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Sample records for affinity iron permease

  1. High Affinity Iron Permease is Required for Virulence of Rhizopus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizopus oryzae is the most common cause of mucormycosis. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to develop mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iro...

  2. The High Affinity Iron Permease is a Key Virulence Factor Required for Rhizopus oryzae Pathogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rhizopus oryzae is the most common cause of mucormycosis, an angioinvasive fungal infection that causes a >/=50% mortality rate despite first-line therapy. Clinical and animal model data clearly demonstrate that the presence of elevated available serum iron predisposes the host to mucormycosis. Th...

  3. Functional mapping and implications of substrate specificity of the yeast high-affinity leucine permease Bap2.

    PubMed

    Usami, Yuki; Uemura, Satsohi; Mochizuki, Takahiro; Morita, Asami; Shishido, Fumi; Inokuchi, Jin-ichi; Abe, Fumiyoshi

    2014-07-01

    Leucine is a major amino acid in nutrients and proteins and is also an important precursor of higher alcohols during brewing. In Saccharomyces cerevisiae, leucine uptake is mediated by multiple amino acid permeases, including the high-affinity leucine permease Bap2. Although BAP2 transcription has been extensively analyzed, the mechanisms by which a substrate is recognized and moves through the permease remain unknown. Recently, we determined 15 amino acid residues required for Tat2-mediated tryptophan import. Here we introduced homologous mutations into Bap2 amino acid residues and showed that 7 residues played a role in leucine import. Residues I109/G110/T111 and E305 were located within the putative α-helix break in TMD1 and TMD6, respectively, according to the structurally homologous Escherichia coli arginine/agmatine antiporter AdiC. Upon leucine binding, these α-helix breaks were assumed to mediate a conformational transition in Bap2 from an outward-open to a substrate-binding occluded state. Residues Y336 (TMD7) and Y181 (TMD3) were located near I109 and E305, respectively. Bap2-mediated leucine import was inhibited by some amino acids according to the following order of severity: phenylalanine, leucine>isoleucine>methionine, tyrosine>valine>tryptophan; histidine and asparagine had no effect. Moreover, this order of severity clearly coincided with the logP values (octanol-water partition coefficients) of all amino acids except tryptophan. This result suggests that the substrate partition efficiency to the buried Bap2 binding pocket is the primary determinant of substrate specificity rather than structural amino acid side chain recognition.

  4. The chloroplast permease PIC1 regulates plant growth and development by directing homeostasis and transport of iron.

    PubMed

    Duy, Daniela; Stübe, Roland; Wanner, Gerhard; Philippar, Katrin

    2011-04-01

    The membrane-spanning protein PIC1 (for permease in chloroplasts 1) in Arabidopsis (Arabidopsis thaliana) was previously described to mediate iron transport across the inner envelope membrane of chloroplasts. The albino phenotype of pic1 knockout mutants was reminiscent of iron-deficiency symptoms and characterized by severely impaired plastid development and plant growth. In addition, plants lacking PIC1 showed a striking increase in chloroplast ferritin clusters, which function in protection from oxidative stress by sequestering highly reactive free iron in their spherical protein shell. In contrast, PIC1-overexpressing lines (PIC1ox) in this study rather resembled ferritin loss-of-function plants. PIC1ox plants suffered from oxidative stress and leaf chlorosis, most likely originating from iron overload in chloroplasts. Later during growth, plants were characterized by reduced biomass as well as severely defective flower and seed development. As a result of PIC1 protein increase in the inner envelope membrane of plastids, flower tissue showed elevated levels of iron, while the content of other transition metals (copper, zinc, manganese) remained unchanged. Seeds, however, specifically revealed iron deficiency, suggesting that PIC1 overexpression sequestered iron in flower plastids, thereby becoming unavailable for seed iron loading. In addition, expression of genes associated with metal transport and homeostasis as well as photosynthesis was deregulated in PIC1ox plants. Thus, PIC1 function in plastid iron transport is closely linked to ferritin and plastid iron homeostasis. In consequence, PIC1 is crucial for balancing plant iron metabolism in general, thereby regulating plant growth and in particular fruit development.

  5. Regulation of high affinity iron uptake in the yeast Saccharomyces cerevisiae. Role of dioxygen and Fe.

    PubMed

    Hassett, R F; Romeo, A M; Kosman, D J

    1998-03-27

    High affinity iron uptake in Saccharomyces cerevisiae requires a metal reductase, a multicopper ferroxidase, and an iron permease. Fet3, the apparent ferroxidase, is proposed to facilitate iron uptake by catalyzing the oxidation of reductase-generated Fe(II) to Fe(III) by O2; in this model, Fe(III) is the substrate for the iron permease, encoded by FTR1 (Kaplan, J., and O'Halloran, T. V. (1996) Science 271, 1510-1512). We show here that dioxygen also plays an essential role in the expression of these iron uptake activities. Cells grown anaerobically exhibited no Fe(III) reductase or high affinity iron uptake activity, even if assayed for these activities under air. Northern blot analysis showed that the amount of those mRNAs encoding proteins associated with this uptake was repressed in anaerobic cultures but was rapidly induced by exposure of the culture to dioxygen. The anaerobic repression was reduced in cells expressing an iron-independent form of the trans-activator, Aft1, a protein that regulates the expression of these proteins. Thus, the effect of oxygenation on this expression appeared due at least in part to the state or distribution of iron in the cells. In support of this hypothesis, the membrane-permeant Fe(II) chelator, 2, 2'-bipyridyl, in contrast to the impermeant chelator bathophenanthroline disulfonate, caused a strong and rapid induction of these transcripts under anaerobic conditions. An increase in the steady-state levels of iron-regulated transcripts upon oxygenation or 2,2'-bipyridyl addition occurred within 5 min, indicating that a relatively small, labile intracellular pool of Fe(II) regulates the expression of these activities. The strength of the anaerobic repression was dependent on the low affinity, Fe(II)-specific iron transporter, encoded by FET4, suggesting that this Fe(II) pool was linked in part to iron brought into the cell via Fet4 protein. The data suggest a model in which dioxygen directly or indirectly modulates the Fe

  6. Crystal structure of lactose permease in complex with an affinity inactivator yields unique insight into sugar recognition

    SciTech Connect

    Chaptal, Vincent; Kwon, Seunghyug; Sawaya, Michael R.; Guan, Lan; Kaback, H. Ronald; Abramson, Jeff

    2011-08-29

    Lactose permease of Escherichia coli (LacY) with a single-Cys residue in place of A122 (helix IV) transports galactopyranosides and is specifically inactivated by methanethiosulfonyl-galactopyranosides (MTS-gal), which behave as unique suicide substrates. In order to study the mechanism of inactivation more precisely, we solved the structure of single-Cys122 LacY in complex with covalently bound MTS-gal. This structure exhibits an inward-facing conformation similar to that observed previously with a slight narrowing of the cytoplasmic cavity. MTS-gal is bound covalently, forming a disulfide bond with C122 and positioned between R144 and W151. E269, a residue essential for binding, coordinates the C-4 hydroxyl of the galactopyranoside moiety. The location of the sugar is in accord with many biochemical studies.

  7. Pressure-induced endocytic degradation of the Saccharomyces cerevisiae low-affinity tryptophan permease Tat1 is mediated by Rsp5 ubiquitin ligase and functionally redundant PPxY motif proteins.

    PubMed

    Suzuki, Asaha; Mochizuki, Takahiro; Uemura, Satoshi; Hiraki, Toshiki; Abe, Fumiyoshi

    2013-07-01

    Cells of Saccharomyces cerevisiae express two tryptophan permeases, Tat1 and Tat2, which have different characteristics in terms of their affinity for tryptophan and intracellular localization. Although the high-affinity permease Tat2 has been well documented in terms of its ubiquitin-dependent degradation, the low-affinity permease Tat1 has not yet been characterized fully. Here we show that a high hydrostatic pressure of 25 MPa triggers a degradation of Tat1 which depends on Rsp5 ubiquitin ligase and the EH domain-containing protein End3. Tat1 was resistant to a 3-h cycloheximide treatment, suggesting that it is highly stable under normal growth conditions. The ubiquitination of Tat1 most likely occurs at N-terminal lysines 29 and 31. Simultaneous substitution of arginine for the two lysines prevented Tat1 degradation, but substitution of either of them alone did not, indicating that the roles of lysines 29 and 31 are redundant. When cells were exposed to high pressure, Tat1-GFP was completely lost from the plasma membrane, while substantial amounts of Tat1(K29R-K31R)-GFP remained. The HPG1-1 (Rsp5(P514T)) and rsp5-ww3 mutations stabilized Tat1 under high pressure, but any one of the rsp5-ww1, rsp5-ww2, and bul1Δ bul2Δ mutations or single deletions of genes encoding arrestin-related trafficking adaptors did not. However, simultaneous loss of 9-arrestins and Bul1/Bul2 prevented Tat1 degradation at 25 MPa. The results suggest that multiple PPxY motif proteins share some essential roles in regulating Tat1 ubiquitination in response to high hydrostatic pressure.

  8. A ferroxidation/permeation iron uptake system is required for virulence in Ustilago maydis.

    PubMed

    Eichhorn, Heiko; Lessing, Franziska; Winterberg, Britta; Schirawski, Jan; Kämper, Jörg; Müller, Philip; Kahmann, Regine

    2006-11-01

    In the smut fungus Ustilago maydis, a tightly regulated cAMP signaling cascade is necessary for pathogenic development. Transcriptome analysis using whole genome microarrays set up to identify putative target genes of the protein kinase A catalytic subunit Adr1 revealed nine genes with putative functions in two high-affinity iron uptake systems. These genes locate to three gene clusters on different chromosomes and include the previously identified complementing siderophore auxotroph genes sid1 and sid2 involved in siderophore biosynthesis. Transcription of all nine genes plus three additional genes associated with the gene clusters was also coregulated by iron through the Urbs1 transcription factor. Two components of a high-affinity iron uptake system were characterized in more detail: fer2, encoding a high-affinity iron permease; and fer1, encoding an iron multicopper oxidase. Fer2 localized to the plasma membrane and complemented an ftr1 mutant of Saccharomyces cerevisiae lacking a high-affinity iron permease. During pathogenic development, fer2 expression was confined to the phase of hyphal proliferation inside the plant. fer2 as well as fer1 deletion mutants were strongly affected in virulence. These data highlight the importance of the high-affinity iron uptake system via an iron permease and a multicopper oxidase for biotrophic development in the U. maydis/maize (Zea mays) pathosystem.

  9. A Ferroxidation/Permeation Iron Uptake System Is Required for Virulence in Ustilago maydis[W

    PubMed Central

    Eichhorn, Heiko; Lessing, Franziska; Winterberg, Britta; Schirawski, Jan; Kämper, Jörg; Müller, Philip; Kahmann, Regine

    2006-01-01

    In the smut fungus Ustilago maydis, a tightly regulated cAMP signaling cascade is necessary for pathogenic development. Transcriptome analysis using whole genome microarrays set up to identify putative target genes of the protein kinase A catalytic subunit Adr1 revealed nine genes with putative functions in two high-affinity iron uptake systems. These genes locate to three gene clusters on different chromosomes and include the previously identified complementing siderophore auxotroph genes sid1 and sid2 involved in siderophore biosynthesis. Transcription of all nine genes plus three additional genes associated with the gene clusters was also coregulated by iron through the Urbs1 transcription factor. Two components of a high-affinity iron uptake system were characterized in more detail: fer2, encoding a high-affinity iron permease; and fer1, encoding an iron multicopper oxidase. Fer2 localized to the plasma membrane and complemented an ftr1 mutant of Saccharomyces cerevisiae lacking a high-affinity iron permease. During pathogenic development, fer2 expression was confined to the phase of hyphal proliferation inside the plant. fer2 as well as fer1 deletion mutants were strongly affected in virulence. These data highlight the importance of the high-affinity iron uptake system via an iron permease and a multicopper oxidase for biotrophic development in the U. maydis/maize (Zea mays) pathosystem. PMID:17138696

  10. Structures of Streptococcus pneumoniae PiaA and Its Complex with Ferrichrome Reveal Insights into the Substrate Binding and Release of High Affinity Iron Transporters

    PubMed Central

    Cheng, Wang; Li, Qiong; Jiang, Yong-Liang; Zhou, Cong-Zhao; Chen, Yuxing

    2013-01-01

    Iron scarcity is one of the nutrition limitations that the Gram-positive infectious pathogens Streptococcus pneumoniae encounter in the human host. To guarantee sufficient iron supply, the ATP binding cassette (ABC) transporter Pia is employed to uptake iron chelated by hydroxamate siderophore, via the membrane-anchored substrate-binding protein PiaA. The high affinity towards ferrichrome enables PiaA to capture iron at a very low concentration in the host. We presented here the crystal structures of PiaA in both apo and ferrichrome-complexed forms at 2.7 and 2.1 Å resolution, respectively. Similar to other class III substrate binding proteins, PiaA is composed of an N-terminal and a C-terminal domain bridged by an α-helix. At the inter-domain cleft, a molecule of ferrichrome is stabilized by a number of highly conserved residues. Upon ferrichrome binding, two highly flexible segments at the entrance of the cleft undergo significant conformational changes, indicating their contribution to the binding and/or release of ferrichrome. Superposition to the structure of Escherichia coli ABC transporter BtuF enabled us to define two conserved residues: Glu119 and Glu262, which were proposed to form salt bridges with two arginines of the permease subunits. Further structure-based sequence alignment revealed that the ferrichrome binding pattern is highly conserved in a series of PiaA homologs encoded by both Gram-positive and negative bacteria, which were predicted to be sensitive to albomycin, a sideromycin antibiotic derived from ferrichrome. PMID:23951167

  11. Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

    PubMed Central

    Silva-Bailão, Mirelle Garcia; Bailão, Elisa Flávia Luiz Cardoso; Lechner, Beatrix Elisabeth; Gauthier, Gregory M.; Lindner, Herbert; Bailão, Alexandre Melo; Haas, Hubertus; de Almeida Soares, Célia Maria

    2014-01-01

    Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity. PMID:25157575

  12. Accurate Electron Affinity of Iron and Fine Structures of Negative Iron ions

    PubMed Central

    Chen, Xiaolin; Luo, Zhihong; Li, Jiaming; Ning, Chuangang

    2016-01-01

    Ionization potential (IP) is defined as the amount of energy required to remove the most loosely bound electron of an atom, while electron affinity (EA) is defined as the amount of energy released when an electron is attached to a neutral atom. Both IP and EA are critical for understanding chemical properties of an element. In contrast to accurate IPs and structures of neutral atoms, EAs and structures of negative ions are relatively unexplored, especially for the transition metal anions. Here, we report the accurate EA value of Fe and fine structures of Fe− using the slow electron velocity imaging method. These measurements yield a very accurate EA value of Fe, 1235.93(28) cm−1 or 153.236(34) meV. The fine structures of Fe− were also successfully resolved. The present work provides a reliable benchmark for theoretical calculations, and also paves the way for improving the EA measurements of other transition metal atoms to the sub cm−1 accuracy. PMID:27138292

  13. Accurate Electron Affinity of Iron and Fine Structures of Negative Iron ions.

    PubMed

    Chen, Xiaolin; Luo, Zhihong; Li, Jiaming; Ning, Chuangang

    2016-05-03

    Ionization potential (IP) is defined as the amount of energy required to remove the most loosely bound electron of an atom, while electron affinity (EA) is defined as the amount of energy released when an electron is attached to a neutral atom. Both IP and EA are critical for understanding chemical properties of an element. In contrast to accurate IPs and structures of neutral atoms, EAs and structures of negative ions are relatively unexplored, especially for the transition metal anions. Here, we report the accurate EA value of Fe and fine structures of Fe(-) using the slow electron velocity imaging method. These measurements yield a very accurate EA value of Fe, 1235.93(28) cm(-1) or 153.236(34) meV. The fine structures of Fe(-) were also successfully resolved. The present work provides a reliable benchmark for theoretical calculations, and also paves the way for improving the EA measurements of other transition metal atoms to the sub cm(-1) accuracy.

  14. Paracoccidioides spp. ferrous and ferric iron assimilation pathways

    PubMed Central

    Bailão, Elisa Flávia L. C.; Lima, Patrícia de Sousa; Silva-Bailão, Mirelle G.; Bailão, Alexandre M.; Fernandes, Gabriel da Rocha; Kosman, Daniel J.; Soares, Célia Maria de Almeida

    2015-01-01

    Iron is an essential micronutrient for almost all organisms, including fungi. Usually, fungi can uptake iron through receptor-mediated internalization of a siderophore or heme, and/or reductive iron assimilation (RIA). Traditionally, the RIA pathway consists of ferric reductases (Fres), ferroxidase (Fet3) and a high-affinity iron permease (Ftr1). Paracoccidioides spp. genomes do not present an Ftr1 homolog. However, this fungus expresses zinc regulated transporter homologs (Zrts), members of the ZIP family of membrane transporters that are able in some organisms to transport zinc and iron. A 2,3,5-triphenyltetrazolium chloride (TTC)-overlay assay indicates that both Pb01 and Pb18 express a ferric reductase activity; however, 59Fe uptake assays indicate that only in Pb18 is this activity coupled to a reductase-dependent iron uptake pathway. In addition, Zrts are up-regulated in iron deprivation, as indicated by RNAseq and qRT-PCR using Pb01 transcripts. RNAseq strategy also demonstrated that transcripts related to siderophore uptake and biosynthesis are up-regulated in iron-deprived condition. The data suggest that the fungus could use both a non-classical RIA, comprising ferric reductases and Fe/Zn permeases (Zrts), and siderophore uptake pathways under iron-limited conditions. The study of iron metabolism reveals novel surface molecules that could function as accessible targets for drugs to block iron uptake and, consequently, inhibit pathogen's proliferation. PMID:26441843

  15. Dual regulation of the Arabidopsis high-affinity root iron uptake system by local and long-distance signals.

    PubMed

    Vert, Grégory A; Briat, Jean-François; Curie, Catherine

    2003-06-01

    Regulation of the root high-affinity iron uptake system by whole-plant signals was investigated at the molecular level in Arabidopsis, through monitoring FRO2 and IRT1 gene expression. These two genes encode the root ferric-chelate reductase and the high-affinity iron transporter, respectively, involved in the iron deficiency-induced uptake system. Recovery from iron-deficient conditions and modulation of apoplastic iron pools indicate that iron itself plays a major role in the regulation of root iron deficiency responses at the mRNA and protein levels. Split-root experiments show that the expression of IRT1 and FRO2 is controlled both by a local induction from the root iron pool and through a systemic pathway involving a shoot-borne signal, both signals being integrated to tightly control production of the root iron uptake proteins. We also show that IRT1 and FRO2 are expressed during the day and down-regulated at night and that this additional control is overruled by iron starvation, indicating that the nutritional status prevails on the diurnal regulation. Our work suggests, for the first time to our knowledge, that like in grasses, the root iron acquisition in strategy I plants may also be under diurnal regulation. On the basis of the new molecular insights provided in this study and given the strict coregulation of IRT1 and FRO2 observed, we present a model of local and long-distance regulation of the root iron uptake system in Arabidopsis.

  16. PHOS-Select Iron Affinity beads enrich peptides for detection of organophosphorus adducts on albumin

    PubMed Central

    Jiang, Wei; Dubrovskii, Yaroslav A; Podolskaya, Ekaterina P; Murashko, Ekaterina A; Babakov, Vladimir; Nachon, Florian; Masson, Patrick; Schopfer, Lawrence M; Lockridge, Oksana

    2013-01-01

    Albumin is covalently modified by organophosphorus toxicants (OP) on tyrosine 411, but less than 1% of albumin is modified in humans by lethal OP doses that inhibit 95% of plasma butyrylcholinesterase. A method that enriches OP-modified albumin peptides could aid analysis of low dose exposures. Soman or chlorpyrifos oxon treated human plasma was digested with pepsin. Albumin peptides were enriched by binding to Fe3+ beads at pH 11 and eluted with pH 2.6 buffer. Similarly, mouse and guinea pig albumin modified by chlorpyrifos oxon were digested with pepsin and enriched by binding to Fe3+ beads. Peptides were identified by MALDI-TOF/TOF mass spectrometry. PHOS-select Iron Affinity beads specifically enriched albumin peptides VRY411TKKVPQVST and LVRY411TKKVPQVST in a pepsin digest of human plasma. The unmodified as well as OP-modified peptides bound to the beads. The binding capacity of 500 μl beads was the pepsin digest of 2.1 μL human plasma. The limit of detection was 0.2% of OP-modified albumin peptide in 0.43 μL plasma. Enrichment of OP-modified albumin peptides by binding to Fe3+ beads is a method with potential application to diagnosis of OP pesticide and nerve agent exposure in humans, mice, and guinea pigs. PMID:24187955

  17. A systematic analysis reveals an essential role for high-affinity iron uptake system, haemolysin and CFEM domain-containing protein in iron homoeostasis and virulence in Candida glabrata.

    PubMed

    Srivastava, Vivek Kumar; Suneetha, Korivi Jyothiraj; Kaur, Rupinder

    2014-10-01

    Iron is an essential nutrient for all living organisms and human pathogens employ a battery of factors to scavenge iron from the high-affinity iron-binding host proteins. In the present study, we have elucidated, via a candidate gene approach, major iron acquisition and homoeostatic mechanisms operational in an opportunistic human fungal pathogen Candida glabrata. Phenotypic, biochemical and molecular analysis of a set of 13 C. glabrata strains, deleted for proteins potentially implicated in iron metabolism, revealed that the high-affinity reductive iron uptake system is required for utilization of alternate carbon sources and for growth under both in vitro iron-limiting and in vivo conditions. Furthermore, we show for the first time that the cysteine-rich CFEM (common in fungal extracellular membranes) domain-containing cell wall structural protein, CgCcw14, and a putative haemolysin, CgMam3, are essential for maintenance of intracellular iron content, adherence to epithelial cells and virulence. Consistent with their roles in iron homoeostasis, mitochondrial aconitase activity was lower and higher in mutants disrupted for high-affinity iron transport, and haemolysin respectively. Additionally, we present evidence that the mitochondrial frataxin, CgYfh1, is pivotal to iron metabolism. Besides yielding insights into major in vitro and in vivo iron acquisition strategies, our findings establish high-affinity iron uptake mechanisms as critical virulence determinants in C. glabrata.

  18. Arabidopsis IRT2 cooperates with the high-affinity iron uptake system to maintain iron homeostasis in root epidermal cells.

    PubMed

    Vert, Grégory; Barberon, Marie; Zelazny, Enric; Séguéla, Mathilde; Briat, Jean-François; Curie, Catherine

    2009-05-01

    Iron is an essential nutrient for all organisms but toxic when present in excess. Consequently, plants carefully regulate their iron uptake, dependent on the FRO2 ferric reductase and the IRT1 transporter, to control its homeostasis. Arabidopsis IRT2 gene, whose expression is induced in root epidermis upon iron deprivation, was shown to encode a functional iron/zinc transporter in yeast, and proposed to function in iron acquisition from the soil. In this study, we demonstrate that, unlike its close homolog IRT1, IRT2 is not involved in iron absorption from the soil since overexpression of IRT2 does not rescue the iron uptake defect of irt1-1 mutant and since a null irt2 mutant shows no chlorosis in low iron. Consistently, an IRT2-green fluorescent fusion protein, transiently expressed in culture cells, localizes to intracellular vesicles. However, IRT2 appears strictly co-regulated with FRO2 and IRT1, supporting the view that IRT2 is an integral component of the root response to iron deficiency in root epidermal cells. We propose a model where IRT2 likely prevents toxicity from IRT1-dependent iron fluxes in epidermal cells, through compartmentalization.

  19. Iron source preference and regulation of iron uptake in Cryptococcus neoformans.

    PubMed

    Jung, Won Hee; Sham, Anita; Lian, Tianshun; Singh, Arvinder; Kosman, Daniel J; Kronstad, James W

    2008-02-08

    The level of available iron in the mammalian host is extremely low, and pathogenic microbes must compete with host proteins such as transferrin for iron. Iron regulation of gene expression, including genes encoding iron uptake functions and virulence factors, is critical for the pathogenesis of the fungus Cryptococcus neoformans. In this study, we characterized the roles of the CFT1 and CFT2 genes that encode C. neoformans orthologs of the Saccharomyces cerevisiae high-affinity iron permease FTR1. Deletion of CFT1 reduced growth and iron uptake with ferric chloride and holo-transferrin as the in vitro iron sources, and the cft1 mutant was attenuated for virulence in a mouse model of infection. A reduction in the fungal burden in the brains of mice infected with the cft1 mutant was observed, thus suggesting a requirement for reductive iron acquisition during cryptococcal meningitis. CFT2 played no apparent role in iron acquisition but did influence virulence. The expression of both CFT1 and CFT2 was influenced by cAMP-dependent protein kinase, and the iron-regulatory transcription factor Cir1 positively regulated CFT1 and negatively regulated CFT2. Overall, these results indicate that C. neoformans utilizes iron sources within the host (e.g., holo-transferrin) that require Cft1 and a reductive iron uptake system.

  20. Metal ion affinity-based biomolecular recognition and conjugation inside synthetic polymer nanopores modified with iron-terpyridine complexes.

    PubMed

    Ali, Mubarak; Nasir, Saima; Nguyen, Quoc Hung; Sahoo, Jugal Kishore; Tahir, Muhammad Nawaz; Tremel, Wolfgang; Ensinger, Wolfgang

    2011-11-02

    Here we demonstrate a novel biosensing platform for the detection of lactoferrin (LFN) via metal-organic frameworks, in which the metal ions have accessible free coordination sites for binding, inside the single conical nanopores fabricated in polymeric membrane. First, monolayer of amine-terminated terpyridine (metal-chelating ligand) is covalently immobilized on the inner walls of the nanopore via carbodiimide coupling chemistry. Second, iron-terpyridine (iron-terPy) complexes are obtained by treating the terpyridine modified-nanopores with ferrous sulfate solution. The immobilized iron-terPy complexes can be used as recognition elements to fabricate biosensing nanodevice. The working principle of the proposed biosensor is based on specific noncovalent interactions between LFN and chelated metal ions in the immobilized terpyridine monolayer, leading to the selective detection of analyte protein. In addition, control experiments proved that the designed biosensor exhibits excellent biospecificity and nonfouling properties. Furthermore, complementary experiments are conducted with multipore membranes containing an array of cylindrical nanopores. We demonstrate that in the presence of LFN in the feed solution, permeation of methyl viologen (MV(2+)) and 1,5-naphthalenedisulphate (NDS(2-)) is drastically suppressed across the iron-terPy modified membranes. On the basis of these findings, we envision that apart from conventional ligand-receptor interactions, the designing and immobilization of alternative functional ligands inside the synthetic nanopores would extend this method for the construction of new metal ion affinity-based biomimetic systems for the specific binding and recognition of other biomolecules.

  1. Characterization of a high-affinity iron transport system in Acinetobacter baumannii.

    PubMed Central

    Echenique, J R; Arienti, H; Tolmasky, M E; Read, R R; Staneloni, R J; Crosa, J H; Actis, L A

    1992-01-01

    Analysis of a clinical isolate of Acinetobacter baumannii showed that this bacterium was able to grow under iron-limiting conditions, using chemically defined growth media containing different iron chelators such as human transferrin, ethylenediaminedi-(o-hydroxyphenyl)acetic acid, nitrilotriacetic acid, and 2,2'-bipyridyl. This iron uptake-proficient phenotype was due to the synthesis and secretion of a catechol-type siderophore compound. Utilization bioassays using the Salmonella typhimurium iron uptake mutants enb-1 and enb-7 proved that this siderophore is different from enterobactin. This catechol siderophore was partially purified from culture supernatants by adsorption chromatography using an XAD-7 resin. The purified component exhibited a chromatographic behavior and a UV-visible light absorption spectrum different from those of 2,3-dihydroxybenzoic acid and other bacterial catechol siderophores. Furthermore, the siderophore activity of this extracellular catechol was confirmed by its ability to stimulate energy-dependent uptake of 55Fe(III) as well as to promote the growth of A. baumannii bacterial cells under iron-deficient conditions imposed by 60 microM human transferrin. Polyacrylamide gel electrophoresis analysis showed the presence of iron-regulated proteins in both inner and outer membranes of this clinical isolate of A. baumannii. Some of these membrane proteins may be involved in the recognition and internalization of the iron-siderophore complexes. Images PMID:1447137

  2. Thermodynamic mechanism for inhibition of lactose permease by the phosphotransferase protein IIAGlc

    PubMed Central

    Hariharan, Parameswaran; Balasubramaniam, Dhandayuthapani; Peterkofsky, Alan; Kaback, H. Ronald

    2015-01-01

    In a variety of bacteria, the phosphotransferase protein IIAGlc plays a key regulatory role in catabolite repression in addition to its role in the vectorial phosphorylation of glucose catalyzed by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS). The lactose permease (LacY) of Escherichia coli catalyzes stoichiometric symport of a galactoside with an H+, using a mechanism in which sugar- and H+-binding sites become alternatively accessible to either side of the membrane. Both the expression (via regulation of cAMP levels) and the activity of LacY are subject to regulation by IIAGlc (inducer exclusion). Here we report the thermodynamic features of the IIAGlc–LacY interaction as measured by isothermal titration calorimetry (ITC). The studies show that IIAGlc binds to LacY with a Kd of about 5 μM and a stoichiometry of unity and that binding is driven by solvation entropy and opposed by enthalpy. Upon IIAGlc binding, the conformational entropy of LacY is restrained, which leads to a significant decrease in sugar affinity. By suppressing conformational dynamics, IIAGlc blocks inducer entry into cells and favors constitutive glucose uptake and utilization. Furthermore, the studies support the notion that sugar binding involves an induced-fit mechanism that is inhibited by IIAGlc binding. The precise mechanism of the inhibition of LacY by IIAGlc elucidated by ITC differs from the inhibition of melibiose permease (MelB), supporting the idea that permeases can differ in their thermodynamic response to binding IIAGlc. PMID:25675534

  3. Iron-dependent oxidative inactivation with affinity cleavage of pyruvate kinase.

    PubMed

    Murakami, Keiko; Tsubouchi, Ryoko; Fukayama, Minoru; Qiao, Shanlou; Yoshino, Masataka

    2009-07-01

    Treatment of rabbit muscle pyruvate kinase with iron/ascorbate caused an inactivation with the cleavage of peptide bond. The inactivation or fragmentation of the enzyme was prevented by addition of Mg2+, catalase, and mannitol, but ADP and PEP the substrates did not show any effect. Protective effect of catalase and mannitol suggests that hydroxyl radical produced through the ferrous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed five pairs of fragments, which were determined to result from the cleavage of the Lys114-Gly115, Glu117-Ile118, Asp177-Gly178, Gly207-Val208, and Phe243-Ile244 bonds of the enzyme by amino-terminal sequencing analysis. Protection of the enzyme by Mg2+ implies the identical binding sites of Fe2+ and Mg2+, but the cleavage sites were discriminated from the cofactor Mg2+-binding sites. Considering amino acid residues interacting with metal ions and tertiary structure, Fe2+ ion may bind to Asp177 neighboring to Gly207 and Glu117 neighboring to Lys114 and Phe243, causing the peptide cleavage by hydroxyl radical. Iron-dependent oxidative inactivation/fragmentation of pyruvate kinase can explain the decreased glycolytic flux under aerobic conditions. Intracellular free Mg2+ concentrations are responsible for the control of cellular respiration and glycolysis.

  4. Amino Acid Permeases and Virulence in Cryptococcus neoformans

    PubMed Central

    Takahashi, Juliana Possato Fernandes; Guerra, Juliana Mariotti; Santos, Dayane Cristina da Silva; Purisco, Sônia Ueda; Melhem, Márcia de Souza Carvalho; Fazioli, Raquel dos Anjos; Phanord, Clerlune; Sartorelli, Patrícia; Vallim, Marcelo A.

    2016-01-01

    Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i

  5. Amino Acid Permeases and Virulence in Cryptococcus neoformans.

    PubMed

    Martho, Kevin Felipe Cruz; de Melo, Amanda Teixeira; Takahashi, Juliana Possato Fernandes; Guerra, Juliana Mariotti; Santos, Dayane Cristina da Silva; Purisco, Sônia Ueda; Melhem, Márcia de Souza Carvalho; Fazioli, Raquel Dos Anjos; Phanord, Clerlune; Sartorelli, Patrícia; Vallim, Marcelo A; Pascon, Renata C

    2016-01-01

    Fungal opportunistic pathogens colonize various environments, from plants and wood to human and animal tissue. Regarding human pathogens, one great challenge during contrasting niche occupation is the adaptation to different conditions, such as temperature, osmolarity, salinity, pressure, oxidative stress and nutritional availability, which may constitute sources of stress that need to be tolerated and overcome. As an opportunistic pathogen, C. neoformans faces exactly these situations during the transition from the environment to the human host, encountering nutritional constraints. Our previous and current research on amino acid biosynthetic pathways indicates that amino acid permeases are regulated by the presence of the amino acids, nitrogen and temperature. Saccharomyces cerevisiae and Candida albicans have twenty-four and twenty-seven genes encoding amino acid permeases, respectively; conversely, they are scarce in number in Basidiomycetes (C. neoformans, Coprinopsis cinerea and Ustilago maydis), where nine to ten permease genes can be found depending on the species. In this study, we have demonstrated that two amino acid permeases are essential for virulence in C. neoformans. Our data showed that C. neoformans uses two global and redundant amino acid permeases, Aap4 and Aap5 to respond correctly to thermal and oxidative stress. Double deletion of these permeases causes growth arrest in C. neoformans at 37°C and in the presence of hydrogen peroxide. The inability to uptake amino acid at a higher temperature and under oxidative stress also led to virulence attenuation in vivo. Our data showed that thermosensitivity caused by the lack of permeases Aap4 and Aap5 can be remedied by alkaline conditions (higher pH) and salinity. Permeases Aap4 and Aap5 are also required during fluconazole stress and they are the target of the plant secondary metabolite eugenol, a potent antifungal inhibitor that targets amino acid permeases. In summary, our work unravels (i

  6. ArsP: a methylarsenite efflux permease

    PubMed Central

    Chen, Jian; Madegowda, Mahendra; Bhattacharjee, Hiranmoy; Rosen, Barry P.

    2015-01-01

    Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3-nitro-4-hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III)>Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus ArsP is the first identified efflux system specific for trivalent organoarsenicals. PMID:26234817

  7. Fob1 and Fob2 Proteins Are Virulence Determinants of Rhizopus oryzae via Facilitating Iron Uptake from Ferrioxamine

    PubMed Central

    Liu, Mingfu; Lin, Lin; Gebremariam, Teclegiorgis; Luo, Guanpingsheng; Skory, Christopher D.; French, Samuel W.; Chou, Tsui-Fen; Edwards, John E.; Ibrahim, Ashraf S.

    2015-01-01

    Dialysis patients with chronic renal failure receiving deferoxamine for treating iron overload are uniquely predisposed for mucormycosis, which is most often caused by Rhizopus oryzae. Although the deferoxamine siderophore is not secreted by Mucorales, previous studies established that Rhizopus species utilize iron from ferrioxamine (iron-rich form of deferoxamine). Here we determined that the CBS domain proteins of Fob1 and Fob2 act as receptors on the cell surface of R. oryzae during iron uptake from ferrioxamine. Fob1 and Fob2 cell surface expression was induced in the presence of ferrioxamine and bound radiolabeled ferrioxamine. A R. oryzae strain with targeted reduced Fob1/Fob2 expression was impaired for iron uptake, germinating, and growing on medium with ferrioxamine as the sole source of iron. This strain also exhibited reduced virulence in a deferoxamine-treated, but not the diabetic ketoacidotic (DKA), mouse model of mucormycosis. The mechanism by which R. oryzae obtains iron from ferrioxamine involves the reductase/permease uptake system since the growth on ferrioxamine supplemented medium is associated with elevated reductase activity and the use of the ferrous chelator bathophenanthroline disulfonate abrogates iron uptake and growth on medium supplemented with ferrioxamine as a sole source of iron. Finally, R. oryzae mutants with reduced copies of the high affinity iron permease (FTR1) or with decreased FTR1 expression had an impaired iron uptake from ferrioxamine in vitro and reduced virulence in the deferoxamine-treated mouse model of mucormycosis. These two receptors appear to be conserved in Mucorales, and can be the subject of future novel therapy to maintain the use of deferoxamine for treating iron-overload. PMID:25974051

  8. Fob1 and Fob2 Proteins Are Virulence Determinants of Rhizopus oryzae via Facilitating Iron Uptake from Ferrioxamine.

    PubMed

    Liu, Mingfu; Lin, Lin; Gebremariam, Teclegiorgis; Luo, Guanpingsheng; Skory, Christopher D; French, Samuel W; Chou, Tsui-Fen; Edwards, John E; Ibrahim, Ashraf S

    2015-05-01

    Dialysis patients with chronic renal failure receiving deferoxamine for treating iron overload are uniquely predisposed for mucormycosis, which is most often caused by Rhizopus oryzae. Although the deferoxamine siderophore is not secreted by Mucorales, previous studies established that Rhizopus species utilize iron from ferrioxamine (iron-rich form of deferoxamine). Here we determined that the CBS domain proteins of Fob1 and Fob2 act as receptors on the cell surface of R. oryzae during iron uptake from ferrioxamine. Fob1 and Fob2 cell surface expression was induced in the presence of ferrioxamine and bound radiolabeled ferrioxamine. A R. oryzae strain with targeted reduced Fob1/Fob2 expression was impaired for iron uptake, germinating, and growing on medium with ferrioxamine as the sole source of iron. This strain also exhibited reduced virulence in a deferoxamine-treated, but not the diabetic ketoacidotic (DKA), mouse model of mucormycosis. The mechanism by which R. oryzae obtains iron from ferrioxamine involves the reductase/permease uptake system since the growth on ferrioxamine supplemented medium is associated with elevated reductase activity and the use of the ferrous chelator bathophenanthroline disulfonate abrogates iron uptake and growth on medium supplemented with ferrioxamine as a sole source of iron. Finally, R. oryzae mutants with reduced copies of the high affinity iron permease (FTR1) or with decreased FTR1 expression had an impaired iron uptake from ferrioxamine in vitro and reduced virulence in the deferoxamine-treated mouse model of mucormycosis. These two receptors appear to be conserved in Mucorales, and can be the subject of future novel therapy to maintain the use of deferoxamine for treating iron-overload.

