in marine organisms result from its accumulation within specific tissues. However, most of these organisms have developed subcellular detoxification processes, including the synthesis of metallothioneins, low-molecular weight, metal-binding proteins. Bivalves have the ability to accumulate and concentrate cadmium to levels several orders of magnitude above those found in their environment. The present study was designed to examine the involvement of metallothionein synthesis in cadmium accumulation and elimination in the bivalve Ruditapes decussata when exposed to a sublethal cadmium concentration (100 [mu]g/l) and to a mixture of cadmium (100 [mu]g/1), copper (50 [mu]g/l) and zinc (50 [mu]g/1). 15 refs., 2 figs., 1 tab.

  1. A Determination of Metallothionein in Larvae of Freshwater Midges (Chironomus riparius) Using Brdicka Reaction

    PubMed Central

    Fabrik, Ivo; Ruferova, Zuzana; Hilscherova, Klara; Adam, Vojtech; Trnkova, Libuse; Kizek, Rene

    2008-01-01

    Among wide spectrum of biomolecules induced by various stress factors low molecular mass protein called metallothionein (MT) is suitable for assessment of the heavy metal environmental pollution. The aim of this work was to determine the metallothionein and total thiols content in larvae of freshwater midges (Chironomus riparius) sampled from laboratory exposure to cadmium(II) ions and from field studies using differential pulse voltammetry Brdicka reaction. Unique electrochemical instrument, stationary electrochemical analyser Autolab coupled with autosampler, was utilized for the analysis of the samples. The detection limit for MT was evaluated as 5 nM. The larvae exposed to two doses (50 ng/g or 50 μg/g) of cadmium(II) ions for fifteen days under laboratory controlled conditions were at the end of the exposure killed, homogenized and analysed. MT content in control samples was 1.2 μM, in larvae exposed to 50 ng Cd/g it was 2.0 μM and in larvae exposed to 50 μg Cd/g 2.9 μM. Moreover at field study chironomid larvae as well as sediment samples have been collected from eight field sites with different levels of pollution by heavy. The metals content (chromium, nickel, copper, zinc, arsenic, molybdenum, cadmium, tin and lead) in the sediment and or MT content in the chironomid larvae were determined by inductively coupled plasma mass spectrometry or Brdicka reaction, respectively. PMID:27879923

  2. Subcellular targets of cadmium nephrotoxicity: cadmium binding to renal membrane proteins in animals with or without protective metallothionein synthesis.

    PubMed Central

    Nordberg, G F; Jin, T; Nordberg, M

    1994-01-01

    Nephrotoxic effects of cadmium exposure are well established in humans and experimental animals. An early manifestation of such toxicity is calciuria a few hours after injection of CdMT in rats. Protection against calciuria and other adverse effects such as proteinuria (occurring later) is offered by pretreatment with Cd, which effectively induces metallothionein synthesis. In the present experiment, one group of animals was given pretreatment with CdCl2 to induce metallothionein synthesis. The comparison group was left without pretreatment. The distribution of Cd from a normally nephrotoxic dose of 109CdMT was studied by gel chromatography in subcellular fractions of kidney cortex in both groups. In the pretreated animals, 109Cd in the plasma membrane and microsome fractions of renal cortical cells was mainly bound to metallothionein and other low molecular weight proteins at 4 hr. In nonpretreated animals the major part of 109Cd was bound to high molecular weight proteins. These findings indicate that membrane proteins may be important targets for Cd when inducing nephrotoxicity and that sequestering of Cd by metallothionein (and other low molecular weight proteins) may be a mechanism of protection. PMID:7843096

  3. Survival and hepatic metallothionein in developing rainbow trout exposed to a mixture of zinc, copper, and cadmium

    SciTech Connect

    Roch, M.; McCarter, J.A.

    1986-02-01

    Rainbow trout (Salmo gairdneri) in Buttle Lake on Vancouver Island, B.C. are exposed to metal contamination originating from a copper and zinc mining operation at Myra Falls near the head of the lake. In order to properly assess the risk to a population of rainbow trout in Buttle Lake, the authors initiated a long-term exposure of rainbow trout from hatch including the swim-up stage. Copper, zinc or cadmium are known to induce metallothionein in mammals and as a mixture of metals, induce hepatic metallothionein in rainbow trout. Investigation of hepatic metallothionein concentrations in wild rainbow trout from Buttle Lake and in lakes of the Campbell River downstream showed a correlation with metal concentrations in the water. Rainbow trout held in situ for 4 weeks showed the same correlation. In this report they determined whether or not the degree of contamination was correlated with concentrations of metallothionein in the livers of rainbow trout exposed to the mixture of metals during the early life stages.

  4. GLUTATHIONE AND METALLOTHIONEIN STATUS IN AN ACUTE RESPONSE BY MERCENARIA MERCENARIA BROWN CELLS TO COPPER IN VIVO

    EPA Science Inventory

    Red glands of Mercenaria mercenaria comprise brown cells that accumulate, detoxify, and excrete copper. Brown cell involvement in metal detoxification is due in part to endogenous glutathione (GSH) and metallothionein (MT).The intent of this study was to test the hypotheses that ...

  5. Contribution of hepatic cytochrome CYP1A and metallothionein mRNA abundance to biomonitoring-A case study with European flounder (Platichthys flesus) from the Gulf of Gdańsk.

    PubMed

    Kopecka-Pilarczyk, Justyna; Schirmer, Kristin

    2016-10-01

    The aim of the research was to explore the contribution of hepatic cytochrome CYP1A and metallothionein (MT) mRNA expression to biological effect monitoring. The study was conducted in the European flounder (Platichthys flesus) from the Gulf of Gdańsk. mRNA abundance was measured using reverse transcriptase polymerase chain reaction (RT-PCR) in liver RNA of fish sampled from three coastal stations and from one offshore station in the inner Gulf. The contribution of the mRNA-based biomarkers to the assessment of the environment was determined in conjunction with a selection of commonly applied biochemical markers: 7-ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), metallothioneins (MT), fluorescent aromatic compounds (FACs), all measured in the same individual fish. The mRNA biomarkers contributed to the separation between the sampling sites, but no correlations between CYP1A mRNA and EROD nor between MT mRNA and MT proteins were found, which should be attributed to the different levels these biomarkers correspond to and to the differences in factors that may affect them. One case of strong correlation between CYP1A mRNA and FACs was encountered. The overall results of this study suggest that biomarkers measured at the mRNA abundance level constitute a valuable addition to biomonitoring studies by providing additional information and contributing to the differentiation of results.

  6. Metal and metallothionein concentrations in Scoter (Melanitta spp.) from the Pacific northwest of Canada, 1989-1994.

    PubMed

    Barjaktarovic, L; Elliott, J E; Scheuhammer, A M

    2002-11-01

    Eighty-six surf ( Melanitta perspicillata) and nine white winged (M. fusca) scoters were collected from 1989-1994 at 11 locations in British Columbia and the Yukon. Their kidney and liver tissues were analyzed for cadmium (Cd), copper (Cu), zinc (Zn), and metallothionein (MT) concentrations. Individual kidney Cd values ranged from 2.4 microg/g dry weight (DW) in birds from northern Vancouver Island to 390.2 microg/g (DW) in birds from the Queen Charlotte Islands, which is in the range of values potentially associated with kidney damage. Birds from the Queen Charlotte Islands also had high kidney Zn concentrations, which were significantly higher than values in birds from Naniamo and Westham Island on the south coast of BC. Cd values were significantly (p < 0.001) correlated with both Zn and MT concentrations in kidney tissue (r = 0.66 and 0.62, respectively). Male surf scoters had significantly higher kidney Cd and Zn levels than females, with mean Cd values of 47.3 and 19.7 microg/g DW (p < 0.002) for males and females, respectively. Mean Zn values were 120.8 and 108.0 microg/g DW (p < 0.05) for males and females, respectively. However, no sex differences were observed for either Cu or MT concentrations in kidney tissue. Individual MT values ranged from 2.7 to 416.8 microg/g wet weight (WW). Individual kidney Cu values ranged from 15.1 to 48.4 microg/g DW for both sexes. Kidney and liver Cd concentrations were significantly correlated (r = 0.90, p < 0.05) with kidney levels almost always higher than liver values. Although Cd accumulation by the Skidegate scoters was high, comparable tissue Cd values have been documented in other seabirds collected from the same general area, indicating elevated Cd concentrations in marine food chains around the Queen Charlotte Islands (Haida Gwaii) region of the Pacific coast.

  7. Transgenic Arabidopsis Gene Expression System

    NASA Technical Reports Server (NTRS)

    Ferl, Robert; Paul, Anna-Lisa

    2009-01-01

    The Transgenic Arabidopsis Gene Expression System (TAGES) investigation is one in a pair of investigations that use the Advanced Biological Research System (ABRS) facility. TAGES uses Arabidopsis thaliana, thale cress, with sensor promoter-reporter gene constructs that render the plants as biomonitors (an organism used to determine the quality of the surrounding environment) of their environment using real-time nondestructive Green Fluorescent Protein (GFP) imagery and traditional postflight analyses.

  8. The comparison of mouse full metallothionein-1 versus alpha and beta domains and metallothionein-1-to-3 mutation following traumatic brain injury reveals different biological motifs.

    PubMed

    Manso, Yasmina; Serra, Montserrat; Comes, Gemma; Giralt, Mercedes; Carrasco, Javier; Cols, Neus; Vasák, Milan; González-Duarte, Pilar; Hidalgo, Juan

    2010-06-01

    Traumatic injury to the brain is one of the leading causes of injury-related death or disability, but current therapies are limited. Previously it has been shown that the antioxidant proteins metallothioneins (MTs) are potent neuroprotective factors in animal models of brain injury. The exogenous administration of MTs causes effects consistent with the roles proposed from studies in knock-out mice. We herewith report the results comparing full mouse MT-1 with the independent alpha and beta domains, alone or together, in a cryoinjury model. The lesion of the cortex caused the mice to perform worse in the horizontal ladder beam and the rota-rod tests; all the proteins showed a modest effect in the former test, while only full MT-1 improved the performance of animals in the rota-rod, and the alpha domain showed a rather detrimental effect. Gene expression analysis by RNA protection assay demonstrated that all proteins may alter the expression of host-response genes such as GFAP, Mac1 and ICAM, in some cases being the beta domain more effective than the alpha domain or even the full MT-1. A MT-1-to-MT-3 mutation blunted some but not all the effects caused by the normal MT-1, and in some cases increased its potency. Thus, splitting the two MT-1 domains do not seem to eliminate all MT functions but certainly modifies them, and different motifs seem to be present in the protein underlying such functions.

  9. A polymorphism in metallothionein 1A (MT1A) is associated with cadmium-related excretion of urinary beta 2‐microglobulin

    SciTech Connect

    Lei, Lijian; Chang, Xiuli; Rentschler, Gerda; Tian, Liting; Zhu, Guoying; Chen, Xiao; Jin, Taiyi; Broberg, Karin

    2012-12-15

    Objectives: Cadmium (Cd) toxicity of the kidney varies between individuals despite similar exposure levels. In humans Cd is mainly bound to metallothioneins (MT), which scavenge its toxic effects. Here we analyzed whether polymorphisms in MT genes MT1A and MT2A influence Cd-related kidney damage. Methods: In a cross-sectional study N = 512 volunteers were selected from three areas in South-Eastern China, which to varying degree were Cd-polluted from a smelter (control area [median Cd in urine U-Cd = 2.67 μg/L], moderately [U-Cd = 4.23 μg/L] and highly [U-Cd = 9.13 μg/L] polluted areas). U-Cd and blood Cd (B-Cd) concentrations were measured by graphite-furnace atomic absorption spectrometry. MT1A rs11076161 (G/A), MT2A rs10636 (G/C) and MT2A rs28366003 (A/G) were determined by Taqman assays; urinary N-Acetyl-beta-(D)-Glucosaminidase (UNAG) by spectrometry, and urinary β2-microglobulin (UB2M) by ELISA. Results: Higher B-Cd (natural log-transformed) with increasing number of MT1A rs11076161 A-alleles was found in the highly polluted group (p-value trend = 0.033; all p-values adjusted for age, sex, and smoking). In a linear model a significant interaction between rs11076161 genotype and B-Cd was found for UNAG (p = 0.001) and UB2M concentrations (p = 0.001). Carriers of the rs11076161 AA genotype showed steeper slopes for the associations between Cd in blood and natural log-transformed UB2M (β = 1.2, 95% CI 0.72–1.6) compared to GG carriers (β = 0.30, 95% CI 0.15–0.45). Also for UNAG (natural log-transformed) carriers of the AA genotype had steeper slopes (β = 0.55, 95% CI 0.27–0.84) compared to GG carriers (β = 0.018, 95% CI − 0.79–0.11). Conclusions: MT1A rs11076161 was associated with B-Cd concentrations and Cd-induced kidney toxicity at high exposure levels. -- Highlights: ► Cadmium is toxic to the kidney but the susceptibility differs between individuals. ► The toxic effect of cadmium is scavenged by metallothioneins. ► A common variant of

  10. Metallothioneins, Unconventional Proteins from Unconventional Animals: A Long Journey from Nematodes to Mammals †

    PubMed Central

    Isani, Gloria; Carpenè, Emilio

    2014-01-01

    Metallothioneins (MTs) are ubiquitous low molecular weight cysteine-rich proteins characterized by high affinity for d10 electron configuration metals, including essential (Zn and Cu) and non-essential (Cd and Hg) trace elements. The biological role of these ancient and well-conserved multifunctional proteins has been debated since MTs were first discovered in 1957. Their main hypothesized functions are: (1) homeostasis of Zn and Cu; (2) detoxification of Cd, and Hg; and (3) free radical scavenging. This review will focus on MTs in unconventional animals, those not traditionally studied in veterinary medicine but of increasing interest in this field of research. Living in different environments, these animals represent an incredible source of physiological and biochemical adaptations still partly unexplored. The study of metal-MT interactions is of great interest for clinicians and researchers working in veterinary medicine, food quality and endangered species conservation. PMID:24970224

  11. Metal concentrations and metallothionein levels in Mytilus galloprovincialis from Elefsis bay (Saronikos gulf, Greece).

    PubMed

    Strogyloudi, Evangelia; Angelidis, Michael O; Christides, Anastassios; Papathanassiou, Evangelos

    2012-12-01

    Spatial and temporal variability of Cd, Cu, Cr, Ni, Zn, Fe and Mn and metallothionein (MT) concentrations were determined in mussels Mytilus galloprovincialis from Elefsis bay (Saronikos gulf, Greece). Higher concentrations of both metal concentrations and MTs were recorded in mussels inhabiting industrial locations (steelworks and shipyard), indicating a markedly higher metal bioavailability. However at these sites, located at the eastern part of the bay, mussel metal concentrations were not always correlated with both seawater metal concentrations and MTs possibly due to different time scales of integration of the metal sources into mussels and/or the participation of other metal regulatory mechanisms except MT induction. The pattern of the temporal variation of mussel metal concentrations and the MT levels was similar among stations with higher values during the winter-spring season and lower during the summer-autumn period. The inverse relationship of flesh condition index with mussel metal concentrations was attributed to the influence of mussel annual reproductive cycle.

  12. Copper accumulation and compartmentalization in mouse fibroblast lacking metallothionein and copper chaperone, Atox1

    SciTech Connect

    Miyayama, Takamitsu; Suzuki, Kazuo T.; Ogra, Yasumitsu

    2009-06-01

    Copper (Cu) is the active center of some enzymes because of its redox-active property, although that property could have harmful effects. Because of this, cells have strict regulation/detoxification systems for this metal. In this study, multi-disciplinary approaches, such as speciation and elemental imaging of Cu, were applied to reveal the detoxification mechanisms for Cu in cells bearing a defect in Cu-regulating genes. Although Cu concentration in metallothionein (MT)-knockout cells was increased by the knockdown of the Cu chaperone, Atox1, the concentrations of the Cu influx pump, Ctr1, and another Cu chaperone, Ccs, were paradoxically increased; namely, the cells responded to the Cu deficiency despite the fact that cellular Cu concentration was actually increased. Cu imaging showed that the elevated Cu was compartmentalized in cytoplasmic vesicles. Together, the results point to the novel roles of MT and cytoplasmic vesicles in the detoxification of Cu in mammalian cells.

  13. Transcription patterns of genes encoding four metallothionein homologs in Daphnia pulex exposed to copper and cadmium are time- and homolog- dependent

    PubMed Central

    Asselman, Jana; Shaw, Joseph R.; Glaholt, Stephen P.; Colbourne, John K.; De Schamphelaere, Karel AC.

    2013-01-01

    Metallothioneins are proteins that play an essential role in metal homeostasis and detoxification in nearly all organisms studied to date. Yet discrepancies between outcomes of chronic and acute exposure experiments hamper the understanding of the regulatory mechanisms of their isoforms following metal exposure. Here, we investigated transcriptional differences among four identified homologs (mt1–mt4) in Daphnia pulex exposed across time to copper and cadmium relative to a control. Transcriptional upregulation of mt1 and mt3 was detected on day four following exposure to cadmium, whereas that of mt2 and mt4 was detected on day two and day eight following exposure to copper. These results confirm temporal and metal-specific differences in the transcriptional induction of genes encoding metallothionein homologs upon metal exposure which should be considered in ecotoxicological monitoring programs of metal-contaminated water bodies. Indeed, the mRNA expression patterns observed here illustrate the complex regulatory system associated with metallothioneins, as these patterns are not only dependent on the metal, but also on exposure time and the homolog studied. Further phylogenetic analysis and analysis of regulatory elements in upstream promoter regions revealed a high degree of similarity between metallothionein genes of Daphnia pulex and Daphnia magna, a species belonging to the same genus. These findings, combined with a limited amount of available expression data for D. magna metallothionein genes, tentatively suggest a potential generalization of the metallothionein response system between these Daphnia species. PMID:24113165

  14. Transcription patterns of genes encoding four metallothionein homologs in Daphnia pulex exposed to copper and cadmium are time- and homolog-dependent.

    PubMed

    Asselman, Jana; Shaw, Joseph R; Glaholt, Stephen P; Colbourne, John K; De Schamphelaere, Karel A C

    2013-10-15

    Metallothioneins are proteins that play an essential role in metal homeostasis and detoxification in nearly all organisms studied to date. Yet discrepancies between outcomes of chronic and acute exposure experiments hamper the understanding of the regulatory mechanisms of their isoforms following metal exposure. Here, we investigated transcriptional differences among four identified homologs (mt1-mt4) in Daphnia pulex exposed across time to copper and cadmium relative to a control. Transcriptional upregulation of mt1 and mt3 was detected on day four following exposure to cadmium, whereas that of mt2 and mt4 was detected on day two and day eight following exposure to copper. These results confirm temporal and metal-specific differences in the transcriptional induction of genes encoding metallothionein homologs upon metal exposure which should be considered in ecotoxicological monitoring programs of metal-contaminated water bodies. Indeed, the mRNA expression patterns observed here illustrate the complex regulatory system associated with metallothioneins, as these patterns are not only dependent on the metal, but also on exposure time and the homolog studied. Further phylogenetic analysis and analysis of regulatory elements in upstream promoter regions revealed a high degree of similarity between metallothionein genes of Daphnia pulex and Daphnia magna, a species belonging to the same genus. These findings, combined with a limited amount of available expression data for D. magna metallothionein genes, tentatively suggest a potential generalization of the metallothionein response system between these Daphnia species.

  15. Role of metallothioneins as danger signals in the pathogenesis of colitis.

    PubMed

    Devisscher, Lindsey; Hindryckx, Pieter; Lynes, Michael A; Waeytens, Anouk; Cuvelier, Claude; De Vos, Filip; Vanhove, Christian; Vos, Martine De; Laukens, Debby

    2014-05-01

    Inflammatory bowel diseases (IBDs) are recurrent intestinal pathologies characterized by a compromised epithelial barrier and an exaggerated immune activation. Mediators of immune cell infiltration may represent new therapeutic opportunities. Metallothioneins (MTs) are stress-responsive proteins with immune-modulating functions. Metallothioneins have been linked to IBDs, but their role in intestinal inflammation is inconclusive. We investigated MT expression in colonic biopsies from IBDs and acute infectious colitis patients and healthy controls and evaluated MT's role in experimental colitis using MT knockout mice and anti-MT antibodies. Antibody potential to target extracellular MT and its mechanism was tested in vitro. Biopsies of patients with active colitis showed infiltration of MT-positive cells in a pattern that correlated with the grade of inflammation. MT knockout mice displayed less severe acute dextran sulphate sodium (DSS)-induced colitis compared to congenic wild-type mice based on survival, weight loss, colon length, histological inflammation and leukocyte infiltration. Chronic DSS-colitis confirmed that Mt1 and Mt2 gene disruption enhances clinical outcome. Blockade of extracellular MT with antibodies reduced F4/80-positive macrophage infiltration in DSS- and trinitrobenzene sulphonic acid-colitis, with a tendency towards a better outcome. Whole-body single-photon emission computer tomography of mice injected with radioactive anti-MT antibodies showed antibody accumulation in the colon during colitis and clearance during recovery. Necrotic and not apoptotic cell death resulted in western blot MT detection in HT29 cell supernatant. In a Boyden chamber migration assay, leukocyte attraction towards the necrotic cell supernatant could be abolished with anti-MT antibody, indicating the chemotactic potential of endogenous released MT. Our results show that human colitis is associated with infiltration of MT-positive inflammatory cells. Since antibody

  16. Physiological, Diurnal and Stress-Related Variability of Cadmium-Metallothionein Gene Expression in Land Snails.

    PubMed

    Pedrini-Martha, Veronika; Niederwanger, Michael; Kopp, Renate; Schnegg, Raimund; Dallinger, Reinhard

    2016-01-01

    The terrestrial Roman snail Helix pomatia has successfully adapted to strongly fluctuating conditions in its natural soil habitat. Part of the snail's stress defense strategy is its ability to express Metallothioneins (MTs). These are multifunctional, cysteine-rich proteins that bind and inactivate transition metal ions (Cd(2+), Zn(2+), Cu(+)) with high affinity. In Helix pomatia a Cadmium (Cd)-selective, inducible Metallothionein Isoform (CdMT) is mainly involved in detoxification of this harmful metal. In addition, the snail CdMT has been shown to also respond to certain physiological stressors. The aim of the present study was to investigate the physiological and diurnal variability of CdMT gene expression in snails exposed to Cd and non-metallic stressors such as desiccation and oxygen depletion. CdMT gene expression was upregulated by Cd exposure and desiccation, whereas no significant impact on the expression of CdMT was measured due to oxygen depletion. Overall, Cd was clearly more effective as an inducer of the CdMT gene expression compared to the applied non-metallic stressors. In unexposed snails, diurnal rhythmicity of CdMT gene expression was observed with higher mRNA concentrations at night compared to daytime. This rhythmicity was severely disrupted in Cd-exposed snails which exhibited highest CdMT gene transcription rates in the morning. Apart from diurnal rhythmicity, feeding activity also had a strong impact on CdMT gene expression. Although underlying mechanisms are not completely understood, it is clear that factors increasing MT expression variability have to be considered when using MT mRNA quantification as a biomarker for environmental stressors.

  17. Metal, metallothionein and glutathione levels in blue crab (Callinectes sp.) specimens from southeastern Brazil.

    PubMed

    Lavradas, Raquel Teixeira; Hauser-Davis, Rachel Ann; Lavandier, Ricardo Cavalcanti; Rocha, Rafael Christian Chávez; Saint' Pierre, Tatiana D; Seixas, Tércia; Kehrig, Helena Amaral; Moreira, Isabel

    2014-09-01

    Metal concentrations (Cu, Pb, Zn and Cd) were determined in muscle, gills, soft tissues and eggs in male, non-ovigerous and ovigerous female Callinectes sp. specimens from a reference site in Southeastern Brazil. Metallothionein (MT) and reduced glutathione (GSH) levels were also determined. Results demonstrate that sex has a significant influence on metal, MT and GSH concentrations. Significant maternal transfer of Pb and Zn from ovigerous females to eggs was verified, while female crabs, both ovigerous and non-ovigerous, showed elevated GSH and MT in viscera when compared to males, indicating possible MT role in excreting metals to eggs in ovigerous females of this species. Several strong statistical correlations between metals and MT indicate MTs role in detoxification of both toxic and essential elements in different organs. Pb and Zn were significantly correlated to GSH, indicating oxidative stress caused by the former and a direct link between Zn and GSH in maintaining homeostasis. Regarding human consumption, metal concentrations were lower than the maximum permissible levels established by international and Brazilian regulatory agencies, indicating that this species is safe for human consumption concerning this parameter. The presence of metals in Callinectes sp., however, is still of importance considering that this is a key species within the studied ecosystem and, therefore, plays a major role in the transference of pollutants to higher trophic levels. In addition, the presence of significant metal concentrations found in eggs must be considered in this context, since crab eggs are eaten by several other species, such as shorebirds, seabirds, and fish. Also, to the best of our knowledge, this is the first study regarding both MT and GSH levels in Callinectes sp. eggs and is of interest in the investigation of molecular mechanisms regarding metal exposure in these crustaceans. Data reported in this study support the conclusions from previous reports

  18. Physiological, Diurnal and Stress-Related Variability of Cadmium-Metallothionein Gene Expression in Land Snails

    PubMed Central

    Pedrini-Martha, Veronika; Niederwanger, Michael; Kopp, Renate; Schnegg, Raimund; Dallinger, Reinhard

    2016-01-01

    The terrestrial Roman snail Helix pomatia has successfully adapted to strongly fluctuating conditions in its natural soil habitat. Part of the snail’s stress defense strategy is its ability to express Metallothioneins (MTs). These are multifunctional, cysteine-rich proteins that bind and inactivate transition metal ions (Cd2+, Zn2+, Cu+) with high affinity. In Helix pomatia a Cadmium (Cd)-selective, inducible Metallothionein Isoform (CdMT) is mainly involved in detoxification of this harmful metal. In addition, the snail CdMT has been shown to also respond to certain physiological stressors. The aim of the present study was to investigate the physiological and diurnal variability of CdMT gene expression in snails exposed to Cd and non-metallic stressors such as desiccation and oxygen depletion. CdMT gene expression was upregulated by Cd exposure and desiccation, whereas no significant impact on the expression of CdMT was measured due to oxygen depletion. Overall, Cd was clearly more effective as an inducer of the CdMT gene expression compared to the applied non-metallic stressors. In unexposed snails, diurnal rhythmicity of CdMT gene expression was observed with higher mRNA concentrations at night compared to daytime. This rhythmicity was severely disrupted in Cd-exposed snails which exhibited highest CdMT gene transcription rates in the morning. Apart from diurnal rhythmicity, feeding activity also had a strong impact on CdMT gene expression. Although underlying mechanisms are not completely understood, it is clear that factors increasing MT expression variability have to be considered when using MT mRNA quantification as a biomarker for environmental stressors. PMID:26935042

  19. Multiplex micro-respiratory measurements of Arabidopsis tissues.

    PubMed

    Sew, Yun Shin; Ströher, Elke; Holzmann, Cristián; Huang, Shaobai; Taylor, Nicolas L; Jordana, Xavier; Millar, A Harvey

    2013-11-01

    Researchers often want to study the respiratory properties of individual parts of plants in response to a range of treatments. Arabidopsis is an obvious model for this work; however, because of its size, it represents a challenge for gas exchange measurements of respiration. The combination of micro-respiratory technologies with multiplex assays has the potential to bridge this gap, and make measurements possible in this model plant species. We show the adaptation of the commercial technology used for mammalian cell respiration analysis to study three critical tissues of interest: leaf sections, root tips and seeds. The measurement of respiration in single leaf discs has allowed the age dependence of the respiration rate in Arabidopsis leaves across the rosette to be observed. The oxygen consumption of single root tips from plate-grown seedlings shows the enhanced respiration of root tips and their time-dependent susceptibility to salinity. The monitoring of single Arabidopsis seeds shows the kinetics of respiration over 48 h post-imbibition, and the effect of the phytohormones gibberellic acid (GA3 ) and abscisic acid (ABA) on respiration during seed germination. These studies highlight the potential for multiplexed micro-respiratory assays to study oxygen consumption in Arabidopsis tissues, and open up new possibilities to screen and study mutants and to identify differences in ecotypes or populations of different plant species.

  20. A predicted interactome for Arabidopsis.

    PubMed

    Geisler-Lee, Jane; O'Toole, Nicholas; Ammar, Ron; Provart, Nicholas J; Millar, A Harvey; Geisler, Matt

    2007-10-01

    The complex cellular functions of an organism frequently rely on physical interactions between proteins. A map of all protein-protein interactions, an interactome, is thus an invaluable tool. We present an interactome for Arabidopsis (Arabidopsis thaliana) predicted from interacting orthologs in yeast (Saccharomyces cerevisiae), nematode worm (Caenorhabditis elegans), fruitfly (Drosophila melanogaster), and human (Homo sapiens). As an internal quality control, a confidence value was generated based on the amount of supporting evidence for each interaction. A total of 1,159 high confidence, 5,913 medium confidence, and 12,907 low confidence interactions were identified for 3,617 conserved Arabidopsis proteins. There was significant coexpression of genes whose proteins were predicted to interact, even among low confidence interactions. Interacting proteins were also significantly more likely to be found within the same subcellular location, and significantly less likely to be found in conflicting localizations than randomly paired proteins. A notable exception was that proteins located in the Golgi were more likely to interact with Golgi, vacuolar, or endoplasmic reticulum sorted proteins, indicating possible docking or trafficking interactions. These predictions can aid researchers by extending known complexes and pathways with candidate proteins. In addition we have predicted interactions for many previously unknown proteins in known pathways and complexes. We present this interactome, and an online Web interface the Arabidopsis Interactions Viewer, as a first step toward understanding global signaling in Arabidopsis, and to whet the appetite for those who are awaiting results from high-throughput experimental approaches.

  1. Functional GFP-metallothionein fusion protein from Tetrahymena thermophila: a potential whole-cell biosensor for monitoring heavy metal pollution and a cell model to study metallothionein overproduction effects

    PubMed Central

    Amaro, Francisco; Turkewitz, Aaron P.; Martín-González, Ana; Gutiérrez, Juan Carlos

    2016-01-01

    The significance of metal(oid)s as environmental pollutants has made them a priority in ecotoxicology, with the aim of minimizing exposure to animals or humans. Therefore, it is necessary to develop sensitive and inexpensive methods that can efficiently detect and monitor these pollutants in the environment. Conventional analytical techniques suffer from the disadvantages of high cost and complexity. Alternatively, prokaryotic or eukaryotic whole-cell biosensors (WCB) are one of the newest molecular tools employed in environmental monitoring that use the cell as an integrated reporter incorporating a reporter gene fused to a heavy metal responsive promoter. In the present paper, we report results from expressing, in the ciliate Tetrahymena thermophila, constructs consisting of the reporter gfp gene fused to the complete MTT1 or MTT5 protein coding regions under the transcriptional control of the MTT1 metallothionein promoter, which plays a critical role in heavy metal stress in this ciliate. When exposed to Cd2+, such cells overexpress both the GFP reporter transgene and the linked metallothionein gene. We report that, for the GFPMTT5 strain, this metallothionein overexpression results in marked resistance to cadmium toxicity (24h LC50 ~ 15 µM of Cd2+), compared to wild type cells (24h LC50 ~ 1.73 µM of Cd2+). These results provide the first experimental evidence that ciliate metallothioneins, like in other organisms, function to protect the cell against toxic metal ions. Because these strains may have novel advantages as WCBs, we have compared their properties to those of other previously reported Tetrahymena WCBs. PMID:24430977

  2. Induction by mercury compounds of metallothioneins in mouse tissues: inorganic mercury accumulation is not a dominant factor for metallothionein induction in the liver.

    PubMed

    Yasutake, Akira; Nakamura, Masaaki

    2011-06-01

    Among the naturally occurring three mercury species, metallic mercury (Hg(0)), inorganic mercury (Hg(II)) and methylmercury (MeHg), Hg(II) is well documented to induce metallothionein (MT) in tissues of injected animals. Although Hg(0) and MeHg are considered to be inert in terms of directly inducing MT, MT can be induced by them after in vivo conversion to Hg(II) in an animal body. In the present study we examined accumulations of inorganic mercury and MT inductions in mouse tissues (brain, liver and kidney) up to 72 hr after treatment by one of three mercury compounds of sub-lethal doses. Exposure to mercury compounds caused significant mercury accumulations in mouse tissues examined, except for the Hg(II)-treated mouse brain. Although MeHg caused the highest total mercury accumulation in all tissues among mercury compounds, the rates of inorganic mercury were less than 10% through the experimental period. MT inductions that depended on the inorganic mercury accumulation were observed in kidney and brain. However, MT induction in the liver could not be accounted for by the inorganic mercury accumulation, but by plasma IL6 levels, marked elevation of which was observed in Hg(II) or MeHg-treated mouse. The present study demonstrated that MT was induced in mouse tissues after each of three mercury compounds, Hg(0), Hg(II) and MeHg, but the induction processes were different among tissues. The induction would occur directly through accumulation of inorganic mercury in brain and kidney, whereas the hepatic MT might be induced secondarily through mercury-induced elevation in the plasma cytokines, rather than through mercury accumulation in the tissue.

  3. Distribution of mercury in metallothionein-null mice after exposure to mercury vapor: amount of metallothionein isoform does not affect accumulation of mercury in the brain.

    PubMed

    Yasutake, Akira; Yoshida, Minoru; Honda, Akiko; Watanabe, Chiho; Satoh, Masahiko

    2012-01-01

    To examine the contribution of metallothionein (MT) to mercury accumulation in mouse tissues, 129 strain female mice and MT null mice were exposed to metallic mercury vapor at a sub-toxic level, and Hg levels in the brain, kidney and liver were determined on 1, 3 and 7 days after the exposure. After exposure to mercury vapor, significant Hg accumulation was observed in the brains of wild-type and MT-I/II null and MT-III null mice, as well as in the liver and kidneys. No strain difference was observed in the tissue Hg accumulations 24 hr after the exposure except for the kidneys, where the highest accumulation was found in MT-III null mice. Although the brains of MT-III null mice showed slightly higher Hg accumulation than the other two strains, no significant difference was observed except in the cerebrum on Day 7. Gel chromatograms of cerebrum soluble fractions revealed that a significant amount of Hg existed as an MT-bound form in all the mouse strains. On the other hand, MT-bound Hg was found as a minor fraction in soluble fractions of the kidneys and livers in wild-type and MT-III null mice. Despite a significant strain difference in total MT levels in the cerebrum, there was no difference among the three strains in the amount of Hg accumulated in the cerebrum and its distribution rates in MT fractions. The present study demonstrated that brain uptake of Hg(0) and its accumulation as Hg(2+) did not depend on the amount of MT isoform in the tissue, at least in the early phase.

  4. Tissue expression analysis of FeMT3, a drought and oxidative stress related metallothionein gene from buckwheat (Fagopyrum esculentum).

    PubMed

    Samardzić, Jelena T; Nikolić, Dragana B; Timotijević, Gordana S; Jovanović, Zivko S; Milisavljević, Mira Đ; Maksimović, Vesna R

    2010-11-01

    Metallothionein type 3 (MT3) expression has previously been detected in leaves, fruits, and developing somatic embryos in different plant species. However, specific tissular and cellular localization of MT3 transcripts have remained unidentified. In this study, in situ RNA-RNA analysis revealed buckwheat metallothionein type 3 (FeMT3) transcript localization in vascular elements, mesophyll and guard cells of leaves, vascular tissue of roots and throughout the whole embryo. Changes in FeMT3 mRNA levels in response to drought and oxidative stress, as well as ROS scavenging abilities of the FeMT3 protein in yeast were also detected, indicating possible involvement of FeMT3 in stress defense and ROS related cellular processes.

  5. Metallothionein, oxidative stress and trace metals in gills and liver of demersal and pelagic fish species from Kuwaits' marine area.

    PubMed

    Beg, M U; Al-Jandal, N; Al-Subiai, S; Karam, Q; Husain, S; Butt, S A; Ali, A; Al-Hasan, E; Al-Dufaileej, S; Al-Husaini, M

    2015-11-30

    Two fish species yellowfin seabream (Acanthopagrus latus) and tonguesole (Cynoglossus arel) were collected from two locations in Kuwait's territorial waters in non-reproductive periods and used as bio-indicator organism for the assessment of metals in the marine environment. Species variation in fish was observed; seabream contained high metal content and metallothionein in liver and gill tissues compared to tonguesole, especially from Kuwait Bay area. Oxidative injury was registered in the gills of both species, but in tonguesole liver was also involved. Consequently, antioxidant enzyme catalase was elevated in tonguesole enabling bottom dwelling fish to combat oxidative assault. The study provided information about the current status of metals in marine sediment and levels of metals accumulated in representative species along with oxidative damage in exposed tissues and the range of biomarker protein metallothionein and enzymes of antioxidant defence mechanism enhancing our understanding about the biological response to the existing marine environment in Kuwait.

  6. Accumulated Metals and Metallothionein Expression in Organs of Hares (Lepus europaeus Pallas) Within Natural Gas Fields of Podravina, Croatia.

    PubMed

    Tota, Marin; Jakovac, Hrvoje; Špirić, Zdravko; Srebočan, Emil; Milin, Čedomila

    2015-01-01

    Environmental impact of natural gas facility near Molve (Podravina, Croatia) was assessed using hares (Lepus europaeus Pallas) as biomonitors. Elevated levels of heavy metals in the environment lead to their accumulation in different tissues of hares. The authors have tested accumulation and distribution of several metals in hare liver, kidney, and muscle tissues. The accumulation of copper in hare liver and kidneys with concomitant decrease of zinc was observed in animals from Podravina region as opposed to the control group of animals (Island Krk, Croatia). Secondly, the expression of metallothioneins was assessed because of their crucial role in metal homeostasis. Observed elevation of metallothionein expression in tested organs emphasizes the possible prolonged negative effects of heavy metals in the surroundings as well as a state of oxidative stress in animals. Further monitoring of the area is necessary for better control of hydrocarbon processing to diminish the possible negative environmental effects.

  7. Cloning, characterization, and expression of cadmium-induced metallothionein-2 gene from earthworm Pheretima aspergillum (E. Perrier).

    PubMed

    Gong, L; Li, W; Li, J; Li, W E; Wu, W R; Yu, L W

    2015-12-14

    Metallothioneins (MTs) are ubiquitous metal-binding, cysteine-rich proteins, associated with metal accumulation and thus providing protection against toxic heavy metals such as cadmium (Cd). To investigate the mechanisms of enrichment of Cd in the earthworm Pheretima aspergillum, we isolated and cloned metallothionein-2 (MT-2) cDNA (538 bp) from P. aspergillum, analyzed its sequence, and examined MT-2 transcription levels by relative quantitative real-time PCR under different concentrations of Cd. The sequence of P. aspergillum MT-2 cDNA and its putative amino acid sequence were highly similar to sequences from other earthworms. The induction with Cd increased the MT-2 gene transcription level in a dose-dependent manner. In addition, earthworm recombinant MT-2 exhibited high Cd bioaccumulation ability in vitro. These results suggested that MT-2 plays an important role in tolerance and accumulation of Cd in P. aspergillum.

  8. Differential Expression of Metallothionein Isoforms in Terrestrial Snail Embryos Reflects Early Life Stage Adaptation to Metal Stress

    PubMed Central

    Baurand, Pierre-Emmanuel; Pedrini-Martha, Veronika; de Vaufleury, Annette; Niederwanger, Michael; Capelli, Nicolas; Scheifler, Renaud; Dallinger, Reinhard

    2015-01-01

    The aim of this study was to analyze the expression of three metallothionein (MT) isoform genes (CdMT, CuMT and Cd/CuMT), already known from adults, in the Early Life Stage (ELS) of Cantareus aspersus. This was accomplished by detection of the MT isoform-specific transcription adopting Polymerase Chain Reaction (PCR) amplification and quantitative Real Time (qRT)-PCR of the three MT genes. Freshly laid eggs were kept for 24 hours under control conditions or exposed to three cadmium (Cd) solutions of increasing concentration (5, 10, and 15 mg Cd/L). The transcription of the three MT isoform genes was detected via PCR in 1, 6 and 12-day-old control or Cd-exposed embryos. Moreover, the transcription of this isoform genes during development was followed by qRT-PCR in 6 and 12-day-old embryos. Our results showed that the CdMT and Cd/CuMT genes, but not the CuMT gene, are expressed in embryos at the first day of development. The transcription of the 3 MT genes in control embryos increased with development time, suggesting that the capacities of metal regulation and detoxification may have gradually increased throughout embryogenesis. However in control embryos, the most highly expressed MT gene was that of the Cd/CuMT isoform, whose transcription levels greatly exceeded those of the other two MT genes. This contrasts with the minor significance of this gene in adult snails and suggests that in embryos, this isoform may play a comparatively more important role in metal physiology compared to adult individuals. This function in adult snails appears not to be related to Cd detoxification. Instead, snail embryos responded to Cd exposure by over-expression of the CdMT gene in a concentration-dependent manner, whereas the expression of the Cd/CuMT gene remained unaffected. Moreover, our study demonstrates the ability of snail embryos to respond very early to Cd exposure by up-regulation of the CdMT gene. PMID:25706953

  9. Arabidopsis thaliana—Aphid Interaction

    PubMed Central

    Louis, Joe; Singh, Vijay; Shah, Jyoti

    2012-01-01

    Aphids are important pests of plants that use their stylets to tap into the sieve elements to consume phloem sap. Besides the removal of photosynthates, aphid infestation also alters source-sink patterns. Most aphids also vector viral diseases. In this chapter, we will summarize on recent significant findings in plant-aphid interaction, and how studies involving Arabidopsis thaliana and Myzus persicae (Sülzer), more commonly known as the green peach aphid (GPA), are beginning to provide important insights into the molecular basis of plant defense and susceptibility to aphids. The recent demonstration that expression of dsRNA in Arabidopsis can be used to silence expression of genes in GPA has further expanded the utility of Arabidopsis for evaluating the contribution of the aphid genome-encoded proteins to this interaction. PMID:22666177

  10. A Plasmid Containing the Human Metallothionein II Gene Can Function as an Antibody-assisted Electrophoretic Biosensor for Heavy Metals

    DTIC Science & Technology

    2015-01-16

    function as an antibody-assisted electrophoretic biosensor for heavy metals 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER...biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human...ARTICLE A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals Dennis C

  11. Copper-metallothioneins in the American lobster, Homarus americanus: potential role as Cu(I) donors to apohemocyanin

    SciTech Connect

    Brouwer, M.; Whaling, P.; Engel, D.W.

    1986-03-01

    The physiological function of copper(I)-metallothionein is not well understood. The respiratory function of hemocyanin, a copper(I)-containing respiratory protein found in the hemolymph of many invertebrates, has been known a long time. However, the mechanism by which Cu(I) is inserted into the oxygen-binding site of apohemocyanin is completely unknown. This investigation tests that hypothesis that copper(I)-metallothionein may act as a Cu(I) donor to apohemocyanin. To this end, copper-binding proteins and hemocyanin were purified from the digestive gland and hemolymph of the American lobster, Homarus americanus. In the presence of ..beta..-mercaptoethanol, the copper-binding proteins can be resolved into three components of DEAE-cellulose. The first two have been characterized as metallothioneins. The cysteine content of the third component is half of that of components I and II. The purified proteins are not capable of transferring Cu(I) to the active sites of completely copper-free apohemocyanin. They are capable, however, of transferring Cu(I) to active sites of hemocyanin containing reduced amounts of Cu(I), suggesting that the conformational state of hemocyanin is the determining factor in the Cu(I) transfer mechanism.

  12. Expression of metallothionein and α-tubulin in heavy metal-tolerant Anopheles gambiae sensu stricto (Diptera: Culicidae)

    PubMed Central

    Mireji, Paul O.; Keating, Joseph; Hassanali, Ahmed; Impoinvil, Daniel E.; Mbogo, Charles M.; Njeri, Martha; Nyambaka, Hudson; Kenya, Eucharia; Githure, John I; Beier, John C.

    2009-01-01

    Anopheles mosquitoes have been shown to adapt to heavy metals in their natural habitats. In this study we explored the possibility of using Anopheles gambiae sensu stricto as bio-reporters for environmental heavy metal pollution through expressions of their metal responsive metallothionein and α-tubulin genes. The study was undertaken with third instar larvae after selection by cadmium, copper, or lead at LC30 through five successive generations. Expression levels were determined in the fifth generation by semi quantitative RT-PCR on the experimental and control populations. The data were analyzed using one-way ANOVA. The highest metallothionein (F3, 11= 4.574, P = 0.038) and α-tubulin (F3,11= 12.961, P = 0.002) responses were observed in cadmium-tolerant treatments. There was significantly higher expression of metallothionein in cadmium or copper treatments relative to the control (P = 0.012), and in cadmium than in lead treatments (P = 0.044). Expressions of α-tubulin were significantly higher in cadmium than in control treatments (P = 0.008). These results demonstrate capacity of An. gambiae s.s. to develop tolerance to increased levels of heavy metal challenge. The results also confirm the potential of heavy metal responsive genes in mosquitoes as possible bio-indicators of heavy metal environmental pollution. How the tolerance and expressions relate to An. gambiae s.s. fitness and vectorial capacity in the environment remains to be elucidated. PMID:19735939

  13. Phenotypic consequences of aneuploidy in Arabidopsis thaliana.

    PubMed

    Henry, Isabelle M; Dilkes, Brian P; Miller, Eric S; Burkart-Waco, Diana; Comai, Luca

    2010-12-01

    Aneuploid cells are characterized by incomplete chromosome sets. The resulting imbalance in gene dosage has phenotypic consequences that are specific to each karyotype. Even in the case of Down syndrome, the most viable and studied form of human aneuploidy, the mechanisms underlying the connected phenotypes remain mostly unclear. Because of their tolerance to aneuploidy, plants provide a powerful system for a genome-wide investigation of aneuploid syndromes, an approach that is not feasible in animal systems. Indeed, in many plant species, populations of aneuploid individuals can be easily obtained from triploid individuals. We phenotyped a population of Arabidopsis thaliana aneuploid individuals containing 25 different karyotypes. Even in this highly heterogeneous population, we demonstrate that certain traits are strongly associated with the dosage of specific chromosome types and that chromosomal effects can be additive. Further, we identified subtle developmental phenotypes expressed in the diploid progeny of aneuploid parent(s) but not in euploid controls from diploid lineages. These results indicate long-term phenotypic consequences of aneuploidy that can persist after chromosomal balance has been restored. We verified the diploid nature of these individuals by whole-genome sequencing and discuss the possibility that trans-generational phenotypic effects stem from epigenetic modifications passed from aneuploid parents to their diploid progeny.

  14. Co-exposure to aluminum and acrylamide disturbs expression of metallothionein, proinflammatory cytokines and induces genotoxicity: Biochemical and histopathological changes in the kidney of adult rats.

    PubMed

    Ghorbel, Imen; Maktouf, Sameh; Fendri, Nesrine; Jamoussi, Kamel; Ellouze Chaabouni, Semia; Boudawara, Tahia; Zeghal, Najiba

    2016-09-01

    The individual toxic effects of aluminum and acrylamide are known but there is no data on their combined effects. The present study investigates the toxic effects after combined exposure to these toxicants on: (i) oxidative stress during combined chronic exposure to aluminum and acrylamide on kidney function (ii) correlation of oxidative stress with metallothionein (MT) and inflammatory cytokines expression, DNA damage, and histopathological changes. Rats were exposed to aluminum (50 mg/kg body weight) in drinking water and acrylamide (20 mg/kg body weight) by gavage either individually or in combination for 3 weeks. Exposure rats to aluminum chloride or acrylamide alone and in combination induced nephrotoxicity, as evidenced by a decrease in the 24-h urine volume and uric acid levels in plasma and an increase of plasma creatinine, urea, and blood urea nitrogen levels. Nephrotoxicity was objectified by a significant increase in malondialdehyde level, advanced oxidation protein, and protein carbonyl contents, whereas reduced glutathione, nonprotein thiol, vitamin C levels, catalase, and glutathione peroxidase activities showed a significant decline. Superoxide dismutase activity and its gene expression were increased. Aluminum and acrylamide co-exposure exhibited synergism in various biochemical variables and also in DNA damage. Kidney total MT levels and genes expression of MT1, MT2, and proinflammatory cytokines were increased. All these changes were supported by histopathological observations. Co-exposure to aluminum and acrylamide exhibited synergism and more pronounced toxic effects compared with their individual effects based on various biochemical variables, genotoxic, and histopathological changes. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1044-1058, 2016.

  15. Handling Arabidopsis plants: growth, preservation of seeds, transformation, and genetic crosses.

    PubMed

    Rivero, Luz; Scholl, Randy; Holomuzki, Nicholas; Crist, Deborah; Grotewold, Erich; Brkljacic, Jelena

    2014-01-01

    Growing healthy plants is essential for the advancement of Arabidopsis thaliana (Arabidopsis) research. Over the last 20 years, the Arabidopsis Biological Resource Center (ABRC) has collected and developed a series of best-practice protocols, some of which are presented in this chapter. Arabidopsis can be grown in a variety of locations, growth media, and environmental conditions. Most laboratory accessions and their mutant or transgenic derivatives flower after 4-5 weeks and set seeds after 7-8 weeks, under standard growth conditions (soil, long day, 23 ºC). Some mutant genotypes, natural accessions, and Arabidopsis relatives require strict control of growth conditions best provided by growth rooms, chambers, or incubators. Other lines can be grown in less-controlled greenhouse settings. Although the majority of lines can be grown in soil, certain experimental purposes require utilization of sterile solid or liquid growth media. These include the selection of primary transformants, identification of homozygous lethal individuals in a segregating population, or bulking of a large amount of plant material. The importance of controlling, observing, and recording growth conditions is emphasized and appropriate equipment required to perform monitoring of these conditions is listed. Proper conditions for seed harvesting and preservation, as well as seed quality control, are also described. Plant transformation and genetic crosses, two of the methods that revolutionized Arabidopsis genetics, are introduced as well.

  16. Induction of hepatic metallothioneins determined at isoprotein and messenger RNA levels in glucocorticoid-treated rats.

    PubMed Central

    Lehman-McKeeman, L D; Andrews, G K; Klaassen, C D

    1988-01-01

    Induction of metallothionein-I (MT-I) and metallothionein-II (MT-II) by glucocorticoids was determined by h.p.l.c. analysis of proteins and Northern-blot analysis of MT mRNAs. Rats were injected with dexamethasone (0.03-10 mumol/kg) and hepatic concentrations of MTs were determined 24 h later. In control rats, only MT-II was detected (9.4 +/- 2.5 micrograms/g of liver), whereas the hepatic concentration of MT-I was below the detection limit (5 micrograms of MT/g). Dexamethasone did not increase MT-I above the detection limit at any dosage tested, but MT-II increased to 2.5 times control values at dosages of 0.30 mumol/kg and higher. Time-course experiments indicated that MT-II reached a maximum at 24 h after a single dosage of dexamethasone and returned to control values by 48 h. To determine whether dexamethasone increased MT-I in liver, samples were saturated with 109Cd, after which the amount of 109Cd in MT-I and MT-II was determined. Results indicated that, by this approach, MT-I and MT-II could be detected in control rats, and there was approx. 1.8 times more 109Cd in MT-II than in MT-I. At 24 h after administration of dexamethasone (1 mumol/kg), there was a small increase in the amount of 109Cd bound to MT-I, whereas the amount of 109Cd bound to MT-II increased to more than 2 times control values. Northern-blot hybridization with mouse cRNA probes indicated that MT-I and MT-II mRNAs increased co-ordinately after administration of dexamethasone. Thus, although glucocorticoids increase both MT-I and MT-II mRNAs, MT-II preferentially accumulates after administration of dexamethasone. Images Fig. 4. PMID:3342021

  17. Metallothionein as a compensatory component prevents intermittent hypoxia-induced cardiomyopathy in mice

    SciTech Connect

    Yin, Xia; Zhou, Shanshan; Zheng, Yang; Tan, Yi; Kong, Maiying; Wang, Bo; Feng, Wenke; Epstein, Paul N.; Cai, Jun; Cai, Lu

    2014-05-15

    Obstructive sleep apnea (OSA) causes chronic intermittent hypoxia (IH) to induce cardiovascular disease, which may be related to oxidative damage. Metallothionein (MT) has been extensively proved to be an endogenous and highly inducible antioxidant protein expressed in the heart. Therefore, we tested the hypotheses that oxidative stress plays a critical role in OSA induced cardiac damage and MT protects the heart from OSA-induced cardiomyopathy. To mimic hypoxia/reoxygenation events that occur in adult OSA patients, mice were exposed to IH for 3 days to 8 weeks. The IH paradigm consisted of alternating cycles of 20.9% O{sub 2}/8% O{sub 2} F{sub I}O{sub 2} (30 episodes per hour) with 20 s at the nadir F{sub I}O{sub 2} for 12 h a day during daylight. IH significantly increased the ratio of heart weight to tibia length at 4 weeks with a decrease in cardiac function from 4 to 8 weeks. Cardiac oxidative damage and fibrosis were observed after 4 and 8 weeks of IH exposures. Endogenous MT expression was up-regulated in response to 3-day IH, but significantly decreased at 4 and 8 weeks of IH. In support of MT as a major compensatory component, mice with cardiac overexpression of MT gene and mice with global MT gene deletion were completely resistant, and highly sensitive, respectively, to chronic IH induced cardiac effects. These findings suggest that chronic IH induces cardiomyopathy characterized by oxidative stress-mediated cardiac damage and the antioxidant MT protects the heart from such pathological and functional changes. - Highlights: • The effect of intermittent hypoxia (IH) on cardiac metallothionein (MT) • Cardiac MT expression was up-regulated in response to 3-day IH. • Exposure to 4- or 8-week IH downregulated cardiac MT expression. • Overexpression of cardiac MT protects from IH-induced cardiac damage. • Global deletion of MT gene made the heart more sensitive to IH damage.

  18. The salty tale of Arabidopsis.

    PubMed

    Sanders, D

    2000-06-29

    High concentrations of sodium chloride are toxic to most plant species. New insights into the mechanisms by which plants tolerate salt have emerged from the identification of genes in Arabidopsis thaliana that play a critical part in physiological resistance to salt.

  19. Araport: the Arabidopsis Information Portal

    PubMed Central

    Krishnakumar, Vivek; Hanlon, Matthew R.; Contrino, Sergio; Ferlanti, Erik S.; Karamycheva, Svetlana; Kim, Maria; Rosen, Benjamin D.; Cheng, Chia-Yi; Moreira, Walter; Mock, Stephen A.; Stubbs, Joseph; Sullivan, Julie M.; Krampis, Konstantinos; Miller, Jason R.; Micklem, Gos; Vaughn, Matthew; Town, Christopher D.

    2015-01-01

    The Arabidopsis Information Portal (https://www.araport.org) is a new online resource for plant biology research. It houses the Arabidopsis thaliana genome sequence and associated annotation. It was conceived as a framework that allows the research community to develop and release ‘modules’ that integrate, analyze and visualize Arabidopsis data that may reside at remote sites. The current implementation provides an indexed database of core genomic information. These data are made available through feature-rich web applications that provide search, data mining, and genome browser functionality, and also by bulk download and web services. Araport uses software from the InterMine and JBrowse projects to expose curated data from TAIR, GO, BAR, EBI, UniProt, PubMed and EPIC CoGe. The site also hosts ‘science apps,’ developed as prototypes for community modules that use dynamic web pages to present data obtained on-demand from third-party servers via RESTful web services. Designed for sustainability, the Arabidopsis Information Portal strategy exploits existing scientific computing infrastructure, adopts a practical mixture of data integration technologies and encourages collaborative enhancement of the resource by its user community. PMID:25414324

  20. Role of ARF6 in internalization of metal-binding proteins, metallothionein and transferrin, and cadmium-metallothionein toxicity in kidney proximal tubule cells

    SciTech Connect

    Wolff, Natascha A.; Lee, Wing-Kee; Abouhamed, Marouan

    2008-07-01

    Filtered metal-protein complexes, such as cadmium-metallothionein-1 (CdMT-1) or transferrin (Tf) are apically endocytosed partly via megalin/cubilin by kidney proximal tubule (PT) cells where CdMT-1 internalization causes apoptosis. Small GTPase ARF (ADP-ribosylation factor) proteins regulate endocytosis and vesicular trafficking. We investigated roles of ARF6, which has been shown to be involved in internalization of ligands and endocytic trafficking in PT cells, following MT-1/CdMT-1 and Tf uptake by PT cells. WKPT-0293 Cl.2 cells derived from rat PT S1 segment were transfected with hemagglutinin-tagged wild-type (ARF6-WT) or dominant negative (ARF6-T27N) forms of ARF6. Using immunofluorescence, endogenous ARF6 was associated with the plasma membrane (PM) as well as juxtanuclear and co-localized with Rab5a and Rab11 involved in early and recycling endosomal trafficking. Immunofluorescence staining of megalin showed reduced surface labelling in ARF6 dominant negative (ARF6-DN) cells. Intracellular Alexa Fluor 546-conjugated MT-1 uptake was reduced in ARF6-DN cells and CdMT-1 (14.8 {mu}M for 24 h) toxicity was significantly attenuated from 27.3 {+-} 3.9% in ARF6-WT to 11.1 {+-} 4.0% in ARF6-DN cells (n = 6, P < 0.02). Moreover, reduced Alexa Fluor 546-conjugated Tf uptake was observed in ARF-DN cells (75.0 {+-} 4.6% versus 3.9 {+-} 3.9% of ARF6-WT cells, n = 3, P < 0.01) and/or remained near the PM (89.3 {+-} 5. 6% versus 45.2 {+-} 14.3% of ARF6-WT cells, n = 3, P < 0.05). In conclusion, the data support roles for ARF6 in receptor-mediated endocytosis and trafficking of MT-1/Tf to endosomes/lysosomes and CdMT-1 toxicity of PT cells.

  1. Functional overlap of the Arabidopsis leaf and root microbiota.

    PubMed

    Bai, Yang; Müller, Daniel B; Srinivas, Girish; Garrido-Oter, Ruben; Potthoff, Eva; Rott, Matthias; Dombrowski, Nina; Münch, Philipp C; Spaepen, Stijn; Remus-Emsermann, Mitja; Hüttel, Bruno; McHardy, Alice C; Vorholt, Julia A; Schulze-Lefert, Paul

    2015-12-17

    Roots and leaves of healthy plants host taxonomically structured bacterial assemblies, and members of these communities contribute to plant growth and health. We established Arabidopsis leaf- and root-derived microbiota culture collections representing the majority of bacterial species that are reproducibly detectable by culture-independent community sequencing. We found an extensive taxonomic overlap between the leaf and root microbiota. Genome drafts of 400 isolates revealed a large overlap of genome-encoded functional capabilities between leaf- and root-derived bacteria with few significant differences at the level of individual functional categories. Using defined bacterial communities and a gnotobiotic Arabidopsis plant system we show that the isolates form assemblies resembling natural microbiota on their cognate host organs, but are also capable of ectopic leaf or root colonization. While this raises the possibility of reciprocal relocation between root and leaf microbiota members, genome information and recolonization experiments also provide evidence for microbiota specialization to their respective niche.

  2. A reference map of the Arabidopsis thaliana mature pollen proteome

    SciTech Connect

    Noir, Sandra; Braeutigam, Anne; Colby, Thomas; Schmidt, Juergen; Panstruga, Ralph . E-mail: panstrug@mpiz-koeln.mpg.de

    2005-12-02

    The male gametophyte (or pollen) plays an obligatory role during sexual reproduction of higher plants. The extremely reduced complexity of this organ renders pollen a valuable experimental system for studying fundamental aspects of plant biology such as cell fate determination, cell-cell interactions, cell polarity, and tip-growth. Here, we present the first reference map of the mature pollen proteome of the dicotyledonous model plant species, Arabidopsis thaliana. Based on two-dimensional gel electrophoresis, matrix-assisted laser desorption/ionization time-of-flight, and electrospray quadrupole time-of-flight mass spectrometry, we reproducibly identified 121 different proteins in 145 individual spots. The presence, subcellular localization, and functional classification of the identified proteins are discussed in relation to the pollen transcriptome and the full protein complement encoded by the nuclear Arabidopsis genome.