  9. Diversity and Evolutionary History of Iron Metabolism Genes in Diatoms

    PubMed Central

    Groussman, Ryan D.; Parker, Micaela S.; Armbrust, E. Virginia

    2015-01-01

    Ferroproteins arose early in Earth’s history, prior to the emergence of oxygenic photosynthesis and the subsequent reduction of bioavailable iron. Today, iron availability limits primary productivity in about 30% of the world’s oceans. Diatoms, responsible for nearly half of oceanic primary production, have evolved molecular strategies for coping with variable iron concentrations. Our understanding of the evolutionary breadth of these strategies has been restricted by the limited number of species for which molecular sequence data is available. To uncover the diversity of strategies marine diatoms employ to meet cellular iron demands, we analyzed 367 newly released marine microbial eukaryotic transcriptomes, which include 47 diatom species. We focused on genes encoding proteins previously identified as having a role in iron management: iron uptake (high-affinity ferric reductase, multi-copper oxidase, and Fe(III) permease); iron storage (ferritin); iron-induced protein substitutions (flavodoxin/ferredoxin, and plastocyanin/cytochrome c6) and defense against reactive oxygen species (superoxide dismutases). Homologs encoding the high-affinity iron uptake system components were detected across the four diatom Classes suggesting an ancient origin for this pathway. Ferritin transcripts were also detected in all Classes, revealing a more widespread utilization of ferritin throughout diatoms than previously recognized. Flavodoxin and plastocyanin transcripts indicate possible alternative redox metal strategies. Predicted localization signals for ferredoxin identify multiple examples of gene transfer from the plastid to the nuclear genome. Transcripts encoding four superoxide dismutase metalloforms were detected, including a putative nickel-coordinating isozyme. Taken together, our results suggest that the majority of iron metabolism genes in diatoms appear to be vertically inherited with functional diversity achieved via possible neofunctionalization of paralogs. This

  10. Diversity and Evolutionary History of Iron Metabolism Genes in Diatoms.

    PubMed

    Groussman, Ryan D; Parker, Micaela S; Armbrust, E Virginia

    2015-01-01

    Ferroproteins arose early in Earth's history, prior to the emergence of oxygenic photosynthesis and the subsequent reduction of bioavailable iron. Today, iron availability limits primary productivity in about 30% of the world's oceans. Diatoms, responsible for nearly half of oceanic primary production, have evolved molecular strategies for coping with variable iron concentrations. Our understanding of the evolutionary breadth of these strategies has been restricted by the limited number of species for which molecular sequence data is available. To uncover the diversity of strategies marine diatoms employ to meet cellular iron demands, we analyzed 367 newly released marine microbial eukaryotic transcriptomes, which include 47 diatom species. We focused on genes encoding proteins previously identified as having a role in iron management: iron uptake (high-affinity ferric reductase, multi-copper oxidase, and Fe(III) permease); iron storage (ferritin); iron-induced protein substitutions (flavodoxin/ferredoxin, and plastocyanin/cytochrome c6) and defense against reactive oxygen species (superoxide dismutases). Homologs encoding the high-affinity iron uptake system components were detected across the four diatom Classes suggesting an ancient origin for this pathway. Ferritin transcripts were also detected in all Classes, revealing a more widespread utilization of ferritin throughout diatoms than previously recognized. Flavodoxin and plastocyanin transcripts indicate possible alternative redox metal strategies. Predicted localization signals for ferredoxin identify multiple examples of gene transfer from the plastid to the nuclear genome. Transcripts encoding four superoxide dismutase metalloforms were detected, including a putative nickel-coordinating isozyme. Taken together, our results suggest that the majority of iron metabolism genes in diatoms appear to be vertically inherited with functional diversity achieved via possible neofunctionalization of paralogs. This

  11. The high-affinity metal Transporters NRAMP1 and IRT1 Team up to Take up Iron under Sufficient Metal Provision

    PubMed Central

    Castaings, Loren; Caquot, Antoine; Loubet, Stéphanie; Curie, Catherine

    2016-01-01

    Iron (Fe) and manganese (Mn) are essential metals which, when scarce in the growth medium, are respectively taken up by the root high affinity transporters IRT1 and NRAMP1 in Arabidopsis thaliana. The molecular bases for low affinity transport however remained unknown. Since IRT1 and NRAMP1 have a broad range of substrates among metals, we tested the hypothesis that they might be functionally redundant by generating nramp1 irt1 double mutants. These plants showed extreme Fe-deficiency symptoms despite optimal provision of the metal. Their phenotype, which includes low Fe and Mn contents and a defect of Fe entry into root cells as revealed by Fe staining, is rescued by high Fe supply. Using a promoter swap-based strategy, we showed that root endodermis retains the ability to carry out high affinity Fe transport and furthermore might be important to high-affinity Mn uptake. We concluded that NRAMP1 plays a pivotal role in Fe transport by cooperating with IRT1 to take up Fe in roots under replete conditions, thus providing the first evidence for a low affinity Fe uptake system in plants. PMID:27849020

  12. The high-affinity metal Transporters NRAMP1 and IRT1 Team up to Take up Iron under Sufficient Metal Provision.

    PubMed

    Castaings, Loren; Caquot, Antoine; Loubet, Stéphanie; Curie, Catherine

    2016-11-16

    Iron (Fe) and manganese (Mn) are essential metals which, when scarce in the growth medium, are respectively taken up by the root high affinity transporters IRT1 and NRAMP1 in Arabidopsis thaliana. The molecular bases for low affinity transport however remained unknown. Since IRT1 and NRAMP1 have a broad range of substrates among metals, we tested the hypothesis that they might be functionally redundant by generating nramp1 irt1 double mutants. These plants showed extreme Fe-deficiency symptoms despite optimal provision of the metal. Their phenotype, which includes low Fe and Mn contents and a defect of Fe entry into root cells as revealed by Fe staining, is rescued by high Fe supply. Using a promoter swap-based strategy, we showed that root endodermis retains the ability to carry out high affinity Fe transport and furthermore might be important to high-affinity Mn uptake. We concluded that NRAMP1 plays a pivotal role in Fe transport by cooperating with IRT1 to take up Fe in roots under replete conditions, thus providing the first evidence for a low affinity Fe uptake system in plants.

  13. Retention of chimeric Tat2-Gap1 permease in the endoplasmic reticulum induces unfolded protein response in Saccharomyces cerevisiae.

    PubMed

    Mochizuki, Takahiro; Kimata, Yukio; Uemura, Satoshi; Abe, Fumiyoshi

    2015-08-01

    In Saccharomyces cerevisiae, high-affinity tryptophan import is performed by subtle mechanisms involving tryptophan permease Tat2. We have shown that Tat2 requires 15 amino acid residues in the transmembrane domains (TMDs) for its import activity, whereas leucine permease Bap2 requires only seven corresponding residues for its leucine import. For this reason, the structure of Tat2 is elaborately designed to transport the hydrophobic and bulky tryptophan. Newly synthesized cell surface proteins first undergo endoplasmic reticulum (ER)-associated quality check before entering the secretory pathway. In this study, we used domain replacement with general amino acid permease Gap1 to show that Tat2 chimeric proteins were dysfunctional when TMD10 or TMD11 was replaced. These chimeras formed large 270-800-kDa protein complexes and were stably retained in the ER membrane without efficient degradation. In contrast, Tat2 chimeras of TMD9 or TMD12 retained some of their tryptophan import activity and underwent vacuolar degradation as observed with wild-type Tat2. Thus, ours results suggest that TMD10 and TMD11 are essential for the correct folding of Tat2, probably because of their interdomain interactions. Notably, overexpression of Tat2-Gap1 chimera of TMD10 activated the unfolded protein response (UPR) element-lacZ reporter, suggesting that ER retention of the protein aggregates induces the UPR.

  14. Reductive iron assimilation and intracellular siderophores assist extracellular siderophore-driven iron homeostasis and virulence.

    PubMed

    Condon, Bradford J; Oide, Shinichi; Gibson, Donna M; Krasnoff, Stuart B; Turgeon, B Gillian

    2014-08-01

    Iron is an essential nutrient and prudent iron acquisition and management are key traits of a successful pathogen. Fungi use nonribosomally synthesized secreted iron chelators (siderophores) or reductive iron assimilation (RIA) mechanisms to acquire iron in a high affinity manner. Previous studies with the maize pathogen Cochliobolus heterostrophus identified two genes, NPS2 and NPS6, encoding different nonribosomal peptide synthetases responsible for biosynthesis of intra- and extracellular siderophores, respectively. Deletion of NPS6 results in loss of extracellular siderophore biosynthesis, attenuated virulence, hypersensitivity to oxidative and iron-depletion stress, and reduced asexual sporulation, while nps2 mutants are phenotypically wild type in all of these traits but defective in sexual spore development when NPS2 is missing from both mating partners. Here, it is reported that nps2nps6 mutants have more severe phenotypes than both nps2 and nps6 single mutants. In contrast, mutants lacking the FTR1 or FET3 genes encoding the permease and ferroxidase components, respectively, of the alternate RIA system, are like wild type in all of the above phenotypes. However, without supplemental iron, combinatorial nps6ftr1 and nps2nps6ftr1 mutants are less virulent, are reduced in growth, and are less able to combat oxidative stress and to sporulate asexually, compared with nps6 mutants alone. These findings demonstrate that, while the role of RIA in metabolism and virulence is overshadowed by that of extracellular siderophores as a high-affinity iron acquisition mechanism in C. heterostrophus, it functions as a critical backup for the fungus.

  15. Iron

    MedlinePlus

    Iron is a mineral that our bodies need for many functions. For example, iron is part of hemoglobin, a protein which carries ... It helps our muscles store and use oxygen. Iron is also part of many other proteins and ...

  16. Substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae.

    PubMed

    Tavoulari, Sotiria; Frillingos, Stathis

    2008-02-22

    We have examined the substrate selectivity of the melibiose permease (MelY) from Enterobacter cloacae in comparison with that of the lactose permease (LacY) from Escherichia coli. Both proteins catalyze active transport of lactose or melibiose with comparable affinity and capacity. However, MelY does not transport the analogue methyl-1-thio-beta,d-galactopyranoside (TMG), which is a very efficient substrate in LacY. We show that MelY binds TMG and conserves Cys148 (helix V) as a TMG binding residue but fails to transport this ligand. Based on homology modeling, organization of the putative MelY sugar binding site is the same as that in LacY and residues irreplaceable for the symport mechanism are conserved. Moreover, only 15% of the residues where a single-Cys mutant is inactivated by site-directed alkylation differ in MelY. Using site-directed mutagenesis at these positions and engineered cross-homolog chimeras, we show that Val367, at the periplasmic end of transmembrane helix XI, contributes in defining the substrate selectivity profile. Replacement of Val367 with the MelY residue (Ala) leads to impairment of TMG uptake. Exchanging domains N6 and C6 between LacY and MelY also leads to impairment of TMG uptake. TMG uptake activity is restored by the re-introduction of a Val367 in the background of chimera N6(LacY)-C6(MelY). Much less prominent effects are found with the same mutants and chimeras for the transport of lactose or melibiose.

  17. In situ affinity purification of his-tagged protein A from Bacillus megaterium cultivation using recyclable superparamagnetic iron oxide nanoparticles.

    PubMed

    Gädke, Johannes; Kleinfeldt, Lennart; Schubert, Chris; Rohde, Manfred; Biedendieck, Rebekka; Garnweitner, Georg; Krull, Rainer

    2017-01-20

    This paper discusses the use of recyclable functionalized nanoparticles for an improved downstream processing of recombinant products. The Gram-positive bacterium Bacillus megaterium was used to secrete recombinant protein A fused to a histidine tag into the culture supernatant in shaker flasks. Superparamagnetic iron oxide nanoparticles functionalized with 3-glycidoxypropyl-trimethoxysilane-coupled-nitrilotriacetic-acid groups (GNTA-SPION) were synthesized and added directly to the growing culture. After 10min incubation time, >85% of the product was adsorbed onto the particles. The particles were magnetically separated using handheld neodymium magnets and the product was eluted. The GNTA-SPION were successfully regenerated and reused in five consecutive cycles. In the one-step purification, the purity of the product reached >99.9% regarding protein A. A very low particle concentration of 0.5g/L was sufficient for effective product separation. Bacterial growth was not influenced negatively by this concentration. Particle analysis showed similar properties between freshly synthesized and regenerated GNTA-SPION. The overall process efficiency was however influenced by partial disintegration of particle agglomerates and thus loss of particles. The demonstration of very fast in situ product removal from growing bacterial culture combined with a very high product purity within one step shows possibilities for automated large scale purification combined with recycling of biomass.

  18. Identification of a site in the phosphocarrier protein, HPr, which influences its interactions with sugar permeases of the bacterial phosphotransferase system: kinetic analyses employing site-specific mutants.

    PubMed Central

    Koch, S; Sutrina, S L; Wu, L F; Reizer, J; Schnetz, K; Rak, B; Saier, M H

    1996-01-01

    The permeases of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system (PTS), the sugar-specific enzymes II, are energized by sequential phosphoryl transfer from phosphoenolpyruvate to (i) enzyme I, (ii) the phosphocarrier protein HPr, (iii) the enzyme IIA domains of the permeases, and (iv) the enzyme IIBC domains of the permeases which transport and phosphorylate their sugar substrates. A number of site-specific mutants of HPr were examined by using kinetic approaches. Most of the mutations exerted minimal effects on the kinetic parameters characterizing reactions involving phosphoryl transfer from phospho-HPr to various sugars. However, when the well-conserved aspartyl 69 residue in HPr was changed to a glutamyl residue, the affinities for phospho-HPr of the enzymes II specific for mannitol, N-acetylglucosamine, and beta-glucosides decreased markedly without changing the maximal reaction rates. The same mutation reduced the spontaneous rate of phosphohistidyl HPr hydrolysis but did not appear to alter the rate of phosphoryl transfer from phospho-enzyme I to HPr. When the adjacent glutamyl residue 70 in HPr was changed to a lysyl residue, the Vmax values of the reactions catalyzed by the enzymes II were reduced, but the Km values remained unaltered. Changing this residue to alanine exerted little effect. Site-specific alterations in the C terminus of the beta-glucoside enzyme II which reduced the maximal reaction rate of phosphoryl transfer about 20-fold did not alter the relative kinetic parameters because of the aforementioned mutations in HPr. Published three-dimensional structural analyses of HPr and the complex of HPr with the glucose-specific enzyme IIA (IIAGlc) (homologous to the beta-glucoside and N-acetylglucosamine enzyme IIA domains) have revealed that residues 69 and 70 in HPr are distant from the active phosphorylation site and the IIAGlc binding interface in HPr. The results reported therefore suggest that residues D-69 and E-70 in

  19. Elevated temperature inhibits recruitment of transferrin-positive vesicles and induces iron-deficiency genes expression in Aiptasia pulchella host-harbored Symbiodinium.

    PubMed

    Song, Po-Ching; Wu, Tsung-Meng; Hong, Ming-Chang; Chen, Ming-Chyuan

    2015-10-01

    Coral bleaching is the consequence of disruption of the mutualistic Cnidaria-dinoflagellate association. Elevated seawater temperatures have been proposed as the most likely cause of coral bleaching whose severity is enhanced by a limitation in the bioavailability of iron. Iron is required by numerous organisms including the zooxanthellae residing inside the symbiosome of cnidarian cells. However, the knowledge of how symbiotic zooxanthellae obtain iron from the host cells and how elevated water temperature affects the association is very limited. Since cellular iron acquisition is known to be mediated through transferrin receptor-mediated endocytosis, a vesicular trafficking pathway specifically regulated by Rab4 and Rab5, we set out to examine the roles of these key proteins in the iron acquisition by the symbiotic Symbiodinium. Thus, we hypothesized that the iron recruitments into symbiotic zooxanthellae-housed symbiosomes may be dependent on rab4/rab5-mediated fusion with vesicles containing iron-bound transferrins and will be retarded under elevated temperature. In this study, we cloned a novel monolobal transferrin (ApTF) gene from the tropical sea anemone Aiptasia pulchella and confirmed that the association of ApTF with A. pulchella Rab4 (ApRab4) or A. pulchella Rab5 (ApRab5) vesicles is inhibited by elevated temperature through immunofluorescence analysis. We confirmed the iron-deficient phenomenon by demonstrating the induced overexpression of iron-deficiency-responsive genes, flavodoxin and high-affinity iron permease 1, and reduced intracellular iron concentration in zooxanthellae under desferrioxamine B (iron chelator) and high temperature treatment. In conclusion, our data are consistent with algal iron deficiency being a contributing factor for the thermal stress-induced bleaching of symbiotic cnidarians.

  20. Proline transport and stress tolerance of ammonia-insensitive mutants of the PUT4-encoded proline-specific permease in yeast.

    PubMed

    Poole, Kate; E Walker, Michelle; Warren, Tristan; Gardner, Jennie; McBryde, Colin; de Barros Lopes, Miguel; Jiranek, Vladimir

    2009-12-01

    The imino amino acid, proline, has roles in both cellular nutrition and response to stress. Proline uptake in Saccharomyces cerevisiae is largely mediated by a high affinity, specific permease, Put4p, and a low affinity general amino acid permease, Gap1p. Both are subject to nitrogen catabolite repression (NCR) and nitrogen catabolite inactivation (NCI). In order for proline to be fully exploited, its transport must be derepressed, as occurs upon depletion of preferred nitrogen sources, and molecular oxygen must be present to allow the first step of catabolism via proline oxidase. This study focuses on the isolation of variants of Put4p, which are insensitive to repression by a preferred nitrogen source (ammonia) and their subsequent effect on proline transport and stress tolerance. Specific amino acid residues in the carboxy-terminal region of Put4p were targeted by site-directed mutagenesis. Substitution at Serine(605), a potential phosphorylation target, led to the amelioration of ammonia-induced down-regulation of Put4p. When combined with a promoter mutation (-160), the S(605)A mutation resulted in increased proline uptake and accumulation. This increase in proline accumulation was associated with increased cell viability in conditions of high temperature and osmotic stress raising possible benefits in industrial fermentation applications.

  1. Substrate-Induced Ubiquitylation and Endocytosis of Yeast Amino Acid Permeases

    PubMed Central

    Ghaddar, Kassem; Merhi, Ahmad; Saliba, Elie; Krammer, Eva-Maria; Prévost, Martine

    2014-01-01

    Many plasma membrane transporters are downregulated by ubiquitylation, endocytosis, and delivery to the lysosome in response to various stimuli. We report here that two amino acid transporters of Saccharomyces cerevisiae, the general amino acid permease (Gap1) and the arginine-specific permease (Can1), undergo ubiquitin-dependent downregulation in response to their substrates and that this downregulation is not due to intracellular accumulation of the transported amino acids but to transport catalysis itself. Following an approach based on permease structural modeling, mutagenesis, and kinetic parameter analysis, we obtained evidence that substrate-induced endocytosis requires transition of the permease to a conformational state preceding substrate release into the cell. Furthermore, this transient conformation must be stable enough, and thus sufficiently populated, for the permease to undergo efficient downregulation. Additional observations, including the constitutive downregulation of two active Gap1 mutants altered in cytosolic regions, support the model that the substrate-induced conformational transition inducing endocytosis involves remodeling of cytosolic regions of the permeases, thereby promoting their recognition by arrestin-like adaptors of the Rsp5 ubiquitin ligase. Similar mechanisms might control many other plasma membrane transporters according to the external concentrations of their substrates. PMID:25266656

  2. Differential D-glucose requirements of the general amino-acid permease and protein synthesis in Saccharomyces cerevisiae var. ellipsoideus.

    PubMed

    Iglesias, R; Ferreras, J M; Muñoz, R; Arias, F J; Rojo, M A; Girbés, T

    1990-01-01

    The dependence of the general aminoacid permease and protein synthesis on the availability of D-glucose as energy source was studied. Stimulation by the sugar was immediate once added to the cell suspensions and seems to be mediated by energy derived directly from glycolysis. The general aminoacid permease was saturated linearly with D-glucose whereas protein synthesis was saturated sigmoidealy requiring much higher concentration of the sugar than the general aminoacid permease.

  3. Siderophore Biosynthesis but Not Reductive Iron Assimilation Is Essential for the Dimorphic Fungus Nomuraea rileyi Conidiation, Dimorphism Transition, Resistance to Oxidative Stress, Pigmented Microsclerotium Formation, and Virulence

    PubMed Central

    Li, Yan; Wang, Zhongkang; Liu, Xuee; Song, Zhangyong; Li, Ren; Shao, Changwen; Yin, Youping

    2016-01-01

    Iron is an indispensable factor for the dimorphic insect pathogenic Nomuraea rileyi to form persistent microsclerotia which can replace conidia or blastospores for commercial mass production. There are two high affinity iron acquisition pathways in N. rileyi, siderophore-assisted iron mobilization and reductive iron assimilation systems. Transcription of the two iron uptake pathways related genes is induced under iron-limiting conditions. Stage-specific iron uptake-related genes expression during microsclerotia development shows siderophore-mediated iron acquisition genes are rigorously upregulated specifically during the formation and mature period while reductive iron assimilation related genes just display a higher expression at the late maturation period. Abrogation of reductive iron assimilation, by the deletion of the high affinity iron permease (NrFtrA), has no visible effect on microsclerotia biogenesis in N. rileyi. In sharp contrast, N. rileyi L-ornithine-N5-monooxygenase (NrSidA), required for synthesis of all siderophores, is absolutely necessary for the development of pigmented microsclerotia. In agreement with the lower intracellular iron contents of microsclerotia in ΔNrSidA strains, not only the pigments, but both the number and the biomass are also noticeably reduced. Certain concentration of ROS is required for promoting microsclerotia biogenesis. Combined with expression pattern analysis of related genes and quantitative of intracellular iron or extracellular siderophore in WT and mutants, these data demonstrate the lack of adequate intracellular iron caused by the loss of the siderophore results in the deficiency of ROS detoxication. Furthermore, ΔNrSidA strains show significantly increased sensitivity to hydrogen peroxide. Besides, NrSidA, but not NrFtrA, play a crucial role in vegetative growth under iron-limiting conditions, conidiation, and dimorphic switching. Remarkably, the slower growth of the ΔNrSidA strains in vivo due to a reduced

  4. The inner interhelix loop 4-5 of the melibiose permease from Escherichia coli takes part in conformational changes after sugar binding.

    PubMed

    Meyer-Lipp, Kerstin; Séry, Natacha; Ganea, Constanta; Basquin, Cécile; Fendler, Klaus; Leblanc, Gérard

    2006-09-08

    Cytoplasmic loop 4-5 of the melibiose permease from Escherichia coli is essential for the process of Na+-sugar translocation (Abdel-Dayem, M., Basquin, C., Pourcher, T., Cordat, E., and Leblanc, G. (2003) J. Biol. Chem. 278, 1518-1524). In the present report, we analyze functional consequences of mutating each of the three acidic amino acids in this loop into cysteines. Among the mutants, only the E142C substitution impairs selectively Na+-sugar translocation. Because R141C has a similar defect, we investigated these two mutants in more detail. Liposomes containing purified mutated melibiose permease were adsorbed onto a solid supported lipid membrane, and transient electrical currents resulting from different substrate concentration jumps were recorded. The currents evoked by a melibiose concentration jump in the presence of Na+, previously assigned to an electrogenic conformational transition (Meyer-Lipp, K., Ganea, C., Pourcher, T., Leblanc, G., and Fendler, K. (2004) Biochemistry 43, 12606-12613), were much smaller for the two mutants than the corresponding signals in cysteineless MelB. Furthermore, in R141C the stimulating effect of melibiose on Na+ affinity was lost. Finally, whereas tryptophan fluorescence spectroscopy revealed impaired conformational changes upon melibiose binding in the mutants, fluorescence resonance energy transfer measurements indicated that the mutants still show cooperative modification of their sugar binding sites by Na+. These data suggest that: 1) loop 4-5 contributes to the coordinated interactions between the ion and sugar binding sites; 2) it participates in an electrogenic conformational transition after melibiose binding that is essential for the subsequent obligatory coupled translocation of substrates. A two-step mechanism for substrate translocation in the melibiose permease is suggested.

  5. TonB-Dependent Heme Iron Acquisition in the Tsetse Fly Symbiont Sodalis glossinidius

    PubMed Central

    Hrusa, Gili; Farmer, William; Weiss, Brian L.; Applebaum, Taylor; Roma, Jose Santinni; Szeto, Lauren; Aksoy, Serap

    2015-01-01

    Sodalis glossinidius is an intra- and extracellular symbiont of the tsetse fly (Glossina sp.), which feeds exclusively on vertebrate blood. S. glossinidius resides in a wide variety of tsetse tissues and may encounter environments that differ dramatically in iron content. The Sodalis chromosome encodes a putative TonB-dependent outer membrane heme transporter (HemR) and a putative periplasmic/inner membrane ABC heme permease system (HemTUV). Because these gene products mediate iron acquisition processes by other enteric bacteria, we characterized their regulation and physiological role in the Sodalis/tsetse system. Our results show that the hemR and tonB genes are expressed by S. glossinidius in the tsetse fly. Furthermore, transcription of hemR in Sodalis is repressed in a high-iron environment by the iron-responsive transcriptional regulator Fur. Expression of the S. glossinidius hemR and hemTUV genes in an Escherichia coli strain unable to use heme as an iron source stimulated growth in the presence of heme or hemoglobin as the sole iron source. This stimulation was dependent on the presence of either the E. coli or Sodalis tonB gene. Sodalis tonB and hemR mutant strains were defective in their ability to colonize the gut of tsetse flies that lacked endogenous symbionts, while wild-type S. glossinidius proliferated in this same environment. Finally, we show that the Sodalis HemR protein is localized to the bacterial membrane and appears to bind hemin. Collectively, this study provides strong evidence that TonB-dependent, HemR-mediated iron acquisition is important for the maintenance of symbiont homeostasis in the tsetse fly, and it provides evidence for the expression of bacterial high-affinity iron acquisition genes in insect symbionts. PMID:25681181

  6. Staphylococcus aureus FepA and FepB proteins drive heme iron utilization in Escherichia coli.

    PubMed

    Turlin, Evelyne; Débarbouillé, Michel; Augustyniak, Katarzyna; Gilles, Anne-Marie; Wandersman, Cécile

    2013-01-01

    EfeUOB-like tripartite systems are widespread in bacteria and in many cases they are encoded by genes organized into iron-regulated operons. They consist of: EfeU, a protein similar to the yeast iron permease Ftrp1; EfeO, an extracytoplasmic protein of unknown function and EfeB, also an extracytoplasmic protein with heme peroxidase activity, belonging to the DyP family. Many bacterial EfeUOB systems have been implicated in iron uptake, but a prefential iron source remains undetermined. Nevertheless, in the case of Escherichia coli, the EfeUOB system has been shown to recognize heme and to allow extracytoplasmic heme iron extraction via a deferrochelation reaction. Given the high level of sequence conservations between EfeUOB orthologs, we hypothesized that heme might be the physiological iron substrate for the other orthologous systems. To test this hypothesis, we undertook characterization of the Staphylococcus aureus FepABC system. Results presented here indicate: i) that the S. aureus FepB protein binds both heme and PPIX with high affinity, like EfeB, the E. coli ortholog; ii) that it has low peroxidase activity, comparable to that of EfeB; iii) that both FepA and FepB drive heme iron utilization, and both are required for this activity and iv) that the E. coli FepA ortholog (EfeO) cannot replace FepA in FepB-driven iron release from heme indicating protein specificity in these activities. Our results show that the function in heme iron extraction is conserved in the two orthologous systems.

  7. Temperature effects on sulfate permease in a thermophile and related mesophile

    SciTech Connect

    Wang, J.L.; Woodin, T.

    1986-05-01

    The activity and stability of a specific membrane function, sulfate permease, from Penicillium duponti (PD) a thermophilic fungus capable of growth between 30 and 58/sup 0/ and from Pencilium chrysogenum (PC) a mesophial capable of growth between 4 and 33/sup 0/ were compared to determine whether such activity reflects actual or optimal growth temperature. Permease was assayed by incubating derepressed mycelia (grown in media containing 1.0 mg/l cysteic acid instead of 1.0 g/l SO/sub 4/) with radioactive sulfate, collecting mycelia at 30 sec intervals and counting the /sup 35/S incorporated into mycelial pellets. Mycelia from cells grown at 8/sup 0/ (PC only), 30/sup 0/ (PC and PD) or 50/sup 0/ (PD only) were assayed. The temperature optimum from PC cells grown at either 8 or 30/sup 0/ is 25/sup 0/, while the temperature optimum from PD cells grown at either 30 or 50/sup 0/ is 45/sup 0/. However the specific activity of the permease, the shape of the temperature optimum curve and the stability of the permease vary dramatically with the growth temperature and growth stage of the mycelia. There is an apparent ability to compensate for growth at lower temperature by either an increase in permease specific activity in 30/sup 0/ grown PD cells or a broadening of the temperature optimum curve for 8/sup 0/ grown PC. Transfer of cells grown in complete media (citrate No. 3 containing 4% glucose and 1 g/l sodium sulfate) to media lacking sulfate also results in derepression for sulfate permease. The time course and maximum amount of derepression observed reflects fungal growth temperature.

  8. A defect in iron uptake enhances the susceptibility of Cryptococcus neoformans to azole antifungal drugs

    PubMed Central

    Kim, Jeongmi; Cho, Yong-Joon; Do, Eunsoo; Choi, Jaehyuk; Hu, Guanggan; Cadieux, Brigitte; Chun, Jongsik; Lee, Younghoon; Kronstad, James W.; Jung, Won Hee

    2015-01-01

    The high-affinity reductive iron uptake system that includes a ferroxidase (Cfo1) and an iron permease (Cft1) is critical for the pathogenesis of Cryptococcus neoformans. In addition, a mutant lacking CFO1 or CFT1 not only has reduced iron uptake but also displays a markedly increased susceptibility to azole antifungal drugs. Altered antifungal susceptibility of the mutants was of particular interest because the iron uptake system has been proposed as an alternative target for antifungal treatment. In this study, we used transcriptome analysis to begin exploring the molecular mechanisms of altered antifungal susceptibility in a cfo1 mutant. The wild-type strain and the cfo1 mutant were cultured with or without the azole antifungal drug fluconazole and their transcriptomes were compared following sequencing with Illumina Genome Analyzer IIx (GAIIx) technology. As expected, treatment of both strains with fluconazole caused elevated expression of genes in the ergosterol biosynthetic pathway that includes the target enzyme Erg11. Additionally, genes differentially expressed in the cfo1 mutant were involved in iron uptake and homeostasis, mitochondrial functions and respiration. The cfo1 mutant also displayed phenotypes consistent with these changes including a reduced ratio of NAD+/NADH and down-regulation of Fe-S cluster synthesis. Moreover, combination treatment of the wild-type strain with fluconazole and the respiration inhibitor diphenyleneiodonium dramatically increased susceptibility to fluconazole. This result supports the hypothesis that down-regulation of genes required for respiration contributed to the altered fluconazole susceptibility of the cfo1 mutant. Overall, our data suggest that iron uptake and homeostasis play a key role in antifungal susceptibility and could be used as novel targets for combination treatment of cryptococcosis. Indeed, we found that iron chelation in combination with fluconazole treatment synergistically inhibited the growth of C

  9. Evidence for phospholipid microdomain formation in liquid crystalline liposomes reconstituted with Escherichia coli lactose permease.

    PubMed Central

    Lehtonen, J Y; Kinnunen, P K

    1997-01-01

    The well-characterized integral membrane protein lactose (lac) permease from Escherichia coli was reconstituted together with trace amounts (molar fraction X = 0.005 of the total phospholipid) of different pyrene-labeled phospholipid analogs into 1-palmitoyl-2-oleoyl-sn-glycero-3-sn-glycero-3-phospho-rac'-glycerol (POPG) liposomes. Effects of lac permease on bilayer lipid dynamics were investigated by measuring the excimer-to-monomer fluorescence intensity ratio IE/IM. Compared to control vesicles, the presence of lac permease (at a protein:phospholipid stoichiometry P/L of 1:4.000) increased the rate of excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by approximately fivefold. Decreasing P/L from approximately 1:4.000 to 1:7.600 decreased the IE/IM for PPDPC from 0.16 to 0.05, respectively. An increase in bilayer fluidity due to permease is unlikely, thus implying that the augmented IE/IM should arise from partial lateral segregation of PPDPC in the vesicles. This notion is supported by the further 38% increase in IE/IM observed for the pyrene-labeled Cys-148 lac permease reconstituted into POPG vesicles at P/L 1:4000. The importance of the length of the lipid-protein boundary is implicated by the reduction in IE/IM resulting from the aggregation of the lac permease in vesicles by a monoclonal antibody. Interestingly, excimer formation by 1-palmitoyl-2[6-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) was enhanced only fourfold in the presence of lac permease. Results obtained with the corresponding pyrenyl phosphatidylglycerols and -methanols were qualitatively similar to those above, thus indicating that lipid headgroup-protein interactions are not involved. Inclusion of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamino-N-(5-fluoresce inthio- carbamoyl) (DPPF, X = 0.005) into reconstituted lactose permease vesicles containing PPDPC caused a nearly 90% decrease in excimer fluorescence, whereas in control

  10. Experimental and theoretical bases of specific affinity, a cytoarchitecture-based formulation of nutrient collection proposed to supercede the Michaelis-Menten paradigm of microbial kinetics.

    PubMed

    Button, D K; Robertson, Betsy; Gustafson, Elizabeth; Zhao, Xiaoming

    2004-09-01

    A theory for solute uptake by whole cells was derived with a focus on the ability of oligobacteria to sequester nutrients. It provided a general relationship that was used to obtain the kinetic constants for in situ marine populations in the presence of naturally occurring substrates. In situ affinities of 0.9 to 400 liters g of cells(-1) h(-1) found were up to 10(3) times smaller than those from a "Marinobacter arcticus " isolate, but springtime values were greatly increased by warming. Affinities of the isolate for usual polar substrates but not for hydrocarbons were diminished by ionophores. A kinetic curve or Monod plot was constructed from the best available data for cytoarchitectural components of the isolate by using the theory together with concepts and calculations from first principles. The order of effect of these components on specific affinity was membrane potential > cytoplasmic enzyme concentration > cytoplasmic enzyme affinity > permease concentration > area of the permease site > translation coefficient > porin concentration. Component balance was influential as well; a small increase in cytoplasmic enzyme concentration gave a large increase in the effect of permease concentration. The effect of permease concentration on specific affinity was large, while the effect on K(m) was small. These results are in contrast to the Michaelis-Menten theory as applied by Monod that has uptake kinetics dependent on the quality of the permease molecules, with K(m) as an independent measure of affinity. Calculations demonstrated that most oligobacteria in the environment must use multiple substrates simultaneously to attain sufficient energy and material for growth, a requirement consistent with communities largely comprising few species.

  11. Structural model of a putrescine-cadaverine permease from Trypanosoma cruzi predicts residues vital for transport and ligand binding.

    PubMed

    Soysa, Radika; Venselaar, Hanka; Poston, Jacqueline; Ullman, Buddy; Hasne, Marie-Pierre

    2013-06-15

    The TcPOT1.1 gene from Trypanosoma cruzi encodes a high affinity putrescine-cadaverine transporter belonging to the APC (amino acid/polyamine/organocation) transporter superfamily. No experimental three-dimensional structure exists for any eukaryotic member of the APC family, and thus the structural determinants critical for function of these permeases are unknown. To elucidate the key residues involved in putrescine translocation and recognition by this APC family member, a homology model of TcPOT1.1 was constructed on the basis of the atomic co-ordinates of the Escherichia coli AdiC arginine/agmatine antiporter crystal structure. The TcPOT1.1 homology model consisted of 12 transmembrane helices with the first ten helices organized in two V-shaped antiparallel domains with discontinuities in the helical structures of transmembrane spans 1 and 6. The model suggests that Trp241 and a Glu247-Arg403 salt bridge participate in a gating system and that Asn245, Tyr148 and Tyr400 contribute to the putrescine-binding pocket. To test the validity of the model, 26 site-directed mutants were created and tested for their ability to transport putrescine and to localize to the parasite cell surface. These results support the robustness of the TcPOT1.1 homology model and reveal the importance of specific aromatic residues in the TcPOT1.1 putrescine-binding pocket.

  12. In vivo expression of the lacY gene in two segments leads to functional lac permease

    SciTech Connect

    Bibi, E.; Kaback, H.R. )

    1990-06-01

    The lacY gene of Escherichia coli was cut into two approximately equal-size fragments with Afl II and subcloned individually or together under separate lac operator/promoters in plasmid pT7-5. Under these conditions, lac permease is expressed in two portions: (i) the N-terminal portion (the N terminus, the first six putative transmembrane helices, and most of putative loop 7) and (ii) the C-terminal portion (the last six putative transmembrane helices and the C terminus). Cells harboring pT7-5 encoding both fragments transport lactose at about 30% the rate of cells expressing intact permease to a comparable steady-state level of accumulation. In contrast, cells expressing either half of the permease independently do not transport lactose. As judged by ({sup 35}S)methionine labeling and immunoblotting, intact permease in completely absent from the membrane of cells expressing lacY fragments either individually or together. Thus, transport activity must result from an association between independently synthesized pieces of lac permease. When the gene fragments are expressed individually, the N-terminal portion of the permease is observed inconsistently, and the C-terminal portion is not observed. When the gene fragments are expressed together, polypeptides identified as the N- and C-terminal moieties of the permease are found in the membrane. It is concluded that the N- or C-terminal halves of lac permease are proteolyzed when synthesized independently and that association between the two complementing polypeptides leads to a more stable, catalytically active complex.

  13. The Role of Amino Acid Permeases and Tryptophan Biosynthesis in Cryptococcus neoformans Survival

    PubMed Central

    Fernandes, João Daniel Santos; Martho, Kevin; Tofik, Veridiana; Vallim, Marcelo A.; Pascon, Renata C.

    2015-01-01

    Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (Nitrogen Catabolism Repression, NCR) and carbon sources (Carbon Catabolism Repression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (Global Amino Acid Control, GAAC). This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro. PMID:26162077

  14. The Role of Amino Acid Permeases and Tryptophan Biosynthesis in Cryptococcus neoformans Survival.

    PubMed

    Fernandes, João Daniel Santos; Martho, Kevin; Tofik, Veridiana; Vallim, Marcelo A; Pascon, Renata C

    2015-01-01

    Metabolic diversity is an important factor during microbial adaptation to different environments. Among metabolic processes, amino acid biosynthesis has been demonstrated to be relevant for survival for many microbial pathogens, whereas the association between pathogenesis and amino acid uptake and recycling are less well-established. Cryptococcus neoformans is an opportunistic fungal pathogen with many habitats. As a result, it faces frequent metabolic shifts and challenges during its life cycle. Here we studied the C. neoformans tryptophan biosynthetic pathway and found that the pathway is essential. RNAi indicated that interruptions in the biosynthetic pathway render strains inviable. However, auxotroph complementation can be partially achieved by tryptophan uptake when a non preferred nitrogen source and lower growth temperature are applied, suggesting that amino acid permeases may be the target of nitrogen catabolism repression (NCR). We used bioinformatics to search for amino acid permeases in the C. neoformans and found eight potential global permeases (AAP1 to AAP8). The transcriptional profile of them revealed that they are subjected to regulatory mechanisms which are known to respond to nutritional status in other fungi, such as (i) quality of nitrogen (Nitrogen Catabolism Repression, NCR) and carbon sources (Carbon Catabolism Repression, CCR), (ii) amino acid availability in the extracellular environment (SPS-sensing) and (iii) nutritional deprivation (Global Amino Acid Control, GAAC). This study shows that C. neoformans has fewer amino acid permeases than other model yeasts, and that these proteins may be subjected to complex regulatory mechanisms. Our data suggest that the C. neoformans tryptophan biosynthetic pathway is an excellent pharmacological target. Furthermore, inhibitors of this pathway cause Cryptococcus growth arrest in vitro.

  15. Extra- and intracellular lactose catabolism in Penicillium chrysogenum: phylogenetic and expression analysis of the putative permease and hydrolase genes.

    PubMed

    Jónás, Ágota; Fekete, Erzsébet; Flipphi, Michel; Sándor, Erzsébet; Jäger, Szilvia; Molnár, Ákos P; Szentirmai, Attila; Karaffa, Levente

    2014-07-01

    Penicillium chrysogenum is used as an industrial producer of penicillin. We investigated its catabolism of lactose, an abundant component of whey used in penicillin fermentation, comparing the type strain NRRL 1951 with the high producing strain AS-P-78. Both strains grew similarly on lactose as the sole carbon source under batch conditions, exhibiting almost identical time profiles of sugar depletion. In silico analysis of the genome sequences revealed that P. chrysogenum features at least five putative β-galactosidase (bGal)-encoding genes at the annotated loci Pc22g14540, Pc12g11750, Pc16g12750, Pc14g01510 and Pc06g00600. The first two proteins appear to be orthologs of two Aspergillus nidulans family 2 intracellular glycosyl hydrolases expressed on lactose. The latter three P. chrysogenum proteins appear to be distinct paralogs of the extracellular bGal from A. niger, LacA, a family 35 glycosyl hydrolase. The P. chrysogenum genome also specifies two putative lactose transporter genes at the annotated loci Pc16g06850 and Pc13g08630. These are orthologs of paralogs of the gene encoding the high-affinity lactose permease (lacpA) in A. nidulans for which P. chrysogenum appears to lack the ortholog. Transcript analysis of Pc22g14540 showed that it was expressed exclusively on lactose, whereas Pc12g11750 was weakly expressed on all carbon sources tested, including D-glucose. Pc16g12750 was co-expressed with the two putative intracellular bGal genes on lactose and also responded on L-arabinose. The Pc13g08630 transcript was formed exclusively on lactose. The data strongly suggest that P. chrysogenum exhibits a dual assimilation strategy for lactose, simultaneously employing extracellular and intracellular hydrolysis, without any correlation to the penicillin-producing potential of the studied strains.