  3. Influence of Cadmium(II) Ions and Brewery Sludge on Metallothionein Level in Earthworms (Eisenia fetida) – Bio-transforming of Toxic Wastes

    PubMed Central

    Huska, Dalibor; Krizkova, Sona; Beklova, Miroslava; Havel, Ladislav; Zehnalek, Josef; Diopan, Vaclav; Adam, Vojtech; Zeman, Ladislav; Babula, Petr; Kizek, Rene

    2008-01-01

    Metallothioneins belong to a group of intracellular, high molecular and cysteine-rich proteins whose content in an organism increase with increasing concentration of a heavy metal. The aim of this work was to apply the electrochemical analysis for the analysis of metallothioneins in earthworms exposed to cadmium ions and brewery sludge. Here we utilized adsorptive transfer technique coupled with differential pulse voltammetry Brdicka reaction to determine metallothionein in different biological samples. By means this very sensitive technique it was possible to analyze metallothionein in concentrations below 1 μmol.l−1 with the standard deviation of 4-5%. We found out that the average MT level in the non-treated earthworms oscillated between 19 and 48 μmol.l−1. When we analysed samples of earthworms treated by cadmium, we observed that the MT content increased with the exposition length and increase dose of cadmium ions. Finally, we attempted to study and compare the toxicity of the raw sludge and its leach by using of earthworms. The raw brewery sludge caused the death of the earthworms quickly. Earthworms held in the presence of leach from brewery sludge increased their weight of 147 % of their original weight because they ingested the nutrients from the sludge. The metallothionein level changes markedly with increasing time of exposition and applied dose of toxic compound. It clearly follows from the obtained results that the MT synthesis is insufficient in the first hours of the exposition and increases after more than 24 h. PMID:27879751

  4. Localization and Spectroscopic Analysis of the Cu(I) Binding Site in Wheat Metallothionein Ec-1

    PubMed Central

    Tarasava, Katsiaryna; Loebus, Jens; Freisinger, Eva

    2016-01-01

    The early cysteine-labeled metallothionein (MT) from Triticum aestivum (common wheat), denoted Ec-1, features two structurally well-defined domains, γ and βE, coordinating two and four Zn(II) ions, respectively. While the protein is currently assumed to function mainly in zinc homeostasis, a low amount of copper ions was also recently detected in a native Ec-1 sample. To evaluate the observed copper binding in more detail, the recombinant Zn6Ec-1 form was exposed to different amounts of Cu(I) ions and the resulting species characterized with spectroscopic methods. Data reveal that the first Cu(I) equivalent coordinates exclusively to the N-terminal γ-domain of the protein and replaces one Zn(II) ion. To analyze the ability of the γ-domain for coordination of monovalent metal ions in more detail, the γ-Ec-1 peptide fragment was incubated with increasing amounts of Cu(I) and the process monitored with UV–VIS, circular dichroism, and luminescence spectroscopy. Closely similar spectra are observed regardless if the apo- or the metal ion-loaded and, hence, pre-folded forms, were used for the titration experiments with Cu(I). The results indicate that low amounts of Cu(I) ions displace the two metal ions subsequently and stoichiometrically, despite the different coordination geometry requirements of Cu(I) and Zn(II). PMID:26978358

  5. Cloning metallothionein gene in Zacco platypus and its potential as an exposure biomarker against cadmium.

    PubMed

    Lee, Sangwoo; Kim, Cheolmin; Kim, Jungkon; Kim, Woo-Keun; Shin, Hyun Suk; Lim, Eun-Suk; Lee, Jin Wuk; Kim, Sunmi; Kim, Ki-Tae; Lee, Sung-Kyu; Choi, Cheol Young; Choi, Kyungho

    2015-07-01

    Zacco platypus, pale chub, is an indigenous freshwater fish of East Asia including Korea and has many useful characteristics as indicator species for water pollution. While utility of Z. platypus as an experimental species has been recognized, genetic-level information is very limited and warrants extensive research. Metallothionein (MT) is widely used and well-known biomarker for heavy metal exposure in many experimental species. In the present study, we cloned MT in Z. platypus and evaluated its utility as a biomarker for metal exposure. For this purpose, we sequenced complete complementary DNA (cDNA) of MT in Z. platypus and carried out phylogenetic analysis with its sequences. The transcription-level responses of MT gene following the exposure to CdCl2 were also assessed to validate the utility of this gene as an exposure biomarker. Analysis of cDNA sequence of MT gene demonstrated high conformity with those of other fish. MT messenger RNA (mRNA) expression and enzymatic MT content significantly increased following CdCl2 exposure in a concentration-dependent manner. The level of CdCl2 that resulted in significant MT changes in Z. platypus was within the range that was reported from other fish. The MT gene of Z. platypus sequenced in the present study can be used as a useful biomarker for heavy metal exposure in the aquatic environment of Korea and other countries where this freshwater fish species represents the ecosystem.

  6. Heavy metal pollution in sediments and mussels: assessment by using pollution indices and metallothionein levels.

    PubMed

    Okay, Oya S; Ozmen, Murat; Güngördü, Abbas; Yılmaz, Atilla; Yakan, Sevil D; Karacık, Burak; Tutak, Bilge; Schramm, Karl-Werner

    2016-06-01

    In the present work, the concentration of eight metals (Cd, Cr, Cu, Fe, Mn, Ni, Pb, Zn) was determined in the sediments and transplanted and native mussels (Mytilus galloprovincialis). The study was conducted in Turkish marinas, shipyards, and shipbreaking yards. The effect of metal pollution was evaluated by determining the levels of metallothionein (MT) in the mussels. The extent of contamination for each single metal was assessed by using the geoaccumulation index (I geo) and enrichment factor (EF). Whereas, to evaluate the overall metal pollution and effect, the pollution load index (PLI), modified contamination degree (mC d), potential toxicity response index (RI), mean effects range median (ERM) quotient (m-ERM-Q), and mean PEL quotient (m-PEL-Q) were calculated. The influence of different background values on the calculations was discussed. The results indicated a significant metal pollution caused by Cu, Pb, and Zn especially in shipyard and shipbreaking sites. Higher concentrations of MT were observed in the ship/breaking yard samples after the transplantation.

  7. The cytotoxicity of mercury chloride to the keratinocytes is associated with metallothionein expression.

    PubMed

    Hwang, Tsann-Long; Chen, Hsiao-Ying; Changchien, Tzu-Tsung; Wang, Chee-Chan; Wu, Chi-Ming

    2013-05-01

    There are trace amounts of heavy metals in cosmetics. Heavy metals such as mercury (Hg), which is added to skin-whitening cosmetics, may cause acute or chronic damage to human cells. The aim of this study was to investigate the cytotoxicity of mercury chloride (HgCl2) to human keratinocytes. The keratinocytes were treated with various concentrations of HgCl2 and the cell survival fractions were found to be 38.08, 17.59, 12.76, 3.29 and 0.77% when the cells were treated with 0.25, 0.5, 0.75, 1 and 1.5 μM of HgCl2, respectively. Moreover, we observed that the greatest damage was to the cell membrane. The metallothionein (MT) protein expression was also investigated. MT expression levels increased with increasing concentrations of HgCl2. The results indicated that MT protects the keratinocytes against HgCl2-induced toxicity.

  8. Purification and characterization of metallothionein-like cadmium binding protein from Asian periwinkle Littorina brevicula.

    PubMed

    Park, Jin-Sung; Chung, Soohee; Park, Il-Seon; Kim, Yangsun; Koh, Chul- Hwan; Lee, In-Sook

    2002-04-01

    Although mussels and oysters in the ocean are known to act as bioconcentrators for contaminants such as heavy metals, their ability to survive in heavily polluted water is relatively limited. The Asian periwinkle, Littorina brevicula, is one species that can accumulate a variety of environmental heavy metals, and the expression of its metal binding protein (MBP) is induced by cadmium. To better characterize this protein and its detoxification mechanism against cadmium, the present work examined the induction of a cadmium binding protein (Cd-BP) in Littorina brevicula exposed to 400 microg/l CdCl(2) for 30 days. The induced Cd-BP was purified by chromatography from the supernatants of homogenized organs (digestive gland, gonad, gill and kidney). This Cd-BP was found to consist of 103 amino acids, was rich in Cys (21 residues), and partial C-terminal sequence obtained by MALDI-TOF MS analysis revealed a Cys-XXX-Cys motif, which resembles a typical feature of mollusc metallothionein (MT). The Cd-BP molecular weight of 9.8 kDa is a little larger than that of other MTs.

  9. Metallothionein-like multinuclear clusters of mercury(II) and sulfur in peat

    USGS Publications Warehouse

    Nagy, K.L.; Manceau, A.; Gasper, J.D.; Ryan, J.N.; Aiken, G.R.

    2011-01-01

    Strong mercury(II)-sulfur (Hg-SR) bonds in natural organic matter, which influence mercury bioavailability, are difficult to characterize. We report evidence for two new Hg-SR structures using X-ray absorption spectroscopy in peats from the Florida Everglades with added Hg. The first, observed at a mole ratio of organic reduced S to Hg (Sred/Hg) between 220 and 1140, is a Hg4Sx type of cluster with each Hg atom bonded to two S atoms at 2.34 ?? and one S at 2.53 ??, and all Hg atoms 4.12 ?? apart. This model structure matches those of metal-thiolate clusters in metallothioneins, but not those of HgS minerals. The second, with one S atom at 2.34 ?? and about six C atoms at 2.97 to 3.28 ??, occurred at S red/Hg between 0.80 and 4.3 and suggests Hg binding to a thiolated aromatic unit. The multinuclear Hg cluster indicates a strong binding environment to cysteinyl sulfur that might impede methylation. Along with a linear Hg(SR)2 unit with Hg - S bond lengths of 2.34 ?? at Sred/Hg of about 10 to 20, the new structures support a continuum in Hg-SR binding strength in natural organic matter. ?? 2011 American Chemical Society.

  10. Effects of chitosan on oxidative stress and metallothioneins in aquatic worm Tubifex tubifex (Oligochaeta, Tubificidae).

    PubMed

    Mosleh, Yahia Y; Paris-Palacios, Séverine; Ahmed, Mohamed T; Mahmoud, F M; Osman, M A; Biagianti-Risbourg, Sylvie

    2007-02-01

    Chitosan is a natural polymer which has the property to elicit the natural defenses mechanism in plant and which can be an interesting biopesticides. It is then necessary to investigate the potential toxicity of chitosan for aquatic animal health. Metallothioneins (MTs) are low molecular weight proteins, mainly implicated in metal ion detoxification. Increase in MTs contents had been considered as a specific biomarker of metal exposure. However recently it has been demonstrated that MTs participate in several cellular functions such as regulation of growth and anti-oxidative defenses. Therefore, the induction of MTs has been investigated in the aquatic worms Tubifex tubifex exposed to chitosan. MTs levels in exposed worm increased significantly (p > 0.05) after 2, 4, and 7 days of exposure to different concentrations of chitosan (maximum + 158.19 +/- 10.2% after 2 days of exposure to 125 mgl(-1) of chitosan). Several antioxidant parameters including glutathione (GSH), glutathione-S-transferase (GST), glutathione reductase (GR), and catalase (CAT) were quantified in T. tubifex after 2, 4, and 7 days of exposure to chitosan. Exposure to chitosan had a negative effect on T. tubifex growth (maximum effect -6.11 +/- 1.6% after 7 days with 125 mgl(-1)) demonstrating the toxic effect of the pesticide. This growth rate decrease was accompanied by a reduction in protein contents. The activity of catalase (CAT), glutathione-S-transferase (GST), and glutathione reductase (GR) increased in response to the chitosan demonstrating an oxidative stress in the worms.

  11. Molecular characterization and expression of metallothionein from freshwater pearl mussel, Hyriopsis schlegelii.

    PubMed

    Wang, Chengyuan; Sheng, Junqing; Hong, Yijiang; Peng, Kou; Wang, Junhua; Wu, Di; Shi, Jianwu; Hu, Beijuan

    2016-07-01

    Two metallothionein genes (HsMT1 and HsMT2) were first identified and described from Hyriopsis schlegelii. The open reading frame of HsMT1 and HsMT2 were 216 and 222 bp, encoding a protein of 71 and 73 amino acid residues. The deduced amino acid sequences showed they contained parts of typical MT characteristics, apart from HsMT2 lacked Cys-Cys motifs. The phylogenetic tree showed HsMT1 shared a high similarity with that of other molluscs, but HsMT2 was split into a distinct group separated from known molluscan MTs. HsMT1 exhibited constitutive expression in all examined tissues and the highest expression occurred in hepatopancreas, however, nearly all HsMT2 was just detected in gonad. After Cd exposure, their mRNA levels presented similar expression patterns. The transgenic bacteria of HsMT1 showed higher tolerance than HsMT2 in Cd environment. It was implied that HsMT1 and HsMT2 were involved in metal response but HsMT2 might have other physiological functions.

  12. Localization of metallothionein in the genital organs of the male rat.

    PubMed

    Nishimura, H; Nishimura, N; Tohyama, C

    1990-07-01

    We studied the immunohistological localization of metallothionein (MT), a low molecular weight metal binding protein, in male rat genital organs (testis, epididymis, ejaculatory duct, seminal vesicle, coagulating gland, and prostate) by use of the avidin-biotin-peroxidase complex method. MT concentrations in testis, seminal vesicle, and prostate ranged from 15-30 micrograms/g tissue. In testis, seminiferous tubules with mature spermatozoa exhibited weak MT staining, whereas the tubules containing differentiating spermatogenic cells but not containing spermatozoa showed strong MT staining. No MT immunostaining was observed in Leydig cells. In growing rat testes, the pattern of MT immunostaining was found to change with development: MT was found in supporting cells only on Day 7, spermatogonia adjacent to basement membrane on Day 14, and spermatocytes localized in the central part of the tubules on Day 21. Strong MT immunostaining in the basal cells was a common feature in other genital tissues, except the ductus efferentes. In prostate, the strongest MT staining was found in the lateral lobe, and MT was localized in apocrine secretions in the dorsal lobe. The present results suggest a close association of MT with cell proliferation and differentiation, as well as possible involvement of MT in supply or storage of zinc ions.

  13. Induction of metallothionein synthesis with preservation of testicular function in rats following long term renal transplantation.

    PubMed

    Cai, L; Deng, D X; Jiang, J; Chen, S; Zhong, R; Cherian, M G; Chakrabarti, S

    2000-04-01

    Metallothionein (MT), as an acute phase or stress-response protein and free radical scavenger, is related to inflammation and cellular protection from oxidative damage. In order to evaluate long-term testicular damage and the role of MT following renal transplant, nine allogenic (Fisher 344 --> Lewis) and seven isogenic (Lewis --> Lewis) renal transplants were performed and the recipient rats were followed for 140 days when allografts develop chronic transplant rejection. Testicular weight, light microscopic morphology, and lactate dehydrogenase-X enzyme activity were assessed. Testicular MT was determined by Cd-heme assay, and was localized immunocytochemically using a polyclonal rabbit antibody. No differences in testis weight, morphology, or LDH-X enzyme activity were found between allograft and isograft recipients. Testicular MT level was significantly increased in the testis of allograft recipients. Testicular zinc (Zn) and copper (Cu) levels, but not iron (Fe) level, were significantly higher in testis with allograft kidney than that with isograft kidney. In addition, Cu/Zn ratio was also significantly high in the allograft group. However, the MT level did not show any significant correlation either with Cu and Zn alone or with Cu/Zn and Fe/Zn ratios. These data suggest that allogenic stimuli may induce MT synthesis in the recipient testis. The increased MT level in an allograft may offer a protective action from oxidative damage in the testis.

  14. Cell specificity and an effect of ras on human metallothionein gene expression.

    PubMed Central

    Schmidt, C J; Hamer, D H

    1986-01-01

    The expression of three human metallothionein (hMT) genes has been compared in various established cell lines and primary liver. The single gene for hMT isoform II is ubiquitously expressed in all cell types in response to cadmium. In contrast, two genes encoding hMT-I isoforms are expressed in a highly specific, reciprocal fashion that correlates with the embryonic germ layer origin of the cells. In one cell line that failed to express detectable amounts of hMT-IE, treatment with the demethylating agent 5-azacytidine led to cadmium-inducible expression of this subtype. The genes for both MT-I isoforms are coordinately inducible by heavy metals but differ in their response to glucocorticoids. Surprisingly, cells transformed with the Ha-ras oncogene contain elevated basal levels of both MT-I and MT-II RNA. The implications of these results for growth-related and developmental functions of MT are discussed. Images PMID:3517857

  15. Resonance light scattering determination of metallothioneins using levofloxacin-palladium complex as a light scattering probe

    NASA Astrophysics Data System (ADS)

    Xue, Jin-Hua; Qian, Qiu-Mei; Wang, Yong-Sheng; Meng, Xia-Ling; Liu, Lu

    2013-02-01

    A novel method of resonance light scattering (RLS) was developed for the analysis of trace metallothioneins (MTs) in human urine. In a CH3COOH-CH3COONa buffer solution of pH 4.5, the formation of a complex between levofloxacin (LEV)-Pd and MTs led to enhance the RLS intensity of the system, and the enhanced RLS intensity at 468 nm was proportional to the concentration of MTs in the range of 0.059-22.4 μg mL-1. The linear regression equation was ΔI = 127.5 ρ (μg mL-1)-88.02 with a correlation coefficient of 0.9992, and the detection limit of 17.8 ng mL-1. The relative standard deviation and the average recovery were 3.8-5.4% (n = 11) and 92.15%, respectively. The proposed method is convenient, reliable and sensitive, and has been used successfully for the determination of trace MTs in human urine samples.

  16. Isolation and characterization of metallothioneins in calves ingesting Zn toxic diets

    SciTech Connect

    Graham, T.W.; Clegg, M.S.; Lonnerdal, B.; Thurmond, M.C.; Keen, C.L.

    1986-03-05

    The authors have recently described an outbreak of Zn toxicosis in 95 Holstein bull calves which occurred as a result of an accidental over-supplementation of the diet with Zn. Signs of Zn toxicosis including anorexia, polydipsia, polyphagia, polyurea and diarrhea, began to appear 23 days after initiation of the diet. Liver, kidney, muscle and brain were collected from animals that died or were euthanized. Multielement analysis indicated that liver had the highest concentration of Zn (362 ..mu..g Zn/g wet wt.) followed by kidney (233 ..mu..g Zn/g wet wt.), muscle (22 ..mu..g Zn/g wet wt.) and brain (10 ..mu..g Zn/g wet wt.). To examine the toxic effects of Zn at the molecular level, liver was fractionated by conventional molecular sieve (Sephadex G75) and anion exchange chromatography (DEAE Sephadex A-25). In addition, Fast Protein Liquid Chromatography (FPLC) was used to verify the results obtained by conventional methods. The principle advantages of FPLC are that the molecular sieve (Superose 12) and anion (Mono Q) steps are performed in 1 h as opposed to several hours, thus substantially minimizing artifacts resulting from oxidative degradation of labile proteins. Zn was primarily associated with two peaks, designated metallothionein (MT) I and MT II, in a ratio of 10 to 1. The results show that one effect of Zn toxicity in calves can be a preferential induction of MT I.

  17. Metals and metallothioneins in Morelet's crocodile (Crocodylus moreletii) from a transboundary river between Mexico and Belize.

    PubMed

    Buenfil-Rojas, A M; Álvarez-Legorreta, T; Cedeño-Vázquez, J R

    2015-02-01

    The aim of this study was to determine concentrations of heavy metals (cadmium [Cd] and mercury [Hg]) and metallothioneins (MTs) in blood plasma and caudal scutes of Morelet's crocodile (Crocodylus moreletii) from Rio Hondo, a river and natural border between Mexico and Belize. Three transects of the river (approximately 20 km each) were surveyed in September 2012 and April 2013, and samples were collected from 24 crocodiles from these areas. In blood plasma, Cd (7.6 ± 9.6 ng/ml) was detected in 69 % of samples (n = 9); Hg (12.2 ± 9.2 ng/ml) was detected in 46 % of samples (n = 6); and MTs (10,900 ± 9,400 ng/ml) were detected in 92 % of samples (n = 12). In caudal scutes samples, Cd (31.7 ± 39.4 ng/g) was detected in 84 % of samples (n = 12) and Hg (374.1 ± 429.4 ng/g) in 83 % of samples (n = 20). No MTs were detected in caudal scutes. Hg concentrations in scutes from the Rio Hondo were 2- to 5-fold greater than those previously reported in scutes from other localities in northern Belize. In blood plasma, a significant positive relationship between Hg and body size was observed. Mean concentrations of Cd and MTs in size classes suggest that MTs may be related to Cd exposure. This is the first report of MT presence in crocodile blood.

  18. Zn tolerance of novel Colocasia esculenta metallothionein and its domains in Escherichia coli and tobacco.

    PubMed

    Kim, Yeon-Ok; Lee, Yoon Gyo; Patel, Darshan H; Kim, Ho Myeong; Ahn, Sung-Ju; Bae, Hyeun-Jong

    2012-11-01

    Contrary to extensive researches on the roles of metallothioneins (MTs) in metal tolerance of animals, the roles of plant MTs in metal tolerance are largely under investigation. In this study, we evaluated the functional role of type 2 MT from Colocasia esculenta (CeMT2b) in Zn tolerance of tobacco and E. coli cells. Under Zn-stress conditions, transgenic tobacco overexpressing CeMT2b displayed much better seedling growth, a significant decrease in the levels of H(2)O(2) and an increase in Zn accumulation compared with the wild type. Overexpression of CeMT2b in E. coli greatly enhanced Zn tolerance and Zn accumulation under Zn stresses compared with control cells. CeMT2b bound 5.38 ± 0.29 atoms of Zn per protein. To identify a structural domain of CeMT2b for Zn binding, we investigated the growth of E. coli expressing each of the N-terminal, C-terminal, and central linker domains or a CNC motif deletion from the C-terminus of full-length CeMT2b. The results showed that the CNC motif is required for Zn tolerance, and the N-terminal domain is more effective in Zn tolerance than the C-terminal domain. Taken together, our results provide direct evidence for functional contributions of CeMT2b in Zn tolerance of tobacco and E. coli cells.

  19. Metal and metallothionein concentrations in Paracentrotus lividus from Amvrakikos gulf (Ionian Sea-Greece).

    PubMed

    Strogyloudi, Evangelia; Pancucci-Papadopoulou, Maria-Antonietta; Papadopoulos, Giorgio L

    2014-09-01

    Concentrations of Cd, Cu, Cr, Ni, Zn, Fe and metallothioneins (MTs) were measured in the gonads of Paracentrotus lividus from Amvrakikos gulf (Ionian Sea, Greece). Three natural populations were selected; two of them, growing inside the gulf (Agios Thomas and Koronisia), presented higher density and smaller body size than the population living in a coastal area just outside the gulf (Mytikas). Metal and MT levels were not elevated, with the exception of Zn, showing high values related to the reproduction stage of the sea urchins. Significant geographical variations were recorded in the concentrations of Cu, Zn, Cd, Cr and MTs. The highest mean and maximum values of Cu, Zn and MTs were recorded in Agios Thomas while Cd and Cr were higher in Mytikas population. Copper, Zn, Fe and MT concentrations were negatively correlated to the sea urchin body size, while a positive concentration-size relationship was observed for Cd. Although all studied populations grow in a low metal level marine environment, urchins with smaller body size living in a food limited marine environment showed higher gonadosomatic index, metal concentrations and MT levels in their gonads (Agios Thomas and Koronisia) than larger specimens growing in a food unlimited area (Mytikas).

  20. Stress to cadmium monitored by metallothionein gene induction in Paracentrotus lividus embryos

    PubMed Central

    Russo, Roberta; Bonaventura, Rosa; Zito, Francesca; Schröder, Heinz-C.; Müller, Isabel; Müller, Werner E. G.; Matranga, Valeria

    2003-01-01

    We used sea urchin embryos as bioindicators to study the effects of exposure to sublethal cadmium concentrations on the expression of the metallothionein (MT) gene stress marker. For this purpose, the complete complementary deoxyribonucleic acid of the species Paracentrotus lividus (Pl) was cloned and sequenced. Northern blot analysis showed that basal levels of Pl-MT messenger ribonucleic acid, having an apparent size of 700 bases, are expressed in all developmental stages analyzed, from early cleavage to pluteus. However, when embryos were continuously cultured in sublethal CdCl2 concentrations and harvested at cleavage, swimming blastula, late gastrula, and pluteus stages (6, 12, 24, and 48 hours after fertilization, respectively), a time- and dose-dependent increase in the transcription levels of the Pl-MT gene was observed. Interestingly, although microscopical inspection revealed the occurrence of abnormalities only after 24 hours of exposure to the pollutant, Northern blot and reverse transcriptase–polymerase chain reaction analyses revealed significant increases in Pl-MT expression levels already after 12 and 6 hours of exposure, respectively. Therefore, this study confirms the validity of MT as marker of exposure and provides evidence that Pl-MT and sea urchin embryos can be a potentially valuable and sensitive model for testing in very short periods of time seawaters heavily contaminated with cadmium. PMID:14984056

  1. Expression of a Neurospora crassa metallothionein and its variants in Escherichia coli

    SciTech Connect

    Romeyer, F.M.; Jacobs, F.A.; Brousseau, R. )

    1990-09-01

    The Neurospora crassa metallothionein (NC) synthesis gene was cloned and expressed in Escherichia coli in two different expression vectors (pING2 and pUA7), both under the regulation of the Salmonella typhimurium arabinose operon. Upon induction with arabinose, the pING2-NC vector expressed as inclusion body-localized AraB{prime}::NC fusion protein of 21 kilodaltons. The pUA7-NC vector expressed a 5.3 kilodalton Lpp::NC fusion protein anchored to the outer membrane of the cell. Cells expressing the NC fusion proteins accumulated Cd{sup 2+} and Cu{sup +} (between 2.3- and 11-fold) compared with nonexpressing cells. To generate novel forms of metal-binding peptides, a set of specific mutant genes for N. crassa NC was designed in which each cysteine residue was replaced with a subset of amino acids implicated in peptide-metal coordination (Asn, Asp, His, Lys, or Tyr residues). These mutant NC sequences were cloned into the two vectors and expressed in E. coli. One of the mutant proteins (containing His residues) showed accumulation of Cd{sup 2+} and Cu{sup +} (threefold) from a mixture of 16 heavy metals species. None of the other heavy metals present in the culture was accumulated.

  2. Allelic diversity of metallothionein in Orchesella cincta (L.): traces of natural selection by environmental pollution.

    PubMed

    Timmermans, M J T N; Ellers, J; Van Straalen, N M

    2007-05-01

    The advances made in statistical methods to detect selection from DNA sequence variation has resulted in an enormous increase in the number of studies reporting positive selection. However, a disadvantage of such statistical tests is that often no insight into the actual source of selection is obtained. Finer understanding of evolution can be obtained when those statistical tests are combined with field observations on allele frequencies. We assessed whether the metallothionein (mt) gene of Orchesella cincta (Collembola), which codes for a metal-binding protein, is subject to selection, by investigating alleles and allele frequencies among European metal-stressed and reference populations. Eight highly divergent alleles were resolved in Northwest Europe. At the nucleotide level, a total of 51 polymorphic sites (five of them implying amino-acid changes) were observed. Although statistical tests applied to the sequences alone showed no indication of selection, a G-test rejected the null hypothesis that alleles are homogeneously distributed over metal-stressed and reference populations. Analysis of molecular variance assigned a small, but significant amount of the total variance to differences between metal-stressed and non-stressed populations. In addition, it was shown that metal-stressed populations tend to be more genetically diversified at this locus than non-stressed ones. These results suggest that the mt gene and its surrounding DNA region are affected by environmental metal contamination. This study illustrates that, in addition to statistical tests, field observations on allele frequencies are needed to gain understanding of selection and adaptive evolution.

  3. Differential protein expression in metallothionein protection from depleted uranium-induced nephrotoxicity

    PubMed Central

    Hao, Yuhui; Huang, Jiawei; Liu, Cong; Li, Hong; Liu, Jing; Zeng, Yiping; Yang, Zhangyou; Li, Rong

    2016-01-01

    The purpose of this study was to investigate the underlying mechanism of metallothionein (MT) protection from depleted uranium (DU) using a proteomics approach to search for a DU toxicity-differential protein. MT−/− and MT+/+ mice were administrated with a single dose of DU (10 mg/kg, i.p.) or equal volume of saline. After 4 days, protein changes in kidney tissues were evaluated using a proteomics approach. A total of 13 differentially expressed proteins were identified using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The validating results showed that the expression of aminoacylase-3 (ACY-3) and the mitochondrial ethylmalonic encephalopathy 1 (ETHE1) decreased significantly after DU exposure; in addition, the reduction in MT−/− mice was more significant than that in MT+/+ mice. The results also showed that exogenous ETHE1 or ACY-3 could increase the survival rate of human embryonic kidney 293 (HEK293) cells after DU exposure. A specific siRNA of ETHE1 significantly increased cell apoptosis rates after DU exposure, whereas exogenous ETHE1 significantly decreased cell apoptosis rates. In summary, ACY-3 and ETHE1 might involve in protection roles of MT. ETHE1 could be a new sensitive molecular target of DU-induced cell apoptosis. PMID:27966587

  4. Influence of zinc on the ontogeny of hepatic metallothionein in the fetal rat

    SciTech Connect

    Charles-Shannon, V.L.; Sasser, L.B.; Burbank, D.K.; Kelman, B.J.

    1981-10-01

    The ontogeny of hepatic metallothioneins (Mt) in fetal tissue as related to dietary and hepatic Zn was investigated. Sixty 6-month-old female rats were divided into two groups and given either double-distilled water or water containing 700 )g of Zn per milliliter. Dams from each group were killed on 16, 19, or 21 days of gestation, and maternal and fetal livers were removed. Mt content of the tissue was estimated by Piotrowski's Hg-saturation method. Results established the presence of an endogenous hepatic Mt in the fetal rat as early as 16 days of gestation. We further demonstrated a marked progressive increase in fetal Mt from Day 16 through gestation accompanied by a decrease in maternal hepatic Mt. It is suggested that Zn increased fetal Mt by inducing fetal synthesis, redistributing fetal Mt, or increasing Mt transport to the fetus, because both fetal and maternal hepatic Mt were increased. Fetal hepatic Mt concentration was several times greater than maternal Mt at corresponding stages of gestation. Mt may serve to either ensure adequate storage of Zn or Cu for fetal development or protect the fetus against metal toxicity, but the significance of these high endogenous levels of fetal Mt are not clear at this time.

  5. Characterization of the role of metallothionein-3 in an animal model of Alzheimer's disease.

    PubMed

    Manso, Yasmina; Carrasco, Javier; Comes, Gemma; Meloni, Gabriele; Adlard, Paul A; Bush, Ashley I; Vašák, Milan; Hidalgo, Juan

    2012-11-01

    Among the dementias, Alzheimer's disease (AD) is the most commonly diagnosed, but there are still no effective drugs available for its treatment. It has been suggested that metallothionein-3 (MT-3) could be somehow involved in the etiology of AD, and in fact very promising results have been found in in vitro studies, but the role of MT-3 in vivo needs further analysis. In this study, we analyzed the role of MT-3 in a mouse model of AD, Tg2576 mice, which overexpress human Amyloid Precursor Protein (hAPP) with the Swedish mutation. MT-3 deficiency partially rescued the APP-induced mortality of females, and mildly affected APP-induced changes in behavior assessed in the hole-board and plus-maze tests in a gender-dependent manner. Amyloid plaque burden and/or hAPP expression were decreased in the cortex and hippocampus of MT-3-deficient females. Interestingly, exogenously administered Zn(7)MT-3 increased soluble Aβ40 and Aβ42 and amyloid plaques and gliosis, particularly in the cortex, and changed several behavioral traits (increased deambulation and exploration and decreased anxiety). These results highlight that the control of the endogenous production and/or action of MT-3 could represent a powerful therapeutic target in AD.

  6. The effects of cadmium of the growth and metallothionein expression of the bivalve larvae, crassostrea virginica

    SciTech Connect

    Craig, K.D.; Burnett, K.; Ringwood, A.; MacDougal, K.; Kendall, L.

    1994-12-31

    Oyster larvae, Crassostrea virginica, were exposed to 20 ppb of cadmium (Cd) and fed (mixture of Isochrysis galbana & Chaetoceros gracilis, 40mL) in the laboratory for 10 days. On the 0, 4, 7 and 10 day the larvae samples were taken and frozen. Then they were homogenized, centrifuged, ultrafiltered through a membrane separation technique used to segregate substances according to the molecular weight and size. The cytosolic protein was first partially purified by gel permeation, then by PAGE (Polyacrylamide Gel Electrophoresis). The controls and metal exposed larvae were evaluated on total wet weight and the metallothioneins (MT) were identified from the preparations using silver staining techniques. No significant changes could be detected in the controls. However, there was a great number dead at the beginning of the experiment. Cd accumulation began at the time of exposure. This suggests that surface area may play a role in determining short-term accumulation rates. Cd effects on growth (wet weight) was slightly different, the exposed weighed less than or equal to the controls. In addition, the Cd uptake via food played an insignificant role compared to direct uptake from sea water. Between day 0 and 7 there was a number of mortalities for the controls and exposed. In addition, there was a major weight change with the exposed, they appeared to weigh less than the controls on day 7, whereas on day 4 they weighed more. So weight is a very sensitive indicator of toxic stress.

  7. Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression.

    PubMed

    Kmiecik, Alicja M; Pula, Bartosz; Suchanski, Jaroslaw; Olbromski, Mateusz; Gomulkiewicz, Agnieszka; Owczarek, Tomasz; Kruczak, Anna; Ambicka, Aleksandra; Rys, Janusz; Ugorski, Maciej; Podhorska-Okolow, Marzena; Dziegiel, Piotr

    2015-01-01

    It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients' shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases.

  8. Acetylcholinesterase and metallothionein in oysters (Crassostrea corteziensis) from a subtropical Mexican Pacific estuary.

    PubMed

    Bernal-Hernández, Y Y; Medina-Díaz, I M; Robledo-Marenco, M L; Velázquez-Fernández, J B; Girón-Pérez, M I; Ortega-Cervantes, L; Maldonado-Vázquez, W A; Rojas-García, A E

    2010-04-01

    Substantial efforts have been devoted to developing and applying biomarkers for ecological risk assessment. Bivalve mollusks, such as mussels and oysters, are commonly used in environmental monitoring programs because of their wide geographical distribution, great sensitivity to environmental pollutants, and ability to accumulate anthropogenically derived chemicals at a high rate. Acetylcholinesterase (AChE) activity and metallothionein (MT's) content are representative specific biomarkers that indicate the presence of anticholinesterasic compounds (like organophosphorus and carbamate pesticides) and metals, respectively. The aim of this study was to evaluate AChE activity and MT's content in Crassostrea corteziensis from Boca de Camichín estuary. The results obtained here showed that AChE activity was 65% lower in oysters from Boca de Camichín than in control organisms. In contrast, MT's content in collected organisms was not statistically different from that in control organisms. AChE activity and MT's content in oysters could be used as early biomarkers of effects and exposure to pesticides and heavy metals, respectively, in aquatic environments.

  9. Metallothioneins and trace elements dyshomeostasis induced by exposure to gasoline vapor in mice.

    PubMed

    Grebić, Damir; Tota, Marin; Jakovac, Hrvoje; Broznić, Dalibor; Marinić, Jelena; Canadi, Gordana; Milin, Cedomila; Radosević-Stasić, Biserka

    2014-03-01

    To investigate the effects of air pollution related with the gasoline/petrochemical industry the expression of metallothionein I (MT-I) mRNA and tissue metals were analyzed in organs of mice, exposed to gasoline (G) vapor in laboratory conditions. Control groups consisted of intact mice and of those exposed in the metabolic chamber to fresh air. The data obtained by RT-PCR and inductively coupled plasma spectrometry have shown that exposure to G vapor leads to upregulation of MT-I mRNA in organs that receive a strong respiratory and olfactory input or participate in gasoline degradation and elimination (lungs, brain, kidney and liver). Besides, in the brain and in the lungs, kidney and liver a decreased tissue content of Zn²⁺ or Cu²⁺ and Mg²⁺ was found (p<0.001). Some of these changes were obtained also in mice closed in the metabolic chamber, pointing to the involvement of stress-induced mechanisms in the transcriptional regulation of MTs.

  10. Metal-induced hydroxyl radical generation by Cu(+)-metallothioneins from LEC rat liver.

    PubMed

    Nakamura, M; Nakayama, K; Shishido, N; Yumino, K; Ohyama, T

    1997-02-24

    Reactions of LEC (Long-Evans rats with a cinnamonlike coat color) rat liver Cu(I)-metallothioneins (MTs) with HgCl2 or K3Fe(CN)6 were investigated by ESR spectroscopy and generation of hydroxyl radicals was demonstrated using the ESR spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). When Cu(I)-MTs were incubated with more than one equivalent mole HgCl2 or K3Fe(CN)6 to Cu+ bound to MTs, strong signals due to Cu2+ appeared. ESR spectra, which were a combination of the DMPO-OH adduct signal and a six-line signal, were observed in the reaction of Cu(I)-MTs with HgCl2, whereas no oxygen radical signal was seen with K3Fe(CN)6. The DMPO-OH signal intensity was greater in the presence of SOD while the signal disappeared in the presence of catalase. The results suggest that addition of HgCl2 causes the liberation of cuprous ions from MTs followed by a reaction with oxygen, leading to hydroxyl radical formation through a Fenton-type Haber-Weiss reaction.

  11. Metallothionein-3 Increases Triple-Negative Breast Cancer Cell Invasiveness via Induction of Metalloproteinase Expression

    PubMed Central

    Suchanski, Jaroslaw; Olbromski, Mateusz; Gomulkiewicz, Agnieszka; Owczarek, Tomasz; Kruczak, Anna; Ambicka, Aleksandra; Rys, Janusz; Ugorski, Maciej; Podhorska-Okolow, Marzena; Dziegiel, Piotr

    2015-01-01

    It has been recently found that metallothionein-3 (MT3) enhances the invasiveness and tumorigenesis of prostate cancer cells. This finding is in contrast to those of earlier studies, which indicated that overexpression of MT3 in breast cancer and prostate cancer cell lines inhibits their growth in vitro. Therefore, to clarify the role of MT3 in breast cancer progression, we analyzed the effect of MT3-overexpression on proliferation, invasiveness, migration, and tumorigenesis of breast cancer MDA-MB-231/BO2 cells. It was found that MDA-MB-231/BO2 cells overexpressing MT3 were characterized by increased invasiveness in vitro, compared to the control cells. Interestingly, this increased invasiveness correlated with a highly increased concentration of MMP3 in the culture supernatants (p<0.0001). Our data suggest that MT3 may regulate breast cancer cell invasiveness by modulating the expression of MMP3. These experimental results, obtained using triple-negative MDA-MB-231/BO2 cells, were further supported by clinical data. It was found that, in triple-negative breast cancer (TNBC), nuclear MT3 immunoreactivity in cancer cells tended to be associated with patients’ shorter disease-specific survival, suggesting that nuclear MT3 expression may be a potential marker of poor prognosis of triple-negative TNBC cases. PMID:25933064

  12. Effect of zinc supplementation on type 2 diabetes parameters and liver metallothionein expressions in Wistar rats.

    PubMed

    Wang, Xue; Li, Hongyan; Fan, Zhe; Liu, Ya

    2012-12-01

    Zinc is a trace metal and acts as an active component of various enzymes. Zinc deficiency has been suggested to be associated with the development of diabetes. The present study investigated the role of zinc supplementation on prevention of diabetic conditions. A double-disease model mimicking hyperlipidemia and type 2 diabetes was created by applying high-fat diet and streptozotocin (STZ) to Wistar rats. We demonstrated that zinc supplementation improved symptoms of diabetes such as polydipsia and increased serum level of high-density lipoprotein cholesterol, indicating that zinc supplementation has a potential beneficial effect on diabetic conditions. The level of maldondialdehyde (MDA), an oxidative stress marker, was reduced in liver by zinc supplementation in high fat-fed rats with or without STZ injection. Meanwhile, we observed an increase in the expression of metallothioneins (MTs) in liver of rats treated with zinc. This suggests that the induction of MTs in liver, which has been shown to be important in scavenging free radicals, could be one of the underlying mechanisms of zinc supplementation on reducing MDA levels in liver. Finally, we found that zinc levels in liver were increased while there was no change in serum zinc levels, indicating that local zinc level might be a critical factor for the induction of MTs. Also, the level of MTs could potentially be an index of zinc bioavailability. Taken together, these results suggest that both zinc and MT could play an important role in balancing nutrition and metabolism to prevent diabetic development.

  13. The Balance between Life and Death of Cells: Roles of Metallothioneins

    PubMed Central

    Nielsen, Allan Evald; Bohr, Adam; Penkowa, Milena

    2006-01-01

    Metallothionein (MT) is a highly conserved, low-molecular-weight, cysteine-rich protein that occurs in 4 isoforms (MT-I to MT-IV), of which MT-I+II are the major and best characterized proteins. This review will focus on mammalian MT-I+II and their functional impact upon cellular survival and death, as seen in two rather contrasting pathological conditions: Neurodegeneration and neoplasms. MT-I+II have analogous functions including: 1) Antioxidant scavenging of reactive oxygen species (ROS); 2) Cytoprotection against degeneration and apoptosis; 3) Stimulation of cell growth and repair including angiogenesis/revascularization, activation of stem/progenitor cells, and neuroregeneration. Thereby, MT-I+II mediate neuroprotection, CNS restoration and clinical recovery during neurodegenerative disorders. Due to the promotion of cell survival, increased MT-I+II levels have been associated with poor tumor prognosis, although the data are less clear and direct causative roles of MT-I+II in oncogenesis remain to be identified. The MT-I+II molecular mechanisms of actions are not fully elucidated. However, their role in metal ion homeostasis might be fundamental in controlling Zn-dependent transcription factors, protein synthesis, cellular energy levels/metabolism and cell redox state. Here, the neuroprotective and regenerative functions of MT-I+II are reviewed, and the presumed link to oncogenesis is critically perused. PMID:19690641

  14. Expression of metallothionein mRNAs on mouse cerebellum microglia cells by thimerosal and its metabolites.

    PubMed

    Minami, Takeshi; Miyata, Eriko; Sakamoto, Yamato; Kohama, Azusa; Yamazaki, Hideo; Ichida, Seiji

    2009-06-30

    Effects of thimerosal and its metabolites, ethyl mercury and thiosalicylate, on the expression of metallothionein (MT) mRNAs in mouse cerebellum microglia cell line, C8-B4 cells, were studied. The level of MT-1 mRNA significantly decreased at early hours and recovered time-dependently 24h after thimerosal was added to the C8-B4 cells. However, MT-2 and MT-3 mRNA expressions did not change from the control group. In contrast, the expression of MT-1 mRNA increased in a mouse neuroblastoma cell line 6h after incubation with thimerosal. In addition, the level of MT-1 mRNA decreased in C8-B4 cells 6h after the addition of thiosalicylate, but ethyl mercury induced MT-1 mRNA expression. When cell viability was compared with thimerosal, thiosalicylate, and ethyl mercury, the viability of C8-B4 cells decreased dose-dependently 24h after either thimerosal or ethyl mercury was added; however, the viability increased dose-dependently until 15 microM thiosalicylate was added. From the present results, it is concluded that the expression of MT-1 mRNA may be mediated by different factors than the expression of MT-2 mRNA in C8-B4 cells. The reduction of MT-1 mRNA level by thiosalicylate may affect the proliferation of C8-B4 cells.

  15. Preparation and reactivity of a tetranuclear Fe(II) core in the metallothionein α-domain.

    PubMed

    Sano, Yohei; Onoda, Akira; Sakurai, Rie; Kitagishi, Hiroaki; Hayashi, Takashi

    2011-05-01

    Metallothioneins (MTs) are small cysteine-rich proteins which exhibit high affinities for various metal ions and play roles in storage of essential metals and detoxification of toxic metals. Studies on the redox properties of MTs have been quite limited. Recently, we focused on the α-domain of MT (MTα) as a protein matrix and incorporated a tetranuclear metal cluster as a reductant. UV-visible, CD and MS data indicate the formation of the stable tetranuclear metal-cysteine cluster in the MTα matrix with Fe(II)(4)-MTα and Co(II)(4)-MTα species existing in water. Furthermore, the Fe(II)(4)-MTα species was found to promote the reduction of met-myoglobin and azobenzene derivatives under mild conditions. Particularly, the stoichiometric reduction of methyl red with Fe(II)(4)-MTα (1:1) was found to proceed with a conversion of 98% over a period of 6h at 25°C. This indicates that all of the four Fe(II) cores contribute to the reduction. In this paper, we describe the preparation and reactivity of the tetranuclear iron cluster in the protein matrix.

  16. Functional characterization of four metallothionein genes in Daphnia pulex exposed to environmental stressors

    PubMed Central

    Asselman, J.; Glaholt, S.P.; Smith, Z.; Smagghe, G.; Janssen, C.R.; Colbourne, J.K.; Shaw, J.R.; De Schamphelaere, K.A.C.

    2014-01-01

    We characterized the metallothionein genes (Mt1, Mt2, Mt3, and Mt4) in Daphnia pulex on both molecular and ecotoxicological level. We therefore conducted a bioinformatical analysis of the gene location and predicted protein sequence, and screened the upstream flanking region for regulatory elements. The number of these elements and their positions relative to the start codon varied strongly among the four genes and even among two gene duplicates (Mt1A and Mt1B), suggesting different roles of the four proteins in the organisms’ response to stress. We subsequently conducted a chronic 16-day exposure of D. pulex to different environmental stressors (at sublethal levels causing approximately 50% reduction in reproduction). Based on prior knowledge, we exposed them to the metals Cd, Cu, and Ni, the moulting hormone hydroxyecdysone (20E), and the oxidative stressors cyanobacteria (Microcystis aeruginosa), and paraquat (Pq). We then compared mRNA expression levels of the four Mt genes under these stress conditions with control conditions in “The Chosen One” clone (TCO), for which the full genome was sequenced and annotated. All together, the mRNA expression results under the different stress regimes indicate that different Mt genes may play different and various roles in the response of D. pulex to stress and that some (but not all) of the differences among the four genes could be related to the pattern of regulatory elements in their upstream flanking region. PMID:22266576

  17. Identification of quantum dots labeled metallothionein by fast scanning laser-induced breakdown spectroscopy

    NASA Astrophysics Data System (ADS)

    Konecna, Marie; Novotny, Karel; Krizkova, Sona; Blazkova, Iva; Kopel, Pavel; Kaiser, Jozef; Hodek, Petr; Kizek, Rene; Adam, Vojtech

    2014-11-01

    The technique described in this paper allows detection of quantum dots (QDs) specifically deposited on the polystyrene surface by laser-induced breakdown spectroscopy (LIBS). Using LIBS, the distribution of QDs or their conjugates with biomolecules deposited on the surface can be observed, regardless of the fact if they exhibit fluorescence or not. QDs deposited on the specific surface of polystyrene microplate in the form of spots are detected by determination of the metal included in the QDs structure. Cd-containing QDs (CdS, CdTe) stabilized with mercaptopropionic (MPA) or mercaptosuccinic (MSA) acid, respectively, alone or in the form of conjugates with metallothionein (MT) biomolecule are determined by using the 508.58 nm Cd emission line. The observed absolute detection limit for Cd in CdTe QDs conjugates with MT in one spot was 3 ng Cd. Due to the high sensitivity of this technique, the immunoanalysis in combination with LIBS was also investigated. Cd spatial distribution in sandwich immunoassay was detected.

  18. Prevention of gastrointestinal lead poisoning using recombinant Lactococcus lactis expressing human metallothionein-I fusion protein.

    PubMed

    Xiao, Xue; Zhang, Changbin; Liu, Dajun; Bai, Weibin; Zhang, Qihao; Xiang, Qi; Huang, Yadong; Su, Zhijian

    2016-04-05

    Low-level lead poisoning is an insidious disease that affects millions of children worldwide, leading to biochemical and neurological dysfunctions. Blocking lead uptake via the gastrointestinal tract is an important prevention strategy. With this in mind, we constructed the recombinant Lactococcus lactis strain pGSMT/MG1363, which constitutively expressed the fusion protein glutathione S-transferase (GST)-small molecule ubiquitin-like modifier protein (SUMO)-metallothionein-I (GST-SUMO-MT). The thermodynamic data indicated that the average number of lead bound to a GST-SUMO-MT molecule was 3.655 and this binding reaction was a spontaneous, exothermic and entropy-increasing process. The total lead-binding capacity of pGSMT/MG1363 was 4.11 ± 0.15 mg/g dry mass. Oral administration of pGSMT/MG1363 (1 × 10(10) Colony-Forming Units) to pubertal male rats that were also treated with 5 mg/kg of lead acetate daily significantly inhibited the increase of blood lead levels, the impairment of hepatic function and the decrease of testosterone concentration in the serum, which were all impaired in rats treated by lead acetate alone. Moreover, the administration of pGSMT/MG1363 for 6 weeks did not affect the serum concentration of calcium, magnesium, potassium or sodium ions. This study provides a convenient and economical biomaterial for preventing lead poisoning via the digestive tract.

  19. Metallothionein response to stress in rats: role in free radical scavenging.

    PubMed

    Hidalgo, J; Campmany, L; Borras, M; Garvey, J S; Armario, A

    1988-10-01

    The possibility that liver metallothionein (MT) can function as an antioxidant in vivo has been studied in the rat. It was found that the stress of food and water deprivation with or without physical immobilization consistently increased liver lipid peroxidation (LLP), suggesting that liver MT induction by stress might be related to the stress-induced LLP. This was supported by results with the lipid peroxidation promoter dimethyl sulfoxide (DMSO) and the natural antioxidant vitamin E. Whereas DMSO administration increased LLP levels in basal and stress situations, vitamin E decreased them. Liver MT levels were increased by DMSO in basal and stress situations, whereas they were decreased by vitamin E during stress. These in vivo results are consistent with an antioxidant role of liver MT suggested by previous in vitro results. However, liver MT preinduction by Zn treatment did not result in a lower MT response to stress. Instead a positive synergistic effect between Zn and stress appeared to be present. This result indicates that the mechanism of action of MT as antioxidant remains unclear.

  20. Crambescin C1 Exerts a Cytoprotective Effect on HepG2 Cells through Metallothionein Induction

    PubMed Central

    Roel, María; Rubiolo, Juan A.; Ternon, Eva; Thomas, Olivier P.; Vieytes, Mercedes R.; Botana, Luis M.

    2015-01-01

    The Mediterranean marine sponge Crambe crambe is the source of two families of guanidine alkaloids known as crambescins and crambescidins. Some of the biological effects of crambescidins have been previously reported while crambescins have undergone little study. Taking this into account, we performed comparative transcriptome analysis to examine the effect of crambescin-C1 (CC1) on human tumor hepatocarcinoma cells HepG2 followed by validation experiments to confirm its predicted biological activities. We report herein that, while crambescin-A1 has a minor effect on these cells, CC1 protects them against oxidative injury by means of metallothionein induction even at low concentrations. Additionally, at high doses, CC1 arrests the HepG2 cell cycle in G0/G1 and thus inhibits tumor cell proliferation. The findings presented here provide the first detailed approach regarding the different effects of crambescins on tumor cells and provide a basis for future studies on other possible cellular mechanisms related to these bioactivities. PMID:26225985

  1. Levels of heavy metals and their binding protein metallothionein in type 2 diabetics with kidney disease.

    PubMed

    Raudenska, Martina; Dvorakova, Veronika; Pacal, Lukas; Chalasova, Katarina; Kratochvilova, Monika; Gumulec, Jaromir; Ruttkay-Nedecky, Branislav; Zitka, Ondrej; Kankova, Katerina; Adam, Vojtech; Masarik, Michal

    2017-01-06

    Hyperglycemia, a major metabolic disturbance present in diabetes, promotes oxidative stress. Activation of antioxidant defense is an important mechanism to prevent cell damage. Levels of heavy metals and their binding proteins can contribute to oxidative stress. Antiradical capacity and levels of metallothionein (MT), metals (zinc and copper), and selected antioxidants (bilirubin, cysteine, and glutathione) were determined in 70 type 2 diabetes mellitus (T2DM) subjects and 80 healthy subjects of Caucasian origin. Single nucleotide polymorphism (rs28366003) in MT gene was detected. Antiradical capacity, conjugated bilirubin, and copper were significantly increased in diabetics, whereas MT and glutathione were decreased. Genotype AA of rs28366003 was associated with higher zinc levels in the diabetic group. The studied parameters were not influenced by renal function. This is the first study comprehensively investigating differences in MT and metals relevant to oxidative stress in T2DM. Ascertained differences indicate increased oxidative stress in T2DM accompanied by abnormalities in non-enzymatic antioxidant defense systems.

  2. Cadmium, zinc, and copper in horse liver and in horse liver metallothionein: comparisons with kidney cortex

    SciTech Connect

    Elinder, C.G.; Nordberg, M.; Palm, B.; Piscator, M.

    1981-10-01

    Cadmium, zinc, and copper were determined in liver and in kidney cortex samples obtained from 33 normal Swedish horses. Cadmium concentrations in liver ranged from 0.002 to 0.165 mmole/kg and in kidney from 0.01 to 2.15 mmole/kg. There was a significant correlation between liver and kidney concentrations of cadmium. The average kidney concentration of cadmium was about 15 times that of liver. Zinc concentrations increased with increasing cadmium concentrations in both liver and kidney. The relative increase of zinc with cadmium was more pronounced in liver than in kidney. However, the absolute increase of zinc was larger in kidney due to the much higher concentration of cadmium in kidney compared to liver. Any significant correlation between copper and cadmium, or copper and zinc, could not be revealed. Sephadex gel filtration was performed on supernatants from homogenates of kidney and liver from 19 of the horses. In both organs the major part of cadmium was recovered in protein fractions corresponding to metallothionein (MT), in which the increase of zinc also took place. The molar ratio between zinc and cadmium was higher in MT fractions obtained from liver than in MT fractions obtained from kidney.

  3. Crambescin C1 Exerts a Cytoprotective Effect on HepG2 Cells through Metallothionein Induction.

    PubMed

    Roel, María; Rubiolo, Juan A; Ternon, Eva; Thomas, Olivier P; Vieytes, Mercedes R; Botana, Luis M

    2015-07-27

    The Mediterranean marine sponge Crambe crambe is the source of two families of guanidine alkaloids known as crambescins and crambescidins. Some of the biological effects of crambescidins have been previously reported while crambescins have undergone little study. Taking this into account, we performed comparative transcriptome analysis to examine the effect of crambescin-C1 (CC1) on human tumor hepatocarcinoma cells HepG2 followed by validation experiments to confirm its predicted biological activities. We report herein that, while crambescin-A1 has a minor effect on these cells, CC1 protects them against oxidative injury by means of metallothionein induction even at low concentrations. Additionally, at high doses, CC1 arrests the HepG2 cell cycle in G0/G1 and thus inhibits tumor cell proliferation. The findings presented here provide the first detailed approach regarding the different effects of crambescins on tumor cells and provide a basis for future studies on other possible cellular mechanisms related to these bioactivities.

  4. Screening of Cd tolerant genotypes and isolation of metallothionein genes in alfalfa (Medicago sativa L.).

    PubMed

    Wang, Xiaojuan; Song, Yu; Ma, Yanhua; Zhuo, Renying; Jin, Liang

    2011-12-01

    In order to evaluate Cd tolerance in wide-ranging sources of alfalfa (Medicago sativa) and to identify Cd tolerant genotypes which may potentially be useful for restoring Cd-contaminated environments, thirty-six accessions of alfalfa were screened under hydroponic culture. Our results showed that the relative root growth rate varied from 0.48 to 1.0, which indicated that different alfalfa accessions had various responses to Cd stress. The candidate fragments derived from differentially expressed metallothionein (MT) genes were cloned from leaves of two Cd tolerant genotypes, YE and LZ. DNA sequence and the deduced protein sequence showed that MsMT2a and MsMT2b had high similarity to those in leguminous plants. DDRT-PCR analysis showed that MsMT2a expressed in both YE and LZ plants under control and Cd stress treatment, but MsMT2b only expressed under Cd stress treatment. This suggested that MsMT2a was universally expressed in leaves of alfalfa but expression of MsMT2b was Cadmium (Cd) inducible.

  5. The Functions of Metallothionein and ZIP and ZnT Transporters: An Overview and Perspective

    PubMed Central

    Kimura, Tomoki; Kambe, Taiho

    2016-01-01

    Around 3000 proteins are thought to bind zinc in vivo, which corresponds to ~10% of the human proteome. Zinc plays a pivotal role as a structural, catalytic, and signaling component that functions in numerous physiological processes. It is more widely used as a structural element in proteins than any other transition metal ion, is a catalytic component of many enzymes, and acts as a cellular signaling mediator. Thus, it is expected that zinc metabolism and homeostasis have sophisticated regulation, and elucidating the underlying molecular basis of this is essential to understanding zinc functions in cellular physiology and pathogenesis. In recent decades, an increasing amount of evidence has uncovered critical roles of a number of proteins in zinc metabolism and homeostasis through influxing, chelating, sequestrating, coordinating, releasing, and effluxing zinc. Metallothioneins (MT) and Zrt- and Irt-like proteins (ZIP) and Zn transporters (ZnT) are the proteins primarily involved in these processes, and their malfunction has been implicated in a number of inherited diseases such as acrodermatitis enteropathica. The present review updates our current understanding of the biological functions of MTs and ZIP and ZnT transporters from several new perspectives. PMID:26959009

  6. Prevention of gastrointestinal lead poisoning using recombinant Lactococcus lactis expressing human metallothionein-I fusion protein

    PubMed Central

    Xiao, Xue; Zhang, Changbin; Liu, Dajun; Bai, Weibin; Zhang, Qihao; Xiang, Qi; Huang, Yadong; Su, Zhijian

    2016-01-01

    Low-level lead poisoning is an insidious disease that affects millions of children worldwide, leading to biochemical and neurological dysfunctions. Blocking lead uptake via the gastrointestinal tract is an important prevention strategy. With this in mind, we constructed the recombinant Lactococcus lactis strain pGSMT/MG1363, which constitutively expressed the fusion protein glutathione S-transferase (GST)–small molecule ubiquitin-like modifier protein (SUMO)–metallothionein-I (GST-SUMO-MT). The thermodynamic data indicated that the average number of lead bound to a GST-SUMO-MT molecule was 3.655 and this binding reaction was a spontaneous, exothermic and entropy-increasing process. The total lead-binding capacity of pGSMT/MG1363 was 4.11 ± 0.15 mg/g dry mass. Oral administration of pGSMT/MG1363 (1 × 1010 Colony-Forming Units) to pubertal male rats that were also treated with 5 mg/kg of lead acetate daily significantly inhibited the increase of blood lead levels, the impairment of hepatic function and the decrease of testosterone concentration in the serum, which were all impaired in rats treated by lead acetate alone. Moreover, the administration of pGSMT/MG1363 for 6 weeks did not affect the serum concentration of calcium, magnesium, potassium or sodium ions. This study provides a convenient and economical biomaterial for preventing lead poisoning via the digestive tract. PMID:27045906

  7. Metallothionein in the central nervous system: roles in protection, regeneration and cognition

    PubMed Central

    West, Adrian K.; Hidalgo, Juan; Eddins, Donnie; Levin, Edward D.; Aschner, Michael

    2008-01-01

    Metallothionein (MT) is an enigmatic protein, and its physiological role remains a matter of intense study and debate fifty years after its discovery. This is particularly true of its function in the central nervous system (CNS), where the challenge remains to link its known biochemical properties of metal binding and free radical scavenging to the intricate workings of brain. In this compilation of four reports, first delivered at the 11th International Neurotoxicology Association (INA-11) meeting, June 2007, the authors present the work of their laboratories, each of which gives an important insight into the actions of MT in the brain. What emerges is that MT has the potential to contribute to a variety of processes, including neuroprotection, regeneration, and even cognitive functions. In this article, the properties and CNS expression of MT are briefly reviewed before Dr Juan Hidalgo describes his pioneering work using transgenic models of MT expression to demonstrate how this protein plays a major role in the defence of the CNS against neurodegenerative disorders and other CNS injuries. His group’s work leads to two further questions, what are the mechanisms at the cellular level by which MT acts, and does this protein influence higher order issues of architecture and cognition. These topics are addressed in the second and third sections of this review by Dr Adrian West, and Drs Edward Levin and Donnie Eddins, respectively. Finally, Dr Michael Aschner examines the ability of MT to protect against a specific toxicant, methymercury, in the CNS. PMID:18313142

  8. Arbuscular mycorrhiza affects nickel translocation and expression of ABC transporter and metallothionein genes in Festuca arundinacea.

    PubMed

    Shabani, Leila; Sabzalian, Mohammad R; Mostafavi pour, Sodabeh

    2016-01-01

    Mycorrhizal fungi are key microorganisms for enhancing phytoremediation of soils contaminated with heavy metals. In this study, the effects of the arbuscular mycorrhizal fungus (AMF) Funneliformis mosseae (=Glomus mosseae) on physiological and molecular mechanisms involved in the nickel (Ni) tolerance of tall fescue (Festuca arundinacea = Schedonorus arundinaceus) were investigated. Nickel addition had a pronounced negative effect on tall fescue growth and photosynthetic pigment contents, as well as on AMF colonization. Phosphorus content increased markedly in mycorrhizal plants (M) compared to non-inoculated (NM) ones. However, no significant difference was observed in root carbohydrate content between AMF-inoculated and non-inoculated plants. For both M and NM plants, Ni concentrations in shoots and roots increased according to the addition of the metal into soil, but inoculation with F. mosseae led to significantly lower Ni translocation from roots to the aboveground parts compared to non-inoculated plants. ABC transporter and metallothionein transcripts accumulated to considerably higher levels in tall fescue plants colonized by F. mosseae than in the corresponding non-mycorrhizal plants. These results highlight the importance of mycorrhizal colonization in alleviating Ni-induced stress by reducing Ni transport from roots to shoots of tall fescue plants.

  9. Survival, growth, metallothionein and glycogen levels of Nucella lapillus (L.) exposed to subchronic cadmium stress: the influence of nutritional state and prey type.

    PubMed

    Leung, K M; Furness, R W

    2001-08-01

    Dogwhelks Nucella lapillus feed mainly on mussels and barnacles, and may experience periods of starvation. We report effects of nutritional state and prey type on the survival, growth, cadmium (Cd) accumulation, metallothionein (MT) induction and glycogen stores in N. lapillus exposed to Cd in water. Adult dogwhelks, with similar shell length (30.0+/-1.5 mm), were either starved or fed to satiation with barnacles Semibalanus balanoides, mussels Mytilus edulis or Cd-dosed M. edulis, and kept in filtered natural seawater (< 0.01 microg Cd 1(-1)) or Cd-contaminated (400 microg Cd 1(-1)) seawater for 80 days. Mortality and individual growth rate were determined. Cd, MT and glycogen were measured in different tissues. Prolonged starvation and exposure to Cd significantly reduced the survivorship of N. lapillus, but feeding could help dogwhelks to combat Cd toxicity and minimise mortality. Extended starvation also caused tissue wastage, leading to higher concentrations of Cd and MT in tissues, whereas fed animals increased in weight and had lower Cd and MT concentrations because of the tissue dilution effect. Prey type significantly affected growth rate of dogwhelks and indirectly influenced Cd accumulation, MT induction and glycogen stores. Eating mussels promoted better growth and higher glycogen reserves than eating barnacles. Individual growth rate decreased with increasing Cd accumulation. Cd-exposed survivors grew faster and consumed more than control animals, implying that these survivors may have better fitness and greater tolerance to Cd toxicity. The use of growth, condition index, MT and glycogen as biomarkers of environmental pollution are discussed. These results indicate a need to incorporate biological data including growth (or at least condition index) and prey type into biomonitoring programmes to allow sound interpretation.

  10. Asparagine Metabolic Pathways in Arabidopsis.

    PubMed

    Gaufichon, Laure; Rothstein, Steven J; Suzuki, Akira

    2016-04-01

    Inorganic nitrogen in the form of ammonium is assimilated into asparagine via multiple steps involving glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AspAT) and asparagine synthetase (AS) in Arabidopsis. The asparagine amide group is liberated by the reaction catalyzed by asparaginase (ASPG) and also the amino group of asparagine is released by asparagine aminotransferase (AsnAT) for use in the biosynthesis of amino acids. Asparagine plays a primary role in nitrogen recycling, storage and transport in developing and germinating seeds, as well as in vegetative and senescence organs. A small multigene family encodes isoenzymes of each step of asparagine metabolism in Arabidopsis, except for asparagine aminotransferase encoded by a single gene. The aim of this study is to highlight the structure of the genes and encoded enzyme proteins involved in asparagine metabolic pathways; the regulation and role of different isogenes; and kinetic and physiological properties of encoded enzymes in different tissues and developmental stages.

  11. Effect of metallothionein 2A gene polymorphism on allele-specific gene expression and metal content in prostate cancer

    SciTech Connect

    Krześlak, Anna; Forma, Ewa; Jóźwiak, Paweł; Szymczyk, Agnieszka; Bryś, Magdalena

    2013-05-01

    Metallothioneins (MTs) are highly conserved, small molecular weight, cysteine rich proteins. The major physiological functions of metallothioneins include homeostasis of essential metals Zn and Cu and protection against cytotoxicity of heavy metals. The aim of this study was to determine whether there is an association between the − 5 A/G single nucleotide polymorphism (SNP; rs28366003) in core promoter region and expression of metallothionein 2A (MT2A) gene and metal concentration in prostate cancer tissues. MT2A polymorphism was determined by the polymerase chain reaction–restriction fragment length polymorphism technique (PCR–RFLP) using 412 prostate cancer tissue samples. MT2A gene expression analysis was performed by real-time RT-PCR method. A significant association between rs28366003 genotype and MT2A expression level was found. The average mRNA level was found to be lower among minor allele carriers (the risk allele) than average expression among homozygotes for the major allele. Metal levels were analyzed by flamed atomic absorption spectrometer system. Highly statistically significant associations were detected between the SNP and Cd, Zn, Cu and Pb levels. The results of Spearman's rank correlation showed that the expressions of MT2A and Cu, Pb and Ni concentrations were negatively correlated. On the basis of the results obtained in this study, we suggest that SNP polymorphism may affect the MT2A gene expression in prostate and this is associated with some metal accumulation. - Highlights: • MT2A gene expression and metal content in prostate cancer tissues • Association between SNP (rs28366003) and expression of MT2A • Significant associations between the SNP and Cd, Zn, Cu and Pb levels • Negative correlation between MT2A gene expression and Cu, Pb and Ni levels.

  12. Cadmium induces alpha(1)collagen (I) and metallothionein II gene and alters the antioxidant system in rat hepatic stellate cells.

    PubMed

    del Carmen, Escobar Ma; Souza, Verónica; Bucio, Leticia; Hernández, Elizabeth; Damián-Matsumura, Pablo; Zaga, Verónica; Gutiérrez-Ruiz, Ma Concepción

    2002-01-15

    The mechanism of cadmium-mediated hepatotoxicity has been the subject of numerous investigations, principally in hepatocytes. Although, some uncertainties persist, sufficient evidence has emerged to provide a reasonable account of the toxic process in parenchymal cells. However, there is no information about the effect of cadmium in other hepatic cell types, such as stellate cells (fat storing cells, Ito cells, perisinusoidal cells, parasinusoidal cells, lipocytes). Hepatic stellate cells (HSC) express a quiescent phenotype in a healthy liver and acquire an activated phenotype in liver injury. These cells play an important role in the fibrogenic process. The objective of this study was to investigate the effect of a 24 h treatment of low Cd concentrations in glutathione content, lipid peroxidation damage, cytosolic free Ca, antioxidant enzyme activities: glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase along with the capacity of this heavy metal to induce metallothionein II and alpha(1)collagen (I) in an hepatic stellate cell line (CFSC-2G). Cd-treated cells increased lipid peroxidation and the content of cytosolic free calcium, decreased glutathione content and superoxide dismutase, glutathione peroxidase and catalase activity. Cd was able to induce the expression of the metallothionein II and alpha(1)collagen (I) gene, that was not described in this cell type. Cadmium may act as a pro-fibrogenic agent in the liver probably by inducing oxidative damage by enhancing lipid peroxidation and altering the antioxidant system of the cells. Although, the exact role metallothionein induction plays in this process is unknown, it probably, provides a cytosolic pool of potential binding sites to sequester ionic Cd, thereby decreasing its toxicity.

  13. Multiple forms of metallothionein from the digestive gland of naturally occurring and cadmium-exposed mussels, Mytilus galloprovincialis

    NASA Astrophysics Data System (ADS)

    Ivanković, Dušica; Pavičić, Jasenka; Kozar, Sonja; Raspor, Biserka

    2002-06-01

    Polymorphism of metallothioneins in the digestive gland of naturally occurring (control) and experimentally Cd-exposed mussels Mytilus galloprovincialis (200 µg Cd l-1; 14 days) was studied by applying the conventional methods of Sephadex column liquid chromatography (G-75 and DEAE A-25), and by an electrochemical method (DPASV) for determination of Cd, Zn and Cu concentrations in chromatographic fractions. In both control and Cd-exposed mussels, two distinct molecular mass components of the metallothioneins, monomeric (MT-10) and dimeric (MT-20), were resolved by Sephadex G-75 gel filtration chromatography. In control mussels, the MT-10 component was predominantly expressed as containing markedly higher constitutive levels of Zn (100×) and Cu (10×) than of Cd. Each of these two molecular mass components was further resolved into seven metal-rich peaks by anion-exchange chromatography. In Cd-exposed mussels the larger proportion of Cd was bound to the MT-20 than to the MT-10 component, suggesting that the dimeric component may be considered as a primarily inducible metallothionein. The elution positions of metal-binding maxima of Cd-exposed and control mussels on the respective DEAE chromatographic profiles were comparable. A great similarity in elution positions of Cd maxima between the composite and single-specimen samples was also observed. Our study confirms a high multiplicity of MT forms in mussels from the Mytilus genus not only under the laboratory high-level metal exposure conditions, but also at a natural seawater metal exposure level. The ecotoxicological significance of dimeric and monomeric MT forms, as well as their possible application in the biomonitoring of seawater for trace metals, has been considered.

  14. Large-scale atlas of microarray data reveals biological landscape of gene expression in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcriptome datasets from thousands of samples of the model plant Arabidopsis thaliana have been collectively generated by multiple individual labs. Although integration and meta-analysis of these samples has become routine in the plant research community, it is often hampered by the lack of metad...

  15. Metallothionein-Like Proteins and Energy Reserve Levels after Ni and Pb Exposure in the Pacific White Prawn Penaeus vannamei

    PubMed Central

    Nunez-Nogueira, Gabriel; Mouneyrac, Catherine; Muntz, Alice; Fernandez-Bringas, Laura

    2010-01-01

    This study analyzed the changes in metallothionein-like proteins (MTLPs) and Energy Reserves (ERs) in hepatopancreas and abdominal muscle of the white prawn Penaeus vannamei. Realistic metal concentration exposure for 10 days to Ni and Pb in solution revealed that juvenile prawns partially induce MTLP in hepatopancreas after Pb exposure. Ni was distributed equally between soluble and insoluble fractions, while Pb was present only in the insoluble fraction, suggesting different detoxification strategy. No changes in lipids and glycogen concentration were detected under these experimental conditions in both tissues analyzed. MTLP could not be considered as a suitable indicator for lead exposure in hepatopancreas. PMID:20862200

  16. Occurrence of metallothionein gene smtA in synechococcus Tx-20 and other blue-green algae

    SciTech Connect

    Robinson, N.J.; Gupta, A.; Huckle, J.W.; Jackson, P.; Whitton, B.A. )

    1990-06-01

    Blue-green algae are often abundant at Zn- and Cd-contaminated sites. In order to understand the mechanisms associated with Zn- and Cd-tolerance, we have isolated a metallothionein gene, designated smtA, in Synechococcus Tx-20 (- Pcc 6301 - Anacystis nidulans), a strain apparently obtained from an unpolluted site. The gene was cloned and sequenced, and its expression investigated in a range of heavy-metal-tolerant strains of the same organism obtained by stepwise adaptation. The polymerase chain reaction was used to probe for the possible presence of the homologous gene in a range of other strains (especially Synechococcus) isolated from sites without and with heavy metal contamination.

  17. Metallothionein-Like Proteins and Energy Reserve Levels after Ni and Pb Exposure in the Pacific White Prawn Penaeus vannamei.

    PubMed

    Nunez-Nogueira, Gabriel; Mouneyrac, Catherine; Muntz, Alice; Fernandez-Bringas, Laura

    2010-01-01

    This study analyzed the changes in metallothionein-like proteins (MTLPs) and Energy Reserves (ERs) in hepatopancreas and abdominal muscle of the white prawn Penaeus vannamei. Realistic metal concentration exposure for 10 days to Ni and Pb in solution revealed that juvenile prawns partially induce MTLP in hepatopancreas after Pb exposure. Ni was distributed equally between soluble and insoluble fractions, while Pb was present only in the insoluble fraction, suggesting different detoxification strategy. No changes in lipids and glycogen concentration were detected under these experimental conditions in both tissues analyzed. MTLP could not be considered as a suitable indicator for lead exposure in hepatopancreas.

  18. Variability and Constancy in Cellular Growth of Arabidopsis Sepals.

    PubMed

    Tauriello, Gerardo; Meyer, Heather M; Smith, Richard S; Koumoutsakos, Petros; Roeder, Adrienne H K

    2015-12-01

    Growth of tissues is highly reproducible; yet, growth of individual cells in a tissue is highly variable, and neighboring cells can grow at different rates. We analyzed the growth of epidermal cell lineages in the Arabidopsis (Arabidopsis thaliana) sepal to determine how the growth curves of individual cell lineages relate to one another in a developing tissue. To identify underlying growth trends, we developed a continuous displacement field to predict spatially averaged growth rates. We showed that this displacement field accurately describes the growth of sepal cell lineages and reveals underlying trends within the variability of in vivo cellular growth. We found that the tissue, individual cell lineages, and cell walls all exhibit growth rates that are initially low, accelerate to a maximum, and decrease again. Accordingly, these growth curves can be represented by sigmoid functions. We examined the relationships among the cell lineage growth curves and surprisingly found that all lineages reach the same maximum growth rate relative to their size. However, the cell lineages are not synchronized; each cell lineage reaches this same maximum relative growth rate but at different times. The heterogeneity in observed growth results from shifting the same underlying sigmoid curve in time and scaling by size. Thus, despite the variability in growth observed in our study and others, individual cell lineages in the developing sepal follow similarly shaped growth curves.

  19. Effect of Maotai liquor in inducing metallothioneins and on hepatic stellate cells

    PubMed Central

    Cheng, Ming-Liang; Wu, Jun; Wang, Hai-Qin; Xue, Lie-Ming; Tan, Ying-Zhi; Ping, Liu; Li, Cheng-Xiu; Huang, Neng-Hui; Yao, Yu-Mei; Ren, Lan-Zheng; Ye, Lan; Li, Ling; Jia, Mei-Lin

    2002-01-01

    AIM: To explore the possible mechanism why drinking Maotai liquor dose not cause hepatic fibrosis. METHODS: After being fed with Maotai for 56 days consecutively, the male SD rats were decollated for detecting the biological indexes, and the livers were harvested to examine the liver indexes and the level of hepatic metallothioneins (MT). Hepatic stellate cells (HSC) proliferation and collagen generation were also observed. RESULTS: Hepatic MT contents were 216.0 ng·g-1 ± 10.8 ng·g-1 in the rats of Maotai group and 10.0 ng·g-1 ± 2.8 ng·g-1 in the normal control group, which was increased obviously in Maotain group (P < 0.05). In the rats with grade CCL2 poisoning induced by Maotai, hepatic MT content was 304.8 ng·g-1 ± 12.1 ng·g-1 whereas in the controls with grade CCL4 poisoning, it was 126.4 ng·g-1 ± 4.8 ng·g-1 (P < 0.05). MDA was 102.0 nmol·g-1 ± 3.4 nmol·g-1 in Maotai group and 150.8 nmol·g-1 ± 6.7 nmol·g-1 in the control group (P < 0.05). When both of the groups were suffering from grade CCL4 poisoning, hepatic MT contents was negatively correlated with MDA (r = -0.8023, n = 20, P < 0.01). The 570 nmA values of each tube with HSC regeneration at concentrations of 0, 10, 50, 100, and 200 g·L-1 of Maotai were 0.818, 0.742, 0.736, 0.72, 0.682, and 0.604, respectively. From the concentration of 10 g·L-1, Maotai began to show obvious inhibitory effects against HSC, and the inhibition was concentration-dependent (P < 0.05, P < 0.01). Type I collagen contents in HSC were 61.4, 59.9, 50.1, 49.2, 48.7, 34.4 μg·g-1 at concentrations of 0, 10, 50, 100, and 200 g·L-1 of Maotai. At the concentration of 100-200 g·L-1, Maotai had obvious inhibitory effect against the secretion of type I collagen (P < 0.05). Gene expression analysis was conducted on cells with Maotai concentrations of 0, 50, 100 g·L-1 respectively and the ash values of β-actin gene expression were 0.88, 0.74, and 0.59, respectively, suggesting that at the concentration of 100 g·L-1

  20. Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

    PubMed Central

    Ullio, Chiara; Brunk, Ulf T; Urani, Chiara; Melchioretto, Pasquale; Bonelli, Gabriella; Baccino, Francesco M; Autelli, Riccardo

    2015-01-01

    Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX. PMID:26566051

  1. The changes of heavy metal and metallothionein distribution in testis induced by cadmium exposure.

    PubMed

    Kusakabe, Takahiko; Nakajima, Katsuyuki; Suzuki, Keiji; Nakazato, Kyoumi; Takada, Hisashi; Satoh, Takahiro; Oikawa, Masakazu; Kobayashi, Kenji; Koyama, Hiroshi; Arakawa, Kazuo; Nagamine, Takeaki

    2008-02-01

    Cadmium (Cd) is known to cause various disorders in the testis, and metallothionein (MT) is known as a protein, which has a detoxification function for heavy metals. However, the changes of Fe, Cu, and Zn distribution in the testis induced by Cd exposure have not been well examined. Moreover, only a few studies have been reported on the localization of MT after Cd exposure. In this study, we have investigated the changes of Fe, Cu, and Zn distribution in Cd-exposed testis by a newly developed in air micro-Particle Induced X-ray Emission (PIXE) method. Also, we examined the distribution of MT expression in testis. In the testis of Cd-treated rats with significant increases of lipid peroxidation, the sertoli cell tight junction was damaged by Cd exposure, resulting from disintegration of the blood testis barrier (BTB). Evaluation by in air micro-PIXE method revealed that Cd and Fe distribution were increased in the interstitial tissues and seminiferous tubules. The histological findings indicated that the testicular tissue damage was advanced, which may have been caused by Fe flowing into seminiferous tubules followed by disintegration of the BTB. As a result, Fe was considered to enhance the tissue damage caused by Cd exposure. MT was detected in spermatogonia, spermatocytes, and Sertoli's cells in the testis of Cd-treated rats, but was not detected in interstitial tissues. These results suggested that MT was induced by Cd in spermatogonia, spermatocytes, and Sertoli's cells, and was involved in the resistance to tissue damage induced by Cd.

  2. Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells.

    PubMed

    Ullio, Chiara; Brunk, Ulf T; Urani, Chiara; Melchioretto, Pasquale; Bonelli, Gabriella; Baccino, Francesco M; Autelli, Riccardo

    2015-01-01

    Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other iron-binding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.

  3. Modification of neurobehavioral effects of mercury by genetic polymorphisms of metallothionein in children.

    PubMed

    Woods, James S; Heyer, Nicholas J; Russo, Joan E; Martin, Michael D; Pillai, Pradeep B; Farin, Federico M

    2013-01-01

    Mercury (Hg) is neurotoxic, and children may be particularly susceptible to this effect. A current major challenge is the identification of children who may be uniquely susceptible to Hg toxicity because of genetic disposition. We examined the hypothesis that genetic variants of metallothionein (MT) that are reported to affect Hg toxicokinetics in adults would modify the neurotoxic effects of Hg in children. Five hundred seven children, 8-12 years of age at baseline, participated in a clinical trial to evaluate the neurobehavioral effects of Hg from dental amalgam tooth fillings. Subjects were evaluated at baseline and at 7 subsequent annual intervals for neurobehavioral performance and urinary Hg levels. Following the completion of the clinical trial, we performed genotyping assays for variants of MT isoforms MT1M (rs2270837) and MT2A (rs10636) on biological samples provided by 330 of the trial participants. Regression modeling strategies were employed to evaluate associations between allelic status, Hg exposure, and neurobehavioral test outcomes. Among girls, few significant interactions or independent main effects for Hg exposure and either of the MT gene variants were observed. In contrast, among boys, numerous significant interaction effects between variants of MT1M and MT2A, alone and combined, with Hg exposure were observed spanning multiple domains of neurobehavioral function. All dose-response associations between Hg exposure and test performance were restricted to boys and were in the direction of impaired performance. These findings suggest increased susceptibility to the adverse neurobehavioral effects of Hg among children with relatively common genetic variants of MT, and may have important public health implications for future strategies aimed at protecting children and adolescents from the potential health risks associated with Hg exposure. We note that because urinary Hg reflects a composite exposure index that cannot be attributed to a specific

  4. Identification of Two Metallothioneins as Novel Inhalative Coffee Allergens Cof a 2 and Cof a 3

    PubMed Central

    Peters, Ulrike; Frenzel, Karsten; Brettschneider, Reinhold; Oldenburg, Marcus; Bittner, Cordula

    2015-01-01

    Background Dust of green coffee beans is known to be a relevant cause for occupational allergic disorders in coffee industry workers. Recently, we described the first coffee allergen (Cof a 1) establishing an allergenic potential of green coffee dust. Objective Our aim was to identify allergenic components of green coffee in order to enhance inhalative coffee allergy diagnosis. Methods A Coffea arabica pJuFo cDNA phage display library was created and screened for IgE binding with sera from allergic coffee workers. Two further coffee allergens were identified by sequence analysis, expressed in E. coli, and evaluated by Western blots. The prevalence of sensitization to recombinant Cof a 1, Cof a 2, and Cof a 3 and to commercially available extract was investigated by ELISA (enzyme-linked immunosorbent assay) respectively CAP (capacity test) screening in 18 sera of symptomatic coffee workers. Results In addition to the previously described chitinase Cof a 1, two Coffea arabica cysteine-rich metallothioneins of 9 and 7 kDa were identified and included in the IUIS Allergen Nomenclature as Cof a 2 and Cof a 3. Serum IgE antibodies to at least one of the recombinant allergens were found in 8 out of 18 symptomatic coffee workers (44%). Only 2 of the analysed sera (11%) had reacted previously to the commercial allergy test. Conclusions In addition to the previously described Cof a 1 we have identified two further coffee proteins to be type I coffee allergens (Cof a 2 and Cof a 3) which may have a relevant potential for the specific diagnosis and/or therapy of coffee allergy. PMID:25962169

  5. Metal accumulation and metallothionein induction in the spotted dogfish Scyliorhinus canicula.

    PubMed

    De Boeck, G; Eyckmans, M; Lardon, I; Bobbaers, R; Sinha, A K; Blust, R

    2010-04-01

    Recent studies indicate that elasmobranch fish respond differently to metal exposure than marine teleosts. Accumulation rates can be high, which despite the fact that normal background levels for metals in the marine environment are low, is worrying due to the long life span and late fecundity of most shark. The goals of the present study were to examine differences in accumulation rates and toxicity of a range of metals at equimolar concentrations (10microM) in the Mediterranean or spotted dogfish, Scyliorhinus canicula. For this purpose, we exposed the dogfish to Ni (587microg/L), Cd (1124microg/L), Pb (2072microg/L), Cu (635microg/L), and Ag (1079microg/L and two additional exposures at 10microg/L and 1microg/L) for one week and measured total metal accumulation, metallothionein induction, and parameters related to osmoregulation. Our study confirms the high toxicity and accumulation rates of Ag for elasmobranch fish, even at levels 100 to 1000 times lower than exposure levels of other metals. Also Pb accumulated readily in all organs, but did not cause any osmoregulatory disturbance at the exposure levels used. Ni and Cd seem to accumulate primarily in the kidney while Cu mainly accumulated in liver. In contrast to Ni and Cd, the three other metals Ag, Cu and Pb accumulated in the rectal gland, an important organ for osmoregulation and possible target organ for metal toxicity. Only Cu succeeded in initiating a protective response by inducing MT synthesis in liver and gills.

  6. Metallothioneins 1 and 2 Modulate Inflammation and Support Remodeling in Ischemic Cardiomyopathy in Mice

    PubMed Central

    Dewald, Daniela; Schmitz, Eva J.; Verfuerth, Luise; Keppel, Katharina; Peigney, Christine; Ghanem, Alexander; Welz, Armin; Dewald, Oliver

    2016-01-01

    Aims. Repetitive brief ischemia and reperfusion (I/R) is associated with left ventricular dysfunction during development of ischemic cardiomyopathy. We investigated the role of zinc-donor proteins metallothionein MT1 and MT2 in a closed-chest murine model of I/R. Methods. Daily 15-minute LAD-occlusion was performed for 1, 3, and 7 days in SV129 (WT)- and MT1/2 knockout (MT−/−)-mice (n = 8–10/group). Hearts were examined with M-mode echocardiography and processed for histological and mRNA studies. Results. Expression of MT1/2 mRNA was transiently induced during repetitive I/R in WT-mice, accompanied by a transient inflammation, leading to interstitial fibrosis with left ventricular dysfunction without infarction. In contrast, MT−/−-hearts presented with enhanced apoptosis and small infarctions leading to impaired global and regional pump function. Molecular analysis revealed maladaptation of myosin heavy chain isoforms and antioxidative enzymes in MT1/2−/−-hearts. Despite their postponed chemokine induction we found a higher total neutrophil density and macrophage infiltration in small infarctions in MT−/−-hearts. Subsequently, higher expression of osteopontin 1 and tenascin C was associated with increased myofibroblast density resulting in predominately nonreversible fibrosis and adverse remodeling in MT1/2−/−-hearts. Conclusion. Cardioprotective effects of MT1/2 seem to be exerted via modulation of contractile elements, antioxidative enzymes, inflammatory response, and myocardial remodeling. PMID:27403038

  7. Metallothionein and bioaccumulation of cadmium in juvenile bluegills exposed to aqueous and sediment-associated cadmium

    SciTech Connect

    Cope, W.G.

    1991-01-01

    The author evaluated metallothionein (MT), free (unbound) hepatic cadmium and whole body cadmium as indicators of cadmium exposure in juvenile bluegills Lepomis macrochirus in laboratory tests. Two types of cadmium exposure were tested; aqueous and sediment-associated. In the aqueous tests, fish were exposed to cadmium (0.0 to 32.3 [mu]g/L) in an intermittent-flow diluter. In the sediment-associated cadmium test, fish were exposed to resuspended river sidment containing 1.3 to 21.4 [mu]g Cd/g (dry weight) at a nominal total suspended solids concentration of 1,000 mg/L in revolving, circular glass exposure chambers. Total cadmium concentrations were measured in various bluegill liver fractions, whole bluegill, water, and resuspended sediment to assess the partitioning and bioaccumulation of cadmium after the tests. Mean concentrations of MT and free cadmium in bluegill livers and concentrations of cadmium in whole bluegills were positively correlated with aqueous cadmium concentration and were equally suitable as indicators of aqueous cadmium exposure. Sediment-associated cadmium was biologically available, but to a lesser extent than aqueous cadmium. Cadmium concentrations in whole bluegills exposed to resuspended river sediment were 1.5- to 3.5-fold the concentrations in bluegills in sediment-free controls. Free cadmium and MT concentrations in bluegill liver and whole-body cadmium concentrations in bluegills were positively correlated with the cadmium concentrations in filtered water, resuspended sediment, and bulk river sediment; however, whole-body cadmim concentrations were a more sensitive indicator of exposure to sediment-associated cadmium than either free cadmium or MT concentratons in liver.

  8. Mutation at Glu23 eliminates the neuron growth inhibitory activity of human metallothionein-3

    SciTech Connect

    Ding Zhichun; Teng Xinchen; Cai Bin; Wang Hui; Zheng Qi; Wang Yang; Zhou Guoming; Zhang Mingjie; Wu Houming; Sun Hongzhe . E-mail: hsun@hku.hk; Huang Zhongxian . E-mail: zxhuang@fudan.edu.cn

    2006-10-20

    Human metallothionein-3 (hMT3), first isolated and identified as a neuronal growth inhibitory factor (GIF), is a metalloprotein expressed predominantly in brain. However, untill now, the exact mechanism of the bioactivity of hMT3 is still unknown. In order to study the influence of acid-base catalysis on S-nitrosylation of hMT3, we constructed the E23K mutant of hMT3. During the course of bioassay, we found out unexpectedly that mutation at E23 of hMT3 eliminates the neuronal growth inhibitory activity completely. To the best of our knowledge, it is First report that other residues, besides the TCPCP motif, in the {beta}-domain can alter the bioactivity of hMT3. In order to figure out the causes for the loss of bioactivity of the E23K mutant, the biochemical properties were characterized by UV-vis spectroscopy, CD spectroscopy, pH titration, DTNB reaction, EDTA reaction, and SNOC reaction. All data demonstrated that stability of the metal-thiolate cluster and overall structure of the E23K mutant were not altered too much. However, the reaction of the E23K mutant with SNOC exhibited biphasic kinetics and the mutant protein released zinc ions much faster than hMT3 in the initial step, while hMT3 exhibited single kinetic process. The 2D [{sup 1}H-{sup 15}N] HSQC was also employed to characterize structural changes during the reaction of hMT3 with varying mounts of nitric oxide. It was shown that the resonance of Glu23 disappeared at a molar ratio of NO to protein of 4. Based on these results, we suggest that mutation at Glu23 may alter the NO metabolism and/or affect zinc homeostasis in brain, thus altering the neuronal growth inhibitory activity.

  9. Gene response profiles for Daphnia pulex exposed to the environmental stressor cadmium reveals novel crustacean metallothioneins

    PubMed Central

    Shaw, Joseph R; Colbourne, John K; Davey, Jennifer C; Glaholt, Stephen P; Hampton, Thomas H; Chen, Celia Y; Folt, Carol L; Hamilton, Joshua W

    2007-01-01

    Background Genomic research tools such as microarrays are proving to be important resources to study the complex regulation of genes that respond to environmental perturbations. A first generation cDNA microarray was developed for the environmental indicator species Daphnia pulex, to identify genes whose regulation is modulated following exposure to the metal stressor cadmium. Our experiments revealed interesting changes in gene transcription that suggest their biological roles and their potentially toxicological features in responding to this important environmental contaminant. Results Our microarray identified genes reported in the literature to be regulated in response to cadmium exposure, suggested functional attributes for genes that share no sequence similarity to proteins in the public databases, and pointed to genes that are likely members of expanded gene families in the Daphnia genome. Genes identified on the microarray also were associated with cadmium induced phenotypes and population-level outcomes that we experimentally determined. A subset of genes regulated in response to cadmium exposure was independently validated using quantitative-realtime (Q-RT)-PCR. These microarray studies led to the discovery of three genes coding for the metal detoxication protein metallothionein (MT). The gene structures and predicted translated sequences of D. pulex MTs clearly place them in this gene family. Yet, they share little homology with previously characterized MTs. Conclusion The genomic information obtained from this study represents an important first step in characterizing microarray patterns that may be diagnostic to specific environmental contaminants and give insights into their toxicological mechanisms, while also providing a practical tool for evolutionary, ecological, and toxicological functional gene discovery studies. Advances in Daphnia genomics will enable the further development of this species as a model organism for the environmental sciences

  10. Production of hydroxyl radicals by copper-containing metallothionein: roles as prooxidant.

    PubMed

    Suzuki, K T; Rui, M; Ueda, J; Ozawa, T

    1996-11-01

    Production of hydroxyl radicals by copper (Cu)-containing metallothionein (MT) and its relation to zinc (Zn) bound to MT were studied in vitro with reference to the mechanism of the Cu toxicity in the liver of LEC rats. Zn-MT prepared from the liver of Zn-injected rats was reacted with cupric ions at various Cu/Zn ratios, and the concentrations of the two metals bound to MT and in the solution, valence states of Cu in the solution, production of hydroxyl radicals were determined. Cupric ions replaced Zn in MT after being reduced by thiol groups, and MT, worked as an antioxidant. Cupric ions added to MT that did not contain Zn were reduced to cuprous ions by thiol groups in Cu-MT, and the Cu bound to MT was liberated in a form of cuprous ions. Hydroxyl radicals were produced in the presence of hydrogen peroxide in proportion to the amount of cuprous ions liberated from MT. Cu-containing MT was proposed to work as a prooxidant until all thiol groups in MT were oxidized when Zn was not present in MT. The results indicate that MT works as an antioxidant as long as Zn is present in Cu-containing MT, while it works as a prooxidant when Zn is not present by liberating 1.5 M equivalents of cuprous ions relative to cupric ions added, and hydroxyl radicals are produced in the presence of hydrogen peroxide. On the other hand, MT not bound by Cu does not work as a prooxidant throughout.

  11. Protection against UVA-induced photooxidative damage in mammalian cell lines expressing increased levels of metallothionein

    SciTech Connect

    Dudek, E.J. Illinois Inst. of Tech., Chicago, IL . Dept. of Biology); Peak, J.G.; Peak, M.J. ); Roth, R.M. . Dept. of Biology)

    1990-01-01

    Metallothionein (MT) is an endogenous low molecular weight protein that is inducible in a variety of eukaryotic cells and has the ability to selectivity bind heavy metal ions such as zinc and the cadmium. Although the exact physiological role of MT is still not understood, there is strong evidence that MT is involved in providing cellular resistance against the damaging effects of heavy metals and in the regulation of intracellular zinc and copper. Recently, it has been demonstrated that MT can scavenge radiation-induced reactive oxygen intermediates in vitro, specifically hydroxyl and superoxide radicals, and because of these observations it has been suggested that MT may provide protection against radiation-induced oxidative stress in vivo. Cell lines expressing increased levels of MT have demonstrated resistance to ionizing radiation, to ultraviolet radiation, and also to various DNA damaging agents including melphalan and cis-diaminedichloroplatinum. It is therefore important to gain some insight into the relationship between cellular MT content and cellular resistance to radiation and other DNA damaging agents. In this study we investigated the role of MT in providing protection against monochromatic 365-nm UVA radiation, which is known to generate intracellular reactive oxygen species that are involved in both DNA damage and cell killing. For this purpose, we used zinc acetate, a potent inducer of MT, to elevate MT levels in V79 Chinese hamster fibroblasts prior to UVA exposure and determined cell survival for uninduced and induced cultures. In order to eliminate any zinc effects other than MT induction, we also isolated and characterized cadmium chloride-resistant clones of V79 cells that have increased steady-state levels of both MT mRNA and protein, and we examined their survival characteristics against 365-nm radiation in the absence of zinc acetate. 14 refs., 3 figs.

  12. Induction and characterization of metallothionein in chicken epiphyseal growth plate cartilage chondrocytes.

    PubMed

    Sauer, G R; Nie, D; Wu, L N; Wuthier, R E

    1998-01-01

    Following exposure to cadmium or zinc, chickens were sacrificed and the liver, kidney, and bone epiphyseal growth plates harvested. When cytosolic extracts of the growth plate cartilage were fractionated by gel filtration chromatography, a protein with high metal-binding capacity and low ultraviolet (UV) absorbance eluted in the same position as liver metallothionein (MT) and a MT standard. Cd or Zn treatment resulted in a 25-fold or 5-fold induction in growth plate MT, respectively. In liver the greatest level of MT induction was seen with short-term Cd exposures. In contrast, MT levels in the growth plate increased as the duration of Cd exposure increased. Induction of MT in growth plate chondrocyte cell cultures was observed for media Cd concentrations of > or = 0.1 microM and Zn concentrations of > or = 100 microM. Basal and inducible levels of MT declined through the culture period and were lowest in the terminally differentiated mineralized late stages of the culture. Alkaline phosphatase activity was also lowest in the late-stage cultures, while total cellular protein increased throughout the culture period. Treatment of chondrocytes with Zn prior to Cd exposure resulted in a protective induction of MT. Pre-treatment of chondrocytes with dexamethasone resulted in suppressed synthesis of MT upon Cd exposure and greater Cd toxicity. Both Cd and Zn resulted in significantly increased levels of MT mRNA in chondrocyte cell cultures. Dexamethasone treatment resulted in an approximate 2- to 3-fold increase in MT mRNA. This is contrary to the finding that MT protein levels were decreased by dexamethasone. The findings suggest that an increased rate of MT degradation in dexamethasone-treated and late-stage chondrocyte cultures may be associated with the terminally differentiated phenotype.

  13. Different patterns of regulation for the copper and cadmium metallothioneins of the ectomycorrhizal fungus Hebeloma cylindrosporum.

    PubMed

    Ramesh, G; Podila, G K; Gay, G; Marmeisse, R; Reddy, M S

    2009-04-01

    Metallothioneins (MTs) are small cysteine-rich peptides involved in metal homeostasis and detoxification. We have characterized two MT genes, HcMT1 and HcMT2, from the ectomycorrhizal fungus Hebeloma cylindrosporum in this study. Expression of HcMT1 and HcMT2 in H. cylindrosporum under metal stress conditions was studied by competitive reverse transcription-PCR analysis. The full-length cDNAs were used to perform functional complementation in mutant strains of Saccharomyces cerevisiae. As revealed by heterologous complementation assays in yeast, HcMT1 and HcMT2 each encode a functional polypeptide capable of conferring increased tolerance against Cd and Cu, respectively. The expression levels of HcMT1 were observed to be at their maximum at 24 h, and they increased as a function of Cu concentration. HcMT2 was also induced by Cu, but the expression levels were lower than those for HcMT1. The mRNA accumulation of HcMT1 was not influenced by Cd, whereas Cd induced the transcription of HcMT2. Zn, Pb, and Ni did not affect the transcription of HcMT1 or of HcMT2. Southern blot analysis revealed that both of these genes are present as a single copy in H. cylindrosporum. While the promoters of both HcMT1 and HcMT2 contained the standard stress response elements implicated in the metal response, the numbers and varieties of potential regulatory elements were different in these promoters. These results show that ectomycorrhizal fungi encode different MTs and that each of them has a particular pattern of expression, suggesting that they play critical specific roles in improving the survival and growth of ectomycorrhizal trees in ecosystems contaminated by heavy metals.

  14. Molecular evolution and functional divergence of the metallothionein gene family in vertebrates.

    PubMed

    Serén, Nina; Glaberman, Scott; Carretero, Miguel A; Chiari, Ylenia

    2014-04-01

    The metallothionein (MT) gene superfamily consists of metal-binding proteins involved in various metal detoxification and storage mechanisms. The evolution of this gene family in vertebrates has mostly been studied in mammals using sparse taxon or gene sampling. Genomic databases and available data on MT protein function and expression allow a better understanding of the evolution and functional divergence of the different MT types. We recovered 77 MT coding sequences from 20 representative vertebrates with annotated complete genomes. We found multiple MT genes, also in reptiles, which were thought to have only one MT type. Phylogenetic and synteny analyses indicate the existence of a eutherian MT1 and MT2, a tetrapod MT3, an amniote MT4, and fish MT. The optimal gene-tree/species-tree reconciliation analyses identified the best root in the fish clade. Functional analyses reveal variation in hydropathic index among protein domains, likely correlated with their distinct flexibility and metal affinity. Analyses of functional divergence identified amino acid sites correlated with functional divergence among MT types. Uncovering the number of genes and sites possibly correlated with functional divergence will help to design cost-effective MT functional and gene expression studies. This will permit further understanding of the distinct roles and specificity of these proteins and to properly target specific MT for different types of functional studies. Therefore, this work presents a critical background on the molecular evolution and functional divergence of vertebrate MTs to carry out further detailed studies on the relationship between heavy metal metabolism and tolerances among vertebrates.

  15. Metallothionein (MT) response after chronic palladium exposure in the zebra mussel, Dreissena polymorpha

    SciTech Connect

    Frank, Sabrina N.; Singer, Christoph

    2008-11-15

    The effects of different exposure concentrations of palladium (Pd) on relative metallothionein (MT) response and bioaccumulation were investigated in zebra mussels (Dreissena polymorpha). The mussels were exposed to 0.05, 5, 50, and 500 {mu}g/L Pd{sup 2+} for 10 weeks under controlled temperature and fasting conditions. Relative MT contents were assessed by a modified Ag-saturation method, which allows to discriminate between MT bound to Pd (Pd-MT) and MT bound to unidentified metals (Ag-MT). Determination of metal contents resulted from atomic absorption spectrometry following a microwave digestion. For unexposed mussels and mussels exposed to 0.05 {mu}g/L Pd no metal accumulation could be detected. All other exposure concentrations resulted in detectable Pd accumulation in mussels with final tissue concentrations of 96 {mu}g/g (500 {mu}g/L), 45 {mu}g/g (50 {mu}g/L), and 9 {mu}g/g (5 {mu}g/L). Compared with initial levels Pd-MT concentrations at the end of the exposure period were 600 (500 {mu}g/L), 160 (50 {mu}g/L), and 27 (5 {mu}g/L) times higher. These results show that an increase in MTs in D. polymorpha already occurs at relatively low aqueous Pd concentrations indicating that there is the need for detoxification of Pd in the mussel. Furthermore, correlations between Ag-MT and Pd accumulation indicate that higher exposure concentrations are associated with adverse effects on the mussels. Thus, harmful effects of chronic Pd exposure of organisms even in lowest concentrations cannot be excluded in the environment.

  16. Metallothionein Is Downstream of Nrf2 and Partially Mediates Sulforaphane Prevention of Diabetic Cardiomyopathy.

    PubMed

    Gu, Junlian; Cheng, Yanli; Wu, Hao; Kong, Lili; Wang, Shudong; Xu, Zheng; Zhang, Zhiguo; Tan, Yi; Keller, Bradley B; Zhou, Honglan; Wang, Yuehui; Xu, Zhonggao; Cai, Lu

    2017-02-01

    We have reported that sulforaphane (SFN) prevented diabetic cardiomyopathy in both type 1 and type 2 diabetes (T2DM) animal models via the upregulation of nuclear transcription factor erythroid 2-related factor 2 (Nrf2) and metallothionein (MT). In this study, we tested whether SFN protects the heart from T2DM directly through Nrf2, MT, or both. Using Nrf2-knockout (KO), MT-KO, and wild-type (WT) mice, T2DM was induced by feeding a high-fat diet for 3 months followed by a small dose of streptozotocin. Age-matched controls were given a normal diet. Both T2DM and control mice were then treated with or without SFN for 4 months by continually feeding a high-fat or normal diet. SFN prevented diabetes-induced cardiac dysfunction as well as diabetes-associated cardiac oxidative damage, inflammation, fibrosis, and hypertrophy, with increases in Nrf2 and MT expressions in the WT mice. Both Nrf2-KO and MT-KO diabetic mice exhibited greater cardiac damage than WT diabetic mice. SFN did not provide cardiac protection in Nrf2-KO mice, but partially or completely protected the heart from diabetes in MT-KO mice. SFN did not induce MT expression in Nrf2-KO mice, but stimulated Nrf2 function in MT-KO mice. These results suggest that Nrf2 plays the indispensable role for SFN cardiac protection from T2DM with significant induction of MT and other antioxidants. MT expression induced by SFN is Nrf2 dependent, but is not indispensable for SFN-induced cardiac protection from T2DM.

  17. Metallothionein isoform 3 overexpression is associated with breast cancers having a poor prognosis.

    PubMed

    Sens, M A; Somji, S; Garrett, S H; Beall, C L; Sens, D A

    2001-07-01

    The third isoform (MT-3) of the metallothionein gene family is unique in that it has a limited tissue distribution, is not induced by metals, has a neuronal growth inhibitory activity, and sequesters zinc more effectively under zinc-depleted conditions. The goal of the present study was to determine whether MT-3 was absent in normal breast tissue, was overexpressed in breast cancers, and if MT-3 overexpression would be associated with disease outcome. A combination of immunohistochemistry and reverse-transcription polymerase chain reaction was used to demonstrate that the normal breast had no detectable expression of MT-3 mRNA or protein. Using immunohistochemistry, it was shown that MT-3 was overexpressed in 25 of 34 cases of breast cancer. In all cases of positive staining, MT-3 was diffusely localized to the cytoplasm. The tumors from these 34 cases were divided as to outcome based on known 5-year survival, with 20 patients being disease free at 5 years (good outcome) and the other 14 having recurring disease within 5 years (bad outcome). When analyzed for MT-3 staining, it was shown that there was a trend for increased MT-3 immunoreactivity in the group having bad outcomes. However, when the tumor subgrouping was further defined on the basis of carcinoma in situ (CIS), there was a marked significant difference in MT-3 staining between patients with good and bad outcomes. Limited to DCIS, MT-3 staining was significantly increased in patients with bad outcomes compared to those with good outcomes. Thus, these studies demonstrate that MT-3 is overexpressed in selected breast cancers and that overexpression is associated with tumors having a poor prognosis.

  18. Effects of organism preparation in metallothionein and metal analysis in marine invertebrates for biomonitoring marine pollution.

    PubMed

    Oaten, J F P; Hudson, M D; Jensen, A C; Williams, I D

    2015-06-15

    Metallothionein (MT) is established as a potentially useful biomarker for monitoring aquatic pollution. This paper addresses widespread inconsistencies in storage conditions, tissue type selection and pre-treatment of samples before MT and metal analysis in biomarker studies. This variation hampers comparability and so the widespread implementation of this monitoring approach. Actively sampled Mytilus edulis in Southampton Water, UK were exposed to different storage temperatures, a variety of tissue types were analysed, and various pre-treatments of transportation on ice, transportation in seawater, depuration, and rapid dissection in the field were examined. Storage temperatures of -20 °C were found to be adequate for periods of at least ten weeks, as MT was not reduced by protein degradation compared with samples kept at -80 °C. Whole tissue and digestive gland concentrations of MT and metals were significantly positively correlated and directly relatable. MT in the digestive gland appeared to be more responsive to metals than in whole tissue, where it may be diluted, masking MT responses. However, longer study periods may suffer the effects of mass changes to the digestive gland, which alters MT concentration, and it may therefore be advisable to measure whole tissue. Depuration and transportation in seawater reduced both MT and metal concentrations in the digestive gland, and few correlations between MT and metals were identified for these treatments. It is therefore recommended that: i) samples are transported to the laboratory on ice and dissected as soon as possible thereafter, ii) depuration should not be used when examining MT response to metal exposure until further research clarifying its utility is reported, iii) either whole tissue or the digestive gland can be used to measure MT, though whole tissue may be preferable on long-term studies, and iv) organisms can be stored at -20 °C before analysis for up to ten weeks. These practices can be applied

  19. Electrospray ionization mass spectrometry of zinc, cadmium, and copper metallothioneins: evidence for metal-binding cooperativity.

    PubMed Central

    Gehrig, P. M.; You, C.; Dallinger, R.; Gruber, C.; Brouwer, M.; Kägi, J. H.; Hunziker, P. E.

    2000-01-01

    Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed. PMID:10716192

  20. Metallothionein-like proteins in the blue crab Callinectes sapidus: effect of water salinity and ions.

    PubMed

    De Martinez Gaspar Martins, Camila; Bianchini, Adalto

    2009-03-01

    The effect of water salinity and ions on metallothionein-like proteins (MTLP) concentration was evaluated in the blue crab Callinectes sapidus. MTLP concentration was measured in tissues (hepatopancreas and gills) of crabs acclimated to salinity 30 ppt and abruptly subjected to a hypo-osmotic shock (salinity 2 ppt). It was also measured in isolated gills (anterior and posterior) of crabs acclimated to salinity 30 ppt. Gills were perfused with and incubated in an isosmotic saline solution (ISS) or perfused with ISS and incubated in a hypo-osmotic saline solution (HSS). The effect of each single water ion on gill MTLP concentration was also analyzed in isolated and perfused gills through experiments of ion substitution in the incubation medium. In vivo, MTLP concentration was higher in hepatopancreas than in gills, being not affected by the hypo-osmotic shock. However, MTLP concentration in posterior and anterior gills significantly increased after 2 and 24 h of hypo-osmotic shock, respectively. In vitro, it was also increased when anterior and posterior gills were perfused with ISS and incubated in HSS. In isolated and perfused posterior gills, MTLP concentration was inversely correlated with the calcium concentration in the ISS used to incubate gills. Together, these findings indicate that an increased gill MTLP concentration in low salinity is an adaptive response of the blue crab C. sapidus to the hypo-osmotic stress. This response is mediated, at least in part, by the calcium concentration in the gill bath medium. The data also suggest that the trigger for this increase is purely branchial and not systemic.

  1. Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions.

    PubMed

    Lee, Sook-Jeong; Park, Mi-Ha; Kim, Hyun-Jae; Koh, Jae-Young

    2010-08-01

    Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.

  2. Effects of metallothionein on zinc metabolism in lethal-milk mutant mice

    SciTech Connect

    Grider, A. Jr.

    1986-01-01

    The lethal-milk mice (C57BL/6J-Im) exhibit various pleiotropic effects, including a congenital otolith defect, production of zinc-deficient milk, and clinical signs of a systemic Zn deficiency by one year of age. The clinical signs include alopecia, dermatitis, and skin lesions. The systemic zinc deficiency may be due to increased levels of metallothionein (MT) in the intestine and/or liver of Im mice. The untreated Im mice contain twice as much intestinal MT as do C57BL/6J-(+/sup im//+ /sup Im/) (B6) controls. This was determined by a sulfhydryl assay, by the /sup 109/Cd-saturation/hemolysate method, and by the /sup 65/Zn-binding assay. Various concentrations of Cd or Zn were added to the drinking water three days before assaying for MT. Compared to B6 mice, the Im mice exhibited more MT in their liver by the /sup 65/Zn-MT binding assay (3-fold) and by the /sup 109/Cd-saturation/hemolysate method (18-fold). The effects of the two zinc treatments did not differ significantly between Im and B6 mice. The retention and excretion of /sup 65/Zn (administered intraperitoneally) were determined over a 14-day period, but the results did not different between the Im and B6 mice. The increased concentrations of MT within the Im mice was not significantly different for the intestine and liver. Based on these data and other studies, the Im mice may exhibit alterations in zinc homeostasis due to some deregulation of MT metabolism, including the inner ear of the fetus, the lactating mammary gland, and the intestine and liver of adults by one year of age.

  3. How does the metallothionein induction in bivalves meet the criteria for biomarkers of metal exposure?

    PubMed

    Le, T T Yen; Zimmermann, Sonja; Sures, Bernd

    2016-05-01

    Metallothionein (MT) concentrations in the whole soft tissue or in a particular tissue of bivalves have widely been used in ecotoxicological studies and biomonitoring programmes. This approach is based on the reported results on the enhancement of MT induction in bivalves in response to metal exposure. The validity of using MT induction as a biomarker is briefly assessed in the present study. The sensitivity of MT induction in these organisms is questionable due to the high basal MT level as well as the high natural variability related to the effects of a number of biotic and abiotic factors, which are not well described yet. Moreover, the relationship between exposure to metals, the toxic effects of that exposure, and the appearance of MT in soft tissue, is not well characterized. A variety of factors may influence the appearance and distribution of MT: 1) the uneven distribution of metals in particular portions of the soft tissue and in particular subcellular compartments; 2) pre-exposure to metals, perhaps at non-toxic levels; 3) metal-metal competition and metal-protein interactions; and 4) tissue-specific induction, functions, and isoforms of MT. Therefore, attention is required when using MT induction in bivalves for assessment of metal exposure or consequent toxic effects. The MT concentration can be a reliable indicator only when it is considered in relation with metal uptake kinetics and subcellular partitioning while specifying the isoform of MT synthesised and considering various confounding factors. The kinetic turnover of MT may provide useful information on metal exposure and biological effects since it covers both the synthesis and breakdown of MT as well as the chemical species of metals accumulated and MT.

  4. Oxidative stress biomarkers and metallothionein in Folsomia candida--responses to Cu and Cd.

    PubMed

    Maria, Vera L; Ribeiro, Maria João; Amorim, Mónica J B

    2014-08-01

    Folsomia candida (Collembola) is a standard soil ecotoxicological species; effect assessment includes survival and reproduction as endpoints. In the present study, and for the first time, a range of oxidative stress biomarkers measurement was optimized and validated. The antioxidant capacity was measured by the activities of catalase (CAT), glutathione reductase (GR), glutathione-s-transferase (GST) and content of total glutathione (TG). The oxidative damage in the lipid membranes was estimated by lipid peroxidation (LPO) and metallothionein (MT) levels. The exposure included the essential and non-essential metals Cu and Cd, in LUFA 2.2 natural standard soil, using a series of sampling times along a 10 days period (0, 2, 4, 6 and 10 days). Exposure concentrations were selected based on their reproduction EC50 values, 60 and 1000 mg/kg soil DW, for Cd and Cu respectively. The protocols were optimized and results show that oxidative stress biomarkers can be successfully used in F. candida, this being highly relevant as complementary information to the mechanistic level. The selected sampling times gave a good indication of the markers dynamic and can be reduced/adapted in future testing. Results showed that both metals caused an increase in the MT levels after 6 days but Cd acted as a stronger oxidant agent compared to Cu, i.e. causing higher damage. In sum, Cd mobilized/activated more antioxidant enzymes, but the increased activities were not enough to prevent LPO. This study confirms that the oxidative stress caused by Cd is higher despite the use of same reproduction EC50 indicating that toxicity seems more reversible for Cu than for Cd. Among others, GST and MT would be a good selection of biomarkers for Cd effect.

  5. An International Bioinformatics Infrastructure to Underpin the Arabidopsis Community

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The future bioinformatics needs of the Arabidopsis community as well as those of other scientific communities that depend on Arabidopsis resources were discussed at a pair of recent meetings held by the Multinational Arabidopsis Steering Committee (MASC) and the North American Arabidopsis Steering C...

  6. Novel Vein Patterns in Arabidopsis Induced by Small Molecules1[OPEN

    PubMed Central

    Cutler, Sean

    2016-01-01

    The critical role of veins in transporting water, nutrients, and signals suggests that some key regulators of vein formation may be genetically redundant and, thus, undetectable by forward genetic screens. To identify such regulators, we screened more than 5000 structurally diverse small molecules for compounds that alter Arabidopsis (Arabidopsis thaliana) leaf vein patterns. Many compound-induced phenotypes were observed, including vein networks with an open reticulum; decreased or increased vein number and thickness; and misaligned, misshapen, or nonpolar vascular cells. Further characterization of several individual active compounds suggests that their targets include hormone cross talk, hormone-dependent transcription, and PIN-FORMED trafficking. PMID:26574596

  7. Using "Arabidopsis" Genetic Sequences to Teach Bioinformatics

    ERIC Educational Resources Information Center

    Zhang, Xiaorong

    2009-01-01

    This article describes a new approach to teaching bioinformatics using "Arabidopsis" genetic sequences. Several open-ended and inquiry-based laboratory exercises have been designed to help students grasp key concepts and gain practical skills in bioinformatics, using "Arabidopsis" leucine-rich repeat receptor-like kinase (LRR…

  8. Metallothionein 1A polymorphisms may influence urine uric acid and N-acetyl-beta-D-glucosaminidase (NAG) excretion in chronic lead-exposed workers.

    PubMed

    Yang, Chen-Cheng; Chen, Hsin-I; Chiu, Yu-Wen; Tsai, Chih-Hung; Chuang, Hung-Yi

    2013-04-05

    Lead is a renal toxin, and susceptibility to lead varies between individuals. Metallothionein (MT) is known for its metal scavenging role. The aim of the study was to investigate the association of blood lead levels, urinary uric acid (UA) and N-acetyl-beta-d-glucosaminidase (NAG) in chronic occupational lead-exposed workers, and to study whether the association was influenced by MT1A gene polymorphisms. In this cross-sectional study, 412 lead-exposed workers participated. Their annual health examination data and renal function markers were collected after the Institutional Review Broad of Kaohsiung Medical University Hospital approved the study and consent letters were obtained. From the blood samples, DNA was extracted and used for real-time PCR typing of 2 MT1A single nucleotide polymorphisms (SNPs): rs11640851 and rs8052394 on exons 2 and 3. Descriptive analysis, one-way ANOVA, and multiple linear regressions were performed. There was a significant inverted relationship of creatinine-adjusted urine UA concentrations and the time-weighted index of cumulative blood lead levels (TWICL) that may be significantly influenced by the AC genotypes of rs11640851 in exon 2 and rs8052394 in exon 3. After controlling for potential confounding factors, the creatinine-adjusted urine NAG concentrations were shown to be influenced by the GG genotype of rs8052394 in exon 3, and were weakly increased with TWICL. Therefore, we concluded that the variations of MT1A SNPs may influence urine UA and NAG excretion in chronic lead-exposed workers, and urine creatinine-adjusted urine UA as a biomarker of lead toxicity should be considered.

  9. Metallothionein 2A core promoter region genetic polymorphism and its impact on the risk, tumor behavior, and recurrences of sinonasal inverted papilloma (Schneiderian papilloma).

    PubMed

    Starska, Katarzyna; Bryś, Magdalena; Forma, Ewa; Olszewski, Jurek; Pietkiewicz, Piotr; Lewy-Trenda, Iwona; Stasikowska-Kanicka, Olga; Danilewicz, Marian; Krześlak, Anna

    2015-11-01

    Inverted papillomas are a unique group of locally aggressive benign epithelial neoplasms in the nasal cavity and paranasal sinuses arising from the Schneiderian mucosa. Metallothioneins are sulfhydryl-rich heavy metal-binding proteins required for metal toxicity protection and regulation of biological mechanisms including proliferation and invasion. The goal of this study was to identify three SNPs at loci -5 A/G (rs28366003) and -209 A/G (rs1610216) in the core promoter region and at locus +838 C/G (rs10636) in 3'UTR region of the MT2A gene with IP risk and with tumor invasiveness according to Krouse staging. Genotyping was performed using the PCR restriction fragment length polymorphism technique in 130 genetically unrelated IP individuals, and 418 randomly selected healthy volunteers. The presence of the rs28366003 SNP was significantly related to the risk of IP within the present population-based case-control study. Compared to homozygous common allele carriers, heterozygosity and homozygosity for the G variant had a significantly increased risk of IP (adjusted odds ratio [OR] = 7.71, 95% confidence interval [CI]: 4.01-14.91, p(dominant) < 0.001). Moreover, risk allele carriers demonstrated higher Krouse stage (pT1 vs. pT2-4) (OR = 19.32; 95% CI, 2.30-173.53; p < 0.0001), diffuse tumor growth (OR = 4.58; 95% CI, 1.70-12.11; p = 0.0008), bone destruction (OR = 4.13; 95% CI, 1.50-11.60; p = 0.003), and higher incidence of tumor recurrences (OR = 5.11; 95% CI, 1.68-15.20; p = 0.001). The findings suggest that MT2A gene variation rs28366003 may be implicated in the etiology of sinonasal inverted papilloma in a Polish population.

  10. Genome Sequencing Reveals the Origin of the Allotetraploid Arabidopsis suecica.

    PubMed

    Novikova, Polina Yu; Tsuchimatsu, Takashi; Simon, Samson; Nizhynska, Viktoria; Voronin, Viktor; Burns, Robin; Fedorenko, Olga M; Holm, Svante; Säll, Torbjörn; Prat, Elisa; Marande, William; Castric, Vincent; Nordborg, Magnus

    2017-04-01

    Polyploidy is an example of instantaneous speciation when it involves the formation of a new cytotype that is incompatible with the parental species. Because new polyploid individuals are likely to be rare, establishment of a new species is unlikely unless polyploids are able to reproduce through self-fertilization (selfing), or asexually. Conversely, selfing (or asexuality) makes it possible for polyploid species to originate from a single individual-a bona fide speciation event. The extent to which this happens is not known. Here, we consider the origin of Arabidopsis suecica, a selfing allopolyploid between Arabidopsis thaliana and Arabidopsis arenosa, which has hitherto been considered to be an example of a unique origin. Based on whole-genome re-sequencing of 15 natural A. suecica accessions, we identify ubiquitous shared polymorphism with the parental species, and hence conclusively reject a unique origin in favor of multiple founding individuals. We further estimate that the species originated after the last glacial maximum in Eastern Europe or central Eurasia (rather than Sweden, as the name might suggest). Finally, annotation of the self-incompatibility loci in A. suecica revealed that both loci carry non-functional alleles. The locus inherited from the selfing A. thaliana is fixed for an ancestral non-functional allele, whereas the locus inherited from the outcrossing A. arenosa is fixed for a novel loss-of-function allele. Furthermore, the allele inherited from A. thaliana is predicted to transcriptionally silence the allele inherited from A. arenosa, suggesting that loss of self-incompatibility may have been instantaneous.

  11. Environmental History Modulates Arabidopsis Pattern-Triggered Immunity in a HISTONE ACETYLTRANSFERASE1-Dependent Manner.

    PubMed

    Singh, Prashant; Yekondi, Shweta; Chen, Po-Wen; Tsai, Chia-Hong; Yu, Chun-Wei; Wu, Keqiang; Zimmerli, Laurent

    2014-06-01

    In nature, plants are exposed to a fluctuating environment, and individuals exposed to contrasting environmental factors develop different environmental histories. Whether different environmental histories alter plant responses to a current stress remains elusive. Here, we show that environmental history modulates the plant response to microbial pathogens. Arabidopsis thaliana plants exposed to repetitive heat, cold, or salt stress were more resistant to virulent bacteria than Arabidopsis grown in a more stable environment. By contrast, long-term exposure to heat, cold, or exposure to high concentrations of NaCl did not provide enhanced protection against bacteria. Enhanced resistance occurred with priming of Arabidopsis pattern-triggered immunity (PTI)-responsive genes and the potentiation of PTI-mediated callose deposition. In repetitively stress-challenged Arabidopsis, PTI-responsive genes showed enrichment for epigenetic marks associated with transcriptional activation. Upon bacterial infection, enrichment of RNA polymerase II at primed PTI marker genes was observed in environmentally challenged Arabidopsis. Finally, repetitively stress-challenged histone acetyltransferase1-1 (hac1-1) mutants failed to demonstrate enhanced resistance to bacteria, priming of PTI, and increased open chromatin states. These findings reveal that environmental history shapes the plant response to bacteria through the development of a HAC1-dependent epigenetic mark characteristic of a primed PTI response, demonstrating a mechanistic link between the primed state in plants and epigenetics.