  16. Characterization of Schizosaccharomyces pombe Malate Permease by Expression in Saccharomyces cerevisiae

    PubMed Central

    Camarasa, Carole; Bidard, Frédérique; Bony, Muriel; Barre, Pierre; Dequin, Sylvie

    2001-01-01

    In Saccharomyces cerevisiae, l-malic acid transport is not carrier mediated and is limited to slow, simple diffusion of the undissociated acid. Expression in S. cerevisiae of the MAE1 gene, encoding Schizosaccharomyces pombe malate permease, markedly increased l-malic acid uptake in this yeast. In this strain, at pH 3.5 (encountered in industrial processes), l-malic acid uptake involves Mae1p-mediated transport of the monoanionic form of the acid (apparent kinetic parameters: Vmax = 8.7 nmol/mg/min; Km = 1.6 mM) and some simple diffusion of the undissociated l-malic acid (Kd = 0.057 min−1). As total l-malic acid transport involved only low levels of diffusion, the Mae1p permease was further characterized in the recombinant strain. l-Malic acid transport was reversible and accumulative and depended on both the transmembrane gradient of the monoanionic acid form and the ΔpH component of the proton motive force. Dicarboxylic acids with stearic occupation closely related to l-malic acid, such as maleic, oxaloacetic, malonic, succinic and fumaric acids, inhibited l-malic acid uptake, suggesting that these compounds use the same carrier. We found that increasing external pH directly inhibited malate uptake, resulting in a lower initial rate of uptake and a lower level of substrate accumulation. In S. pombe, proton movements, as shown by internal acidification, accompanied malate uptake, consistent with the proton/dicarboxylate mechanism previously proposed. Surprisingly, no proton fluxes were observed during Mae1p-mediated l-malic acid import in S. cerevisiae, and intracellular pH remained constant. This suggests that, in S. cerevisiae, either there is a proton counterflow or the Mae1p permease functions differently from a proton/dicarboxylate symport. PMID:11526017

  17. Salmonella Utilizes d-Glucosaminate via a Mannose Family Phosphotransferase System Permease and Associated Enzymes

    PubMed Central

    Miller, Katherine A.; Phillips, Robert S.; Mrázek, Jan

    2013-01-01

    Salmonella enterica is a globally significant bacterial food-borne pathogen that utilizes a variety of carbon sources. We report here that Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) uses d-glucosaminate (2-amino-2-deoxy-d-gluconic acid) as a carbon and nitrogen source via a previously uncharacterized mannose family phosphotransferase system (PTS) permease, and we designate the genes encoding the permease dgaABCD (d-glucosaminate PTS permease components EIIA, EIIB, EIIC, and EIID). Two other genes in the dga operon (dgaE and dgaF) were required for wild-type growth of S. Typhimurium with d-glucosaminate. Transcription of dgaABCDEF was dependent on RpoN (σ54) and an RpoN-dependent activator gene we designate dgaR. Introduction of a plasmid bearing dgaABCDEF under the control of the lac promoter into Escherichia coli strains DH5α, BL21, and JM101 allowed these strains to grow on minimal medium containing d-glucosaminate as the sole carbon and nitrogen source. Biochemical and genetic data support a catabolic pathway in which d-glucosaminate, as it is transported across the cell membrane, is phosphorylated at the C-6 position by DgaABCD. DgaE converts the resulting d-glucosaminate-6-phosphate to 2-keto-3-deoxygluconate 6-phosphate (KDGP), which is subsequently cleaved by the aldolase DgaF to form glyceraldehyde-3-phosphate and pyruvate. DgaF catalyzes the same reaction as that catalyzed by Eda, a KDGP aldolase in the Entner-Doudoroff pathway, and the two enzymes can substitute for each other in their respective pathways. Examination of the Integrated Microbial Genomes database revealed that orthologs of the dga genes are largely restricted to certain enteric bacteria and a few species in the phylum Firmicutes. PMID:23836865

  18. The lysP gene encodes the lysine-specific permease.

    PubMed Central

    Steffes, C; Ellis, J; Wu, J; Rosen, B P

    1992-01-01

    Escherichia coli transports lysine by two distinct systems, one of which is specific for lysine (LysP) and the other of which is inhibited by arginine ornithine. The activity of the lysine-specific system increases with growth in acidic medium, anaerobiosis, and high concentrations of lysine. It is inhibited by the lysine analog S-(beta-aminoethyl)-L-cysteine (thiosine). Thiosine-resistant (Tsr) mutants were isolated by using transpositional mutagenesis with TnphoA. A Tsr mutant expressing alkaline phosphatase activity in intact cells was found to lack lysine-specific transport. This lysP mutation was mapped to about 46.5 min on the E. coli chromosome. The lysP-phoA fusion was cloned and used as a probe to clone the wild-type lysP gene. The nucleotide sequence of the 2.7-kb BamHI fragment was determined. An open reading frame from nucleotides 522 to 1989 was observed. The translation product of this open reading frame is predicted to be a hydrophobic protein of 489 residues. The lysP gene product exhibits sequence similarity to a family of amino acid transport proteins found in both prokaryotes and eukaryotes, including the aromatic amino acid permease of E. coli (aroP) and the arginine permease of Saccharomyces cerevisiae (CAN1). Cells carrying a plasmid with the lysP gene exhibited a 10- to 20-fold increase in the rate of lysine uptake above wild-type levels. These results demonstrate that the lysP gene encodes the lysine-specific permease. Images PMID:1315732

  19. Changes in the activity of the general amino acid permease from Saccharomyces cerevisiae var. ellipsoideus during fermentation.

    PubMed

    Lglesias, R; Ferreras, J M; Arias, F J; Muñoz, R; Girbès, T

    1990-10-20

    The evolution of the activity of the general amino acid permease and ethanol and glucose concentrations in the medium were studied in a mild fermentation process carried out by a wine strain of Saccharomyces cerevisiae var. ellipsoideus isolated from grape musts in spontaneous fermentation. The cells displayed a reduction in the activity of the general amino acid permease parallel to the increase of ethanol in the medium. This ethanol increase was not enough to promote a substantial inhibition on the total polypeptide synthesis measured as polyuridylic-acid-directed polyphenylalanine synthesis.

  20. The oligopeptide permease (Opp) of the plant pathogen Xanthomonas axonopodis pv. citri.

    PubMed

    Moutran, Alexandre; Quaggio, Ronaldo Bento; Balan, Andrea; Ferreira, Luis Carlos de Souza; Ferreira, Rita de Cássia Café

    2004-05-01

    The oligopeptide permease (Opp), a protein-dependent ABC transporter, has been found in the genome of Xanthomonas axonopodis pv. citri ( Xac), but not in Xanthomonas campestris pv. campestris ( Xcc). Sequence analysis indicated that 4 opp genes ( oppA, oppB, oppC, oppD/F), located in a 33.8-kbp DNA fragment present only in the Xac genome, are arranged in an operon-like structure and share highest sequence similarities with Streptomyces roseofulvus orthologs. Nonetheless, analyses of the GC content, codon usage, and transposon positioning suggested that the Xac opp operon does not have an exogenous origin. The presence of a stop codon at one of the ATP-binding domains of OppD/F would render the uptake system nonfunctional, but detection of a single polycistronic mRNA and periplasmic OppA in actively growing bacteria suggests that the Opp permease is active and could contribute to the distinct nutritional requirements and host specificities of the two Xanthomonas species.

  1. An expanding role for purine uptake permease-like transporters in plant secondary metabolism

    PubMed Central

    Jelesko, John G.

    2012-01-01

    For the past decade, our understanding of the plant purine uptake permease (PUP) transporter family was primarily oriented on purine nucleobase substrates and their tissue-specific expression patterns in Arabidopsis. However, a tobacco PUP-like homolog demonstrating nicotine uptake permease activity was recently shown to affect both nicotine metabolism and root cell growth. These new findings expand the physiological role for PUP-like transporters to include plant secondary metabolism. Molecular evolution analyses of PUP-like transporters indicate they are distinct group within an ancient super family of drug and metabolite transporters (DMTs). The PUP-like family originated during terrestrial plant evolution sometime between the bryophytes and the lycophytes. A phylogenetic analysis indicates that the PUP-like transporters were likely derived from a pre-existing nucleotide-sugar transporter family within the DMT super family. Within the lycophyte Selaginella, there are three paralogous groups of PUP-like transporters. One of the three PUP-like paralogous groups showed an extensive pattern of gene duplication and diversification within the angiosperm lineage, whereas the more ancestral PUP-like paralogous groups did not. Biochemical characterization of four closely related PUP-like paralogs together with model-based phylogenetic analyses indicate both subfunctionalization and neofunctionalization during the molecular evolution of angiosperm PUP-like transporters. These findings suggest that members of the PUP-like family of DMT transporters are likely involved in diverse primary and secondary plant metabolic pathways. PMID:22639664

  2. Reductive iron assimilation and intracellular siderophores assist extracellular siderophore-driven iron homeostasis and virulence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Iron is an essential nutrient and prudent iron acquisition and management are key traits of a successful pathogen. Fungi use nonribosomally synthesized secreted iron chelators (siderophores) or Reductive Iron Assimilation (RIA) mechanisms to acquire iron in a high affinity manner. Previous studies...

  3. Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli.

    PubMed Central

    Sahin-Tóth, M.; Kaback, H. R.

    1993-01-01

    Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335). Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules. Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity. All single-Cys mutants except Gly-296 --> Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques. In contrast, mutant Gly-296 --> Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from T7 promoter. Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val-315 --> Cys permease is enhanced at least 10-fold in the presence of beta-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or an H+ electrochemical gradient (delta mu-H+). The results demonstrate that only three residues in this region of the permease -Arg-302, His-322, and Glu-325-are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val-315 --> Cys mutant toward NEM in the presence of TDG or delta mu-H+ probably reflects a conformational alteration induced by either substrate binding or delta mu-H+. PMID

  4. Staphylococcus aureus NorD, a putative efflux pump coregulated with the Opp1 oligopeptide permease, contributes selectively to fitness in vivo.

    PubMed

    Ding, Yanpeng; Fu, Yingmei; Lee, Jean C; Hooper, David C

    2012-12-01

    Staphylococcus aureus readily infects humans, causing infections from mild superficial skin infections to lethal bacteremia and endocarditis. Transporters produced by S. aureus allow the pathogen to adapt to a variety of settings, including survival at sites of infection and in the presence of antibiotics. The native functions of many transporters are unknown, but their potential dual contribution to fitness and antimicrobial resistance highlights their importance in staphylococcal infections. Here, we show that S. aureus NorD, a newly recognized efflux pump of the major facilitator superfamily, contributes to fitness in a murine subcutaneous abscess model. In community-associated methicillin-resistant S. aureus (CA-MRSA) strain MW2, norD was selectively upregulated 36-fold at the infection site relative to growth in vitro, and the norD mutant demonstrated significant fitness impairment in abscesses, with fitness 20- to 40-fold lower than that of the parent MW2 strain. Plasmid-encoded NorD could complement the fitness defect of the MW2 norD mutant. Chromosomal norD expression is polycistronic with the upstream oligopeptide permease genes (opp1ABCDF), which encode an ABC oligopeptide transporter. Both norD and opp1 were upregulated in abscesses and iron-restricted culture medium and negatively regulated by Fur, but only NorD contributed to fitness in the murine abscess model.

  5. Iron and fungal pathogenesis: a case study with Cryptococcus neoformans.

    PubMed

    Jung, Won Hee; Kronstad, James W

    2008-02-01

    The acquisition of iron from mammalian hosts is an important aspect of infection because microbes must compete with the host for this nutrient and iron perception often regulates virulence factor expression. For example, iron levels are known to influence the elaboration of two major virulence factors, the polysaccharide capsule and melanin, in the pathogenic fungus Cryptococcus neoformans. This pathogen, which causes meningoencephalitis in immunocompromised people, acquires iron through the use of secreted reductants, cell surface reductases, a permease/ferroxidase uptake system and siderophore transporters. In addition, a master regulator, Cir1, integrates iron sensing with the expression of virulence factors, with growth at 37 degrees C and with signalling pathways that also influence virulence. The challenge ahead is to develop mechanistic views of the iron acquisition functions and regulatory schemes that operate when C. neoformans is in host tissue. Achieving these goals may contribute to an understanding of the notable predilection of the fungus for the mammalian central nervous system.

  6. Genetic evidence that high noninduced maltase and maltose permease activities, governed by MALx3-encoded transcriptional regulators, determine efficiency of gas production by baker's yeast in unsugared dough.

    PubMed

    Higgins, V J; Braidwood, M; Bell, P; Bissinger, P; Dawes, I W; Attfield, P V

    1999-02-01

    Strain selection and improvement in the baker's yeast industry have aimed to increase the speed of maltose fermentation in order to increase the leavening activity of industrial baking yeast. We identified two groups of baker's strains of Saccharomyces cerevisiae that can be distinguished by the mode of regulation of maltose utilization. One group (nonlagging strains), characterized by rapid maltose fermentation, had at least 12-fold more maltase and 130-fold-higher maltose permease activities than maltose-lagging strains in the absence of inducing sugar (maltose) and repressing sugar (glucose). Increasing the noninduced maltase activity of a lagging strain 13-fold led to an increase in CO2 production in unsugared dough. This increase in CO2 production also was seen when the maltose permease activity was increased 55-fold. Only when maltase and maltose permease activities were increased in concert was CO2 production by a lagging strain similar to that of a nonlagging strain. The noninduced activities of maltase and maltose permease constitute the largest determinant of whether a strain displays a nonlagging or a lagging phenotype and are dependent upon the MALx3 allele. Previous strategies for strain improvement have targeted glucose derepression of maltase and maltose permease expression. Our results suggest that increasing noninduced maltase and maltose permease levels is an important target for improved maltose metabolism in unsugared dough.

  7. Sequential truncation of the lactose permease over a three-amino acid sequence near the carboxyl terminus leads to progressive loss of activity and stability

    SciTech Connect

    McKenna, E.; Hardy, D.; Pastore, J.C.; Kaback, H.R. )

    1991-04-15

    Previous experiments are consistent with the notion that residues 396-401 (...SVFTLS...) at the carboxyl terminus of the last putative transmembrane helix of the lactose (lac) permease of Escherichia coli are important for protection against proteolytic degradation and suggest that this region of the permease may be necessary for proper folding. Stop codons (TAA) have now been substituted sequentially for amino acid codons 396-401 in the lacY gene, and the termination mutants were expressed from the plasmid pT7-5. With respect to transport, permease truncated at residue 396-or 397 is completely defective, while molecules truncated at residues 398, 399, 400, and 401, respectively, exhibit 15-25%, 30-40%, 40-45%, and 70-100% of wild-type activity. As judged by pulse-chase experiments with ({sup 35}S)methionine, wild-type permease or permease truncated at residue 401 is stable, while permease molecules truncated at position 400, 399, 398, 397, or 396 are degraded at increasingly rapid rates. The findings indicate that either the last turn of putative helix XII or the region immediately distal to helix XII is important for proper folding and protection against proteolytic degradation.

  8. Secondary structure components and properties of the melibiose permease from Escherichia coli: a fourier transform infrared spectroscopy analysis.

    PubMed Central

    Dave, N; Troullier, A; Mus-Veteau, I; Duñach, M; Leblanc, G; Padrós, E

    2000-01-01

    The structure of the melibiose permease from Escherichia coli has been investigated by Fourier transform infrared spectroscopy, using the purified transporter either in the solubilized state or reconstituted in E. coli lipids. In both instances, the spectra suggest that the permease secondary structure is dominated by alpha-helical components (up to 50%) and contains beta-structure (20%) and additional components assigned to turns, 3(10) helix, and nonordered structures (30%). Two distinct and strong absorption bands are recorded at 1660 and 1653 cm(-1), i.e., in the usual range of absorption of helices of membrane proteins. Moreover, conditions that preserve the transporter functionality (reconstitution in liposomes or solubilization with dodecyl maltoside) make possible the detection of two separate alpha-helical bands of comparable intensity. In contrast, a single intense band, centered at approximately 1656 cm(-1), is recorded from the inactive permease in Triton X-100, or a merged and broader signal is recorded after the solubilized protein is heated in dodecyl maltoside. It is suggested that in the functional permease, distinct signals at 1660 and 1653 cm(-1) arise from two different populations of alpha-helical domains. Furthermore, the sodium- and/or melibiose-induced changes in amide I line shape, and in particular, in the relative amplitudes of the 1660 and 1653 cm(-1) bands, indicate that the secondary structure is modified during the early step of sugar transport. Finally, the observation that approximately 80% of the backbone amide protons can be exchanged suggests high conformational flexibility and/or a large accessibility of the membrane domains to the aqueous solvent. PMID:10920008

  9. Alteration of Sugar-Induced Conformational Changes of the Melibiose Permease by Mutating Arg141 in Loop 4-5

    PubMed Central

    León, Xavier; Leblanc, Gérard; Padrós, Esteve

    2009-01-01

    Abstract The melibiose permease (MelB) from Escherichia coli couples the uptake of melibiose to that of Na+, Li+, or H+. In this work, we applied attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy to obtain information about the structural changes involved in substrate interaction with the R141C mutant and with the wild-type MelB reacted with N-ethylmaleimide (NEM). These modified permeases have the ability to bind the substrates but fail to transport them. It is shown that the sugar-induced ATR-FTIR difference spectra of the R141C mutant are different from those corresponding to the Cys-less permease from which it is derived. There are alterations of peaks assigned to turns and β-structures located most likely in loop 4-5. In addition, and quite notably, a peak at 1659 cm−1, assigned to changes at the level of one α-helix subpopulation, disappears in the melibiose-induced difference spectrum in the presence of Na+, suggesting a reduction of the conformational change capacity of the mutated MelB. These helices may involve structural components that couple the cation- and sugar-binding sites. On the other hand, MelB-NEM difference spectra are proportionally less disrupted than the R141C ones. Hence, the transport cycle of these two permeases, modified at two different loops, is most likely impaired at a different stage. It is proposed that the R141C mutant leads to the generation of a partially defective ternary complex that is unable to catalyze the subsequent conformational change necessary for substrate translocation. PMID:19527646

  10. The antifungal eugenol perturbs dual aromatic and branched-chain amino acid permeases in the cytoplasmic membrane of yeast.

    PubMed

    Darvishi, Emad; Omidi, Mansoor; Bushehri, Ali Akbar Shahnejat; Golshani, Ashkan; Smith, Myron L

    2013-01-01

    Eugenol is an aromatic component of clove oil that has therapeutic potential as an antifungal drug, although its mode of action and precise cellular target(s) remain ambiguous. To address this knowledge gap, a chemical-genetic profile analysis of eugenol was done using ∼4700 haploid Saccharomyces cerevisiae gene deletion mutants to reveal 21 deletion mutants with the greatest degree of susceptibility. Cellular roles of deleted genes in the most susceptible mutants indicate that the main targets for eugenol include pathways involved in biosynthesis and transport of aromatic and branched-chain amino acids. Follow-up analyses showed inhibitory effects of eugenol on amino acid permeases in the yeast cytoplasmic membrane. Furthermore, phenotypic suppression analysis revealed that eugenol interferes with two permeases, Tat1p and Gap1p, which are both involved in dual transport of aromatic and branched-chain amino acids through the yeast cytoplasmic membrane. Perturbation of cytoplasmic permeases represents a novel antifungal target and may explain previous observations that exposure to eugenol results in leakage of cell contents. Eugenol exposure may also contribute to amino acid starvation and thus holds promise as an anticancer therapeutic drug. Finally, this study provides further evidence of the usefulness of the yeast Gene Deletion Array approach in uncovering the mode of action of natural health products.

  11. Chemical rescue of Asp237-->Ala and Lys358-->Ala mutants in the lactose permease of Escherichia coli.

    PubMed

    Frillingos, S; Kaback, H R

    1996-10-15

    Asp237 (helix VII) and Lys358 (helix XI) form a salt bridge in the lactose permease, and neutral replacement of either residue inactivates. Remarkably, noncovalent neutralization of the unpaired Asp or Lys residue, respectively, with n-alkylsulfonates or n-alkylamines of appropriate size restores active transport to high levels in the mutants. Saturation with respect to the concentration of the alkylamines and different size preferences suggest that the alkylamines bind sterically at position 358. Rescue of Asp237-->Ala by alkylsulfonates is apparently more indiscriminate, since methane-, ethane-, or propane-sulfonate have comparable effects. Sodium and chloride, respectively, are also effective in rescuing the Lys358-->Ala and Asp237-->Ala mutants, while various other compounds are ineffective. In marked contrast to Asp237-->Ala or Lys358-->Ala permease, alkylsulfonates or alkylamines have no effect whatsoever on the activity of mutants with neutral replacements for Asp240, Glu269, Arg302, Lys319, His322, or Glu325. The results support the conclusion that neutral replacement of one member of the charge pair between Asp237 and Lys358 leads to inactivation because of an unpaired charge in the low dielectric of the membrane. In addition, the findings are consistent with the idea that interactions between Arg302 and Glu325, His 322 and Glu269, and Asp240 and Lys319 play important roles in the mechanism of the permease, which is not the case for either Asp237 or Lys358 or the salt bridge between the two residues.

  12. Localization of a filarial phosphate permease that is up-regulated in response to depletion of essential Wolbachia endobacteria.

    PubMed

    Arumugam, Sridhar; Hoerauf, Achim; Pfarr, Kenneth M

    2014-03-01

    Wolbachia of filarial nematodes are essential, obligate endobacteria. When depleted by doxycycline worm embryogenesis, larval development and worm survival are inhibited. The molecular basis governing the endosymbiosis between Wolbachia and their filarial host is still being deciphered. In rodent filarial nematode Litomosoides sigmodontis, a nematode encoded phosphate permease gene (Ls-ppe-1) was up-regulated at the mRNA level in response to Wolbachia depletion and this gene promises to have an important role in Wolbachia-nematode endosymbiosis. To further characterize this gene, the regulation of phosphate permease during Wolbachia depletion was studied at the protein level in L. sigmodontis and in the human filaria Onchocerca volvulus. And the localization of phosphate permease (PPE) and Wolbachia in L. sigmodontis and O. volvulus was investigated in untreated and antibiotic treated worms. Depletion of Wolbachia by tetracycline (Tet) resulted in up-regulation of Ls-ppe-1 in L. sigmodontis. On day 36 of Tet treatment, compared to controls (Con), >98% of Wolbachia were depleted with a 3-fold increase in mRNA levels of Ls-ppe-1. Anti-Ls-PPE serum used in Western blots showed up-regulation of Ls-PPE at the protein level in Tet worms on day 15 and 36 of treatment. Immunohistology revealed the localization of Wolbachia and Ls-PPE in the embryos, microfilariae and hypodermis of L. sigmodontis female worms and up-regulation of Ls-PPE in response to Wolbachia depletion. Expression of O. volvulus phosphate permease (Ov-PPE) studied using anti-Ov-PPE serum, showed up-regulation of Ov-PPE at the protein level in doxycycline treated Wolbachia depleted O. volvulus worms and immunohistology revealed localization of Ov-PPE and Wolbachia and up-regulation of Ov-PPE in the hypodermis and embryos of doxycycline treated worms. Ls-PPE and Ov-PPE are upregulated upon Wolbachia depletion in same tissues and regions where Wolbachia are located in untreated worms, reinforcing a link

  13. Bioinformatic Characterization of the 4-Toluene Sulfonate Uptake Permease (TSUP) Family of Transmembrane Proteins

    PubMed Central

    Shlykov, Maksim A.; Zheng, Wei Hao; Chen, Jonathan S.; Saier, Milton H.

    2012-01-01

    The ubiquitous sequence diverse 4-Toluene Sulfonate Uptake Permease (TSUP) family contains few characterized members and is believed to catalyze the transport of several sulfur-based compounds. Prokaryotic members of the TSUP family outnumber the eukaryotic members substantially, and in prokaryotes, but not eukaryotes, extensive lateral gene transfer occurred during family evolution. Despite unequal representation, homologues from the three taxonomic domains of life share well-conserved motifs. We show that the prototypical eight TMS topology arose from an intragenic duplication of a four TMS unit. Possibly, a two TMS α-helical hairpin structure was the precursor of the 4 TMS repeat unit. Genome context analyses confirmed the proposal of a sulfur-based compound transport role for many TSUP homologues, but functional outliers appear to be prevalent as well. Preliminary results suggest that the TSUP family is a member of a large novel superfamily that includes rhodopsins, integral membrane chaperone proteins, transmembrane electron flow carriers and several transporter families. All of these proteins probably arose via the same pathway: 2 → 4 → 8 TMSs followed by loss of a TMS either at the N- or C-terminus, depending on the family, to give the more frequent 7 TMS topology. PMID:22192777

  14. Coarse-grained Simulations of Sugar Transport and Conformational Changes of Lactose Permease

    NASA Astrophysics Data System (ADS)

    Liu, Jin; Jewel, S. M. Yead; Dutta, Prashanta

    2016-11-01

    Escherichia coli lactose permease (LacY) actively transports lactose and other galactosides across cell membranes through lactose/H+ symport process. Lactose/H+ symport is a highly complex process that involves sugar translocation, H+ transfer, as well as large-scale protein conformational changes. The complete picture of lactose/H+ symport is largely unclear due to the complexity and multiscale nature of the process. In this work, we develop the force field for sugar molecules compatible with PACE, a hybrid and coarse-grained force field that couples the united-atom protein models with the coarse-grained MARTINI water/lipid. After validation, we implement the new force field to investigate the transport of a β-D-galactopyranosyl-1-thio- β-D-galactopyranoside (TDG) molecule across a wild-type LacY during lactose/H+ symport process. Results show that the local interactions between TDG and LacY at the binding pocket are consistent with the X-ray experiment. Protonation of Glu325 stabilizes the TDG and inward-facing conformation of LacY. Protonation of Glu269 induces a dramatic protein structural reorganization and causes the expulsion of TDG from LacY to both sides of the membrane. The structural changes occur primarily in the N-terminal domain of LacY. This work is supported by NSF Grants: CBET-1250107 and CBET -1604211.

  15. Amino acids regulate the intracellular trafficking of the general amino acid permease of Saccharomycescerevisiae.

    PubMed

    Chen, Esther J; Kaiser, Chris A

    2002-11-12

    The delivery to the plasma membrane of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae is regulated by the quality of the nitrogen source in the growth medium. In an effort to define how different nitrogen sources control Gap1p sorting, we find that mutations in GDH1 and GLN1 that decrease the flux through the glutamate and glutamine synthesis pathways result in increased Gap1p sorting to the plasma membrane. Conversely, deletion of MKS1, which increases glutamate and glutamine synthesis, decreases Gap1p sorting to the plasma membrane. Glutamate and glutamine are not unusual in their ability to regulate Gap1p sorting, because the addition of all natural amino acids and many amino acid analogs to the growth medium results in increased Gap1p sorting to the vacuole. Importantly, amino acids have the capacity to signal Gap1p sorting to the vacuole regardless of whether they can be used as a source of nitrogen. Finally, we show that rapamycin does not affect Gap1p sorting, indicating that Gap1p sorting is not directly influenced by the TOR pathway. Together, these data show that amino acids are a signal for sorting Gap1p to the vacuole and imply that the nitrogen-regulated Gap1p sorting machinery responds to amino acid-like compounds rather than to the overall nutritional status associated with growth on a particular nitrogen source.

  16. High-throughput genome sequencing of lichenizing fungi to assess gene loss in the ammonium transporter/ammonia permease gene family

    PubMed Central

    2013-01-01

    Background Horizontal gene transfer has shaped the evolution of the ammonium transporter/ammonia permease gene family. Horizontal transfers of ammonium transporter/ammonia permease genes into the fungi include one transfer from archaea to the filamentous ascomycetes associated with the adaptive radiation of the leotiomyceta. The horizontally transferred gene has subsequently been lost in most of the group but has been selectively retained in lichenizing fungi. However, some groups of lichens appear to have secondarily lost the archaeal ammonium transporter. Definitive assessment of gene loss can only be made via whole genome sequencing. Results Ammonium transporter/ammonia permease gene sequences were recovered from the assembled genomes of eight lichenizing fungi in key clades including the Caliciales, the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacterium as a primary photobiont or lichens living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the lichens from the Acarosporomycetidae, Chaetotheriomycetes and Arthoniomycetes. In addition, the four ammonium transporter/ammonia permease genes from Cladonia grayi were shown to be functional by expressing the lichen genes in a strain of Saccharomyces cerevisiae in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source. Conclusions Given sufficient coverage, next-generation sequencing technology can definitively address the loss of a gene in a genome when using environmental DNA isolated from lichen thalli collected from their natural habitats. Lichen-forming fungi have been losing ammonium transporters

  17. Time-resolved study of the inner space of lactose permease.

    PubMed Central

    Nachliel, E; Pollak, N; Huppert, D; Gutman, M

    2001-01-01

    Pyranine (8-hydroxy pyrene-1,3,6-trisulfonate) is a commonly used photoacid that discharges a proton when excited to its first electronic singlet state. Follow-up of its dissociation kinetics reveals the physicochemical properties of its most immediate environment. At vanishing ionic strength the dye adsorbs to the Escherichia coli lactose permease with stoichiometry of 1:1 and an association constant of 2.5 x 10(5) M(-1). The reversal of the binding at high ionic strength and the lower pK value of the bound dye imply that positive charge(s) stabilize the dye in its site. The fluorescence decay curve of the bound dye was measured by time-correlated single photon counting and the measured transient was subjected to kinetic analysis based on the geminate recombination model. The analysis indicated that the binding domain is a cleft (between 9 and 17 A deep) characterized by low activity of water (a((water)) = 0.71), reduced diffusivity of protons, and enhanced electrostatic potential. The binding of pyranine and a substrate are not mutually exclusive; however, when the substrate is added, the dye-binding environment is better solvated. These properties, if attributed to the substrate-conducting pathway, may explain some of the forces operating on the substrate in the cavity. The reduced activities of the water strips the substrate from some of its solvation water molecules and replace them by direct interaction with the protein. In parallel, the lower dielectric constant enhances the binding of the proton to the protein, thus keeping a tight seal that prevents protons from diffusing. PMID:11222310

  18. Phosphatidylethanolamine-lactose permease interaction: a comparative study based on FRET.

    PubMed

    Suárez-Germà, Carme; Loura, Luís M S; Domènech, Oscar; Montero, M Teresa; Vázquez-Ibar, José Luís; Hernández-Borrell, Jordi

    2012-12-06

    In this work we have investigated the selectivity of lactose permease (LacY) of Escherichia coli (E. coli) for its surrounding phospholipids when reconstituted in binary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-Palmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) with 1-palmitoyl-2-oleoyl-sn-glycero-3-(phospho-rac-(1-glycerol)) (POPG). Förster resonance energy transfer (FRET) measurements have been performed to investigate the selectivity between a single tryptophan mutant of LacY used as donor (D), and two analogues of POPE and POPG labeled with pyrene in the acyl chains (Pyr-PE and Pyr-PG) used as acceptors. As a difference from previous works, now the donor has been single-W151/C154G/D68C LacY. It has been reported that the replacement of the aspartic acid in position 68 by cysteine inhibits active transport in LacY. The objectives of this work were to elucidate the phospholipid composition of the annular region of this mutant and to determine whether the mutation performed, D68C, induced changes in the protein-lipid selectivity. FRET efficiencies for Pyr-PE were always higher than for Pyr-PG. The values of the probability of each site in the annular ring being occupied by a label (μ) were similar at the studied temperatures (24 °C and 37 °C), suggesting that the lipid environment is not significantly affected when increasing the temperature. By comparing the results with those obtained for single-W151/C154G LacY, we observe that the mutation in the 68 residue indeed changes the selectivity of the protein for the phospholipids. This might be probably due to a change in the conformational dynamics of LacY.

  19. Endocytosis of a maltose permease is induced when amylolytic enzyme production is repressed in Aspergillus oryzae.

    PubMed

    Hiramoto, Tetsuya; Tanaka, Mizuki; Ichikawa, Takanori; Matsuura, Yuka; Hasegawa-Shiro, Sachiko; Shintani, Takahiro; Gomi, Katsuya

    2015-09-01

    In the filamentous fungus Aspergillus oryzae, amylolytic enzyme production is induced by the presence of maltose. Previously, we identified a putative maltose permease (MalP) gene in the maltose-utilizing cluster of A. oryzae. malP disruption causes a significant decrease in α-amylase activity and maltose consumption, indicating that MalP is a maltose transporter required for amylolytic enzyme production in A. oryzae. Although the expression of amylase genes and malP is repressed by the presence of glucose, the effect of glucose on the abundance of functional MalP is unknown. In this study, we examined the effect of glucose and other carbon sources on the subcellular localization of green fluorescence protein (GFP)-tagged MalP. After glucose addition, GFP-MalP at the plasma membrane was internalized and delivered to the vacuole. This glucose-induced internalization of GFP-MalP was inhibited by treatment with latrunculin B, an inhibitor of actin polymerization. Furthermore, GFP-MalP internalization was inhibited by repressing the HECT ubiquitin ligase HulA (ortholog of yeast Rsp5). These results suggest that MalP is transported to the vacuole by endocytosis in the presence of glucose. Besides glucose, mannose and 2-deoxyglucose also induced the endocytosis of GFP-MalP and amylolytic enzyme production was inhibited by the addition of these sugars. However, neither the subcellular localization of GFP-MalP nor amylolytic enzyme production was influenced by the addition of xylose or 3-O-methylglucose. These results imply that MalP endocytosis is induced when amylolytic enzyme production is repressed.

  20. Characterization of the Candida albicans Amino Acid Permease Family: Gap2 Is the Only General Amino Acid Permease and Gap4 Is an S-Adenosylmethionine (SAM) Transporter Required for SAM-Induced Morphogenesis

    PubMed Central

    Kraidlova, Lucie; Schrevens, Sanne; Tournu, Hélène; Van Zeebroeck, Griet; Sychrova, Hana

    2016-01-01

    ABSTRACT Amino acids are key sources of nitrogen for growth of Candida albicans. In order to detect and take up these amino acids from a broad range of different and changing nitrogen sources inside the host, this fungus must be able to adapt via its expression of genes for amino acid uptake and further metabolism. We analyzed six C. albicans putative general amino acid permeases based on their homology to the Saccharomyces cerevisiae Gap1 general amino acid permease. We generated single- and multiple-deletion strains and found that, based on growth assays and transcriptional or posttranscriptional regulation, Gap2 is the functional orthologue to ScGap1, with broad substrate specificity. Expression analysis showed that expression of all GAP genes is under control of the Csy1 amino acid sensor, which is different from the situation in S. cerevisiae, where the expression of ScGAP1 is not regulated by Ssy1. We show that Gap4 is the functional orthologue of ScSam3, the only S-adenosylmethionine (SAM) transporter in S. cerevisiae, and we report that Gap4 is required for SAM-induced morphogenesis. IMPORTANCE Candida albicans is a commensal organism that can thrive in many niches in its human host. The environmental conditions at these different niches differ quite a bit, and this fungus must be able to sense these changes and adapt its metabolism to them. Apart from glucose and other sugars, the uptake of amino acids is very important. This is underscored by the fact that the C. albicans genome encodes 6 orthologues of the Saccharomyces. cerevisiae general amino acid permease Gap1 and many other amino acid transporters. In this work, we characterize these six permeases and we show that C. albicans Gap2 is the functional orthologue of ScGap1 and that C. albicans Gap4 is an orthologue of ScSam3, an S-adenosylmethionine (SAM) transporter. Furthermore, we show that Gap4 is required for SAM-induced morphogenesis, an important virulence factor of C. albicans. PMID

  1. A dielectric affinity microbiosensor

    NASA Astrophysics Data System (ADS)

    Huang, Xian; Li, Siqi; Schultz, Jerome S.; Wang, Qian; Lin, Qiao

    2010-01-01

    We present an affinity biosensing approach that exploits changes in dielectric properties of a polymer due to its specific, reversible binding with an analyte. The approach is demonstrated using a microsensor comprising a pair of thin-film capacitive electrodes sandwiching a solution of poly(acrylamide-ran-3-acrylamidophenylboronic acid), a synthetic polymer with specific affinity to glucose. Binding with glucose induces changes in the permittivity of the polymer, which can be measured capacitively for specific glucose detection, as confirmed by experimental results at physiologically relevant concentrations. The dielectric affinity biosensing approach holds the potential for practical applications such as long-term continuous glucose monitoring.

  2. Affinity in electrophoresis.

    PubMed

    Heegaard, Niels H H

    2009-06-01

    The journal Electrophoresis has greatly influenced my approaches to biomolecular affinity studies. The methods that I have chosen as my main tools to study interacting biomolecules--native gel and later capillary zone electrophoresis--have been the topic of numerous articles in Electrophoresis. Below, the role of the journal in the development and dissemination of these techniques and applications reviewed. Many exhaustive reviews on affinity electrophoresis and affinity CE have been published in the last few years and are not in any way replaced by the present deliberations that are focused on papers published by the journal.

  3. Affine dynamics with torsion

    NASA Astrophysics Data System (ADS)

    Gültekin, Kemal

    2016-03-01

    In this study, we give a thorough analysis of a general affine gravity with torsion. After a brief exposition of the affine gravities considered by Eddington and Schrödinger, we construct and analyze different affine gravities based on the determinants of the Ricci tensor, the torsion tensor, the Riemann tensor, and their combinations. In each case we reduce equations of motion to their simplest forms and give a detailed analysis of their solutions. Our analyses lead to the construction of the affine connection in terms of the curvature and torsion tensors. Our solutions of the dynamical equations show that the curvature tensors at different points are correlated via non-local, exponential rescaling factors determined by the torsion tensor.

  4. Lectin affinity electrophoresis.

    PubMed

    Kobayashi, Yuka

    2014-01-01

    An interaction or a binding event typically changes the electrophoretic properties of a molecule. Affinity electrophoresis methods detect changes in the electrophoretic pattern of molecules (mainly macromolecules) that occur as a result of biospecific interactions or complex formation. Lectin affinity electrophoresis is a very effective method for the detection and analysis of trace amounts of glycobiological substances. It is particularly useful for isolating and separating the glycoisomers of target molecules. Here, we describe a sensitive technique for the detection of glycoproteins separated by agarose gel-lectin affinity electrophoresis that uses antibody-affinity blotting. The technique is tested using α-fetoprotein with lectin (Lens culinaris agglutinin and Phaseolus vulgaris agglutinin)-agarose gels.

  5. Iron Test

    MedlinePlus

    ... detect and help diagnose iron deficiency or iron overload. In people with anemia , these tests can help ... also be ordered when iron deficiency or iron overload is suspected. Early iron deficiency often goes unnoticed. ...

  6. Iron starvation induces apoptosis in Rhizopus oryzae in vitro.

    PubMed

    Shirazi, Fazal; Kontoyiannis, Dimitrios P; Ibrahim, Ashraf S

    2015-01-01

    Mortality associated with mucormycosis remains high despite current antifungals. Iron-starvation strategies have been shown to have promising activity against Mucorales. We hypothesized that iron starvation enhances apoptosis in Rhizopus oryzae. Apoptosis was characterized in R. oryzae transformed with RNAi plasmid targeting FTR1 expression (iron permease mutant) or empty plasmid grown in iron rich (0.125% FeCl3) and iron depleted media (YNB+1mM ferrozine and 1 mM ascorbic acid). Increased apoptosis was observed with dihydrorhodamine-123 and rhodamine-123 staining in the iron starved mutant FTR1 when compared to empty plasmid, followed by increased extracellular ATP levels. In addition, DNA fragmentation and metacaspase activity were prominent in FTR1. In contrast, Rhizopus strains grown in iron-rich medium displayed minimal apoptosis. Our results demonstrate a metacaspase dependent apoptotic process in iron deprived condition and further support the role of iron starvation strategies as an adjunct treatment for mucormycosis, a mechanism by which iron starvation affects R. oryzae.

  7. Cloning, nucleotide sequence, and characterization of mtr, the structural gene for a tryptophan-specific permease of Escherichia coli K-12.

    PubMed Central

    Heatwole, V M; Somerville, R L

    1991-01-01

    The mtr gene of Escherichia coli K-12 encodes an L-tryptophan-specific permease. This gene was originally identified through the isolation of mutations in the 69-min region of the chromosome, closely linked to argG. Cells with lesions in mtr display a phenotype of 5-methyltryptophan resistance. The mtr gene was cloned by using the mini-Mu system. The amino acid sequence of Mtr (414 codons), deduced by DNA sequence analysis, was found to be 33% identical to that of another single-component transport protein, the tyrosine-specific permease, TyrP. The hydropathy plots of the two permeases were similar. Possible operator sites for the tyrosine and tryptophan repressors are situated within the region of DNA that is likely to be the mtr promoter. PMID:1987112

  8. Effect of L-azetidine 2-carboxilic acid on the activity of the general amino-acid permease from Saccharomyces cerevisiae var. ellipsoideus.