  12. High cadmium-binding ability of a novel Colocasia esculenta metallothionein increases cadmium tolerance in Escherichia coli and tobacco.

    PubMed

    Kim, Yeon-Ok; Patel, Darshan H; Lee, Dae-Seok; Song, Younho; Bae, Hyeun-Jong

    2011-01-01

    Experimental evidence in vivo as to the functional roles and binding properties to cadmium (Cd) of type-2 plants metallothionein (MT) has been limited thus far. We investigated the biological role of metallothionein from Colocasia esculenta (CeMT2b) in Escherichia coli and tobacco, and developed a new model for the relationship between Cd tolerance and Cd-binding ability. Heterologous expression of CeMT2b in Escherichia coli greatly enhanced Cd tolerance and accumulated Cd content as compared to control cells. The molecular weight of CeMT2b increased with Cd, and CeMT2b bound up to 5.96±1 molar ratio (Cd/protein). Under Cd stress, transgenic tobacco plants displayed much better seedling growth and high Cd accumulation than the wild type. The presence of an extra CXC motif in CeMT2b contributed to the enhanced Cd-tolerance. The present study provides the first insight into the ability of type-2 plant MT to bind physiological Cd.

  13. Characterization of calcineurin-dependent response element binding protein and its involvement in copper-metallothionein gene expression in Neurospora

    SciTech Connect

    Kumar, Kalari Satish; Ravi Kumar, B.; Siddavattam, Dayananda; Subramanyam, Chivukula . E-mail: csubramanyam@hotmail.com

    2006-07-07

    In continuation of our recent observations indicating the presence of a lone calcineurin-dependent response element (CDRE) in the -3730 bp upstream region of copper-induced metallothionein (CuMT) gene of Neurospora [K.S. Kumar, S. Dayananda, C. Subramanyam, Copper alone, but not oxidative stress, induces copper-metallothionein gene in Neurospora crassa, FEMS Microbiol. Lett. 242 (2005) 45-50], we isolated and characterized the CDRE-binding protein. The cloned upstream region of CuMT gene was used as the template to specifically amplify CDRE element, which was immobilized on CNBr-activated Sepharose 4B for use as the affinity matrix to purify the CDRE binding protein from nuclear extracts obtained from Neurospora cultures grown in presence of copper. Two-dimensional gel electrophoresis of the affinity purified protein revealed the presence of a single 17 kDa protein, which was identified and characterized by MALDI-TOF. Peptide mass finger printing of tryptic digests and analysis of the 17 kDa protein matched with the regulatory {beta}-subunit of calcineurin (Ca{sup 2+}-calmodulin dependent protein phosphatase). Parallel identification of nuclear localization signals in this protein by in silico analysis suggests a putative role for calcineurin in the regulation of CuMT gene expression.

  14. Coordinated responses of phytochelatin synthase and metallothionein genes in black mangrove, Avicennia germinans, exposed to cadmium and copper.

    PubMed

    Gonzalez-Mendoza, Daniel; Moreno, Adriana Quiroz; Zapata-Perez, Omar

    2007-08-01

    To evaluate the role of phytochelatins and metallothioneins in heavy metal tolerance of black mangrove Avicennia germinans, 3-month-old seedlings were exposed to cadmium or copper for 30 h, under hydroponic conditions. Degenerate Mt2 and PCS primers were synthesized based on amino acid and nucleotide alignment sequences reported for Mt2 and PCS in other plant species found in GenBank. Total RNA was isolated from A. germinans leaves and two partial fragments of metallothionein and phytochelatin synthase genes were isolated. Gene expression was evaluated with reverse transcripatase-polymerase chain reaction (RT-PCR) amplification technique. Temporal analysis showed that low Cd2+ and Cu2+ concentrations caused a slight (but not significant) increase in AvMt2 expression after a 16 h exposure time, while AvPCS expression showed a significant increase under the same conditions but only after 4h. Results strongly suggest that the rapid increase in AvPCS expression may contribute to Cd2+ and Cu2+ detoxification. Moreover, we found that A. germinans has the capacity to over-express both genes (AvMt2 and AvPCS), which may constitute a coordinated detoxification response mechanism targeting non-essential metals. Nonetheless, our results confirm that AvPCS was the most active gene involved in the regulation of essential metals (e.g., Cu2+) in A. germinans leaves.

  15. Whole‐cell biosensors for detection of heavy metal ions in environmental samples based on metallothionein promoters from Tetrahymena thermophila

    PubMed Central

    Amaro, Francisco; Turkewitz, Aaron P.; Martín‐González, Ana; Gutiérrez, Juan‐Carlos

    2011-01-01

    Summary Heavy metals are among the most serious pollutants, and thus there is a need to develop sensitive and rapid biomonitoring methods for heavy metals in the environment. Critical parameters such as bioavailability, toxicity and genotoxicity cannot be tested using chemical analysis, but only can be assayed using living cells. A whole‐cell biosensor uses the whole cell as a single reporter incorporating both bioreceptor and transducer elements. In the present paper, we report results with two gene constructs using the Tetrahymena thermophila MTT1 and MTT5 metallothionein promoters linked with the eukaryotic luciferase gene as a reporter. This is the first report of a ciliated protozoan used as a heavy metal whole‐cell biosensor. T. thermophila transformed strains were created as heavy metal whole‐cell biosensors, and turn on bioassays were designed to detect, in about 2 h, the bioavailable heavy metals in polluted soil or aquatic samples. Validation of these whole‐cell biosensors was carried out using both artificial and natural samples, including methods for detecting false positives and negatives. Comparison with other published cell biosensors indicates that the Tetrahymena metallothionein promoter‐based biosensors appear to be the most sensitive eukaryotic metal biosensors and compare favourably with some prokaryotic biosensors as well. PMID:21366892

  16. Pseudomonas aeruginosa strain WI-1 from Mandovi estuary possesses metallothionein to alleviate lead toxicity and promotes plant growth.

    PubMed

    Naik, Milind Mohan; Pandey, Anju; Dubey, Santosh Kumar

    2012-05-01

    A bacterial isolate from Mandovi estuary Goa, India, which can resist 0.6mM lead nitrate in Tris-buffered minimal medium was identified as Pseudomonas aeruginosa and designated as strain WI-1. PCR amplification clearly revealed presence of bmtA gene encoding bacterial metallothionein responsible for metal sequestration and AAS analysis proved intracellular bioaccumulation of 26.5mg lead/gram dry weight of cells. SDS-PAGE analysis confirmed lead induced bacterial metallothionein with molecular weight 11 kDa, which corresponds to the predicted bmtA gene. Significant growth inhibition of phytopathogenic fungi Fusarium oxysporum NCIM 1008 by siderophore-rich culture supernatant was also observed. Pot experiment with Pisum sativum L inoculated with this strain revealed higher seed germination percentage and significant growth promotion than uninoculated seeds in a soil amended with 7.704 g/kg lead, which indicates amelioration of lead toxicity. This lead resistant strain showed cross tolerance to cadmium, mercury and Tributyltin chloride (TBTC) along with resistance to multiple antibiotics.

  17. Cell Surface Display of Four Types of Solanum nigrum Metallothionein on Saccharomyces cerevisiae for Biosorption of Cadmium.

    PubMed

    Wei, Qinguo; Zhang, Honghai; Guo, Dongge; Ma, Shisheng

    2016-05-28

    We displayed four types of Solanum nigrum metallothionein (SMT) for the first time on the surface of Saccharomyces cerevisiae using an α-agglutinin-based display system. The SMT genes were amplified by RT-PCR. The plasmid pYES2 was used to construct the expression vector. Transformed yeast strains were confirmed by PCR amplification and custom sequencing. Surface-expressed metallothioneins were indirectly indicated by the enhanced cadmium sorption capacity. Flame atomic absorption spectrophotometry was used to examine the concentration of Cd(2+) in this study. The transformed yeast strains showed much higher resistance ability to Cd(2+) compared with the control. Strikingly, their Cd(2+) accumulation was almost twice as much as that of the wild-type yeast cells. Furthermore, surface-engineered yeast strains could effectively adsorb ultra-trace cadmium and accumulate Cd(2+) under a wide range of pH levels, from 3 to 7, without disturbing the Cu(2+) and Hg(2+). Four types of surfaceengineered Saccharomyces cerevisiae strains were constructed and they could be used to purify Cd(2+)-contaminated water and adsorb ultra-trace cadmium effectively. The surface-engineered Saccharomyces cerevisiae strains would be useful tools for the bioremediation and biosorption of environmental cadmium contaminants.

  18. Expression of metallothioneins I and II related to oxidative stress in the liver of aluminium-treated rats.

    PubMed

    Ghorbel, Imen; Chaabane, Mariem; Elwej, Awatef; Boudawara, Ons; Abdelhedi, Sameh; Jamoussi, Kamel; Boudawara, Tahya; Zeghal, Najiba

    2016-10-01

    Hepatotoxicity, induced by aluminium chloride (AlCl3), has been well studied but there are no reports about liver metallothionein (MT) genes induction. Therefore, it is of interest to establish the mechanism involving the relation between MT gene expression levels and the oxidative stress status in hepatic cells of aluminium-treated rats. Aluminium (Al) was administered to rats in their drinking water at a dose of 50 mg/kg body weight for three weeks. AlCl3 provoked hepatotoxicity objectified by an increase in malondialdehyde (MDA), hydrogen peroxide (H2O2), advanced oxidation protein products (AOPP), protein carbonyls (PCO) and a decrease in reduced glutathione (GSH), non-protein thiols (NPSH) and vitamin C. CAT and Glutathione peroxidase (GPx) activities were decreased while Mn-SOD gene expression, total Metallothionein content and MT I and MT II genes induction were increased. There are changes in plasma of some trace elements, albumin levels, transaminases, LDH and ALP activities. All these changes were supported by histopathological observations.

  19. Effects of pergolide mesilate on metallothionein mRNAs expression in a mouse model for Parkinson disease.

    PubMed

    Ono, Shin-ichi; Hirai, Kohji; Tokuda, Ei-ichi

    2009-10-01

    Dopamine agonists have neuroprotective properties in addition to their original pharmacologic function. We examined the effects of pergolide mesilate (PM) on the levels of metallothionein mRNA expression and lipid peroxidation in the corpus striata of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonian mice. Mice were administered normal saline (vehicle as a control), PM, or MPTP. A consecutive 7-d administration of MPTP via a gastric tube at a dose of 30 mg/kg significantly decreased metallothionein (MT)-I mRNA expression but did not influence MT-III mRNA expression. Lipid peroxidation, measured as the production of malondialdehyde reactive substances, did not increase after MPTP treatment. Although PM administration alone did not effect MT-I expression, an additional consecutive 7-d administration of PM (30 mug/kg) following MPTP treatment recovered the decreased MT-I level and increased MT-III expression. Lipid peroxidation was significantly suppressed. These results suggest that PM exerts an antioxidative property through the induction of MT-I and MT-III mRNAs simultaneously in response to cellular and/or tissue injury.

  20. Cadmium binding studies to the earthworm Lumbricus rubellus metallothionein by electrospray mass spectrometry and circular dichroism spectroscopy

    SciTech Connect

    Ngu, Thanh T.; Sturzenbaum, Stephen R.; Stillman, Martin J. . E-mail: Martin.Stillman@uwo.ca

    2006-12-08

    The earthworm Lumbricus rubellus has been found to inhabit cadmium-rich soils and accumulate cadmium within its tissues. Two metallothionein (MT) isoforms (1 and 2) have been identified and cloned from L. rubellus. In this study, we address the metalation status, metal coordination, and structure of recombinant MT-2 from L. rubellus using electrospray ionization mass spectrometry (ESI-MS), UV absorption, and circular dichroism (CD) spectroscopy. This is the first study to show the detailed mass and CD spectral properties for the important cadmium-containing earthworm MT. We report that the 20-cysteine L. rubellus MT-2 binds seven Cd{sup 2+} ions. UV absorption and CD spectroscopy and ESI-MS pH titrations show a distinct biphasic demetalation reaction, which we propose results from the presence of two metal-thiolate binding domains. We propose stoichiometries of Cd{sub 3}Cys{sub 9} and Cd{sub 4}Cys{sub 11} based on the presence of 20 cysteines split into two isolated regions of the sequence with 11 cysteines in the N-terminal and 9 cysteines in the C-terminal. The CD spectrum reported is distinctly different from any other metallothionein known suggesting quite different binding site structure for the peptide.

  1. Cloning, characterization and gene expression of a metallothionein isoform in the edible cockle Cerastoderma edule after cadmium or mercury exposure.

    PubMed

    Paul-Pont, Ika; Gonzalez, Patrice; Montero, Natalia; de Montaudouin, Xavier; Baudrimont, Magalie

    2012-01-01

    Metallothionein (MT) genes encode crucial metal-binding proteins ubiquitously expressed in living organisms and which play important roles in homeostasis of essential metals and detoxification processes. Here, the molecular organization of the first metallothionein gene of the edible cockle Cerastoderma edule and its expression after cadmium (Cd) or mercury (Hg) exposures were determined. The resulting sequence (Cemt1) exhibits unusual features. The full length cDNA encodes a protein of 73 amino acids with nine classical Cys-X((1-3))-Cys motifs, but also one Cys-Cys not generally found in molluscan MT. Moreover, characterization of the molecular organization of the Cemt1 gene revealed two different alleles (A1 and A2) with length differences due to large deletion events in their intronic sequences involving direct Short Interspersed repeated Elements (SINE), while their exonic sequences were identical. To our knowledge, such large excision mechanisms have never before been reported in a bivalve gene sequence. After 10 days of Cd exposure at environmentally relevant doses, quantitative real-time PCR revealed a strong induction of Cemt1 in gills of C. edule. Surprisingly, neither induction of the Cemt1 gene nor of MT protein was shown after Hg exposure, despite the fact that this organism is able to bioaccumulate a high amount of this trace metal which is theoretically one of the most powerful inducers of MT biosynthesis.

  2. Influence of cadmium on metallothionein expression and products of lipid peroxidation in the organs of hares (Lepus europaeus Pallas).

    PubMed

    Linšak, Dijana Tomić; Linšak, Zeljko; Spirić, Zdravko; Srebočan, Emil; Glad, Marin; Cenov, Arijana; Jakovac, Hrvoje; Milin, Cedomila

    2014-03-01

    Cadmium occurs naturally in the environment and as an anthropogenic pollutant. Exposure to low concentrations of cadmium is inevitable and may produce toxic effects. Another important aspect of cadmium toxicity is its interaction, often antagonistic, with essential elements such as selenium. The aim of this study was to highlight the risks of long-term exposure to low cadmium concentrations, using a scientific and chemical approach and hares (Lepus europaeus Pallas) as model organisms in a field study. Two study areas were monitored. Levels of cadmium and selenium were quantified in the organs of hares, the expression of metallothioneins I + II and the products of lipid peroxidation were determined. The median cadmium concentrations (wet weight) in the muscle, liver, kidney and brain of hares from an exposed group ranged from 0.033 to 0.037, 0.763 to 1.054, 3.090 to 16.594 and 0.016 to 0.087 µg g(-1), respectively; whereas, the median selenium concentrations (wet weight) ranged from 0.100 to 0.108, 0.153 to 0.332, 0.677 to 0.701 and 0.078 to 0.116 µg g(-1), respectively. Expression of the metallothioneins I + II proteins was observed in tissues. Lipid peroxidation (LPO) levels, measured as malondialdehyde (MDA) equivalents, increased with the cadmium concentration. Further research on long-term exposure to low concentrations of cadmium in the environment is needed.

  3. A comparison of the effects of penicillamine, trientine, and trithiomolybdate on ( sup 35 S)-labeled metallothionein in vitro; implications for Wilson's disease therapy

    SciTech Connect

    McQuaid, A.; Mason, J. )

    1991-02-01

    The synthesis of radiolabeled metallothionein was induced in rats in vivo by the injection of CuSO{sub 4} and ({sup 35}S)-cysteine. Treatment of 'cold' rat liver cytosol 'spiked' with purified ({sup 35}S) metallothionein with Penicillamine and Trientine showed that even at relatively high concentrations (up to 50 mg/g liver, wet weight), these compounds had no effect on the copper peak or the position of the ({sup 35S}) label in the cytosol eluate after Sephadex G-75 gel filtration. By contrast, incubation of the 'spiked' liver cytosol with Trithiomolybdate, even at relatively low concentrations (0.5 mg/g liver, wet weight), resulted in a transfer of metallothionein copper to high molecular weight protein fractions; the position of the ({sup 35}S) apoprotein was unaffected. This copper 'stripping' effect on metallothionein supports clinical and other evidence that thiomolybdates have a genuine decoppering effect in vivo whereas Penicillamine and Trientine have another mode of action and indicates that thiomolybdates might provide a more rational alternate therapy for Wilson's disease patients.

  4. Evaluation of metallothionein formation as a proxy for zinc absorption in an in vitro digestion/caco-2 cell culture model

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Caco-2 cell metallothionein (MT) formation was studied to determine if MT could be used as a proxy for zinc (Zn) absorption in a cell culture model. MT intracellular concentration was determined by using a cadmium/hemoglobin affinity assay. Cellular Zn uptake was determined in acid digests (5% HNO3)...

  5. Two Metallothionein Genes in Oxya chinensis: Molecular Characteristics, Expression Patterns and Roles in Heavy Metal Stress

    PubMed Central

    Liu, Yaoming; Wu, Haihua; Kou, Lihua; Liu, Xiaojian; Zhang, Jianzhen; Guo, Yaping; Ma, Enbo

    2014-01-01

    Metallothioneins (MTs) are small, cysteine-rich, heavy metal-binding proteins involved in metal homeostasis and detoxification in living organisms. In the present study, we cloned two MT genes (OcMT1 and OcMT2) from Oxya chinensis, analyzed the expression patterns of the OcMT transcripts in different tissues and at varying developmental stages using real-time quantitative PCR (RT-qPCR), evaluated the functions of these two MTs using RNAi and recombinant proteins in an E. coli expression system. The full-length cDNAs of OcMT1 and OcMT2 encoded 40 and 64 amino acid residues, respectively. We found Cys-Cys, Cys-X-Cys and Cys-X-Y-Z-Cys motifs in OcMT1 and OcMT2. These motifs might serve as primary chelating sites, as in other organisms. These characteristics suggest that OcMT1 and OcMT2 may be involved in heavy metal detoxification by capturing the metals. Two OcMT were expressed at all developmental stages, and the highest levels were found in the eggs. Both transcripts were expressed in all eleven tissues examined, with the highest levels observed in the brain and optic lobes, followed by the fat body. The expression of OcMT2 was also relatively high in the ovaries. The functions of OcMT1 and OcMT2 were explored using RNA interference (RNAi) and different concentrations and treatment times for the three heavy metals. Our results indicated that mortality increased significantly from 8.5% to 16.7%, and this increase was both time- and dose-dependent. To evaluate the abilities of these two MT proteins to confer heavy metal tolerance to E. coli, the bacterial cells were transformed with pET-28a plasmids containing the OcMT genes. The optical densities of both the MT-expressing and control cells decreased with increasing concentrations of CdCl2. Nevertheless, the survival rates of the MT-overexpressing cells were higher than those of the controls. Our results suggest that these two genes play important roles in heavy metal detoxification in O. chinensis. PMID:25391131

  6. Toxicity, silver accumulation and metallothionein induction in freshwater rainbow trout during exposure to different silver salts

    SciTech Connect

    Hogstrand, C.; Galvez, F.; Wood, C.M.

    1996-07-01

    Static-renewal 168-h toxicity tests of silver nitrate (AgNO{sub 3}), silver chloride (AgCl{sub n}), and silver thiosulfate (Ag(S{sub 2}O{sub 3}){sub n}) with juvenile rainbow trout (Oncorhyncus mykiss) were performed by standard methods. Because of low solubility of AgCl(s), bioassays for AgCl{sub n} were performed in two separate ways. In one test series, AgCl(s) was added to freshwater and in another, AgCl{sub n}(aq) was generated by adding AgNO{sub 3} to freshwater supplemented with 50 mM NaCl. Concentrations of Ag and metallothionein (MT) were analyzed in gills and livers of fish that survived the exposures. Although Ag added as AgNO{sub 3} was found to be highly toxic to rainbow trout (168-h LC50 = 9.1 {micro}g Ag L{sup {minus}1}), the toxicities of the other Ag salts were low. The 168-h LC50 for Ag(S{sub 2}O{sub 3}){sub n} was 137,000 {micro}g Ag L{sup {minus}1} and no mortality was observed in AgCl{sub n} (100,000 {micro}g Ag L{sup {minus}1}). Exposure to AgNO{sub 3}, Ag(S{sub 2}O{sub 3}){sub n}, or AgCl{sub n} caused accumulation of Ag and induction of MT. Highest Ag levels were found in livers of trout exposed to 164,000 {micro}g Ag L{sup {minus}1} as Ag(S{sub 2}O{sub 3}){sub n}. In these fish, the hepatic Ag concentration was increased 335 times from the control value. The MT levels in gills and liver increased with the water Ag concentration and the highest level of MT was found in liver of fish exposed to Ag(S{sub 2}O{sub 3}){sub n}.

  7. Metal binding and antioxidant properties of chimeric tri- and tetra-domained metallothioneins.

    PubMed

    Moreau, Jean-Luc; Baudrimont, Magalie; Carrier, Patrick; Peltier, Gilles; Bourdineaud, Jean-Paul

    2008-05-01

    An unusual tri-domained (alpha-beta-beta) natural oyster metallothionein (MT) is known, and non-oxidative MT dimers occur in vivo in mollusk species and in mammals. To assess the respective role of the MT domains, two chimeric MTs were constructed: a tetra-domained oyster MT corresponding to the alpha-beta-alpha-beta structure, in order to mimic the natural non-oxidative dimeric form, and a tri-domained alpha-beta-alpha oyster MT. Metal binding and putative antioxidant properties of these two chimeric MTs were investigated using expression of the related genes in the bacteria Escherichia coli. In a wild-type strain these MTs could efficiently bind Cd. In a superoxide dismutase (sodA sodB) null mutant, the tri-domained MT was found to exacerbate Cd toxicity whereas the tetra-domained MT efficiently protected bacteria from Cd. The paradoxical toxicity displayed by the tri-domained MT upon Cd contamination was linked to the generation of superoxide radicals generated by a mechanism which most probably involves a copper-redox cycling reaction, since a Cd-contaminated sodA sodB strain expressing this MT produced 4 times more O2(-) than the control bacteria, and MT toxicity disappeared in the presence of bathocuproine disulfonic acid, a copper chelator. In contrast, the tetra-domained form did not. Interestingly, in bacteria producing superoxide dismutase but hypersensitive to oxidative stress due to either mutations in thioredoxin and glutathione reductase pathways (WM104 mutant) or to a lack of gamma-glutamylcysteine synthetase (gshA mutant), both chimeric MTs were protecting against Cd toxicity. However, an unexpected lack of antioxidant function was observed for both chimeric MTs, which were found to enhance the toxicity of hydrogen peroxide in WM104, or that of menadione in QC1726. Altogether, our results suggest that superoxide dismutase activity counteracts the potential prooxidative effect of the tri-domained MT mediated by Cu ions and that the tetra

  8. Induction of metallothionein in mouse cerebellum and cerebrum with low-dose thimerosal injection.

    PubMed

    Minami, Takeshi; Miyata, Eriko; Sakamoto, Yamato; Yamazaki, Hideo; Ichida, Seiji

    2010-04-01

    Thimerosal, an ethyl mercury compound, is used worldwide as a vaccine preservative. We previously observed that the mercury concentration in mouse brains did not increase with the clinical dose of thimerosal injection, but the concentration increased in the brain after the injection of thimerosal with lipopolysaccharide, even if a low dose of thimerosal was administered. Thimerosal may penetrate the brain, but is undetectable when a clinical dose of thimerosal is injected; therefore, the induction of metallothionein (MT) messenger RNA (mRNA) and protein was observed in the cerebellum and cerebrum of mice after thimerosal injection, as MT is an inducible protein. MT-1 mRNA was expressed at 6 and 9 h in both the cerebrum and cerebellum, but MT-1 mRNA expression in the cerebellum was three times higher than that in the cerebrum after the injection of 12 microg/kg thimerosal. MT-2 mRNA was not expressed until 24 h in both organs. MT-3 mRNA was expressed in the cerebellum from 6 to 15 h after the injection, but not in the cerebrum until 24 h. MT-1 and MT-3 mRNAs were expressed in the cerebellum in a dose-dependent manner. Furthermore, MT-1 protein was detected from 6 to 72 h in the cerebellum after 12 microg/kg of thimerosal was injected and peaked at 10 h. MT-2 was detected in the cerebellum only at 10 h. In the cerebrum, little MT-1 protein was detected at 10 and 24 h, and there were no peaks of MT-2 protein in the cerebrum. In conclusion, MT-1 and MT-3 mRNAs but not MT-2 mRNA are easily expressed in the cerebellum rather than in the cerebrum by the injection of low-dose thimerosal. It is thought that the cerebellum is a sensitive organ against thimerosal. As a result of the present findings, in combination with the brain pathology observed in patients diagnosed with autism, the present study helps to support the possible biological plausibility for how low-dose exposure to mercury from thimerosal-containing vaccines may be associated with autism.

  9. The Fungus Tremella mesenterica Encodes the Longest Metallothionein Currently Known: Gene, Protein and Metal Binding Characterization

    PubMed Central

    Lin, Weiyu; Calatayud, Sara; Palacios, Òscar; Capdevila, Mercè; Atrian, Sílvia

    2016-01-01

    Fungal Cu-thioneins, and among them, the paradigmatic Neurospora crassa metallothionein (MT) (26 residues), were once considered as the shortest MTs -the ubiquitous, versatile metal-binding proteins- among all organisms, and thus representatives of their primeval forms. Nowadays, fungal MTs of diverse lengths and sequence features are known, following the huge heterogeneity of the Kingdom of Fungi. At the opposite end of N. crassa MT, the recently reported Cryptococcus neoformans CnMT1 and CnMT2 (122 and 186 aa) constitute the longest reported fungal MTs, having been identified as virulence factors of this pathogen. CnMTs are high-capacity Cu-thioneins that appear to be built by tandem amplification of a basic unit, a 7-Cys segment homologous to N. crassa MT. Here, we report the in silico, in vivo and in vitro study of a still longer fungal MT, belonging to Tremella mesenterica (TmMT), a saprophytic ascomycete. The TmMT gene has 10 exons, and it yields a 779-bp mature transcript that encodes a 257 residue-long protein. This MT is also built by repeated fragments, but of variable number of Cys: six units of the 7-Cys building blocks-CXCX3CSCPPGXCXCAXCP-, two fragments of six Cys, plus three Cys at the N-terminus. TmMT metal binding abilities have been analyzed through the spectrophotometric and spectrometric characterization of its recombinant Zn-, Cd- and Cu-complexes. Results allow it to be unambiguous classified as a Cu-thionein, also of extraordinary coordinating capacity. According to this feature, when the TmMT cDNA is expressed in MT-devoid yeast cells, it is capable of restoring a high Cu tolerance level. Since it is not obvious that T. mesenterica shares the same physiological needs for a high capacity Cu-binding protein with C. neoformans, the existence of this peculiar MT might be better explained on the basis of a possible role in Cu-handling for the Cu-enzymes responsible in lignin degradation pathways. PMID:26882011

  10. The Fungus Tremella mesenterica Encodes the Longest Metallothionein Currently Known: Gene, Protein and Metal Binding Characterization.

    PubMed

    Iturbe-Espinoza, Paul; Gil-Moreno, Selene; Lin, Weiyu; Calatayud, Sara; Palacios, Òscar; Capdevila, Mercè; Atrian, Sílvia

    2016-01-01

    Fungal Cu-thioneins, and among them, the paradigmatic Neurospora crassa metallothionein (MT) (26 residues), were once considered as the shortest MTs--the ubiquitous, versatile metal-binding proteins--among all organisms, and thus representatives of their primeval forms. Nowadays, fungal MTs of diverse lengths and sequence features are known, following the huge heterogeneity of the Kingdom of Fungi. At the opposite end of N. crassa MT, the recently reported Cryptococcus neoformans CnMT1 and CnMT2 (122 and 186 aa) constitute the longest reported fungal MTs, having been identified as virulence factors of this pathogen. CnMTs are high-capacity Cu-thioneins that appear to be built by tandem amplification of a basic unit, a 7-Cys segment homologous to N. crassa MT. Here, we report the in silico, in vivo and in vitro study of a still longer fungal MT, belonging to Tremella mesenterica (TmMT), a saprophytic ascomycete. The TmMT gene has 10 exons, and it yields a 779-bp mature transcript that encodes a 257 residue-long protein. This MT is also built by repeated fragments, but of variable number of Cys: six units of the 7-Cys building blocks--CXCX3CSCPPGXCXCAXCP-, two fragments of six Cys, plus three Cys at the N-terminus. TmMT metal binding abilities have been analyzed through the spectrophotometric and spectrometric characterization of its recombinant Zn-, Cd- and Cu-complexes. Results allow it to be unambiguous classified as a Cu-thionein, also of extraordinary coordinating capacity. According to this feature, when the TmMT cDNA is expressed in MT-devoid yeast cells, it is capable of restoring a high Cu tolerance level. Since it is not obvious that T. mesenterica shares the same physiological needs for a high capacity Cu-binding protein with C. neoformans, the existence of this peculiar MT might be better explained on the basis of a possible role in Cu-handling for the Cu-enzymes responsible in lignin degradation pathways.

  11. Intracellular sequestration of zinc, cadmium and silver in Hebeloma mesophaeum and characterization of its metallothionein genes.

    PubMed

    Sácký, Jan; Leonhardt, Tereza; Borovička, Jan; Gryndler, Milan; Briksí, Aleš; Kotrba, Pavel

    2014-06-01

    Sequestration of intracellular heavy metals in eukaryotes involves compartmentalization and binding with cytosolic, cysteine-rich metallothionein (MT) peptides. We examined the roles of these processes in handling of zinc (Zn), cadmium (Cd) and silver (Ag) in sporocarps and a metal-exposed extraradical mycelium of Hebeloma mesophaeum, the Zn-accumulating ectomycorrhizal (EM) species frequently associated with metal disturbed sites. Size exclusion chromatography revealed that the majority of Zn and Cd in the sporocarps and mycelium was contained in a low molecular mass fraction attributable to compartmentalized metal. The staining of hyphal cells with the Zn-specific Zinquin and Cd-specific Leadmium fluorescent tracers labeled Zn and Cd in small, punctuated vesicles and vacuoles, respectively. By contrast, the sporocarp and mycelium Ag was associated with cysteine-rich, 5-kDa peptides. The peptides of the same size were also identified in minor Zn and Cd complexes from the metal-exposed mycelium. We have further isolated and characterized HmMT1, HmMT2 and HmMT3 genes coding for different 5-kDa MTs of H. mesophaeum collected at a lead smelter site. Heterologous complementation assays in metal-sensitive yeast mutants indicated that HmMTs encode functional, metal-specific peptides: only HmMT1 was able to complement sensitivity to Zn; HmMT1 conferred higher tolerance to Cd and Cu than HmMT2 or HmMT3; and both HmMT2 and HmMT3, but not HmMT1, conferred increased tolerance to Ag. The presence of HmMT1 and HmMT3, but not HmMT2, was also confirmed in a H. mesophaeum isolate from an unpolluted site. Gene expression analysis in the extraradical mycelium of this isolate revealed that the transcription of HmMT1 was preferentially induced in the presence of Zn and Cd, while Ag was a stronger inducer of HmMT3. Altogether, these results improve our understanding of the handling of intracellular Zn, Cd and Ag in Hebeloma and represent the first evidence suggesting involvement of MTs

  12. Arabidopsis thaliana as Bioindicator of Fungal VOCs in Indoor Air

    PubMed Central

    Hung, Richard; Yin, Guohua; Klich, Maren A.; Grimm, Casey; Bennett, Joan W.

    2016-01-01

    In this paper, we demonstrate the ability of Arabidopsis thaliana to detect different mixtures of volatile organic compounds (VOCs) emitted by the common indoor fungus, Aspergillus versicolor, and demonstrate the potential usage of the plant as a bioindicator to monitor fungal VOCs in indoor air. We evaluated the volatile production of Aspergillus versicolor strains SRRC 108 (NRRL 3449) and SRRC 2559 (ATCC 32662) grown on nutrient rich fungal medium, and grown under conditions to mimic the substrate encountered in the built environment where fungi would typically grow indoors (moist wallboard and ceiling tiles). Using headspace solid phase microextraction/gas chromatography-mass spectrometry, we analyzed VOC profiles of the two strains. The most abundant compound produced by both strains on all three media was 1-octen-3-ol. Strain SRRC 2559 made several terpenes not detected from strain SRRC 108. Using a split-plate bioassay, we grew Arabidopsis thaliana in a shared atmosphere with VOCs from the two strains of Aspergillus versicolor grown on yeast extract sucrose medium. The VOCs emitted by SRRC 2559 had an adverse impact on seed germination and plant growth. Chemical standards of individual VOCs from the Aspergillus versicolor mixture (2-methyl-1-butanol, 3-methyl-1-butanol, 1-octen-3-ol, limonene, and β-farnesene), and β-caryophyllene were tested one by one in seed germination and vegetative plant growth assays. The most inhibitory compound to both seed germination and plant growth was 1-octen-3-ol. Our data suggest that Arabidopsis is a useful model for monitoring indoor air quality as it is sensitive to naturally emitted fungal volatile mixtures as well as to chemical standards of individual compounds, and it exhibits relatively quick concentration- and duration-dependent responses. PMID:27790067

  13. Taxonomy and Phylogeny of Arabidopsis (Brassicaceae)

    PubMed Central

    Al-Shehbaz, Ihsan A.; O'Kane, Steve L.

    2002-01-01

    Detailed taxonomic, cytological, and phylogenetic accounts of Arabidopsis are presented. As currently delimited, the genus consists of nine species all of which are indigenous to Europe, with the ranges of two species extending into northern and eastern Asia and North American into central United States. A survey of chromosome numbers in the genus is presented, and the country of origin for each count is given. Detailed descriptions of all species and subspecies and keys to all taxa are provided. Generic assignments are updated for the 50 species previously included in Arabidopsis. A cladogram of the species of Arabidopsis based on molecular phylogenetic studies by the authors is given. PMID:22303187

  14. Arabidopsis thaliana life without phytochromes

    PubMed Central

    Strasser, Bárbara; Sánchez-Lamas, Maximiliano; Yanovsky, Marcelo J.; Casal, Jorge J.; Cerdán, Pablo D.

    2010-01-01

    Plants use light as a source of energy for photosynthesis and as a source of environmental information perceived by photoreceptors. Testing whether plants can complete their cycle if light provides energy but no information about the environment requires a plant devoid of phytochromes because all photosynthetically active wavelengths activate phytochromes. Producing such a quintuple mutant of Arabidopsis thaliana has been challenging, but we were able to obtain it in the flowering locus T (ft) mutant background. The quintuple phytochrome mutant does not germinate in the FT background, but it germinates to some extent in the ft background. If germination problems are bypassed by the addition of gibberellins, the seedlings of the quintuple phytochrome mutant exposed to red light produce chlorophyll, indicating that phytochromes are not the sole red-light photoreceptors, but they become developmentally arrested shortly after the cotyledon stage. Blue light bypasses this blockage, rejecting the long-standing idea that the blue-light receptors cryptochromes cannot operate without phytochromes. After growth under white light, returning the quintuple phytochrome mutant to red light resulted in rapid senescence of already expanded leaves and severely impaired expansion of new leaves. We conclude that Arabidopsis development is stalled at several points in the presence of light suitable for photosynthesis but providing no photomorphogenic signal. PMID:20176939

  15. Sulfenome mining in Arabidopsis thaliana

    PubMed Central

    Waszczak, Cezary; Akter, Salma; Eeckhout, Dominique; Persiau, Geert; Wahni, Khadija; Bodra, Nandita; Van Molle, Inge; De Smet, Barbara; Vertommen, Didier; Gevaert, Kris; De Jaeger, Geert; Van Montagu, Marc; Messens, Joris; Van Breusegem, Frank

    2014-01-01

    Reactive oxygen species (ROS) have been shown to be potent signaling molecules. Today, oxidation of cysteine residues is a well-recognized posttranslational protein modification, but the signaling processes steered by such oxidations are poorly understood. To gain insight into the cysteine thiol-dependent ROS signaling in Arabidopsis thaliana, we identified the hydrogen peroxide (H2O2)-dependent sulfenome: that is, proteins with at least one cysteine thiol oxidized to a sulfenic acid. By means of a genetic construct consisting of a fusion between the C-terminal domain of the yeast (Saccharomyces cerevisiae) AP-1–like (YAP1) transcription factor and a tandem affinity purification tag, we detected ∼100 sulfenylated proteins in Arabidopsis cell suspensions exposed to H2O2 stress. The in vivo YAP1-based trapping of sulfenylated proteins was validated by a targeted in vitro analysis of DEHYDROASCORBATE REDUCTASE2 (DHAR2). In DHAR2, the active site nucleophilic cysteine is regulated through a sulfenic acid-dependent switch, leading to S-glutathionylation, a protein modification that protects the protein against oxidative damage. PMID:25049418

  16. The fifth international conference on Arabidopsis research

    SciTech Connect

    Hangarter, R.; Scholl, R.; Davis, K.; Feldmann, K.

    1993-12-31

    This volume contains abstracts of oral and poster presentations made in conjunction with the Fifth International Conference on Arabidopsis Research held August 19--22, 1993 at the Ohio State University, Columbus, Ohio.

  17. PAIR: the predicted Arabidopsis interactome resource.

    PubMed

    Lin, Mingzhi; Shen, Xueling; Chen, Xin

    2011-01-01

    The predicted Arabidopsis interactome resource (PAIR, http://www.cls.zju.edu.cn/pair/), comprised of 5990 experimentally reported molecular interactions in Arabidopsis thaliana together with 145,494 predicted interactions, is currently the most comprehensive data set of the Arabidopsis interactome with high reliability. PAIR predicts interactions by a fine-tuned support vector machine model that integrates indirect evidences for interaction, such as gene co-expressions, domain interactions, shared GO annotations, co-localizations, phylogenetic profile similarities and homologous interactions in other organisms (interologs). These predictions were expected to cover 24% of the entire Arabidopsis interactome, and their reliability was estimated to be 44%. Two independent example data sets were used to rigorously validate the prediction accuracy. PAIR features a user-friendly query interface, providing rich annotation on the relationships between two proteins. A graphical interaction network browser has also been integrated into the PAIR web interface to facilitate mining of specific pathways.

  18. Individual Education.

    ERIC Educational Resources Information Center

    Corsini, Raymond

    1981-01-01

    Paper presented at the 66th Convention of the International Association of Pupil Personnel Workers, October 20, 1980, Baltimore, Maryland, describes individual education based on the principles of Alfred Adler. Defines six advantages of individual education, emphasizing student responsibility, mutual respect, and allowing students to progress at…

  19. Functional analysis of the buckwheat metallothionein promoter: tissue specificity pattern and up-regulation under complex stress stimuli.

    PubMed

    Bratić, Ana M; Majić, Dragana B; Samardzić, Jelena T; Maksimović, Vesna R

    2009-06-01

    To shed light on expression regulation of the metallothionein gene from buckwheat (FeMT3), functional promoter analysis was performed with a complete 5' regulatory region and two deletion variants, employing stably transformed tobacco plants. Histochemical GUS assay of transgenic tobacco lines showed the strongest signals in vascular elements of leaves and in pollen grains, while somewhat weaker staining was observed in the roots of mature plants. This tissue specificity pattern implies a possible function of buckwheat MT3 in those tissues. Quantitative GUS assay showed strong up-regulation of all three promoter constructs (proportional to the length of the regulatory region) in leaves submerged in liquid MS medium containing sucrose, after a prolonged time period. This represented a complex stress situation composed of several synergistically related stress stimuli. These findings suggest complex transcriptional regulation of FeMT3, requiring interactions among a number of different factors.

  20. Role of pCeMT, a putative metallothionein from Colocasia esculenta, in response to metal stress.

    PubMed

    Kim, Yeon-Ok; Jung, Sera; Kim, Kyounghyoun; Bae, Hyeun-Jong

    2013-03-01

    Metallothioneins (MTs) play a major role in metal homeostasis and/or detoxification in plants. In this study, a novel gene, pCeMT, was isolated from Colocasia esculenta and characterized. Our results indicate that Escherichia coli cells expressing pCeMT exhibited enhanced Cd, Cu, and Zn tolerance and accumulation compared with control cells. Furthermore, pCeMT-overexpressing tobacco seedlings displayed better growth under Cd, Cu, and Zn stresses and accumulated more Cd and Zn compared with the wild type. Interestingly, transgenic tobacco displayed markedly decreased hydrogen peroxide (H(2)O(2)) and lipid peroxidation levels under Cd, Cu, and Zn treatments. These results suggest that pCeMT could play an important role in the protection of plant cells from oxidative stress by reactive oxygen species (ROS) scavenging and in the detoxification of free metals by metal binding, leading to improved plant metal tolerance.

  1. Effect of thermal processing and canning on cadmium and lead levels in California market squid: the role of metallothioneins.

    PubMed

    Galitsopoulou, A; Georgantelis, D; Kontominas, M G

    2013-01-01

    The effects of two common seafood preparation practices (roasting and industrial canning) on the heavy metal content--cadmium (Cd) and lead (Pb)--of various tissues of California market squid were studied. Emphasis was placed on the role of metallothioneins (MT) in Cd and Pb behaviour during processing. Cd and Pb analysis was conducted by a Zeeman GTA-AAS atomic absorption spectrometry system; MT analysis was performed by a mercury saturation assay. Results showed that Cd levels in the mantle and whole squid were considerably affected by both processing practices, reaching a 240% increase in mantle and a 40% increase in whole squid. Interestingly, Cd behaviour was associated with MT changes during squid processing. On the other hand, Pb content was not affected from either processing or associated with MT content in the raw or processed squid. Therefore, processing operations may affect Cd and Pb content differently due to the specific metal bioaccumulation and chemical features of each heavy metal type.

  2. The role of metallothioneins, selenium and transfer to offspring in mercury detoxification in Franciscana dolphins (Pontoporia blainvillei).

    PubMed

    Romero, M B; Polizzi, P; Chiodi, L; Das, K; Gerpe, M

    2016-08-15

    The concentrations of mercury (Hg), selenium (Se) and metallothioneins (MT) were evaluated in fetuses, calves, juveniles and adults of the endangered coastal Franciscana dolphin (Pontoporia blainvillei) from Argentina. Mercury concentrations varied among analyzed tissues (liver, kidney, muscle and brain), with liver showing the higher concentrations in all specimens. An age-dependent accumulation was found in liver, kidney and brain. No significant relationship between Hg and MT concentrations was found for all tissues analyzed. Hepatic Hg molar concentrations were positively correlated with those of Se, indicating a great affinity between these two elements. Furthermore, dark granules of HgSe were observed in Kupffer cells in the liver by electron microscopy, suggesting the role of this macrophage in the detoxification of Hg. A transfer of Hg through placenta was proved. The presence of Hg in brain in all age classes did not show concentrations associated with neurotoxicity.

  3. Trace-elements, methylmercury and metallothionein levels in Magellanic penguin (Spheniscus magellanicus) found stranded on the Southern Brazilian coast.

    PubMed

    Kehrig, Helena A; Hauser-Davis, Rachel A; Seixas, Tércia G; Fillmann, Gilberto

    2015-07-15

    Magellanic penguins have been reported as good biomonitors for several types of pollutants, including trace-elements. In this context, selenium (Se), total mercury, methylmercury, inorganic mercury (Hg(inorg)), cadmium (Cd) and lead (Pb), as well as metallothionein (MT) levels, were evaluated in the feathers, liver and kidney of juvenile Magellanic penguins found stranded along the coast of Southern Brazil. The highest concentrations of all trace-elements and methylmercury were found in internal organs. Concentrations of Cd and Se in feathers were extremely low in comparison with their concentrations in soft tissues. The results showed that both Se and MT are involved in the detoxification of trace-elements (Cd, Pb and Hg(inorg)) since statistically significant relationships were found in liver. Conversely, hepatic Se was shown to be the only detoxifying agent for methylmercury.

  4. Vermicomposting of Tea Factory Coal Ash: metal accumulation and metallothionein response in Eisenia fetida (Savigny) and Lampito mauritii (Kinberg).

    PubMed

    Goswami, L; Sarkar, S; Mukherjee, S; Das, S; Barman, S; Raul, P; Bhattacharyya, P; Mandal, N C; Bhattacharya, S; Bhattacharya, S S

    2014-08-01

    Earthworms can accumulate heavy metals in their intestines to a great extent. Impact of feed materials and duration of metal exposure on natural activity of earthworms are rather unclear; this investigation therefore addresses the impact of metal rich Tea Factory Coal Ash (TFCA) on reproduction, composting and metal accumulation ability of Eisenia fetida and Lampito mauritii. Earthworm count and cocoon production increased significantly during vermicomposting. pH of the vermicomposted mixtures shifted toward neutrality, total organic C decreased substantially and total N enhanced significantly compared to composting. High heavy metal (Mn, Zn, Cu, As) accumulation was recorded in the intestine of both the earthworm species. Moreover, gradual increase in the metal-inducible metallothionein concentration indicated the causal mechanism of metal accumulation in these species. TFCA+cow dung (CD) (1:1) were most favorable feed mixture for E. fetida and TFCA+CD (1:2) were good for L. mauritii in regard to metal accumulation and compost quality.

  5. Metallothionein 2A affects the cell respiration by suppressing the expression of mitochondrial protein cytochrome c oxidase subunit II.

    PubMed

    Bragina, Olga; Gurjanova, Karina; Krishtal, Jekaterina; Kulp, Maria; Karro, Niina; Tõugu, Vello; Palumaa, Peep

    2015-06-01

    Metallothioneins (MT) are involved in a broad range of cellular processes and play a major role in protection of cells towards various stressors. Two functions of MTs, namely the maintaining of the homeostasis of transition metal ions and the redox balance, are directly linked to the functioning of mitochondria. Dyshomeostasis of MTs is often related with malfunctioning of mitochondria; however, the mechanism by which MTs affect the mitochondrial respiratory chain is still unknown. We demonstrated that overexpression of MT-2A in HEK cell line decreased the oxidative phosphorylation capacity of the cells. HEK cells overexpressing MT-2A demonstrated reduced oxygen consumption and lower cellular ATP levels. MT-2A did not affect the number of mitochondria, but reduced specifically the level of cytochrome c oxidase subunit II protein, which resulted in lower activity of the complex IV.

  6. Analysis of the effects of overexpression of metallothionein-I in transgenic mice on the reproductive toxicology of cadmium

    SciTech Connect

    Dalton, T.; Kai Fu; Andrews, G.K.; Enders, G.C.; Palmiter, R.D.

    1996-01-01

    Exposure to low levels of cadmium reduces fertility. In male mice spermatogensis is highly sensitive to cadmium, whereas in females the peri-implantation period of pregnancy is sensitive. To examine the potential roles of the cadmium-binding protein, metallothionein (MT), in the reproductive toxicology of cadmium, we examined a transgenic mouse strain that overexpresses metallothionein-I (MT-I). These mice had dramatically increased steady-state levels of MT-I mRNA and MT in the testes and in the female reproductive tract during the peri-implantation period of pregnancy, and this overexpression occurred in a cell-specific and temporally regulated manner similar to that of the endogenous MT-I gene. Transgenic and control males were injected with cadmium, and the histology of the testes was examined. An injection of 7.5 {mu}mol Cd/Kg had no effect on histology of the testes in either transgenic or control mice. In contrast, an injection of 10 {mu}mol Cd/kg caused rapid changes in the histology of the testes and resulted in pronounced testicular necrosis in both control and transgenic mice. Female transgenic and control mice were mated and then injected with cadmium (30-45 {mu}mol Cd/kg) on the day of blastocyst implantation (day 4). In both of these groups, injection of cadmium reduced pregnancy rate, and no dramatic protection was afforded by maternal and/or embryonic overexpression of MT. Thus, overexpression of MT-I does not significantly protect against either of these cadmium-induced effects on fertility. 65 refs., 6 figs., 1 tab.

  7. Enhanced metallothionein gene expression is associated with protection from cadmium-induced genotoxity in cultured rat liver cells

    SciTech Connect

    Coogan, T.P.; Bare, R.M.; Bjornson, E.J.; Waalkes, M.P. )

    1994-01-01

    Metallothioneins (MTs) are low-molecular-weight, cysteine-rich proteins that appear to play an important role in the cellular defense system against cadmium toxicity. Although substantial evidence exists demonstrating a reduction in cadmium toxicity concomitant with MT induction, little is known about the possible effects of stimulation of MT synthesis on cadmium-induced genotoxicity. Thus, the alkaline elution technique was used to assess single-strand DNA damage (SSD) in TRL-1215 cells, a liver-derived cell line shown to have inducible MT Gene expression. The SSD accumulated over a 2-h time period in a time-dependent manner following exposure to 500 [mu]M CdCl[sub 2]. Low concentration cadmium pretreatment (10 [mu]M CdCl[sub 2], 24 h) provided protection against the genotoxicity of high-concentration cadmium (500 [mu]M CdCl[sub 2], 2 h). A 2-h exposure to 500 [mu]M CdCl[sub 2], had no effect on viability, as assessed using a tetrazolium-dye based assay, in cells from either the pretreated or nonpretreated group. Metallothionein was induced in a time-dependent manner by low-concentration cadmium pretreatment: Exposure for 24 and 48 h resulted in 3.3- and 6.4-fold increases, respectively. In addition, a 24-h exposure to low-concentration cadmium resulted in an increase in MT-I gene expression. Cadmium accumulation was 2.6-fold greater in low-concentration cadmium-pretreated cells as compared to non-pretreated cells. These data demonstrate that low-concentration cadmium pretreatment provides protection against cadmium-induced single-strand DNA damage and support the hypothesis that this protection is due to stimulation of MT gene expression. 38 refs., 6 figs.

  8. Resonant X-Ray Scattering and Absorption for the Global and Local Structures of Cu-modified Metallothioneins in Solution

    PubMed Central

    Li, Meiyi; Huang, Yu-Shan; Jeng, U-Ser; Hsu, I-Jui; Sermon Wu, YewChung; Lai, Ying-Huang; Su, Chiu-Hun; Lee, Jyh-Fu; Wang, Yu; Chang, Chia-Ching

    2009-01-01

    Abstract With Cd and Zn metal ions removed from the native rabbit-liver metallothionein upon unfolding, Cu-modified metallothioneins (Cu-MTs) were obtained during refolding in solutions containing CuI or CuII ions. X-ray absorption near-edge spectroscopic results confirm the respectively assigned oxidation states of the copper ions in CuI-MT and CuII-MT. Global and local structures of the Cu-MTs were subsequently characterized by anomalous small-angle x-ray scattering (ASAXS) and extended x-ray absorption fine structure. Energy-dependent ASAXS results indicate that the morphology of CuII-MT resembles that of the native MT, whereas CuI-MT forms oligomers with a higher copper content. Both dummy-residue simulation and model-shape fitting of the ASAXS data reveal consistently rodlike morphology for CuII-MT. Clearly identified Cu-S, Cu-O, and Cu-Cu contributions in the extended x-ray absorption fine structure analysis indicate that both CuI and CuII ions are bonded with O and S atoms of nearby amino acids in a four-coordination environment, forming metal clusters smaller than metal thiolate clusters in the native MT. It is demonstrated that a combination of resonant x-ray scattering and x-ray absorption can be particularly useful in revealing complementary global and local structures of metalloproteins due to the atom specific characteristics of the two techniques. PMID:19619476

  9. Effect of quercetin on metallothionein, nitric oxide synthases and cyclooxygenase-2 expression on experimental chronic cadmium nephrotoxicity in rats

    SciTech Connect

    Morales, Ana I.; Vicente-Sanchez, Cesar; Jerkic, Mirjana; Santiago, Jose M.; Sanchez-Gonzalez, Penelope D.; Perez-Barriocanal, Fernando; Lopez-Novoa, Jose M. . E-mail: jmlnovoa@usal.es

    2006-01-15

    Inflammation can play a key role in Cd-induced dysfunctions. Quercetin is a potent oxygen free radical scavenger and a metal chelator. Our aim was to study the effect of quercetin on Cd-induced kidney damage and metallothionein expression. The study was performed in Wistar rats that were administered during 9 weeks with either cadmium (1.2 mg Cd/kg/day, s.c.), quercetin (50 mg/kg/day, i.p.) or cadmium + quercetin. Renal toxicity was evaluated by measuring blood urea nitrogen concentration and urinary excretion of enzymes marker of tubular damage. Endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) renal expression were assessed by Western blot. Renal expression of metallothionein 1 and 2 (MT-1, MT-2) and eNOS mRNA was assessed by Northern blot. Our data demonstrated that Cd-induced renal toxicity was markedly reduced in rats that also received quercetin. MT-1 and MT-2 mRNA levels in kidney were substantially increased during treatment with Cd, being even higher when the animals received Cd and quercetin. Renal eNOS expression was significantly higher in rats receiving Cd and quercetin than in animals receiving Cd alone or in control rats. In the group that received Cd, COX-2 and iNOS expression was markedly higher than in control rats. In the group Cd + quercetin, no changes in COX-2 and iNOS expression were observed compared with the control group. Our results demonstrate that quercetin treatment prevents Cd-induced overexpression of iNOS and COX-2, and increases MT expression. These effects can explain the protection by quercetin of Cd-induced nephrotoxicity.

  10. Deleterious Effects of Minocycline after in vivo Target Deprivation of Thalamocortical Neurons in the Immature, Metallothionein-Deficient Mouse Brain

    PubMed Central

    Potter, Emily G.; Cheng, Ying; Natale, JoAnne E.

    2015-01-01

    Compared to adults, immature metallothionein I & II knockout (MT−/−) mice incur greater neuronal loss and a more rapid rate of microglia accumulation following target deprivation-induced injury. Since minocycline has been proposed to inhibit microglial activation and associated production of neuroinflammatory factors, we investigated its ability to promote neuronal survival in the immature, metallothionein-deficient brain. Following ablation of the visual cortex, 10-day-old MT−/− mice were treated with minocycline or saline and sacrificed 24 or 48 hours after injury. Using stereological methods, the number of microglia and neurons were estimated in the ipsilateral dorsal lateral geniculate nucleus (dLGN) by an investigator blinded to the treatment. No effect on neuronal survival was observed at 24 hours, but 48 hours after injury an unanticipated but significant minocycline-mediated increase in neuronal loss was detected. Further, while failing to inhibit microglial accumulation, minocycline treatment increased the proportion of amoeboid microglia in the ipsilateral dLGN. To understand the molecular mechanisms underlying this neurotoxic response, we identified minocycline-mediated changes in the expression of three potentially pro-apoptotic/ inflammatory genes: growth arrest- and DNA damage-inducible gene 45γ (GADD45γ); interferon-inducible protein 1 (IFI1) and cytokine induced growth factor (CTGF). We also observed increased mitogen-activated protein kinase (MAPK) p38 phosphorylation with minocycline treatment. Although minocycline inhibited calpain activity at 12 hours post-injury, this effect was not sustained at 24 hours. Together, these results help to explain how minocycline has a deleterious effect on neuronal survival in this injury model. PMID:19115404

  11. Cell Polarity Signaling in Arabidopsis

    PubMed Central

    Yang, Zhenbiao

    2009-01-01

    Cell polarization is intimately linked to plant development, growth, and responses to the environment. Major advances have been made in our understanding of the signaling pathways and networks that regulate cell polarity in plants owing to recent studies on several model systems, e.g., tip growth in pollen tubes, cell morphogenesis in the leaf epidermis, and polar localization of PINs. From these studies we have learned that plant cells use conserved mechanisms such as Rho family GTPases to integrate both plant-specific and conserved polarity cues and to coordinate the cytoskeketon dynamics/reorganization and vesicular trafficking required for polarity establishment and maintenance. This review focuses upon signaling mechanisms for cell polarity formation in Arabidopsis, with an emphasis on Rho GTPase signaling in polarized cell growth and how these mechanisms compare with those for cell polarity signaling in yeast and animal systems. PMID:18837672

  12. Apoplastic Diffusion Barriers in Arabidopsis

    PubMed Central

    Schreiber, Lukas; Franke, Rochus Benni; Geldner, Niko; Reina-Pinto, José J.; Kunst, Ljerka

    2013-01-01

    During the development of Arabidopsis and other land plants, diffusion barriers are formed in the apoplast of specialized tissues within a variety of plant organs. While the cuticle of the epidermis is the primary diffusion barrier in the shoot, the Casparian strips and suberin lamellae of the endodermis and the periderm represent the diffusion barriers in the root. Different classes of molecules contribute to the formation of extracellular diffusion barriers in an organ- and tissue-specific manner. Cutin and wax are the major components of the cuticle, lignin forms the early Casparian strip, and suberin is deposited in the stage II endodermis and the periderm. The current status of our understanding of the relationships between the chemical structure, ultrastructure and physiological functions of plant diffusion barriers is discussed. Specific aspects of the synthesis of diffusion barrier components and protocols that can be used for the assessment of barrier function and important barrier properties are also presented. PMID:24465172

  13. Acute and subacute pulmonary toxicity caused by a single intratracheal instillation of colloidal silver nanoparticles in mice: pathobiological changes and metallothionein responses.

    PubMed

    Kaewamatawong, Theerayuth; Banlunara, Wijit; Maneewattanapinyo, Pattwat; Thammachareon, Chuchaat; Ekgasit, Sanong

    2014-01-01

    To study the acute and subacute pulmonary toxicity of colloidal silver nanoparticles (Ag-NPs), 0 or 100 ppm of Ag-NPs were instilled intratracheally in mice. Cellular and biochemical parameters in bronchoalveolar lavage fluid (BALF) and histological alterations were determined 1, 3, 7, 15, and 30 days after instillation. Ag-NPs induced moderate pulmonary inflammation and injury on BALF indices during the acute period; however, these changes gradually regressed in a time-dependent manner. Concomitant histopathological and laminin immunohistochemical findings generally correlated to BALF data. Superoxide dismutase and metallothionein expression occurred in particle-laden macrophages and alveolar epithelial cells, which correlated to lung lesions in mice treated with Ag-NPs. These findings suggest that instillation of Ag-NPs causes transient moderate acute lung inflammation and tissue damage. Oxidative stress may underlie the induction of injury to lung tissue. Moreover, the expression of metallothionein in tissues indicated the protective response to exposure to Ag-NPs.

  14. Study of Interactions between Metallothionein and Cisplatin by using Differential Pulse Voltammetry Brdickás reaction and Quartz Crystal Microbalance

    PubMed Central

    Huska, Dalibor; Fabrik, Ivo; Baloun, Jiri; Adam, Vojtech; Masarik, Michal; Hubalek, Jaromir; Vasku, Anna; Trnkova, Libuse; Horna, Ales; Zeman, Ladislav; Kizek, Rene

    2009-01-01

    Treatment strategies for tumour diseases are progressively focusing on personalization of medicine. However, this focus requires methods revealing the early general biological mechanisms, including the formation anti-cancer drugs’ resistance. The low molecular mass protein metallothionein is thought to be the crucial for the formation of resistance in tumour treatment based on the platinum-cytostatics. The interactions between metallothionein (MT) and cisplatin were determined by the adsorptive transfer stripping technique coupled with the differential pulse votlammetry Brdickás reaction. The signals related to the MT-cisplatin complex appeared at −0.9 V. The formation of this complex depended on the time of interaction between cisplatin and MT. The complex formation was consequently confirmed by quartz crystal microbalance analyses. The formation of this complex was detectable even after a 20 s long interaction. Moreover, we detected presence of MT-cisplatin complex in the blood of male rats treated with this drug. PMID:22573958

  15. Multigene Engineering of Triacylglycerol Metabolism Boosts Seed Oil Content in Arabidopsis1[W][OPEN

    PubMed Central

    van Erp, Harrie; Kelly, Amélie A.; Menard, Guillaume; Eastmond, Peter J.