    PubMed

    Iglesias, R; Ferreras, J M; Arias, F J; Muñoz, R; Rojo, M A; Girbés, T

    1991-01-01

    Addition of the L-proline analogue L-azetidine 2-carboxylic acid to growing cultures of Saccharomyces cerevisiae var. ellipsoideus promoted fast deactivation of the general aminoacid permease, measured as L-valine uptake, without an immediate decrease in the growth rate. Cells preincubated with the analogue for 3 h were unable to restore either growth ability or general aminoacid permease activity in analogue-free medium. Eadie-Hofstee plots of L-valine uptake in the presence of the analogue are consistent with a strong reduction in the number of active molecules of the general amino-acid permease located in the plasma membrane. Inhibitory effects on protein synthesis were seen after preincubations of the yeast with the analogue for 3 h although a 30 min preincubation had no effect.

  9. Probing of the substrate binding domain of lactose permease by a proton pulse.

    PubMed

    Nachliel, E; Gutman, M

    2001-09-03

    The lactose permease of Escherichia coli coupled proton transfer across the bacterial inner membrane with the uptake of beta-galactosides. In the present study we have used the cysteine-less C148 mutant that was selectively labeled by fluorescein maleimide on the C148 residue, which is an active component of the substrate transporting cavity. Measurements of the protonation dynamics of the bound pH indicator in the time resolved domain allowed us to probe the binding site by a free diffusing proton. The measured signal was reconstructed by numeric integration of differential rate equations that comply with the detailed balance principle and account for all proton transfer reactions taking place in the reaction mixture. This analysis yields the rate constants and pK values of all residues participating in the fast proton transfer reaction between the bulk and the protein's surface, revealing the exposed residues that react with free protons in a diffusion controlled reaction and how they transfer protons among themselves. The magnitudes of these rate constants were finally evaluated by comparison with the rate predicted by the Debye-Smoluchowski equation. The analysis of the kinetic and pK values indicated that the protein-fluorescein adduct assumes two conformation states. One is dominant above pH 7.4, while the other exists only below 7.1. In the high pH range, the enzyme assumes a constrained configuration and the rate constant of the reaction of a free diffusing proton with the bound dye is 10 times slower than a diffusion controlled reaction. In this state, the carboxylate moiety of residue E126 is in close proximity to the dye and exchanges a proton with it at a very fast rate. Below pH 7.1, the substrate binding domain is in a relaxed configuration and freely accessed by bulk protons, and the rate of proton exchange between the dye and E126 is 100,000 times slower. The relevance of these observations to the catalytic cycle is discussed.

  10. How iron controls iron.

    PubMed

    Kühn, Lukas C

    2009-12-01

    Cells regulate iron homeostasis by posttranscriptional regulation of proteins responsible for iron uptake and storage. This requires RNA-binding activity of iron-regulatory proteins, IRP1 and IRP2. Two studies recently published in Science by Vashisht et al. (2009) and Salahudeen et al. (2009) reveal how cells adjust IRP2 activity.

  11. The mae1 gene of Schizosaccharomyces pombe encodes a permease for malate and other C4 dicarboxylic acids.

    PubMed

    Grobler, J; Bauer, F; Subden, R E; Van Vuuren, H J

    1995-12-01

    The mae1 gene of the yeast Schizosaccharomyces pombe was identified on the basis of its ability to complement a mutant defective in the transport of malic acid. Analysis of the DNA sequence revealed an open reading frame of 1314 base pairs, encoding a polypeptide of 438 amino acids with a predicted molecular weight of 49 kDa. A hydropathy profile of the predicted amino acid sequence revealed a protein with ten membrane-spanning or associated domains and hydrophilic N- and C- termini. The predicted secondary structure of the protein in similar to models proposed for other integral membrane proteins from both prokaryotes and eukaryotes. The S. pombe mae1 gene encodes a single mRNA of 1.5 kb. The mea1 gene is expressed constitutively and is not subject to catabolite repression as was previously reported for the malate permease systems of Candida utilis and Hansenula anomala. The mae1 gene was mapped 2842 bp 5' to the MFml gene on chromosome I. Transport assays revealed that the mae1 gene encodes a permease involved in the uptake of L-malate, succinate and malonic acid.

  12. Affine Sphere Relativity

    NASA Astrophysics Data System (ADS)

    Minguzzi, E.

    2017-03-01

    We investigate spacetimes whose light cones could be anisotropic. We prove the equivalence of the structures: (a) Lorentz-Finsler manifold for which the mean Cartan torsion vanishes, (b) Lorentz-Finsler manifold for which the indicatrix (observer space) at each point is a convex hyperbolic affine sphere centered on the zero section, and (c) pair given by a spacetime volume and a sharp convex cone distribution. The equivalence suggests to describe (affine sphere) spacetimes with this structure, so that no algebraic-metrical concept enters the definition. As a result, this work shows how the metric features of spacetime emerge from elementary concepts such as measure and order. Non-relativistic spacetimes are obtained replacing proper spheres with improper spheres, so the distinction does not call for group theoretical elements. In physical terms, in affine sphere spacetimes the light cone distribution and the spacetime measure determine the motion of massive and massless particles (hence the dispersion relation). Furthermore, it is shown that, more generally, for Lorentz-Finsler theories non-differentiable at the cone, the lightlike geodesics and the transport of the particle momentum over them are well defined, though the curve parametrization could be undefined. Causality theory is also well behaved. Several results for affine sphere spacetimes are presented. Some results in Finsler geometry, for instance in the characterization of Randers spaces, are also included.

  13. The expression of superoxide dismutase (SOD) and a putative ABC transporter permease is inversely correlated during biofilm formation in Listeria monocytogenes 4b G

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about the molecular basis of biofilm formation in Listeria monocytogenes. The superoxide dismutase (SOD) of the deletion mutant of lm.G_1771 gene, which encodes for a putative ABC_transporter permease, is highly expressed in biofilm. In this study, the sod gene deletion mutant delta ...

  14. Iron Chelation

    MedlinePlus

    ... iron overload and need treatment. What is iron overload? Iron chelation therapy is used when you have ... may want to perform: How quickly does iron overload happen? This is different for each person. It ...

  15. Hydrogen-producing Escherichia coli strains overexpressing lactose permease: FT-IR analysis of the lactose-induced stress.

    PubMed

    Grube, Mara; Dimanta, Ilze; Gavare, Marita; Strazdina, Inese; Liepins, Janis; Juhna, Talis; Kalnenieks, Uldis

    2014-01-01

    The lactose permease gene (lacY) was overexpressed in the septuple knockout mutant of Escherichia coli, previously engineered for hydrogen production from glucose. It was expected that raising the lactose transporter activity would elevate the intracellular lactose concentration, inactivate the lactose repressor, induce the lactose operon, and as a result stimulate overall lactose consumption and conversion. However, overexpression of the lactose transporter caused a considerable growth delay in the recombinant strain on lactose, resembling to some extent the "lactose killing" phenomenon. Therefore, the recombinant strain was subjected to selection on lactose-containing media. Selection on plates with 3% lactose yielded a strain with a decreased content of the recombinant plasmid but with an improved ability to grow and produce hydrogen on lactose. Macromolecular analysis of its biomass by means of Fourier transform-infrared spectroscopy demonstrated that increase of the cellular polysaccharide content might contribute to the adaptation of E. coli to lactose stress.

  16. Role of the Oligopeptide Permease ABC Transporter of Moraxella catarrhalis in Nutrient Acquisition and Persistence in the Respiratory Tract

    PubMed Central

    Jones, Megan M.; Johnson, Antoinette; Koszelak-Rosenblum, Mary; Kirkham, Charmaine; Brauer, Aimee L.; Malkowski, Michael G.

    2014-01-01

    Moraxella catarrhalis is a strict human pathogen that causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults, resulting in significant worldwide morbidity and mortality. M. catarrhalis has a growth requirement for arginine; thus, acquiring arginine is important for fitness and survival. M. catarrhalis has a putative oligopeptide permease ABC transport operon (opp) consisting of five genes (oppB, oppC, oppD, oppF, and oppA), encoding two permeases, two ATPases, and a substrate binding protein. Thermal shift assays showed that the purified recombinant substrate binding protein OppA binds to peptides 3 to 16 amino acid residues in length regardless of the amino acid composition. A mutant in which the oppBCDFA gene cluster is knocked out showed impaired growth in minimal medium where the only source of arginine came from a peptide 5 to 10 amino acid residues in length. Whether methylated arginine supports growth of M. catarrhalis is important in understanding fitness in the respiratory tract because methylated arginine is abundant in host tissues. No growth of wild-type M. catarrhalis was observed in minimal medium in which arginine was present only in methylated form, indicating that the bacterium requires l-arginine. An oppA knockout mutant showed marked impairment in its capacity to persist in the respiratory tract compared to the wild type in a mouse pulmonary clearance model. We conclude that the Opp system mediates both uptake of peptides and fitness in the respiratory tract. PMID:25156736

  17. Regulators of pseudohyphal differentiation in Saccharomyces cerevisiae identified through multicopy suppressor analysis in ammonium permease mutant strains.

    PubMed Central

    Lorenz, M C; Heitman, J

    1998-01-01

    Nitrogen-starved diploid cells of the yeast Saccharomyces cerevisiae differentiate into a filamentous, pseudohyphal growth form. Recognition of nitrogen starvation is mediated, at least in part, by the ammonium permease Mep2p and the Galpha subunit Gpa2p. Genetic activation of the pheromone-responsive MAP kinase cascade, which is also required for filamentous growth, only weakly suppresses the filamentation defect of Deltamep2/Deltamep2 and Deltagpa2/Deltagpa2 strain. Surprisingly, deletion of Mep1p, an ammonium permease not previously thought to regulate differentiation, significantly enhances the potency of MAP kinase activation, such that the STE11-4 allele induces filamentation to near wild-type levels in Deltamep1/Deltamep1 Deltamep2/Deltamep2 and Deltamep1/Deltamep1 Deltagpa2/Deltagpa2 strains. To identify additional regulatory components, we isolated high-copy suppressors of the filamentation defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant. Multicopy expression of TEC1, PHD1, PHD2 (MSS10/MSN1/FUP4), MSN5, CDC6, MSS11, MGA1, SKN7, DOT6, HMS1, HMS2, or MEP2 each restored filamentation in a Deltamep1/Deltamep1 Deltamep2/Deltamep2 strain. Overexpression of SRK1 (SSD1), URE2, DAL80, MEP1, or MEP3 suppressed only the growth defect of the Deltamep1/Deltamep1 Deltamep2/Deltamep2 mutant strain. Characterization of these genes through deletion analysis and epistasis underscores the complexity of this developmental pathway and suggests that stress conditions other than nitrogen deprivation may also promote filamentous growth. PMID:9832522

  18. [Iron and invasive fungal infection].

    PubMed

    Álvarez, Florencio; Fernández-Ruiz, Mario; Aguado, José María

    2013-01-01

    Iron is an essential factor for both the growth and virulence of most of microorganisms. As a part of the innate (or nutritional) immune system, mammals have developed different mechanisms to store and transport this element in order to limit free iron bioavailability. To survive in this hostile environment, pathogenic fungi have specific uptake systems for host iron sources, one of the most important of which is based on the synthesis of siderophores-soluble, low-molecular-mass, high-affinity iron chelators. The increase in free iron that results from iron-overload conditions is a well-established risk factor for invasive fungal infection (IFI) such as mucormycosis or aspergillosis. Therefore, iron chelation may be an appealing therapeutic option for these infections. Nevertheless, deferoxamine -the first approved iron chelator- paradoxically increases the incidence of IFI, as it serves as a xeno-siderophore to Mucorales. On the contrary, the new oral iron chelators (deferiprone and deferasirox) have shown to exert a deleterious effect on fungal growth both in vitro and in animal models. The present review focuses on the role of iron metabolism in the pathogenesis of IFI and summarises the preclinical data, as well as the limited clinical experience so far, in the use of new iron chelators as treatment for mucormycosis and invasive aspergillosis.

  19. Stoichiometry and Substrate Affinity of the Mannitol Transporter, EnzymeIImtl, from Escherichia coli

    PubMed Central

    Veldhuis, Gertjan; Broos, Jaap; Poolman, Bert; Scheek, Ruud M.

    2005-01-01

    Uptake and consecutive phosphorylation of mannitol in Escherichia coli is catalyzed by the mannitol permease EnzymeIImtl. The substrate is bound at an extracellular-oriented binding site, translocated to an inward-facing site, from where it is phosphorylated, and subsequently released into the cell. Previous studies have shown the presence of both a high- and a low-affinity binding site with KD-values in the nano- and micromolar range, respectively. However, reported KD-values in literature are highly variable, which casts doubts about the reliability of the measurements and data analysis. Using an optimized binding measurement system, we investigated the discrepancies reported in literature, regarding both the variability in KD-values and the binding stoichiometry. By comparing the binding capacity obtained with flow dialysis with different methods to determine the protein concentration (UV-protein absorption, Bradford protein detection, and a LDH-linked protein assay to quantify the number of phosphorylation sites), we proved the existence of only one mannitol binding site per dimeric species of unphosphorylated EnzymeIImtl. Furthermore, the affinity of EnzymeIImtl for mannitol appeared to be dependent on the protein concentration and seemed to reflect the presence of an endogenous ligand. The dependency could be simulated assuming that >50% of the binding sites were occupied with a ligand that shows an affinity for EnzymeIImtl in the same range as mannitol. PMID:15879478

  20. Ammonium-induced internalisation of UapC, the general purine permease from Aspergillus nidulans.

    PubMed

    Valdez-Taubas, Javier; Harispe, Laura; Scazzocchio, Claudio; Gorfinkiel, Lisette; Rosa, Alberto L

    2004-01-01

    The Aspergillus nidulans UapC protein is a high-affinity, moderate-capacity, uric acid-xanthine transporter, which also displays a low transport capacity for hypoxanthine, adenine, and guanine. It has been previously shown that a functional UapC-GFP fusion protein localises at the plasma membrane. Here, we demonstrate that ammonium, a preferred nitrogen source, dramatically changes the subcellular distribution of UapC. After addition of ammonium, UapC-GFP is removed from the plasma membrane and is concentrated into the vacuolar compartment. A chimeric gene construct in which an inducible promoter, insensitive to nitrogen repression, drives the expression of UapC-GFP, allowed us to demonstrate that the ammonium-dependent redistribution of UapC can be dissociated from the transcriptional repression of the gene. These results provide further support for the occurrence of endocytosis and the lysosomal-endosomal function of the vacuolar compartment in A. nidulans.

  1. Targeted suppression of the ferroxidase and iron trafficking activities of the multicopper oxidase Fet3p from Saccharomyces cerevisiae.

    PubMed

    Wang, Tzu-Pin; Quintanar, Liliana; Severance, Scott; Solomon, Edward I; Kosman, Daniel J

    2003-07-01

    The Fet3 protein in Saccharomyces cerevisiae is a multicopper oxidase tethered to the outer surface of the yeast plasma membrane. Fet3p catalyzes the oxidation of Fe(2+) to Fe(3+); this ferroxidation reaction is an obligatory first step in high-affinity iron uptake through the permease Ftr1p. Here, kinetic analyses of several Fet3p mutants identify residues that contribute to the specificity that Fet3p has for Fe(2+), one of which is essential also to the coupling of the ferroxidase and uptake processes. The spectral and kinetic properties of the D278A, E185D and A, Y354F and A, and E185A/Y354A mutants of a soluble form of Fet3p showed that all of the mutants exhibited the normal absorbance at 330 nm and 608 nm due to the type 3 and type 1 copper sites in Fet3p, respectively. The EPR spectra of the mutants were also equivalent to wild-type, showing that the type 1 and type 2 Cu(II) sites in the proteins were not perturbed. The only marked kinetic defects measured in vitro were increases in K(M) for Fe(2+) exhibited by the D278A, E185A, Y354A, and E185A/Y354A mutants. These results suggest that these three residues contribute to the ferroxidase specificity site in Fet3p. In vivo analysis of these mutant proteins in their membrane-bound form showed that only E185 mutants exhibited kinetic defects in (59)Fe uptake. For the Fet3p(E185D) mutant, K(M) for iron was 300-fold greater than the wild-type K(M), while Fet3p(E185A) was completely inactive in support of iron uptake. In situ fluorescence demonstrated that all of the mutant Fet3 proteins, in complex with an Ftr1p:YFP fusion protein, were trafficked normally to the plasma membrane. These results suggest that E185 contributes to Fe(2+ )binding to Fet3p and to the subsequent trafficking of the Fe(3+) produced to Ftr1p.

  2. Kernel Affine Projection Algorithms

    NASA Astrophysics Data System (ADS)

    Liu, Weifeng; Príncipe, José C.

    2008-12-01

    The combination of the famed kernel trick and affine projection algorithms (APAs) yields powerful nonlinear extensions, named collectively here, KAPA. This paper is a follow-up study of the recently introduced kernel least-mean-square algorithm (KLMS). KAPA inherits the simplicity and online nature of KLMS while reducing its gradient noise, boosting performance. More interestingly, it provides a unifying model for several neural network techniques, including kernel least-mean-square algorithms, kernel adaline, sliding-window kernel recursive-least squares (KRLS), and regularization networks. Therefore, many insights can be gained into the basic relations among them and the tradeoff between computation complexity and performance. Several simulations illustrate its wide applicability.

  3. Enterococcus faecalis Uses a Phosphotransferase System Permease and a Host Colonization-Related ABC Transporter for Maltodextrin Uptake.

    PubMed

    Sauvageot, Nicolas; Mokhtari, Abdelhamid; Joyet, Philippe; Budin-Verneuil, Aurélie; Blancato, Víctor S; Repizo, Guillermo D; Henry, Céline; Pikis, Andreas; Thompson, John; Magni, Christian; Hartke, Axel; Deutscher, Josef

    2017-05-01

    Maltodextrin is a mixture of maltooligosaccharides, which are produced by the degradation of starch or glycogen. They are mostly composed of α-1,4- and some α-1,6-linked glucose residues. Genes presumed to code for the Enterococcus faecalis maltodextrin transporter were induced during enterococcal infection. We therefore carried out a detailed study of maltodextrin transport in this organism. Depending on their length (3 to 7 glucose residues), E. faecalis takes up maltodextrins either via MalT, a maltose-specific permease of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), or the ATP binding cassette (ABC) transporter MdxEFG-MsmX. Maltotriose, the smallest maltodextrin, is primarily transported by the PTS permease. A malT mutant therefore exhibits significantly reduced growth on maltose and maltotriose. The residual uptake of the trisaccharide is catalyzed by the ABC transporter, because a malT mdxF double mutant no longer grows on maltotriose. The trisaccharide arrives as maltotriose-6″-P in the cell. MapP, which dephosphorylates maltose-6'-P, also releases Pi from maltotriose-6″-P. Maltotetraose and longer maltodextrins are mainly (or exclusively) taken up via the ABC transporter, because inactivation of the membrane protein MdxF prevents growth on maltotetraose and longer maltodextrins up to at least maltoheptaose. E. faecalis also utilizes panose and isopanose, and we show for the first time, to our knowledge, that in contrast to maltotriose, its two isomers are primarily transported via the ABC transporter. We confirm that maltodextrin utilization via MdxEFG-MsmX affects the colonization capacity of E. faecalis, because inactivation of mdxF significantly reduced enterococcal colonization and/or survival in kidneys and liver of mice after intraperitoneal infection.IMPORTANCE Infections by enterococci, which are major health care-associated pathogens, are difficult to treat due to their increasing resistance to clinically

  4. Genome-wide Screening Identifies Phosphotransferase System Permease BepA to Be Involved in Enterococcus faecium Endocarditis and Biofilm Formation.

    PubMed

    Paganelli, Fernanda L; Huebner, Johannes; Singh, Kavindra V; Zhang, Xinglin; van Schaik, Willem; Wobser, Dominique; Braat, Johanna C; Murray, Barbara E; Bonten, Marc J M; Willems, Rob J L; Leavis, Helen L

    2016-07-15

    Enterococcus faecium is a common cause of nosocomial infections, of which infective endocarditis is associated with substantial mortality. In this study, we used a microarray-based transposon mapping (M-TraM) approach to evaluate a rat endocarditis model and identified a gene, originally annotated as "fruA" and renamed "bepA," putatively encoding a carbohydrate phosphotransferase system (PTS) permease (biofilm and endocarditis-associated permease A [BepA]), as important in infective endocarditis. This gene is highly enriched in E. faecium clinical isolates and absent in commensal isolates that are not associated with infection. Confirmation of the phenotype was established in a competition experiment of wild-type and a markerless bepA mutant in a rat endocarditis model. In addition, deletion of bepA impaired biofilm formation in vitro in the presence of 100% human serum and metabolism of β-methyl-D-glucoside. β-glucoside metabolism has been linked to the metabolism of glycosaminoglycans that are exposed on injured heart valves, where bacteria attach and form vegetations. Therefore, we propose that the PTS permease BepA is directly implicated in E. faecium pathogenesis.

  5. Cooperative binding of lactose and the phosphorylated phosphocarrier protein HPr(Ser-P) to the lactose/H+ symport permease of Lactobacillus brevis.

    PubMed Central

    Ye, J J; Saier, M H

    1995-01-01

    Lactobacillus brevis accumulates lactose and nonmetabolizable lactose analogues via sugar/H+ symport, but addition of glucose to the extracellular medium results in rapid efflux of the free sugar from the cells due to the uncoupling of sugar transport from proton transport. By using vesicles of L. brevis cells, we recently showed that these regulatory/effects could be attributed to the metabolite-activated ATP-dependent protein kinase-catalyzed phosphorylation of serine-46 in the phosphocarrier protein HPr [HPr(Ser-P)] of the phosphotransferase system and that a mutant form of HPr with the serine-46-->aspartate replacement ([S46D]HPr) is apparently locked in the seryl phosphorylated conformation. We here demonstrate that [S46D]HPr binds directly to inside-out membrane vesicles of L. brevis that contain the lactose permease. Sugar substrates of the permease markedly and specifically stimulate binding of [S46D]HPr to the membranes while certain transport inhibitors such as N-ethylmaleimide block binding. The pH dependency for binding follows that for transport. Wild-type HPr and the [S46A]HPr mutant protein did not appreciably compete with [S46D]HPr for binding to the permease. These results provide evidence for the direct interaction of HPr(Ser-P) with an allosteric site on the lactose/proton symporter of L. brevis for the purpose of regulating sugar accumulation in response to the metabolic needs of the cell. PMID:7831302

  6. Interrelated Effects of Cold Shock and Osmotic Pressure on the Permeability of the Escherichia coli Membrane to Permease Accumulated Substrates1

    PubMed Central

    Leder, Irwin G.

    1972-01-01

    Permease studies are generally carried out by incubating cells in growth medium with labeled substrate, collecting the cells on microporous membrane filters, and washing them free from extracellular radioactivity with ice-cold medium. Studies of thiomethylgalactoside, valine, and galactose accumulation indicate that in several strains of Escherichia coli the bacterial membrane is exquisitely sensitive to isosmotic cold shock. Substrate pools formed at 25 C may suffer almost total loss if the cells are rapidly chilled to approximately 0 C during sampling. In glycerol-grown cells, this rapid efflux of substrate is prevented or minimized if the cells are subjected at the moment of cold shock to a simultaneous hyperosmotic transition. Because of this protective effect, the apparent size of a permease accumulated substrate pool is extremely sensitive to the osmotic composition of the incubation medium and may appear to be increased as much as 10-fold when the osmolarity is reduced from approximately 0.3 to 0.1 osmolar. These differences vanish when sampling and washing are carried out with medium at room temperature. It is suggested that isosmotic cold shock causes crystallization of the liquid-like lipids within the membrane. The hydrophilic channels created in this process would facilitate the rapid efflux of permease accumulated substrates. The imposition of a simultaneous hyperosmotic transition by dehydrating the cell periphery would cause increased lipid interaction, thus preserving the integrity of the cells membrane. PMID:4591477

  7. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

    We study affine fusion with the adjoint representation. For simple Lie algebras, elementary and universal formulas determine the decomposition of a tensor product of an integrable highest-weight representation with the adjoint representation. Using the (refined) affine depth rule, we prove that equally striking results apply to adjoint affine fusion. For diagonal fusion, a coefficient equals the number of nonzero Dynkin labels of the relevant affine highest weight, minus 1. A nice lattice-polytope interpretation follows and allows the straightforward calculation of the genus-1 1-point adjoint Verlinde dimension, the adjoint affine fusion tadpole. Explicit formulas, (piecewise) polynomial in the level, are written for the adjoint tadpoles of all classical Lie algebras. We show that off-diagonal adjoint affine fusion is obtained from the corresponding tensor product by simply dropping non-dominant representations.

  8. Coregulation of the Kluyveromyces lactis lactose permease and beta-galactosidase genes is achieved by interaction of multiple LAC9 binding sites in a 2.6 kbp divergent promoter.

    PubMed Central

    Gödecke, A; Zachariae, W; Arvanitidis, A; Breunig, K D

    1991-01-01

    The coregulated genes LAC4 and LAC12 encoding beta-galactosidase and lactose permease, respectively, are responsible for the ability of the milk yeast Kluyveromyces lactis to utilise lactose. They are divergently transcribed and separated by an unusually large intergenic region of 2.6 kbp. Mapping of the upstream border of the beta-galactosidase gene (LAC4) promoter by introduction of mutations at the chromosomal locus showed that LAC4 and LAC12 share the same upstream activation sites (UAS). The UASs represent binding sites for the trans-activator LAC9, a K. lactis homologue of GAL4, conforming to the consensus sequence 5'-CGG(N5)A/T(N5)CCG-3'. Two binding sites are located in front of each of the genes at almost symmetrical positions. beta-galactosidase activity measurements as well as quantitation of LAC4 and LAC12 mRNA levels demonstrated that all four sites are required for full induction. LAC4 proximal and a LAC12 proximal sites cooperate in activating transcription of both genes. These sites are more than 1.7 kbp apart and the distal site is located more than 2.3 kbp upstream of the respective start of transcription. Thus, the distance between interacting sites is larger than in any of the well characterised yeast promoters. The contribution to gene activation differs for individual binding sites and correlates with the relative affinity of LAC9 for these sites in vitro suggesting that LAC9 binding is a rate limiting step for LAC promoter function. Images PMID:1923819

  9. Metagenomic Study of Iron Homeostasis in Iron Depositing Hot Spring Cyanobacterial Community

    NASA Technical Reports Server (NTRS)

    Brown, I.; Franklin H.; Tringe, S. G.; Klatt, C. G.; Bryant, D. A.; Sarkisova, S. A.; Guevara, M.

    2010-01-01

    Introduction: It is not clear how an iron-rich thermal hydrosphere could be hospitable to cyanobacteria, since reduced iron appears to stimulate oxidative stress in all domains of life and particularly in oxygenic phototrophs. Therefore, metagenomic study of cyanobacterial community in iron-depositing hot springs may help elucidate how oxygenic prokaryotes can withstand the extremely high concentrations of reactive oxygen species (ROS) produced by interaction between environmental Fe2+ and O2. Method: Anchor proteins from various species of cyanobacteria and some anoxygenic phototrophs were selected on the basis of their hypothetical role in Fe homeostasis and the suppression of oxidative stress and were BLASTed against the metagenomes of iron-depositing Chocolate Pots and freshwater Mushroom hot springs. Results: BLASTing proteins hypothesized to be involved in Fe homeostasis against the microbiomes from the two springs revealed that iron-depositing hot spring has a greater abundance of defensive proteins such as bacterioferritin comigratory protein (Bcp) and DNA-binding Ferritin like protein (Dps) than a fresh-water hot spring. One may speculate that the abundance of Bcp and Dps in an iron-depositing hot spring is connected to the need to suppress oxidative stress in bacteria inhabiting environments with high Fe2+ concnetration. In both springs, Bcp and Dps are concentrated within the cyanobacterial fractions of the microbial community (regardless of abundance). Fe3+ siderophore transport (from the transport system permease protein query) may be less essential to the microbial community of CP because of the high [Fe]. Conclusion: Further research is needed to confirm that these proteins are unique to photoautotrophs such as those living in iron-depositing hot spring.

  10. SMU.746-SMU.747, a putative membrane permease complex, is involved in aciduricity, acidogenesis, and biofilm formation in Streptococcus mutans.

    PubMed

    Król, Jaroslaw E; Biswas, Saswati; King, Clay; Biswas, Indranil

    2014-01-01

    Dental caries induced by Streptococcus mutans is one of the most prevalent chronic infectious diseases worldwide. The pathogenicity of S. mutans relies on the bacterium's ability to colonize tooth surfaces and survive a strongly acidic environment. We performed an ISS1 transposon mutagenesis to screen for acid-sensitive mutants of S. mutans and identified an SMU.746-SMU.747 gene cluster that is needed for aciduricity. SMU.746 and SMU.747 appear to be organized in an operon and encode a putative membrane-associated permease. SMU.746- and SMU.747-deficient mutants showed a reduced ability to grow in acidified medium. However, the short-term or long-term acid survival capacity and F1F0 ATPase activity remained unaffected in the mutants. Furthermore, deletion of both genes did not change cell membrane permeability and the oxidative and heat stress responses. Growth was severely affected even with slight acidification of the defined medium (pH 6.5). The ability of the mutant strain to acidify the defined medium during growth in the presence of glucose and sucrose was significantly reduced, although the glycolysis rate was only slightly affected. Surprisingly, deletion of the SMU.746-SMU.747 genes triggered increased biofilm formation in low-pH medium. The observed effects were more striking in a chemically defined medium. We speculate that the SMU.746-SMU.747 complex is responsible for amino acid transport, and we discuss its possible role in colonization and survival in the oral environment.

  11. Long non-coding RNA-mediated transcriptional interference of a permease gene confers drug tolerance in fission yeast.

    PubMed

    Ard, Ryan; Tong, Pin; Allshire, Robin C

    2014-11-27

    Most long non-coding RNAs (lncRNAs) encoded by eukaryotic genomes remain uncharacterized. Here we focus on a set of intergenic lncRNAs in fission yeast. Deleting one of these lncRNAs exhibited a clear phenotype: drug sensitivity. Detailed analyses of the affected locus revealed that transcription of the nc-tgp1 lncRNA regulates drug tolerance by repressing the adjacent phosphate-responsive permease gene transporter for glycerophosphodiester 1 (tgp1(+)). We demonstrate that the act of transcribing nc-tgp1 over the tgp1(+) promoter increases nucleosome density, prevents transcription factor access and thus represses tgp1(+) without the need for RNA interference or heterochromatin components. We therefore conclude that tgp1(+) is regulated by transcriptional interference. Accordingly, decreased nc-tgp1 transcription permits tgp1(+) expression upon phosphate starvation. Furthermore, nc-tgp1 loss induces tgp1(+) even in repressive conditions. Notably, drug sensitivity results directly from tgp1(+) expression in the absence of the nc-tgp1 RNA. Thus, transcription of an lncRNA governs drug tolerance in fission yeast.

  12. Synergistic interaction of glyceraldehydes-3-phosphate dehydrogenase and ArsJ, a novel organoarsenical efflux permease, confers arsenate resistance.

    PubMed

    Chen, Jian; Yoshinaga, Masafumi; Garbinski, Luis D; Rosen, Barry P

    2016-06-01

    Microbial biotransformations are major contributors to the arsenic biogeocycle. In parallel with transformations of inorganic arsenic, organoarsenicals pathways have recently been recognized as important components of global cycling of arsenic. The well-characterized pathway of resistance to arsenate is reduction coupled to arsenite efflux. Here, we describe a new pathway of arsenate resistance involving biosynthesis and extrusion of an unusual pentavalent organoarsenical. A number of arsenic resistance (ars) operons have two genes of unknown function that are linked in these operons. One, gapdh, encodes the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. The other, arsJ, encodes a major facilitator superfamily (MFS) protein. The two genes were cloned from the chromosome of Pseudomonas aeruginosa. When expressed together, but not alone, in Escherichia coli, gapdh and arsJ specifically conferred resistance to arsenate and decreased accumulation of As(V). Everted membrane vesicles from cells expressing arsJ accumulated As(V) in the presence of purified GAPDH, D-glceraldehylde 3-phosphate (G3P) and NAD(+) . GAPDH forms the unstable organoarsenical 1-arseno-3-phosphoglycerate (1As3PGA). We propose that ArsJ is an efflux permease that extrudes 1As3PGA from cells, where it rapidly dissociates into As(V) and 3-phosphoglycerate (3PGA), creating a novel pathway of arsenate resistance.

  13. Characterization of a purine permease family gene OsPUP7 involved in growth and development control in rice.

    PubMed

    Qi, Zhuyun; Xiong, Lizhong

    2013-11-01

    In this study, PUP-type cytokinin transporter genes were identified in rice (Oryza sativa L.). The Oryza sativa purine permease (OsPUP) family has 12 members that show similar predicted protein sequences with AtPUPs. To reveal the functions of OsPUP genes, we searched the T-DNA mutant library of rice and found one mutant for the member OsPUP7. The T-DNA insertion caused a new transcript that encodes a protein with 26 amino acids different from the native OsPUP7 at the C-terminus. The mutant showed multiple phenotypic changes including increased plant height, big seeds, and delayed flowering. The mutant also showed increased sensitivity to drought and salt stresses and treatments with kinetin and abscisic acid. OsPUP7 is expressed mainly in the vascular bundle, pistil, and stamens. The measurement of cytokinins (CKs) showed that CK content in the mutant spikelets accumulated higher than that in the wild type. Moreover, uptake experiment in the yeast fcy2 mutant suggested that OsPUP7 has the ability to transport caffeine, a CK derivative. Our results indicate that the PUP transport system also exists in rice, and OsPUP7 has an important role in the transport of CK, thus affecting developmental process and stress responses.

  14. The ergosterol biosynthesis inhibitor zaragozic acid promotes vacuolar degradation of the tryptophan permease Tat2p in yeast.

    PubMed

    Daicho, Katsue; Maruyama, Hironori; Suzuki, Asuka; Ueno, Masaru; Uritani, Masahiro; Ushimaru, Takashi

    2007-07-01

    Ergosterol is the yeast functional equivalent of cholesterol in mammalian cells. Deletion of the ERG6 gene, which encodes an enzyme catalyzing a late step of ergosterol biosynthesis, impedes targeting of the tryptophan permease Tat2p to the plasma membrane, but does not promote vacuolar degradation. It is unknown whether similar features appear when other steps of ergosterol biogenesis are inhibited. We show herein that the ergosterol biosynthesis inhibitor zaragozic acid (ZA) evoked massive vacuolar degradation of Tat2p, accompanied by a decrease in tryptophan uptake. ZA inhibits squalene synthetase (SQS, EC 2.5.1.21), which catalyzes the first committed step in the formation of cholesterol/ergosterol. The degradation of Tat2p was dependent on the Rsp5p-mediated ubiquitination of Tat2p and was not suppressed by deletions of VPS1, VPS27, VPS45 or PEP12. We will discuss ZA-mediated Tat2p degradation in the context of lipid rafts.

  15. Overexpression of ubiquitin and amino acid permease genes in association with antimony resistance in Leishmania tropica field isolates.

    PubMed

    Kazemi-Rad, Elham; Mohebali, Mehdi; Khadem-Erfan, Mohammad Bagher; Hajjaran, Homa; Hadighi, Ramtin; Khamesipour, Ali; Rezaie, Sassan; Saffari, Mojtaba; Raoofian, Reza; Heidari, Mansour

    2013-08-01

    The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime®) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

  16. rFTR1 is Required for Pathogenesis, and appears to be an Essential Gene, of Rhizopus oryzae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND: Rhizopus oryzae is a multinucleated fungus responsible for the majority of cases of mucormycosis. The high affinity iron permease gene (rFTR1) is required for R. oryzae iron transport in iron-limited environments. We sought to disrupt the gene to define its role in virulence. METHODS: ...

  17. Electron Affinity Calculations for Thioethers

    NASA Technical Reports Server (NTRS)

    Sulton, Deley L.; Boothe, Michael; Ball, David W.; Morales, Wilfredo

    1997-01-01

    Previous work indicated that polyphenyl thioethers possessed chemical properties, related to their electron affinities, which could allow them to function as vapor phase lubricants (VPL). Indeed, preliminary tribological tests revealed that the thioethers could function as vapor phase lubricants but not over a wide temperature and hertzian pressure range. Increasing the electron affinity of the thioethers may improve their VPL properties over this range. Adding a substituent group to the thioether will alter its electron affinity in many cases. Molecular orbital calculations were undertaken to determine the effect of five different substituent groups on the electron affinity of polyphenyl thioethers. It was found that the NO2, F, and I groups increased the thioethers electron affinity by the greatest amount. Future work will involve the addition of these groups to the thioethers followed by tribological testing to assess their VPL properties.

  18. Iron acquisition and regulation systems in Streptococcus species.

    PubMed

    Ge, Ruiguang; Sun, Xuesong

    2014-05-01

    Gram-positive Streptococcus species are responsible for millions of cases of meningitis, bacterial pneumonia, endocarditis, erysipelas and necrotizing fasciitis. Iron is essential for the growth and survival of Streptococcus in the host environment. Streptococcus species have developed various mechanisms to uptake iron from an environment with limited available iron. Streptococcus can directly extract iron from host iron-containing proteins such as ferritin, transferrin, lactoferrin and hemoproteins, or indirectly by relying on the employment of specialized secreted hemophores (heme chelators) and small siderophore molecules (high affinity ferric chelators). This review presents the most recent discoveries in the iron acquisition system of Streptococcus species - the transporters as well as the regulators.

  19. A mitochondrial GABA permease connects the GABA shunt and the TCA cycle, and is essential for normal carbon metabolism.

    PubMed

    Michaeli, Simon; Fait, Aaron; Lagor, Kelly; Nunes-Nesi, Adriano; Grillich, Nicole; Yellin, Ayelet; Bar, Dana; Khan, Munziba; Fernie, Alisdair R; Turano, Frank J; Fromm, Hillel

    2011-08-01

    In plants, γ-aminobutyric acid (GABA) accumulates in the cytosol in response to a variety of stresses. GABA is transported into mitochondria, where it is catabolized into TCA cycle or other intermediates. Although there is circumstantial evidence for mitochondrial GABA transporters in eukaryotes, none have yet been identified. Described here is an Arabidopsis protein similar in sequence and topology to unicellular GABA transporters. The expression of this protein complements a GABA-transport-deficient yeast mutant. Thus the protein was termed AtGABP to indicate GABA-permease activity. In vivo localization of GABP fused to GFP and immunobloting of subcellular fractions demonstrate its mitochondrial localization. Direct [(3) H]GABA uptake measurements into isolated mitochondria revealed impaired uptake into mitochondria of a gabp mutant compared with wild-type (WT) mitochondria, implicating AtGABP as a major mitochondrial GABA carrier. Measurements of CO(2) release, derived from radiolabeled substrates in whole seedlings and in isolated mitochondria, demonstrate impaired GABA-derived input into the TCA cycle, and a compensatory increase in TCA cycle activity in gabp mutants. Finally, growth abnormalities of gabp mutants under limited carbon availability on artificial media, and in soil under low light intensity, combined with their metabolite profiles, suggest an important role for AtGABP in primary carbon metabolism and plant growth. Thus, AtGABP-mediated transport of GABA from the cytosol into mitochondria is important to ensure proper GABA-mediated respiration and carbon metabolism. This function is particularly essential for plant growth under conditions of limited carbon.

  20. Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos.

    PubMed Central

    Labarre, C; Guzzo, J; Cavin, J F; Diviès, C

    1996-01-01

    Using degenerated primers from conserved regions of the protein sequences of malic enzymes, we amplified a 324-bp DNA fragment by PCR from Leuconostoc oenos and used this fragment as a probe for screening a Leuconostoc oenos genomic bank. Of the 2,990 clones in the genomic bank examined, 7 with overlapping fragments were isolated by performing colony hybridization experiments. Sequencing 3,453 bp from overlapping fragments revealed two open reading frames that were 1,623 and 942 nucleotides long and were followed by a putative terminator structure. The first deduced protein (molecular weight, 59,118) is very similar (level of similarity, 66%) to the malolactic enzyme of Lactococcus lactis; as in several malic enzymes, highly conserved protein regions are present. The synthesis of a protein with an apparent molecular mass of 60 kDa was highlighted by the results of labelling experiments performed with Escherichia coli minicells. The gene was expressed in E. coli and Saccharomyces cerevisiae and conferred "malolactic activity" to these species. The second open reading frame encodes a putative 34,190-Da protein which has the characteristics of a carrier protein and may have 10 membrane-spanning segments organized around a central hydrophilic core. Energy-dependent L-[14C]malate transport was observed with E. coli dicarboxylic acid transport-deficient mutants carrying the malate permease-expressing vector. Our results suggest that in Leuconostoc oenos the genes that encode the malolactic enzyme and a malate carrier protein are organized in a cluster. PMID:8919788

  1. Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli.

    PubMed

    Bogdanov, Mikhail; Heacock, Philip; Guan, Ziqiang; Dowhan, William

    2010-08-24

    Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function.

  2. Plasticity of lipid-protein interactions in the function and topogenesis of the membrane protein lactose permease from Escherichia coli

    PubMed Central

    Bogdanov, Mikhail; Heacock, Philip; Guan, Ziqiang; Dowhan, William

    2010-01-01

    Phosphatidylcholine (PC) has been widely used in place of naturally occurring phosphatidylethanolamine (PE) in reconstitution of bacterial membrane proteins. However, PC does not support native structure or function for several reconstituted transport proteins. Lactose permease (LacY) of Escherichia coli, when reconstituted in E. coli phospholipids, exhibits energy-dependent uphill and energy-independent downhill transport function and proper conformation of periplasmic domain P7, which is tightly linked to uphill transport function. LacY expressed in cells lacking PE and containing only anionic phospholipids exhibits only downhill transport and lacks native P7 conformation. Reconstitution of LacY in the presence of E. coli-derived PE, but not dioleoyl-PC, results in uphill transport. We now show that LacY exhibits uphill transport and native conformation of P7 when expressed in a mutant of E. coli in which PC completely replaces PE even though the structure is not completely native. E. coli-derived PC and synthetic PC species containing at least one saturated fatty acid also support the native conformation of P7 dependent on the presence of anionic phospholipids. Our results demonstrate that the different effects of PE and PC species on LacY structure and function cannot be explained by differences in the direct interaction of the lipid head groups with specific amino acid residues alone but are due to more complex effects of the physical and chemical properties of the lipid environment on protein structure. This conclusion is supported by the effect of different lipids on the proper folding of domain P7, which indirectly influences uphill transport function. PMID:20696931

  3. Comprehensive Mutational Analysis of Sucrose-Metabolizing Pathways in Streptococcus mutans Reveals Novel Roles for the Sucrose Phosphotransferase System Permease

    PubMed Central

    Zeng, Lin

    2013-01-01

    Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand the control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD (encoding glucan exopolysaccharide synthases), ftf (encoding the fructan exopolysaccharide synthase), and the scrAB pathway (sugar-phosphotransferase system [PTS] permease and sucrose-6-PO4 hydrolase) constitute the majority of the sucrose-catabolizing activity; however, mutations in any one of these genes alone did not affect planktonic growth on sucrose. The multiple-sugar metabolism pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on fructooligosaccharides, due in large part to increased expression of the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction system as a model for carbohydrate-dependent gene expression in strains lacking extracellular sucrases, a PlevD-cat (EIIALev) reporter was activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activation by the cognate LevQRST sugars fructose or mannose. Sucrose-dependent catabolite repression was also evident in strains containing an intact sucrose PTS. Collectively, these results reveal a novel regulatory circuitry for the control of sucrose catabolism, with a central role for ScrA. PMID:23222725

  4. Comprehensive mutational analysis of sucrose-metabolizing pathways in Streptococcus mutans reveals novel roles for the sucrose phosphotransferase system permease.

    PubMed

    Zeng, Lin; Burne, Robert A

    2013-02-01

    Sucrose is perhaps the most efficient carbohydrate for the promotion of dental caries in humans, and the primary caries pathogen Streptococcus mutans encodes multiple enzymes involved in the metabolism of this disaccharide. Here, we engineered a series of mutants lacking individual or combinations of sucrolytic pathways to understand the control of sucrose catabolism and to determine whether as-yet-undisclosed pathways for sucrose utilization were present in S. mutans. Growth phenotypes indicated that gtfBCD (encoding glucan exopolysaccharide synthases), ftf (encoding the fructan exopolysaccharide synthase), and the scrAB pathway (sugar-phosphotransferase system [PTS] permease and sucrose-6-PO(4) hydrolase) constitute the majority of the sucrose-catabolizing activity; however, mutations in any one of these genes alone did not affect planktonic growth on sucrose. The multiple-sugar metabolism pathway (msm) contributed minimally to growth on sucrose. Notably, a mutant lacking gtfBC, which cannot produce water-insoluble glucan, displayed improved planktonic growth on sucrose. Meanwhile, loss of scrA led to growth stimulation on fructooligosaccharides, due in large part to increased expression of the fruAB (fructanase) operon. Using the LevQRST four-component signal transduction system as a model for carbohydrate-dependent gene expression in strains lacking extracellular sucrases, a PlevD-cat (EIIA(Lev)) reporter was activated by pulsing with sucrose. Interestingly, ScrA was required for activation of levD expression by sucrose through components of the LevQRST complex, but not for activation by the cognate LevQRST sugars fructose or mannose. Sucrose-dependent catabolite repression was also evident in strains containing an intact sucrose PTS. Collectively, these results reveal a novel regulatory circuitry for the control of sucrose catabolism, with a central role for ScrA.

  5. Allantoin accumulation mediated by allantoinase downregulation and transport by Ureide Permease 5 confers salt stress tolerance to Arabidopsis plants.

    PubMed

    Lescano, Carlos Ignacio; Martini, Carolina; González, Claudio Alejandro; Desimone, Marcelo

    2016-07-01

    Allantoin, a metabolite generated in the purine degradation pathway, was primarily considered an intermediate for recycling of the abundant nitrogen assimilated in plant purines. More specifically, tropical legumes utilize allantoin and allantoic acid as major nodule-to-shoot nitrogen transport compounds. In other species, an increase in allantoin content was observed under different stress conditions, but the underlying molecular mechanisms remain poorly understood. In this work, Arabidopsis thaliana was used as a model system to investigate the effects of salt stress on allantoin metabolism and to know whether its accumulation results in plant protection. Plant seedlings treated with NaCl at different concentrations showed higher allantoin and lower allantoic acid contents. Treatments with NaCl favored the expression of genes involved in allantoin synthesis, but strongly repressed the unique gene encoding allantoinase (AtALN). Due to the potential regulatory role of this gene for allantoin accumulation, AtALN promoter activity was studied using a reporter system. GUS mediated coloration was found in specific plant tissues and was diminished with increasing salt concentrations. Phenotypic analysis of knockout, knockdown and stress-inducible mutants for AtALN revealed that allantoin accumulation is essential for salt stress tolerance. In addition, the possible role of allantoin transport was investigated. The Ureide Permease 5 (UPS5) is expressed in the cortex and endodermis of roots and its transcription is enhanced by salt treatment. Ups5 knockout plants under salt stress presented a susceptible phenotype and altered allantoin root-to-shoot content ratios. Possible roles of allantoin as a protectant compound in oxidative events or signaling are discussed.

  6. Contractions of affine spherical varieties

    SciTech Connect

    Arzhantsev, I V

    1999-08-31

    The language of filtrations and contractions is used to describe the class of G-varieties obtainable as the total spaces of the construction of contraction applied to affine spherical varieties, which is well-known in invariant theory. These varieties are local models for arbitrary affine G-varieties of complexity 1 with a one-dimensional categorical quotient. As examples, reductive algebraic semigroups and three-dimensional SL{sub 2}-varieties are considered.

  7. Iron, transferrin and myelinogenesis

    NASA Astrophysics Data System (ADS)

    Sergeant, C.; Vesvres, M. H.; Devès, G.; Baron, B.; Guillou, F.

    2003-09-01

    Transferrin (Tf), the iron binding protein of vertebrates serum, is known to be synthesized by oligodendrocytes (Ols) in the central nervous system. It has been postulated that Tf is involved in Ols maturation and myelinogenesis. This link is particularly important in the understanding of a severe human pathology: the multiple sclerosis, which remains without efficient treatment. We generated transgenic mice containing the complete human Tf gene and extensive regulatory sequences from the 5 ' and 3 ' untranslated regions that specifically overexpress Tf in Ols. Brain cytoarchitecture of the transgenic mice appears to be normal in all brain regions examined, total myelin content is increased by 30% and motor coordination is significantly improved when compared with non-transgenic littermates. Tf role in the central nervous system may be related to its affinity for metallic cations. Normal and transgenic mice were used for determination of trace metals (iron, copper and zinc) and minerals (potassium and calcium) concentration in cerebellum and corpus callosum. The freeze-dried samples were prepared to allow proton-induced X-ray emission and Rutherford backscattering spectrometry analyses with the nuclear microprobe in Bordeaux. Preliminary results were obtained and carbon distribution was revealed as a very good analysis to distinguish precisely the white matter region. A comparison of metallic and mineral elements contents in brain between normal and transgenic mice shows that iron, copper and zinc levels remained constant. This result provides evidence that effects of Tf overexpression in the brain do not solely relate to iron transport.

  8. Ferrous iron uptake in Cryptococcus neoformans.

    PubMed

    Jacobson, E S; Goodner, A P; Nyhus, K J

    1998-09-01

    Previous studies have implicated ferric reduction in the iron uptake pathway of the opportunistic pathogen Cryptococcus neoformans. Here we studied iron uptake directly, using 55Fe in the presence of reductants. Uptake was linear with respect to time and number of yeast cells. The plot of uptake versus concentration exhibited a steep rise up to about 1 microM, a plateau between 1 and 25 microM, and a second steep rise above 25 microM, consistent with high- and low-affinity uptake systems. A Km for high-affinity uptake was estimated to be 0.6 microM Fe(II); 1 microM was used for standardized uptake assays. At this concentration, the uptake rate was 110 +/- 3 pmol/10(6) cells/h. Iron repletion (15 microM) and copper starvation drastically decreased high-affinity iron uptake. Incubation at 0 degreesC or in the presence of 2 mM KCN abolished high-affinity iron uptake, suggesting that uptake requires metabolic energy. When exogenous reducing agents were not supplied and the culture was washed free of secreted reductants, uptake was reduced by 46%; the remaining uptake activity presumably was dependent upon the cell membrane ferric reductase. Further decreases in free Fe(II) levels achieved by trapping with bathophenanthroline disulfonate or reoxidizing with potassium nitrosodisulfonate reduced iron uptake very drastically, suggesting that it is the Fe(II) species which is transported by the high-affinity transporter. The uptake of Fe was stimulated two- to threefold by deferoxamine, but this increment could be abolished by copper starvation or inhibition of the ferric reductase by Pt, indicating that Fe solubilized by this molecule also entered the reductive iron uptake pathway.

  9. Characterization of a Dipartite Iron Uptake System from Uropathogenic Escherichia coli Strain F11*

    PubMed Central

    Koch, Doreen; Chan, Anson C. K.; Murphy, Michael E. P.; Lilie, Hauke; Grass, Gregor; Nies, Dietrich H.

    2011-01-01

    In the uropathogenic Escherichia coli strain F11, in silico genome analysis revealed the dicistronic iron uptake operon fetMP, which is under iron-regulated control mediated by the Fur regulator. The expression of fetMP in a mutant strain lacking known iron uptake systems improved growth under iron depletion and increased cellular iron accumulation. FetM is a member of the iron/lead transporter superfamily and is essential for iron uptake by the Fet system. FetP is a periplasmic protein that enhanced iron uptake by FetM. Recombinant FetP bound Cu(II) and the iron analog Mn(II) at distinct sites. The crystal structure of the FetP dimer reveals a copper site in each FetP subunit that adopts two conformations: CuA with a tetrahedral geometry composed of His44, Met90, His97, and His127, and CuB, a second degenerate octahedral geometry with the addition of Glu46. The copper ions of each site occupy distinct positions and are separated by ∼1.3 Å. Nearby, a putative additional Cu(I) binding site is proposed as an electron source that may function with CuA/CuB displacement to reduce Fe(III) for transport by FetM. Together, these data indicate that FetMP is an additional iron uptake system composed of a putative iron permease and an iron-scavenging and potentially iron-reducing periplasmic protein. PMID:21596746

  10. Internal Amino Acids Promote Gap1 Permease Ubiquitylation via TORC1/Npr1/14-3-3-Dependent Control of the Bul Arrestin-Like Adaptors

    PubMed Central

    Merhi, Ahmad

    2012-01-01

    Ubiquitylation of many plasma membrane proteins promotes their endocytosis followed by degradation in the lysosome. The yeast general amino acid permease, Gap1, is ubiquitylated and downregulated when a good nitrogen source like ammonium is provided to cells growing on a poor nitrogen source. This ubiquitylation requires the Rsp5 ubiquitin ligase and the redundant arrestin-like Bul1 and Bul2 adaptors. Previous studies have shown that Gap1 ubiquitylation involves the TORC1 kinase complex, which inhibits the Sit4 phosphatase. This causes inactivation of the protein kinase Npr1, which protects Gap1 against ubiquitylation. However, the mechanisms inducing Gap1 ubiquitylation after Npr1 inactivation remain unknown. We here show that on a poor nitrogen source, the Bul adaptors are phosphorylated in an Npr1-dependent manner and bound to 14-3-3 proteins that protect Gap1 against downregulation. After ammonium is added and converted to amino acids, the Bul proteins are dephosphorylated, dissociate from the 14-3-3 proteins, and undergo ubiquitylation. Furthermore, dephosphorylation of Bul requires the Sit4 phosphatase, which is essential to Gap1 downregulation. The data support the emerging concept that permease ubiquitylation results from activation of the arrestin-like adaptors of the Rsp5 ubiquitin ligase, this coinciding with their dephosphorylation, dissociation from the inhibitory 14-3-3 proteins, and ubiquitylation. PMID:22966204

  11. Bordetella pertussis FbpA binds both unchelated iron and iron siderophore complexes.

    PubMed

    Banerjee, Sambuddha; Weerasinghe, Aruna J; Parker Siburt, Claire J; Kreulen, R Timothy; Armstrong, Sandra K; Brickman, Timothy J; Lambert, Lisa A; Crumbliss, Alvin L

    2014-06-24

    Bordetella pertussis is the causative agent of whooping cough. This pathogenic bacterium can obtain the essential nutrient iron using its native alcaligin siderophore and by utilizing xeno-siderophores such as desferrioxamine B, ferrichrome, and enterobactin. Previous genome-wide expression profiling identified an iron repressible B. pertussis gene encoding a periplasmic protein (FbpABp). A previously reported crystal structure shows significant similarity between FbpABp and previously characterized bacterial iron binding proteins, and established its iron-binding ability. Bordetella growth studies determined that FbpABp was required for utilization of not only unchelated iron, but also utilization of iron bound to both native and xeno-siderophores. In this in vitro solution study, we quantified the binding of unchelated ferric iron to FbpABp in the presence of various anions and importantly, we demonstrated that FbpABp binds all the ferric siderophores tested (native and xeno) with μM affinity. In silico modeling augmented solution data. FbpABp was incapable of iron removal from ferric xeno-siderophores in vitro. However, when FbpABp was reacted with native ferric-alcaligin, it elicited a pronounced change in the iron coordination environment, which may signify an early step in FbpABp-mediated iron removal from the native siderophore. To our knowledge, this is the first time the periplasmic component of an iron uptake system has been shown to bind iron directly as Fe(3+) and indirectly as a ferric siderophore complex.

  12. Bordetella pertussis FbpA Binds Both Unchelated Iron and Iron Siderophore Complexes

    PubMed Central

    2015-01-01

    Bordetella pertussis is the causative agent of whooping cough. This pathogenic bacterium can obtain the essential nutrient iron using its native alcaligin siderophore and by utilizing xeno-siderophores such as desferrioxamine B, ferrichrome, and enterobactin. Previous genome-wide expression profiling identified an iron repressible B. pertussis gene encoding a periplasmic protein (FbpABp). A previously reported crystal structure shows significant similarity between FbpABp and previously characterized bacterial iron binding proteins, and established its iron-binding ability. Bordetella growth studies determined that FbpABp was required for utilization of not only unchelated iron, but also utilization of iron bound to both native and xeno-siderophores. In this in vitro solution study, we quantified the binding of unchelated ferric iron to FbpABp in the presence of various anions and importantly, we demonstrated that FbpABp binds all the ferric siderophores tested (native and xeno) with μM affinity. In silico modeling augmented solution data. FbpABp was incapable of iron removal from ferric xeno-siderophores in vitro. However, when FbpABp was reacted with native ferric-alcaligin, it elicited a pronounced change in the iron coordination environment, which may signify an early step in FbpABp-mediated iron removal from the native siderophore. To our knowledge, this is the first time the periplasmic component of an iron uptake system has been shown to bind iron directly as Fe3+ and indirectly as a ferric siderophore complex. PMID:24873326

  13. Direct evidence of iron uptake by the Gram-positive siderophore-shuttle mechanism without iron reduction.

    PubMed

    Fukushima, Tatsuya; Allred, Benjamin E; Raymond, Kenneth N

    2014-09-19

    Iron is an essential element for all organisms, and microorganisms produce small molecule iron-chelators, siderophores, to efficiently acquire Fe(III). Gram-positive bacteria possess lipoprotein siderophore-binding proteins (SBPs) on the membrane. Some of the SBPs bind both apo-siderophores (iron-free) and Fe-siderophore (iron-chelated) and only import Fe-siderophores. When the SBP initially binds an apo-siderophore, the SBP uses the Gram-positive siderophore-shuttle mechanism (the SBPs exchange Fe(III) from a Fe-siderophore to the apo-siderophore bound to the protein) and/or displacement mechanism (the apo-siderophore bound to the SBP is released and a Fe-siderophore is then bound to the protein) to import the Fe-siderophore. Previously, we reported that the Bacillus cereus SBP, YxeB, exchanges Fe(III) from a ferrioxamine B (FO) to a desferrioxamine B (DFO) bound to YxeB using the siderophore-shuttle mechanism although the iron exchange was indirectly elucidated. Synthetic Cr-DFO (inert metal FO analog) and Ga-DFO (nonreducible FO analog) are bound to YxeB and imported via YxeB and the corresponding permeases and ATPase. YxeB exchanges Fe(III) from FO and Ga(III) from Ga-DFO to DFO bound to the protein, indicating that the metal-exchange occurs without metal reduction. YxeB also binds DFO derivatives including acetylated DFO (apo-siderophore) and acetylated FO (AcFO, Fe-siderophore). The iron from AcFO is transferred to DFO when bound to YxeB, giving direct evidence of iron exchange. Moreover, YxeB also uses the displacement mechanism when ferrichrome (Fch) is added to the DFO:YxeB complex. Uptake by the displacement mechanism is a minor pathway compared to the shuttle mechanism.

  14. Chemical binding affinity estimation using MSB

    NASA Astrophysics Data System (ADS)

    Weaver, John B.; Rauwerdink, Adam M.

    2011-03-01

    Binding affinity can be estimated in several ways in the laboratory but there is no viable way to estimate binding affinity in vivo without assumptions on the number of binding sites. Magnetic spectroscopy of nanoparticle Brownian motion, MSB, measures the rotational Brownian motion. The MSB signal is affected by nanoparticle binding affinity so it provides a mechanism to measure the chemical binding affinity. We present a possible mechanism to quantify the binding affinity and test that mechanism using viscous solutions.

  15. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

    Celik, D.; Ozdemir, Y.; Ureyen, M.

    2007-01-01

    Contractions play a considerable role in the theory of fractals. However, it is not easy to find contractions which are not similitudes. In this study, it is shown by counter examples that an affine transformation of the plane carrying a given triangle onto another triangle may not be a contraction even if it contracts edges, heights or medians.…

  16. Affinity-aware checkpoint restart

    SciTech Connect

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; Hargrove, Paul; Roman, Eric

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core maps and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.

  17. Affinity-aware checkpoint restart

    DOE PAGES

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

  18. ELECTRON AFFINITIES OF INORGANIC RADICALS.

    DTIC Science & Technology

    energy in the latter compound is 110 kcals/mole, distinctly higher than in ammonia. Cyanogen (CN)2 and hydrocyanic acid (HCN) yield values for the...ions very readily, and the electron affinity is 49 kcals/mole. A comparison with the results from thiocyanic acid (HNCS) indicates that the H-N bond

  19. Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway.

    PubMed Central

    Cánovas, D; Vargas, C; Csonka, L N; Ventosa, A; Nieto, J J

    1996-01-01

    The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl-glucose-M63 plates, demonstrating the utilization of these compounds as osmoprotectants. Moreover, betaine and choline stimulated the growth of H. elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salinities below and above the optimum. We found that H. elongata DSM 3043 has at least one high-affinity transport system for betaine (K(m) = 3.06 microM and Vmax = 9.96 nmol of betaine min(-1) mg of protein(-1)). Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease. Finally, thin-layer chromatography and 13C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H. elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium. PMID:8955405

  20. Iron refractory iron deficiency anemia

    PubMed Central

    De Falco, Luigia; Sanchez, Mayka; Silvestri, Laura; Kannengiesser, Caroline; Muckenthaler, Martina U.; Iolascon, Achille; Gouya, Laurent; Camaschella, Clara; Beaumont, Carole

    2013-01-01

    Iron refractory iron deficiency anemia is a hereditary recessive anemia due to a defect in the TMPRSS6 gene encoding Matriptase-2. This protein is a transmembrane serine protease that plays an essential role in down-regulating hepcidin, the key regulator of iron homeostasis. Hallmarks of this disease are microcytic hypochromic anemia, low transferrin saturation and normal/high serum hepcidin values. The anemia appears in the post-natal period, although in some cases it is only diagnosed in adulthood. The disease is refractory to oral iron treatment but shows a slow response to intravenous iron injections and partial correction of the anemia. To date, 40 different Matriptase-2 mutations have been reported, affecting all the functional domains of the large ectodomain of the protein. In vitro experiments on transfected cells suggest that Matriptase-2 cleaves Hemojuvelin, a major regulator of hepcidin expression and that this function is altered in this genetic form of anemia. In contrast to the low/undetectable hepcidin levels observed in acquired iron deficiency, in patients with Matriptase-2 deficiency, serum hepcidin is inappropriately high for the low iron status and accounts for the absent/delayed response to oral iron treatment. A challenge for the clinicians and pediatricians is the recognition of the disorder among iron deficiency and other microcytic anemias commonly found in pediatric patients. The current treatment of iron refractory iron deficiency anemia is based on parenteral iron administration; in the future, manipulation of the hepcidin pathway with the aim of suppressing it might become an alternative therapeutic approach. PMID:23729726

  1. Iron binding activity is essential for the function of IscA in iron-sulphur cluster biogenesis.

    PubMed

    Landry, Aaron P; Cheng, Zishuo; Ding, Huangen

    2013-03-07

    Iron-sulphur cluster biogenesis requires coordinated delivery of iron and sulphur to scaffold proteins, followed by transfer of the assembled clusters from scaffold proteins to target proteins. This complex process is accomplished by a group of dedicated iron-sulphur cluster assembly proteins that are conserved from bacteria to humans. While sulphur in iron-sulphur clusters is provided by L-cysteine via cysteine desulfurase, the iron donor(s) for iron-sulphur cluster assembly remains largely elusive. Here we report that among the primary iron-sulphur cluster assembly proteins, IscA has a unique and strong binding activity for mononuclear iron in vitro and in vivo. Furthermore, the ferric iron centre tightly bound in IscA can be readily extruded by l-cysteine, followed by reduction to ferrous iron for iron-sulphur cluster biogenesis. Substitution of the highly conserved residue tyrosine 40 with phenylalanine (Y40F) in IscA results in a mutant protein that has a diminished iron binding affinity but retains the iron-sulphur cluster binding activity. Genetic complementation studies show that the IscA Y40F mutant is inactive in vivo, suggesting that the iron binding activity is essential for the function of IscA in iron-sulphur cluster biogenesis.

  2. Multiple horizontal gene transfers of ammonium transporters/ammonia permeases from prokaryotes to eukaryotes: toward a new functional and evolutionary classification.

    PubMed

    McDonald, Tami R; Dietrich, Fred S; Lutzoni, François

    2012-01-01

    The proteins of the ammonium transporter/methylammonium permease/Rhesus factor family (AMT/MEP/Rh family) are responsible for the movement of ammonia or ammonium ions across the cell membrane. Although it has been established that the Rh proteins are distantly related to the other members of the family, the evolutionary history of the AMT/MEP/Rh family remains unclear. Here, we use phylogenetic analysis to infer the evolutionary history of this family of proteins across 191 genomes representing all main lineages of life and to provide a new classification of the proteins in this family. Our phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). We show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: The AMT family as defined here exhibits vertical gene transfer (i.e., standard parent-to-offspring inheritance), whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs). These ancient HGT events include a gene replacement during the early evolution of the fungi, which could be a defining trait for the kingdom Fungi, a gene gain from hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina) that was subsequently lost in most lineages but retained in even distantly related lichenized fungi. This recircumscription of the ammonium transporters/ammonia permeases family into MEP and AMT families informs the debate on the mechanism of transport in these proteins and on the nature of the transported molecule because published crystal structures of proteins from the MEP and Rh clades may not be representative of the AMT clade. The clades as depicted in this phylogenetic study appear to correspond to

  3. Multiple Horizontal Gene Transfers of Ammonium Transporters/Ammonia Permeases from Prokaryotes to Eukaryotes: Toward a New Functional and Evolutionary Classification

    PubMed Central

    McDonald, Tami R.; Dietrich, Fred S.; Lutzoni, François

    2012-01-01

    The proteins of the ammonium transporter/methylammonium permease/Rhesus factor family (AMT/MEP/Rh family) are responsible for the movement of ammonia or ammonium ions across the cell membrane. Although it has been established that the Rh proteins are distantly related to the other members of the family, the evolutionary history of the AMT/MEP/Rh family remains unclear. Here, we use phylogenetic analysis to infer the evolutionary history of this family of proteins across 191 genomes representing all main lineages of life and to provide a new classification of the proteins in this family. Our phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). We show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: The AMT family as defined here exhibits vertical gene transfer (i.e., standard parent-to-offspring inheritance), whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs). These ancient HGT events include a gene replacement during the early evolution of the fungi, which could be a defining trait for the kingdom Fungi, a gene gain from hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina) that was subsequently lost in most lineages but retained in even distantly related lichenized fungi. This recircumscription of the ammonium transporters/ammonia permeases family into MEP and AMT families informs the debate on the mechanism of transport in these proteins and on the nature of the transported molecule because published crystal structures of proteins from the MEP and Rh clades may not be representative of the AMT clade. The clades as depicted in this phylogenetic study appear to correspond to

  4. Theoretical proton affinity and fluoride affinity of nerve agent VX.

    PubMed

    Bera, Narayan C; Maeda, Satoshi; Morokuma, Keiji; Viggiano, Al A

    2010-12-23

    Proton affinity and fluoride affinity of nerve agent VX at all of its possible sites were calculated at the RI-MP2/cc-pVTZ//B3LYP/6-31G* and RI-MP2/aug-cc-pVTZ//B3LYP/6-31+G* levels, respectively. The protonation leads to various unique structures, with H(+) attached to oxygen, nitrogen, and sulfur atoms; among which the nitrogen site possesses the highest proton affinity of -ΔE ∼ 251 kcal/mol, suggesting that this is likely to be the major product. In addition some H(2), CH(4) dissociation as well as destruction channels have been found, among which the CH(4) + [Et-O-P(═O)(Me)-S-(CH(2))(2)-N(+)(iPr)═CHMe] product and the destruction product forming Et-O-P(═O)(Me)-SMe + CH(2)═N(+)(iPr)(2) are only 9 kcal/mol less stable than the most stable N-protonated product. For fluoridization, the S-P destruction channel to give Et-O-P(═O)(Me)(F) + [S-(CH(2))(2)-N-(iPr)(2)](-) is energetically the most favorable, with a fluoride affinity of -ΔE ∼ 44 kcal. Various F(-) ion-molecule complexes are also found, with the one having F(-) interacting with two hydrogen atoms in different alkyl groups to be only 9 kcal/mol higher than the above destruction product. These results suggest VX behaves quite differently from surrogate systems.

  5. Iron, melanin and dopamine interaction: relevance to Parkinson's disease.

    PubMed

    Ben-Shachar, D; Youdim, M B

    1993-01-01

    1. Interaction between iron and melanin may provide a reasonable explanation for the vulnerability of the melanin containing dopaminergic neurons in the substantia nigra (SN) to neurodegeneration in Parkinson's disease (PD). 2. Scatchard analysis of the binding of iron to synthetic dopamine melanin revealed a high-affinity (KD = 13 nM) and a lower affinity (KD = 200 nM) binding sites. 3. The binding of iron to melanin is dependent on the concentration of melanin and on pH. 4. Iron chelators, U74500A, desferrioxamine and to a lesser extent 1,10-phenanthroline and chlorpromazine could displace iron from melanin. In contrast, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its metabolite 1-methyl-4-phenyl-pyridinium (MPP+), which cause Parkinsonism, were unable to displace iron. 5. Melanin alone reduced lipid peroxidation in rat cortical membrane preparations. However, iron induced lipid peroxidation, which could be inhibited by desferrioxamine, was potentiated by melanin. 6. Iron bound to neuromelanin in melanized dopamine neurons was detected only in parkinsonian brains and not in controls. The interaction of iron with neuromelanin as identified by x-ray defraction technique was identical to iron interaction with synthetic dopamine melanin. 7. In the absence of an identified exogenous or endogenous neurotoxin in idiopathic Parkinson's disease, iron-melanin interaction in the SN may serve as a candidate for the oxygen-radical induced neurodegeneration of the melanin containing dopaminergic neurons.

  6. Overexpression of Sna3 stabilizes tryptophan permease Tat2, potentially competing for the WW domain of Rsp5 ubiquitin ligase with its binding protein Bul1.

    PubMed

    Hiraki, Toshiki; Abe, Fumiyoshi

    2010-01-04

    Tryptophan permease Tat2 in Saccharomyces cerevisiae undergoes Rsp5-dependent degradation upon exposure to high hydrostatic pressure and it limits the growth of tryptophan auxotrophs. Overexpression of SNA3 encoding an endosomal/vacuolar protein possessing the PPAY motif allowed growth at 25 MPa, which was potentiated by marked stabilization of Tat2. This appeared to depend on the PPAY motif, which interacted with the WW domain of Rsp5. Subcellular localization of Rsp5 was unchanged by overexpression of either SNA3 or SNA3-AAAY. While the loss of Bul1, a binding protein of Rsp5, or the rsp5-ww3 mutation allowed high-pressure growth, overexpression of BUL1 abolished the Sna3-mediated growth at 25 MPa. These results suggest that Sna3 and Bul1 compete for the WW domain of Rsp5 upon Tat2 ubiquitination.

  7. Gastrins, iron and colorectal cancer.

    PubMed

    Baldwin, Graham S

    2009-09-01

    This minireview explores the connections between circulating gastrins, iron status and colorectal cancer. The peptide hormone gastrin is a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. Gastrins bind two ferric ions with μM affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity. The ferric ion ligands have been identified as glutamates 7, 8 and 9 in the 18 amino acid peptide glycine-extended gastrin. An interaction between gastrin and transferrin was first demonstrated by covalent crosslinking techniques, and has been recently confirmed by surface plasmon resonance. We have therefore proposed that gastrins act as catalysts in the loading of transferrin with iron. Several recent lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease haemochromatosis, and that transferrin saturation positively correlates with circulating gastrin concentrations, suggest that gastrins may be involved in iron homeostasis. In addition the recognition that ferric ions may play an unexpected role in the biological activity of non-amidated gastrins may assist in the development of new therapies for colorectal carcinoma.

  8. METABOLISM OF IRON STORES

    PubMed Central

    SAITO, HIROSHI

    2014-01-01

    ABSTRACT Remarkable progress was recently achieved in the studies on molecular regulators of iron metabolism. Among the main regulators, storage iron, iron absorption, erythropoiesis and hepcidin interact in keeping iron homeostasis. Diseases with gene-mutations resulting in iron overload, iron deficiency, and local iron deposition have been introduced in relation to the regulators of storage iron metabolism. On the other hand, the research on storage iron metabolism has not advanced since the pioneering research by Shoden in 1953. However, we recently developed a new method for determining ferritin iron and hemosiderin iron by computer-assisted serum ferritin kinetics. Serum ferritin increase or decrease curves were measured in patients with normal storage iron levels (chronic hepatitis C and iron deficiency anemia treated by intravenous iron injection), and iron overload (hereditary hemochromatosis and transfusion dependent anemia). We thereby confirmed the existence of two iron pathways where iron flows followed the numbered order (1) labile iron, (2) ferritin and (3) hemosiderin in iron deposition and mobilization among many previously proposed but mostly unproven routes. We also demonstrated the increasing and decreasing phases of ferritin iron and hemosiderin iron in iron deposition and mobilization. The author first demonstrated here the change in proportion between pre-existing ferritin iron and new ferritin iron synthesized by removing iron from hemosiderin in the course of iron removal. In addition, the author disclosed the cause of underestimation of storage iron turnover rate which had been reported by previous investigators in estimating storage iron turnover rate of normal subjects. PMID:25741033

  9. Essential role of K(+) uptake permease (Kup) for resistance to sucrose-induced stress in Gluconacetobacter diazotrophicus PAl 5.

    PubMed

    de Oliveira, Marcos V V; Intorne, Aline C; Vespoli, Luciano de S; Andrade, Leandro F; Pereira, Leandro de M; Rangel, Patrícia L; de Souza Filho, Gonçalo A

    2017-04-01

    Microorganisms are constantly challenged by stressful conditions, such as sugar-rich environments. Such environments can cause an imbalance of biochemical activities and compromise cell multiplication. Gluconacetobacter diazotrophicus PAl 5 is among the most sugar-tolerant bacteria, capable of growing in the presence of up to 876 mM sucrose. However, the molecular mechanisms involved in its response to high sucrose remain unknown. The present work aimed to identify sucrose-induced stress resistance genes in G. diazotrophicus PAl 5. Screening of a Tn5 transposon insertion library identified a mutant that was severely compromised in its resistance to high sucrose concentrations. Molecular characterization revealed that the mutation affected the kupA gene, which encodes a K(+) uptake transporter (KupA). Functional complementation of the mutant with the wild type kupA gene recovered the sucrose-induced stress resistance phenotype. High sucrose resistance assay, under different potassium concentrations, revealed that KupA acts as a high-affinity K(+) transporter, which is essential for resistance to sucrose-induced stress, when extracellular potassium levels are low. This study is the first to show the essential role of the KupA protein for resistance to sucrose-induced stress in bacteria by acting as a high-affinity potassium transporter in G. diazotrophicus PAl 5.

  10. Acquisition, transport, and storage of iron by pathogenic fungi.

    PubMed

    Howard, D H

    1999-07-01

    Iron is required by most living systems. A great variety of means of acquisition, avenues of uptake, and methods of storage are used by pathogenic fungi to ensure a supply of the essential metal. Solubilization of insoluble iron polymers is the first step in iron assimilation. The two methods most commonly used by microorganisms for solubilization of iron are reduction and chelation. Reduction of ferric iron to ferrous iron by enzymatic or nonenzymatic means is a common mechanism among pathogenic yeasts. Under conditions of iron starvation, many fungi synthesize iron chelators known as siderophores. Two classes of compounds that function in iron gathering are commonly observed: hydroxamates and polycarboxylates. Two major responses to iron stress in fungi are a high-affinity ferric iron reductase and siderophore synthesis. Regulation of these two mechanisms at the molecular level has received attention. Uptake of siderophores is a diverse process, which varies among the different classes of compounds. Since free iron is toxic, it must be stored for further metabolic use. Polyphosphates, ferritins, and siderophores themselves have been described as storage molecules. The iron-gathering mechanisms used by a pathogen in an infected host are largely unknown and can only be posited on the basis of in vitro studies at present.

  11. Identification and characterization of an iron ABC transporter operon in Gluconacetobacter diazotrophicus Pal 5.

    PubMed

    Urzúa, Lucia Soto; Vázquez-Candanedo, Ada P; Sánchez-Espíndola, Adriana; Ramírez, Carlos Ávila; Baca, Beatriz E

    2013-06-01

    Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane. We have cloned and sequenced the genes coding for the components of the iron ABC-type acquisition system of G. diazotrophicus. Sequence analysis revealed three ORFs, (feuA, feuB, and feuC) organized as an operon and encoding polypeptides of 346 (38 kDa), 342 (34.2 kDa), and 240 (26 kDa) amino acids, respectively. The deduced translation products of the feu operon showed similarity with a periplasmic solute-binding protein (FeuA), permease (FeuB), and ATPase (FeuC) involved in Fe transport. The role of FeuB in the survival of G. diazotrophicus under iron depletion was evaluated by comparing the ability of wild-type and FeuB-Km(R) -mutant strains in a medium without iron supplementation and in a medium containing 2, 2'-dipyridyl (DP). Growth of the mutant was affected in the medium containing DP. The operon was expressed at higher levels in cells depleted for iron than in those that contained the metal. A decrease in nitrogenase activity was observed with the FeuB-Km(R) -mutant strain that with the wild-type under iron deficiency conditions, suggesting that the Feu operon play role in Fe nutrition of G. diazotrophicus.

  12. Production and Characterization of Desmalonichrome Relative Binding Affinity for Uranyl Ions in Relation to Other Siderophores

    SciTech Connect

    Mo, Kai-For; Dai, Ziyu; Wunschel, David S.

    2016-06-24

    Siderophores are Fe binding secondary metabolites that have been investigated for their uranium binding properties. Much of the previous work has focused on characterizing hydroxamate types of siderophores, such as desferrioxamine B, for their uranyl binding affinity. Carboxylate forms of these metabolites hold potential to be more efficient chelators of uranyl, yet they have not been widely studied and are more difficult to obtain. Desmalonichrome is a carboxylate siderophore which is not commercially available and so was obtained from the ascomycete fungus Fusarium oxysporum cultivated under Fe depleted conditions. The relative affinity for uranyl binding of desmalonichrome was investigated using a competitive analysis of binding affinities between uranyl acetate and different concentrations of iron(III) chloride using electrospray ionization mass spectrometry (ESI-MS). In addition to desmalonichrome, three other siderophores, including two hydroxamates (desferrioxamine B and desferrichrome) and one carboxylate (desferrichrome A) were studied to understand their relative affinities for the uranyl ion at two pH values. The binding affinities of hydroxymate siderophores to uranyl ion were found to decrease to a greater degree at lower pH as the concentration of Fe (III) ion increases. On the other hand, lowering pH has little impact on the binding affinities between carboxylate siderophores and uranyl ion. Desmalonichrome was shown to have the greatest relative affinity for uranyl at any pH and Fe(III) concentration. These results suggest that acidic functional groups in the ligands are critical for strong chelation with uranium at lower pH.

  13. Iron Sucrose Injection

    MedlinePlus

    Iron sucrose injection is used treat iron-deficiency anemia (a lower than normal number of red blood cells due ... and may cause the kidneys to stop working). Iron sucrose injection is in a class of medications called iron ...