    2014-01-01

    Increasing the yield of oilseed crops is an important objective for biotechnologists. A number of individual genes involved in triacylglycerol metabolism have previously been reported to enhance the oil content of seeds when their expression is altered. However, it has yet to be established whether specific combinations of these genes can be used to achieve an additive effect and whether this leads to enhanced yield. Using Arabidopsis (Arabidopsis thaliana) as an experimental system, we show that seed-specific overexpression of WRINKLED1 (a transcriptional regulator of glycolysis and fatty acid synthesis) and DIACYLGLYCEROL ACYLTRANSFERASE1 (a triacylglycerol biosynthetic enzyme) combined with suppression of the triacylglycerol lipase SUGAR-DEPENDENT1 results in a higher percentage seed oil content and greater seed mass than manipulation of each gene individually. Analysis of total seed yield per plant suggests that, despite a reduction in seed number, the total yield of oil is also increased. PMID:24696520

  16. Rhizobacteria-induced priming in Arabidopsis is dependent on ethylene, jasmonic acid, and NPR1.

    PubMed

    Ahn, Il-Pyung; Lee, Sang-Woo; Suh, Seok-Cheol

    2007-07-01

    A nonpathogenic rhizobacterium, Pseudomonas putida LSW17S, elicited systemic protection against Fusarium wilt and pith necrosis caused by Fusarium oxysporum f. sp. lycopersici and P. corrugata in tomato (Lycopersicon esculentum L.). LSW17S also confers disease resistance against P. syringae pv. tomato DC3000 (DC3000) on Arabidopsis ecotype Col-0. To investigate mechanisms underlying disease protection, expression patterns of defense-related genes PR1, PR2, PR5, and PDF1.2 and cellular defense responses such as hydrogen peroxide accumulation and callose deposition were investigated. LSW17S treatment exhibited the typical phenomena of priming. Strong and faster transcription of defense-related genes was induced and hydrogen peroxide or callose were accumulated in Arabidopsis treated with LSW17S and infected with DC3000. In contrast, individual actions of LSW17S and DC3000 did not elicit rapid molecular and cellular defense responses. Priming by LSW17S was translocated systemically and retained for more than 10 days. Treatment with LSW17S reduced pathogen proliferation in Arabidopsis ecotype Col-0 expressing bacterial NahG; however, npr1, etr1, and jar1 mutations impaired inhibition of pathogen growth. Cellular and molecular priming responses support these results. In sum, LSW17S primes Arabidopsis for NPR1-, ethylene-, and jasmonic acid-dependent disease resistance, and efficient molecular and cellular defense responses.

  17. Quantitative divergence of the bacterial root microbiota in Arabidopsis thaliana relatives.

    PubMed

    Schlaeppi, Klaus; Dombrowski, Nina; Oter, Ruben Garrido; Ver Loren van Themaat, Emiel; Schulze-Lefert, Paul

    2014-01-14

    Plants host at the contact zone with soil a distinctive root-associated bacterial microbiota believed to function in plant nutrition and health. We investigated the diversity of the root microbiota within a phylogenetic framework of hosts: three Arabidopsis thaliana ecotypes along with its sister species Arabidopsis halleri and Arabidopsis lyrata, as well as Cardamine hirsuta, which diverged from the former ∼ 35 Mya. We surveyed their microbiota under controlled environmental conditions and of A. thaliana and C. hirsuta in two natural habitats. Deep 16S rRNA gene profiling of root and corresponding soil samples identified a total of 237 quantifiable bacterial ribotypes, of which an average of 73 community members were enriched in roots. The composition of this root microbiota depends more on interactions with the environment than with host species. Interhost species microbiota diversity is largely quantitative and is greater between the three Arabidopsis species than the three A. thaliana ecotypes. Host species-specific microbiota were identified at the levels of individual community members, taxonomic groups, and whole root communities. Most of these signatures were observed in the phylogenetically distant C. hirsuta. However, the branching order of host phylogeny is incongruent with interspecies root microbiota diversity, indicating that host phylogenetic distance alone cannot explain root microbiota diversification. Our work reveals within 35 My of host divergence a largely conserved and taxonomically narrow root microbiota, which comprises stable community members belonging to the Actinomycetales, Burkholderiales, and Flavobacteriales.

  18. Dyskerin is a component of the Arabidopsis telomerase RNP required for telomere maintenance.

    PubMed

    Kannan, Kalpana; Nelson, Andrew D L; Shippen, Dorothy E

    2008-04-01

    Dyskerin binds the H/ACA box of human telomerase RNA and is a core telomerase subunit required for RNP biogenesis and enzyme function in vivo. Missense mutations in dyskerin result in dyskeratosis congenita, a complex syndrome characterized by bone marrow failure, telomerase enzyme deficiency, and progressive telomere shortening. Here we demonstrate that dyskerin also contributes to telomere maintenance in Arabidopsis thaliana. We report that both AtNAP57, the Arabidopsis dyskerin homolog, and AtTERT, the telomerase catalytic subunit, accumulate in the plant nucleolus, and AtNAP57 associates with active telomerase RNP particles in an RNA-dependent manner. Furthermore, AtNAP57 interacts in vitro with AtPOT1a, a novel component of Arabidopsis telomerase. Although a null mutation in AtNAP57 is lethal, AtNAP57, like AtTERT, is not haploinsufficient for telomere maintenance in Arabidopsis. However, introduction of an AtNAP57 allele containing a T66A mutation decreased telomerase activity in vitro, disrupted telomere length regulation on individual chromosome ends in vivo, and established a new, shorter telomere length set point. These results imply that T66A NAP57 behaves as a dominant-negative inhibitor of telomerase. We conclude that dyskerin is a conserved component of the telomerase RNP complex in higher eukaryotes that is required for maximal enzyme activity in vivo.

  19. Inactivation of GSK-3β by Metallothionein Prevents Diabetes-Related Changes in Cardiac Energy Metabolism, Inflammation, Nitrosative Damage, and Remodeling

    PubMed Central

    Wang, Yuehui; Feng, Wenke; Xue, Wanli; Tan, Yi; Hein, David W.; Li, Xiao-Kun; Cai, Lu

    2009-01-01

    OBJECTIVE Glycogen synthase kinase (GSK)-3β plays an important role in cardiomyopathies. Cardiac-specific metallothionein-overexpressing transgenic (MT-TG) mice were highly resistant to diabetes-induced cardiomyopathy. Therefore, we investigated whether metallothionein cardiac protection against diabetes is mediated by inactivation of GSK-3β. RESEARCH DESIGN AND METHODS Diabetes was induced with streptozotocin in both MT-TG and wild-type mice. Changes of energy metabolism–related molecules, lipid accumulation, inflammation, nitrosative damage, and fibrotic remodeling were examined in the hearts of diabetic mice 2 weeks, 2 months, and 5 months after the onset of diabetes with Western blotting, RT-PCR, and immunohistochemical assays. RESULTS Activation (dephosphorylation) of GSK-3β was evidenced in the hearts of wild-type diabetic mice but not MT-TG diabetic mice. Correspondingly, cardiac glycogen synthase phosphorylation, hexokinase II, PPARα, and PGC-1α expression, which mediate glucose and lipid metabolisms, were significantly changed along with cardiac lipid accumulation, inflammation (TNF-α, plasminogen activator inhibitor 1 [PAI-1], and intracellular adhesion molecule 1 [ICAM-1]), nitrosative damage (3-nitrotyrosin accumulation), and fibrosis in the wild-type diabetic mice. The above pathological changes were completely prevented either by cardiac metallothionein in the MT-TG diabetic mice or by inhibition of GSK-3β activity in the wild-type diabetic mice with a GSK-3β–specific inhibitor. CONCLUSIONS These results suggest that activation of GSK-3β plays a critical role in diabetes-related changes in cardiac energy metabolism, inflammation, nitrosative damage, and remodeling. Metallothionein inactivation of GSK-3β plays a critical role in preventing diabetic cardiomyopathy. PMID:19324938

  20. Individualizing Medicare.

    PubMed

    Chollet, D J

    1999-05-01

    Despite the enactment of significant changes to the Medicare program in 1997, Medicare's Hospital Insurance trust fund is projected to be exhausted just as the baby boom enters retirement. To address Medicare's financial difficulties, a number of reform proposals have been offered, including several to individualize Medicare financing and benefits. These proposals would attempt to increase Medicare revenues and reduce Medicare expenditures by having individuals bear risk--investment market risk before retirement and insurance market risk after retirement. Many fundamental aspects of these proposals have yet to be worked out, including how to guarantee a baseline level of saving for health insurance after retirement, how retirees might finance unanticipated health insurance price increases after retirement, the potential implications for Medicaid of inadequate individual saving, and whether the administrative cost of making the system fair and adequate ultimately would eliminate any rate-of-return advantages from allowing workers to invest their Medicare contributions in corporate stocks and bonds.

  1. Functional divergence in tandemly duplicated Arabidopsis thaliana trypsin inhibitor genes.

    PubMed Central

    Clauss, M J; Mitchell-Olds, T

    2004-01-01

    In multigene families, variation among loci and alleles can contribute to trait evolution. We explored patterns of functional and genetic variation in six duplicated Arabidopsis thaliana trypsin inhibitor (ATTI) loci. We demonstrate significant variation in constitutive and herbivore-induced transcription among ATTI loci that show, on average, 65% sequence divergence. Significant variation in ATTI expression was also found between two molecularly defined haplotype classes. Population genetic analyses for 17 accessions of A. thaliana showed that six ATTI loci arranged in tandem within 10 kb varied 10-fold in nucleotide diversity, from 0.0009 to 0.0110, and identified a minimum of six recombination events throughout the tandem array. We observed a significant peak in nucleotide and indel polymorphism spanning ATTI loci in the interior of the array, due primarily to divergence between the two haplotype classes. Significant deviation from the neutral equilibrium model for individual genes was interpreted within the context of intergene linkage disequilibrium and correlated patterns of functional differentiation. In contrast to the outcrosser Arabidopsis lyrata for which recombination is observed even within ATTI loci, our data suggest that response to selection was slowed in the inbreeding, annual A. thaliana because of interference among functionally divergent ATTI loci. PMID:15082560

  2. A role for seed storage proteins in Arabidopsis seed longevity.

    PubMed

    Nguyen, Thu-Phuong; Cueff, Gwendal; Hegedus, Dwayne D; Rajjou, Loïc; Bentsink, Leónie

    2015-10-01

    Proteomics approaches have been a useful tool for determining the biological roles and functions of individual proteins and identifying the molecular mechanisms that govern seed germination, vigour and viability in response to ageing. In this work the dry seed proteome of four Arabidopsis thaliana genotypes, that carry introgression fragments at the position of seed longevity quantitative trait loci and as a result display different levels of seed longevity, was investigated. Seeds at two physiological states, after-ripened seeds that had the full germination ability and aged (stored) seeds of which the germination ability was severely reduced, were compared. Aged dry seed proteomes were markedly different from the after-ripened and reflected the seed longevity level of the four genotypes, despite the fact that dry seeds are metabolically quiescent. Results confirmed the role of antioxidant systems, notably vitamin E, and indicated that protection and maintenance of the translation machinery and energy pathways are essential for seed longevity. Moreover, a new role for seed storage proteins (SSPs) was identified in dry seeds during ageing. Cruciferins (CRUs) are the most abundant SSPs in Arabidopsis and seeds of a triple mutant for three CRU isoforms (crua crub cruc) were more sensitive to artificial ageing and their seed proteins were highly oxidized compared with wild-type seeds. These results confirm that oxidation is involved in seed deterioration and that SSPs buffer the seed from oxidative stress, thus protecting important proteins required for seed germination and seedling formation.

  3. A role for seed storage proteins in Arabidopsis seed longevity

    PubMed Central

    Nguyen, Thu-Phuong; Cueff, Gwendal; Hegedus, Dwayne D; Rajjou, Loïc; Bentsink, Leónie

    2015-01-01

    Proteomics approaches have been a useful tool for determining the biological roles and functions of individual proteins and identifying the molecular mechanisms that govern seed germination, vigour and viability in response to ageing. In this work the dry seed proteome of four Arabidopsis thaliana genotypes, that carry introgression fragments at the position of seed longevity quantitative trait loci and as a result display different levels of seed longevity, was investigated. Seeds at two physiological states, after-ripened seeds that had the full germination ability and aged (stored) seeds of which the germination ability was severely reduced, were compared. Aged dry seed proteomes were markedly different from the after-ripened and reflected the seed longevity level of the four genotypes, despite the fact that dry seeds are metabolically quiescent. Results confirmed the role of antioxidant systems, notably vitamin E, and indicated that protection and maintenance of the translation machinery and energy pathways are essential for seed longevity. Moreover, a new role for seed storage proteins (SSPs) was identified in dry seeds during ageing. Cruciferins (CRUs) are the most abundant SSPs in Arabidopsis and seeds of a triple mutant for three CRU isoforms (crua crub cruc) were more sensitive to artificial ageing and their seed proteins were highly oxidized compared with wild-type seeds. These results confirm that oxidation is involved in seed deterioration and that SSPs buffer the seed from oxidative stress, thus protecting important proteins required for seed germination and seedling formation. PMID:26184996

  4. Plasticity of the Arabidopsis root system under nutrient deficiencies.

    PubMed

    Gruber, Benjamin D; Giehl, Ricardo F H; Friedel, Swetlana; von Wirén, Nicolaus

    2013-09-01

    Plant roots show a particularly high variation in their morphological response to different nutrient deficiencies. Although such changes often determine the nutrient efficiency or stress tolerance of plants, it is surprising that a comprehensive and comparative analysis of root morphological responses to different nutrient deficiencies has not yet been conducted. Since one reason for this is an inherent difficulty in obtaining nutrient-deficient conditions in agar culture, we first identified conditions appropriate for producing nutrient-deficient plants on agar plates. Based on a careful selection of agar specifically for each nutrient being considered, we grew Arabidopsis (Arabidopsis thaliana) plants at four levels of deficiency for 12 nutrients and quantified seven root traits. In combination with measurements of biomass and elemental concentrations, we observed that the nutritional status and type of nutrient determined the extent and type of changes in root system architecture (RSA). The independent regulation of individual root traits further pointed to a differential sensitivity of root tissues to nutrient limitations. To capture the variation in RSA under different nutrient supplies, we used principal component analysis and developed a root plasticity chart representing the overall modulations in RSA under a given treatment. This systematic comparison of RSA responses to nutrient deficiencies provides a comprehensive view of the overall changes in root plasticity induced by the deficiency of single nutrients and provides a solid basis for the identification of nutrient-sensitive steps in the root developmental program.

  5. Natural variation in herbivore-induced volatiles in Arabidopsis thaliana

    PubMed Central

    Snoeren, Tjeerd A. L.; Broekgaarden, Colette; Mumm, Roland; Dicke, Marcel; Bouwmeester, Harro J.

    2010-01-01

    To study whether natural variation in Arabidopsis thaliana could be used to dissect the genetic basis of responses to herbivory in terms of induced volatile emissions, nine accessions were characterized upon herbivory by biting-chewing Pieris rapae caterpillars or after treatment with the phytohormone jasmonic acid (JA). Analysis of 73 compounds in the headspace showed quantitative differences in the emission rates of several individual compounds among the accessions. Moreover, variation in the emission of volatile compounds after JA treatment was reflected in the behaviour of the parasitoid Diadegma semiclausum when they were offered the headspace volatiles of several combinations of accessions in two-choice experiments. Accessions also differ in transcript levels of genes that are associated with the emission of plant volatiles. The genes BSMT1 and Cyp72A13 could be connected to the emission of methyl salicylate and (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT), respectively. Overall, Arabidopsis showed interesting phenotypic variations with respect to the volatile blend emitted in response to herbivory that can be exploited to identify genes and alleles that underlie this important plant trait. PMID:20488836

  6. [Individualizing Education.

    ERIC Educational Resources Information Center

    Horrigan, William J.

    The individually guided education (IGE) program developed by the Kettering Foundation was implemented in September of 1973 at the John F. Kennedy Memorial Junior High School in Woburn, Massachusetts. The components of the program described in this speech include pupil and teacher scheduling, physical layout, pupil selection and adjustment,…

  7. Identifying essential genes in Arabidopsis thaliana.

    PubMed

    Meinke, David; Muralla, Rosanna; Sweeney, Colleen; Dickerman, Allan

    2008-09-01

    Eight years after publication of the Arabidopsis genome sequence and two years before completing the first phase of an international effort to characterize the function of every Arabidopsis gene, plant biologists remain unable to provide a definitive answer to the following basic question: what is the minimal gene set required for normal growth and development? The purpose of this review is to summarize different strategies employed to identify essential genes in Arabidopsis, an important component of the minimal gene set in plants, to present an overview of the datasets and specific genes identified to date, and to discuss the prospects for future saturation of this important class of genes. The long-term goal of this collaborative effort is to facilitate basic research in plant biology and complement ongoing research with other model organisms.

  8. Differentiation of programmed Arabidopsis cells

    PubMed Central

    Xie, De-Yu; Shi, Ming-Zhu

    2012-01-01

    Plants express genes that encode enzymes that catalyse reactions to form plant secondary metabolites in specific cell types. However, the mechanisms of how plants decide their cellular metabolic fate and how cells diversify and specialise their specific secondary metabolites remains largely unknown. Additionally, whether and how an established metabolic program impacts genome-wide reprogramming of plant gene expression is unclear. We recently isolated PAP1-programmed anthocyanin-producing (red) and -free (white) cells from Arabidopsis thaliana; our previous studies have indicated that the PAP1 expression level is similar between these two different cell types. Transcriptional analysis showed that the red cells contain the TTG1-GL3/TT8-PAP1 regulatory complex, which controls anthocyanin biosynthesis; in contrast, the white cells and the wild-type cells lack this entire complex. These data indicate that different regulatory programming underlies the different metabolic states of these cells. In addition, our previous transcriptomic comparison indicated that there is a clear difference in the gene expression profiles of the red and wild-type cells, which is probably a consequence of cell-specific reprogramming. Based on these observations, in this report we discuss the potential mechanisms that underlie the programming and reprogramming of gene expression involved in anthocyanin biosynthesis. PMID:22126737

  9. Individualized Communications

    NASA Technical Reports Server (NTRS)

    1997-01-01

    IntelliWeb and IntelliPrint, products from MicroMass Communications, utilize C Language Integrated Production System (CLIPS), a development and delivery expert systems tool developed at Johnson Space Center. IntelliWeb delivers personalized messages by dynamically creating single web pages or entire web sites based on information provided by each website visitor. IntelliPrint is a product designed to create tailored, individualized messages via printed media. The software uses proprietary technology to generate printed messages that are personally relevant and tailored to meet each individual's needs. Intelliprint is in use in many operations including Brystol-Myers Squibb's personalized newsletter, "Living at Your Best," geared to each recipient based on a health and lifestyle survey taken earlier; and SmithKline Beecham's "Nicorette Committed Quitters Program," in which customized motivational materials support participants in their attempt to quit smoking.

  10. Properties of tobacco (Nicotiana tabacum) cadmium-binding peptide(s). Unique non-metallothionein cadmium ligands.

    PubMed Central

    Reese, R N; Wagner, G J

    1987-01-01

    The chemical and physical characteristics of Cd-binding peptides isolated from tobacco (Nicotiana tabacum) leaves and suspension-cultured tobacco cells were determined and compared with properties of rat liver Cd,Zn-thionein. Some emphasis was placed on metal-binding and specificity properties. Cd-peptides of apparent Mr 6000 and 2000 were induced in tobacco leaves by growth of plants with 90 microM-Cd. Only the apparent-Mr-2000 Cd-peptide was induced in the leaves of tobacco plants grown in the presence of 3 microM-Cd. In cultured tobacco cells exposed to a wide range of Cd levels (3-180 microM), a peptide of apparent Mr 2000 was observed. Under denaturing conditions [6 M-guanidinium chloride (GdmCl) with or without 100 mM-2-mercaptoethanol], all of the above forms were shown to have an Mr of approx. 1300, compared with an Mr of 6000 for Cd,Zn-thionein. The apparent disaggregation of the Mr-6000 form by GdmCl to what appears to be the unit Cd-binding peptide was reversible. Tobacco-derived Cd-peptide contained approx. 40, 35 and 15 residues of glutamate/glutamine, cysteine and glycine respectively, with serine, lysine, and aromatic residues being absent. Tobacco Cd-peptide had an isoelectric point (pI) of 3.15, which is lower than the pI greater than or equal to 4 reported for metallothionein. A 50% dissociation of Cd occurred at pH 5 and 3.5 for the tobacco Cd-peptide and Cd,Zn-thionein respectively, and GdmCl was shown to cause Cd dissociation from tobacco peptide, but not from metallothionein. No evidence was obtained for Zn induction in vivo of, or Zn binding in vitro to, tobacco Cd-peptide. Copper induced a low-Mr metal-binding component in cultured tobacco cells which did not appear to be identical with the peptide induced by Cd. Properties of tobacco Cd-peptide and Cd,Zn-thionein, including metal affinity and selectivity, are greatly different, except for the common presence of 30 residues of cysteine/100 residues. PMID:3593213

  11. The role of metallothionein IIa in defending lens epithelial cells against cadmium and TBHP induced oxidative stress

    PubMed Central

    Hawse, John R.; Padgaonkar, Vanita A.; Leverenz, Victor R.; Pelliccia, Sara E.; Kantorow, Marc; Giblin, Frank J.

    2007-01-01

    Purpose Heavy metals and other forms of oxidative stress have been implicated as key factors in the formation of age-related cataract in humans. Metallothioneins are a group of proteins known to play important roles in defending cells against the cytotoxic effects of heavy metals. However, little is known about their involvement in defending against other forms of oxidative stress. Here, we examined the ability of metallothionein IIa (MTIIa) to protect human lens epithelial cells against cadmium and tertiary butyl hydroperoxide (TBHP)-induced oxidative stress. Methods MTIIa over-expressing human lens epithelial cells (SRA01/04) were created by retroviral mediated gene transfer. Normal and MTIIa over-expressing cells were exposed to various concentrations of cadmium and TBHP and subsequently monitored for cell death, changes in cell phenotype and differences in growth rate. In addition, expression levels of three other important antioxidant genes, heme oxygenase-1, thioredoxin reductase-1, and manganese superoxide dismutase were monitored by real-time RT-PCR following exposure to TBHP. Results Analysis of the over expressing cell lines revealed an approximate 3–4 fold increase in MTIIa expression relative to control cells, resulting in as much as 20% protection against cadmium-induced oxidative stress (p<0.001). The MTIIa over expressing cells were also significantly more resistant to TBHP treatment while control cells exhibited significant shrinking and rounding-up following 3–6 h TBHP treatment, no changes were observed in TBHP-treated over expressing cells. When control cells were treated for 3 h or overnight with TBHP, 40–45% cell death occurred by day three. However, no cell death was observed at this time for the treated MTIIa over-expressing cell line. In addition, TBHP induced the expression of MTIIa, heme oxygenase-1, thioredoxin reductase-1, and MnSOD in both normal and MTIIa over-expressed cell lines. Interestingly the latter three genes were

  12. Analysis of Unfolded Protein Response in Arabidopsis

    PubMed Central

    Chen, Yani; Brandizzi, Federica

    2014-01-01

    The unfolded protein response (UPR) is fundamental for development and adaption in eukaryotic cells. Arabidopsis has become one of the best model systems to uncover conserved mechanisms of the UPR in multicellular eukaryotes as well as organism-specific regulation of the UPR in plants. Monitoring the UPR in planta is an elemental approach to identifying regulatory components and to revealing molecular mechanisms of the plant UPR. In this chapter, we provide protocols for the induction and analyses of plant UPR at a molecular level in Arabidopsis. Three kinds of ER stress treatment methods and quantitation of the plant UPR activation are described here. PMID:23913037

  13. One, two, three...models for trichome patterning in Arabidopsis?

    PubMed

    Pesch, Marina; Hülskamp, Martin

    2009-10-01

    Trichome patterning in Arabidopsis rosette leaves serves as a model system to study how individual cells are determined in a regular spacing pattern from initially equivalent cells. A conserved gene cassette regulates this spacing pattern. bHLH, MYB, and WD40 factors are positive regulators of trichome development that are inhibited by R3 single-repeat MYB proteins. One positive regulator, the WD40-protein, and the negative regulators are mobile and are transported in opposite directions: the WD40 factor moves to the negative regulators away from trichome initials. This movement behavior and the genetic and molecular interactions between the contributing genes and proteins make it very difficult to derive the mechanistic core behind pattern formation. Attempts to develop mathematical models toward this end are critically discussed.

  14. A mutational analysis of leaf morphogenesis in Arabidopsis thaliana.

    PubMed Central

    Berná, G; Robles, P; Micol, J L

    1999-01-01

    As a contribution to a better understanding of the developmental processes that are specific to plants, we have begun a genetic analysis of leaf ontogeny in the model system Arabidopsis thaliana by performing a large-scale screening for mutants with abnormal leaves. After screening 46,159 M2 individuals, arising from 5770 M1 parental seeds exposed to EMS, we isolated 1926 M2 putative leaf mutants, 853 of which yielded viable M3 inbred progeny. Mutant phenotypes were transmitted with complete penetrance and small variations in expressivity in 255 lines. Most of them were inherited as recessive monogenic traits, belonging to 94 complementation groups, which suggests that we did not reach saturation of the genome. We discuss the nature of the processes presumably perturbed in the phenotypic classes defined among our mutants. PMID:10353913

  15. Assessment of potential biomarkers, metallothionein and vitellogenin mRNA expressions in various chemically exposed benthic Chironomus riparius larvae

    NASA Astrophysics Data System (ADS)

    Park, Kiyun; Kwak, Inn-Sil

    2012-12-01

    The objective of this study was conducted to identify the possibility of using Chironomus metallothionein (MT) and vitellogenin (VTG) as biomarkers of stress caused by endocrinedisrupting chemicals (EDCs), heavy metals, herbicides and veterinary antibiotics. We characterized the MT and VTG cDNA in Chironomus riparius and evaluated their mRNA expression profiles following exposure to different environmental pollutants. The gene expression analysis showed that the MT mRNA levels increased significantly after long-term exposure to cadmium (Cd), copper (Cu), Lead (Pb), di(2-ethylhexyl) phthalate (DEHP), and 2,4-dichlorophenoxyacetic acid (2,4-D). Moreover, the VTG mRNA expression increased significantly in C. riparius larvae exposed to BPA, NP, DEHP, Cd, 2,4-D and fenbendazole. Evaluation of the long-term effects of environmental pollutants revealed up regulation of Chironomus MT mRNA in response to DEHP exposure among EDCs, and the level of the VTG mRNA was increased significantly following treatment with Cd and herbicide 2,4-D at all concentrations in a dose-dependent manner. These results indicate that VTG could be used as a potential biomarker of herbicide and Cd as well as EDCs, while MT was a potential biomarker of heavy metals such as Cd, Cu, and Pb in aquatic environments.

  16. Primary structure of a copper-binding metallothionein from mantle tissue of the terrestrial gastropod Helix pomatia L.

    PubMed Central

    Berger, B; Dallinger, R; Gehrig, P; Hunziker, P E

    1997-01-01

    A novel copper-binding metallothionein (MT) has been purified from mantle tissue of the terrestrial snail Helix pomatia using gel-permeation chromatography, ion-exchange chromatography and reverse-phase HPLC. Copper was removed from the thionein by addition of ammonium tetrathiomolybdate. The resulting apothionein (molecular mass 6247 Da) was S-methylated and digested with trypsin, endoproteinase Arg-C and endoproteinase Lys-C. Amino acid sequences of the resulting peptides were determined by collision-induced dissociation tandem MS. The protein is acetylated at its N-terminus, and consists of 64 amino acids, 18 of which are cysteine residues. A comparison with the cadmium-binding MT isolated from the midgut gland of the same species shows an identical arrangement of the cysteines, but an unexpectedly high variability in the other amino acids. The two MT isoforms differ in total length and at 26 positions of their peptide chains. We suggest that the copper-binding MT isoform from the mantle of H. pomatia is responsible for regulatory functions in favour of copper, probably in connection with the metabolism of the copper-bearing protein, haemocyanin. PMID:9359856

  17. Evidence for nuclear interaction of a cytoskeleton protein (OsIFL) with metallothionein and its role in salinity stress tolerance

    PubMed Central

    Soda, Neelam; Sharan, Ashutosh; Gupta, Brijesh K.; Singla-Pareek, Sneh L.; Pareek, Ashwani

    2016-01-01

    Soil salinity is being perceived as a major threat to agriculture. Plant breeders and molecular biologist are putting their best efforts to raise salt-tolerant crops. The discovery of the Saltol QTL, a major QTL localized on chromosome I, responsible for salt tolerance at seedling stage in rice has given new hopes for raising salinity tolerant rice genotypes. In the present study, we have functionally characterized a Saltol QTL localized cytoskeletal protein, intermediate filament like protein (OsIFL), of rice. Studies related to intermediate filaments are emerging in plants, especially with respect to their involvement in abiotic stress response. Our investigations clearly establish that the heterologous expression of OsIFL in three diverse organisms (bacteria, yeast and tobacco) provides survival advantage towards diverse abiotic stresses. Screening of rice cDNA library revealed OsIFL to be strongly interacting with metallothionein protein. Bimolecular fluorescence complementation assay further confirmed this interaction to be occurring inside the nucleus. Overexpression of OsIFL in transgenic tobacco plants conferred salinity stress tolerance by maintaining favourable K+/Na+ ratio and thus showed protection from salinity stress induced ion toxicity. This study provides the first evidence for the involvement of a cytoskeletal protein in salinity stress tolerance in diverse organisms. PMID:27708383

  18. Effect of metal accumulation on metallothionein level and condition of the periwinkle Littorina littorea along the Scheldt estuary (the Netherlands).

    PubMed

    Van den Broeck, Heidi; De Wolf, Hans; Backeljau, Thierry; Blust, Ronny

    2010-05-01

    Metal (i.e. Ag, As, Ca, Cd, Co, Cu, Mn, Pb and Zn) and metallothionein (MT) concentrations in the soft tissue of Littorina littorea were measured along the heavily polluted Western Scheldt (WS) and relatively clean Eastern Scheldt (ES) estuary. Along the WS metal and MT levels in periwinkles reflected the known downstream decreasing pollution gradient. Surprisingly in ES animals As, Mn and Zn concentrations decreased from east to west reflecting past pollution. Compared to the WS metal concentrations of ES periwinkles were significantly lower and both estuaries were maximally discriminated from each other based on their Cd soft tissue concentration using a canonical discriminant analysis. Furthermore, no overall difference was found in MT levels among animals from both estuaries. Using previously obtained condition data (i.e. dry/wet weight ratio and lipid content) the relation between soft tissue metal concentration (i.e. Cd, Cu and Zn) and fitness indicators (i.e. MT and condition data) was examined using a canonical correlation analysis. Periwinkles with a high metal load (i.e. Cd and Zn) also had high MT levels but were in a relatively poor condition.

  19. Freezing and anoxia stresses induce expression of metallothionein in the foot muscle and hepatopancreas of the marine gastropod Littorina littorea.

    PubMed

    English, Tamara E; Storey, Kenneth B

    2003-07-01

    Differential screening of cDNA libraries constructed from the foot muscle of marine snails Littorina littorea revealed several cDNAs that are upregulated during anoxia or freezing exposures, environmental stresses that are naturally endured by this species. One full-length clone of 1196 nucleotides (GenBank accession number AY034179) hybridized with a 1200-nucleotide band on northern blots and encoded a 100-amino-acid protein that was identified as belonging to the metallothionein (MT) family. L. littorea MT shared 45% and 56% identity with the copper- and cadmium-binding MT isoforms, respectively, from another gastropod, Helix pomatia and 43-47% identity with marine bivalve MTs. The L. littorea sequence included the mollusc-specific C-terminal motif Cys-X-Cys-X(3)-Cys-Thr-Gly-X(3)-Cys-X-Cys-X(3)-Cys-X-Cys-Lys that identifies it as a family 2 (mollusc) MT. Northern blot analysis showed that L. littorea MT was upregulated in both foot muscle and hepatopancreas in response to both freezing and anoxia stresses; within 1 h of the beginning of the stress transcript levels rose 2.5- to sixfold of control levels, reaching maximal levels at 12 or 24 h. After 24 h recovery from either stress, transcript levels were reduced again in three cases but remained elevated in hepatopancreas from anoxia-treated snails. Upregulation of MT during environmental stress could serve one or more possible roles, including a function in antioxidant defense.

  20. Effects of the herbicide isoproturon on metallothioneins, growth, and antioxidative defenses in the aquatic worm Tubifex tubifex (Oligochaeta, Tubificidae).

    PubMed

    Mosleh, Yahia Y; Paris-Palacios, Séverine; Couderchet, Michel; Biagianti-Risbourg, Sylvie; Vernet, Guy

    2005-07-01

    Metallothioneins (MTs) are low molecular weight proteins, mainly implicated in metal ion detoxification. Increase in MT contents is considered to be a specific biomarker of metal exposure. Recently it has been demonstrated that MTs participate in several cellular functions such as regulation of growth, and antioxidative defenses. Therefore, the induction of MTs as biomarkers of exposure to the pesticide isoproturon has been investigated in the aquatic worms Tubifex tubifex. MT levels in exposed worms increased significantly (p < 0.05) after 2, 4, and 7 days of exposure to different concentrations of isoproturon (maximum increase compared to unexposed controls: +148.56% for 10 mg l(-1) after 4 days of exposure). In response to isoproturon, the activity of glutathione-S-transferase (max. +52%), glutathione-reductase (max. +100%), and catalase (max. +117%) increased, demonstrating the occurrence of an oxidative stress response to the herbicide. Thus, the increase in MT contents caused by isoproturon was interpreted as a defense response towards increased oxidative stress generated by the herbicide. Residues of isoproturon and its metabolites, 1-(4-isopropylphenyl)-3-methylurea, 1-(4-isopropylphenyl) urea, and 4-isopropylanilin were detected in the worm growth medium. Half-life of the herbicide was shorter at a low (0.1 mg l(-1)) initial concentration. The herbicide accumulated in T. tubifex but no metabolite could be detected.

  1. Simultaneous induction of Pb-metallothionein-like protein and Zn-thionein in the liver of rats given lead acetate.

    PubMed Central

    Ikebuchi, H; Teshima, R; Suzuki, K; Terao, T; Yamane, Y

    1986-01-01

    Administration of a sublethal dose of lead acetate to rats induced the simultaneous synthesis of a Pb-metallothionein (Pb-MT)-like protein (Pb-BP) and Zn-thionein (Zn-BP) in the liver. The Pb-BP had an apparent molecule mass of 6900 Da and seemed to bind preferentially to lead in the liver cytosol. The Zn-BP was identified by comparison of the Mr, elution profiles from Sephadex G-75 and DEAE-Sephadex A-25 columns, and polyacrylamide-gel-electrophoretic mobility, with those of rat liver Zn-MT-II. The Pb-BP accumulated in the liver to a maximum 6 h after the intraperitoneal injection of lead acetate and accounted for about 60% of the lead in the liver cytosol at this stage. However, after that, it gradually decreased in the liver, until it was close to the basal amount 24 h after the induction. In contrast, the amount of Zn-MT increased gradually, reached a maximum 12 h after the administration of lead acetate and maintained a constant value until at least 24 h after the induction. Amino acid analysis of the Pb-BP indicated that it contained about 28% half-cysteine. These results strongly suggest that lead acetate induces the synthesis of Pb-MT as well as Zn-MT in rat liver. Images Fig. 6. PMID:3954751

  2. Is the digestive gland of Mytilus galloprovincialis a tissue of choice for estimating cadmium exposure by means of metallothioneins?

    PubMed

    Raspor, Biserka; Dragun, Zrinka; Erk, Marijana; Ivanković, Dusica; Pavicić, Jasenka

    2004-10-15

    A study performed over 12 months with caged mussels Mytilus galloprovincialis in the coastal marine zone, which is under urban pressure, reveals a temporal variation of digestive gland mass, which causes "biological dilution" of cytosolic metallothionein (MT) and trace metal (Cd, Cu, Zn, Fe, Mn) concentrations. The dilution effect was corrected by expressing the cytosolic MT and metal concentrations as the tissue content. Consequently, the changes of the average digestive gland mass coincide with the changes of MT and trace metal contents. From February to June, MT contents are nearly twice and trace metal contents nearly three times higher than those of the other months. The period of increased average digestive gland mass, of MT and trace metal contents probably overlaps with the sexual maturation of mussels (gametogenesis) and enhanced food availability. Since natural factors contribute more to the MT content than the sublethal levels of Cd, the digestive gland of M. galloprovincialis is not considered as a tissue of choice for estimating Cd exposure by means of MTs.

  3. Partial Gene Sequencing of CYP1A, Vitellogenin, and Metallothionein in Mosquitofish Gambusia yucatana and Gambusia sexradiata.

    PubMed

    Vázquez-Euán, Roberto; Escalante-Herrera, Karla S; Rodríguez-Fuentes, Gabriela

    2017-01-01

    Ground characteristics in the Yucatan Peninsula make recovery and treatment of wastewater very expensive. This situation has contributed to an increase of pollutants in the aquifer. Unfortunately, studies related to the effects of those pollutants in native organisms are scarce. The aim of this work was to obtain partial sequences of widely known genes used as biomarkers of pollutant effect in Gambusia yucatana and Gambusia sexradiata. The studied genes were: cytochrome P450 1A (CYP1A); vitellogenin (VTG); metallothionein (MT), and two housekeeping genes, 18S and β-actin. From reported sequences of Gambusia affinis, primers were designed and amplification was done in the local Gambusia species exposed for 48 h to gasoline (100 µL/L, stirred for 24 h pre-exposure). Preliminary results revealed partial sequences of all genes with an approximate average length of 200 bp. BLAST analysis of found sequences indicated a minimum of 97% identity with reported sequences for G. affinis or Gambusia holbrooki showing great similarity.

  4. Localization of genes encoding metallothionein-like protein (mt2 and smtb) in the brain of zebrafish.

    PubMed

    Teoh, Seong Lin; Ogawa, Satoshi; Parhar, Ishwar S

    2015-12-01

    Metallothionein (MT) is a small cysteine-rich heavy metal-binding protein involved in metal homeostasis, detoxification and free radical-scavenging. MT is ubiquitously expressed in several tissues, but its role in the central nervous system is not well understood. In this study, we identified two MT homologous genes (mt2 and smtb) in the zebrafish. Digoxigenin-in situ hybridization showed the expression of mt2 and smtb genes in the ventricular layers in the telencephalon, diencephalon, mesencephalon and rhombencephalon, most of which are cell proliferating regions in the brain of zebrafish. Cellular characteristics of MT genes expressing cells were examined by double-labelling with markers for neurons (HuC/D) and astrocytes (glial fibrillary acidic protein, GFAP and S100 protein) and cell proliferation marker (PCNA). mt2 and smtb mRNAs are expressed in neurons and not in astrocytes, and they were co-localized with PCNA. These results suggest that mt2 and smtb may play an important role in neurogenesis and neuroprotection.

  5. Metallothionein mRNA expression and cadmium tolerance in metal-stressed and reference populations of the springtail Orchesella cincta.

    PubMed

    Timmermans, Martijn J T N; Ellers, Jacintha; Roelofs, Dick; van Straalen, Nico M

    2005-10-01

    Metal contamination in soil ecosystems is a permanent and often strong selection pressure. The present study investigates metal tolerance in 17 Orchesella cincta (Collembola) populations from metal-contaminated and reference sites, and combines analyses at the phenotypic and molecular level. Metal tolerance was phenotypically assayed by measuring survival times of laboratory cultures during exposure to cadmium. Comparisons of survival curves showed that five out of eight metal-stressed populations tested evolved increased cadmium tolerance (Stolberg, Plombieres, Hoboken, Hygum and Gusum). In addition, the role of the metallothionein (MT) gene in cadmium tolerance of O. cincta was studied by means of quantitative RT-PCR. The constitutive and Cd-induced MT mRNA expression of the laboratory cultures was measured. Results show that the mean constitutive MT mRNA expression of populations from polluted sites was significantly higher than of populations from reference sites. However, no correlation between MT mRNA expression levels after laboratory exposure to cadmium and field cadmium concentrations was observed. Furthermore, no relation between survival rate during exposure to cadmium and MT mRNA expression was detected. Our results suggest that constitutive MT mRNA expression plays a role in early protection against cadmium toxicity, and indicate that mechanisms other then MT up-regulation are involved in tolerance to prolonged exposure to cadmium.

  6. Metallothionein-I/II Knockout Mice Aggravate Mitochondrial Superoxide Production and Peroxiredoxin 3 Expression in Thyroid after Excessive Iodide Exposure

    PubMed Central

    Zhang, Na; Wang, Lingyan; Duan, Qi; Lin, Laixiang; Ahmed, Mohamed; Wang, Tingting; Yao, Xiaomei

    2015-01-01

    Purpose. We aim to figure out the effect of metallothioneins on iodide excess induced oxidative stress in the thyroid. Methods. Eight-week-old MT-I/II knockout (MT-I/II KO) mice and background-matched wild-type (WT) mice were used. Mitochondrial superoxide production and peroxiredoxin (Prx) 3 expression were measured. Results. In in vitro study, more significant increases in mitochondrial superoxide production and Prx 3 expression were detected in the MT-I/II KO groups. In in vivo study, significantly higher concentrations of urinary iodine level were detected in MT-I/II KO mice in 100 HI group. Compared to the NI group, there was no significant difference existing in serum thyroid hormones level in either groups (P > 0.05), while the mitochondrial superoxide production was significantly increased in 100 HI groups with significantly increased LDH activity and decreased relative cell viability. Compared to WT mice, more significant changes were detected in MT-I/II KO mice in 100 HI groups. No significant differences were detected between the NI group and 10 HI group in both the MT-I/II KO and WT mice groups (P > 0.05). Conclusions. Iodide excess in a thyroid without MT I/II protection may result in strong mitochondrial oxidative stress, which further leads to the damage of thyrocytes. PMID:26101557

  7. Investigations into the possible use of marine flatfish metallothionein concentrations as an indicator of toxic metal exposure

    SciTech Connect

    Overnell, J.; Fletcher, T.C.; McIntosh, R. )

    1988-09-01

    Marine flatfish would seem to be a useful class of fish because they are potential targets for pollution. Flounders tend to stay within their estuaries and a species of dab is the most common fish feeding at least one sewage dumpsite. However, before it is worth starting a monitoring program they need more information about this class of fish. The authors need to known whether metallothionein levels can be elevated by metals, the effect of non-metal stress-associated factors and the seasonal variation of the endogenous levels. If MT is elevated then how long does the effect persist They also need to know the effect of dietary metals since a number of studies on zinc and copper uptake into fish have indicated, that except in extreme conditions such as downstream from a copper mine, quantitatively the most important route for the metals zinc and copper is not directly from the water but through the diet. They have attempted to answer some of these questions.

  8. Clinical utility of copper, ceruloplasmin, and metallothionein plasma determinations in human neurodegenerative patients and their first-degree relatives.

    PubMed

    Arnal, Nathalie; Cristalli, Diana Olga; de Alaniz, María J T; Marra, Carlos Alberto

    2010-03-10

    The concentration of plasma copper, ceruloplasmin (CRP), non-ceruloplasmin-bound Cu (NCBC), and metallothioneins (MTs) were studied as putative biomarkers for neurodegenerative diseases in patients and in their first-degree relatives. We found increased levels of Cu in the plasma of Alzheimer's disease (AD), Parkinson's disease (PD), and vascular dementia (VD) patients, and the increase observed in VD group was linked to the evolution of the disease. CRP was also elevated in response to the inflammatory component of the diseases, however, a correlation with illness progression was only observed in VD patients. The level of MTs is proportional to the evolution of VD. The Cu/CRP and Cu/MTs ratios are both indicative of disease progression for AD patients but not for those with PD or VD. Moreover, there is a correlation between the NCBC levels and the cognitive impairment estimated through the Mini-mental State Examination (MMSE) scale. This dependence is linear for AD and PD patients and non-linear for the VD ones. The relative values of NCBC showed dependence on the disease duration, especially for AD. Copper measurement and the Cu/CRP ratio may be predictive markers of risk for the first-degree relatives of AD patients. We believe that these results are valuable as a reliable clinical tool.

  9. Genetic variation in metallothionein and metal-regulatory transcription factor 1 in relation to urinary cadmium, copper, and zinc

    PubMed Central

    Adams, Scott V.; Barrick, Brian; Freney, Emily P.; Shafer, Martin M.; Makar, Karen; Song, Xiaoling; Lampe, Johanna; Vilchis, Hugo; Ulery, April; Newcomb, Polly A.

    2015-01-01

    Background Metallothionein (MT) proteins play critical roles in the physiological handling of both essential (Cu and Zn) and toxic (Cd) metals. MT expression is regulated by metal-regulatory transcription factor 1 (MTF1). Hence, genetic variation in the MT gene family and MTF1 might therefore influence excretion of these metals. Methods 321 women were recruited in Seattle, WA and Las Cruces, NM and provided demographic information, urine samples for measurement of metal concentrations by mass spectrometry and creatinine, and blood or saliva for extraction of DNA. Forty-one single nucleotide polymorphisms (SNPs) within the MTF1 gene region and the region of chromosome 16 encoding the MT gene family were selected for genotyping in addition to an ancestry informative marker panel. Linear regression was used to estimate the association of SNPs with urinary Cd, Cu, and Zn, adjusted for age, urinary creatinine, smoking history, study site, and ancestry. Results Minor alleles of rs28366003 and rs10636 near the MT2A gene were associated with lower urinary Cd, Cu, and Zn. Minor alleles of rs8044719 and rs1599823, near MT1A and MT1B, were associated with lower urinary Cd and Zn, respectively. Minor alleles of rs4653329 in MTF1 was associated with lower urinary Cd. Conclusions These results suggest that genetic variation in the MT gene region and MTF1 influences urinary Cd, Cu, and Zn excretion. PMID:26529669

  10. Expression and function analysis of the metallothionein-like (MT-like) gene from Festuca rubra in Chlamydomonas reinhardtii chloroplast.

    PubMed

    Han, SiHai; Hu, ZhangLi; Lei, AnPing

    2008-12-01

    The cDNA of the metallothionein-like (MT-like) gene from Festuca rubra cv. Merlin was optimized with bias codon of Chlamydomonas reinhardtii chloroplast genome. The optimized MT-like gene was delivered into C. reinhardtii chloroplast and the transgenic strains expressing MT-like gene was obtained. PCR-Southern blot and RT-PCR-Southern blot analysis demonstrated that the MT-like gene was integrated into chloroplast genome of C. reinhardtii and expressed at the transcriptional level. The cadmium binding capacity of the transgenic C. reinhardtii was determined by hydride generation-atomic fluorescence spectrometry (HG-AFS) and the binding properties were analyzed. Results showed that the transgenic C. reinhardtii expressing the MT-like gene exhibited remarkably higher Cd(2+) binding capacity and grew to higher densities at toxic Cd(2+) concentrations (40-100 micromol/L) than the wild type strain, and that the IC(50) of Cd(2+) (3-d treating) to algal cell growth of transgenic strain was 55.43% higher than that of the wild type strain, indicating that the Cd(2+) binding capacity and Cd(2+) tolerance of C. reinhardtii was enhanced through the expression of the foreign MT-like gene in chloroplast.

  11. Preventive effects of metallothionein against DNA and lipid metabolic damages in dyslipidemic mice under repeated mild stress.

    PubMed

    Higashimoto, Minoru; Isoyama, Naohiro; Ishibashi, Satoshi; Ogawa, Naoko; Takiguchi, Masufumi; Suzuki, Shinya; Ohnishi, Yoshinari; Sato, Masao

    2013-01-01

    The effects of repeated mild stress on DNA and lipid metabolic damages in multiple organs of dyslipidemic mice, and the preventive role of metallothionein (MT) were investigated. Female adult wild-type and MT-null mice fed high-fat diet (HFD) or standard diet (STD) were repeatedly subjected to fasting or restraint for three weeks. The liver, pancreas, spleen, bone marrow and serum samples were taken for evaluating DNA damage, MT, glutathione (GSH), corticosterone, carnitine and adiponectin. Body weights of restraint groups were reduced with the intensity of stress increased, even if the energy intakes were higher than those of STD group. Hepatic GSH levels were reduced in HFD control group and were further reduced in stress groups, especially in restraint groups, while the hepatic MT and serum corticosterone levels were increased in concert with the intensity of stress. Cellular DNA damages were generally increased by the restraint stress, especially in MT-null mice. Hepatic carnitine levels of MT-null mice were markedly lower than those of wild-type mice. The data suggest that MT plays a preventive role by acting as an antioxidant in corporation with GSH decreased by repeated stress and that MT may be an essential factor for inducing carnitine under the stress.

  12. Structural study of the zinc and cadmium complexes of a type 2 plant (Quercus suber) metallothionein: insights by vibrational spectroscopy.

    PubMed

    Domènech, Jordi; Tinti, Anna; Capdevila, Mercè; Atrian, Silvia; Torreggiani, Armida

    2007-06-15

    Zn- and Cd-complexes of Quercus suber metallothionein (QsMT) were obtained by in vivo-synthesis, in order to obtain physiologically representative aggregates, and characterized by spectrometric and spectroscopic methods. The secondary structure elements and the coordination environments of the metal binding sites of the two aggregates were determined, as well as the main metal-containing species formed. The results obtained from the analysis of the Raman and IR spectra reveal that these metal-MT complexes predominantly contain beta-sheet elements (about 60%), whereas they lack alpha-helices. These structural features slightly depend on the divalent metal bound. In particular, Cd(II) binding to QsMT induces a slight increase of the beta-sheet percentage, as well as a decrease in beta-turn elements with respect to Zn(II) binding. Conversely, the in vivo capability of QsMT to inglobe metal and sulfide ions is metal-depending. Spectroscopic vibrational data also confirm the presence of sulfide ligands in the metal clusters of both Zn- and Cd-QsMT, while the participation of the spacer His residue in metal coordination was only found in Cd-QsMT, in agreement with the CD results. Overall data suggest different coordination environments for Zn(II) and Cd(II) ions in QsMT.

  13. Metallothioneins and heat shock proteins 70 in marine mussels as sensors of environmental pollution in Northern Adriatic Sea.

    PubMed

    Mićović, Vladimir; Bulog, Aleksandar; Kučić, Natalia; Jakovac, Hrvoje; Radošević-Stašić, Biserka

    2009-11-01

    In an attempt to assess the intensity of environmental pollution in industrial zones of Kvarnerian Bay in Northern Adriatic Sea and the reactivity of Mytilus galloprovincialis to these changes, in this study we estimated the concentration of heavy metals at four locations in both sea-sediment and in the mussels. Further we tried to correlate these changes with seasonal variations in environmental temperature, pH and salinity, as well as with the expression of metallothioneins (MTs) and heat shock proteins (HSPs) in the digestive tract of the mussels. Sampling in vivo was performed monthly, during the year 2008, while under the laboratory conditions the reactivity of acclimated mussels were tested to increasing concentrations of CdCl(2) and to thermal stress. The data have shown that the induction of MTs and HSP isoforms of the 70-kDa size class were highly affected by model agents treatment including contamination of sea-sediment by Pb, Hg and Cd, implying that these stress proteins might be power biomarkers of marine pollution.

  14. Prooxidative effect of copper--metallothionein in the acute cytotoxicity of hydrogen peroxide in Ehrlich ascites tumour cells.

    PubMed

    Suntres, Zacharias E; Lui, Edmund M K

    2006-01-16

    This study was concerned with the role of copper (Cu) and Cu-metallothionein (Cu-MT) in oxidative stress. Hydrogen peroxide (H(2)O(2))-induced oxidative injury was examined in Ehrlich ascites tumour cells isolated from host mice pretreated with 0, 1 or 2mg of CuSO(4) (ip) 24h earlier. Control Ehrlich cells contained low levels of Cu and Cu treatment produced dose-related increases in cellular Cu and Cu-MT levels and corresponding increases in sensitivity to oxidative toxicity of H(2)O(2) (LC(50), cell blebbing, lipid peroxidation, GSH depletion, and increase in intracellular free [Ca(2+)](i)). Hydrogen peroxide treatment also resulted in the oxidation of MT thiolates, reduction in the binding of Cu to MT resulting in translocation of Cu to other subcellular sites. d-penicillamine, a Cu-chelating agent, obliterated the sensitization effect of Cu-pretreatment and reduced the redistribution of MT-bound Cu, suggesting the participation of Cu ions derived from MT in promoting oxidant stress. Additional experiments with desferoxamine and mannitol have revealed the involvement of a Cu-dependent Fenton reaction in the mediation of the prooxidative effect of Cu-MT. These data suggest that cells with high levels of Cu-MT may be particularly susceptible to oxidative stress.

  15. Exposure of cultured human proximal tubular cells to cadmium, mercury, zinc and bismuth: toxicity and metallothionein induction.

    PubMed

    Rodilla, V; Miles, A T; Jenner, W; Hawksworth, G M

    1998-08-14

    The kidney, in particular the proximal convoluted tubule, is a major target site for the toxic effects of various metals. However, little is known about the early effects of these metals after acute exposure in man. In the present study we have evaluated the toxicity of several inorganic metal compounds (CdCl2, HgCl2, ZnCl2, and Bi(NO3)3) and the induction of metallothionein by these compounds in cultured human proximal tubular (HPT) cells for up to 4 days. The results showed that bismuth was not toxic even at the highest dose (100 microM) used, while zinc, cadmium and mercury exhibited varying degrees of toxicity, zinc being the least toxic and mercury the most potent. A significant degree of interindividual variation between the different isolates used in these experiments was also observed. All metals used in the present study induced MT, as revealed by immunocytochemistry. All metals showed maximal induction between 1 and 3 days after treatment. Although a certain amount of constitutive MT was present in the cultures, the intensity of the staining varied with time in culture and between the different isolates studied. No correlation could be made between the intensity of the staining in control cultures (indicating total amount of constitutive MT) and the susceptibility of a given isolate to metal toxicity. Furthermore, no correlation could be made between metal-induced MT and the susceptibility of a given isolate to that particular metal.

  16. Transcriptional response of lysozyme, metallothionein, and superoxide dismutase to combined exposure to heavy metals and bacteria in Mactra veneriformis.

    PubMed

    Fang, Yan; Yang, Hongsheng; Liu, Baozhong; Zhang, Libing

    2013-01-01

    The response of the defense components lysozyme (LYZ), metallothionein (MT), and superoxide dismutase (SOD) to combined exposure to heavy metals and bacteria was assessed at transcriptional level in the surf clam Mactra veneriformis. First, the full-length LYZ cDNA containing 808 nucleotides and encoding 194 deduced amino acids was identified from the clam. Multiple alignments revealed that MvLYZ had a high identity with invertebrate-type LYZs from other mollusks. Next, clams were exposed to Vibrio parahaemolyticus and a mixture of cadmium and mercury, alone or in combination, for 7 days. Cumulative mortality of clams and mRNA expressions of the three defense components were analyzed. The highest cumulative mortality took place in the combined treatment on day 7. The expression of the three genes was up-regulated in response to treatments compared to the control with different response times and transcriptional levels; the response to combined exposure occurred earlier than to single exposure. Among the experimental groups, MvLYZ expression and MvSOD expression peaked in the combined treatment on day 3, whereas MvMT expression peaked in heavy metals treatment on day 5. Furthermore, interactive effects of heavy metals and Vibrio on transcriptional response changed over the exposure time. Therefore, transcriptional regulation of the three genes under combined exposure was more complex than under single exposure.

  17. Hypoxia drives apoptosis independently of p53 and metallothionein transcript levels in hemocytes of the whiteleg shrimp Litopenaeus vannamei.

    PubMed

    Felix-Portillo, Monserrath; Martínez-Quintana, José A; Arenas-Padilla, Marina; Mata-Haro, Verónica; Gómez-Jiménez, Silvia; Yepiz-Plascencia, Gloria

    2016-10-01

    The cellular mechanisms used by the shrimp Litopenaeus vannamei to respond to hypoxia have been studied from the energetic metabolism and antioxidant angles. We herein investigated the participation of p53 and metallothionein (MT) in the apoptotic process in response to hypoxia in shrimp hemocytes. The Lvp53 or LvMT genes were efficiently silenced by injection of double stranded RNA for p53 or MT. The effects of silencing on apoptosis were measured as caspase-3 activity and flow cytometry in hemocytes after 24 and 48 h of hypoxia (1.5 mg DO L(-1)). Hemocytes from unsilenced animals had significantly higher apoptosis levels upon both times of hypoxia. The apoptotic levels were diminished but not suppressed in dsp53-silenced but not dsMT-silenced hemocytes after 24 h of hypoxia, indicating a contribution of Lvp53 to apoptosis. Apoptosis in normoxia was significantly higher in dsp53-and dsMT-silenced animals compared to the unsilenced controls, pointing to a possible cytoprotective role of LvMT and Lvp53 during the basal apoptotic program in normoxia. Overall, these results indicate that hypoxia augments apoptosis in shrimp hemocytes and high mRNA levels of Lvp53 and LvMT are not necessary for this response.

  18. An Adsorptive Transfer Technique Coupled with Brdicka Reaction to Reveal the Importance of Metallothionein in Chemotherapy with Platinum Based Cytostatics

    PubMed Central

    Krizkova, Sona; Fabrik, Ivo; Huska, Dalibor; Adam, Vojtech; Babula, Petr; Hrabeta, Jan; Eckschlager, Tomas; Pochop, Pavel; Darsova, Denisa; Kukacka, Jiri; Prusa, Richard; Trnkova, Libuse; Kizek, Rene

    2010-01-01

    The drugs based on platinum metals represent one of the oldest, but also one of the most effective groups of chemotherapeutic agents. Thanks to many clinical studies it is known that resistance of tumor cells to drugs is a frequent cause of chemotherapy failure. With regard to platinum based drugs, multidrug resistance can also be connected with increased expression of low-molecular weight protein metallothionein (MT). This study aimed at investigating the interactions of MT with cisplatin or carboplatin, using the adsorptive transfer technique coupled with differential pulse voltammetry Brdicka reaction (AdTS DPV Brdicka reaction), and a comparison of in vitro results with results obtained in vivo. The results obtained from the in vitro study show a strong affinity between platinum based drugs and MT. Further, we analyzed extracts of neuroblastoma cell lines treated with cisplatin or carboplatin. It is clear that neuroblastoma UKF-NB-4 cisplatin-resistant and cisplatin-sensitive cell lines unlikely respond to the presence of the platinum-based cytostatics cisplatin and carboplatin. Finally, we determined the level of MT in samples from rabbits treated with carboplatin and patients with retinoblastoma treated with the same drug. PMID:21614176

  19. Cloning and characterization of metallothionein gene (HcMT) from Halostachys caspica and its expression in E. coli.

    PubMed

    Liu, Zhongyuan; Meng, Hongen; Abdulla, Hasiyatihan; Zhang, Fuchun; Mao, Xinfang

    2016-07-10

    Halostachys caspica is a short shrub distributed in the semi-arid and saline-alkali area, which evolved various mechanisms for modulating salt and metal level. In the present study, a Type 2 metallothionein (HcMT) gene was cloned from the salt induced suppression subtractive hybridization (SSH) cDNA library of H.caspica. Quantitative real time PCR (qRT-PCR) analysis indicated that HcMT gene was up-regulated under the stress of Cu(2+), Zn(2+) and Cd(2+), and the tolerance of E. coli strain harboring with the recombinant HcMT (pET-32a-HcMT) to Cu(2+), Zn(2+) and Cd(2+) was enhanced compared to strain with control vector (pET-32a). Moreover, the purified TrxA-HcMT fusion protein from E. coli cells grown in the presence of 0.3mM CuSO4, 0.3mM ZnSO4, or 0.1mM CdCl2 could bind more metal ions than TrxA alone. The predicted 3D structure showed that HcMT could form a single metal-thiolate cluster, which confers the ability to bind five divalent metal ions through fourteen cysteine residues. These data indicate that HcMT may be involved in processes of metal tolerance in H. caspica and could be employed as a potential candidate for heavy metal phytoremediation.

  20. Utilizing of Adsorptive Transfer Stripping Technique Brdicka Reaction for Determination of Metallothioneins Level in Melanoma Cells, Blood Serum and Tissues.

    PubMed

    Krizkova, Sona; Fabrik, Ivo; Adam, Vojtech; Kukacka, Jiri; Prusa, Richard; Chavis, Grace J; Trnkova, Libuse; Strnadel, Jan; Horak, Vratislav; Kizek, Rene

    2008-05-10

    In the paper we utilized the adsorptive transfer stripping differential pulse voltammetry Brdicka reaction for the determination of metallothioneins (MT) in melanoma cells, animal melanoma tissues (MeLiM miniature pig) and blood serum of patients with malignant melanoma. Primarily we attempted to investigate the influence of dilution of real sample on MT electrochemical response. Dilution of samples of 1 000 times was chosen the most suitable for determination of MT level in biological samples. Then we quantified the MT level in the melanoma cells, the animal melanoma tissues and the blood serum samples. The MT content in the cells varied within the range from 4.2 to 11.2 μM. At animal melanoma tissues (melanomas localized on abdomen, back limb and dorsum) the highest content of MT was determined in the tumour sampled on the back of the animal and was nearly 500 μg of MTs per gram of a tissue. We also quantified content of MT in metastases, which was found in liver, spleen and lymph nodes. Moreover the average MT level in the blood serum samples from patients with melanoma was 3.0 ± 0.8 μM. MT levels determined at melanoma samples were significantly (p < 0.05) higher compared to control ones at cells, tissues and blood serum.

  1. Hepatic and renal metallothionein concentrations in Commerson's dolphins (Cephalorhynchus commersonii) from Tierra del Fuego, South Atlantic Ocean.