  14. The Organization of Controller Motifs Leading to Robust Plant Iron Homeostasis.

    PubMed

    Agafonov, Oleg; Selstø, Christina Helen; Thorsen, Kristian; Xu, Xiang Ming; Drengstig, Tormod; Ruoff, Peter

    2016-01-01

    Iron is an essential element needed by all organisms for growth and development. Because iron becomes toxic at higher concentrations iron is under homeostatic control. Plants face also the problem that iron in the soil is tightly bound to oxygen and difficult to access. Plants have therefore developed special mechanisms for iron uptake and regulation. During the last years key components of plant iron regulation have been identified. How these components integrate and maintain robust iron homeostasis is presently not well understood. Here we use a computational approach to identify mechanisms for robust iron homeostasis in non-graminaceous plants. In comparison with experimental results certain control arrangements can be eliminated, among them that iron homeostasis is solely based on an iron-dependent degradation of the transporter IRT1. Recent IRT1 overexpression experiments suggested that IRT1-degradation is iron-independent. This suggestion appears to be misleading. We show that iron signaling pathways under IRT1 overexpression conditions become saturated, leading to a breakdown in iron regulation and to the observed iron-independent degradation of IRT1. A model, which complies with experimental data places the regulation of cytosolic iron at the transcript level of the transcription factor FIT. Including the experimental observation that FIT induces inhibition of IRT1 turnover we found a significant improvement in the system's response time, suggesting a functional role for the FIT-mediated inhibition of IRT1 degradation. By combining iron uptake with storage and remobilization mechanisms a model is obtained which in a concerted manner integrates iron uptake, storage and remobilization. In agreement with experiments the model does not store iron during its high-affinity uptake. As an iron biofortification approach we discuss the possibility how iron can be accumulated even during high-affinity uptake.

  15. The Organization of Controller Motifs Leading to Robust Plant Iron Homeostasis

    PubMed Central

    Agafonov, Oleg; Selstø, Christina Helen; Thorsen, Kristian; Xu, Xiang Ming; Drengstig, Tormod; Ruoff, Peter

    2016-01-01

    Iron is an essential element needed by all organisms for growth and development. Because iron becomes toxic at higher concentrations iron is under homeostatic control. Plants face also the problem that iron in the soil is tightly bound to oxygen and difficult to access. Plants have therefore developed special mechanisms for iron uptake and regulation. During the last years key components of plant iron regulation have been identified. How these components integrate and maintain robust iron homeostasis is presently not well understood. Here we use a computational approach to identify mechanisms for robust iron homeostasis in non-graminaceous plants. In comparison with experimental results certain control arrangements can be eliminated, among them that iron homeostasis is solely based on an iron-dependent degradation of the transporter IRT1. Recent IRT1 overexpression experiments suggested that IRT1-degradation is iron-independent. This suggestion appears to be misleading. We show that iron signaling pathways under IRT1 overexpression conditions become saturated, leading to a breakdown in iron regulation and to the observed iron-independent degradation of IRT1. A model, which complies with experimental data places the regulation of cytosolic iron at the transcript level of the transcription factor FIT. Including the experimental observation that FIT induces inhibition of IRT1 turnover we found a significant improvement in the system’s response time, suggesting a functional role for the FIT-mediated inhibition of IRT1 degradation. By combining iron uptake with storage and remobilization mechanisms a model is obtained which in a concerted manner integrates iron uptake, storage and remobilization. In agreement with experiments the model does not store iron during its high-affinity uptake. As an iron biofortification approach we discuss the possibility how iron can be accumulated even during high-affinity uptake. PMID:26800438

  16. Iron acquisition from Fe-pyoverdine by Arabidopsis thaliana.

    PubMed

    Vansuyt, Gérard; Robin, Agnès; Briat, Jean-François; Curie, Catherine; Lemanceau, Philippe

    2007-04-01

    Taking into account the strong iron competition in the rhizosphere and the high affinity of pyoverdines for Fe(III), these molecules are expected to interfere with the iron nutrition of plants, as they do with rhizospheric microbes. The impact of Fe-pyoverdine on iron content of Arabidopsis thaliana was compared with that of Fe-EDTA. Iron chelated to pyoverdine was incorporated in a more efficient way than when chelated to EDTA, leading to increased plant growth of the wild type. A transgenic line of A. thaliana overexpressing ferritin showed a higher iron content than the wild type when supplemented with Fe-EDTA but a lower iron content when supplemented with Fe-pyoverdine despite its increased reductase activity, suggesting that this activity was not involved in the iron uptake from pyoverdine. A mutant knock-out iron transporter IRT1 showed lower iron and chlorophyll contents when supplemented with Fe-EDTA than the wild type but not when supplemented with Fe-pyoverdine, indicating that, in contrast to iron from EDTA, iron from pyoverdine was not incorporated through the IRT1 transporter. Altogether these data suggest that iron from Fe-pyoverdine was not incorporated in planta through the strategy I, which is based on reductase activity and IRT1 transporter. This is supported by the presence of pyoverdine in planta as shown by enzyme-linked immunosorbent assay and by tracing 15N of 15N-pyoverdine.

  17. Iron and alloys of iron. [lunar resources

    NASA Technical Reports Server (NTRS)

    Sastri, Sankar

    1992-01-01

    All lunar soil contains iron in the metallic form, mostly as an iron-nickel alloy in concentrations of a few tenths of 1 percent. Some of this free iron can be easily separated by magnetic means. It is estimated that the magnetic separation of 100,000 tons of lunar soil would yield 150-200 tons of iron. Agglutinates contain metallic iron which could be extracted by melting and made into powder metallurgy products. The characteristics and potential uses of the pure-iron and iron-alloy lunar products are discussed. Processes for working iron that might be used in a nonterrestrial facility are also addressed.

  18. Pressure-induced differential regulation of the two tryptophan permeases Tat1 and Tat2 by ubiquitin ligase Rsp5 and its binding proteins, Bul1 and Bul2.

    PubMed

    Abe, Fumiyoshi; Iida, Hidetoshi

    2003-11-01

    Tryptophan uptake appears to be the Achilles' heel in yeast physiology, since under a variety of seemingly diverse toxic conditions, it becomes the limiting factor for cell growth. When growing cells of Saccharomyces cerevisiae are subjected to high hydrostatic pressure, tryptophan uptake is down-regulated, leading to cell cycle arrest in the G(1) phase. Here we present evidence that the two tryptophan permeases Tat1 and Tat2 are differentially regulated by Rsp5 ubiquitin ligase in response to high hydrostatic pressure. Analysis of high-pressure growth mutants revealed that the HPG1 gene was allelic to RSP5. The HPG1 mutation or the bul1Delta bul2Delta double mutation caused a marked increase in the steady-state level of Tat2 but not of Tat1, although both permeases were degraded at high pressure in an Rsp5-dependent manner. There were marked differences in subcellular localization. Tat1 localized predominantly in the plasma membrane, whereas Tat2 was abundant in the internal membranes. Moreover, Tat1 was associated with lipid rafts, whereas Tat2 localized in bulk lipids. Surprisingly, Tat2 became associated with lipid rafts upon the occurrence of a ubiquitination defect. These results suggest that ubiquitination is an important determinant of the localization and regulation of these tryptophan permeases. Determination of the activation volume (DeltaV( not equal )) for Tat1- and Tat2-mediated tryptophan uptake (89.3 and 50.8 ml/mol, respectively) revealed that both permeases are highly sensitive to membrane perturbation and that Tat1 rather than Tat2 is likely to undergo a dramatic conformational change during tryptophan import. We suggest that hydrostatic pressure is a unique tool for elucidating the dynamics of integral membrane protein functions as well as for probing lipid microenvironments where they localize.

  19. Ligand binding processes in hemoglobin. Chemical reactivity of iron studied by XANES spectroscopy.

    PubMed Central

    Pin, S; Valat, P; Cortes, R; Michalowicz, A; Alpert, B

    1985-01-01

    K-absorption edge of coordinated ions exhibits a fine structure (through the use of XANES, or x-ray absorption near edge structures) that reflects the electronic repartition and the chemical reactivity of these ions. Comparative analysis of iron K-absorption-edge shape for hemoglobin derivatives with different ligand affinity suggests strongly that in hemoglobin, iron-forms with high and low affinity are highly improbable. PMID:4092074

  20. Affinity membrane introduction mass spectrometry

    SciTech Connect

    Xu, C.; Patrick, J.S.; Cooks, R.G. )

    1995-02-15

    A new technique, affinity membrane introduction mass spectrometry, is described. In this method, a chemically modified membrane is used to selectively adsorb analytes bearing a particular functional group and concentrate them from solution. Release of the bound analyte results in its transfer across the membrane and allows it to be monitored mass spectrometrically, using, in the present case, a benchtop ion trap instrument. Alkylamine-modified cellulose membranes are used to bind substituted benzaldehydes through imine formation at high pH. Release of the bound aldehyde is achieved by acid hydrolysis of the surface-bound imine. Benzaldehyde is detected with excellent specificity at 10 ppm in a complex mixture using this method. Using the enrichment capability of the membrane, a full mass spectrum of benzaldehyde can be measured at a concentration of 10 ppb. The behavior of a variety of other aldehydes is also discussed to illustrate the capabilities of the method. 21 refs., 5 figs., 2 tabs.

  1. Antisymmetric tensor generalizations of affine vector fields

    PubMed Central

    Morisawa, Yoshiyuki; Tomoda, Kentaro

    2016-01-01

    Tensor generalizations of affine vector fields called symmetric and antisymmetric affine tensor fields are discussed as symmetry of spacetimes. We review the properties of the symmetric ones, which have been studied in earlier works, and investigate the properties of the antisymmetric ones, which are the main theme in this paper. It is shown that antisymmetric affine tensor fields are closely related to one-lower-rank antisymmetric tensor fields which are parallelly transported along geodesics. It is also shown that the number of linear independent rank-p antisymmetric affine tensor fields in n-dimensions is bounded by (n + 1)!/p!(n − p)!. We also derive the integrability conditions for antisymmetric affine tensor fields. Using the integrability conditions, we discuss the existence of antisymmetric affine tensor fields on various spacetimes. PMID:26858463

  2. Conformal field theory on affine Lie groups

    SciTech Connect

    Clubok, Kenneth Sherman

    1996-04-01

    Working directly on affine Lie groups, we construct several new formulations of the WZW model, the gauged WZW model, and the generic affine-Virasoro action. In one formulation each of these conformal field theories (CFTs) is expressed as a one-dimensional mechanical system whose variables are coordinates on the affine Lie group. When written in terms of the affine group element, this formulation exhibits a two-dimensional WZW term. In another formulation each CFT is written as a two-dimensional field theory, with a three- dimensional WZW term, whose fields are coordinates on the affine group. On the basis of these equivalent formulations, we develop a translation dictionary in which the new formulations on the affine Lie group are understood as mode formulations of the conventional formulations on the Lie group. Using this dictionary, we also express each CFT as a three-dimensional field theory on the Lie group with a four-dimensional WZW term. 36 refs.

  3. Iron and iron derived radicals

    SciTech Connect

    Borg, D.C.; Schaich, K.M.

    1987-04-01

    We have discussed some reactions of iron and iron-derived oxygen radicals that may be important in the production or treatment of tissue injury. Our conclusions challenge, to some extent, the usual lines of thought in this field of research. Insofar as they are born out by subsequent developments, the lessons they teach are two: Think fastexclamation Think smallexclamation In other words, think of the many fast reactions that can rapidly alter the production and fate of highly reactive intermediates, and when considering the impact of competitive reactions on such species, think how they affect the microenvironment (on the molecular scale) ''seen'' by each reactive molecule. 21 refs., 3 figs., 1 tab.

  4. A Novel Vertex Affinity for Community Detection

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    We propose a novel vertex affinity measure in this paper. The new vertex affinity quantifies the proximity between two vertices in terms of their clustering strength and is ideal for such graph analytics applications as community detection. We also developed a framework that combines simple graph searches and resistance circuit formulas to compute the vertex affinity efficiently. We study the properties of the new affinity measure empirically in comparison to those of other popular vertex proximity metrics. Our results show that the existing metrics are ill-suited for community detection due to their lack of fundamental properties that are essential for correctly capturing inter- and intra-cluster vertex proximity.

  5. Structural determinants of sigma receptor affinity

    SciTech Connect

    Largent, B.L.; Wikstroem, H.G.; Gundlach, A.L.; Snyder, S.H.

    1987-12-01

    The structural determinants of sigma receptor affinity have been evaluated by examining a wide range of compounds related to opioids, neuroleptics, and phenylpiperidine dopaminergic structures for affinity at sigma receptor-binding sites labeled with (+)-(/sup 3/H)3-PPP. Among opioid compounds, requirements for sigma receptor affinity differ strikingly from the determinants of affinity for conventional opiate receptors. Sigma sites display reverse stereoselectivity to classical opiate receptors. Multi-ringed opiate-related compounds such as morphine and naloxone have negligible affinity for sigma sites, with the highest sigma receptor affinity apparent for benzomorphans which lack the C ring of opioids. Highest affinity among opioids and other compounds occurs with more lipophilic N-substituents. This feature is particularly striking among the 3-PPP derivatives as well as the opioids. The butyrophenone haloperidol is the most potent drug at sigma receptors we have detected. Among the series of butyrophenones, receptor affinity is primarily associated with the 4-phenylpiperidine moiety. Conformational calculations for various compounds indicate a fairly wide range of tolerance for distances between the aromatic ring and the amine nitrogen, which may account for the potency at sigma receptors of structures of considerable diversity. Among the wide range of structures that bind to sigma receptor-binding sites, the common pharmacophore associated with high receptor affinity is a phenylpiperidine with a lipophilic N-substituent.

  6. Gastrins, iron homeostasis and colorectal cancer.

    PubMed

    Kovac, Suzana; Anderson, Gregory J; Baldwin, Graham S

    2011-05-01

    The peptide hormone gastrin has been identified as a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. The importance of gastric acid in the absorption of dietary iron first became evident 50 years ago when iron deficiency anemia was recognized as a long-term consequence of partial gastrectomy. This review summarizes the connections between circulating gastrins, iron status and colorectal cancer. Gastrins bind two ferric ions with micromolar affinity and, in the case of non-amidated forms of the hormone, iron binding is essential for biological activity in vitro and in vivo. The demonstration of an interaction between gastrin and transferrin by biochemical techniques led to the proposal that gastrins catalyze the loading of transferrin with iron. Several lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron overload disease hemochromatosis and that transferrin saturation positively correlates with circulating gastrin concentration, suggest the potential involvement of gastrins in iron homeostasis. Conversely, recognition that ferric ions play an unexpected role in the biological activity of gastrins may assist in the development of useful therapies for colorectal carcinoma and other disorders of mucosal proliferation in the gastrointestinal tract. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

  7. Gastrins, Iron Homeostasis and Colorectal Cancer

    PubMed Central

    Kovac, Suzana; Anderson, Gregory J.; Baldwin, Graham S.

    2011-01-01

    The peptide hormone gastrin has been identified as a major regulator of acid secretion and a potent mitogen for normal and malignant gastrointestinal cells. The importance of gastric acid in the absorption of dietary iron first became evident 50 years ago when iron-deficiency anemia was recognised as a long-term consequence of partial gastrectomy. This review summarises the connections between circulating gastrins, iron status and colorectal cancer. Gastrins bind two ferric ions with micromolar affinity and, in the case of non-amidated forms of the hormone, iron-binding is essential for biological activity in vitro and in vivo. The demonstration of an interaction between gastrin and transferrin by biochemical techniques led to the proposal that gastrins catalyse the loading of transferrin with iron. Several lines of evidence, including the facts that the concentrations of circulating gastrins are increased in mice and humans with the iron-overload disease hemochromatosis and that transferrin saturation positively correlates with circulating gastrin concentration, suggest the potential involvement of gastrins in iron homeostasis. Conversely, recognition that ferric ions play an unexpected role in the biological activity of gastrins may assist in the development of useful therapies for colorectal carcinoma and other disorders of mucosal proliferation in the gastrointestinal tract. PMID:21320535

  8. The General Amino Acid Permease FfGap1 of Fusarium fujikuroi Is Sorted to the Vacuole in a Nitrogen-Dependent, but Npr1 Kinase-Independent Manner

    PubMed Central

    Pfannmüller, Andreas; Wagner, Dominik; Sieber, Christian; Schönig, Birgit; Boeckstaens, Mélanie; Marini, Anna Maria; Tudzynski, Bettina

    2015-01-01

    The rice pathogenic fungus Fusarium fujikuroi is well known for the production of a broad spectrum of secondary metabolites (SMs) such as gibberellic acids (GAs), mycotoxins and pigments. The biosynthesis of most of these SMs strictly depends on nitrogen availability and of the activity of permeases of nitrogen sources, e.g. the ammonium and amino acid permeases. One of the three ammonium permeases, MepB, was recently shown to act not only as a transporter but also as a nitrogen sensor affecting the production of nitrogen-repressed SMs. Here we describe the identification of a general amino acid permease, FfGap1, among the 99 putative amino acid permeases (AAPs) in the genome of F. fujikuroi. FfGap1 is able to fully restore growth of the yeast gap1∆ mutant on several amino acids including citrulline and tryptophane. In S. cerevisiae, Gap1 activity is regulated by shuttling between the plasma membrane (nitrogen limiting conditions) and the vacuole (nitrogen sufficiency), which we also show for FfGap1. In yeast, the Npr1 serine/threonine kinase stabilizes the Gap1 position at the plasma membrane. Here, we identified and characterized three NPR1-homologous genes, encoding the putative protein kinases FfNpr1-1, FfNpr1-2 and FfNpr1-3 with significant similarity to yeast Npr1. Complementation of the yeast npr1Δ mutant with each of the three F. fujikuroi NPR1 homologues, resulted in partial restoration of ammonium, arginine and proline uptake by FfNPR1-1 while none of the three kinases affect growth on different nitrogen sources and nitrogen-dependent sorting of FfGap1 in F. fujikuroi. However, exchange of the putative ubiquitin-target lysine 9 (K9A) and 15 (K15A) residues of FfGap1 resulted in extended localization to the plasma membrane and increased protein stability independently of nitrogen availability. These data suggest a similar regulation of FfGap1 by nitrogen-dependent ubiquitination, but differences regarding the role of Fusarium Npr1 homologues compared to

  9. Pseudohyphal growth of Cryptococcus neoformans is a reversible dimorphic transition in response to ammonium that requires Amt1 and Amt2 ammonium permeases.

    PubMed

    Lee, Soo Chan; Phadke, Sujal; Sun, Sheng; Heitman, Joseph

    2012-11-01

    Cryptococcus neoformans is a human-pathogenic basidiomycete that commonly infects HIV/AIDS patients to cause meningoencephalitis (7, 19). C. neoformans grows as a budding yeast during vegetative growth or as hyphae during sexual reproduction. Pseudohyphal growth of C. neoformans has been observed rarely during murine and human infections but frequently during coculture with amoeba; however, the genetics underlying pseudohyphal growth are largely unknown. Our studies found that C. neoformans displays pseudohyphal growth under nitrogen-limiting conditions, especially when a small amount of ammonium is available as a sole nitrogen source. Pseudohyphal growth was observed with Cryptococcus neoformans serotypes A and D and Cryptococcus gattii. C. neoformans pseudohyphae bud to produce yeast cells and normal smooth hemispherical colonies when transferred to complete media, indicating that pseudohyphal growth is a conditional developmental stage. Subsequent analysis revealed that two ammonium permeases encoded by the AMT1 and AMT2 genes are required for pseudohyphal growth. Both amt1 and amt2 mutants are capable of forming pseudohyphae; however, amt1 amt2 double mutants do not form pseudohyphae. Interestingly, C. gattii pseudohypha formation is irreversible and involves a RAM pathway mutation that drives pseudohyphal development. We also found that pseudohyphal growth is related to the invasive growth into the medium. These results demonstrate that pseudohyphal growth is a common reversible growth pattern in C. neoformans but a mutational genetic event in C. gattii and provide new insights into understanding pseudohyphal growth of Cryptococcus.

  10. Metabolism of D-galactose is dispensable for the induction of the beta-galactosidase (bgaD) and lactose permease (lacpA) genes in Aspergillus nidulans.

    PubMed

    Orosz, Anita; Fekete, Erzsébet; Flipphi, Michel; Karaffa, Levente

    2014-10-01

    In this study, we analyze the expression of the Aspergillus nidulans bgaD-lacpA gene couple (encoding an intracellular beta-galactosidase and a lactose permease) in the presence of D-galactose. This monosaccharide can be catabolized via alternative, independent pathways in this model organism. The inductive capabilities of intermediates of the two alternative routes of D-galactose utilization were addressed in loss-of-function mutants defective in a defined step in one of the two pathways. In a galactokinase (galE9) mutant, the cluster is strongly induced by D-galactose, suggesting that formation of Leloir pathway intermediates is not required. The expression profiles of bgaD and lacpA were similar in wild type, L-arabinitol dehydrogenase (araA1), and hexokinase (hxkA1) negative backgrounds, indicating that intermediates of the oxido-reductive pathway downstream of galactitol are not necessary either. Furthermore, bgaD-lacpA transcription was not induced in any of the tested strains when galactitol was provided as the growth substrate. An hxkA1/galE9 double mutant cannot grow on d-galactose at all, but still produced bgaD and lacpA transcripts upon transfer to d-galactose. We therefore concluded that the physiological inducer of the bgaD-lacpA gene cluster upon growth on D-galactose is the nonmetabolized sugar itself.

  11. WW domains of Rsp5p define different functions: determination of roles in fluid phase and uracil permease endocytosis in Saccharomyces cerevisiae.

    PubMed

    Gajewska, B; Kamińska, J; Jesionowska, A; Martin, N C; Hopper, A K; Zoładek, T

    2001-01-01

    Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions. To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30 degrees. Thus, individual WW domains are not essential. Each rsp5-w mutation caused temperature-sensitive growth. Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is important for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis. Thus, various WW domains play different roles in the endocytosis of these two substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle. Altering WW domains does not change the location of Rsp5p.

  12. The N- and C-terminal mutations in tryptophan permease Tat2 confer cell growth in Saccharomyces cerevisiae under high-pressure and low-temperature conditions.

    PubMed

    Nagayama, Ai; Kato, Chiaki; Abe, Fumiyoshi

    2004-04-01

    Tryptophan uptake appears to be the limiting factor in growth of tryptophan auxotrophic Saccharomyces cerevisiae strains under the conditions of high hydrostatic pressure and low temperature. When the cells are subjected to a pressure of 25 MPa, tryptophan permease Tat2 is degraded in a manner dependent on ubiquitination by Rsp5. One of the high-pressure growth-conferring genes, HPG2, was shown to be allelic to TAT2. The HPG2-1 (Tat2(E27F)) mutation site is located within the ExKS motif in the N-terminus, and the HPG2-2 (Tat2(D563N)) and HPG2-3 (Tat2(E570K)) mutation sites are located at the KQEIAE sequence in the C-terminus. The HPG2 mutations enhance the stability of Tat2 during high-pressure or low-temperature incubation, leading to cell growth under these stressful conditions. These results suggest that the cytoplasmic tails are involved in Rsp5-mediated ubiquitination of Tat2 under high-pressure or low-temperature conditions.

  13. The lysine biosynthetic enzyme Lys4 influences iron metabolism, mitochondrial function and virulence in Cryptococcus neoformans.

    PubMed

    Do, Eunsoo; Park, Minji; Hu, Guanggan; Caza, Mélissa; Kronstad, James W; Jung, Won Hee

    2016-09-02

    The lysine biosynthesis pathway via α-aminoadipate in fungi is considered an attractive target for antifungal drugs due to its absence in mammalian hosts. The iron-sulfur cluster-containing enzyme homoaconitase converts homocitrate to homoisocitrate in the lysine biosynthetic pathway, and is encoded by LYS4 in the model yeast Saccharomyces cerevisiae. In this study, we identified the ortholog of LYS4 in the human fungal pathogen, Cryptococcus neoformans, and found that LYS4 expression is regulated by iron levels and by the iron-related transcription factors Hap3 and HapX. Deletion of the LYS4 gene resulted in lysine auxotrophy suggesting that Lys4 is essential for lysine biosynthesis. Our study also revealed that lysine uptake was mediated by two amino acid permeases, Aap2 and Aap3, and influenced by nitrogen catabolite repression (NCR). Furthermore, the lys4 mutant showed increased sensitivity to oxidative stress, agents that challenge cell wall/membrane integrity, and azole antifungal drugs. We showed that these phenotypes were due in part to impaired mitochondrial function as a result of LYS4 deletion, which we propose disrupts iron homeostasis in the organelle. The combination of defects are consistent with our observation that the lys4 mutant was attenuated virulence in a mouse inhalation model of cryptococcosis.

  14. Iron-refractory iron deficiency anemia (IRIDA).

    PubMed

    Heeney, Matthew M; Finberg, Karin E

    2014-08-01

    Iron deficiency anemia is a common global problem whose etiology is typically attributed to acquired inadequate dietary intake and/or chronic blood loss. However, in several kindreds multiple family members are affected with iron deficiency anemia that is unresponsive to oral iron supplementation and only partially responsive to parenteral iron therapy. The discovery that many of these cases harbor mutations in the TMPRSS6 gene led to the recognition that they represent a single clinical entity: iron-refractory iron deficiency anemia (IRIDA). This article reviews clinical features of IRIDA, recent genetic studies, and insights this disorder provides into the regulation of systemic iron homeostasis.

  15. Structure of classical affine and classical affine fractional W-algebras

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

    We introduce a classical BRST complex (See Definition 3.2.) and show that one can construct a classical affine W-algebra via the complex. This definition clarifies that classical affine W-algebras can be considered as quasi-classical limits of quantum affine W-algebras. We also give a definition of a classical affine fractional W-algebra as a Poisson vertex algebra. As in the classical affine case, a classical affine fractional W-algebra has two compatible λ-brackets and is isomorphic to an algebra of differential polynomials as a differential algebra. When a classical affine fractional W-algebra is associated to a minimal nilpotent, we describe explicit forms of free generators and compute λ-brackets between them. Provided some assumptions on a classical affine fractional W-algebra, we find an infinite sequence of integrable systems related to the algebra, using the generalized Drinfel’d and Sokolov reduction.

  16. Bioavailable affinity label for collagen prolyl 4-hydroxylase

    PubMed Central

    Vasta, James D.; Higgin, Joshua J.; Kersteen, Elizabeth A.

    2013-01-01

    Collagen is the most abundant protein in animals. Its prevalent 4-hydroxyproline residues contribute greatly to its conformational stability. The hydroxyl groups arise from a post-translational modification catalyzed by the non-heme iron-dependent enzyme, collagen prolyl 4-hydroxylase (P4H). Here, we report that 4-oxo-5,6-epoxyhexanoate, a mimic of the α-ketoglutarate co-substrate, inactivates human P4H. The inactivation installs a ketone functionality in P4H, providing a handle for proteomic experiments. Caenorhabditis elegans exposed to the esterified epoxy ketone displays the phenotype of a worm lacking P4H. Thus, this affinity label can be used to mediate collagen stability in an animal, as is desirable in the treatment of a variety of fibrotic diseases. PMID:23702396

  17. Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways.

    PubMed

    Schalk, Isabelle J; Guillon, Laurent

    2013-05-01

    Siderophore production and utilization is one of the major strategies deployed by bacteria to get access to iron, a key nutrient for bacterial growth. The biological function of siderophores is to solubilize iron in the bacterial environment and to shuttle it back to the cytoplasm of the microorganisms. This uptake process for Gram-negative species involves TonB-dependent transporters for translocation across the outer membranes. In Escherichia coli and many other Gram-negative bacteria, ABC transporters associated with periplasmic binding proteins import ferrisiderophores across cytoplasmic membranes. Recent data reveal that in some siderophore pathways, this step can also be carried out by proton-motive force-dependent permeases, for example the ferrichrome and ferripyochelin pathways in Pseudomonas aeruginosa. Iron is then released from the siderophores in the bacterial cytoplasm by different enzymatic mechanisms depending on the nature of the siderophore. Another strategy has been reported for the pyoverdine pathway in P. aeruginosa: iron is released from the siderophore in the periplasm and only siderophore-free iron is transported into the cytoplasm by an ABC transporter having two atypical periplasmic binding proteins. This review presents recent findings concerning both ferrisiderophore and siderophore-free iron transport across bacterial cytoplasmic membranes and considers current knowledge about the mechanisms involved in iron release from siderophores.

  18. Improving image segmentation by learning region affinities

    SciTech Connect

    Prasad, Lakshman; Yang, Xingwei; Latecki, Longin J

    2010-11-03

    We utilize the context information of other regions in hierarchical image segmentation to learn new regions affinities. It is well known that a single choice of quantization of an image space is highly unlikely to be a common optimal quantization level for all categories. Each level of quantization has its own benefits. Therefore, we utilize the hierarchical information among different quantizations as well as spatial proximity of their regions. The proposed affinity learning takes into account higher order relations among image regions, both local and long range relations, making it robust to instabilities and errors of the original, pairwise region affinities. Once the learnt affinities are obtained, we use a standard image segmentation algorithm to get the final segmentation. Moreover, the learnt affinities can be naturally unutilized in interactive segmentation. Experimental results on Berkeley Segmentation Dataset and MSRC Object Recognition Dataset are comparable and in some aspects better than the state-of-art methods.

  19. Transdermal iron replenishment therapy.

    PubMed

    Modepalli, Naresh; Shivakumar, H N; Kanni, K L Paranjothy; Murthy, S Narasimha

    2015-01-01

    Iron deficiency anemia is one of the major nutritional deficiency disorders. Iron deficiency anemia occurs due to decreased absorption of iron from diet, chronic blood loss and other associated diseases. The importance of iron and deleterious effects of iron deficiency anemia are discussed briefly in this review followed by the transdermal approaches to deliver iron. Transdermal delivery of iron would be able to overcome the side effects associated with conventional oral and parenteral iron therapy and improves the patient compliance. During preliminary investigations, ferric pyrophosphate and iron dextran were selected as iron sources for transdermal delivery. Different biophysical techniques were explored to assess their efficiency in delivering iron across the skin, and in vivo studies were carried out using anemic rat model. Transdermal iron delivery is a promising approach that could make a huge positive impact on patients suffering with iron deficiency.

  20. [Iron dysregulation and anemias].

    PubMed

    Ikuta, Katsuya

    2015-10-01

    Most iron in the body is utilized as a component of hemoglobin that delivers oxygen to the entire body. Under normal conditions, the iron balance is tightly regulated. However, iron dysregulation does occasionally occur; total iron content reductions cause iron deficiency anemia and overexpression of the iron regulatory peptide hepcidin disturbs iron utilization resulting in anemia of chronic disease. Conversely, the presence of anemia may ultimately lead to iron overload; for example, thalassemia, a common hereditary anemia worldwide, often requires transfusion, but long-term transfusions cause iron accumulation that leads to organ damage and other poor outcomes. On the other hand, there is a possibility that iron overload itself can cause anemia; iron chelation therapy for the post-transfusion iron overload observed in myelodysplastic syndrome or aplastic anemia improves dependency on transfusions in some cases. These observations reflect the extremely close relationship between anemias and iron metabolism.

  1. Characterization of the Plesiomonas shigelloides Genes Encoding the Heme Iron Utilization System

    PubMed Central

    Henderson, D. P.; Wyckoff, E. E.; Rashidi, C. E.; Verlei, H.; Oldham, A. L.

    2001-01-01

    Plesiomonas shigelloides is a gram-negative pathogen which can utilize heme as an iron source. In previous work, P. shigelloides genes which permitted heme iron utilization in a laboratory strain of Escherichia coli were isolated. In the present study, the cloned P. shigelloides sequences were found to encode ten potential heme utilization proteins: HugA, the putative heme receptor; TonB and ExbBD; HugB, the putative periplasmic binding protein; HugCD, the putative inner membrane permease; and the proteins HugW, HugX, and HugZ. Three of the genes, hugA, hugZ, and tonB, contain a Fur box in their putative promoters, indicating that the genes may be iron regulated. When the P. shigelloides genes were tested in E. coli K-12 or in a heme iron utilization mutant of P. shigelloides, hugA, the TonB system genes, and hugW, hugX, or hugZ were required for heme iron utilization. When the genes were tested in a hemA entB mutant of E. coli, hugWXZ were not required for utilization of heme as a porphyrin source, but their absence resulted in heme toxicity when the strains were grown in media containing heme as an iron source. hugA could replace the Vibrio cholerae hutA in a heme iron utilization assay, and V. cholerae hutA could complement a P. shigelloides heme utilization mutant, suggesting that HugA is the heme receptor. Our analyses of the TonB system of P. shigelloides indicated that it could function in tonB mutants of both E. coli and V. cholerae and that it was similar to the V. cholerae TonB1 system in the amino acid sequence of the proteins and in the ability of the system to function in high-salt medium. PMID:11292789

  2. Pharmacology of iron transport.

    PubMed

    Byrne, Shaina L; Krishnamurthy, Divya; Wessling-Resnick, Marianne

    2013-01-01

    Elucidating the molecular basis for the regulation of iron uptake, storage, and distribution is necessary to understand iron homeostasis. Pharmacological tools are emerging to identify and distinguish among different iron transport pathways. Stimulatory or inhibitory small molecules with effects on iron uptake can help characterize the mechanistic elements of iron transport and the roles of the transporters involved in these processes. In particular, iron chelators can serve as potential pharmacological tools to alleviate diseases of iron overload. This review focuses on the pharmacology of iron transport, introducing iron transport membrane proteins and known inhibitors.

  3. Pharmacology of Iron Transport

    PubMed Central

    Byrne, Shaina L.; Krishnamurthy, Divya; Wessling-Resnick, Marianne

    2013-01-01

    Elucidating the molecular basis for the regulation of iron uptake, storage, and distribution is necessary to understand iron homeostasis. Pharmacological tools are emerging to identify and distinguish among different iron transport pathways. Stimulatory or inhibitory small molecules with effects on iron uptake can help characterize the mechanistic elements of iron transport and the roles of the transporters involved in these processes. In particular, iron chelators can serve as potential pharmacological tools to alleviate diseases of iron overload. This review focuses on the pharmacology of iron transport, introducing iron transport membrane proteins and known inhibitors. PMID:23020294

  4. Iron acquisition in Pasteurella haemolytica: expression and identification of a bovine-specific transferrin receptor.

    PubMed Central

    Ogunnariwo, J A; Schryvers, A B

    1990-01-01

    Seven type 1 field isolates of Pasteurella haemolytica were screened for their ability to use different transferrins as a source of iron for growth. All seven strains were capable of using bovine but not human, porcine, avian, or equine transferrin. A screening assay failed to detect siderophore production in any of the strains tested. Iron-deficient cells from these strains expressed a binding activity, specific for bovine transferrin, that was regulated by the level of iron in the medium. Inhibition of expression by translation and transcription inhibitors suggested that iron regulation was occurring at the gene level. Affinity isolation of receptor proteins from all seven strains with biotinylated bovine transferrin identified a 100-kilodalton iron-regulated outer membrane protein as the bovine transferrin receptor. Iron-regulated outer membrane proteins of 71 and 77 kilodaltons were isolated along with the 100-kilodalton protein when less stringent washing procedures were employed in the affinity isolation procedure. Images PMID:2365453

  5. Affinity purification of copper chelating peptides from chickpea protein hydrolysates.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-05-16

    Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

  6. The affinity of magnetic microspheres for Schistosoma eggs.

    PubMed

    Candido, Renata R F; Favero, Vivian; Duke, Mary; Karl, Stephan; Gutiérrez, Lucía; Woodward, Robert C; Graeff-Teixeira, Carlos; Jones, Malcolm K; St Pierre, Timothy G

    2015-01-01

    Schistosomiasis is a chronic parasitic disease of humans, with two species primarily causing the intestinal infection: Schistosoma mansoni and Schistosoma japonicum. Traditionally, diagnosis of schistosomiasis is achieved through direct visualisation of eggs in faeces using techniques that lack the sensitivity required to detect all infections, especially in areas of low endemicity. A recently developed method termed Helmintex™ is a very sensitive technique for detection of Schistosoma eggs and exhibits 100% sensitivity at 1.3 eggs per gram of faeces, enough to detect even low-level infections. The Helminthex™ method is based on the interaction of magnetic microspheres and schistosome eggs. Further understanding the underlying egg-microsphere interactions would enable a targeted optimisation of egg-particle binding and may thus enable a significant improvement of the Helmintex™ method and diagnostic sensitivity in areas with low infection rates. We investigated the magnetic properties of S. mansoni and S. japonicum eggs and their interactions with microspheres with different magnetic properties and surface functionalization. Eggs of both species exhibited higher binding affinity to the magnetic microspheres than the non-magnetic microspheres. Binding efficiency was further enhanced if the particles were coated with streptavidin. Schistosoma japonicum eggs bound more microspheres compared with S. mansoni. However, distinct differences within eggs of each species were also observed when the distribution of the number of microspheres bound per egg was modelled with double Poisson distributions. Using this approach, both S. japonicum and S. mansoni eggs fell into two groups, one having greater affinity for magnetic microspheres than the other, indicating that not all eggs of a species exhibit the same binding affinity. Our observations suggest that interaction between the microspheres and eggs is more likely to be related to surface charge-based electrostatic

  7. The Cutting Edge of Affinity Electrophoresis Technology

    PubMed Central

    Kinoshita, Eiji; Kinoshita-Kikuta, Emiko; Koike, Tohru

    2015-01-01

    Affinity electrophoresis is an important technique that is widely used to separate and analyze biomolecules in the fields of biology and medicine. Both quantitative and qualitative information can be gained through affinity electrophoresis. Affinity electrophoresis can be applied through a variety of strategies, such as mobility shift electrophoresis, charge shift electrophoresis or capillary affinity electrophoresis. These strategies are based on changes in the electrophoretic patterns of biological macromolecules that result from interactions or complex-formation processes that induce changes in the size or total charge of the molecules. Nucleic acid fragments can be characterized through their affinity to other molecules, for example transcriptional factor proteins. Hydrophobic membrane proteins can be identified by means of a shift in the mobility induced by a charged detergent. The various strategies have also been used in the estimation of association/disassociation constants. Some of these strategies have similarities to affinity chromatography, in that they use a probe or ligand immobilized on a supported matrix for electrophoresis. Such methods have recently contributed to profiling of major posttranslational modifications of proteins, such as glycosylation or phosphorylation. Here, we describe advances in analytical techniques involving affinity electrophoresis that have appeared during the last five years. PMID:28248262

  8. Ferritin couples iron and fatty acid metabolism.

    PubMed

    Bu, Weiming; Liu, Renyu; Cheung-Lau, Jasmina C; Dmochowski, Ivan J; Loll, Patrick J; Eckenhoff, Roderic G

    2012-06-01

    A physiological relationship between iron, oxidative injury, and fatty acid metabolism exists, but transduction mechanisms are unclear. We propose that the iron storage protein ferritin contains fatty acid binding sites whose occupancy modulates iron uptake and release. Using isothermal microcalorimetry, we found that arachidonic acid binds ferritin specifically and with 60 μM affinity. Arachidonate binding by ferritin enhanced iron mineralization, decreased iron release, and protected the fatty acid from oxidation. Cocrystals of arachidonic acid and horse spleen apoferritin diffracted to 2.18 Å and revealed specific binding to the 2-fold intersubunit pocket. This pocket shields most of the fatty acid and its double bonds from solvent but allows the arachidonate tail to project well into the ferrihydrite mineralization site on the ferritin L-subunit, a structural feature that we implicate in the effects on mineralization by demonstrating that the much shorter saturated fatty acid, caprylate, has no significant effects on mineralization. These combined effects of arachidonate binding by ferritin are expected to lower both intracellular free iron and free arachidonate, thereby providing a previously unrecognized mechanism for limiting lipid peroxidation, free radical damage, and proinflammatory cascades during times of cellular stress.

  9. Iron and Your Child

    MedlinePlus

    ... get iron by eating foods like meat and dark green leafy vegetables. Iron is also added to ... tofu dried beans and peas dried fruits leafy dark green vegetables iron-fortified breakfast cereals, breads, and ...

  10. Iron metabolism and toxicity

    SciTech Connect

    Papanikolaou, G.; Pantopoulos, K. . E-mail: kostas.pantopoulos@mcgill.ca

    2005-01-15

    Iron is an essential nutrient with limited bioavailability. When present in excess, iron poses a threat to cells and tissues, and therefore iron homeostasis has to be tightly controlled. Iron's toxicity is largely based on its ability to catalyze the generation of radicals, which attack and damage cellular macromolecules and promote cell death and tissue injury. This is lucidly illustrated in diseases of iron overload, such as hereditary hemochromatosis or transfusional siderosis, where excessive iron accumulation results in tissue damage and organ failure. Pathological iron accumulation in the liver has also been linked to the development of hepatocellular cancer. Here we provide a background on the biology and toxicity of iron and the basic concepts of iron homeostasis at the cellular and systemic level. In addition, we provide an overview of the various disorders of iron overload, which are directly linked to iron's toxicity. Finally, we discuss the potential role of iron in malignant transformation and cancer.

  11. Tobacco Nicotine Uptake Permease Regulates the Expression of a Key Transcription Factor Gene in the Nicotine Biosynthesis Pathway1[C][W

    PubMed Central

    2014-01-01

    The down-regulation of a tobacco (Nicotiana tabacum) plasma membrane-localized nicotine uptake permease, NUP1, was previously reported to reduce total alkaloid levels in tobacco plants. However, it was unclear how this nicotine transporter affected the biosynthesis of the alkaloid nicotine. When NUP1 expression was suppressed in cultured tobacco cells treated with jasmonate, which induces nicotine biosynthesis, the NICOTINE2-locus transcription factor gene ETHYLENE RESPONSE FACTOR189 (ERF189) and its target structural genes, which function in nicotine biosynthesis and transport, were strongly suppressed, resulting in decreased total alkaloid levels. Conversely, NUP1 overexpression had the opposite effect. In these experiments, the expression levels of the MYC2 transcription factor gene and its jasmonate-inducible target gene were not altered. Inhibiting tobacco alkaloid biosynthesis by suppressing the expression of genes encoding enzymes in the nicotine pathway did not affect the expression of ERF189 and other nicotine pathway genes, indicating that ERF189 is not regulated by cellular alkaloid levels. Suppressing the expression of jasmonate signaling components in cultured tobacco cells showed that NUP1 acts downstream of the CORONATINE INSENSITIVE1 receptor and MYC2, but upstream of ERF189. These results suggest that although jasmonate-activated expression of MYC2 induces the expression of both NUP1 and ERF189, expression of ERF189 may actually be mediated by NUP1. Furthermore, NUP1 overexpression in tobacco plants inhibited the long-range transport of nicotine from the roots to the aerial parts. Thus, NUP1 not only mediates the uptake of tobacco alkaloids into root cells, but also positively controls the expression of ERF189, a key gene in the biosynthesis of these alkaloids. PMID:25344505

  12. Proper Fatty Acid Composition Rather than an Ionizable Lipid Amine Is Required for Full Transport Function of Lactose Permease from Escherichia coli*

    PubMed Central

    Vitrac, Heidi; Bogdanov, Mikhail; Dowhan, William

    2013-01-01

    Energy-dependent uphill transport but not energy-independent downhill transport by lactose permease (LacY) is impaired when expressed in Escherichia coli cells or reconstituted in liposomes lacking phosphatidylethanolamine (PE) and containing only anionic phospholipids. The absence of PE results in inversion of the N-terminal half and misfolding of periplasmic domain P7, which are required for uphill transport of substrates. Replacement of PE in vitro by lipids with no net charge (phosphatidylcholine (PC), monoglucosyl diacylglycerol (GlcDAG), or diglucosyl diacylglycerol (GlcGlcDAG)) supported wild type transmembrane topology of the N-terminal half of LacY. The restoration of uphill transport in vitro was dependent on LacY native topology and proper folding of P7. Support of uphill transport by net neutral lipids in vitro (PE > PC ≫ GlcDAG ≠ GlcGlcDAG provided that PE or PC contained one saturated fatty acid) paralleled the results observed previously in vivo (PE = PC > GlcDAG ≠ GlcGlcDAG). Therefore, a free amino group is not required for uphill transport as previously concluded based on the lack of in vitro uphill transport when fully unsaturated PC replaced E. coli-derived PE. A close correlation was observed in vivo and in vitro between the ability of LacY to carry out uphill transport, the native conformation of P7, and the lipid headgroup and fatty acid composition. Therefore, the headgroup and the fatty acid composition of lipids are important for defining LacY topological organization and catalytically important structural features, further illustrating the direct role of lipids, independent of other cellular factors, in defining membrane protein structure/function. PMID:23322771

  13. Four-locus phylogeny of Fusarium avenaceum and related species and their species-specific identification based on partial phosphate permease gene sequences.

    PubMed

    Stakheev, Alexander A; Khairulina, Dina R; Zavriev, Sergey K

    2016-05-16

    The fungus Fusarium avenaceum and its closest relatives are responsible for contamination of agricultural plants and their products by mycotoxins such as enniatins and moniliformin. Precise identification of mycotoxin producers is necessary for estimation of the accumulation risk of those compounds and for preventing the consumption of highly contaminated products. Nucleic acids amplification-based techniques proved to be the most rapid and reliable approach for pathogen diagnostics and identification. In this study partial phosphate permease gene (PHO) sequences were determined for Fusarium avenaceum (including one isolate identified as F. arthrosporioides), F. tricinctum, F. acuminatum and F. torulosum. Phylogenetic analysis of 40 isolates of those species from different climates and geographical regions of Russia and some neighboring countries based on sequences of PHO, translation elongation factor 1 alpha (TEF1α), beta-tubulin (β-TUB), enniatin synthetase (Esyn1) genes and combined data set demonstrated that the PHO gene possesses the highest rate of variability among them and can be considered as an informative marker for phylogenetic studies of these species. According to the combined data set phylogeny, the isolates of each species formed clusters with a high bootstrap support. Analysis of PHO sequences revealed a high intraspecific variability of F. avenaceum: there were 5 independent clusters on the dendrogram, including one cluster which was closer to F. torulosum than to other F. avenaceum isolates. Variable sites in PHO sequences have been used for the design of species-specific primers and a fluorescent hydrolysis probe. The specificity of the assay was shown for DNA samples extracted from 68 isolates of 23 Fusarium species. Quantitative PCR approach was applied to estimate the contamination rate of 17 naturally infected oat and barley samples, previously characterized by microbiological procedures.

  14. Expression patterns of Brassica napus genes implicate IPT, CKX, sucrose transporter, cell wall invertase, and amino acid permease gene family members in leaf, flower, silique, and seed development.

    PubMed

    Song, Jiancheng; Jiang, Lijun; Jameson, Paula Elizabeth

    2015-08-01

    Forage brassica (Brassica napus cv. Greenland) is bred for vegetative growth and biomass production, while its seed yield remains to be improved for seed producers without affecting forage yield and quality. Cytokinins affect seed yield by influencing flower, silique and seed number, and seed size. To identify specific cytokinin gene family members as targets for breeding, as well as genes associated with yield and/or quality, a B. napus transcriptome was obtained from a mixed sample including leaves, flower buds and siliques of various stages. Gene families for cytokinin biosynthesis (BnIPT1, 2, 3, 5, 7, 8 and 9), cytokinin degradation (BnCKX1 to BnCKX7), cell wall invertase (BnCWINV1 to BnCWINV6), sugar transporter (BnSUT1 to BnSUT6) and amino acid permease (BnAAP1 to BnAAP8) were identified. As B. napus is tetraploid, homoeologues of each gene family member were sought. Using multiple alignments and phylogenetic analysis, the parental genomes of the two B. napus homoeologues could be differentiated. RT-qPCR was then used to determine the expression of gene family members and their homoeologues in leaves, flowers, siliques and seeds of different developmental stages. The expression analysis showed both temporal and organ-specific expression profiles among members of these multi-gene families. Several pairs of homoeologues showed differential expression, both in terms of level of expression and differences in temporal or organ-specificity. BnCKX2 and 4 were identified as targets for TILLING, EcoTILLING and MAS.

  15. Expression patterns of Brassica napus genes implicate IPT, CKX, sucrose transporter, cell wall invertase, and amino acid permease gene family members in leaf, flower, silique, and seed development

    PubMed Central

    Song, Jiancheng; Jiang, Lijun; Jameson, Paula Elizabeth

    2015-01-01

    Forage brassica (Brassica napus cv. Greenland) is bred for vegetative growth and biomass production, while its seed yield remains to be improved for seed producers without affecting forage yield and quality. Cytokinins affect seed yield by influencing flower, silique and seed number, and seed size. To identify specific cytokinin gene family members as targets for breeding, as well as genes associated with yield and/or quality, a B. napus transcriptome was obtained from a mixed sample including leaves, flower buds and siliques of various stages. Gene families for cytokinin biosynthesis (BnIPT1, 2, 3, 5, 7, 8 and 9), cytokinin degradation (BnCKX1 to BnCKX7), cell wall invertase (BnCWINV1 to BnCWINV6), sugar transporter (BnSUT1 to BnSUT6) and amino acid permease (BnAAP1 to BnAAP8) were identified. As B. napus is tetraploid, homoeologues of each gene family member were sought. Using multiple alignments and phylogenetic analysis, the parental genomes of the two B. napus homoeologues could be differentiated. RT-qPCR was then used to determine the expression of gene family members and their homoeologues in leaves, flowers, siliques and seeds of different developmental stages. The expression analysis showed both temporal and organ-specific expression profiles among members of these multi-gene families. Several pairs of homoeologues showed differential expression, both in terms of level of expression and differences in temporal or organ-specificity. BnCKX2 and 4 were identified as targets for TILLING, EcoTILLING and MAS. PMID:25873685

  16. GABA (γ-Aminobutyric Acid) Uptake Via the GABA Permease GabP Represses Virulence Gene Expression in Pseudomonas syringae pv. tomato DC3000.

    PubMed

    McCraw, S L; Park, D H; Jones, R; Bentley, M A; Rico, A; Ratcliffe, R G; Kruger, N J; Collmer, A; Preston, G M

    2016-12-01

    The nonprotein amino acid γ-aminobutyric acid (GABA) is the most abundant amino acid in the tomato (Solanum lycopersicum) leaf apoplast and is synthesized by Arabidopsis thaliana in response to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (hereafter called DC3000). High levels of exogenous GABA have previously been shown to repress the expression of the type III secretion system (T3SS) in DC3000, resulting in reduced elicitation of the hypersensitive response (HR) in the nonhost plant tobacco (Nicotiana tabacum). This study demonstrates that the GABA permease GabP provides the primary mechanism for GABA uptake by DC3000 and that the gabP deletion mutant ΔgabP is insensitive to GABA-mediated repression of T3SS expression. ΔgabP displayed an enhanced ability to elicit the HR in young tobacco leaves and in tobacco plants engineered to produce increased levels of GABA, which supports the hypothesis that GABA uptake via GabP acts to regulate T3SS expression in planta. The observation that P. syringae can be rendered insensitive to GABA through loss of gabP but that gabP is retained by this bacterium suggests that GabP is important for DC3000 in a natural setting, either for nutrition or as a mechanism for regulating gene expression. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

  17. A Putative ABC Transporter Permease Is Necessary for Resistance to Acidified Nitrite and EDTA in Pseudomonas aeruginosa under Aerobic and Anaerobic Planktonic and Biofilm Conditions

    PubMed Central

    McDaniel, Cameron; Su, Shengchang; Panmanee, Warunya; Lau, Gee W.; Browne, Tristan; Cox, Kevin; Paul, Andrew T.; Ko, Seung-Hyun B.; Mortensen, Joel E.; Lam, Joseph S.; Muruve, Daniel A.; Hassett, Daniel J.

    2016-01-01

    Pseudomonas aeruginosa (PA) is an important airway pathogen of cystic fibrosis and chronic obstructive disease patients. Multiply drug resistant PA is becoming increasing prevalent and new strategies are needed to combat such insidious organisms. We have previously shown that a mucoid, mucA22 mutant PA is exquisitely sensitive to acidified nitrite (A-NO2−, pH 6.5) at concentrations that are well tolerated in humans. Here, we used a transposon mutagenesis approach to identify PA mutants that are hypersensitive to A-NO2−. Among greater than 10,000 mutants screened, we focused on PA4455, in which the transposon was found to disrupt the production of a putative cytoplasmic membrane-spanning ABC transporter permease. The PA4455 mutant was not only highly sensitive to A-NO2−, but also the membrane perturbing agent, EDTA and the antibiotics doxycycline, tigecycline, colistin, and chloramphenicol, respectively. Treatment of bacteria with A-NO2− plus EDTA, however, had the most dramatic and synergistic effect, with virtually all bacteria killed by 10 mM A-NO2−, and EDTA (1 mM, aerobic, anaerobic). Most importantly, the PA4455 mutant was also sensitive to A-NO2− in biofilms. A-NO2− sensitivity and an anaerobic growth defect was also noted in two mutants (rmlC and wbpM) that are defective in B-band LPS synthesis, potentially indicating a membrane defect in the PA4455 mutant. Finally, this study describes a gene, PA4455, that when mutated, allows for dramatic sensitivity to the potential therapeutic agent, A-NO2− as well as EDTA. Furthermore, the synergy between the two compounds could offer future benefits against antibiotic resistant PA strains. PMID:27064218

  18. Genetics Home Reference: iron-refractory iron deficiency anemia

    MedlinePlus

    ... refractory iron deficiency anemia iron-refractory iron deficiency anemia Enable Javascript to view the expand/collapse boxes. ... All Close All Description Iron-refractory iron deficiency anemia is one of many types of anemia , which ...

  19. Parenteral iron therapy options.

    PubMed

    Silverstein, Scott B; Rodgers, George M

    2004-05-01

    Parenteral iron therapy is occasionally necessary for patients intolerant or unresponsive to oral iron therapy, for receiving recombinant erythropoietin therapy, or for use in treating functional iron deficiency. There are now three parenteral iron products available: iron dextran, ferric gluconate, and iron sucrose. We summarize the advantages and disadvantages of each product, including risk of anaphylaxis and hypersensitivity, dosage regimens, and costs. The increased availability of multiple parenteral iron preparations should decrease the need to use red cell transfusions in patients with iron-deficiency anemia.

  20. Visualizing Antibody Affinity Maturation in Germinal Centers

    PubMed Central

    Tas, Jeroen M.J.; Mesin, Luka; Pasqual, Giulia; Targ, Sasha; Jacobsen, Johanne T.; Mano, Yasuko M.; Chen, Casie S.; Weill, Jean-Claude; Reynaud, Claude-Agnès; Browne, Edward P.; Meyer-Hermann, Michael; Victora, Gabriel D.

    2016-01-01

    Antibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC, and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with non-immunodominant specificities must be elicited, as is the case for HIV-1 and influenza. PMID:26912368

  1. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    EPA Science Inventory

    Despite the amount of resources that have been invested by national and international academic, government, and commercial sectors to develop affinity-based biosensor products, little obvious success has been realized through commercialization of these devices for specific applic...

  2. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-04-01

    PDZ domains function in nature as protein-binding domains within scaffold and membrane-associated proteins. They comprise approximately 90 residues and undergo specific, high-affinity interactions with complementary C-terminal peptide sequences, other PDZ domains, and/or phospholipids. We have previously shown that the specific, strong interactions of PDZ domains with their ligands make them well suited for use in affinity chromatography. This unit provides protocols for the PDZ affinity chromatography procedure that are applicable for the purification of proteins that contain PDZ domains or PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We detail the preparation of affinity resins composed of PDZ domains or PDZ domain peptide ligands coupled to solid supports. These resins can be used to purify proteins containing endogenous or genetically introduced PDZ domains or ligands, eluting the proteins with free PDZ domain peptide ligands.

  3. Affinity Electrophoresis Using Ligands Attached To Polymers

    NASA Technical Reports Server (NTRS)

    Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

    1990-01-01

    In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

  4. Nucleotide-specific recognition of iron-responsive elements by iron regulatory protein 1.

    PubMed

    Selezneva, Anna I; Walden, William E; Volz, Karl W

    2013-09-23

    IRP1 [iron regulatory protein (IRP) 1] is a bifunctional protein with mutually exclusive end-states. In one mode of operation, IRP1 binds iron-responsive element (IRE) stem-loops in messenger RNAs encoding proteins of iron metabolism to control their rate of translation. In its other mode, IRP1 serves as cytoplasmic aconitase to correlate iron availability with the energy and oxidative stress status of the cell. IRP1/IRE binding occurs through two separate interfaces, which together contribute about two-dozen hydrogen bonds. Five amino acids make base-specific contacts and are expected to contribute significantly to binding affinity and specificity of this protein:RNA interaction. In this mutagenesis study, each of the five base-specific amino acids was changed to alter binding at each site. Analysis of IRE binding affinity and translational repression activity of the resulting IRP1 mutants showed that four of the five contact points contribute uniquely to the overall binding affinity of the IRP1:IRE interaction, while one site was found to be unimportant. The stronger-than-expected effect on binding affinity of mutations at Lys379 and Ser681, residues that make contact with the conserved nucleotides G16 and C8, respectively, identified them as particularly critical for providing specificity and stability to IRP1:IRE complex formation. We also show that even though the base-specific RNA-binding residues are not part of the aconitase active site, their substitutions can affect the aconitase activity of holo-IRP1, positively or negatively.

  5. Iron-refractory iron deficiency anemia.

    PubMed

    Yılmaz Keskin, Ebru; Yenicesu, İdil

    2015-03-05

    Iron is essential for life because it is indispensable for several biological reactions, such as oxygen transport, DNA synthesis, and cell proliferation. Over the past few years, our understanding of iron metabolism and its regulation has changed dramatically. New disorders of iron metabolism have emerged, and the role of iron as a cofactor in other disorders has begun to be recognized. The study of genetic conditions such as hemochromatosis and iron-refractory iron deficiency anemia (IRIDA) has provided crucial insights into the molecular mechanisms controlling iron homeostasis. In the future, these advances may be exploited to improve treatment of both genetic and acquired iron disorders. IRIDA is caused by mutations in TMPRSS6, the gene encoding matriptase-2, which downregulates hepcidin expression under conditions of iron deficiency. The typical features of this disorder are hypochromic, microcytic anemia with a very low mean corpuscular volume of erythrocytes, low transferrin saturation, no (or inadequate) response to oral iron, and only a partial response to parenteral iron. In contrast to classic iron deficiency anemia, serum ferritin levels are usually low-normal, and serum or urinary hepcidin levels are inappropriately high for the degree of anemia. Although the number of cases reported thus far in the literature does not exceed 100, this disorder is considered the most common of the "atypical" microcytic anemias. The aim of this review is to share the current knowledge on IRIDA and increase awareness in this field.

  6. Iron-Refractory Iron Deficiency Anemia

    PubMed Central

    Yılmaz Keskin, Ebru; Yenicesu, İdil

    2015-01-01

    Iron is essential for life because it is indispensable for several biological reactions, such as oxygen transport, DNA synthesis, and cell proliferation. Over the past few years, our understanding of iron metabolism and its regulation has changed dramatically. New disorders of iron metabolism have emerged, and the role of iron as a cofactor in other disorders has begun to be recognized. The study of genetic conditions such as hemochromatosis and iron-refractory iron deficiency anemia (IRIDA) has provided crucial insights into the molecular mechanisms controlling iron homeostasis. In the future, these advances may be exploited to improve treatment of both genetic and acquired iron disorders. IRIDA is caused by mutations in TMPRSS6, the gene encoding matriptase-2, which downregulates hepcidin expression under conditions of iron deficiency. The typical features of this disorder are hypochromic, microcytic anemia with a very low mean corpuscular volume of erythrocytes, low transferrin saturation, no (or inadequate) response to oral iron, and only a partial response to parenteral iron. In contrast to classic iron deficiency anemia, serum ferritin levels are usually low-normal, and serum or urinary hepcidin levels are inappropriately high for the degree of anemia. Although the number of cases reported thus far in the literature does not exceed 100, this disorder is considered the most common of the “atypical” microcytic anemias. The aim of this review is to share the current knowledge on IRIDA and increase awareness in this field. PMID:25805669

  7. The copper-iron connection in biology: Structure of the metallo-oxidase Fet3p

    SciTech Connect

    Taylor, A. B.; Stoj, C. S.; Ziegler, L.; Kosman, D. J.; Hart, P. J.

    2005-10-17

    Fet3p is a multicopper-containing glycoprotein localized to the yeast plasma membrane that catalyzes the oxidation of Fe(II) to Fe(III). This ferrous iron oxidation is coupled to the reduction of O2 to H2O and is termed the ferroxidase reaction. Fet3p-produced Fe(III) is transferred to the permease Ftr1p for import into the cytosol. The posttranslational insertion of four copper ions into Fet3p is essential for its activity, thus linking copper and iron homeostasis. The mammalian ferroxidases ceruloplasmin and hephaestin are homologs of Fet3p. Loss of the Fe(II) oxidation catalyzed by these proteins results in a spectrum of pathological states, including death. Here, we present the structure of the Fet3p extracellular ferroxidase domain and compare it with that of human ceruloplasmin and other multicopper oxidases that are devoid of ferroxidase activity. The Fet3p structure delineates features that underlie the unique reactivity of this and homologous multicopper oxidases that support the essential trafficking of iron in diverse eukaryotic organisms. The findings are correlated with biochemical and physiological data to cross-validate the elements of Fet3p that define it as both a ferroxidase and cuprous oxidase.

  8. Potential of Phytase-Mediated Iron Release from Cereal-Based Foods: A Quantitative View

    PubMed Central

    Nielsen, Anne V. F.; Tetens, Inge; Meyer, Anne S.

    2013-01-01

    The major part of iron present in plant foods such as cereals is largely unavailable for direct absorption in humans due to complexation with the negatively charged phosphate groups of phytate (myo-inositol (1,2,3,4,5,6)-hexakisphosphate). Human biology has not evolved an efficient mechanism to naturally release iron from iron phytate complexes. This narrative review will evaluate the quantitative significance of phytase-catalysed iron release from cereal foods. In vivo studies have shown how addition of microbially derived phytases to cereal-based foods has produced increased iron absorption via enzyme-catalysed dephosphorylation of phytate, indicating the potential of this strategy for preventing and treating iron deficiency anaemia. Despite the immense promise of this strategy and the prevalence of iron deficiency worldwide, the number of human studies elucidating the significance of phytase-mediated improvements in iron absorption and ultimately in iron status in particularly vulnerable groups is still low. A more detailed understanding of (1) the uptake mechanism for iron released from partially dephosphorylated phytate chelates, (2) the affinity of microbially derived phytases towards insoluble iron phytate complexes, and (3) the extent of phytate dephosphorylation required for iron release from inositol phosphates is warranted. Phytase-mediated iron release can improve iron absorption from plant foods. There is a need for development of innovative strategies to obtain better effects. PMID:23917170

  9. Iron and Your Child

    MedlinePlus

    ... sure to teach kids that iron is an important part of a healthy diet. Foods rich in iron include: beef, pork, poultry, and seafood tofu dried beans and peas dried fruits leafy dark green vegetables iron-fortified breakfast cereals, breads, and pastas (Note: Iron from animal ...

  10. Iron stress in plants.

    PubMed

    Connolly, Erin L; Guerinot, Mary

    2002-07-30

    Although iron is an essential nutrient for plants, its accumulation within cells can be toxic. Plants, therefore, respond to both iron deficiency and iron excess by inducing expression of different gene sets. Here, we review recent advances in the understanding of iron homeostasis in plants gained through functional genomic approaches

  11. Gas-phase nitronium ion affinities.

    PubMed Central

    Cacace, F; de Petris, G; Pepi, F; Angelelli, F

    1995-01-01

    Evaluation of nitronium ion-transfer equilibria, L1NO2+ + L2 = L2NO2+ + L1 (where L1 and L2 are ligands 1 and 2, respectively) by Fourier-transform ion cyclotron resonance mass spectrometry and application of the kinetic method, based on the metastable fragmentation of L1(NO2+)L2 nitronium ion-bound dimers led to a scale of relative gas-phase nitronium ion affinities. This scale, calibrated to a recent literature value for the NO2+ affinity of water, led for 18 ligands, including methanol, ammonia, representative ketones, nitriles, and nitroalkanes, to absolute NO2+ affinities, that fit a reasonably linear general correlation when plotted vs. the corresponding proton affinities (PAs). The slope of the plot depends to a certain extent on the specific nature of the ligands and, hence, the correlations between the NO2+ affinities, and the PAs of a given class of compounds display a better linearity than the general correlation and may afford a useful tool for predicting the NO2+ affinity of a molecule based on its PA. The NO2+ binding energies are considerably lower than the corresponding PAs and well below the binding energies of related polyatomic cations, such as NO+, a trend consistent with the available theoretical results on the structure and the stability of simple NO2+ complexes. The present study reports an example of extension of the kinetic method to dimers, such as L1(NO2+)L2, bound by polyatomic ions, which may considerably widen its scope. Finally, measurement of the NO2+ affinity of ammonia allowed evaluation of the otherwise inaccessible PA of the amino group of nitramide and, hence, direct experimental verification of previous theoretical estimates. PMID:11607578

  12. Parenteral iron dextran therapy.

    PubMed

    Kumpf, V J; Holland, E G

    1990-02-01

    Parenteral iron therapy is indicated in patients with iron-deficiency anemia associated with conditions that interfere with the ingestion or absorption of oral iron. Replacement doses of iron required to replenish iron stores are based on body weight and the observed hemoglobin value. Methods of administering iron dextran are reviewed, including intramuscular and intravenous injections of the undiluted drug, intravenous infusion of a diluted preparation, and as an addition to parenteral nutrition solutions. The overall incidence of adverse reactions associated with the parenteral administration of iron is low, but the potential for an anaphylactic reaction requires that an initial test dose be given followed by careful patient observation.

  13. Parenteral iron supplementation.

    PubMed

    Kumpf, V J

    1996-08-01

    Indications for the use of parenteral iron are limited to conditions in which the oral supplementation of iron is not possible or fails. An overview of iron balance and iron requirements is presented to describe situations in which iron supplementation may be required. When parenteral iron supplementation is required, careful attention to proper dosing and administration is necessary to optimize efficacy and safety. The purpose of this article is to review the literature regarding the clinical use of parenteral iron therapy and provide guidelines on dosing and administration. Methods of iron dextran administration, including the IV and intramuscular injection of undiluted drug and total dose infusion, are compared. Complications associated with the use of parenteral iron are also be reviewed. Finally, the use of iron supplementation in patients receiving parenteral nutrition care explored.

  14. Stability of flavin semiquinones in the gas phase: the electron affinity, proton affinity, and hydrogen atom affinity of lumiflavin.

    PubMed

    Zhang, Tianlan; Papson, Kaitlin; Ochran, Richard; Ridge, Douglas P

    2013-11-07

    Examination of electron transfer and proton transfer reactions of lumiflavin and proton transfer reactions of the lumiflavin radical anion by Fourier transform ion cyclotron resonance mass spectrometry is described. From the equilibrium constant determined for electron transfer between 1,4-naphthoquinone and lumiflavin the electron affinity of lumiflavin is deduced to be 1.86 ± 0.1 eV. Measurements of the rate constants and efficiencies for proton transfer reactions indicate that the proton affinity of the lumiflavin radical anion is between that of difluoroacetate (331.0 kcal/mol) and p-formyl-phenoxide (333.0 kcal/mol). Combining the electron affinity of lumiflavin with the proton affinity of the lumiflavin radical anion gives a lumiflavin hydrogen atom affinity of 59.7 ± 2.2 kcal/mol. The ΔG298 deduced from these results for adding an H atom to gas phase lumiflavin, 52.1 ± 2.2 kcal/mol, is in good agreement with ΔG298 for adding an H atom to aqueous lumiflavin from electrochemical measurements in the literature, 51.0 kcal/mol, and that from M06-L density functional calculations in the literature, 51.2 kcal/mol, suggesting little, if any, solvent effect on the H atom addition. The proton affinity of lumiflavin deduced from the equilibrium constant for the proton transfer reaction between lumiflavin and 2-picoline is 227.3 ± 2.0 kcal mol(-1). Density functional theory calculations on isomers of protonated lumiflavin provide a basis for assigning the most probable site of protonation as position 1 on the isoalloxazine ring and for estimating the ionization potentials of lumiflavin neutral radicals.

  15. Proton Affinity Calculations with High Level Methods.

    PubMed

    Kolboe, Stein

    2014-08-12

    Proton affinities, stretching from small reference compounds, up to the methylbenzenes and naphthalene and anthracene, have been calculated with high accuracy computational methods, viz. W1BD, G4, G3B3, CBS-QB3, and M06-2X. Computed and the currently accepted reference proton affinities are generally in excellent accord, but there are deviations. The literature value for propene appears to be 6-7 kJ/mol too high. Reported proton affinities for the methylbenzenes seem 4-5 kJ/mol too high. G4 and G3 computations generally give results in good accord with the high level W1BD. Proton affinity values computed with the CBS-QB3 scheme are too low, and the error increases with increasing molecule size, reaching nearly 10 kJ/mol for the xylenes. The functional M06-2X fails markedly for some of the small reference compounds, in particular, for CO and ketene, but calculates methylbenzene proton affinities with high accuracy.

  16. Classification of neocortical interneurons using affinity propagation.

    PubMed

    Santana, Roberto; McGarry, Laura M; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits.

  17. Classification of neocortical interneurons using affinity propagation

    PubMed Central

    Santana, Roberto; McGarry, Laura M.; Bielza, Concha; Larrañaga, Pedro; Yuste, Rafael

    2013-01-01

    In spite of over a century of research on cortical circuits, it is still unknown how many classes of cortical neurons exist. In fact, neuronal classification is a difficult problem because it is unclear how to designate a neuronal cell class and what are the best characteristics to define them. Recently, unsupervised classifications using cluster analysis based on morphological, physiological, or molecular characteristics, have provided quantitative and unbiased identification of distinct neuronal subtypes, when applied to selected datasets. However, better and more robust classification methods are needed for increasingly complex and larger datasets. Here, we explored the use of affinity propagation, a recently developed unsupervised classification algorithm imported from machine learning, which gives a representative example or exemplar for each cluster. As a case study, we applied affinity propagation to a test dataset of 337 interneurons belonging to four subtypes, previously identified based on morphological and physiological characteristics. We found that affinity propagation correctly classified most of the neurons in a blind, non-supervised manner. Affinity propagation outperformed Ward's method, a current standard clustering approach, in classifying the neurons into 4 subtypes. Affinity propagation could therefore be used in future studies to validly classify neurons, as a first step to help reverse engineer neural circuits. PMID:24348339

  18. Identity, Affinity, Reality: Making the Case for Affinity Groups in Elementary School

    ERIC Educational Resources Information Center

    Parsons, Julie; Ridley, Kimberly

    2012-01-01

    Affinity groups are places where students build connections and process "ouch" moments from their classes. Children talk about the isolation they sometimes feel. The relationships students gain through race-based affinity groups enable them to feel less alone with their emotions and help them build a stronger sense of self. At the same…

  19. Stepparents' Affinity-Seeking and Affinity-Maintaining Strategies with Stepchildren.

    ERIC Educational Resources Information Center

    Ganong, Lawrence; Coleman, Marilyn; Fine, Mark; Martin, Patricia

    1999-01-01

    Examines the strategies that stepparents use to develop and maintain affinity with stepchildren and the effects that these strategies have on the development of stepparent-stepchildren relationships. Thirty-one affinity-seeking strategies are identified. Results show that dyadic activities worked best, but it is important that stepchildren…

  20. Phosphorylation of a conserved Thr357 in yeast Nedd4-like ubiquitin ligase Rsp5 is involved in down-regulation of the general amino acid permease Gap1.

    PubMed

    Sasaki, Toshiya; Takagi, Hiroshi

    2013-06-01

    Rsp5, an essential HECT-type ubiquitin ligase, is the only yeast Saccharomyces cerevisiae member of the Nedd4 family. Rsp5 triggers the ubiquitination-dependent endocytosis of the general amino acid permease Gap1 in response to a good nitrogen source. Previously, we showed that the Thr357Ala/Lys764Glu variant Rsp5 induces the constitutive inactivation of Gap1, which is mainly involved in uptake of the toxic proline analogue, l-azetidine-2-carboxylate (AZC). Here, our experimental results indicated that the Thr357Ala substitution in the substrate-recognizing WW2 domain of Rsp5 constitutively causes the down-regulation of four proline permeases (Gap1, Put4, Agp1 and Gnp1), leading to AZC tolerance to yeast cells. In RSP5(T357A) cells, Gap1 was highly ubiquitinated and constantly delivered to the vacuole from the Golgi without sorting to the plasma membrane. Analyses of RSP5 mutants using antiphosphopeptide antibody suggest that Thr phosphorylation occurred in all three WW domains and, interestingly, that Thr357 in the WW2 domain was phosphorylated, in agreement with the in vitro result for the mouse Rsp5 orthologue. Furthermore, the phosphorylation-mimic mutant (Thr357Asp) showed strong sensitivity to AZC. From these results, we propose a possible mechanism involved in the regulation of Rsp5 activity for Gap1 down-regulation via the phosphorylation of a conserved Thr357 in the Nedd4 family.

  1. Affine coherent states and Toeplitz operators

    NASA Astrophysics Data System (ADS)

    Hutníková, Mária; Hutník, Ondrej

    2012-06-01

    We study a parameterized family of Toeplitz operators in the context of affine coherent states based on the Calderón reproducing formula (= resolution of unity on L_2( {R})) and the specific admissible wavelets (= affine coherent states in L_2( {R})) related to Laguerre functions. Symbols of such Calderón-Toeplitz operators as individual coordinates of the affine group (= upper half-plane with the hyperbolic geometry) are considered. In this case, a certain class of pseudo-differential operators, their properties and their operator algebras are investigated. As a result of this study, the Fredholm symbol algebras of the Calderón-Toeplitz operator algebras for these particular cases of symbols are described. This article is part of a special issue of Journal of Physics A: Mathematical and Theoretical devoted to ‘Coherent states: mathematical and physical aspects’.

  2. Non-affine elasticity in jammed systems

    NASA Astrophysics Data System (ADS)

    Maloney, Craig

    2006-03-01

    Symmetry dictates that perfect crystals should deform homogeneously, or affinely, under external load, and computing the elastic moduli from the underlying interaction potential is then straightforward. For disordered materials no such simple procedure exists, and recent numerical works have demonstrated that non-affine corrections can dramatically reduce the naive expectation for the shear modulus in a broad class of disordered systems and may control rigidity loss in the zero pressure limit in purely repulsive systems, i.e. the unjamming transition (c.f. [O'Hern et. al. PRE 68, 011306 (2003)]). We present numerical results and an analytical framework for the study of these non-affine corrections to the elastic response of disordered packings.

  3. Biomimetic affinity ligands for protein purification.

    PubMed

    Sousa, Isabel T; Taipa, M Angela

    2014-01-01

    The development of sophisticated molecular modeling software and new bioinformatic tools, as well as the emergence of data banks containing detailed information about a huge number of proteins, enabled the de novo intelligent design of synthetic affinity ligands. Such synthetic compounds can be tailored to mimic natural biological recognition motifs or to interact with key surface-exposed residues on target proteins and are designated as "biomimetic ligands." A well-established methodology for generating biomimetic or synthetic affinity ligands integrates rational design with combinatorial solid-phase synthesis and screening, using the triazine scaffold and analogues of amino acids side chains to create molecular diversity.Triazine-based synthetic ligands are nontoxic, low-cost, highly stable compounds that can replace advantageously natural biological ligands in the purification of proteins by affinity-based methodologies.

  4. Iron metabolism and related genetic diseases: A cleared land, keeping mysteries.

    PubMed

    Brissot, Pierre; Loréal, Olivier

    2016-02-01

    Body iron has a very close relationship with the liver. Physiologically, the liver synthesizes transferrin, in charge of blood iron transport; ceruloplasmin, acting through its ferroxidase activity; and hepcidin, the master regulator of systemic iron. It also stores iron inside ferritin and serves as an iron reservoir, both protecting the cell from free iron toxicity and ensuring iron delivery to the body whenever needed. The liver is first in line for receiving iron from the gut and the spleen, and is, therefore, highly exposed to iron overload when plasma iron is in excess, especially through its high affinity for plasma non-transferrin bound iron. The liver is strongly involved when iron excess is related either to hepcidin deficiency, as in HFE, hemojuvelin, hepcidin, and transferrin receptor 2 related haemochromatosis, or to hepcidin resistance, as in type B ferroportin disease. It is less involved in the usual (type A) form of ferroportin disease which targets primarily the macrophagic system. Hereditary aceruloplasminemia raises important pathophysiological issues in light of its peculiar organ iron distribution.

  5. Use of Affinity Diagrams as Instructional Tools in Inclusive Classrooms.

    ERIC Educational Resources Information Center

    Haselden, Polly G.

    2003-01-01

    This article describes how the affinity diagram, a tool for gathering information and organizing it into natural groupings, can be used in inclusive classrooms. It discusses how students can be taught to use an affinity diagram, how affinity diagrams can be used to reflect many voices, and how affinity diagrams can be used to plan class projects.…

  6. Cation affinity numbers of Lewis bases.

    PubMed

    Lindner, Christoph; Tandon, Raman; Maryasin, Boris; Larionov, Evgeny; Zipse, Hendrik

    2012-01-01

    Using selected theoretical methods the affinity of a large range of Lewis bases towards model cations has been quantified. The range of model cations includes the methyl cation as the smallest carbon-centered electrophile, the benzhydryl and trityl cations as models for electrophilic substrates encountered in Lewis base-catalyzed synthetic procedures, and the acetyl cation as a substrate model for acyl-transfer reactions. Affinities towards these cationic electrophiles are complemented by data for Lewis-base addition to Michael acceptors as prototypical neutral electrophiles.

  7. New unitary affine-Virasoro constructions

    SciTech Connect

    Halpern, M.B.; Kiritsis, E.; Obers, N.A.; Poratti, M. ); Yamron, J.P. )

    1990-06-20

    This paper reports on a quasi-systematic investigation of the Virasoro master equation. The space of all affine-Virasoro constructions is organized by K-conjugation into affine-Virasoro nests, and an estimate of the dimension of the space shows that most solutions await discovery. With consistent ansatze for the master equation, large classes of new unitary nests are constructed, including quadratic deformation nests with continuous conformal weights, and unitary irrational central charge nests, which may dominate unitary rational central charge on compact g.

  8. On the electron affinity of B2

    SciTech Connect

    Glezakou, Vanda A.; Taylor, Peter

    2009-02-02

    We present the results of high-level ab initio calculations on the electron affinity of B2. Our new best estimate of 1.93±0.03 eV is in agreement with previous calculations as well as the sole existing experimental estimate of 1.8 eV, as derived from quantities with an uncertainty of 0.4 eV. The electron affinity of atomic boron, which is much smaller, is also calculated for comparison, and again found to be in good agreement with experiment. Pacific Northwest National Laboratory is operated by Battelle for the US Department of Energy.

  9. Negative Electron Affinity Mechanism for Diamond Surfaces

    NASA Technical Reports Server (NTRS)

    Krainsky, I. L.; Asnin, V. M.

    1998-01-01

    The energy distribution of the secondary electrons for chemical vacuum deposited diamond films with Negative Electron Affinity (NEA) was investigated. It was found that while for completely hydrogenated diamond surfaces the negative electron affinity peak in the energy spectrum of the secondary electrons is present for any energy of the primary electrons, for partially hydrogenated diamond surfaces there is a critical energy above which the peak is present in the spectrum. This critical energy increases sharply when hydrogen coverage of the diamond surface diminishes. This effect was explained by the change of the NEA from the true type for the completely hydrogenated surface to the effective type for the partially hydrogenated surfaces.

  10. Evidence of multi-affinity in the Japanese stock market

    NASA Astrophysics Data System (ADS)

    Katsuragi, Hiroaki

    2000-04-01

    Fluctuations of the Japanese stock market (Tokyo Stock Price Index: TOPIX) are analyzed using a multi-affine analysis method. In the research to date, only some simulated self-affine models have shown multi-affinity. In most experiments using observations of self-affine fractal profiles, multi-affinity has not been found. However, we find evidence of multi-affinity in fluctuations of the Japanese stock market (TOPIX). The qth-order Hurst exponent Hq varies with changes in q. This multi-affinity indicates that there are plural mechanisms that affect the same time scale as stock market price fluctuation dynamics.

  11. Can we accurately quantify nanoparticle associated proteins when constructing high-affinity MRI molecular imaging probes?