    PubMed

    Cáceres-Saez, Iris; Polizzi, Paula; Romero, Belén; Dellabianca, Natalia A; Ribeiro Guevara, Sergio; Goodall, R Natalie P; Cappozzo, H Luis; Gerpe, Marcela

    2016-07-15

    The Commerson's dolphin is the most common endemic odontocete of subantarctic waters of Tierra del Fuego, Argentina incidentally caught in fishing nets. The species is classified as "Data Deficient" by the IUCN. Metallothioneins (MTs) are considered as suitable biomarkers for health and environmental monitoring. The aims of the study were to assess MT concentrations in the liver and kidney of bycaught specimens. Moreover, correlations with Zn, Se, Cd, Ag and Hg, and the molar ratios of MT:metals were estimated to evaluate if there is an indication of their respective protective role against metal toxicity in tissues. Hepatic and renal MT concentrations were similar, ranging from 11.6 to 29.1nmol·g(-1) WW, and Kidney/Liver ratios ranging from 0.73 to 1.93 corresponded to normal ranges. Results suggest that MTs are related to physiological ranges for the species. This information constitutes the first MT report on Commerson's dolphins and possibly considered as baseline for species' conservation.

  2. Quantum mechanical treatment of As(3+)-thiol model compounds: implication for the core structure of As(III)-metallothionein.

    PubMed

    Garla, Roobee; Kaur, Narinder; Bansal, Mohinder Pal; Garg, Mohan Lal; Mohanty, Biraja Prasad

    2017-03-01

    Exposure to inorganic arsenic (As) is one of the major health concerns in several regions around the world. Binding of As(III) with thiols is central to the mechanisms related to its toxicity, detoxification, and therapeutic effects. Due to its high thiol content, metallothionein (MT) is presumed to play an important role in case of arsenic toxicity. Consequences of these As-thiol interactions are not yet clear due to various difficulties in the characterization of arsenic bound proteins by spectroscopic techniques. Computational modeling can be a reliable approach in predicting the molecular structures of such complexes. This paper presents the results of a systematic study on different As(III)-thiol model compounds conducted by both ab initio and DFT methods with different Gaussian type basis sets. Proficiency of these theoretical methods has been evaluated in terms of bond lengths, bond angles, free energy, partial atomic charges, computational cost, and comparison with the experimental data. It has been demonstrated that the DFT-B3LYP/6-311+G(3df) functional offers better accuracy in predicting the structure and the UV absorption spectra of As(III)-thiol complexes. The results of the present study also helps in defining the boundaries for the core of arsenic bound MT so that quantum mechanical/molecular mechanical (QM/MM) methods can be employed to predict the structural and functional aspects of the protein. Graphical Abstract Optimized structural parameters of As(3+)-thiol model compounds.

  3. [Effects of the expression of mouse metallothionein-I gene in human HeLa cell line on drug resistance].

    PubMed

    Li, X; Lü, W; Yin, S; Li, L

    2000-07-01

    Metallothionein-I (MT-I) gene was inserted into EcoRI site by using pSV2-neo plasmid vector. Recombiant plasmid was transfected into HeLa cells by DNA-calcium phosphate precipitation technique. MT-I expression colones were growing in medium including G418. The amount of MT-I expression in transfected cells was found 2.6 times higher than that of non-transfected ones. In order to observe the relationship between the expression of MT-I gene in cells and drug resistance, cells were treated with different concentrations of cisplatin and adriamycin respectively. The results indicated that cisplatin (0.1 mumol/ml) inhibited the growth of both transfected and non-transfected cells. The inhibitory rates were 34% and 82% respectively(P < 0.05). IC50(50% inhibitory concentration for cell growing) was 0.144 mumol/ml and 0.061 mumol/ml and the ratio of them was 2.36: 1 after the treatment of cisplatin 72 h later. The cells were treated with adriamycin 72 h later, the inhibitory rates of transfected and non-transfected cells were 18% and 25% separately. The rates showed no significant difference (P > 0.05). The results indicated that MT was related to drug resistance of tumor cells.

  4. Utilizing of Adsorptive Transfer Stripping Technique Brdicka Reaction for Determination of Metallothioneins Level in Melanoma Cells, Blood Serum and Tissues

    PubMed Central

    Krizkova, Sona; Fabrik, Ivo; Adam, Vojtech; Kukacka, Jiri; Prusa, Richard; Chavis, Grace J.; Trnkova, Libuse; Strnadel, Jan; Horak, Vratislav; Kizek, Rene

    2008-01-01

    In the paper we utilized the adsorptive transfer stripping differential pulse voltammetry Brdicka reaction for the determination of metallothioneins (MT) in melanoma cells, animal melanoma tissues (MeLiM miniature pig) and blood serum of patients with malignant melanoma. Primarily we attempted to investigate the influence of dilution of real sample on MT electrochemical response. Dilution of samples of 1 000 times was chosen the most suitable for determination of MT level in biological samples. Then we quantified the MT level in the melanoma cells, the animal melanoma tissues and the blood serum samples. The MT content in the cells varied within the range from 4.2 to 11.2 μM. At animal melanoma tissues (melanomas localized on abdomen, back limb and dorsum) the highest content of MT was determined in the tumour sampled on the back of the animal and was nearly 500 μg of MTs per gram of a tissue. We also quantified content of MT in metastases, which was found in liver, spleen and lymph nodes. Moreover the average MT level in the blood serum samples from patients with melanoma was 3.0 ± 0.8 μM. MT levels determined at melanoma samples were significantly (p < 0.05) higher compared to control ones at cells, tissues and blood serum. PMID:27879868

  5. Metal induction of a Pisolithus albus metallothionein and its potential involvement in heavy metal tolerance during mycorrhizal symbiosis.

    PubMed

    Reddy, M Sudhakara; Kour, Manpreet; Aggarwal, Sipla; Ahuja, Shanky; Marmeisse, Roland; Fraissinet-Tachet, Laurence

    2016-09-01

    Metallothioneins (MTs) are small, cysteine-rich peptides involved in intracellular sequestration of heavy metals in eukaryotes. We examined the role in metal homeostasis and detoxification of an MT from the ectomycorrhizal fungus Pisolithus albus (PaMT1). PaMT1 encodes a 35 amino acid-long polypeptide, with 7 cysteine residues; most of them part of a C-x-C motif found in other known basidiomycete MTs. The expression levels of PaMT1 increased as a function of increased external Cu and Cd concentrations and were higher with Cu than with Cd. Heterologous complementation assays in metal-sensitive yeast mutants indicated that PaMT1 encodes a polypeptide capable of conferring higher tolerance to both Cu and Cd. Eucalyptus tereticornis plantlets colonized with P. albus grown in the presence of Cu and Cd showed better growth compared with those with non-mycorrhizal plants. Higher PaMT1 expression levels were recorded in mycorrhizal plants grown in the presence of Cu and Cd compared with those in control mycorrhizal plants not exposed to heavy metals. These data provide the first evidence to our knowledge that fungal MTs could protect ectomycorrhizal fungi from heavy metal stress and in turn help the plants to establish in metal-contaminated sites.

  6. Imaging lipid droplets in Arabidopsis mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Confocal fluorescence microscopy was adapted for the imaging of neutral lipids in plant leaves with defects in normal lipid metabolism using two different fluorescent dyes. Disruptions in a gene locus, At4g24160, yielded Arabidopsis thaliana plants with a preponderance of oil bodies in their leaves ...

  7. Engineering calcium oxalate crystal formation in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many plants accumulate crystals of calcium oxalate. Just how these crystals form remains unknown. To gain insight into the mechanisms regulating calcium oxalate crystal formation, a crystal engineering approach was initiated utilizing the non-crystal accumulating plant, Arabidopsis. The success of t...

  8. Arabidopsis, the Rosetta stone of flowering time?

    PubMed

    Simpson, Gordon G; Dean, Caroline

    2002-04-12

    Multiple environmental and endogenous inputs regulate when plants flower. The molecular genetic dissection of flowering time control in Arabidopsis has identified an integrated network of pathways that quantitatively control the timing of this developmental switch. This framework provides the basis to understand the evolution of different reproductive strategies and how floral pathways interact through seasonal progression.

  9. Shotgun Proteomic Analysis of Arabidopsis thaliana Leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two shotgun tandem mass spectrometry proteomics approaches, Multidimensional Protein Identification Technology (MudPIT) and 1D-Gel-LC-MS/MS, were used to identify Arabidopsis thaliana leaf proteins. These methods utilize different protein/peptide separation strategies. Detergents not compatible wit...

  10. Arabidopsis gene expression patterns during spaceflight

    NASA Astrophysics Data System (ADS)

    Paul, A.-L.; Ferl, R. J.

    The exposure of Arabidopsis thaliana (Arabidopsis) plants to spaceflight environments resulted in the differential expression of hundreds of genes. A 5 day mission on orbiter Columbia in 1999 (STS-93) carried transgenic Arabidopsis plants engineered with a transgene composed of the alcohol dehydrogenase (Adh) gene promoter linked to the β -Glucuronidase (GUS) reporter gene. The plants were used to evaluate the effects of spaceflight on two fronts. First, expression patterns visualized with the Adh/GUS transgene were used to address specifically the possibility that spaceflight induces a hypoxic stress response, and to assess whether any spaceflight response was similar to control terrestrial hypoxia-induced gene expression patterns. (Paul et al., Plant Physiol. 2001, 126:613). Second, genome-wide patterns of native gene expression were evaluated utilizing the Affymetrix ATH1 GeneChip? array of 8,000 Arabidopsis genes. As a control for the veracity of the array analyses, a selection of genes identified with the arrays was further characterized with quantitative Real-Time RT PCR (ABI - TaqmanTM). Comparison of the patterns of expression for arrays of hybridized with RNA isolated from plants exposed to spaceflight compared to the control arrays revealed hundreds of genes that were differentially expressed in response to spaceflight, yet most genes that are hallmarks of hypoxic stress were unaffected. These results will be discussed in light of current models for plant responses to the spaceflight environment, and with regard to potential future flight opportunities.

  11. An improved method for the visualization of conductive vessels in Arabidopsis thaliana inflorescence stems

    PubMed Central

    Jupa, Radek; Didi, Vojtěch; Hejátko, Jan; Gloser, Vít

    2015-01-01

    Dye perfusion is commonly used for the identification of conductive elements important for the study of xylem development as well as precise hydraulic estimations. The tiny size of inflorescence stems, the small amount of vessels in close arrangement, and high hydraulic resistivity delimit the use of the method for quantification of the water conductivity of Arabidopsis thaliana, one of the recently most extensively used plant models. Here, we present an extensive adjustment to the method in order to reliably identify individual functional (conductive) vessels. Segments of inflorescence stems were sealed in silicone tubes to prevent damage and perfused with a dye solution. Our results showed that dyes often used for staining functional xylem elements (safranin, fuchsine, toluidine blue) failed with Arabidopsis. In contrast, Fluorescent Brightener 28 dye solution perfused through segments stained secondary cell walls of functional vessels, which were clearly distinguishable in native cross sections. When compared to identification based on the degree of development of secondary cell walls, identification with the help of dye perfusion revealed a significantly lower number of functional vessels and values of theoretical hydraulic conductivity. We found that lignified but not yet functional vessels form a substantial portion of the xylem in apical and basal segments of Arabidopsis and, thus, significantly affect the analyzed functional parameters of xylem. The presented methodology enables reliable identification of individual functional vessels, allowing thus estimations of hydraulic conductivities to be improved, size distributions and vessel diameters to be refined, and data variability generally to be reduced. PMID:25914701

  12. The Prx Q protein of Arabidopsis thaliana is a member of the luminal chloroplast proteome.

    PubMed

    Petersson, Ulrika A; Kieselbach, Thomas; García-Cerdán, José G; Schröder, Wolfgang P

    2006-11-13

    Peroxiredoxins have been discovered in many organisms ranging from eubacteria to mammals, and their known biological functions include both oxidant defense and signal transduction. The genome of Arabidopsis thaliana encodes for ten individual peroxiredoxins, of which four are located in the chloroplast. The best-characterized member of the chloroplast peroxiredoxins is 2-Cys Prx that is associated with the stroma side of the thylakoid membrane and is considered to participate in antioxidant defense and protection of photosynthesis. This study addressed the chloroplast peroxiredoxin Prx Q and showed that its subcellular location is the lumen of the thylakoid membrane. To get insight in the biological function of the Prx Q protein of Arabidopsis, the protein levels of the Prx Q protein in thylakoid membranes were studied under different light conditions and oxidative stress. A T-DNA knockout mutant of Prx Q did not show any visible phenotype and had normal photosynthetic performance with a slightly increased oxygen evolving activity.

  13. High-throughput, high-sensitivity analysis of gene expression in Arabidopsis.

    PubMed

    Kris, Richard Martin; Felder, Stephen; Deyholos, Michael; Lambert, Georgina M; Hinton, James; Botros, Ihab; Martel, Ralph; Seligmann, Bruce; Galbraith, David W

    2007-07-01

    High-throughput gene expression analysis of genes expressed during salt stress was performed using a novel multiplexed quantitative nuclease protection assay that involves customized DNA microarrays printed within the individual wells of 96-well plates. The levels of expression of the transcripts from 16 different genes were quantified within crude homogenates prepared from Arabidopsis (Arabidopsis thaliana) plants also grown in a 96-well plate format. Examples are provided of the high degree of reproducibility of quantitative dose-response data and of the sensitivity of detection of changes in gene expression within limiting amounts of tissue. The lack of requirement for RNA purification renders the assay particularly suited for high-throughput gene expression analysis and for the discovery of novel chemical compounds that specifically modulate the expression of endogenous target genes.

  14. [Individual consciousness].

    PubMed

    Chaĭlakhian, L M

    2009-01-01

    The main modern concepts on the consciousness nature are considered. Together with the dualistic concepts, there exist concepts the adherents of which find it possible to get to know the origin of consciousness on the basis of natural science. A critical analysis of those concepts brings the author to the conclusion that they do not solve the main problem of individual consciousness: how subjective elements of consciousness arise in the brain as a result of objectively registered processes. The main reason of failures to solve said problem is considered by the author in the fact that the subjective categories of consciousness are not really subject to science. Nevertheless, it does not mean the dualism is to be inevitably accepted. In fact, the subjective categories arise in the limits of a life the area of which is substantially wider than that of science. An original information and physical hypothesis is being set up that provides for necessary premises and conditions enabling the origination of subjective categories of consciousness during the progressive natural evolution of living systems.

  15. 17th International Conference on Arabidopsis Research

    SciTech Connect

    Bender, Judith

    2006-07-02

    The 17th International Conference on Arabidopsis Research was held at the University of Madison, Wisconsin from June 27- July 2, 2006. ICAR-2006 included approximately 625 scientists from across the world. The scientific program was of excellent quality featuring 73 talks, including 30 from invited speakers. There were also 6 community-organized workshops (facilitated by conference staff) featuring additional talks on topics including ‘Submitting data to long-term repositories,’ ‘TAIR introductory workshop,’ ‘Web services and demonstration,’ ‘Public engagement: broadening the impact of your research,’ ‘Systems biology approaches to analysis of metabolic and regulatory networks of Arabidopsis,’ and ‘Mechanotransduction in Arabidopsis.’ Approximately 440 posters were presented in general topic areas including, among others, Development, Modeling/Other Systems, Energy, Environment, and Genetic/Epigenetic mechanisms. Graduate students, postdoctoral researchers, junior faculty, and underrepresented minorities made up a significant portion of the oral presentations thereby promoting the training of young scientists and facilitating important career development opportunities for speakers. Several poster sessions provided an opportunity for younger participants to freely meet with more established scientists. The North American Arabidopsis Steering Committee (NAASC) continued its outreach effort and again sponsored two special luncheons to encourage personal and professional development of young scientists and also underrepresented minorities. The ‘Emerging Scientists Luncheon’ featured 10 graduate students selected on the basis of scientific excellence of their submitted research abstracts. The ‘Minority Funding Luncheon,’ featured 8 awardees selected by the NAASC through a widely-publicized application process. This luncheon was established specifically to provide an opportunity for underrepresented minorities, and/or scientists from

  16. A zinc-binding citrus protein metallothionein can act as a plant defense factor by controlling host-selective ACR-toxin production.

    PubMed

    Nishimura, Satoshi; Tatano, Satoshi; Miyamoto, Yoko; Ohtani, Kouhei; Fukumoto, Takeshi; Gomi, Kenji; Tada, Yasuomi; Ichimura, Kazuya; Akimitsu, Kazuya

    2013-01-01

    Metallothionein is a small cysteine-rich protein known to have a metal-binding function. We isolated three different lengths of rough lemon cDNAs encoding a metallothionein (RlemMT1, RlemMT2 and RlemMT3), and only RlemMT1-recombinant protein had zinc-binding activity. Appropriate concentration of zinc is an essential micronutrient for living organisms, while excess zinc is toxic. Zinc also stimulates the production of host-selective ACR-toxin for citrus leaf spot pathogen of Alternaria alternata rough lemon pathotype. Trapping of zinc by RlemMT1-recombinant protein or by a zinc-scavenging agent in the culture medium caused suppression of ACR-toxin production by the fungus. Since ACR-toxin is the disease determinant for A. alternata rough lemon pathotype, addition of RlemMT1 to the inoculum suspension led to a significant decrease in symptoms on rough lemon leaves as a result of reduced ACR-toxin production from the zinc trap around infection sites. RlemMT1-overexpression mutant of A. alternata rough lemon pathotype also produced less ACR-toxin and reduced virulence on rough lemon. This suppression was caused by an interruption of zinc absorption by cells from the trapping of the mineral by RlemMT1 and an excess supplement of ZnSO(4) restored toxin production and pathogenicity. Based on these results, we propose that zinc adsorbents including metallothionein likely can act as a plant defense factor by controlling toxin biosynthesis via inhibition of zinc absorption by the pathogen.

  17. Regulation of Sulfate Assimilation in Arabidopsis and Beyond

    PubMed Central

    KOPRIVA, STANISLAV

    2006-01-01

    • Background and Aims Sulfate assimilation is a pathway used by prokaryotes, fungi and photosynthetic organisms to convert inorganic sulfate to sulfide, which is further incorporated into carbon skeletons of amino acids to form cysteine or homocysteine. The pathway is highly regulated in a demand-driven manner; however, this regulation is not necessarily identical in various plant species. Therefore, our knowledge of the regulation of sulfate assimilation is reviewed here in detail with emphasis on different plant species. • Scope Although demand-driven control plays an essential role in regulation of sulfate assimilation in all plants, the molecular mechanisms of the regulation and the effects of various treatments on the individual enzymes and metabolites are often different. This review summarizes (1) the molecular regulation of sulfate assimilation in Arabidopsis thaliana, especially recent data derived from platform technologies and functional genomics, (2) the co-ordination of sulfate, nitrate and carbon assimilations in Lemna minor, (3) the role of sulfate assimilation and glutathione in plant–Rhizobia symbiosis, (4) the cell-specific distribution of sulfate reduction and glutathione synthesis in C4 plants, (5) the regulation of glutathione biosynthesis in poplar, (6) the knock-out of the adenosine 5′phosphosulfate reductase gene in Physcomitrella patens and identification of 3′-phosphoadenosyl 5′-phosphosulfate reductase in plants, and (7) the sulfur sensing mechanism in green algae. • Conclusions As the molecular mechanisms of regulation of the sulfate assimilation pathway are not known, the role of Arabidopsis as a model plant will be further strengthened. However, this review demonstrates that investigations of other plant species will still be necessary to address specific questions of regulation of sulfur nutrition. PMID:16464881

  18. Rethinking Transcriptional Activation in the Arabidopsis Circadian Clock

    PubMed Central

    Fogelmark, Karl; Troein, Carl

    2014-01-01

    Circadian clocks are biological timekeepers that allow living cells to time their activity in anticipation of predictable daily changes in light and other environmental factors. The complexity of the circadian clock in higher plants makes it difficult to understand the role of individual genes or molecular interactions, and mathematical modelling has been useful in guiding clock research in model organisms such as Arabidopsis thaliana. We present a model of the circadian clock in Arabidopsis, based on a large corpus of published time course data. It appears from experimental evidence in the literature that most interactions in the clock are repressive. Hence, we remove all transcriptional activation found in previous models of this system, and instead extend the system by including two new components, the morning-expressed activator RVE8 and the nightly repressor/activator NOX. Our modelling results demonstrate that the clock does not need a large number of activators in order to reproduce the observed gene expression patterns. For example, the sequential expression of the PRR genes does not require the genes to be connected as a series of activators. In the presented model, transcriptional activation is exclusively the task of RVE8. Predictions of how strongly RVE8 affects its targets are found to agree with earlier interpretations of the experimental data, but generally we find that the many negative feedbacks in the system should discourage intuitive interpretations of mutant phenotypes. The dynamics of the clock are difficult to predict without mathematical modelling, and the clock is better viewed as a tangled web than as a series of loops. PMID:25033214

  19. Gene Transposition Causing Natural Variation for Growth in Arabidopsis thaliana

    PubMed Central

    Vlad, Daniela; Rappaport, Fabrice; Simon, Matthieu; Loudet, Olivier

    2010-01-01

    A major challenge in biology is to identify molecular polymorphisms responsible for variation in complex traits of evolutionary and agricultural interest. Using the advantages of Arabidopsis thaliana as a model species, we sought to identify new genes and genetic mechanisms underlying natural variation for shoot growth using quantitative genetic strategies. More quantitative trait loci (QTL) still need be resolved to draw a general picture as to how and where in the pathways adaptation is shaping natural variation and the type of molecular variation involved. Phenotypic variation for shoot growth in the Bur-0 × Col-0 recombinant inbred line set was decomposed into several QTLs. Nearly-isogenic lines generated from the residual heterozygosity segregating among lines revealed an even more complex picture, with major variation controlled by opposite linked loci and masked by the segregation bias due to the defective phenotype of SG3 (Shoot Growth-3), as well as epistasis with SG3i (SG3-interactor). Using principally a fine-mapping strategy, we have identified the underlying gene causing phenotypic variation at SG3: At4g30720 codes for a new chloroplast-located protein essential to ensure a correct electron flow through the photosynthetic chain and, hence, photosynthesis efficiency and normal growth. The SG3/SG3i interaction is the result of a structural polymorphism originating from the duplication of the gene followed by divergent paralogue's loss between parental accessions. Species-wide, our results illustrate the very dynamic rate of duplication/transposition, even over short periods of time, resulting in several divergent—but still functional—combinations of alleles fixed in different backgrounds. In predominantly selfing species like Arabidopsis, this variation remains hidden in wild populations but is potentially revealed when divergent individuals outcross. This work highlights the need for improved tools and algorithms to resolve structural variation

  20. Augmented hepatic injury followed by impaired regeneration in metallothionein-I/II knockout mice after treatment with thioacetamide

    SciTech Connect

    Oliver, Jordan R.; Jiang, Sean; Cherian, M. George . E-mail: mcherian@uwo.ca

    2006-02-01

    A previous study (Oliver, J.R., Mara, T.W., Cherian, M.G. 2005. Impaired hepatic regeneration in metallothionein-I/II knockout mice after partial hepatectomy. Exp. Biol. Med. 230, 61-67) has shown an impairment of liver regeneration following partial hepatectomy (PH) in metallothionein (MT)-I and MT-II gene knockout (MT-null) mice, thus suggesting a requirement for MT in cellular growth. The present study was undertaken to investigate whether MT may play a similar role in hepatic injury and regeneration after acute treatment with thioacetamide (TAA). Hepatotoxicity of TAA is caused by the generation of oxidative stress. TAA was injected ip to both wild-type (WT) and MT-null mice. Mice were killed at 6, 12, 24, 48, 60, and 72 h after injection of TAA (125 mg/kg) or 48 h after injection of saline (vehicle control), and different parameters of hepatic injury were measured. The levels of hepatic lipid peroxidation were increased at 12 h in both types of mice; however, lipid peroxidation was significantly less in WT mice than MT-null mice at 48 h after injection of TAA. Analysis of hepatic glutathione (GSH) levels after TAA injection showed depletion of GSH at 12 h in WT mice and at 6 h in MT-null mice; however, significantly more GSH was depleted early (6-24 h) in MT-null mice than WT mice. An increase in hepatic iron (Fe) levels was observed in both types of mice after injection of TAA, but Fe levels were significantly higher in MT-null mice than WT mice at 6-60 h. The levels of hepatic copper (Cu) and zinc (Zn) were significantly higher in WT mice than MT-null mice at 6-60 h for Cu, and at 24 h and 60 h for Zn, respectively. Histopathological examination showed hemorrhagic necrosis in the liver of both types of mice at 12-72 h, with hepatic injury being more prominent in MT-null mice than WT mice. The hepatic MT levels were increased in WT mice after injection of TAA, and were highest at 24-72 h. Immunohistochemical staining for MT in WT mice indicated the presence

  1. Metallothionein is crucial for safe intracellular copper storage and cell survival at normal and supra-physiological exposure levels.

    PubMed Central

    Tapia, Lucía; González-Agüero, Mauricio; Cisternas, Mónica F; Suazo, Miriam; Cambiazo, Verónica; Uauy, Ricardo; González, Mauricio

    2004-01-01

    MTs (metallothioneins) increase the resistance of cells to exposure to high Cu (copper) levels. Characterization of the MT-Cu complex suggests that MT has an important role in the cellular storage and/or delivery of Cu ions to cuproenzymes. In this work we investigate how these properties contribute to Cu homoeostasis by evaluating the uptake, accumulation and efflux of Cu in wild-type and MT I/II null rat fibroblast cell lines. We also assessed changes in the expression of Cu metabolism-related genes in response to Cu exposure. At sub-physiological Cu levels (0.4 microM), the metal content was not dependent on MT; however, when extracellular Cu was increased to physiological levels (10 microM), MTs were required for the cell's ability to accumulate the metal. The subcellular localization of the accumulated metal in the cytoplasm was MT-dependent. Following supra-physiological Cu exposure (>50 microM), MT null cells had a decreased capacity for Cu storage and an elevated sensitivity to a minor increment in intracellular metal levels, suggesting that intracellular Cu toxicity is due not to the metal content but to the interactions of the metal with cellular components. Moreover, MT null cells failed to show increased levels of mRNAs encoding MT I, SOD1 (superoxide dismutase 1) and Ccs1 (Cu chaperone for SOD) in response to Cu exposure. These results support a role for MT in the storage of Cu in a safe compartment and in sequestering an intracellular excess of Cu in response to supra-physiological Cu exposure. Gene expression analysis suggests the necessity of having MT as part of the signalling pathway that induces gene expression in response to Cu. PMID:14627437

  2. Characterization and Expression of DNA Sequences Encoding Putative Type-II Metallothioneins in the Seagrass Posidonia oceanica1

    PubMed Central

    Giordani, Tommaso; Natali, Lucia; Maserti, Bianca Elena; Taddei, Sonia; Cavallini, Andrea

    2000-01-01

    Posidonia oceanica is a marine phanerogam, largely widespread in the Mediterranean sea, representing an important food substrate for many marine organisms. A progressive reduction of P. oceanica meadows has been reported, due to anthropogenic coastal activity. Studying mechanisms by which this species responds to environmental stresses, three DNA sequences putatively encoding metallothioneins (MTs) have been isolated, by PCR. Two sequences, Pomt2a (accession no. AJ249603) and Pomt2b (accession no. AJ249602), show high similarities with genes encoding type-II MTs and are interrupted by two and one intron, respectively. The third sequence, Pomt2c (accession no. AJ249604), is supposed to be a pseudogene, originated by retrotranscription of the Pomt2b mRNA. These sequences belong to a multigene family with at least five members. Northern hybridizations indicated that MT transcripts accumulation is constitutive and seasonally regulated. MT encoding RNAs increase after rhyzome harvesting and (at a lesser extent) after 15 d of cultivation in an aquarium. As for animal MTs, transcripts accumulation is observed also after exposure to trace metals such as copper and cadmium. In the case of copper, the effect depends on concentration. Finally, taking into consideration the great interest in studying the biogeochemical cycle of mercury in the Mediterranean basin and since P. oceanica is commonly considered a bioindicator of this metal, the effect of mercury treatments on the accumulation of MT transcripts has been analyzed: in only a few experiments a small increase in the level of transcripts was recorded, suggesting that MTs are not key elements in the mercury accumulation by this species. PMID:10938373

  3. Metallothionein differentially affects the host response to Listeria infection both with and without an additional stress from cold-restraint.

    PubMed

    Emeny, Rebecca T; Kasten-Jolly, Jane; Mondal, Tapan; Lynes, Michael A; Lawrence, David A

    2015-11-01

    Acute stress alters anti-bacterial defenses, but the neuroimmunological mechanisms underlying this association are not yet well understood. Metallothionein (MT), a cysteine-rich protein, is a stress response protein that is induced by a variety of chemical, biological, and psychological stressors, and MT has been shown to influence immune activities. We investigated MT's role in the management of anti-bacterial responses that occur during stress, using a C57BL/6 (B6) strain that has targeted disruptions of the Mt1 and Mt2 genes (B6-MTKO), and a B6 strain that has additional copies of Mt (B6-MTTGN). The well-characterized listeriosis model was used to examine immune mechanisms that are altered by a 1-h stress treatment (cold-restraint, CR) administered just prior to bacterial infection. Intriguingly, MT gene doses both greater and lower than that of wild-type (WT) B6 mice were associated with improved host defenses against Listeria monocytogenes (LM). This augmented protection was diminished by CR stress in the MTKO mice, but transgenic mice with additional MT copies had no CR stress-induced increase in their listerial burden. During the transition from innate to adaptive immunity, on day 3 after infection, oxidative burst and apoptosis were assessed by flow cytometric methods, and cytokine transcription was measured by real-time quantitative PCR. MT gene expression and CR-stress affected the expression of IL-6 and TNFα. Additionally, these genetic and environmental modulations altered the generation of ROS responses as well as the number of apoptotic cells in livers and spleens. Although the level of MT altered the listerial response, MT expression was equally elevated by listerial infection with or without CR stress. These results indicate the ability of MT to regulate immune response mechanisms and demonstrate that increased amounts of MT can eliminate the immunosuppression induced by CR.

  4. Cadmium-binding proteins of rat testes. Characterization of a low-molecular-mass protein that lacks identity with metallothionein.

    PubMed Central

    Waalkes, M P; Chernoff, S B; Klaassen, C D

    1984-01-01

    Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein. PMID:6466305

  5. Mitochondrial electron transport is inhibited by disappearance of metallothionein in human bronchial epithelial cells following exposure to silver nitrate.

    PubMed

    Miyayama, Takamitsu; Arai, Yuta; Suzuki, Noriyuki; Hirano, Seishiro

    2013-03-08

    Silver (Ag) possesses antibacterial activity and has been used in wound dressings and deodorant powders worldwide. However, the metabolic behavior and biological roles of Ag in mammals have not been well characterized. In the present study, we exposed human bronchial epithelial cells (BEAS-2B) to AgNO3 and investigated uptake and intracellular distribution of Ag, expression of metallothionein (MT), generation of reactive oxygen species (ROS), and changes in mitochondrial respiration. The culture medium concentration of Ag decreased with time and stabilized at 12h. The concentration of both Ag and MT in the soluble cellular fraction increased up to 3h and then decreased, indicating that cytosolic Ag relocated to the insoluble fraction of the cells. The levels of mRNAs for the major human MT isoforms MT-I and MT-II paralleled with the protein levels of Ag-MT. The intensity of fluorescence derived from ROS was elevated in the mitochondrial region at 24h. Ag decreased mitochondrial oxygen consumption in a dose-dependent manner and the activity of mitochondrial complex I-IV enzymes was significantly inhibited following exposure to Ag. In a separate experiment, we found that hydrogen peroxide (H2O2) at concentrations as low as 0.001% (equivalent to the concentration of H2O2 in Ag-exposed cells) removed Ag from MT. These results suggest MT was decomposed by cytosolic H2O2, and then Ag released from MT relocated to insoluble cellular fractions and inhibited electron chain transfer of mitochondrial complexes, which eventually led to cell damage.

  6. Enhanced seizures and hippocampal neurodegeneration following kainic acid-induced seizures in metallothionein-I + II-deficient mice.

    PubMed

    Carrasco, J; Penkowa, M; Hadberg, H; Molinero, A; Hidalgo, J

    2000-07-01

    Metallothioneins (MTs) are major zinc binding proteins in the CNS that could be involved in the control of zinc metabolism as well as in protection against oxidative stress. Mice lacking MT-I and MT-II (MT-I + II deficient) because of targeted gene inactivation were injected with kainic acid (KA), a potent convulsive agent, to examine the neurobiological importance of these MT isoforms. At 35 mg/kg KA, MT-I + II deficient male mice showed a higher number of convulsions and a longer convulsion time than control mice. Three days later, KA-injected mice showed gliosis and neuronal injury in the hippocampus. MT-I + II deficiency decreased both astrogliosis and microgliosis and potentiated neuronal injury and apoptosis as shown by terminal deoxynucleotidyl transferase-mediated in situ end labelling (TUNEL), detection of single stranded DNA (ssDNA) and by increased interleukin-1beta-converting enzyme (ICE) and caspase-3 levels. Histochemically reactive zinc in the hippocampus was increased by KA to a greater extent in MT-I + II-deficient compared with control mice. KA-induced seizures also caused increased oxidative stress, as suggested by the malondialdehyde (MDA) and protein tyrosine nitration (NITT) levels and by the expression of MT-I + II, nuclear factor-kappaB (NF-kappaB), and Cu/Zn-superoxide dismutase (Cu/Zn-SOD). MT-I + II deficiency potentiated the oxidative stress caused by KA. Both KA and MT-I + II deficiency significantly affected the expression of MT-III, granulocyte-macrophage colony stimulating factor (GM-CSF) and its receptor (GM-CSFr). The present results indicate MT-I + II as important for neuron survival during KA-induced seizures, and suggest that both impaired zinc regulation and compromised antioxidant activity contribute to the observed neuropathology of the MT-I + II-deficient mice.

  7. A study of metal concentrations and metallothionein binding capacity in liver, kidney and brain tissues of three Arctic seal species.

    PubMed

    Sonne, Christian; Aspholm, Ole; Dietz, Rune; Andersen, Steen; Berntssen, Marc H G; Hylland, Ketil

    2009-12-01

    Arctic seals are known to accumulate relatively high concentrations of potential toxic heavy metals in their vital organs, such as livers and kidneys, as well as in their central nervous system. We therefore decided to determine whether mercury, copper, cadmium and zinc levels in liver, kidney and brain tissues of three Arctic seal species were associated with the intracellular metal-binding protein metallothionein (MT) as a sign of toxic exposure. Samples from four ringed (Phoca hispida), five harp (P.groenlandica) and five hooded (Cystophora cristata) seals taken during field trips to Central West Greenland (Godhavn) and the Barents Sea in the spring of 1999 were used for the present study. In all three seal species concentrations of mercury, zinc and copper were highest in the liver, except for cadmium which was highest in the kidneys. Metal concentrations increased significantly in the order: ringed seal

  8. Metallothionein treatment reduces proinflammatory cytokines IL-6 and TNF-alpha and apoptotic cell death during experimental autoimmune encephalomyelitis (EAE).

    PubMed

    Penkowa, M; Hidalgo, J

    2001-07-01

    Experimental autoimmune encephalomyelitis (EAE) is an animal model for the human autoimmune disease multiple sclerosis (MS). Proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are considered important for induction and pathogenesis of EAE/MS disease, which is characterized by significant inflammation and neuroglial damage. We have recently shown that the exogenous administration of the antioxidant protein zinc-metallothionein-II (Zn-MT-II) significantly decreased the clinical symptoms, mortality, and leukocyte infiltration of the CNS during EAE. However, it is not known how EAE progression is regulated nor how cytokine production and cell death can be reduced. We herewith demonstrate that treatment with Zn-MT-II significantly decreased the CNS expression of IL-6 and TNF-alpha during EAE. Zn-MT-II treatment could also significantly reduce apoptotic cell death of neurons and oligodendrocytes during EAE, as judged by using TUNEL and immunoreactivity for cytochrome c and caspases 1 and 3. In contrast, the number of apoptotic lymphocytes and macrophages was less affected by Zn-MT-II treatment. The Zn-MT-II-induced decrease in proinflammatory cytokines and apoptosis during EAE could contribute to the reported diminution of clinical symptoms and mortality in EAE-immunized rats receiving Zn-MT-II treatment. Our results demonstrate that MT-II reduces the CNS expression of proinflammatory cytokines and the number of apoptotic neurons during EAE in vivo and that MT-II might be a potentially useful factor for treatment of EAE/MS.

  9. Alterations in metal toxicity and metal-induced metallothionein gene expression elicited by growth medium calcium concentration.

    PubMed

    Singh, Rajendra K; Albrecht, Amy L; Somji, Seema; Sens, Mary Ann; Sens, Donald A; Garrett, Scott H

    2008-06-01

    The calcium content of the growth medium has been shown to influence the growth and differentiation of primary epithelial cells in culture. The goal of the present study was to determine if growth medium calcium concentration could influence the susceptibility to metal toxicity and metallothionein gene expression of an immortalized human prostate-derived epithelial cell line (RWPE-1). The RWPE-1 cell line was grown in medium containing either 0.1 or 1.4 mM calcium. Confluent cells were exposed to either Zn(+2) (50, 100, or 150 microM) or Cd(+2) (3, 6, or 12 microM) for 13 days, and cell toxicity and MT gene expression were determined along the time course of exposure. It was demonstrated that the calcium content of the growth medium had a marked influence on Zn(+2) toxicity and a lesser but significant effect on Cd(+2) toxicity to the RWPE-1 cells. Calcium concentration of the growth medium was also shown to alter the accumulation of MT-1/2 protein and MT-1E, MT-1X, and MT-2A mRNAs. It was shown that MT-1/2 protein was markedly increased for metal-exposed cells grown in medium containing 0.1 mM calcium; however, the increased expression did not cause an increase in the resistance of the cells to Zn(+2) or Cd(+2) exposure. These observations show that growth medium calcium concentration can influence metal toxicity and the pattern of expression of the MT mRNAs and protein for RWPE-1 cells. The results suggest that caution should be exercised when comparing toxicological responses between cell lines that may be grown in growth formulations differing in calcium concentration.

  10. Renoprotection From Diabetic Complications in OVE Transgenic Mice by Endothelial Cell Specific Overexpression of Metallothionein: A TEM Stereological Analysis

    PubMed Central

    Chhoun, Jennifer M.; Grove, Bryon D.; Laturnus, Donna I.; Zheng, Shirong; Epstein, Paul N.; Tan, Yi

    2017-01-01

    ABSTRACT We previously demonstrated that OVE transgenic diabetic mice are susceptible to chronic complications of diabetic nephropathy (DN) including substantial oxidative damage to the renal glomerular filtration barrier (GFB). Importantly, the damage was mitigated significantly by overexpression of the powerful antioxidant, metallothionein (MT) in podocytes. To test our hypothesis that GFB damage in OVE mice is the result of endothelial oxidative insult, a new JTMT transgenic mouse was designed in which MT overexpression was targeted specifically to endothelial cells. At 60 days of age, JTMT mice were crossed with age‐matched OVE diabetic mice to produce bi‐transgenic OVE‐JTMT diabetic progeny that carried the endothelial targeted JTMT transgene. Renal tissues from the OVE‐JTMT progeny were examined by unbiased TEM stereometry for possible GFB damage and other alterations from chronic complications of DN. In 150 day‐old OVE‐JTMT mice, blood glucose and HbA1c were indistinguishable from age‐matched OVE mice. However, endothelial‐specific MT overexpression in OVE‐JTMT mice mitigated several DN complications including significantly increased non‐fenestrated glomerular endothelial area, and elimination of glomerular basement membrane thickening. Significant renoprotection was also observed outside of endothelial cells, including reduced podocyte effacement, and increased podocyte and total glomerular cell densities. Moreover, when compared to OVE diabetic animals, OVE‐JTMT mice showed significant mitigation of nephromegaly, glomerular hypertrophy, increased mesangial cell numbers and increased total glomerular cell numbers. These results confirm the importance of oxidative stress to glomerular damage in DN, and show the central role of endothelial cell injury to the pathogenesis of chronic complications of diabetes. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:560–576, 2017. © 2016 The Authors. The Anatomical Record published

  11. Ultraviolet irradiation increases the sensitivity of cultured human skin cells to cadmium probably through the inhibition of metallothionein gene expression.

    PubMed

    Yamada, Hirotomo; Murata, Mie; Suzuki, Kaoru; Koizumi, Shinji

    2004-11-01

    We previously developed an apparatus that can irradiate cultured cells with monochromatic ultraviolet (UV) rays to exactly assess the biological effects of UV components on mammalian cells. Using this device, we studied the effects of UV in and near the UVB region on the general as well as specific protein synthesis of the human skin-derived NB1RGB cells. We found that Cd-induced synthesis of metallothioneins (MTs), which are the proteins involved in the protection against heavy metals and oxidative stress, is inhibited by UV at 280 nm more extensively than total protein synthesis. Such an inhibition was observed when MTs were induced by different inducers such as Cd, Zn, and dexamethasone in three human cell lines, indicating that it is not an event specific to a certain inducer or a certain cell type. By contrast, UV at 300 or 320 nm showed only a marginal effect. UV at 280 nm was likely to block MT gene transcription because Cd-induced increase of MT mRNA was strongly inhibited by irradiation. Cd induction of 70-kDa heat shock protein mRNA was also inhibited by UV irradiation, suggesting that the expression of inducible genes are commonly sensitive to UV. Furthermore, we observed that the irradiation of UV at 280 nm renders NB1RGB cells extremely susceptible to Cd, probably due to the reduced MT synthesis. These observations strongly suggest that UV at 280 nm severely damages cellular inducible protective functions, warning us of a new risk of UV exposure.

  12. Bioaccumulation and metallothionein response in the Asiatic clam (Corbicula fluminea) after experimental exposure to cadmium and inorganic mercury

    SciTech Connect

    Baudrimont, M.; Metivaud, J.; Maury-Brachet, R.; Ribeyre, F.; Boudou, A.

    1997-10-01

    The involvement of metallothioneins (MTs) in cadmium (Cd) and inorganic mercury (Hg[II]) bioaccumulation by the freshwater bivalve Corbicula fluminea was experimentally investigated after 0, 15, 30 and 45 d of exposure from the water column source. Three levels of contamination were studied for each metal: 0, 5, and 35 {micro}g Cd/L and 0, 1.45, and 5 {micro}g Hg/L, with two replicates per condition. Forty eight experimental units (EUs) were conducted simultaneously. The mollusks were fed twice a week by additions of phytoplanktonic algae. Quantification of MTs was done by Hg-saturation assay, using cold Hg(II). A partial purification of these proteins was conducted by gel-filtration chromatography, followed by Cd determinations in the different eluted fractions. Results at the whole organism (soft tissues) and organ or tissue group (gills, mantle, foot, visceral mass) levels show high metal concentrations, with a fourfold greater accumulation of inorganic Hg than Cd after 30 d exposure at the same concentration of 5 {micro}g/L. Gills and visceral mass were the principal storage compartments. A significant increase in MT concentrations was revealed in these two organs after exposure to Cd: ratios between the MT concentrations in contaminated and control mollusks were 2.4 and 2.8, respectively, for 5 and 35 {micro}g Cd/L. Cd burdens in the cytosol and in {le}18-kDa protein fractions, similar to purified mammal MTs, correspond to 30 and 14% of the total Cd accumulated in the whole organisms. No significant increase in MT biosynthesis was observed after exposure to inorganic Hg, despite the high metal concentrations in the organs.

  13. Autocrine induction of tumor protease production and invasion by a metallothionein-regulated TGF-beta 1 (Ser223, 225).

    PubMed Central

    Samuel, S K; Hurta, R A; Kondaiah, P; Khalil, N; Turley, E A; Wright, J A; Greenberg, A H

    1992-01-01

    An expression vector was constructed in which TGF-beta 1 was placed under the control of the metallothionein promoter. Cys223 and Cys225 in the TGF-beta 1 propeptide were converted to serines, mutations which result in dissociation of the pro-peptide and secretion of bioactive TGF-beta 1 [Brunner, A.M., Marquardt, H., Malacko, A.R., Lioubin, M.N. and Purchio, A.F. (1989) J. Biol. Chem., 264, 13660-13664]. A fibrosarcoma was transfected with this plasmid and a clone (17.18) was selected in which TGF-beta 1 mRNA was able to be induced six-fold following zinc sulphate treatment. These cells increased the secretion of bioactive TGF-beta 1 14-fold and exhibited a coincidental increase in jun-B mRNA expression, suggesting that secreted TGF-beta 1 was acting to induce this early response gene by autocrine activation. Following zinc sulphate induction, the tumor cells became progressively more motile and able to invade collagen gels. In contrast to parental tumor not bearing the TGF-beta 1 expression vector, zinc sulphate stimulation of clone 17.18 enhanced collagenase IV and procathepsin L mRNA levels and enhanced the secretion of many collagenolytic proteases into the medium. Since the action of TGF-beta generally decreases proteolysis by suppression of protease transcription, we compared the response of normal parental fibroblasts to ras-transformed fibrosarcomas and confirmed that TGF-beta could greatly enhance collagenase IV and procathepsin L mRNA levels while having little effect on non-transformed fibroblasts. These experiments indicate that induction of TGF-beta secretion can enhance motility and protease production through autocrine activation, thus increasing the invasion potential of fibrosarcomas. Images PMID:1314170

  14. High positive frequency of antibodies to metallothionein and heat shock protein 70 in sera of patients with metal allergy

    PubMed Central

    JIN, G-B; NAKAYAMA, H; SHMYHLO, M; INOUE, S; KONDO, M; IKEZAWA, Z; OUCHI, Y; CYONG, J-C

    2003-01-01

    Two principal types of stress protein, heat shock proteins (hsps) and metallothionein (MT), are induced in cells responding to a variety of stresses. They play an important role in protecting cells from these stresses. However, many reports indicate that antibodies to hsps are present in human serum and are associated with several autoimmunity diseases. Metals, which are commonly allergenic to humans, induce both MT and hsp70 (one of the hsps family). Until now, there has been no report of any antibody to MT in human serum. In the present study, serum samples from healthy controls (Group I), and patients suffering from atopic dermatitis without (Group II) or with (Group III) metal allergy, were measured for antibodies to MT and hsp70, using an enzyme-linked immunosorbent assay (ELISA). Metal allergy was confirmed by patch testing. We first found that antibody to MT exists in human serum. We also found a high positive frequency of antibody to MT (51·3%) and to hsp70 (43·6%) in the sera of Group III, compared to those of Group I (3·8% and 5·1%) or Group II (6·4% and 5·1%). Furthermore, there was a strong positive correlation between antibody to MT and antibody to hsp70 in Group III (P = 0·0013), but not in Group I and Group II. Our results indicate that antibody to MT exists in human serum, as do antibodies to hsps, and suggest that elevated levels of MT and hsp70 antibodies are associated with metal allergy in atopic patients. PMID:12562388

  15. Metallothionein proteins expression, copper and zinc concentrations, and lipid peroxidation level in a rodent model for amyotrophic lateral sclerosis.

    PubMed

    Tokuda, Eiichi; Ono, Shin-Ichi; Ishige, Kumiko; Naganuma, Akira; Ito, Yoshihisa; Suzuki, Takashi

    2007-01-05

    It has been hypothesized that copper-mediated oxidative stress contributes to the pathogenesis of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease in humans. To verify this hypothesis, we examined the copper and zinc concentrations and the amounts of lipid peroxides, together with that of the expression of metallothionein (MT) isoforms in a mouse model [superoxide dismutase1 transgenic (SOD1 Tg) mouse] of ALS. The expression of MT-I and MT-II (MT-I/II) isoforms were measured together with Western blotting, copper level, and lipid peroxides amounts increased in an age-dependent manner in the spinal cord, the region responsible for motor paralysis. A significant increase was already seen as early as 8-week-old SOD1 Tg mice, at which time the mice had not yet exhibited motor paralysis, and showed a further increase at 16 weeks of age, when paralysis was evident. Inversely, the spinal zinc level had significantly decreased at both 8 and 16 weeks of age. The third isoform, the MT-III level, remained at the same level as an 8-week-old wild-type mouse, finally increasing to a significant level at 16 weeks of age. It has been believed that a mutant SOD1 protein, encoded by a mutant SOD1, gains a novel cytotoxic function while maintaining its original enzymatic activity, and causes motor neuron death (gain-of-toxic function). Copper-mediated oxidative stress seems to be a probable underlying pathogenesis of gain-of-toxic function. Taking the above current concepts and the classic functions of MT into account, MTs could have a disease modifying property: the MT-I/II isoform for attenuating the gain-of-toxic function at the early stage of the disease, and the MT-III isoform at an advanced stage.

  16. Induction of metallothionein I by phenolic antioxidants requires metal-activated transcription factor 1 (MTF-1) and zinc.

    PubMed Central

    Bi, Yongyi; Palmiter, Richard D; Wood, Kristi M; Ma, Qiang

    2004-01-01

    Phenolic antioxidants, such as tBHQ [2,5-di-(t-butyl)-1,4-hydroquinone], induce Mt1 (metallothionein 1) gene expression and accumulation of MT protein. Induction of Mt1 mRNA does not depend on protein synthesis, and correlates with oxidation-reduction functions of the antioxidants. In the present study, we analysed the biochemical pathway of the induction. Induction depends on the presence of MTF-1 (metal-activated transcription factor 1), a transcription factor that is required for metal-induced transcription of Mt1, but does not require nuclear factor erythroid 2-related factor 2, a tBHQ-activated CNC bZip (cap 'n' collar basic leucine zipper) protein, that is responsible for regulating genes encoding phase II drug-metabolizing enzymes. Moreover, tBHQ induces the expression of MRE-beta Geo, a reporter gene driven by five metal response elements that constitute an optimal MTF-1 binding site. Reconstitution of Mtf1 -null cells with MTF-1 restores induction by both zinc and tBHQ. Unlike activation of phase II genes by tBHQ, induction of Mt1 expression does not occur in the presence of EDTA, when cells are cultured in zinc-depleted medium, or in cells with reduced intracellular 'free' zinc due to overexpression of ZnT1, a zinc-efflux transporter, indicating that induction requires zinc. In addition, fluorescence imaging reveals that tBHQ increases cytoplasmic free zinc concentration by mobilizing intracellular zinc pools. These findings establish that phenolic antioxidants activate Mt1 transcription by a zinc-dependent mechanism, which involves MTF-1 binding to metal regulator elements in the Mt1 gene promoter. PMID:14998373

  17. Identification and response to metals of metallothionein in two ancient fishes: white sturgeon (Acipenser transmontanus) and lake sturgeon (Acipenser fulvescens).

    PubMed

    Doering, Jon A; Beitel, Shawn C; Eisner, Bryanna K; Heide, Timon; Hollert, Henner; Giesy, John P; Hecker, Markus; Wiseman, Steve B

    2015-05-01

    White sturgeon (Acipenser transmontanus) are among the most sensitive species of fishes to Cu, Cd, and Zn, but there is no information about sensitivity of lake sturgeon (Acipenser fulvescens). To begin to elucidate molecular mechanism(s) of sensitivity of sturgeons to metals a cDNA encoding metallothionein (MT) was amplified from livers of white sturgeon (WS-MT) and lake sturgeon (LS-MT), and expression in response to Cu, Cd, or Zn was characterized in liver explants from each species. The primary structure of WS-MT and LS-MT contained 20 cysteine residues, which is the same as MTs of teleost fishes. However, the primary structure of WS-MT and LS-MT contained 63 amino acids, which is longer than any MT identified in teleost fishes. Abundance of transcripts of WS-MT in explants exposed to 0.3, 3, 30, or 100 μg/L of Cu was 1.7-, 1.7-, 2.1-, and 2.6-fold less than in controls, respectively. In contrast, abundances of transcripts of WS-MT were 3.3- and 2.4-fold greater in explants exposed to 30 μg/L of Cd and 1000 μg/L of Zn, respectively. Abundance of transcripts of LS-MT was not significantly different at any concentration of Cu, Cd, or Zn. MT is hypothesized to represent a critical mechanism for detoxification of metals. Therefore, results of this study suggest that sensitivity of sturgeons to exposure to Cu, Cd, or Zn might be a result of the relatively lesser maximal response of MT to metals. The study also suggestslake sturgeon might be more sensitive than white sturgeon to metals.

  18. Effect of dietary zinc deficiency on the accumulation of cadmium and metallothionein in selected tissues of the rat

    SciTech Connect

    Waalkes, M.P.

    1986-01-01

    The effect of continuous dietary zinc deficiency on the metabolism of the toxic heavy metal cadmium has not been widely studied. This investigation was designed to assess the effects of subadequate dietary zinc intake on the accumulation of dietary cadmium and on metallothionein (MT) and zinc concentrations in target organs of cadmium toxicity. Adult male Wistar rats (180-200 g) were allowed, ad libitum, diets either adequate (60 ppm) or deficient (7 ppm) in zinc for a total of 9 wk. The zinc-deficient diet resulted in an approximately 40% reduction in plasma zinc (assessed at 3, 6, and 9 wk) in the absence of overt signs of zinc deficiency (i.e., reduced weight gain, alopecia, etc.). Separate groups of rats were also maintained on zinc-defined diets for a total of 9 wk, but cadmium was added to the diet (0, 12.5, 25, 50, 100, and 200 ppm) a the end of wk 3 and maintained at that level throughout the remaining 6 wk of the study, when the rats were killed. The feeding of the zinc-deficient diet markedly enhanced the accumulation of cadmium in the liver, kidney, and testes. Hepatic, renal, and testicular zinc concentrations were not affected by suboptimal zinc intake alone. However, marked reductions in renal and testicular zinc concentrations were caused by zinc deficiency in concert with cadmium exposure. MT levels, when related to tissue cadmium concentrations, were elevated to a significantly lesser extent in the kidneys of zinc-deficient animals. These results indicate that marginal zinc deficiency markedly increases cadmium accumulation in various organs and reduces zinc content and MT induction in some organs.

  19. A plasmid containing the human metallothionein II gene can function as an antibody-assisted electrophoretic biosensor for heavy metals.

    PubMed

    Wooten, Dennis C; Starr, Clarise R; Lyon, Wanda J

    2016-01-01

    Different forms of heavy metals affect biochemical systems in characteristic ways that cannot be detected with typical metal analysis methods like atomic absorption spectrometry. Further, using living systems to analyze interaction of heavy metals with biochemical systems can be laborious and unreliable. To generate a reliable easy-to-use biologically-based biosensor system, the entire human metallothionein-II (MT-II) gene was incorporated into a plasmid (pUC57-MT) easily replicated in Escherichia coli. In this system, a commercial polyclonal antibody raised against human metal-responsive transcription factor-1 protein (MTF-1 protein) could modify the electrophoretic migration patterns (i.e. cause specific decreases in agarose gel electrophoretic mobility) of the plasmid in the presence or absence of heavy metals other than zinc (Zn). In the study here, heavy metals, MTF-1 protein, and polyclonal anti-MTF-1 antibody were used to assess pUC57-MT plasmid antibody-assisted electrophoretic mobility. Anti-MTF-1 antibody bound both MTF-1 protein and pUC57-MT plasmid in a non-competitive fashion such that it could be used to differentiate specific heavy metal binding. The results showed that antibody-inhibited plasmid migration was heavy metal level-dependent. Zinc caused a unique mobility shift pattern opposite to that of other metals tested, i.e. Zn blocked the antibody ability to inhibit plasmid migration, despite a greatly increased affinity for DNA by the antibody when Zn was present. The Zn effect was reversed/modified by adding MTF-1 protein. Additionally, antibody inhibition of plasmid mobility was resistant to heat pre-treatment and trypsinization, indicating absence of residual DNA extraction-resistant bacterial DNA binding proteins. DNA binding by anti-DNA antibodies may be commonly enhanced by xenobiotic heavy metals and elevated levels of Zn, thus making them potentially effective tools for assessment of heavy metal bioavailability in aqueous solutions and

  20. A PU.1 suppressive target gene, metallothionein 1G, inhibits retinoic acid-induced NB4 cell differentiation.

    PubMed

    Hirako, Naomi; Nakano, Hiroko; Takahashi, Shinichiro

    2014-01-01

    We recently revealed that myeloid master regulator SPI1/PU.1 directly represses metallothionein (MT) 1G through its epigenetic activity of PU.1, but the functions of MT1G in myeloid differentiation remain unknown. To clarify this, we established MT1G-overexpressing acute promyelocytic leukemia NB4 (NB4MTOE) cells, and investigated whether MT1G functionally contributes to all-trans retinoic acid (ATRA)-induced NB4 cell differentiation. Real-time PCR analyses demonstrated that the inductions of CD11b and CD11c and reductions in myeloperoxidase and c-myc by ATRA were significantly attenuated in NB4MTOE cells. Morphological examination revealed that the percentages of differentiated cells induced by ATRA were reduced in NB4MTOE cells. Since G1 arrest is a hallmark of ATRA-induced NB4 cell differentiation, we observed a decrease in G1 accumulation, as well as decreases in p21WAF1/CIP1 and cyclin D1 inductions, by ATRA in NB4MTOE cells. Nitroblue tetrazolium (NBT) reduction assays revealed that the proportions of NBT-positive cells were decreased in NB4MTOE cells in the presence of ATRA. Microarray analyses showed that the changes in expression of several myeloid differentiation-related genes (GATA2, azurocidin 1, pyrroline-5-carboxylate reductase 1, matrix metallopeptidase -8, S100 calcium-binding protein A12, neutrophil cytosolic factor 2 and oncostatin M) induced by ATRA were disturbed in NB4MTOE cells. Collectively, overexpression of MT1G inhibits the proper differentiation of myeloid cells.

  1. Comparative Raman study of four plant metallothionein isoforms: Insights into their Zn(II) clusters and protein conformations.

    PubMed

    Tomas, Mireia; Tinti, Anna; Bofill, Roger; Capdevila, Mercè; Atrian, Silvia; Torreggiani, Armida

    2016-03-01

    Four Metallothioneins (MTs) from soybean (Glycine max) were heterologously synthesized and comparatively analysed by Raman spectroscopy. The participation of protein donor groups (S-thiol and N-imidazol) in Zn(II) chelation, as well as the presence of secondary structure elements was comparatively analysed. Metal clusters with different geometry can be hypothesised for the four GmMTs: a cubane-like or an adamantane-like metal cluster in Zn-GmMT1, and dinuclear Zn-S clusters in Zn-GmMT2, Zn-GmMT3 and Zn-GmMT4. The latter have also a similar average Cys/Zn content, whereas a lower ratio is present in Zn-GmMT1. This is possible thanks to the involvement in metal coordination of a greater number of bridging Cys, as well as of some carboxylate groups. As regards secondary structure elements, a large content of β-turn segments is present in all four Zn-GmMTs, especially for isoforms 1 and 4. β-strands give a contribution to the folding of three GmMTs isoforms, and the highest percentage was found in Zn-GmMT2 (~45%). Conversely, the α-helix content is negligible in all the GmMTs except in Zn-GmMT3, where this peculiar feature coincides with the possible involvement of the two His residues in metal coordination. Conversely, His is predominantly free and present as tautomer I in Zn-GmMT4. In conclusion, this work illustrates the attractive potential of Raman spectroscopy, combined with other techniques, to be a very informative tool for evidencing structural differences among in vivo synthesized metal-MT complexes.

  2. Differential metallothionein, reduced glutathione and metal levels in Perna perna mussels in two environmentally impacted tropical bays in southeastern Brazil.

    PubMed

    Lavradas, Raquel T; Rocha, Rafael C C; Bordon, Isabella C A C; Saint'Pierre, Tatiana D; Godoy, José M; Hauser-Davis, Rachel A

    2016-07-01

    Mussel farming is an important economic activity in Brazil, and these organisms are consumed by the majority of the population in most coastal zones in the country. However, despite the increasing pollution of aquatic ecosystems in Brazil, little is known about the biochemical activity in mussels in response to metal exposure. In this context, the aim of the present study was to investigate metal and metalloid exposure effects in Perna perna mussels, by determining metal levels, the induction of metallothionein (MT) synthesis, and oxidative stress, in the form of reduced glutathione (GSH) in 3 contaminated areas from the Guanabara Bay in comparison to a reference site, Ilha Grande Bay, both in summer and winter. Metal and metalloid concentrations were also compared to Brazilian and international guidelines, to verify potential health risks to human consumers. Mussels from all sampling sites were shown to be improper for human consumption due to metal contamination, including Ilha Grande Bay, which has previously been considered a reference site. Several statistically significant correlations and seasonal differences were observed between MT, GSH and metals and metalloids in both analyzed tissues. A Discriminant Canonical Analysis indicated that the digestive gland is a better bioindicator for environmental contamination by metals and metalloids in this species and offers further proof that MT variations observed are due to metal exposure and not oxidative stress, since GSH influence for both muscle tissue and the digestive glands was non-significant in this analysis. These results show that P. perna mussels are an adequate sentinel species for metal contamination with significant effects on oxidative stress and metal exposure biomarkers. To the best of our knowledge, this is the first study to report metals, metalloids, MT and GSH levels in the muscle tissue of this species.

  3. Metallothionein-like peptides involved in sequestration of Zn in the Zn-accumulating ectomycorrhizal fungus Russula atropurpurea.

    PubMed

    Leonhardt, Tereza; Sácký, Jan; Šimek, Pavel; Šantrůček, Jiří; Kotrba, Pavel

    2014-09-01

    Homeostatic mechanisms preventing the toxicity of free Zn ions in cells involve, among others, cytosolic Zn-binding ligands, particularly the cysteine-rich metallothioneins (MTs). Here we examined the Zn-binding peptides of Russula atropurpurea, an ectomycorrhizal fungus known for its ability to accumulate high amounts of Zn in its sporocarps. The Zn complexes and their peptide ligands were characterized using chromatography, electrophoresis after fluorescent labeling of cysteine residues, and tandem mass spectrometry. Functional complementation assays in Saccharomyces cerevisiae were used to obtain and characterize cDNA sequences. Zn-speciation analysis showed that nearly 80% of the Zn extracted from the sporocarps was associated with cysteine-containing peptides in a 5 kDa complex. Screening of an R. atropurpurea cDNA library for sequences encoding peptides capable of sequestering divalent heavy metals was conducted in the Cd-hypersensitive ycf1Δ yeast. This allowed identification of two cDNAs, RaZBP1 and RaZBP2, which protected the metal-sensitive yeast mutants against Cd and Zn, but not Co, Mn or Cu, toxicity. The corresponding RaZBP1 and RaZBP2 peptides consisting of 53 amino acid (AA) residues and sharing 77% identity showed only a limited sequence similarity to known MTs, particularly due to the absence of multiple Cys-AA-Cys motifs. Both RaZBPs were detected in a native Zn-complex of R. atropurpurea and the recombinant RaZBP1 was found associated with Zn and Cd in yeasts. Altogether, the results point to an important role of RaZBPs in the handling of a substantial portion of the Zn pool in R. atropurpurea.

  4. Evolutionary Divergence of Arabidopsis thaliana Classical Peroxidases.

    PubMed

    Kupriyanova, E V; Mamoshina, P O; Ezhova, T A

    2015-10-01

    Polymorphisms of 62 peroxidase genes derived from Arabidopsis thaliana were investigated to evaluate evolutionary dynamics and divergence of peroxidase proteins. By comparing divergence of duplicated genes AtPrx53-AtPrx54 and AtPrx36-AtPrx72 and their products, nucleotide and amino acid substitutions were identified that were apparently targets of positive selection. These substitutions were detected among paralogs of 461 ecotypes from Arabidopsis thaliana. Some of these substitutions are conservative and matched paralogous peroxidases in other Brassicaceae species. These results suggest that after duplication, peroxidase genes evolved under the pressure of positive selection, and amino acid substitutions identified during our study provided divergence of properties and physiological functions in peroxidases. Our predictions regarding functional significance for amino acid residues identified in variable sites of peroxidases may allow further experimental assessment of evolution of peroxidases after gene duplication.

  5. Root gravitropism in maize and Arabidopsis

    NASA Technical Reports Server (NTRS)

    Evans, Michael L.

    1993-01-01

    Research during the period 1 March 1992 to 30 November 1993 focused on improvements in a video digitizer system designed to automate the recording of surface extension in plants responding to gravistimulation. The improvements included modification of software to allow detailed analysis of localized extension patterns in roots of Arabidopsis. We used the system to analyze the role of the postmitotic isodiametric growth zone (a region between the meristem and the elongation zone) in the response of maize roots to auxin, calcium, touch and gravity. We also used the system to analyze short-term auxin and gravitropic responses in mutants of Arabidopsis with reduced auxin sensitivity. In a related project, we studied the relationship between growth rate and surface electrical currents in roots by examining the effects of gravity and thigmostimulation on surface potentials in maize roots.

  6. DYn-2 Based Identification of Arabidopsis Sulfenomes*

    PubMed Central

    Akter, Salma; Huang, Jingjing; Bodra, Nandita; De Smet, Barbara; Wahni, Khadija; Rombaut, Debbie; Pauwels, Jarne; Gevaert, Kris; Carroll, Kate; Van Breusegem, Frank; Messens, Joris

    2015-01-01

    Identifying the sulfenylation state of stressed cells is emerging as a strategic approach for the detection of key reactive oxygen species signaling proteins. Here, we optimized an in vivo trapping method for cysteine sulfenic acids in hydrogen peroxide (H2O2) stressed plant cells using a dimedone based DYn-2 probe. We demonstrated that DYn-2 specifically detects sulfenylation events in an H2O2 dose- and time-dependent way. With mass spectrometry, we identified 226 sulfenylated proteins after H2O2 treatment of Arabidopsis cells, residing in the cytoplasm (123); plastid (68); mitochondria (14); nucleus (10); endoplasmic reticulum, Golgi and plasma membrane (7) and peroxisomes (4). Of these, 123 sulfenylated proteins have never been reported before to undergo cysteine oxidative post-translational modifications in plants. All in all, with this DYn-2 approach, we have identified new sulfenylated proteins, and gave a first glance on the locations of the sulfenomes of Arabidopsis thaliana. PMID:25693797

  7. High-throughput TILLING for Arabidopsis.

    PubMed

    Till, Bradley J; Colbert, Trenton; Codomo, Christine; Enns, Linda; Johnson, Jessica; Reynolds, Steven H; Henikoff, Jorja G; Greene, Elizabeth A; Steine, Michael N; Comai, Luca; Henikoff, Steven

    2006-01-01

    Targeting induced local lesions in genomes (TILLING) is a general strategy for identifying induced point mutations that can be applied to almost any organism. In this chapter, we describe the basic methodology for high-throughput TILLING. Gene segments are amplified using fluorescently tagged primers, and products are denatured and reannealed to form heteroduplexes between the mutated sequence and its wild-type counterpart. These heteroduplexes are substrates for cleavage by the endonuclease CEL I. Following cleavage, products are analyzed on denaturing polyacrylamide gels using the LI-COR DNA analyzer system. High-throughput TILLING has been adopted by the Arabidopsis TILLING Project (ATP) to provide allelic series of point mutations for the general Arabidopsis community.

  8. Comparative transcriptomics of Arabidopsis sperm cells.

    PubMed

    Borges, Filipe; Gomes, Gabriela; Gardner, Rui; Moreno, Nuno; McCormick, Sheila; Feijó, José A; Becker, Jörg D

    2008-10-01

    In flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize (Zea mays) and Plumbago and in lily (Lilium longiflorum) generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis (Arabidopsis thaliana) were hindered, because no method to isolate sperm cells was available. Here, we used fluorescence-activated cell sorting to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of their transcriptome at a genome-wide level. Comparative analysis of the sperm cell transcriptome with those of representative sporophytic tissues and of pollen showed that sperm has a distinct and diverse transcriptional profile. Functional classifications of genes with enriched expression in sperm cells showed that DNA repair, ubiquitin-mediated proteolysis, and cell cycle progression are overrepresented Gene Ontology categories. Moreover, analysis of the small RNA and DNA methylation pathways suggests that distinct mechanisms might be involved in regulating the epigenetic state of the paternal genome. We identified numerous candidate genes whose involvement in sperm cell development and fertilization can now be directly tested in Arabidopsis. These results provide a roadmap to decipher the role of sperm-expressed proteins.

  9. Molecular analysis of menadione-induced resistance against biotic stress in Arabidopsis.

    PubMed

    Borges, Andrés A; Dobon, Albor; Expósito-Rodríguez, Marino; Jiménez-Arias, David; Borges-Pérez, Andrés; Casañas-Sánchez, Verónica; Pérez, Jose A; Luis, Juan C; Tornero, Pablo

    2009-10-01

    Menadione sodium bisulphite (MSB) is a water-soluble derivative of vitamin K3, or menadione, and has been previously demonstrated to function as a plant defence activator against several pathogens in several plant species. However, there are no reports of the role of this vitamin in the induction of resistance in the plant model Arabidopsis thaliana. In the current study, we demonstrate that MSB induces resistance by priming in Arabidopsis against the virulent strain Pseudomonas syringae pv. tomato DC3000 (Pto) without inducing necrosis or visible damage. Changes in gene expression in response to 0.2 mm MSB were analysed in Arabidopsis at 3, 6 and 24 h post-treatment using microarray technology. In general, the treatment with MSB does not correlate with other publicly available data, thus MSB produces a unique molecular footprint. We observed 158 differentially regulated genes among all the possible trends. More up-regulated genes are included in categories such as 'response to stress' than the background, and the behaviour of these genes in different treatments confirms their role in response to biotic and abiotic stress. In addition, there is an over-representation of the G-box in their promoters. Some interesting functions are represented among the individual up-regulated genes, such as glutathione S-transferases, transcription factors (including putative regulators of the G-box) and cytochrome P450s. This work provides a wide insight into the molecular cues underlying the effect of MSB as a plant resistance inducer.

  10. Regulatory Proteolysis in Arabidopsis-Pathogen Interactions.

    PubMed

    Pogány, Miklós; Dankó, Tamás; Kámán-Tóth, Evelin; Schwarczinger, Ildikó; Bozsó, Zoltán

    2015-09-24

    Approximately two and a half percent of protein coding genes in Arabidopsis encode enzymes with known or putative proteolytic activity. Proteases possess not only common housekeeping functions by recycling nonfunctional proteins. By irreversibly cleaving other proteins, they regulate crucial developmental processes and control responses to environmental changes. Regulatory proteolysis is also indispensable in interactions between plants and their microbial pathogens. Proteolytic cleavage is simultaneously used both by plant cells, to recognize and inactivate invading pathogens, and by microbes, to overcome the immune system of the plant and successfully colonize host cells. In this review, we present available results on the group of proteases in the model plant Arabidopsis thaliana whose functions in microbial pathogenesis were confirmed. Pathogen-derived proteolytic factors are also discussed when they are involved in the cleavage of host metabolites. Considering the wealth of review papers available in the field of the ubiquitin-26S proteasome system results on the ubiquitin cascade are not presented. Arabidopsis and its pathogens are conferred with abundant sets of proteases. This review compiles a list of those that are apparently involved in an interaction between the plant and its pathogens, also presenting their molecular partners when available.

  11. Analysis of the Arabidopsis Mitochondrial Proteome1

    PubMed Central

    Millar, A. Harvey; Sweetlove, Lee J.; Giegé, Philippe; Leaver, Christopher J.

    2001-01-01

    The complete set of nuclear genes that encode proteins targeted to mitochondria in plants is currently undefined and thus the full range of mitochondrial functions in plants is unknown. Analysis of two-dimensional gel separations of Arabidopsis cell culture mitochondrial protein revealed approximately 100 abundant proteins and 250 low-abundance proteins. Comparison of subfractions of mitochondrial protein on two-dimensional gels provided information on the soluble, membrane, or integral membrane locations of this protein set. A total of 170 protein spots were excised, trypsin-digested, and matrix-assisted laser desorption ionization/time of flight mass spectrometry spectra obtained. Using this dataset, 91 of the proteins were identified by searching translated Arabidopsis genomic databases. Of this set, 81 have defined functions based on sequence comparison. These functions include respiratory electron transport, tricarboxylic acid cycle metabolism, amino acid metabolism, protein import, processing, and assembly, transcription, membrane transport, and antioxidant defense. A total of 10 spectra were matched to Arabidopsis putative open reading frames for which no specific function has been determined. A total of 64 spectra did not match to an identified open reading frame. Analysis of full-length putative protein sequences using bioinformatic tools to predict subcellular targeting (TargetP, Psort, and MitoProt) revealed significant variation in predictions, and also a lack of mitochondrial targeting prediction for several characterized mitochondrial proteins. PMID:11743115

  12. Arabidopsis alternative oxidase sustains Escherichia coli respiration.

    PubMed Central

    Kumar, A M; Söll, D

    1992-01-01

    Glutamyl-tRNA reductase, encoded by the hemA gene, is the first enzyme in porphyrin biosynthesis in many organisms. Hemes, important porphyrin derivatives, are essential components of redox enzymes, such as cytochromes. Thus a hemA Escherichia coli strain (SASX41B) is deficient in cytochrome-mediated aerobic respiration. Upon complementation of this strain with an Arabidopsis thaliana cDNA library, we isolated a clone which permitted the SASX41B strain to grow aerobically. The clone encodes the gene for Arabidopsis alternative oxidase, whose deduced amino acid sequence was found to have 71% identity with that of the enzyme from the voodoo lily, Sauromatum guttatum. The Arabidopsis protein is expressed as a 31-kDa protein in E. coli and confers on this organism cyanide-resistant growth, which in turn is sensitive to salicylhydroxamate. This implies that a single polypeptide is sufficient for alternative oxidase activity. Based on these observations we propose that a cyanide-insensitive respiratory pathway operates in the transformed E. coli hemA strain. Introduction of this pathway now opens the way to genetic/molecular biological investigations of alternative oxidase and its cofactor. Images PMID:1438286

  13. Phosphorylation of plastoglobular proteins in Arabidopsis thaliana

    PubMed Central

    Lohscheider, Jens N.; Friso, Giulia; van Wijk, Klaas J.

    2016-01-01

    Plastoglobules (PGs) are plastid lipid–protein particles with a small specialized proteome and metabolome. Among the 30 core PG proteins are six proteins of the ancient ABC1 atypical kinase (ABC1K) family and their locations in an Arabidopsis mRNA-based co-expression network suggested central regulatory roles. To identify candidate ABC1K targets and a possible ABC1K hierarchical phosphorylation network within the chloroplast PG proteome, we searched Arabidopsis phosphoproteomics data from publicly available sources. Evaluation of underlying spectra and/or associated information was challenging for a variety of reasons, but supported pSer sites and a few pThr sites in nine PG proteins, including five FIBRILLINS. PG phosphorylation motifs are discussed in the context of possible responsible kinases. The challenges of collection and evaluation of published Arabidopsis phosphorylation data are discussed, illustrating the importance of deposition of all mass spectrometry data in well-organized repositories such as PRIDE and ProteomeXchange. This study provides a starting point for experimental testing of phosho-sites in PG proteins and also suggests that phosphoproteomics studies specifically designed toward the PG proteome and its ABC1K are needed to understand phosphorylation networks in these specialized particles. PMID:26962209

  14. [Imprinting genes and it's expression in Arabidopsis].

    PubMed

    Zhang, Hong-Yu; Xu, Pei-Zhou; Yang, Hua; Wu, Xian-Jun

    2010-07-01

    Genomic imprinting refers to the phenomenon that the expression of a gene copy depends on its parent of origin. The Arabidopsis imprinted FIS (Fertilisation-independent seed) genes, mea, fis2, and fie, play essential roles in the repression of central cell and the regulation of early endosperm development. fis mutants display two phenotypes: autonomous diploid endosperm development when fertilization is absent and un-cellularised endosperm formation when fertilization occurs. The FIS Polycomb protein complex including the above three FIS proteins catalyzes histone H3 K27 tri-methylation on target loci. DME (DEMETER), a DNA glycosylase, and AtMET1 (Methyltransferase1), a DNA methyltransferase, are involved in the regulation of imprinted expression of both mea and fis2. This review summarizes the studies on the Arabidopsis imprinted FIS genes and other related genes. Recent works have shown that the insertion of transposons may affect nearby gene expression, which may be the main driving force behind the evolution of genomic imprinting. This summary covers the achievements on Arabidopsis imprinted genes will provide important information for studies on genomic imprinting in the important crops such as rice and maize.

  15. TBP-associated factors in Arabidopsis.

    PubMed

    Lago, Clara; Clerici, Elena; Mizzi, Luca; Colombo, Lucia; Kater, Martin M

    2004-11-24

    Initiation of transcription mediated by RNA polymerase II requires a number of transcription factors among which TFIID is the major core promoter recognition factor. TFIID is composed of highly conserved factors which include the TATA-binding protein (TBP) and about 14 TBP-associated factors (TAFs). Since TAFs play important roles in transcription they have been extensively studied in organisms like yeast, Drosophila and human. Surprisingly, TAFs have been poorly characterized in plants. With the completion of the Arabidopsis genome sequence, it is possible to search for TAFs, since many of them have conserved amino acid sequences. Mining the genome of Arabidopsis for TAFs resulted in the identification of 18 putative Arabidopsis TAFs (AtTAFs). We have analyzed their protein structure and their genomic localisation. Expression profiling by RT-PCR showed that these TAFs are expressed in all parts of the plant which is in agreement with their general role in transcription. These analyses in combination with their evolutionary conservation with TAFs of other organisms are discussed.

  16. Phytochrome regulation of branching in Arabidopsis.

    PubMed

    Finlayson, Scott A; Krishnareddy, Srirama R; Kebrom, Tesfamichael H; Casal, Jorge J

    2010-04-01

    The red light:far-red light ratio perceived by phytochromes controls plastic traits of plant architecture, including branching. Despite the significance of branching for plant fitness and productivity, there is little quantitative and mechanistic information concerning phytochrome control of branching responses in Arabidopsis (Arabidopsis thaliana). Here, we show that in Arabidopsis, the negative effects of the phytochrome B mutation and of low red light:far-red light ratio on branching were largely due to reduced bud outgrowth capacity and an increased degree of correlative inhibition acting on the buds rather than due to a reduced number of leaves and buds available for branching. Phytochrome effects on the degree of correlative inhibition required functional BRANCHED1 (BRC1), BRC2, AXR1, MORE AXILLARY GROWTH2 (MAX2), and MAX4. The analysis of gene expression in selected buds indicated that BRC1 and BRC2 are part of different gene networks. The BRC1 network is linked to the growth capacity of specific buds, while the BRC2 network is associated with coordination of growth among branches. We conclude that the branching integrators BRC1 and BRC2 are necessary for responses to phytochrome, but they contribute differentially to these responses, likely acting through divergent pathways.

  17. Sodium Influx and Accumulation in Arabidopsis1

    PubMed Central

    Essah, Pauline A.; Davenport, Romola; Tester, Mark

    2003-01-01

    Arabidopsis is frequently used as a genetic model in plant salt tolerance studies, however, its physiological responses to salinity remain poorly characterized. This study presents a characterization of initial Na+ entry and the effects of Ca2+ on plant growth and net Na+ accumulation in saline conditions. Unidirectional Na+ influx was measured carefully using very short influx times in roots of 12-d-old seedlings. Influx showed three components with distinct sensitivities to Ca2+, diethylpyrocarbonate, and osmotic pretreatment. Pharmacological agents and known mutants were used to test the contribution of different transport pathways to Na+ uptake. Influx was stimulated by 4-aminobutyric acid and glutamic acid; was inhibited by flufenamate, quinine, and cGMP; and was insensitive to modulators of K+ and Ca2+ channels. Influx did not differ from wild type in akt1 and hkt1 insertional mutants. These data suggested that influx was mediated by several different types of nonselective cation channels. Na+ accumulation in plants grown in 50 mm NaCl was strongly reduced by increasing Ca2+ activity (from 0.05-3.0 mm), and plant survival was improved. However, plant biomass was not affected by shoot Na+ concentration, suggesting that in Arabidopsis Na+ toxicity is not dependent on shoot Na+ accumulation. These data suggest that Arabidopsis is a good model for investigation of Na+ transport, but may be of limited utility as a model for the study of Na+ toxicity. PMID:12970496

  18. Variability and Constancy in Cellular Growth of Arabidopsis Sepals1[OPEN

    PubMed Central

    Tauriello, Gerardo; Meyer, Heather M.; Smith, Richard S.

    2015-01-01

    Growth of tissues is highly reproducible; yet, growth of individual cells in a tissue is highly variable, and neighboring cells can grow at different rates. We analyzed the growth of epidermal cell lineages in the Arabidopsis (Arabidopsis thaliana) sepal to determine how the growth curves of individual cell lineages relate to one another in a developing tissue. To identify underlying growth trends, we developed a continuous displacement field to predict spatially averaged growth rates. We showed that this displacement field accurately describes the growth of sepal cell lineages and reveals underlying trends within the variability of in vivo cellular growth. We found that the tissue, individual cell lineages, and cell walls all exhibit growth rates that are initially low, accelerate to a maximum, and decrease again. Accordingly, these growth curves can be represented by sigmoid functions. We examined the relationships among the cell lineage growth curves and surprisingly found that all lineages reach the same maximum growth rate relative to their size. However, the cell lineages are not synchronized; each cell lineage reaches this same maximum relative growth rate but at different times. The heterogeneity in observed growth results from shifting the same underlying sigmoid curve in time and scaling by size. Thus, despite the variability in growth observed in our study and others, individual cell lineages in the developing sepal follow similarly shaped growth curves. PMID:26432876

  19. The Arabidopsis TAC Position Viewer: a high-resolution map of transformation-competent artificial chromosome (TAC) clones aligned with the Arabidopsis thaliana Columbia-0 genome.

    PubMed

    Hirose, Yoshitsugu; Suda, Kunihiro; Liu, Yao-Guang; Sato, Shusei; Nakamura, Yukino; Yokoyama, Koji; Yamamoto, Naoki; Hanano, Shigeru; Takita, Eiji; Sakurai, Nozomu; Suzuki, Hideyuki; Nakamura, Yasukazu; Kaneko, Takakazu; Yano, Kentaro; Tabata, Satoshi; Shibata, Daisuke

    2015-09-01

    We present a high-resolution map of genomic transformation-competent artificial chromosome (TAC) clones extending over all Arabidopsis thaliana (Arabidopsis) chromosomes. The Arabidopsis genomic TAC clones have been valuable genetic tools. Previously, we constructed an Arabidopsis genomic TAC library consisting of more than 10,000 TAC clones harboring large genomic DNA fragments extending over the whole Arabidopsis genome. Here, we determined 13,577 end sequences from 6987 Arabidopsis TAC clones and mapped 5937 TAC clones to precise locations, covering approximately 90% of the Arabidopsis chromosomes. We present the large-scale data set of TAC clones with high-resolution mapping information as a Java application tool, the Arabidopsis TAC Position Viewer, which provides ready-to-go transformable genomic DNA clones corresponding to certain loci on Arabidopsis chromosomes. The TAC clone resources will accelerate genomic DNA cloning, positional walking, complementation of mutants and DNA transformation for heterologous gene expression.

  20. Expansive evolution of the trehalose-6-phosphate phosphatase gene family in Arabidopsis.

    PubMed

    Vandesteene, Lies; López-Galvis, Lorena; Vanneste, Kevin; Feil, Regina; Maere, Steven; Lammens, Willem; Rolland, Filip; Lunn, John E; Avonce, Nelson; Beeckman, Tom; Van Dijck, Patrick

    2012-10-01

    Trehalose is a nonreducing sugar used as a reserve carbohydrate and stress protectant in a variety of organisms. While higher plants typically do not accumulate high levels of trehalose, they encode large families of putative trehalose biosynthesis genes. Trehalose biosynthesis in plants involves a two-step reaction in which trehalose-6-phosphate (T6P) is synthesized from UDP-glucose and glucose-6-phosphate (catalyzed by T6P synthase [TPS]), and subsequently dephosphorylated to produce the disaccharide trehalose (catalyzed by T6P phosphatase [TPP]). In Arabidopsis (Arabidopsis thaliana), 11 genes encode proteins with both TPS- and TPP-like domains but only one of these (AtTPS1) appears to be an active (TPS) enzyme. In addition, plants contain a large family of smaller proteins with a conserved TPP domain. Here, we present an in-depth analysis of the 10 TPP genes and gene products in Arabidopsis (TPPA-TPPJ). Collinearity analysis revealed that all of these genes originate from whole-genome duplication events. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that all encode active TPP enzymes with an essential role for some conserved residues in the catalytic domain. These results suggest that the TPP genes function in the regulation of T6P levels, with T6P emerging as a novel key regulator of growth and development in higher plants. Extensive gene expression analyses using a complete set of promoter-β-glucuronidase/green fluorescent protein reporter lines further uncovered cell- and tissue-specific expression patterns, conferring spatiotemporal control of trehalose metabolism. Consistently, phenotypic characterization of knockdown and overexpression lines of a single TPP, AtTPPG, points to unique properties of individual TPPs in Arabidopsis, and underlines the intimate connection between trehalose metabolism and abscisic acid signaling.

  1. Phytoplasma Effector SAP54 Induces Indeterminate Leaf-Like Flower Development in Arabidopsis Plants1[C][W][OA

    PubMed Central

    MacLean, Allyson M.; Sugio, Akiko; Makarova, Olga V.; Findlay, Kim C.; Grieve, Victoria M.; Tóth, Réka; Nicolaisen, Mogens; Hogenhout, Saskia A.

    2011-01-01

    Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches’ broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host. PMID:21849514

  2. Acclimation-induced changes in toxicity and induction of metallothionein-like proteins in the fathead minnow following sublethal exposure to cobalt, silver, and zinc

    SciTech Connect

    Hobson, J.F.

    1986-01-01

    Increases in tolerance and resistance to metal toxicity by aquatic organisms have been linked to elevated levels of low-molecular-weight metal-binding proteins (e.g., metallothioneins). Acclimation-induced changes in toxic response and the concentration of metallothionein-like proteins (MTP) were studied in laboratory populations of the fathead minnow, Pimephales promelas, following sublethal exposure to Co, Ag, and Zn. Following 7 and 14 days of sublethal exposure, tolerance and resistance, as measured by acute toxicity values, were altered in a dose dependent fashion. Acute toxicity values returned to control levels after 21 days of continuous exposure. Tolerance and resistance of Co- and Zn-acclimated animals were depressed after a 7-day post-acclimation period in control water. Tolerance and resistance of Ag-acclimated animals were temporarily enhanced after 7 days post-acclimation and returned to control levels after 14 days. Accumulation of Co, Ag, and Zn measured as wholebody residues appeared to be regulated in 4 of 6 exposure regimes with residues reaching stable levels after 7 to 14 days of exposure. MTP was induced by exposure to 1.8 mg Zn/L and 0.01 mg Ag/L, however, no sustained (i.e., post 21 days) tolerance or resistance were observed at these dose levels indicating that these two biological responses may not be directly related.

  3. Sequestration of cadmium and copper by recombinant rainbow trout and human metallothioneins and by chimeric (mermaid and fishman) proteins with interchanged domains.

    PubMed

    Kille, P; Lees, W E; Darke, B M; Winge, D R; Dameron, C T; Stephens, P E; Kay, J

    1992-04-25

    A family of synthetic genes was constructed encoding a rainbow trout metallothionein (MT), a human MT, and two chimeric molecules which contained respectively (i) the N-terminal (or head) domain of human MT followed by the C-terminal (or tail) domain of a fish MT (termed mermaid MT) and (ii) the head domain of fish MT fused with the tail domain of human MT (denoted fishman MT). These were expressed in Escherichia coli and the four recombinant proteins were purified to homogeneity therefrom. All four were found to bind 7 g atoms of Cd(II) per mol; at pH 7.0, but not at pH 8.6, four Cd(II) ions were sequestered preferentially in the tail (or alpha) domain. Reciprocally, copper was found to bind preferentially in the head (or beta) domain. The human and fishman MTs displayed a stoichiometry of 12 g atoms of Cu(I) per mol, while rainbow trout and mermaid MTs bound only 10. The significance of these findings is discussed in relation to the different positional organization of cysteine residues close to the N and C termini of mammalian and piscine metallothioneins.

  4. Effects of heavy metals on the expression of a zinc-inducible metallothionein-III gene and antioxidant enzyme activities in Crassostrea gigas.

    PubMed

    Cong, Ming; Wu, Huifeng; Liu, Xiaoli; Zhao, Jianmin; Wang, Xuan; Lv, Jiasen; Hou, Lin

    2012-10-01

    Sequestration by metallothioneins and antioxidant defense are two kinds of important defense mechanisms employed by mollusks to minimize adverse effects caused by heavy metal contaminants in marine environment. In the present study, a novel metallothionein gene, CgMT-III, was cloned from Crassostrea gigas, consisting of eighteen conserved cysteine residues and encoding a MT III-like protein with two tandem β domains. The expression level of CgMT-III transcript induced by zinc was much higher than that induced by cadmium exposure. It suggested that CgMT-III was perhaps mainly involved in homeostatic control of zinc metabolism, which was distinct from previously identified MTs in C. gigas. Among the tested antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), SOD and GPx showed varying up-regulations in a tissue-specific manner, while CAT activities were inhibited in both gill and hepatopancreas from C. gigas exposed to heavy metals. It can be inferred that CgMT-III was mainly involved in zinc homeostasis, and CgMT-III gene together with CAT enzyme could be potential biomarkers to indicate heavy metal, especially zinc pollution in marine organisms.

  5. Effect of temperature and season on reproduction, neutral red retention and metallothionein responses of earthworms exposed to metals in field soils.

    PubMed

    Svendsen, Claus; Hankard, Peter K; Lister, Lindsay J; Fishwick, Samantha K; Jonker, Martijs J; Spurgeon, David J

    2007-05-01

    This study investigated the short-term survival, reproduction and physiological (lysosomal membrane stability, metallothionein transcript copy number, body tissue metal concentrations) responses of Lumbricus rubellus exposed to metal contaminated field soils under different laboratory temperatures (10, 15 and 20 degrees C) and physiological responses of earthworms collected from the field in three different seasons (spring, autumn, winter). In the laboratory, metal contaminated soils had significant effects on reproduction (p<0.001), metallothionein-2 (MT-2) expression (p=0.033) and earthworm As (p=0.003), Cd (p=0.001), Pb (p<0.001) and Zn (p<0.001) concentration, but not lysosomal membrane stability and tissue Hg and Cu. No effect of temperature was found for any parameter. Principal component analysis of extractable and tissue metal concentrations indicated PC1 as a measure of metal stress. Both cocoon production (r=-0.75) and MT-2 induction (r=0.41) were correlated with PC1. A correlation was also found between cocoon production and MT-2 expression (r=-0.41). Neutral red retention and MT-2 measurements in worms collected from the field sites in three seasons confirmed the absence of a temperature effect on these responses.

  6. Extremely low-frequency electromagnetic field induces neural differentiation of hBM-MSCs through regulation of (Zn)-metallothionein-3.

    PubMed

    Aikins, Anastasia Rosebud; Hong, Sung-Won; Kim, Hyun-Jung; Yoon, Cheol-Ho; Chung, Joo-Hee; Kim, MiJung; Kim, Chan-Wha

    2017-03-29

    Extremely low-frequency electromagnetic field (ELFEMF) can stimulate neural differentiation in human bone marrow-derived mesenchymal cells (hBM-MSCs), and this provides an opportunity for research on neurodegenerative diseases such as Alzheimer's disease (AD). Metallothionein-3 (MT3), an isoform of the metal-binding proteins, metallothioneins, involved in maintaining intracellular zinc (Zn) homeostasis and the deregulation of zinc homeostasis, has separately been implicated in AD. Here, we investigated the effect of ELFEMF-induced neural differentiation of hBM-MSCs on Zn-MT3 homeostatic interaction. Exposure to ELFEMF induced neural differentiation of hBM-MSCs, which was characterized by decreased proliferation and enhanced neural-like morphology. We observed expression of neuronal markers such as β-tubulin3, pleiotrophin, and neurofilament-M at the mRNA level and MAP2 at the protein level. ELFEMF-induced neural differentiation correlated with decreased expression of metal-response element-transcription factor 1 and MT3, as well as decreased intracellular Zn concentration. In addition, upregulation of dihydropyrimidinase-related protein 2 was observed, but there was no change in γ-enolase expression. These data indicate a possible regulatory mechanism for MT3 during neural differentiation. Our findings provide considerable insight into molecular mechanisms involved in neural differentiation, which is useful for developing new treatments for neurodegenerative diseases. Bioelectromagnetics. 2017;9999:XX-XX. © 2017 Wiley Periodicals, Inc.

  7. Heterologous expression of a rice metallothionein isoform (OsMTI-1b) in Saccharomyces cerevisiae enhances cadmium, hydrogen peroxide and ethanol tolerance.

    PubMed

    Ansarypour, Zahra; Shahpiri, Azar

    2017-02-04

    Metallothioneins are a superfamily of low-molecular-weight, cysteine (Cys)-rich proteins that are believed to play important roles in protection against metal toxicity and oxidative stress. The main purpose of this study was to investigate the effect of heterologous expression of a rice metallothionein isoform (OsMTI-1b) on the tolerance of Saccharomyces cerevisiae to Cd(2+), H2O2 and ethanol stress. The gene encoding OsMTI-1b was cloned into p426GPD as a yeast expression vector. The new construct was transformed to competent cells of S. cerevisiae. After verification of heterologous expression of OsMTI-1b, the new strain and control were grown under stress conditions. In comparison to control strain, the transformed S. cerevisiae cells expressing OsMTI-1b showed more tolerance to Cd(2+) and accumulated more Cd(2+) ions when they were grown in the medium containing CdCl2. In addition, the heterologous expression of GST-OsMTI-1b conferred H2O2 and ethanol tolerance to S. cerevisiae cells. The results indicate that heterologous expression of plant MT isoforms can enhance the tolerance of S. cerevisiae to multiple stresses.

  8. A survey of dominant mutations in Arabidopsis thaliana.

    PubMed

    Meinke, David W

    2013-02-01

    Following the recent publication of a comprehensive dataset of 2400 genes with a loss-of-function mutant phenotype in Arabidopsis (Arabidopsis thaliana), questions remain concerning the diversity of dominant mutations in Arabidopsis. Most of these dominant phenotypes are expected to result from inappropriate gene expression, novel protein function, or disrupted protein complexes. This review highlights the major classes of dominant mutations observed in model organisms and presents a collection of 200 Arabidopsis genes associated with a dominant or semidominant phenotype. Emphasis is placed on mutants identified through forward genetic screens of mutagenized or activation-tagged populations. These datasets illustrate the variety of genetic changes and protein functions that underlie dominance in Arabidopsis and may ultimately contribute to phenotypic variation in flowering plants.

  9. The Arabidopsis Zinc Finger-Homeodomain Genes Encode Proteins with Unique Biochemical Properties That Are Coordinately Expressed during Floral Development1

    PubMed Central

    Tan, Queenie K.-G.; Irish, Vivian F.

    2006-01-01

    Arabidopsis (Arabidopsis thaliana) contains approximately 100 homeobox genes, many of which have been shown to play critical roles in various developmental processes. Here we characterize the zinc finger-homeodomain (ZF-HD) subfamily of homeobox genes, consisting of 14 members in Arabidopsis. We demonstrate that the HDs of the ZF-HD proteins share some similarities with other known HDs in Arabidopsis, but they contain distinct features that cluster them as a unique class of plant HD-containing proteins. We have carried out mutational analyses to show that the noncanonical residues present in the HDs of this family of proteins are important for function. Yeast (Saccharomyces cerevisiae) two-hybrid matrix analyses of the ZF-HD proteins reveal that these proteins both homo- and heterodimerize, which may contribute to greater selectivity in DNA binding. These assays also show that most of these proteins do not contain an intrinsic activation domain, suggesting that interactions with other factors are required for transcriptional activation. We also show that the family members are all expressed predominantly or exclusively in floral tissue, indicating a likely regulatory role during floral development. Furthermore, we have identified loss-of-function mutations for six of these genes that individually show no obvious phenotype, supporting the idea that the encoded proteins have common roles in floral development. Based on these results, we propose the ZF-HD gene family encodes a group of transcriptional regulators with unique biochemical activities that play overlapping regulatory roles in Arabidopsis floral development. PMID:16428600

  10. Metal Dealing at the Origin of the Chordata Phylum: The Metallothionein System and Metal Overload Response in Amphioxus

    PubMed Central

    Capdevila, Mercè; Palacios, Òscar; Atrian, Sílvia

    2012-01-01

    Non-vertebrate chordates, specifically amphioxus, are considered of the utmost interest for gaining insight into the evolutionary trends, i.e. differentiation and specialization, of gene/protein systems. In this work, MTs (metallothioneins), the most important metal binding proteins, are characterized for the first time in the cephalochordate subphylum at both gene and protein level, together with the main features defining the amphioxus response to cadmium and copper overload. Two MT genes (BfMT1 and BfMT2) have been identified in a contiguous region of the genome, as well as several ARE (antioxidant response element) and MRE (metal response element) located upstream the transcribed region. Their corresponding cDNAs exhibit identical sequence in the two lancelet species (B. floridae and B. lanceolatum), BfMT2 cDNA resulting from an alternative splicing event. BfMT1 is a polyvalent metal binding peptide that coordinates any of the studied metal ions (Zn, Cd or Cu) rendering complexes stable enough to last in physiological environments, which is fully concordant with the constitutive expression of its gene, and therefore, with a metal homeostasis housekeeping role. On the contrary, BfMT2 exhibits a clear ability to coordinate Cd(II) ions, while it is absolutely unable to fold into stable Cu (I) complexes, even as mixed species. This identifies it as an essential detoxification agent, which is consequently only induced in emergency situations. The cephalochordate MTs are not directly related to vertebrate MTs, neither by gene structure, protein similarity nor metal-binding behavior of the encoded peptides. The closest relative is the echinoderm MT, which confirm proposed phylogenetic relationships between these two groups. The current findings support the existence in most organisms of two types of MTs as for their metal binding preferences, devoted to different biological functions: multivalent MTs for housekeeping roles, and specialized MTs that evolve either as Cd

  11. Idiorrhythmic zinc dose-rate induction of intestinal metallothionein in rats depends upon their nutritional zinc status(dagger).

    PubMed

    Momcilovic, B; Reeves, P G.

    2001-04-01

    The idiorrhythmic dose-rate feeding experimental model was used to study the induction of intestinal metallothionein (iMT) by zinc (Zn) in the gastrointestinal (GIT) mucosa of young growing male rats relative to their nutritional Zn status. The idiorrhythmic approach requires that the average dietary Zn concentration, referred to as modulo (M), is kept constant across different groups over the whole experimental epoch (E). This is done by adjusting the Zn concentration of the supplemented diet to compensate for the reduction in the number of days on which this diet is fed, the latter being spread evenly over the whole experiment. Idiorrhythms (I) involve offering the diet with n times the overall Zn concentration (M) only every nth day with a Zn-deficient diet offered on other days. We studied three modulos (low-Zn, M3; adequate-Zn, M12; and high-Zn, M48), each M having 8 analogous idiorrhythms (I = Mx/1 to 8Mx/8); every I was fed over a 48-d idiorrhythmic E. Over the wide range of peak doses of dietary Zn (3-384 mg Zn/kg diet), the higher the modulo, the greater the capacity for iMT to be induced (M3 < M12 < M48; P < 0.05). Also, the ability of Zn to induce iMT increased proportionally with the progression of the idiorrhythms from I = Mx/1 to 8Mx/8 (P < 0.001). When rats were fed M3, less Zn was required to induce iMT than when they were fed M12 or M48. Thus, within the M and E limits of this study, the better the nutritional Zn status of the animal, the more Zn is required to induce iMT and vice versa. The fact that iMT was increased means that the amount of available Zn was not proportional with the actual steady state of its metabolism. This indicates that for any Zn supplementation program to be effective, it should progress gradually from a lower to a higher Zn dose relative to the given nutritional Zn status.

  12. Metal dealing at the origin of the Chordata phylum: the metallothionein system and metal overload response in amphioxus.

    PubMed

    Guirola, Maria; Pérez-Rafael, Sílvia; Capdevila, Mercè; Palacios, Oscar; Atrian, Sílvia

    2012-01-01

    Non-vertebrate chordates, specifically amphioxus, are considered of the utmost interest for gaining insight into the evolutionary trends, i.e. differentiation and specialization, of gene/protein systems. In this work, MTs (metallothioneins), the most important metal binding proteins, are characterized for the first time in the cephalochordate subphylum at both gene and protein level, together with the main features defining the amphioxus response to cadmium and copper overload. Two MT genes (BfMT1 and BfMT2) have been identified in a contiguous region of the genome, as well as several ARE (antioxidant response element) and MRE (metal response element) located upstream the transcribed region. Their corresponding cDNAs exhibit identical sequence in the two lancelet species (B. floridae and B. lanceolatum), BfMT2 cDNA resulting from an alternative splicing event. BfMT1 is a polyvalent metal binding peptide that coordinates any of the studied metal ions (Zn, Cd or Cu) rendering complexes stable enough to last in physiological environments, which is fully concordant with the constitutive expression of its gene, and therefore, with a metal homeostasis housekeeping role. On the contrary, BfMT2 exhibits a clear ability to coordinate Cd(II) ions, while it is absolutely unable to fold into stable Cu (I) complexes, even as mixed species. This identifies it as an essential detoxification agent, which is consequently only induced in emergency situations. The cephalochordate MTs are not directly related to vertebrate MTs, neither by gene structure, protein similarity nor metal-binding behavior of the encoded peptides. The closest relative is the echinoderm MT, which confirm proposed phylogenetic relationships between these two groups. The current findings support the existence in most organisms of two types of MTs as for their metal binding preferences, devoted to different biological functions: multivalent MTs for housekeeping roles, and specialized MTs that evolve either as Cd

  13. Changes in metallothionein levels in freshwater mussels exposed to urban wastewaters: effects from exposure to heavy metals?

    PubMed

    Gagné, F; Gagnon, C; Turcotte, P; Blaise, C

    2007-03-29

    Municipal effluents are complex mixtures of compounds such as heavy metals, aromatic and aliphatic hydrocarbons, and micro-organisms and are released in aquatic ecosystems. The purpose of this study was to verify whether changes in metallothioneins (MT) were associated with the accumulation of labile metals in tissue of freshwater mussels exposed to the dispersion plume of a major municipal effluent. Mussels were placed in experimental cages deployed at sites 1.5 km upstream, 8 km downstream and 12 km downstream of the outfall of a major, primary-treated municipal effluent in the St. Lawrence River (Québec, Canada). Mussels were analysed for MT and labile zinc levels in their gonads, gills and digestive glands. Lipogenic enzyme (isocitrate and glucose-6-phosphate dehydrogenase) and arachidonic acid cyclooxygenase (COX) activities were also measured in gonad and gill tissues. Although MT was induced in all the tissues examined, the results showed that labile zinc levels were significantly reduced in gill and gonad tissues, with an increase observed only at the 12 km downstream site in the digestive gland. COX activity was readily induced in gills and gonads. Glucose-6-phosphate dehydrogenase activity was reduced at both downstream sites, but isocitrate dehydrogenase activity was significantly induced at the farthest (12 km) site. Analysis of covariance revealed that MT levels in gills were more influenced by COX activity than with distance in the dispersion plume and was negatively correlated with labile zinc levels. In conclusion, MT induction was inversely related to the levels of labile zinc but positively so with the inflammation biomarker COX. Hence, the induction of MT in mussels exposed to the municipal effluent of a large city appears to be associated with either inflammatory processes or as compensation for the loss of labile essential metals. We propose that the simple and complimentary parameters of labile zinc and COX evaluations be used to link MT

  14. Subcellular partitioning profiles and metallothionein levels in indigenous clams Moerella iridescens from a metal-impacted coastal bay.

    PubMed

    Wang, Zaosheng; Feng, Chenglian; Ye, Chun; Wang, Youshao; Yan, Changzhou; Li, Rui; Yan, Yijun; Chi, Qiaoqiao

    2016-07-01

    In this study, the effect of environmental metal exposure on the accumulation and subcellular distribution of metals in the digestive gland of clams with special emphasis on metallothioneins (MTs) was investigated. Specimens of indigenous Moerella iridescens were collected from different natural habitats in Maluan Bay (China), characterized by varying levels of metal contamination. The digestive glands were excised, homogenized and six subcellular fractions were separated by differential centrifugation procedures and analyzed for their Cu, Zn, Cd and Pb contents. MTs were quantified independently by spectrophotometric measurements of thiols. Site-specific differences were observed in total metal concentrations in the tissues, correlating well with variable environmental metal concentrations and reflecting the gradient trends in metal contamination. Concentrations of the non-essential Cd and Pb were more responsive to environmental exposure gradients than were tissue concentrations of the essential metals, Cu and Zn. Subcellular partitioning profiles for Cu, Zn and Cd were relatively similar, with the heat-stable protein (HSP) fraction as the dominant metal-binding compartment, whereas for Pb this fraction was much less important. The variations in proportions and concentrations of metals in this fraction along with the metal bioaccumulation gradients suggested that the induced MTs play an important role in metal homeostasis and detoxification for M. iridescens in the metal-contaminated bay. Nevertheless, progressive accumulation of non-essential metals (Cd, and especially Pb) resulting from "spillover" was observed in putative metal- sensitive (e.g., mitochondria and heat-denaturable protein (HDP)) or lysosome/microsome fractions, demonstrating that metal detoxification was incomplete and increased the toxicological risk to M. iridescens inhabiting the metal-impacted environments. Through multiple stepwise regression analysis, the induction of MTs was statistically

  15. Plant-in-chip: Microfluidic system for studying root growth and pathogenic interactions in Arabidopsis

    NASA Astrophysics Data System (ADS)

    Parashar, Archana; Pandey, Santosh

    2011-06-01

    We report a microfluidic platform for the hydroponic growth of Arabidopsis plants with high-resolution visualization of root development and root-pathogen interactions. The platform comprises a set of parallel microchannels with individual input/output ports where 1-day old germinated seedlings are initially placed. Under optimum conditions, a root system grows in each microchannel and its images are recorded over a 198-h period. Different concentrations of plant growth media show different root growth characteristics. Later, the developed roots are inoculated with two plant pathogens (nematodes and zoospores) and their physicochemical interactions with the live root systems are observed.

  16. Rice DB: an Oryza Information Portal linking annotation, subcellular location, function, expression, regulation, and evolutionary information for rice and Arabidopsis.

    PubMed

    Narsai, Reena; Devenish, James; Castleden, Ian; Narsai, Kabir; Xu, Lin; Shou, Huixia; Whelan, James

    2013-12-01

    Omics research in Oryza sativa (rice) relies on the use of multiple databases to obtain different types of information to define gene function. We present Rice DB, an Oryza information portal that is a functional genomics database, linking gene loci to comprehensive annotations, expression data and the subcellular location of encoded proteins. Rice DB has been designed to integrate the direct comparison of rice with Arabidopsis (Arabidopsis thaliana), based on orthology or 'expressology', thus using and combining available information from two pre-eminent plant models. To establish Rice DB, gene identifiers (more than 40 types) and annotations from a variety of sources were compiled, functional information based on large-scale and individual studies was manually collated, hundreds of microarrays were analysed to generate expression annotations, and the occurrences of potential functional regulatory motifs in promoter regions were calculated. A range of computational subcellular localization predictions were also run for all putative proteins encoded in the rice genome, and experimentally confirmed protein localizations have been collated, curated and linked to functional studies in rice. A single search box allows anything from gene identifiers (for rice and/or Arabidopsis), motif sequences, subcellular location, to keyword searches to be entered, with the capability of Boolean searches (such as AND/OR). To demonstrate the utility of Rice DB, several examples are presented including a rice mitochondrial proteome, which draws on a variety of sources for subcellular location data within Rice DB. Comparisons of subcellular location, functional annotations, as well as transcript expression in parallel with Arabidopsis reveals examples of conservation between rice and Arabidopsis, using Rice DB (http://ricedb.plantenergy.uwa.edu.au).

  17. Rice DB: an Oryza Information Portal linking annotation, subcellular location, function, expression, regulation, and evolutionary information for rice and Arabidopsis

    PubMed Central

    Narsai, Reena; Devenish, James; Castleden, Ian; Narsai, Kabir; Xu, Lin; Shou, Huixia; Whelan, James

    2013-01-01

    Omics research in Oryza sativa (rice) relies on the use of multiple databases to obtain different types of information to define gene function. We present Rice DB, an Oryza information portal that is a functional genomics database, linking gene loci to comprehensive annotations, expression data and the subcellular location of encoded proteins. Rice DB has been designed to integrate the direct comparison of rice with Arabidopsis (Arabidopsis thaliana), based on orthology or ‘expressology’, thus using and combining available information from two pre-eminent plant models. To establish Rice DB, gene identifiers (more than 40 types) and annotations from a variety of sources were compiled, functional information based on large-scale and individual studies was manually collated, hundreds of microarrays were analysed to generate expression annotations, and the occurrences of potential functional regulatory motifs in promoter regions were calculated. A range of computational subcellular localization predictions were also run for all putative proteins encoded in the rice genome, and experimentally confirmed protein localizations have been collated, curated and linked to functional studies in rice. A single search box allows anything from gene identifiers (for rice and/or Arabidopsis), motif sequences, subcellular location, to keyword searches to be entered, with the capability of Boolean searches (such as AND/OR). To demonstrate the utility of Rice DB, several examples are presented including a rice mitochondrial proteome, which draws on a variety of sources for subcellular location data within Rice DB. Comparisons of subcellular location, functional annotations, as well as transcript expression in parallel with Arabidopsis reveals examples of conservation between rice and Arabidopsis, using Rice DB (http://ricedb.plantenergy.uwa.edu.au). PMID:24147765

  18. Deciphering the responses of root border-like cells of Arabidopsis and flax to pathogen-derived elicitors.

    PubMed

    Plancot, Barbara; Santaella, Catherine; Jaber, Rim; Kiefer-Meyer, Marie Christine; Follet-Gueye, Marie-Laure; Leprince, Jérôme; Gattin, Isabelle; Souc, Céline; Driouich, Azeddine; Vicré-Gibouin, Maïté

    2013-12-01

    Plant pathogens including fungi and bacteria cause many of the most serious crop diseases. The plant innate immune response is triggered upon recognition of microbe-associated molecular patterns (MAMPs) such as flagellin22 and peptidoglycan. To date, very little is known of MAMP-mediated responses in roots. Root border cells are cells that originate from root caps and are released individually into the rhizosphere. Root tips of Arabidopsis (Arabidopsis thaliana) and flax (Linum usitatissimum) release cells known as "border-like cells." Whereas root border cells of pea (Pisum sativum) are clearly involved in defense against fungal pathogens, the function of border-like cells remains to be established. In this study, we have investigated the responses of root border-like cells of Arabidopsis and flax to flagellin22 and peptidoglycan. We found that both MAMPs triggered a rapid oxidative burst in root border-like cells of both species. The production of reactive oxygen species was accompanied by modifications in the cell wall distribution of extensin epitopes. Extensins are hydroxyproline-rich glycoproteins that can be cross linked by hydrogen peroxide to enhance the mechanical strength of the cell wall. In addition, both MAMPs also caused deposition of callose, a well-known marker of MAMP-elicited defense. Furthermore, flagellin22 induced the overexpression of genes involved in the plant immune response in root border-like cells of Arabidopsis. Our findings demonstrate that root border-like cells of flax and Arabidopsis are able to perceive an elicitation and activate defense responses. We also show that cell wall extensin is involved in the innate immunity response of root border-like cells.

  19. The yellow catfish, Pelteobagrus fulvidraco (Siluriformes) metallothionein cDNA: molecular cloning and transcript expression level in response to exposure to the heavy metals Cd, Cu, and Zn.

    PubMed

    Kim, Jin-Hyoung; Rhee, Jae-Sung; Dahms, Hans-Uwe; Lee, Young-Mi; Han, Kyung-Nam; Lee, Jae-Seong

    2012-10-01

    Metallothionein (MT) has been used extensively as a potential molecular biomarker to detect heavy metal pollution in aquatic organisms. In order to investigate the modulation effect of heavy metals and to establish suitable biomarkers for the monitoring of heavy metal pollution, Pelteobagrus fulvidraco metallothionein gene was characterized as the first report in the family Bagridae. Pf-MT transcript was detected at high levels in liver, gonad, kidney, and brain compared to other tissues. A time-course study in response to waterborne Cd (5 ppm) revealed that a significant increase in the Pf-MT transcript abundance was observed at 6 h in gill, kidney, and liver. These elevated levels were kept for 96 h, implying that Cd distributed fast into different organs and was involved in the tissue-specific induction pattern. We observed a significant Pf-MT transcript increase in liver tissues at 48 h, followed by gill at 12 h and intestine at 48 h after Cd exposure. This indicates hepatic MT expression as a potential biomarker of acute Cd exposure in this species. Cd-binding ability of recombinant Pf-MT protein provided evidence for sensitivity to Cd and other heavy metal exposure. In the case of Zn exposure (1 ppm), a significant increase in Pf-MT transcript abundance was observed at 12 h, and a peak induction level reaching sixfold at 24 h was kept until 48 h, showing similar transcript induction patterns with Cd. A high level of Pf-MT mRNA after exposure to Cu (1 ppm) was observed at 12 h that gradually increased until 96 h with a 12-fold induction, revealing a long-lasting induction and somewhat dissimilar pattern compared to other metals in liver. Our results demonstrate that Pf-MT can be induced by heavy metals in a tissue-specific and metal-specific manner and plays probably a conserved role in metal detoxification. This study provides new information on P. fulvidraco metallothionein gene for the use of biomarkers indicating metal pollution in fish.

  20. Live confocal imaging of Arabidopsis flower buds.

    PubMed

    Prunet, Nathanaël; Jack, Thomas P; Meyerowitz, Elliot M

    2016-11-01

    Recent advances in confocal microscopy, coupled with the development of numerous fluorescent reporters, provide us with a powerful tool to study the development of plants. Live confocal imaging has been used extensively to further our understanding of the mechanisms underlying the formation of roots, shoots and leaves. However, it has not been widely applied to flowers, partly because of specific challenges associated with the imaging of flower buds. Here, we describe how to prepare and grow shoot apices of Arabidopsis in vitro, to perform both single-point and time-lapse imaging of live, developing flower buds with either an upright or an inverted confocal microscope.

  1. RNA in situ hybridization in Arabidopsis.

    PubMed

    Wu, Miin-Feng; Wagner, Doris

    2012-01-01

    RNA in situ hybridization using digoxigenin-labeled riboprobes on tissue sections is a powerful technique for revealing microscopic spatial gene expression. Here, we describe an in situ hybridization method commonly practiced in Arabidopsis research labs. The highly stringent hybridization condition eliminates the usage of Ribonlucease A and gives highly specific signals. This also allows the use of longer probes which enhance signal strength without cross hybridization to closely related genes. In addition, using spin columns in template and riboprobe purification greatly reduces background signals.

  2. Effects of vertical rotation on Arabidopsis development

    NASA Technical Reports Server (NTRS)

    Brown, A. H.; Chapman, D. K.; Dahl, A. O.

    1975-01-01

    Various gross morphological end points of Arabidopsis development are examined in an attempt to separate the effects of growth on the horizontal clinostat into a component caused by rotation alone and another component caused by the altered position with respect to the direction of the g-vector. In a series of tests which involved comparisons between vertical stationary plants, vertical rotated plants, and plants rotated on clinostats, certain characters were consistently influenced by vertical rotation alone. The characters for which this effect was statistically significant were petiole length and leaf blade width.

  3. Carotenoid Biosynthesis in Arabidopsis: A Colorful Pathway

    PubMed Central

    Ruiz-Sola, M. Águila; Rodríguez-Concepción, Manuel

    2012-01-01

    Plant carotenoids are a family of pigments that participate in light harvesting and are essential for photoprotection against excess light. Furthermore, they act as precursors for the production of apocarotenoid hormones such as abscisic acid and strigolactones. In this review, we summarize the current knowledge on the genes and enzymes of the carotenoid biosynthetic pathway (which is now almost completely elucidated) and on the regulation of carotenoid biosynthesis at both transcriptional and post-transcriptional levels. We also discuss the relevance of Arabidopsis as a model system for the study of carotenogenesis and how metabolic engineering approaches in this plant have taught important lessons for carotenoid biotechnology. PMID:22582030

  4. Linkage maps for Arabidopsis lyrata subsp. lyrata and Arabidopsis lyrata subsp. petraea combining anonymous and Arabidopsis thaliana-derived markers.

    PubMed

    Beaulieu, Julien; Jean, Martine; Belzile, François

    2007-02-01

    Arabidopsis lyrata, a close relative of the model plant Arabidopsis thaliana, is 1 of a few plant species for which the genome is to be entirely sequenced, which promises to yield important insights into genome evolution. Only 2 sparse linkage maps have been published, and these were based solely on markers derived from the A. thaliana genome. Because the genome of A. lyrata is practically twice as large as that of A. thaliana, the extent of map coverage of the A. lyrata genome remains uncertain. In this study, a 2-way pseudo-testcross strategy was used to construct genetic linkage maps of A. lyrata subsp. petraea and A. lyrata subsp. lyrata, using simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) markers from the A. thaliana genome, and anonymous amplified fragment length polymorphism (AFLP) markers that could potentially uncover regions unique to the A. lyrata genome. The SSR and CAPS markers largely confirmed the relationships between linkage groups in A. lyrata and A. thaliana. AFLP markers slightly increased the coverage of the A. lyrata maps, but mostly increased marker density on the linkage groups. We noted a much lower level of polymorphism and a greater segregation distortion in A. lyrata subsp. lyrata markers. The implications of these findings for the sequencing of the A. lyrata genome are discussed.

  5. Genetic architecture of nonadditive inheritance in Arabidopsis thaliana hybrids

    PubMed Central

    Seymour, Danelle K.; Chae, Eunyoung; Grimm, Dominik G.; Martín Pizarro, Carmen; Habring-Müller, Anette; Vasseur, François; Rakitsch, Barbara; Borgwardt, Karsten M.

    2016-01-01

    The ubiquity of nonparental hybrid phenotypes, such as hybrid vigor and hybrid inferiority, has interested biologists for over a century and is of considerable agricultural importance. Although examples of both phenomena have been subject to intense investigation, no general model for the molecular basis of nonadditive genetic variance has emerged, and prediction of hybrid phenotypes from parental information continues to be a challenge. Here we explore the genetics of hybrid phenotype in 435 Arabidopsis thaliana individuals derived from intercrosses of 30 parents in a half diallel mating scheme. We find that nonadditive genetic effects are a major component of genetic variation in this population and that the genetic basis of hybrid phenotype can be mapped using genome-wide association (GWA) techniques. Significant loci together can explain as much as 20% of phenotypic variation in the surveyed population and include examples that have both classical dominant and overdominant effects. One candidate region inherited dominantly in the half diallel contains the gene for the MADS-box transcription factor AGAMOUS-LIKE 50 (AGL50), which we show directly to alter flowering time in the predicted manner. Our study not only illustrates the promise of GWA approaches to dissect the genetic architecture underpinning hybrid performance but also demonstrates the contribution of classical dominance to genetic variance. PMID:27803326

  6. Temporal heterogeneity of cold acclimation phenotypes in Arabidopsis leaves.

    PubMed

    Gorsuch, Peter A; Pandey, Subedar; Atkin, Owen K

    2010-02-01

    To predict the effects of temperature changes on plant growth and performance, it is crucial to understand the impact of thermal history on leaf morphology, anatomy and physiology. Here, we document a comprehensive range of leaf phenotypes in 25/20 degrees C-grown Arabidopsis thaliana plants that were shifted to 5 degrees C for up to 2 months. When warm-grown, pre-existing (PE) leaves were exposed to cold, leaf thickness increased due to an increase in mesophyll cell size. Leaves that were entirely cold-developed (CD) were twice as thick (eight cell layers) as their warm-developed (WD) counterparts (six layers), and also had higher epidermal and stomatal cell densities. After 4 d of cold, PE leaves accumulated high levels of total non-structural carbohydrates (TNC). However, glucose and starch levels declined thereafter, and after 45 d in the cold, PE leaves exhibited similar TNC to CD leaves. A similar phenomenon was observed in delta(13)C and a range of photosynthetic parameters. In cold-treated PE leaves, an increase in respiration (R(dark)) with cold exposure time was evident when measured at 25 degrees C but not 5 degrees C. Cold acclimation was associated with a large increase in the ratio of leaf R(dark) to photosynthesis. The data highlight the importance of understanding developmental thermal history in determining individual phenotypic traits.

  7. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes.

    PubMed

    Riechmann, J L; Heard, J; Martin, G; Reuber, L; Jiang, C; Keddie, J; Adam, L; Pineda, O; Ratcliffe, O J; Samaha, R R; Creelman, R; Pilgrim, M; Broun, P; Zhang, J Z; Ghandehari, D; Sherman, B K; Yu, G

    2000-12-15

    The completion of the Arabidopsis thaliana genome sequence allows a comparative analysis of transcriptional regulators across the three eukaryotic kingdoms. Arabidopsis dedicates over 5% of its genome to code for more than 1500 transcription factors, about 45% of which are from families specific to plants. Arabidopsis transcription factors that belong to families common to all eukaryotes do not share significant similarity with those of the other kingdoms beyond the conserved DNA binding domains, many of which have been arranged in combinations specific to each lineage. The genome-wide comparison reveals the evolutionary generation of diversity in the regulation of transcription.

  8. Transcription Analysis of Arabidopsis Membrane Transporters and Hormone Pathways during Developmental and Induced Leaf Senescence1[W

    PubMed Central

    van der Graaff, Eric; Schwacke, Rainer; Schneider, Anja; Desimone, Marcelo; Flügge, Ulf-Ingo; Kunze, Reinhard

    2006-01-01

    A comparative transcriptome analysis for successive stages of Arabidopsis (Arabidopsis thaliana) developmental leaf senescence (NS), darkening-induced senescence of individual leaves attached to the plant (DIS), and senescence in dark-incubated detached leaves (DET) revealed many novel senescence-associated genes with distinct expression profiles. The three senescence processes share a high number of regulated genes, although the overall number of regulated genes during DIS and DET is about 2 times lower than during NS. Consequently, the number of NS-specific genes is much higher than the number of DIS- or DET-specific genes. The expression profiles of transporters (TPs), receptor-like kinases, autophagy genes, and hormone pathways were analyzed in detail. The Arabidopsis TPs and other integral membrane proteins were systematically reclassified based on the Transporter Classification system. Coordinate activation or inactivation of several genes is observed in some TP families in all three or only in individual senescence types, indicating differences in the genetic programs for remobilization of catabolites. Characteristic senescence type-specific differences were also apparent in the expression profiles of (putative) signaling kinases. For eight hormones, the expression of biosynthesis, metabolism, signaling, and (partially) response genes was investigated. In most pathways, novel senescence-associated genes were identified. The expression profiles of hormone homeostasis and signaling genes reveal additional players in the senescence regulatory network. PMID:16603661

  9. Effects of dietary copper supplementation of rats on the occurrence of metallothionein-I in liver and its secretion into blood, bile and urine.

    PubMed Central

    Bremner, I; Mehra, R K; Morrison, J N; Wood, A M

    1986-01-01

    The appearance and excretion of metallothionein-I (MT-I) was studied in rats given a diet containing 1000 mg of Cu/kg for several weeks. No significant increase in MT-I concentrations in liver, plasma or bile was detected in rats with liver copper concentrations less than 600 micrograms of Cu/g fresh wt. Above this concentration, liver MT-I concentrations increased in proportion to the increase in hepatic copper content. Plasma and bile MT-I concentrations were directly related to those in the liver and were about 10 times those in normal rats. Urinary MT-I concentration also increased 10-fold within 1 week. Fractionation of bile and urine on Sephadex G-50 revealed the presence of monomeric MT-I and a range of possible degradation products of the isoprotein. PMID:3753441

  10. A baseline study of the metallothioneins induction and its reversibility in Neohelice granulata from the Bahía Blanca Estuary (Argentina).

    PubMed

    Buzzi, N S; Marcovecchio, J E

    2016-11-15

    Contamination by heavy metals causes serious effects in marine systems. Nowadays, the combination of chemical and biological data is recommended in monitoring programs. Metallothioneins (MT) are early-warning signals of metal exposure and are widely used in biomonitoring. The present research evaluates the heavy metals levels in sediments and the MT synthesis in the crab Neohelice granulata from the Bahía Blanca Estuary (BBE). Then, the recovery capabilities of N. granulata followed by a depuration phase are assessed. Results demonstrate a slow decline in the level of metals in the study area. In relation to MT, female crabs showed elevated MT when compared to males. Furthermore, MT synthesis diminished after the depuration phase. These data constitute the first MT information in organisms from the BBE and may be considered as baseline for future studies in this matter. Although pollution level found was low, further biomonitoring considering both types of data is recommended.

  11. The immune responses and expression of metallothionein (MT) gene and heat shock protein 70 (HSP 70) in juvenile rockfish, Sebastes schlegelii, exposed to waterborne arsenic (As(3+)).

    PubMed

    Kim, Jun-Hwan; Kang, Ju-Chan

    2016-10-01

    Juvenile rockfish, Sebastes schlegelii (mean length 16.4±1.9cm, and mean weight 71.6±6.4g) were exposed for 20days with the different levels of waterborne arsenic concentration (0, 50, 100, 200 and 400μg/L). The plasma cortisol of S. schlegelii was significantly increased by the waterborne arsenit exposure. In the immune responses, the immunoglobulin M (Ig M) and lysozyme activity of S. schlegelii were significantly increased by the waterborne arsenic exposure. The acetylcholinesterase (AChE) activity of S. schlegelii was inhibited by the waterborne arsenic exposure. The substantial increases in the gene expression such as metallothionein (MT) and heat shock protein 70 (HSP 70) were observed by the waterborne arsenic exposure. The results demonstrated that waterborne arsenic exposure can induce the significant alterations in the immune responses and specific gene expression of S. schlegelii.

  12. Changes in abundance of an abscisic acid-responsive, early cysteine-labeled metallothionein transcript during pollen embryogenesis in bread wheat (Triticum aestivum).

    PubMed

    Reynolds, T L; Crawford, R L

    1996-12-01

    A clone for an embryoid-abundant, early cysteine-labeled metallothionein (EcMt) gene has been isolated from a wheat pollen embryoid cDNA library. The transcript of this gene was only expressed in embryogenic microspores, pollen embryoids, and developing zygotic embryos of wheat. Accumulation of the EcMt mRNA showed a direct and positive correlation with an increase of the plant hormone, abscisic acid (ABA) in developing pollen embryoids. Treating cultures with an inhibitor of ABA biosynthesis, fluridone, suppressed not only ABA accumulation but also the appearance of the EcMt gene transcript and the ability of microspores to form embryoids. These results suggest that the EcMt gene may act as a molecular marker for pollen embryogenesis because ABA biosynthesis is accompanied by the increased expression of the EcMt transcript that coincides with the differentiation of pollen embryoids in wheat anther cultures.

  13. Effects of low protein diet and sex difference on the amounts of metallothionein in liver and kidney of cadmium-administered rats

    SciTech Connect

    Ninomiya, R.; Inoue, Y.; Koizumi, N.; Tsukamoto, T.

    1985-01-01

    Cadmium (Cd) was orally administered in a dose of 100 ..mu..g daily a total of 100 times to investigate the effects of the intake of low (5%) protein diet and sex difference on the amounts of metallothionein (MT) in the liver and kidney. The amount of MT in the liver was significantly increased by the intake of low protein diet. In females, the increase in the amount of MT was proportional to the amount of Cd accumulated. The concentration of copper-thionein in the liver was higher in females than in males and further increased after intake of low protein diet. The levels of MT, cadmium-thionein, zinc-thionein and copper-thionein in the kidney were not influenced by the intake of low protein diet nor did show a sex difference.

  14. Evaluation of induction of metallothionein-like proteins (MTLPs) in the polychaetes for biomonitoring of heavy metal pollution in marine sediments.

    PubMed

    Won, Eun-Ji; Raisuddin, Sheikh; Shin, Kyung-Hoon

    2008-01-01

    Polychaetes are suitable organisms for evaluation of impact of sediment pollution. We evaluated toxicity of cadmium and copper and measured metallothionein-like proteins (MTLPs) in the polychaete Perinereis nuntia. At the same concentration ranges copper was unexpectedly more toxic than cadmium. Copper also caused no significant increase in MTLPs in the polychaetes. When P. nuntia and another polychaete species, Cirratulus cirratus were cultured in the contaminated sediments collected from Lake Sihwa (Korea), a high mortality of 80% was observed on day 6 in P. nuntia in the sediment with the highest metal concentration. However, no mortality was observed up to 35 days in C. cirratus in any sediment. MTLP contents between two species also varied. These findings suggest that MTLP induction response in the polychaetes varies with the metal type and species and it may be used as a biomarker of sediment pollution in the polychaetes after further validation and field trials.

  15. Heavy metal content in liver and kidneys of grey seals (Halichoerus grypus) in various life stages correlated with metallothionein levels: Some metal-binding characteristics of this protein

    SciTech Connect

    Teigen, S.W.; Andersen, R.A.; Daae, H.L.; Skaare, J.U.

    1999-10-01

    Liver and kidneys from grey seals (Halichoerus grypus) in their natural habitats in Norwegian waters contain high levels of Cd and Hg as well as metallothionein (MT). The data show significant placental transfer of heavy metals in seals. The MT concentrations were higher in fetus liver and kidneys than in the liver and kidneys of juveniles and adults. A significantly higher proportion of the total Cd and Cu, as compared to Zn and Hg, in fetal liver was associated with MT. Only low amounts of metals and MT were found in juveniles. In kidneys, little metal was associated with MT, although high amounts of Cd were bound in fetal stages. Positive correlations were found between total metal, MT content, and MT-bound metals, particularly for Cd and Zn. Grey seal MT may have higher affinity to Zn than to Cd. The use of seal MT as a biomarker for environmental heavy metal exposure seems promising.

  16. Requirement of proline synthesis during Arabidopsis reproductive development

    PubMed Central

    2012-01-01

    Background Gamete and embryo development are crucial for successful reproduction and seed set in plants, which is often the determining factor for crop yield. Proline accumulation was largely viewed as a specific reaction to overcome stress conditions, while recent studies suggested important functions of proline metabolism also in reproductive development. Both the level of free proline and proline metabolism were proposed to influence the transition to flowering, as well as pollen and embryo development. Results In this study, we performed a detailed analysis of the contribution of individual proline biosynthetic enzymes to vegetative development and reproductive success in Arabidopsis. In contrast to previous reports, we found that pyrroline-5-carboxylate (P5C) synthetase 2 (P5CS2) is not essential for sexual reproduction although p5cs2 mutant plants were retarded in vegetative development and displayed reduced fertility under long-day conditions. Single mutant plants devoid of P5CS1 did not show any developmental defects. Simultaneous absence of both P5CS isoforms resulted in pollen sterility, while fertile egg cells could still be produced. Expression of P5C reductase (P5CR) was indispensable for embryo development but surprisingly not needed for pollen or egg cell fertility. The latter observation could be explained by an extreme stability of P5CR activity, which had a half-life time of greater than 3 weeks in vitro. Expression of P5CR-GFP under the control of the endogenous P5CR promoter was able to restore growth of homozygous p5cr mutant embryos. The analysis of P5CR-GFP-fluorescence in planta supported an exclusively cytoplasmatic localisation of P5CR. Conclusions Our results demonstrate that potential alternative pathways for proline synthesis or inter-generation transfer of proline are not sufficient to overcome a defect in proline biosynthesis from glutamate during pollen development. Proline biosynthesis through P5CS2 and P5CR is limiting for

  17. Arabidopsis MET1 cytosine methyltransferase mutants.

    PubMed Central

    Kankel, Mark W; Ramsey, Douglas E; Stokes, Trevor L; Flowers, Susan K; Haag, Jeremy R; Jeddeloh, Jeffrey A; Riddle, Nicole C; Verbsky, Michelle L; Richards, Eric J

    2003-01-01

    We describe the isolation and characterization of two missense mutations in the cytosine-DNA-methyltransferase gene, MET1, from the flowering plant Arabidopsis thaliana. Both missense mutations, which affect the catalytic domain of the protein, led to a global reduction of cytosine methylation throughout the genome. Surprisingly, the met1-2 allele, with the weaker DNA hypomethylation phenotype, alters a well-conserved residue in methyltransferase signature motif I. The stronger met1-1 allele caused late flowering and a heterochronic delay in the juvenile-to-adult rosette leaf transition. The distribution of late-flowering phenotypes in a mapping population segregating met1-1 indicates that the flowering-time phenotype is caused by the accumulation of inherited defects at loci unlinked to the met1 mutation. The delay in flowering time is due in part to the formation and inheritance of hypomethylated fwa epialleles, but inherited defects at other loci are likely to contribute as well. Centromeric repeat arrays hypomethylated in met1-1 mutants are partially remethylated when introduced into a wild-type background, in contrast to genomic sequences hypomethylated in ddm1 mutants. ddm1 met1 double mutants were constructed to further our understanding of the mechanism of DDM1 action and the interaction between two major genetic loci affecting global cytosine methylation levels in Arabidopsis. PMID:12663548

  18. Diuretics prime plant immunity in Arabidopsis thaliana.

    PubMed

    Noutoshi, Yoshiteru; Ikeda, Mika; Shirasu, Ken

    2012-01-01

    Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application.

  19. Epigenetic natural variation in Arabidopsis thaliana.

    PubMed

    Vaughn, Matthew W; Tanurdzić, Milos; Lippman, Zachary; Jiang, Hongmei; Carrasquillo, Robert; Rabinowicz, Pablo D; Dedhia, Neilay; McCombie, W Richard; Agier, Nicolas; Bulski, Agnès; Colot, Vincent; Doerge, R W; Martienssen, Robert A

    2007-07-01

    Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F(2) families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means.

  20. Epigenetic Natural Variation in Arabidopsis thaliana

    PubMed Central

    Jiang, Hongmei; Carrasquillo, Robert; Rabinowicz, Pablo D; Dedhia, Neilay; McCombie, W. Richard; Agier, Nicolas; Bulski, Agnès; Colot, Vincent; Doerge, R.W; Martienssen, Robert A

    2007-01-01

    Cytosine methylation of repetitive sequences is widespread in plant genomes, occurring in both symmetric (CpG and CpNpG) as well as asymmetric sequence contexts. We used the methylation-dependent restriction enzyme McrBC to profile methylated DNA using tiling microarrays of Arabidopsis Chromosome 4 in two distinct ecotypes, Columbia and Landsberg erecta. We also used comparative genome hybridization to profile copy number polymorphisms. Repeated sequences and transposable elements (TEs), especially long terminal repeat retrotransposons, are densely methylated, but one third of genes also have low but detectable methylation in their transcribed regions. While TEs are almost always methylated, genic methylation is highly polymorphic, with half of all methylated genes being methylated in only one of the two ecotypes. A survey of loci in 96 Arabidopsis accessions revealed a similar degree of methylation polymorphism. Within-gene methylation is heritable, but is lost at a high frequency in segregating F 2 families. Promoter methylation is rare, and gene expression is not generally affected by differences in DNA methylation. Small interfering RNA are preferentially associated with methylated TEs, but not with methylated genes, indicating that most genic methylation is not guided by small interfering RNA. This may account for the instability of gene methylation, if occasional failure of maintenance methylation cannot be restored by other means. PMID:17579518

  1. Epigenetic regulation of gene responsiveness in Arabidopsis

    PubMed Central

    To, Taiko K.; Kim, Jong Myong

    2014-01-01

    The regulation of chromatin structure is inevitable for proper transcriptional response in eukaryotes. Recent reports in Arabidopsis have suggested that gene responsiveness is modulated by particular chromatin status. One such feature is H2A.Z, a histone variant conserved among eukaryotes. In Arabidopsis, H2A.Z is enriched within gene bodies of transcriptionally variable genes, which is in contrast to genic DNA methylation found within constitutive genes. In the absence of H2A.Z, the genes normally harboring H2A.Z within gene bodies are transcriptionally misregulated, while DNA methylation is unaffected. Therefore, H2A.Z may promote variability of gene expression without affecting genic DNA methylation. Another epigenetic information that could be important for gene responsiveness is trimethylation of histone H3 lysine 4 (H3K4me3). The level of H3K4me3 increases when stress responsive genes are transcriptionally activated, and it decreases after recovery from the stress. Even after the recovery, however, H3K4me3 is kept at some atypical levels, suggesting possible role of H3K4me3 for a stress memory. In this review, we summarize and discuss the growing evidences connecting chromatin features and gene responsiveness. PMID:24432027

  2. MTHFD1 controls DNA methylation in Arabidopsis

    PubMed Central

    Groth, Martin; Moissiard, Guillaume; Wirtz, Markus; Wang, Haifeng; Garcia-Salinas, Carolina; Ramos-Parra, Perla A.; Bischof, Sylvain; Feng, Suhua; Cokus, Shawn J.; John, Amala; Smith, Danielle C.; Zhai, Jixian; Hale, Christopher J.; Long, Jeff A.; Hell, Ruediger; Díaz de la Garza, Rocío I.; Jacobsen, Steven E.

    2016-01-01

    DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases. PMID:27291711

  3. Local Evolution of Seed Flotation in Arabidopsis

    PubMed Central

    Saez-Aguayo, Susana; Rondeau-Mouro, Corinne; Macquet, Audrey; Kronholm, Ilkka; Ralet, Marie-Christine; Berger, Adeline; Sallé, Christine; Poulain, Damien; Granier, Fabienne; Botran, Lucy; Loudet, Olivier; de Meaux, Juliette; Marion-Poll, Annie; North, Helen M.

    2014-01-01

    Arabidopsis seeds rapidly release hydrophilic polysaccharides from the seed coat on imbibition. These form a heavy mucilage layer around the seed that makes it sink in water. Fourteen natural Arabidopsis variants from central Asia and Scandinavia were identified with seeds that have modified mucilage release and float. Four of these have a novel mucilage phenotype with almost none of the released mucilage adhering to the seed and the absence of cellulose microfibrils. Mucilage release was modified in the variants by ten independent causal mutations in four different loci. Seven distinct mutations affected one locus, coding the MUM2 β-D-galactosidase, and represent a striking example of allelic heterogeneity. The modification of mucilage release has thus evolved a number of times independently in two restricted geographical zones. All the natural mutants identified still accumulated mucilage polysaccharides in seed coat epidermal cells. Using nuclear magnetic resonance (NMR) relaxometry their production and retention was shown to reduce water mobility into internal seed tissues during imbibition, which would help to maintain seed buoyancy. Surprisingly, despite released mucilage being an excellent hydrogel it did not increase the rate of water uptake by internal seed tissues and is more likely to play a role in retaining water around the seed. PMID:24625826

  4. Brassinosteroid functions in Arabidopsis seed development

    PubMed Central

    Jiang, Wen-Bo; Lin, Wen-Hui

    2013-01-01

    Seed development of flowering plant is a complicated process controlled by a signal network. Double fertilization generates 2 zygotic products (embryo and endosperm). Embryo gives rise to a daughter plant while endosperm provides nutrients for embryo during embryogenesis and germination. Seed coat differentiates from maternally derived integument and encloses embryo and endosperm. Seed size/mass and number comprise final seed yield, and seed shape also contributes to seed development and weight. Seed size is coordinated by communication among endosperm, embryo, and integument. Seed number determination is more complex to investigate and shows differencies between monocot and eudicot. Total seed number depends on sillique number and seed number per sillique in Arabidopsis. Seed comes from fertilized ovule, hence the ovule number per flower determines the maximal seed number per sillique. Early studies reported that engineering BR levels increased the yield of ovule and seed; however the molecular mechanism of BR regulation in seed development still remained unclear. Our recent studies demonstrated that BR regulated seed size, shape, and number by transcriptionally modulating specific seed developmental pathways. This review summarizes roles of BR in Arabidopsis seed development and gives clues for future application of BR in agricultural production. PMID:24270689

  5. Local evolution of seed flotation in Arabidopsis.

    PubMed

    Saez-Aguayo, Susana; Rondeau-Mouro, Corinne; Macquet, Audrey; Kronholm, Ilkka; Ralet, Marie-Christine; Berger, Adeline; Sallé, Christine; Poulain, Damien; Granier, Fabienne; Botran, Lucy; Loudet, Olivier; de Meaux, Juliette; Marion-Poll, Annie; North, Helen M

    2014-03-01

    Arabidopsis seeds rapidly release hydrophilic polysaccharides from the seed coat on imbibition. These form a heavy mucilage layer around the seed that makes it sink in water. Fourteen natural Arabidopsis variants from central Asia and Scandinavia were identified with seeds that have modified mucilage release and float. Four of these have a novel mucilage phenotype with almost none of the released mucilage adhering to the seed and the absence of cellulose microfibrils. Mucilage release was modified in the variants by ten independent causal mutations in four different loci. Seven distinct mutations affected one locus, coding the MUM2 β-D-galactosidase, and represent a striking example of allelic heterogeneity. The modification of mucilage release has thus evolved a number of times independently in two restricted geographical zones. All the natural mutants identified still accumulated mucilage polysaccharides in seed coat epidermal cells. Using nuclear magnetic resonance (NMR) relaxometry their production and retention was shown to reduce water mobility into internal seed tissues during imbibition, which would help to maintain seed buoyancy. Surprisingly, despite released mucilage being an excellent hydrogel it did not increase the rate of water uptake by internal seed tissues and is more likely to play a role in retaining water around the seed.

  6. Polyploidization increases meiotic recombination frequency in Arabidopsis

    PubMed Central

    2011-01-01

    Background Polyploidization is the multiplication of the whole chromosome complement and has occurred frequently in vascular plants. Maintenance of stable polyploid state over generations requires special mechanisms to control pairing and distribution of more than two homologous chromosomes during meiosis. Since a minimal number of crossover events is essential for correct chromosome segregation, we investigated whether polyploidy has an influence on the frequency of meiotic recombination. Results Using two genetically linked transgenes providing seed-specific fluorescence, we compared a high number of progeny from diploid and tetraploid Arabidopsis plants. We show that rates of meiotic recombination in reciprocal crosses of genetically identical diploid and autotetraploid Arabidopsis plants were significantly higher in tetraploids compared to diploids. Although male and female gametogenesis differ substantially in meiotic recombination frequency, both rates were equally increased in tetraploids. To investigate whether multivalent formation in autotetraploids was responsible for the increased recombination rates, we also performed corresponding experiments with allotetraploid plants showing strict bivalent pairing. We found similarly increased rates in auto- and allotetraploids, suggesting that the ploidy effect is independent of chromosome pairing configurations. Conclusions The evolutionary success of polyploid plants in nature and under domestication has been attributed to buffering of mutations and sub- and neo-functionalization of duplicated genes. Should the data described here be representative for polyploid plants, enhanced meiotic recombination, and the resulting rapid creation of genetic diversity, could have also contributed to their prevalence. PMID:21510849

  7. Defining the core Arabidopsis thaliana root microbiome

    PubMed Central

    Gehring, Jase; Malfatti, Stephanie; Tremblay, Julien; Engelbrektson, Anna; Kunin, Victor; del Rio, Tijana Glavina; Edgar, Robert C.; Eickhorst, Thilo; Ley, Ruth E.; Hugenholtz, Philip; Tringe, Susannah Green; Dangl, Jeffery L.

    2014-01-01

    Land plants associate with a root microbiota distinct from the complex microbial community present in surrounding soil. The microbiota colonizing therhizosphere(immediately surroundingthe root) and the endophytic compartment (within the root) contribute to plant growth, productivity, carbon sequestration and phytoremediation1-3. Colonization of the root occurs despite a sophisticated plant immune system4,5, suggesting finely tuned discrimination of mutualists and commensals from pathogens. Genetic principles governing the derivation of host-specific endophyte communities from soil communities are poorly understood. Here we report the pyrosequencing of the bacterial 16S ribosomal RNA gene of more than 600 Arabidopsis thaliana plants to test the hypotheses that the root rhizosphere and endophytic compartment microbiota of plants grown under controlled conditions in natural soils are sufficiently dependent on the host to remain consistent across different soil types and developmental stages, and sufficiently dependent on host genotype to vary between inbred Arabidopsis accessions. We describe different bacterial communities in two geochemically distinct bulk soils and in rhizosphere and endophytic compartments prepared from roots grown in these soils. The communities in each compartment are strongly influenced by soil type. Endophytic compartments from both soils feature overlapping, low-complexity communities that are markedly enriched in Actinobacteria and specific families from other phyla, notably Proteobacteria. Some bacteria vary quantitatively between plants of different developmental stage and genotype. Our rigorous definition of an endophytic compartment microbiome should facilitate controlled dissection of plantmicrobe interactions derived from complex soil communities. PMID:22859206

  8. Photoperiodic flowering regulation in Arabidopsis thaliana

    PubMed Central

    Golembeski, Greg S.; Kinmonth-Schultz, Hannah A.; Song, Young Hun; Imaizumi, Takato

    2015-01-01

    Photoperiod, or the duration of light in a given day, is a critical cue that flowering plants utilize to effectively assess seasonal information and coordinate their reproductive development in synchrony with the external environment. The use of the model plant, Arabidopsis thaliana, has greatly improved our understanding of the molecular mechanisms that determine how plants process and utilize photoperiodic information to coordinate a flowering response. This mechanism is typified by the transcriptional activation of FLOWERING LOCUS T (FT) gene by the transcription factor CONSTANS (CO) under inductive long-day conditions in Arabidopsis. FT protein then moves from the leaves to the shoot apex, where floral meristem development can be initiated. As a point of integration from a variety of environmental factors in the context of a larger system of regulatory pathways that affect flowering, the importance of photoreceptors and the circadian clock in CO regulation throughout the day has been a key feature of the photoperiodic flowering pathway. In addition to these established mechanisms, the recent discovery of a photosynthate derivative trehalose-6-phosphate as an activator of FT in leaves has interesting implications for the involvement of photosynthesis in the photoperiodic flowering response that were suggested from previous physiological experiments in flowering induction. PMID:25684830

  9. Zinc rescues obesity-induced cardiac hypertrophy via stimulating metallothionein to suppress oxidative stress-activated BCL10/CARD9/p38 MAPK pathway.

    PubMed

    Wang, Shudong; Gu, Junlian; Xu, Zheng; Zhang, Zhiguo; Bai, Tao; Xu, Jianxiang; Cai, Jun; Barnes, Gregory; Liu, Qiu-Ju; Freedman, Jonathan H; Wang, Yonggang; Liu, Quan; Zheng, Yang; Cai, Lu

    2017-02-03

    Obesity often leads to obesity-related cardiac hypertrophy (ORCH), which is suppressed by zinc-induced inactivation of p38 mitogen-activated protein kinase (p38 MAPK). In this study, we investigated the mechanisms by which zinc inactivates p38 MAPK to prevent ORCH. Mice (4-week old) were fed either high fat diet (HFD, 60% kcal fat) or normal diet (ND, 10% kcal fat) containing variable amounts of zinc (deficiency, normal and supplement) for 3 and 6 months. P38 MAPK siRNA and the p38 MAPK inhibitor SB203580 were used to suppress p38 MAPK activity in vitro and in vivo, respectively. HFD activated p38 MAPK and increased expression of B-cell lymphoma/CLL 10 (BCL10) and caspase recruitment domain family member 9 (CARD9). These responses were enhanced by zinc deficiency and attenuated by zinc supplement. Administration of SB203580 to HFD mice or specific siRNA in palmitate-treated cardiomyocytes eliminated the HFD and zinc deficiency activation of p38 MAPK, but did not significantly impact the expression of BCL10 and CARD9. In cultured cardiomyocytes, inhibition of BCL10 expression by siRNA prevented palmitate-induced increased p38 MAPK activation and atrial natriuretic peptide (ANP) expression. In contrast, inhibition of p38 MAPK prevented ANP expression, but did not affect BCL10 expression. Deletion of metallothionein abolished the protective effect of zinc on palmitate-induced up-regulation of BCL10 and phospho-p38 MAPK. HFD and zinc deficiency synergistically induce ORCH by increasing oxidative stress-mediated activation of BCL10/CARD9/p38 MAPK signalling. Zinc supplement ameliorates ORCH through activation of metallothionein to repress oxidative stress-activated BCL10 expression and p38 MAPK activation.

  10. Rhenium and technetium tricarbonyl, {M(CO)3} (+) (M = Tc, Re), binding to mammalian metallothioneins: new insights into chemical and radiopharmaceutical implications.

    PubMed

    Lecina, Joan; Palacios, Òscar; Atrian, Sílvia; Capdevila, Mercè; Suades, Joan

    2015-04-01

    This paper deals with the binding of the four mammalian metallothioneins (MTs) to the organometallic metal fragment {fac-M(CO)3}(+) (M = (99)Tc, Re), which is highly promising for the preparation of second-generation radiopharmaceuticals. The study of the transmetallation reaction between zinc and rhenium in Zn7-MT1 by means of UV-vis and CD spectroscopy demonstrated the incorporation of the {fac-Re(CO)3}(+) fragment to the MTs. This reaction should be performed at 70 °C to accelerate the reaction rate, a result that is consistent with the reported reactivity of the rhenium fragment. ESI-TOF MS demonstrated the formation of mixed-metal species as Zn6,{Re(CO)3}-MT, Zn6,{Re(CO)3}2-MT, and Zn5,{Re(CO)3}3-MT, as well as the different reactivity of the four MT isoforms. Hence, Zn-MT3 showed the highest reactivity, in agreement with its high Cu-thionein character, whereas Zn-MT2 exhibited the lowest reactivity, in line with its high Zn-thionein character. The reactivity of the Zn-loaded forms of MT1 and MT4 is intermediate between those of MT3 and MT2. The study of the binding of the {fac-(99)Tc(CO)3}(+) fragment to MTs showed a significant and very interesting different reactivity in relation to rhenium. The transmetallation reaction is much more effective with technetium than with rhenium and significant amounts of mixed Zn x ,{(99)Tc(CO)3} y -MT species were formed with the four MT isoforms whereas only MT3 rendered similar amounts of rhenium derivatives. The results obtained in this study support the possible use of technetium for labelling mammalian metallothioneins and also for possible radiopharmaceutical applications.

  11. Cloning and characterization of HbMT2a, a metallothionein gene from Hevea brasiliensis Muell. Arg differently responds to abiotic stress and heavy metals

    SciTech Connect

    Li, Yan; Chen, Yue Yi; Yang, Shu Guang; Tian, Wei Min

    2015-05-22

    Metallothioneins (MTs) are of low molecular mass, cysteine-rich proteins. They play an important role in the detoxification of heavy metals and homeostasis of intracellular metal ions, and protecting against intracellular oxidative damages. In this study a full-length cDNA of type 2 plant metallothioneins, HbMT2a, was isolated from 25 mM Polyethyleneglycol (PEG) stressed leaves of Hevea brasiliensis by RACE. The HbMT2a was 372 bp in length and had a 237 bp open reading frame (ORF) encoding for a protein of 78 amino acid residues with molecular mass of 7.772 kDa. The expression of HbMT2a in the detached leaves of rubber tree clone RY7-33-97 was up-regulated by Me-JA, ABA, PEG, H{sub 2}O{sub 2}, Cu{sup 2+} and Zn{sup 2+}, but down-regulated by water. The role of HbMT2a protein in protecting against metal toxicity was demonstrated in vitro. PET-28a-HbMT2-beared Escherichia coli. Differential expression of HbMT2a upon treatment with 10 °C was observed in the detached leaves of rubber tree clone 93-114 which is cold-resistant and Reken501 which is cold-sensitive. The expression patterns of HbMT2a in the two rubber tree clones may be ascribed to a change in the level of endogenous H{sub 2}O{sub 2}. - Highlights: • Cloning an HbMT2a gene from rubber tree. • Analyzing expression patterns of HbMT2a upon abiotic stress and heavy metal stress. • Finding different expression patterns of HbMT2a among two Hevea germplasm. • The expressed protein of HbMT2a enhances copper and zinc tolerance in Escherichia coli.

  12. Involvement of DNA hypermethylation in down-regulation of the zinc transporter ZIP8 in cadmium-resistant metallothionein-null cells

    SciTech Connect

    Fujishiro, Hitomi; Okugaki, Satomi; Yasumitsu, Saori; Enomoto, Shuichi; Himeno, Seiichiro

    2009-12-01

    The Zrt/Irt-related protein 8 (ZIP8) encoded by slc39a8 is now emerging as an important zinc transporter involved in cellular cadmium incorporation. We have previously shown that mRNA and protein levels of ZIP8 were decreased in cadmium-resistant metallothionein-null (A7) cells, leading to a decrease in cadmium accumulation. However, the mechanism by which ZIP8 expression is suppressed in these cells remains to be elucidated. In the present study, we investigated the possibility that epigenetic silencing of the slc39a8 gene by DNA hypermethylation is involved in the down-regulation of ZIP8 expression. A7 cells showed a higher mRNA level of DNA methyltransferase 3b than parental cells. Hypermethylation of the CpG island of the slc39a8 gene was detected in A7 cells. Treatment of A7 cells with 5-aza-deoxycytidine, an inhibitor of DNA methyltransferase, caused demethylation of the CpG island of the slc39a8 gene and enhancement of mRNA and protein levels of ZIP8. In response to the recovery of ZIP8 expression, A7 cells treated with 5-aza-deoxycytidine showed an increase in cadmium accumulation and consequently an increase in sensitivity to cadmium. These results suggest that epigenetic silencing of the slc39a8 gene by DNA hypermethylation plays an important role in the down-regulation of ZIP8 in cadmium-resistant metallothionein-null cells.

  13. Association of metals (mercury, cadmium and zinc) with metallothionein-like proteins in storage organs of stranded dolphins from the Mediterranean sea (Southern Italy).

    PubMed

    Decataldo, Alba; Leo, Antonella Di; Giandomenico, Santina; Cardellicchio, Nicola

    2004-04-01

    Selected toxic and essential metals (mercury, Hg; cadmium, Cd; and zinc, Zn) were determined in the liver and in the kidney collected from 13 dolphins (Stenella coeruleoalba and Tursiops truncatus) stranded along the southern coasts of Italy from 1991 to 1999. Liver samples were also analysed for their methyl mercury (MeHg) and selenium (Se) contents. For subcellular fractionation, liver and kidney samples were homogenized in Tris-HCl buffer; after centrifugation, the supernatant (cytosol) was separated from pellets (insoluble fraction), heated at 80 degrees C for 10 min and centrifuged in order to separate the precipitate containing the thermolabile high molecular weight proteins. The cytosol heat-stable fraction, including metallothionein-like proteins (MTLPs), was then purified by gel filtration chromatography on Sephadex G-75 column. The three subcellular fractions collected (insoluble fraction, thermolabile fraction and purified heat-stable fraction) were analysed for their Hg, Cd and Zn contents. The analytical results confirm previous similar studies on toothed whales in showing that: (1) in the liver, as well as in the kidney, Hg was mainly found in the insoluble fraction, therefore, metallothioneins seem to have no role in the Hg detoxification; (2) in the liver, the molar ratio between Se and inorganic Hg was very close to 1; this suggests that the final compound of MeHg detoxification could be HgSe (tiemannite); (3) in almost all the samples, Cd and Zn were detected in the purified heat-stable fraction (including MTLPs). The mechanism of Cd detoxification and Zn homeostasis is also discussed.

  14. Cloning and characterization of HbMT2a, a metallothionein gene from Hevea brasiliensis Muell. Arg differently responds to abiotic stress and heavy metals.

    PubMed

    Li, Yan; Chen, Yue Yi; Yang, Shu Guang; Tian, Wei Min

    2015-05-22

    Metallothioneins (MTs) are of low molecular mass, cysteine-rich proteins. They play an important role in the detoxification of heavy metals and homeostasis of intracellular metal ions, and protecting against intracellular oxidative damages. In this study a full-length cDNA of type 2 plant metallothioneins, HbMT2a, was isolated from 25 mM Polyethyleneglycol (PEG) stressed leaves of Hevea brasiliensis by RACE. The HbMT2a was 372bp in length and had a 237bp open reading frame (ORF) encoding for a protein of 78 amino acid residues with molecular mass of 7.772 kDa. The expression of HbMT2a in the detached leaves of rubber tree clone RY7-33-97 was up-regulated by Me-JA, ABA, PEG, H2O2, Cu(2+) and Zn(2+), but down-regulated by water. The role of HbMT2a protein in protecting against metal toxicity was demonstrated in vitro. PET-28a-HbMT2-beared Escherichia coli. Differential expression of HbMT2a upon treatment with 10 °C was observed in the detached leaves of rubber tree clone 93-114 which is cold-resistant and Reken501 which is cold-sensitive. The expression patterns of HbMT2a in the two rubber tree clones may be ascribed to a change in the level of endogenous H2O2.

  15. ScMT2-1-3, a metallothionein gene of sugarcane, plays an important role in the regulation of heavy metal tolerance/accumulation.

    PubMed

    Guo, Jinlong; Xu, Liping; Su, Yachun; Wang, Hengbo; Gao, Shiwu; Xu, Jingsheng; Que, Youxiong

    2013-01-01

    Plant metallothioneins (MTs), which are cysteine-rich, low-molecular-weight, and metal-binding proteins, play important roles in detoxification, metal ion homeostasis, and metal transport adjustment. In this study, a novel metallothionein gene, designated as ScMT2-1-3 (GenBank Accession number JQ627644), was identified from sugarcane. ScMT2-1-3 was 700 bp long, including a 240 bp open reading frame (ORF) encoding 79 amino acid residues. A His-tagged ScMT2-1-3 protein was successfully expressed in Escherichia coli system which had increased the host cell's tolerance to Cd(2+), Cu(2+), PEG, and NaCl. The expression of ScMT2-1-3 was upregulated under Cu(2+) stress but downregulated under Cd(2+) stress. Real-time qPCR demonstrated that the expression levels of ScMT2-1-3 in bud and root were over 14 times higher than those in stem and leaf, respectively. Thus, both the E. coli assay and sugarcane plantlets assay suggested that ScMT2-1-3 is significantly involved in the copper detoxification and storage in the cell, but its functional mechanism in cadmium detoxification and storage in sugarcane cells needs more testification though its expressed protein could obviously increase the host E. coli cell's tolerance to Cd(2+). ScMT2-1-3 constitutes thus a new interesting candidate for elucidating the molecular mechanisms of MTs-implied plant heavy metal tolerance/accumulation and for developing sugarcane phytoremediator varieties.

  16. Genome-Wide Analysis of Arabidopsis Pentatricopeptide Repeat Proteins Reveals Their Essential Role in Organelle BiogenesisW⃞

    PubMed Central

    Lurin, Claire; Andrés, Charles; Aubourg, Sébastien; Bellaoui, Mohammed; Bitton, Frédérique; Bruyère, Clémence; Caboche, Michel; Debast, Cédrig; Gualberto, José; Hoffmann, Beate; Lecharny, Alain; Le Ret, Monique; Martin-Magniette, Marie-Laure; Mireau, Hakim; Peeters, Nemo; Renou, Jean-Pierre; Szurek, Boris; Taconnat, Ludivine; Small, Ian

    2004-01-01

    The complete sequence of the Arabidopsis thaliana genome revealed thousands of previously unsuspected genes, many of which cannot be ascribed even putative functions. One of the largest and most enigmatic gene families discovered in this way is characterized by tandem arrays of pentatricopeptide repeats (PPRs). We describe a detailed bioinformatic analysis of 441 members of the Arabidopsis PPR family plus genomic and genetic data on the expression (microarray data), localization (green fluorescent protein and red fluorescent protein fusions), and general function (insertion mutants and RNA binding assays) of many family members. The basic picture that arises from these studies is that PPR proteins play constitutive, often essential roles in mitochondria and chloroplasts, probably via binding to organellar transcripts. These results confirm, but massively extend, the very sparse observations previously obtained from detailed characterization of individual mutants in other organisms. PMID:15269332

  17. Overexpression of poplar wounding-inducible genes in Arabidopsis caused improved resistance against Helicoverpa armigera (Hübner) larvae

    PubMed Central

    Hu, Rongfeng; Wang, Jiehua; Ji, Yan; Song, Yingjin; Yang, Shaohui

    2012-01-01

    Four highly inducible genes of poplar trees, PtdKTI5, PtdWIN4, PtdPOP3 from hybrid poplar (Populus trichocarpa × P. deltoides) and PtKTI2 from trembling aspen (Populus tremuloides Michx.) have been individually transformed into Arabidopsis thaliana for overexpression. High transcriptional level of each transgene in transgenic Arabidopsis lines was confirmed by RT-PCR analysis. The development, body weight and survivorship of cotton bollworm (Helicoverpa armigera) fed on four types of transgenic Arabidopis plants were evaluated in the laboratory. Our data indicated that these four Populus defense-related genes exhibited various degree of insectital activity on larval and postlarval development of cotton bollworm and may be utilized for herbivore resistance improvement in plant genetic engineering. PMID:23226090

  18. Metallothioneins (I+II) and thyroid-thymus axis efficiency in old mice: role of corticosterone and zinc supply.

    PubMed

    Mocchegiani, Eugenio; Giacconi, Robertina; Cipriano, Catia; Gasparini, Nazzarena; Orlando, Fiorenza; Stecconi, Rosalia; Muzzioli, Mario; Isani, Gloria; Carpenè, Emilio

    2002-03-31

    Thymic atrophy or thymus absence causes depressed thyroid-thymus axis (TTA) efficiency in old, young propyl-thiouracil (PTU) (experimental hypothyroidism) and in young-adult thymectomised (Tx) mice, respectively. Altered zinc turnover may be also involved in depressed TTA efficiency. Zinc turnover is under the control of zinc-bound metallothioneins (Zn-MTs) synthesis. Thyroid hormones, corticosterone and nutritional zinc affect Zn-MT induction. Zn-MT releases zinc in young-adult age during transient oxidative stress for prompt immune response. In constant oxidative stress (ageing and liver regeneration after partial hepatectomy), high liver Zn-MTs, low zinc ion bioavailability and depressed TTA efficiency appear. This last finding suggested that MT might not release zinc during constant oxidative stress leading to impaired TTA efficiency. The aim of this work/study is to clarify the role of Zn-MTs (I+II) in TTA efficiency during development and ageing. The main results are (1) Old and PTU mice display high corticosterone, enhanced liver MTmRNA, low zinc and depressed TTA efficiency restored by zinc supply. Increased survival and no significant increments in basal liver Zn-MTs proteins occur in old and PTU mice after zinc supply. (2) Lot of zinc ions bound with MT in the liver from old mice than young (HPLC). (3) Young-adult Tx mice, evaluated at 15 days from thymectomy, display high MTmRNA and nutritional-endocrine-immune damage restored by zinc supply or by thymus grafts from old zinc-treated mice. (4) Young-adult Tx mice, but evaluated at 40 days from thymectomy, display natural normalisation in MTmRNA and nutritional-endocrine-immune profile with survival similar to normal mice. (5) Stressed (constant dark for 10 days) mice overexpressing MT display low zinc, depressed immunity, reduced thymic cortex, high corticosterone, altered thyroid hormones turnover showing a likeness with old mice. These findings, taken altogether, show that corticosterone is pivotal in

  19. Comprehensive Assessment of Transcriptional Regulation Facilitates Metabolic Engineering of Isoprenoid Accumulation in Arabidopsis.

    PubMed

    Lange, Iris; Poirier, Brenton C; Herron, Blake K; Lange, Bernd Markus

    2015-11-01

    In plants, two spatially separated pathways provide the precursors for isoprenoid biosynthesis. We generated transgenic Arabidopsis (Arabidopsis thaliana) lines with modulated levels of expression of each individual gene involved in the cytosolic/peroxisomal mevalonate and plastidial methylerythritol phosphate pathways. By assessing the correlation of transgene expression levels with isoprenoid marker metabolites (gene-to-metabolite correlation), we determined the relative importance of transcriptional control at each individual step of isoprenoid precursor biosynthesis. The accumulation patterns of metabolic intermediates (metabolite-to-gene correlation) were then used to infer flux bottlenecks in the sterol pathway. The extent of metabolic cross talk, the exchange of isoprenoid intermediates between compartmentalized pathways, was assessed by a combination of gene-to-metabolite and metabolite-to-metabolite correlation analyses. This strategy allowed the selection of genes to be modulated by metabolic engineering, and we demonstrate that the overexpression of predictable combinations of genes can be used to significantly enhance flux toward specific end products of the sterol pathway. Transgenic plants accumulating increased amounts of sterols are characterized by significantly elevated biomass, which can be a desirable trait in crop and biofuel plants.

  20. Single molecule PCR reveals similar patterns of non-homologous DSB repair in tobacco and Arabidopsis.

    PubMed

    Lloyd, Andrew H; Wang, Dong; Timmis, Jeremy N

    2012-01-01

    DNA double strand breaks (DSBs) occur constantly in eukaryotes. These potentially lethal DNA lesions are repaired efficiently by two major DSB repair pathways: homologous recombination and non-homologous end joining (NHEJ). We investigated NHEJ in Arabidopsis thaliana and tobacco (Nicotiana tabacum) by introducing DNA double-strand breaks through inducible expression of I-SceI, followed by amplification of individual repair junction sequences by single-molecule PCR. Using this process over 300 NHEJ repair junctions were analysed in each species. In contrast to previously published variation in DSB repair between Arabidopsis and tobacco, the two species displayed similar DSB repair profiles in our experiments. The majority of repair events resulted in no loss of sequence and small (1-20 bp) deletions occurred at a minority (25-45%) of repair junctions. Approximately ~1.5% of the observed repair events contained larger deletions (>20 bp) and a similar percentage contained insertions. Strikingly, insertion events in tobacco were associated with large genomic deletions at the site of the DSB that resulted in increased micro-homology at the sequence junctions suggesting the involvement of a non-classical NHEJ repair pathway. The generation of DSBs through inducible expression of I-SceI, in combination with single molecule PCR, provides an effective and efficient method for analysis of individual repair junctions and will prove a useful tool in the analysis of NHEJ.

  1. Comprehensive Assessment of Transcriptional Regulation Facilitates Metabolic Engineering of Isoprenoid Accumulation in Arabidopsis1[OPEN

    PubMed Central

    Lange, Iris; Poirier, Brenton C.; Herron, Blake K.; Lange, Bernd Markus

    2015-01-01

    In plants, two spatially separated pathways provide the precursors for isoprenoid biosynthesis. We generated transgenic Arabidopsis (Arabidopsis thaliana) lines with modulated levels of expression of each individual gene involved in the cytosolic/peroxisomal mevalonate and plastidial methylerythritol phosphate pathways. By assessing the correlation of transgene expression levels with isoprenoid marker metabolites (gene-to-metabolite correlation), we determined the relative importance of transcriptional control at each individual step of isoprenoid precursor biosynthesis. The accumulation patterns of metabolic intermediates (metabolite-to-gene correlation) were then used to infer flux bottlenecks in the sterol pathway. The extent of metabolic cross talk, the exchange of isoprenoid intermediates between compartmentalized pathways, was assessed by a combination of gene-to-metabolite and metabolite-to-metabolite correlation analyses. This strategy allowed the selection of genes to be modulated by metabolic engineering, and we demonstrate that the overexpression of predictable combinations of genes can be used to significantly enhance flux toward specific end products of the sterol pathway. Transgenic plants accumulating increased amounts of sterols are characterized by significantly elevated biomass, which can be a desirable trait in crop and biofuel plants. PMID:26282236

  2. Single and multiple CH (calponin homology) domain containing multidomain proteins in Arabidopsis and Saccharomyces: an inventory.

    PubMed

    Friedberg, Felix

    2011-01-01

    Genes for individual domains such as CH, lim, ankyrin, PH and RhoGAP, IQ motif, Ig_FLMN, spectrin, and EF hand probably existed in early evolution before there were plants, fungi or animals so that when we examine multidomain proteins in Arabidopsis, Saccharomyces, Dictyostelium or Homo Sapiens we encounter various combinations of such domains. While all of these four species express Fimbrin and EB1, the lists of CH containing multidomain proteins, however, differ in number and in type for each of them. There was no further great increase in the number of new single domain proteins. Still many new multidomain genes evolved--but far more so in metazoans--than in plants or fungi. In both plants and fungi only singlet CH domains but no doublets (other than those forming the Fimbrin quadruplet) were incorporated. That is in these two branches one finds no alpha actinin, dystrophin or filamin even though the individual building blocks (i.e. domains such as spectrin or IG-FLMN) were available in Arabidopsis. Possibly transposons create new chimeric multidomain genes by mixing and matching genes or gene fragments.

  3. Characterization of five polyamine oxidase isoforms in Arabidopsis thaliana.

    PubMed

    Takahashi, Yoshihiro; Cong, Runzi; Sagor, G H M; Niitsu, Masaru; Berberich, Thomas; Kusano, Tomonobu

    2010-09-01

    The genome of Arabidopsis thaliana contains five genes (AtPAO1 to AtPAO5) encoding polyamine oxidase (PAO) which is an enzyme responsible for polyamine catabolism. To understand the individual roles of the five AtPAOs, here we characterized their tissue-specific and space-temporal expression. AtPAO1 seems to have a specific function in flower organ. AtPAO2 was expressed in shoot meristem and root tip of seedlings, and to a higher extent in the later growth stage within restricted parts of the organs, such as shoot meristem, leaf petiole and also in anther. The expression of AtPAO3 was constitutive, but highest in flower organ. AtPAO3 promoter activity was detected in cotyledon, distal portion of root, boundary region of mature rosette leaf and in filaments of flower. AtPAO4 was expressed at higher level all over young seedlings including roots, and in the mature stage its expression was ubiquitous with rather lower level in stem. AtPAO5 expression was observed in the whole plant body throughout various growth stages. Its highest expression was in flowers, particularly in sepals, but not in petals. Furthermore, we determined the substrate specificity of AtPAO1 to AtPAO4. None of the AtPAO enzymes recognized putrescine (Put). AtPAO2 and AtPAO3 showed almost similar substrate recognition patterns in which the most preferable substrate is spermidine (Spd) followed by less specificity to other tetraamines tested. AtPAO4 seemed to be spermine (Spm)-specific. More interestingly, AtPAO1 preferred thermospermine (T-Spm) and norspermine (NorSpm) to Spm, but did not recognize Spd. Based on the results, the individual function of AtPAOs is discussed.

  4. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, William C.; Brown, Christopher S.

    1994-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional sodium doedocyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  5. 3D Gel Map of Arabidopsis Complex I

    PubMed Central

    Peters, Katrin; Belt, Katharina; Braun, Hans-Peter

    2013-01-01

    Complex I has a unique structure in plants and includes extra subunits. Here, we present a novel study to define its protein constituents. Mitochondria were isolated from Arabidopsis thaliana cell cultures, leaves, and roots. Subunits of complex I were resolved by 3D blue-native (BN)/SDS/SDS-PAGE and identified by mass spectrometry. Overall, 55 distinct proteins were found, seven of which occur in pairs of isoforms. We present evidence that Arabidopsis complex I consists of 49 distinct types of subunits, 40 of which represent homologs of bovine complex I. The nine other subunits represent special proteins absent in the animal linage of eukaryotes, most prominently a group of subunits related to bacterial gamma-type carbonic anhydrases. A GelMap http://www.gelmap.de/arabidopsis-3d-complex-i/ is presented for promoting future complex I research in Arabidopsis thaliana. PMID:23761796

  6. Protein expression in Arabidopsis thaliana after chronic clinorotation

    NASA Technical Reports Server (NTRS)

    Piastuch, W. C.; Brown, C. S.

    1995-01-01

    Soluble protein expression in Arabidopsis thaliana L. (Heynh.) leaf and stem tissue was examined after chronic clinorotation. Seeds of Arabidopsis were germinated and plants grown to maturity on horizontal or vertical slow-rotating clinostats (1 rpm) or in stationary vertical control units. Total soluble proteins and in vivo-labeled soluble proteins isolated from these plants were analyzed by two-dimensional SDS PAGE and subsequent fluorography. Visual and computer analysis of the resulting protein patterns showed no significant differences in either total protein expression or in active protein synthesis between horizontal clinorotation and vertical controls in the Arabidopsis leaf and stem tissue. These results show chronic clinorotation does not cause gross changes in protein expression in Arabidopsis.

  7. VIZARD: analysis of Affymetrix Arabidopsis GeneChip data

    NASA Technical Reports Server (NTRS)

    Moseyko, Nick; Feldman, Lewis J.

    2002-01-01

    SUMMARY: The Affymetrix GeneChip Arabidopsis genome array has proved to be a very powerful tool for the analysis of gene expression in Arabidopsis thaliana, the most commonly studied plant model organism. VIZARD is a Java program created at the University of California, Berkeley, to facilitate analysis of Arabidopsis GeneChip data. It includes several integrated tools for filtering, sorting, clustering and visualization of gene expression data as well as tools for the discovery of regulatory motifs in upstream sequences. VIZARD also includes annotation and upstream sequence databases for the majority of genes represented on the Affymetrix Arabidopsis GeneChip array. AVAILABILITY: VIZARD is available free of charge for educational, research, and not-for-profit purposes, and can be downloaded at http://www.anm.f2s.com/research/vizard/ CONTACT: moseyko@uclink4.berkeley.edu.

  8. The development of Arabidopsis as a model plant.

    PubMed

    Koornneef, Maarten; Meinke, David

    2010-03-01

    Twenty-five years ago, Arabidopsis thaliana emerged as the model organism of choice for research in plant biology. A consensus was reached about the need to focus on a single organism to integrate the classical disciplines of plant science with the expanding fields of genetics and molecular biology. Ten years after publication of its genome sequence, Arabidopsis remains the standard reference plant for all of biology. We reflect here on the major advances and shared resources that led to the extraordinary growth of the Arabidopsis research community. We also underscore the importance of continuing to expand and refine our detailed knowledge of Arabidopsis while seeking to appreciate the remarkable diversity that characterizes the plant kingdom.

  9. Light responses in Photoperiodism in Arabidopsis thaliana

    SciTech Connect

    Anthony R. Cashmore

    2006-08-01

    ADO1: An Arabidopsis blue light photoreceptor We have reported the characterization of an Arabidopsis gene encoding the ADAGIO 1 (ADO1) protein (Jarillo et al., 2001a). ADO1 contains a LOV domain, similar to WHITE COLLAR 1 (WC1), a photoreceptor for entrainment of Neurospora circadian rhythms (Froehlich et al., 2002), as well as PHOT1 and PHOT2, the blue light photoreceptors for phototropism (Briggs et al., 2001; Christie et al., 1998; Jarillo et al., 2001b; Kinoshita et al., 2001). Loss of function ado1 mutants show an unusually long periodicity for their free running circadian rhythm (Jarillo et al., 2001a). This observation holds for plants grown under white light as well as blue light and surprisingly, plants grown under red light also show altered circadian properties. The similarity of the LOV domain of ADO1 to those of PHOT1, PHOT2 and WC1 (known flavoprotein photoreceptors) as well as the genetic and molecular properties of ADO1, indicate that ADO1 is likely a new class of blue light photoreceptor. Indeed, the LOV domain of the related FKF1/ADO3 has been shown to bind FMN, and exhibit the in vitro photochemistry characteristic of PHOT1 (Imaizumi et al., 2003). Furthermore, ZTL/ADO1 has been shown to participate in the circadian and proteasome mediated degradation of the Arabidopsis clock protein, TOC1 (Mas et al., 2003). We also showed that the ado1 mutation selectively confers hypersensitivity to red light — when grown under red light (but not blue light) the ado1 mutant possesses an unusually short hypocotyl. This red light hypersensivity is even more severe in a triple ado1 ado2 ado3 mutant — ADO2 and ADO3 being the two other members of this ADAGIO gene family. This finding of a mutant phenotype under red light is somewhat unexpected for a protein thought to function as a photoreceptor for blue light. We have pursued our studies of ADO1 by preparing a mutant gene for which we have altered the codon for the cysteine residue conserved in all LOV

  10. Using Arabidopsis to study shoot branching in biomass willow.

    PubMed

    Ward, Sally P; Salmon, Jemma; Hanley, Steven J; Karp, Angela; Leyser, Ottoline

    2013-06-01

    The success of the short-rotation coppice system in biomass willow (Salix spp.) relies on the activity of the shoot-producing meristems found on the coppice stool. However, the regulation of the activity of these meristems is poorly understood. In contrast, our knowledge of the mechanisms behind axillary meristem regulation in Arabidopsis (Arabidopsis thaliana) has grown rapidly in the past few years through the exploitation of integrated physiological, genetic, and molecular assays. Here, we demonstrate that these assays can be directly transferred to study the control of bud activation in biomass willow and to assess similarities with the known hormone regulatory system in Arabidopsis. Bud hormone response was found to be qualitatively remarkably similar in Salix spp. and Arabidopsis. These similarities led us to test whether Arabidopsis hormone mutants could be used to assess allelic variation in the cognate Salix spp. hormone genes. Allelic differences in Salix spp. strigolactone genes were observed using this approach. These results demonstrate that both knowledge and assays from Arabidopsis axillary meristem biology can be successfully applied to Salix spp. and can increase our understanding of a fundamental aspect of short-rotation coppice biomass production, allowing more targeted breeding.

  11. Effects of surfactants on the contents of metallothionein, heme and hemoproteins and on the activities of heme oxygenase and drug-metabolizing enzymes in rats pretreated with phenobarbital or. beta. -naphthoflavone

    SciTech Connect

    Ariyoshi, Toshihiko; Hasegawa, Hiroyuki; Matsumoto, Hideki; Arizono, Koji )

    1991-01-01

    Synthetic surfactants as major constituent of detergent products are widely used in consumer and industrial fields, and hence environmental and toxicological investigations of surfactants are numerous. In the previous study, the authors observed that intraperitoneal administration of surfactants such as sodium dodecyl sulfate (SDS), sodium n-dodecylbenzenesulfonate (LAS) and polyoxyethyleneglycol nonylphenyl ether (Emulgen 913) to rats depressed the content of microsomal cytochrome P-450, while they enhanced markedly the activity of heme oxygenase, the first and rate-limiting enzyme in heme degradation. In addition, they noted an increase of metallothionein content in the liver of rats treated with LAS. In this study, the authors investigated the effects of surfactants on metallothionein, heme, hemoproteins, heme oxygenase and drug-metabolizing enzymes in the liver of rats pretreated with phenobarbital or {beta}-naphthoflavone.

  12. The ethylene signal transduction pathway in Arabidopsis

    NASA Technical Reports Server (NTRS)

    Kieber, J. J.; Evans, M. L. (Principal Investigator)

    1997-01-01

    The gaseous hormone ethylene is an important regulator of plant growth and development. Using a simple response of etiolated seedlings to ethylene as a genetic screen, genes involved in ethylene signal transduction have been identified in Arabidopsis. Analysis of two of these genes that have been cloned reveals that ethylene signalling involves a combination of a protein (ETR1) with similarity to bacterial histidine kinases and a protein (CTR1) with similarity to Raf-1, a protein kinase involved in multiple signalling cascades in eukaryotic cells. Several lines of investigation provide compelling evidence that ETR1 encodes an ethylene receptor. For the first time there is a glimpse of the molecular circuitry underlying the signal transduction pathway for a plant hormone.

  13. ELF3 controls thermoresponsive growth in Arabidopsis.

    PubMed

    Box, Mathew S; Huang, B Emma; Domijan, Mirela; Jaeger, Katja E; Khattak, Asif Khan; Yoo, Seong Jeon; Sedivy, Emma L; Jones, D Marc; Hearn, Timothy J; Webb, Alex A R; Grant, Alastair; Locke, James C W; Wigge, Philip A

    2015-01-19

    Plant development is highly responsive to ambient temperature, and this trait has been linked to the ability of plants to adapt to climate change. The mechanisms by which natural populations modulate their thermoresponsiveness are not known. To address this, we surveyed Arabidopsis accessions for variation in thermal responsiveness of elongation growth and mapped the corresponding loci. We find that the transcriptional regulator EARLY FLOWERING3 (ELF3) controls elongation growth in response to temperature. Through a combination of modeling and experiments, we show that high temperature relieves the gating of growth at night, highlighting the importance of temperature-dependent repressors of growth. ELF3 gating of transcriptional targets responds rapidly and reversibly to changes in temperature. We show that the binding of ELF3 to target promoters is temperature dependent, suggesting a mechanism where temperature directly controls ELF3 activity.

  14. Cation channels in the Arabidopsis plasma membrane.

    PubMed

    Véry, Anne Aliénor; Sentenac, Hervé

    2002-04-01

    In vivo analyses have identified different functional types of ion channels in various plant tissues and cells. The Arabidopsis genome contains approximately 70 genes for ion channels, of which 57 might be cation-selective channels (K(+), Ca(2+) or poorly discriminating channels). Here, we describe the different families of (putative) cation channels: the Shakers, the two-P-domain and Kir K(+) channels (encoded by the KCO genes), the cyclic-nucleotide-gated channels, the glutamate receptors, and the Ca(2+) channel TPC1. We also compare molecular data with the data obtained in planta, which should lead to a better understanding of the identity of these channels and provide clues about their roles in plant nutrition and cell signalling.

  15. Crystal structure of Arabidopsis thaliana cytokinin dehydrogenase

    SciTech Connect

    Bae, Euiyoung; Bingman, Craig A.; Bitto, Eduard; Aceti, David J.; Phillips, Jr., George N.

    2008-08-13

    Since first discovered in Zea mays, cytokinin dehydrogenase (CKX) genes have been identified in many plants including rice and Arabidopsis thaliana, which possesses CKX homologues (AtCKX1-AtCKX7). So far, the three-dimensional structure of only Z. mays CKX (ZmCKX1) has been determined. The crystal structures of ZmCKX1 have been solved in the native state and in complex with reaction products and a slowly reacting substrate. The structures revealed four glycosylated asparagine residues and a histidine residue covalently linked to FAD. Combined with the structural information, recent biochemical analyses of ZmCKX1 concluded that the final products of the reaction, adenine and a side chain aldehyde, are formed by nonenzymatic hydrolytic cleavage of cytokinin imine products resulting directly from CKX catalysis. Here, we report the crystal structure of AtCKX7 (gene locus At5g21482.1, UniProt code Q9FUJ1).

  16. The Arabidopsis Cyclophilin Gene Family1

    PubMed Central

    Romano, Patrick G.N.; Horton, Peter; Gray, Julie E.

    2004-01-01

    Database searching has allowed the identification of a number of previously unreported single and multidomain isoform members of the Arabidopsis cyclophilin gene family. In addition to the cyclophilin-like peptidyl-prolyl cis-trans isomerase domain, the latter contain a variety of other domains with characterized functions. Transcriptional analysis showed they are expressed throughout the plant, and differe