    PubMed

    Rimkus, Gabriella; Bremer-Streck, Sibylle; Grüttner, Cordula; Kaiser, Werner Alois; Hilger, Ingrid

    2011-01-01

    Targeted magnetic resonance contrast agents (e.g. iron oxide nanoparticles) have the potential to become highly selective imaging tools. In this context, quantification of the coupled amount of protein is essential for the design of antibody- or antibody fragment-conjugated nanoparticles. Nevertheless, the presence of magnetic iron oxide nanoparticles is still an unsolved problem for this task. The aim of the present work was to clarify whether proteins can be reliably quantified directly in the presence of magnetic iron oxide nanoparticles without the use of fluorescence or radioactivity. Protein quantification via Bradford was not influenced by the presence of magnetic iron oxide nanoparticles (0-17.2 mmol Fe l(-1) ). Instead, bicinchoninic acid based assay was, indeed, distinctly affected by the presence of nanoparticle-iron in suspension (0.1-17.2 mmol Fe l(-1) ), although the influence was linear. This observation allowed for adequate mathematical corrections with known iron content of a given nanoparticle. The applicability of our approach was demonstrated by the determination of bovine serum albumin (BSA) content coupled to dextrane-coated magnetic nanoparticles, which was found with the QuantiPro Bicinchoninic acid assay to be of 1.5 ± 0.2 µg BSA per 1 mg nanoparticle. Both Bradford and bicinchoninic acid assay protein assays allow for direct quantification of proteins in the presence of iron oxide containing magnetic nanoparticles, without the need for the introduction of radioactivity or fluorescence modules. Thus in future it should be possible to make more precise estimations about the coupled protein amount in high-affinity targeted MRI probes for the identification of specific molecules in living organisms, an aspect which is lacking in corresponding works published so far. Additionally, the present protein coupling procedures can be drastically improved by our proposed protein quantification method.

  12. Lack of Plasma Protein Hemopexin Results in Increased Duodenal Iron Uptake

    PubMed Central

    Fiorito, Veronica; Geninatti Crich, Simonetta; Silengo, Lorenzo; Aime, Silvio; Altruda, Fiorella; Tolosano, Emanuela

    2013-01-01

    Purpose The body concentration of iron is regulated by a fine equilibrium between absorption and losses of iron. Iron can be absorbed from diet as inorganic iron or as heme. Hemopexin is an acute phase protein that limits iron access to microorganisms. Moreover, it is the plasma protein with the highest binding affinity for heme and thus it mediates heme-iron recycling. Considering its involvement in iron homeostasis, it was postulated that hemopexin may play a role in the physiological absorption of inorganic iron. Methods and Results Hemopexin-null mice showed elevated iron deposits in enterocytes, associated with higher duodenal H-Ferritin levels and a significant increase in duodenal expression and activity of heme oxygenase. The expression of heme-iron and inorganic iron transporters was normal. The rate of iron absorption was assessed by measuring the amount of 57Fe retained in tissues from hemopexin-null and wild-type animals after administration of an oral dose of 57FeSO4 or of 57Fe-labelled heme. Higher iron retention in the duodenum of hemopexin-null mice was observed as compared with normal mice. Conversely, iron transfer from enterocytes to liver and bone marrow was unaffected in hemopexin-null mice. Conclusions The increased iron level in hemopexin-null duodenum can be accounted for by an increased iron uptake by enterocytes and storage in ferritins. These data indicate that the lack of hemopexin under physiological conditions leads to an enhanced duodenal iron uptake thus providing new insights to our understanding of body iron homeostasis. PMID:23826373

  13. Recovery of uranium from material containing iron, arsenic and siliceous matter

    SciTech Connect

    Weir, D.R.; Genik-Sas-Berezowsky, R.M.; Masters, I.M.

    1983-09-27

    A process is disclosed for the recovery of uranium values from uranium-containing material which also contains iron, arsenic and siliceous matter. The process includes leaching the uranium-containing material in aqueous sulphuric acid solution under conditions to provide dissolved iron present in the resultant leach solution as predominantly ferrous iron rather than ferric iron and/or to provide a sulphuric acid concentration in the leach solution sufficiently high to substantially prevent the precipitation of arsenates. Uranium values are recovered from the leach solution by solvent extraction agent which has little affinity for arsenic.

  14. Iron-induced turnover of the Arabidopsis IRON-REGULATED TRANSPORTER1 metal transporter requires lysine residues.

    PubMed

    Kerkeb, Loubna; Mukherjee, Indrani; Chatterjee, Iera; Lahner, Brett; Salt, David E; Connolly, Erin L

    2008-04-01

    Iron is an essential micronutrient but is toxic if accumulated at high levels. Thus, iron uptake and distribution in plants are controlled by precise regulatory mechanisms. IRON-REGULATED TRANSPORTER1 (IRT1) is the major high affinity iron transporter responsible for iron uptake from the soil in Arabidopsis (Arabidopsis thaliana). Previously, we showed that IRT1 is subject to posttranscriptional regulation; when expressed from the constitutive cauliflower mosaic virus 35S promoter, IRT1 protein accumulates only in iron-deficient roots. IRT1 contains an intracellular loop that may be critical for posttranslational regulation by metals. Of particular interest are a histidine (His) motif (HGHGHGH) that might bind metals and two lysine residues that could serve as attachment sites for ubiquitin. We constructed a set of mutant IRT1 alleles: IRT1H154Q, IRT1H156Q, IRT1H158Q, IRT1H160Q, IRT14HQ (quadruple His mutant), IRT1K146R, IRT1K171R, and a double mutant (IRT1K146R,K171R). Mutation of the His or lysine residues did not eliminate the ability of IRT1 to transport iron or zinc. Expression of each of the IRT1 variants and an IRT1intact construct in plants from the 35S promoter revealed that either K146 or K171 is required for iron-induced protein turnover, and 35S-IRT1K146R,K171R plants contain higher levels of iron as compared to 35S-IRT1 and wild type. Furthermore, accumulation of metals in 35S-IRT1K146R,K171R plants was not associated with an increase in ferric chelate reductase activity; this result indicates that, at least under conditions when iron is abundant, reduction of ferric iron may not be the rate-limiting step in iron uptake by strategy I plants such as Arabidopsis.

  15. Siderophore-Based Iron Acquisition and Pathogen Control

    PubMed Central

    Miethke, Marcus; Marahiel, Mohamed A.

    2007-01-01

    Summary: High-affinity iron acquisition is mediated by siderophore-dependent pathways in the majority of pathogenic and nonpathogenic bacteria and fungi. Considerable progress has been made in characterizing and understanding mechanisms of siderophore synthesis, secretion, iron scavenging, and siderophore-delivered iron uptake and its release. The regulation of siderophore pathways reveals multilayer networks at the transcriptional and posttranscriptional levels. Due to the key role of many siderophores during virulence, coevolution led to sophisticated strategies of siderophore neutralization by mammals and (re)utilization by bacterial pathogens. Surprisingly, hosts also developed essential siderophore-based iron delivery and cell conversion pathways, which are of interest for diagnostic and therapeutic studies. In the last decades, natural and synthetic compounds have gained attention as potential therapeutics for iron-dependent treatment of infections and further diseases. Promising results for pathogen inhibition were obtained with various siderophore-antibiotic conjugates acting as “Trojan horse” toxins and siderophore pathway inhibitors. In this article, general aspects of siderophore-mediated iron acquisition, recent findings regarding iron-related pathogen-host interactions, and current strategies for iron-dependent pathogen control will be reviewed. Further concepts including the inhibition of novel siderophore pathway targets are discussed. PMID:17804665

  16. Investigating the Affinities and Persistence of VX Nerve Agent in Environmental Matrices

    SciTech Connect

    Love, A H; Vance, A L; Reynolds, J G; Davisson, M L

    2004-03-09

    Laboratory experiments were conducted to determine environmental variables that affect the affinities and persistence of the nerve agent O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX) at dilute concentrations in environmental matrices. Quantitative analyses of VX and its degradation products were performed using LC-MS. Batch hydrolysis experiments demonstrated an increasing hydrolysis rate as pH increased, as shown in previous studies, but also indicated that dissolved aqueous constituents can cause significant differences in the absolute hydrolysis rate. Adsorption isotherms from batch aqueous experiments revealed that VX has a high affinity for hydrophobic organics, a moderate affinity for montmorillonite clay, and a very low affinity for an iron-oxyhydroxide soil mineral, goethite. The adsorption on goethite was increased with the presence of dissolved organic matter in solution. VX degraded rapidly when dried onto goethite, when an inner-sphere complex was forced. No enhanced degradation occurred with goethite in small amounts water. These results suggest that aqueous conditions have important controls on VX adsorption and degradation in the environment and a more mechanistic understanding of these controls is needed in order to enable accurate predictions of its long-term fate and persistence.

  17. Investigating the affinities and persistence of VX nerve agent in environmental matrices.

    PubMed

    Love, Adam H; Vance, Andrew L; Reynolds, John G; Davisson, M Lee

    2004-12-01

    Laboratory experiments were conducted to determine environmental variables that affect the affinities and persistence of the nerve agent O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX) at dilute concentrations in environmental matrices. Quantitative analyses of VX and its degradation products were performed using LC-MS. Batch hydrolysis experiments demonstrated an increasing hydrolysis rate as pH increased, as shown in previous studies, but also indicated that dissolved aqueous constituents can cause significant differences in the absolute hydrolysis rate. Adsorption isotherms from batch aqueous experiments revealed that VX has a high affinity for hydrophobic organics, a moderate affinity for montmorillonite clay, and a very low affinity for an iron-oxyhydroxide soil mineral, goethite. The adsorption on goethite was increased with the presence of dissolved organic matter in solution. VX degraded rapidly when dried onto goethite, when specific adsorption was forced. No enhanced degradation occurred with goethite in small amounts of water. These results suggest that aqueous conditions have important controls on VX adsorption and degradation in the environment and a more mechanistic understanding of these controls is needed in order to enable accurate predictions of its long-term fate and persistence.

  18. Kinetics of iron acquisition from ferric siderophores by Paracoccus denitrificans

    SciTech Connect

    Bergeron, R.J.; Weimar, W.R. )

    1990-05-01

    The kinetics of iron accumulation by iron-starved Paracoccus denitrificans during the first 2 min of exposure to 55Fe-labeled ferric siderophore chelates is described. Iron is acquired from the ferric chelate of the natural siderophore L-parabactin in a process exhibiting biphastic kinetics by Lineweaver-Burk analysis. The kinetic data for 1 microM less than (Fe L-parabactin) less than 10 microM fit a regression line which suggests a low-affinity system (Km = 3.9 +/- 1.2 microM, Vmax = 494 pg-atoms of 55Fe min-1 mg of protein-1), whereas the data for 0.1 microM less than or equal to (Fe L-parabactin) less than or equal to 1 microM fit another line consistent with a high-affinity system (Km = 0.24 +/- 0.06 microM, Vmax = 108 pg-atoms of 55Fe min-1 mg of protein-1). The Km of the high-affinity uptake is comparable to the binding affinity we had previously reported for the purified ferric L-parabactin receptor protein in the outer membrane. In marked contrast, ferric D-parabactin data fit a single regression line corresponding to a simple Michaelis-Menten process with comparatively low affinity (Km = 3.1 +/- 0.9 microM, Vmax = 125 pg-atoms of 55Fe min-1 mg of protein-1). Other catecholamide siderophores with an intact oxazoline ring derived from L-threonine (L-homoparabactin, L-agrobactin, and L-vibriobactin) also exhibit biphasic kinetics with a high-affinity component similar to ferric L-parabactin. Circular dichroism confirmed that these ferric chelates, like ferric L-parabactin, exist as the lambda enantiomers.

  19. On modality and complexity of affine embeddings

    SciTech Connect

    Arzhantsev, I V

    2001-08-31

    Let G be a reductive algebraic group and let H be a reductive subgroup of G. The modality of a G-variety X is the largest number of the parameters in a continuous family of G-orbits in X. A precise formula for the maximum value of the modality over all affine embeddings of the homogeneous space G/H is obtained.

  20. Modern affinity reagents: Recombinant antibodies and aptamers.

    PubMed

    Groff, Katherine; Brown, Jeffrey; Clippinger, Amy J

    2015-12-01

    Affinity reagents are essential tools in both basic and applied research; however, there is a growing concern about the reproducibility of animal-derived monoclonal antibodies. The need for higher quality affinity reagents has prompted the development of methods that provide scientific, economic, and time-saving advantages and do not require the use of animals. This review describes two types of affinity reagents, recombinant antibodies and aptamers, which are non-animal technologies that can replace the use of animal-derived monoclonal antibodies. Recombinant antibodies are protein-based reagents, while aptamers are nucleic-acid-based. In light of the scientific advantages of these technologies, this review also discusses ways to gain momentum in the use of modern affinity reagents, including an update to the 1999 National Academy of Sciences monoclonal antibody production report and federal incentives for recombinant antibody and aptamer efforts. In the long-term, these efforts have the potential to improve the overall quality and decrease the cost of scientific research.

  1. Validation of affinity reagents using antigen microarrays.

    PubMed

    Sjöberg, Ronald; Sundberg, Mårten; Gundberg, Anna; Sivertsson, Asa; Schwenk, Jochen M; Uhlén, Mathias; Nilsson, Peter

    2012-06-15

    There is a need for standardised validation of affinity reagents to determine their binding selectivity and specificity. This is of particular importance for systematic efforts that aim to cover the human proteome with different types of binding reagents. One such international program is the SH2-consortium, which was formed to generate a complete set of renewable affinity reagents to the SH2-domain containing human proteins. Here, we describe a microarray strategy to validate various affinity reagents, such as recombinant single-chain antibodies, mouse monoclonal antibodies and antigen-purified polyclonal antibodies using a highly multiplexed approach. An SH2-specific antigen microarray was designed and generated, containing more than 6000 spots displayed by 14 identical subarrays each with 406 antigens, where 105 of them represented SH2-domain containing proteins. Approximately 400 different affinity reagents of various types were analysed on these antigen microarrays carrying antigens of different types. The microarrays revealed not only very detailed specificity profiles for all the binders, but also showed that overlapping target sequences of spotted antigens were detected by off-target interactions. The presented study illustrates the feasibility of using antigen microarrays for integrative, high-throughput validation of various types of binders and antigens.

  2. Stabilization of the Motion of Affine Systems

    NASA Astrophysics Data System (ADS)

    Babenko, E. A.; Martynyuk, A. A.

    2016-07-01

    Sufficient conditions for the stability of a nonlinear affine system subject to interval initial conditions are established. These conditions are based on new estimates of the norms of the solutions of the systems of perturbed equations of motion. This stabilization method is used to analyze an electromechanical system with permanent magnet

  3. Fan Affinity Laws from a Collision Model

    ERIC Educational Resources Information Center

    Bhattacharjee, Shayak

    2012-01-01

    The performance of a fan is usually estimated using hydrodynamical considerations. The calculations are long and involved and the results are expressed in terms of three affinity laws. In this paper we use kinetic theory to attack this problem. A hard sphere collision model is used, and subsequently a correction to account for the flow behaviour…

  4. Vygotsky's and Buber's Pedagogical Perspectives: Some Affinities

    ERIC Educational Resources Information Center

    Bartholo, Roberto; Tunes, Elizabeth; Tacca, Maria Carmen Villela Rosa

    2010-01-01

    The purpose of this paper is to examine the dialogical and creative character of pedagogic work by analyzing the affinities between Martin Buber's "I-Thou relation" and Lev Semenovich Vygotsky's "Zone of Proximal Development". Backed up by empirical studies on the teacher-student relation, we understand that education can only result in students'…

  5. Iron Therapy for Preterm Infants

    PubMed Central

    Rao, Raghavendra; Georgieff, Michael K.

    2009-01-01

    SYNOPSIS Preterm infants are at risk for both iron deficiency and iron overload. The role of iron in multiple organ functions suggests that iron supplementation is essential for the preterm infant. Conversely, the potential for iron overload and the poorly developed anti-oxidant measures in the preterm infant argues against indiscriminate iron supplementation in this population. The purpose of this article is to review the predisposing factors and consequences of iron deficiency and iron overload in the preterm infant, the current recommendation for iron supplementation and its appropriateness, and describe potential management strategies that strike a balance between iron deficiency and iron toxicity. PMID:19161863

  6. Iron deprivation results in a rapid but not sustained increase of the expression of genes involved in iron metabolism and sulfate uptake in tomato (Solanum lycopersicum L.) seedlings.

    PubMed

    Paolacci, Anna Rita; Celletti, Silvia; Catarcione, Giulio; Hawkesford, Malcolm J; Astolfi, Stefania; Ciaffi, Mario

    2014-01-01

    Characterization of the relationship between sulfur and iron in both Strategy I and Strategy II plants, has proven that low sulfur availability often limits plant capability to cope with iron shortage. Here it was investigated whether the adaptation to iron deficiency in tomato (Solanum lycopersicum L.) plants was associated with an increased root sulfate uptake and translocation capacity, and modified dynamics of total sulfur and thiols accumulation between roots and shoots. Most of the tomato sulfate transporter genes belonging to Groups 1, 2, and 4 were significantly upregulated in iron-deficient roots, as it commonly occurs under S-deficient conditions. The upregulation of the two high affinity sulfate transporter genes, SlST1.1 and SlST1.2, by iron deprivation clearly suggests an increased root capability to take up sulfate. Furthermore, the upregulation of the two low affinity sulfate transporter genes SlST2.1 and SlST4.1 in iron-deficient roots, accompanied by a substantial accumulation of total sulfur and thiols in shoots of iron-starved plants, likely supports an increased root-to-shoot translocation of sulfate. Results suggest that tomato plants exposed to iron-deficiency are able to change sulfur metabolic balance mimicking sulfur starvation responses to meet the increased demand for methionine and its derivatives, allowing them to cope with this stress.

  7. Defluoridation by Bacteriogenic Iron Oxides: Sorption Studies

    NASA Astrophysics Data System (ADS)

    Evans, K.; Ferris, F.

    2009-05-01

    At concentrations above 1 mg/L, fluoride in drinking water can lead to dental and skeletal fluorosis, a disease that causes mottling of the teeth, calcification of ligaments, crippling bone deformities and many other physiological disorders that can, ultimately, lead to death. Conservative estimates are that fluorosis afflicts tens of millions of people worldwide. As there is no treatment for fluorosis, prevention is the only means of controlling the disease. While numerous defluoridation techniques have been explored, no single method has been found to be both effective and inexpensive enough to implement widely. Our research began in India, with a large-scale geochemical study of the groundwater in a fluoride-contaminated region of Orissa. Having developed a better understanding of the geochemical relationships that exist between fluoride and other parameters present in an affected area, as well as the complex relationships that arise among those parameters that can impact the presence of fluoride, we began investigating certain remediation scenarios involving iron oxides. A common approach to remediation involves the partitioning of fluoride from groundwater by sorption onto a variety of materials, one of the most effective of which is iron oxide whose surface area acts as a scavenger for fluoride. In the presence of iron oxidizing bacteria, the oxidation rate of iron has been shown to be ˜6 times greater than in their absence; fluoride should, therefore, be removed from an aqueous environment by bacteriogenic iron oxides (BIOS) much more quickly than by abiotic iron oxides. Most recently, sorption studies have been conducted using both BIOS and synthetic hydrous ferric oxides in order to compare the behavior between biotic and abiotic sorbents. These studies have provided sorption isotherms that allow comparison of fluoride removed by sorption to BIOS versus synthetic iron oxides. Sorption affinity constants have also been determined, which allow for the

  8. Iron losses in sweat

    SciTech Connect

    Brune, M.; Magnusson, B.; Persson, H.; Hallberg, L.

    1986-03-01

    The losses of iron in whole body cell-free sweat were determined in eleven healthy men. A new experimental design was used with a very careful cleaning procedure of the skin and repeated consecutive sampling periods of sweat in a sauna. The purpose was to achieve a steady state of sweat iron losses with minimal influence from iron originating from desquamated cells and iron contaminating the skin. A steady state was reached in the third sauna period (second sweat sampling period). Iron loss was directly related to the volume of sweat lost and amounted to 22.5 micrograms iron/l sweat. The findings indicate that iron is a physiological constituent of sweat and derived not only from contamination. Present results imply that variations in the amount of sweat lost will have only a marginal effect on the variation in total body iron losses.

  9. Iron in diet

    MedlinePlus

    The best sources of iron include: Dried beans Dried fruits Eggs (especially egg yolks) Iron-fortified cereals Liver Lean red meat (especially beef) Oysters Poultry, dark red meat Salmon Tuna Whole ...

  10. Serum iron test

    MedlinePlus

    ... GM. Disorders of iron homeostasis: iron deficiency and overload. In: Hoffman R, Benz EJ Jr, Silberstein LE, ... to achieve this important distinction for online health information and services. Learn more about A.D.A. ...

  11. Total iron binding capacity

    MedlinePlus

    ... GM. Disorders of iron homeostasis: iron deficiency and overload. In: Hoffman R, Benz EJ Jr, Silberstein LE, ... to achieve this important distinction for online health information and services. Learn more about A.D.A. ...

  12. Iron deficiency anemia

    MedlinePlus

    ... GM. Disorders of iron homeostasis: iron deficiency and overload. In: Hoffman R, Benz EJ Jr, Silberstein LE, ... to achieve this important distinction for online health information and services. Learn more about A.D.A. ...

  13. Iron supplements (image)

    MedlinePlus

    The mineral iron is an essential nutrient for humans because it is part of blood cells, which carry oxygen to all body cells. There is no conclusive evidence that iron supplements contribute to heart attacks.

  14. Siderophore-based microbial adaptations to iron scarcity across the eastern Pacific Ocean

    PubMed Central

    Mende, Daniel R.; Hawco, Nicholas J.; McIlvin, Matthew R.; Fitzsimmons, Jessica N.; Saito, Mak A.; Sedwick, Peter N.; DeLong, Edward F.; Repeta, Daniel J.

    2016-01-01

    Nearly all iron dissolved in the ocean is complexed by strong organic ligands of unknown composition. The effect of ligand composition on microbial iron acquisition is poorly understood, but amendment experiments using model ligands show they can facilitate or impede iron uptake depending on their identity. Here we show that siderophores, organic compounds synthesized by microbes to facilitate iron uptake, are a dynamic component of the marine ligand pool in the eastern tropical Pacific Ocean. Siderophore concentrations in iron-deficient waters averaged 9 pM, up to fivefold higher than in iron-rich coastal and nutrient-depleted oligotrophic waters, and were dominated by amphibactins, amphiphilic siderophores with cell membrane affinity. Phylogenetic analysis of amphibactin biosynthetic genes suggests that the ability to produce amphibactins has transferred horizontally across multiple Gammaproteobacteria, potentially driven by pressures to compete for iron. In coastal and oligotrophic regions of the eastern Pacific Ocean, amphibactins were replaced with lower concentrations (1–2 pM) of hydrophilic ferrioxamine siderophores. Our results suggest that organic ligand composition changes across the surface ocean in response to environmental pressures. Hydrophilic siderophores are predominantly found across regions of the ocean where iron is not expected to be the limiting nutrient for the microbial community at large. However, in regions with intense competition for iron, some microbes optimize iron acquisition by producing siderophores that minimize diffusive losses to the environment. These siderophores affect iron bioavailability and thus may be an important component of the marine iron cycle. PMID:27911777

  15. Siderophore-based microbial adaptations to iron scarcity across the eastern Pacific Ocean.

    PubMed

    Boiteau, Rene M; Mende, Daniel R; Hawco, Nicholas J; McIlvin, Matthew R; Fitzsimmons, Jessica N; Saito, Mak A; Sedwick, Peter N; DeLong, Edward F; Repeta, Daniel J

    2016-12-13

    Nearly all iron dissolved in the ocean is complexed by strong organic ligands of unknown composition. The effect of ligand composition on microbial iron acquisition is poorly understood, but amendment experiments using model ligands show they can facilitate or impede iron uptake depending on their identity. Here we show that siderophores, organic compounds synthesized by microbes to facilitate iron uptake, are a dynamic component of the marine ligand pool in the eastern tropical Pacific Ocean. Siderophore concentrations in iron-deficient waters averaged 9 pM, up to fivefold higher than in iron-rich coastal and nutrient-depleted oligotrophic waters, and were dominated by amphibactins, amphiphilic siderophores with cell membrane affinity. Phylogenetic analysis of amphibactin biosynthetic genes suggests that the ability to produce amphibactins has transferred horizontally across multiple Gammaproteobacteria, potentially driven by pressures to compete for iron. In coastal and oligotrophic regions of the eastern Pacific Ocean, amphibactins were replaced with lower concentrations (1-2 pM) of hydrophilic ferrioxamine siderophores. Our results suggest that organic ligand composition changes across the surface ocean in response to environmental pressures. Hydrophilic siderophores are predominantly found across regions of the ocean where iron is not expected to be the limiting nutrient for the microbial community at large. However, in regions with intense competition for iron, some microbes optimize iron acquisition by producing siderophores that minimize diffusive losses to the environment. These siderophores affect iron bioavailability and thus may be an important component of the marine iron cycle.

  16. Siderophore-based microbial adaptations to iron scarcity across the eastern Pacific Ocean

    NASA Astrophysics Data System (ADS)

    Boiteau, Rene M.; Mende, Daniel R.; Hawco, Nicholas J.; McIlvin, Matthew R.; Fitzsimmons, Jessica N.; Saito, Mak A.; Sedwick, Peter N.; DeLong, Edward F.; Repeta, Daniel J.

    2016-12-01

    Nearly all iron dissolved in the ocean is complexed by strong organic ligands of unknown composition. The effect of ligand composition on microbial iron acquisition is poorly understood, but amendment experiments using model ligands show they can facilitate or impede iron uptake depending on their identity. Here we show that siderophores, organic compounds synthesized by microbes to facilitate iron uptake, are a dynamic component of the marine ligand pool in the eastern tropical Pacific Ocean. Siderophore concentrations in iron-deficient waters averaged 9 pM, up to fivefold higher than in iron-rich coastal and nutrient-depleted oligotrophic waters, and were dominated by amphibactins, amphiphilic siderophores with cell membrane affinity. Phylogenetic analysis of amphibactin biosynthetic genes suggests that the ability to produce amphibactins has transferred horizontally across multiple Gammaproteobacteria, potentially driven by pressures to compete for iron. In coastal and oligotrophic regions of the eastern Pacific Ocean, amphibactins were replaced with lower concentrations (1–2 pM) of hydrophilic ferrioxamine siderophores. Our results suggest that organic ligand composition changes across the surface ocean in response to environmental pressures. Hydrophilic siderophores are predominantly found across regions of the ocean where iron is not expected to be the limiting nutrient for the microbial community at large. However, in regions with intense competition for iron, some microbes optimize iron acquisition by producing siderophores that minimize diffusive losses to the environment. These siderophores affect iron bioavailability and thus may be an important component of the marine iron cycle.

  17. Magnetic particles as affinity matrix for purification of antithrombin

    NASA Astrophysics Data System (ADS)

    Mercês, A. A. D.; Maciel, J. C.; Carvalho Júnior, L. B.

    2015-11-01

    Immobilization of biomolecules onto insoluble supports is an important tool for the fabrication of a diverse range of functional materials. It provides advantages: enhanced stability and easy separation. In this work two different magnetic composites were synthesized (MAG-PANI-HS and mDAC-HS) to human antithrombin purification. The magnetic particles (MAG) were obtained by co-precipitation method of iron salts II and III and subsequently coated with polyaniline (MAG-PANI particles). Dacron (polyethylene terephthalate) suffered a hydrazinolysis reaction to obtain a powder (Dacron hydrazide) which was subsequently magnetized (mDAC particles) also by co-precipitation method. Heparan sulfate (HS) was immobilized to MAG-PANI and mDAC retained respectively 35μg and 38.6μg per of support. The magnetic composite containing HS immobilized (MAG-PANI-HS and mDAC-HS) was incubated with human blood plasma (1mL) and then washed with NaCl gradients. Electrophoresis of proteins present in eluates showed bands of antithrombin (58kDa). A reduction in the antithrombin activity was detected in plasma that were incubated in the composites magnetic with HS immobilized, suggesting that the antithrombin was removed of the human blood plasma and then purified. Therefore, the above results suggest that both preparations: MAG-PANI-HS and mDAC-HS are able to affinity purify antithrombin, an important component of blood coagulation.

  18. Functionalization of magnetic nanoparticles with high-binding capacity for affinity separation of therapeutic proteins

    NASA Astrophysics Data System (ADS)

    Masthoff, Ingke-Christine; David, Florian; Wittmann, Christoph; Garnweitner, Georg

    2014-01-01

    Magnetic nanoparticles with immobilized metal ligands were prepared for the separation of antibody fragments. First, iron oxide nanoparticles were produced in a solvothermal synthesis using triethylene glycol as solvent and iron(III) acetylacetonate as organic precursor. Via functionalization of the particles with priorly reacted 3-glycidoxypropyltrimethoxysilane and N α, N α-bis(carboxymethyl)- l-lysine (NTA), and charging with Ni2+, magnetic affinity adsorbents were obtained. The particles were applied to separate a His-tagged antibody fragment from a heterogeneous protein mixture of a microbial cultivation supernatant. Binding properties and specificity for purification of the target product ABF D1.3 scFv were optimized regarding the GNTA concentration and were found superior as compared to commercially available systems. A molar ratio of 1:2 Fe2O3:GNTA was most beneficial for the specific purification of the antibody fragment.

  19. Iron and Prochlorococcus

    DTIC Science & Technology

    2009-06-01

    iron deprivation in cyanobacteria include loss of pigmentation (chlorosis), changes in the fluorescence/ absorbance wavelengths of chlorophyll a...77 Figure 3: MED4 photosynthetic efficiency and isiB expression during iron stress. ……………………….79 Figure 4: Global expression response of...cyanobacteria, iron is best known for its role as a cofactor in photosynthetic electron transfer. In fact, 22 atoms of iron are considered necessary for the

  20. Atypical iron storage in marine brown algae: a multidisciplinary study of iron transport and storage in Ectocarpus siliculosus

    PubMed Central

    Matzanke, Berthold F.; Küpper, Frithjof C.; Carrano, Carl J.

    2012-01-01

    Iron is an essential element for all living organisms due to its ubiquitous role in redox and other enzymes, especially in the context of respiration and photosynthesis. The iron uptake and storage systems of terrestrial/higher plants are now reasonably well understood, with two basic strategies for iron uptake being distinguished: strategy I plants use a mechanism involving induction of Fe(III)-chelate reductase (ferrireductase) and Fe(II) transporter proteins, while strategy II plants utilize high-affinity, iron-specific, binding compounds called phytosiderophores. In contrast, little is known about the corresponding systems in marine, plant-like lineages, particularly those of multicellular algae (seaweeds). Herein the first study of the iron uptake and storage mechanisms in the brown alga Ectocarpus siliculosus is reported. Genomic data suggest that Ectocarpus may use a strategy I approach. Short-term radio-iron uptake studies verified that iron is taken up by Ectocarpus in a time- and concentration-dependent manner consistent with an active transport process. Upon long-term exposure to 57Fe, two metabolites have been identified using a combination of Mössbauer and X-ray absorption spectroscopies. These include an iron–sulphur cluster accounting for ~26% of the total intracellular iron pool and a second component with spectra typical of a polymeric (Fe3+O6) system with parameters similar to the amorphous phosphorus-rich mineral core of bacterial and plant ferritins. This iron metabolite accounts for ~74% of the cellular iron pool and suggests that Ectocarpus contains a non-ferritin but mineral-based iron storage pool. PMID:22945940

  1. Role of Nitrosomonas europaea NitABC iron transporter in the uptake of Fe3+-siderophore complexes.

    PubMed

    Vajrala, Neeraja; Sayavedra-Soto, Luis A; Bottomley, Peter J; Arp, Daniel J

    2010-11-01

    Nitrosomonas europaea has a single three-gene operon (nitABC) encoding an iron ABC transporter system (NitABC). Phylogenetic analysis clustered the subunit NitB with Fe(3+)-ABC transporter permease components from other organisms. The N. europaea strain deficient in nitB (nitB::kan) grew well in either Fe-replete or Fe-limited media and in Fe-limited medium containing the catecholate-type siderophore, enterobactin or the citrate-based dihydroxamate-type siderophore, aerobactin. However, the nitB::kan mutant strain was unable to grow in Fe-limited media containing either the hydroxamate-type siderophores, ferrioxamine and ferrichrome or the mixed-chelating type siderophore, pyoverdine. Exposure of N. europaea cells to a ferrichrome analog coupled to the fluorescent moiety naphthalic diimide (Fhu-NI) led to increase in fluorescence in the wild type but not in nitB::kan mutant cells. Spheroplasts prepared from N. europaea wild type exposed to Fhu-NI analog retained the fluorescence, while spheroplasts of the nitB::kan mutant were not fluorescent. NitABC transports intact Fe(3+)-ferrichrome complex into the cytoplasm and is an atypical ABC type iron transporter for Fe(3+) bound to ferrioxamine, ferrichrome or pyoverdine siderophores into the cytoplasm. The mechanisms to transport iron in either the Fe(3+) or Fe(2+) forms or Fe(3+) associated with enterobactin or aerobactin siderophores into the cell across the cytoplasmic membrane are as yet undetermined.

  2. Optimal Affine-Invariant Point Matching

    NASA Astrophysics Data System (ADS)

    Costa, Mauro S.; Haralick, Robert M.; Phillips, Tsaiyun I.; Shapiro, Linda G.

    1989-03-01

    The affine-transformation matching scheme proposed by Hummel and Wolfson (1988) is very efficient in a model-based matching system, not only in terms of the computational complexity involved, but also in terms of the simplicity of the method. This paper addresses the implementation of the affine-invariant point matching, applied to the problem of recognizing and determining the pose of sheet metal parts. It points out errors that can occur with this method due to quantization, stability, symmetry, and noise problems. By beginning with an explicit noise model which the Hummel and Wolfson technique lacks, we can derive an optimal approach which overcomes these problems. We show that results obtained with the new algorithm are clearly better than the results from the original method.

  3. Affinity Chromatography in Nonionic Detergent Solutions

    NASA Astrophysics Data System (ADS)

    Robinson, Jack B.; Strottmann, James M.; Wick, Donald G.; Stellwagen, Earle

    1980-10-01

    Anionic dye affinity chromatography is commonly unproductive in the presence of nonionic detergents used to extract particulate proteins. Using lactate dehydrogenase as a model protein, Cibacron blue F3GA as a model dye, and Triton X-100 as a model detergent, we find that the dye is encapsulated in nonionic detergent micelles, rendering the dye incapable of ligation with the enzyme. However, the dye can be liberated from the micelles without altering the nonionic detergent concentration by addition of an anionic detergent, such as deoxycholate or sodium dodecyl sulfate, forming mixed anionic/nonionic micelles that displace the anionic dye. Encapsulation of the anionic detergents prevents their activity as protein denaturants. These observations have been successfully translated to the dye affinity chromatography of a detergent extract of brain particulate cyclic nucleotide phosphodiesterase.

  4. Iron, radiation, and cancer.

    PubMed Central

    Stevens, R G; Kalkwarf, D R

    1990-01-01

    Increased iron content of cells and tissue may increase the risk of cancer. In particular, high available iron status may increase the risk of a radiation-induced cancer. There are two possible mechanisms for this effect: iron can catalyze the production of oxygen radicals, and it may be a limiting nutrient to the growth and development of a transformed cell in vivo. Given the high available iron content of the western diet and the fact that the world is changing to the western model, it is important to determine if high iron increases the risk of cancer. PMID:2269234

  5. Negative affinity X-ray photocathodes

    NASA Technical Reports Server (NTRS)

    Vanspeybroeck, L.; Kellogg, E.; Murray, S.; Duckett, S.

    1974-01-01

    A new X-ray image intensifier is described. The device should eventually have a quantum efficiency which is an order of magnitude greater than that of presently available high spatial resolution X-ray detectors, such as microchannel plates. The new intesifier is based upon a GaAs crystal photocathode which is activated to achieve negative electron affinity. Details concerning the detector concept are discussed together with the theoretical relations involved, X-ray data, and optical data.

  6. On constructing purely affine theories with matter

    NASA Astrophysics Data System (ADS)

    Cervantes-Cota, Jorge L.; Liebscher, D.-E.

    2016-08-01

    We explore ways to obtain the very existence of a space-time metric from an action principle that does not refer to it a priori. Although there are reasons to believe that only a non-local theory can viably achieve this goal, we investigate here local theories that start with Schrödinger's purely affine theory (Schrödinger in Space-time structure. Cambridge UP, Cambridge, 1950), where he gave reasons to set the metric proportional to the Ricci curvature aposteriori. When we leave the context of unified field theory, and we couple the non-gravitational matter using some weak equivalence principle, we can show that the propagation of shock waves does not define a lightcone when the purely affine theory is local and avoids the explicit use of the Ricci tensor in realizing the weak equivalence principle. When the Ricci tensor is substituted for the metric, the equations seem to have only a very limited set of solutions. This backs the conviction that viable purely affine theories have to be non-local.

  7. Phosphopeptide Enrichment by Immobilized Metal Affinity Chromatography.

    PubMed

    Thingholm, Tine E; Larsen, Martin R

    2016-01-01

    Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. This problem is mainly caused by highly acidic peptides that also share high binding affinity towards these metal ions. By lowering the pH of the loading buffer nonspecific binding can be reduced significantly, however with the risk of reducing specific binding capacity. After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.

  8. FbpA iron storage and release are governed by periplasmic microenvironments.

    PubMed

    Sensoy, Ozge; Atilgan, Ali Rana; Atilgan, Canan

    2017-02-22

    Ferric binding protein (FbpA) is part of an elaborate iron piracy mechanism evolved in Gram-negative bacteria, shuttling iron in the periplasmic space, from the outer to the cytoplasmic membrane side. We address how the dissociation process of iron is facilitated, since the binding constant of iron is on the order of 10(18) M(-1) at 6.5 pH and 200 mM ionic strength (IS). We monitor the conformational preferences of FbpA by extensive molecular dynamics (MD) simulations under conditions where IS, charge states of iron coordinating tyrosines and pH are varied, as well as when a mutation is introduced at an allosteric site. Steered MD is utilized to predict the binding affinity of iron. After triggering lobe opening by changing the charge states of tyrosines, the conformations adopted and the iron binding affinity still depend on pH, IS and allosteric interactions. To relate the observed conformational changes to the environmental conditions that might be encountered in the periplasmic space, we offer a plausible model that couples electrostatic potential distribution to the mechanical motions invoked. Although low pH/IS and allosteric perturbations decrease the affinity of iron, it remains high for spontaneous dissociation. However, the conformational changes modulated by the environmental conditions expose iron for chelation. Our study provides a quantitative dimension and molecular details to interpret the contribution of possible environmental conditions present in the periplasmic space to iron dissociation from FbpA, opening up the opportunity of modulating function via allosteric mutations or altering environmental conditions, thus offering a new route to developing strategies towards antibiotic resistance by targeting nutritional requirements.

  9. Haemopexin affects iron distribution and ferritin expression in mouse brain

    PubMed Central

    Morello, Noemi; Tonoli, Elisabetta; Logrand, Federica; Fiorito, Veronica; Fagoonee, Sharmila; Turco, Emilia; Silengo, Lorenzo; Vercelli, Alessandro; Altruda, Fiorella; Tolosano, Emanuela

    2009-01-01

    Haemopexin (Hx) is an acute phase plasma glycoprotein, mainly produced by the liver and released into plasma where it binds heme with high affinity and delivers it to the liver. This system provides protection against free heme-mediated oxidative stress, limits access by pathogens to heme and contributes to iron homeostasis by recycling heme iron. Hx protein has been found in the sciatic nerve, skeletal muscle, retina, brain and cerebrospinal fluid (CSF). Recently, a comparative proteomic analysis has shown an increase of Hx in CSF from patients with Alzheimer’s disease, thus suggesting its involvement in heme detoxification in brain. Here, we report that Hx is synthesised in brain by the ventricular ependymal cells. To verify whether Hx is involved in heme scavenging in brain, and consequently, in the control of iron level, iron deposits and ferritin expression were analysed in cerebral regions known for iron accumulation. We show a twofold increase in the number of iron-loaded oligodendrocytes in the basal ganglia and thalamus of Hx-null mice compared to wild-type controls. Interestingly, there was no increase in H- and L-ferritin expression in these regions. This condition is common to several human neurological disorders such as Alzheimer’s disease and Parkinson’s disease in which iron loading is not associated with an adequate increase in ferritin expression. However, a strong reduction in the number of ferritin-positive cells was observed in the cerebral cortex of Hx-null animals. Consistent with increased iron deposits and inadequate ferritin expression, malondialdehyde level and Cu–Zn superoxide dismutase-1 expression were higher in the brain of Hx-null mice than in that of wild-type controls. These data demonstrate that Hx plays an important role in controlling iron distribution within brain, thus suggesting its involvement in iron-related neurodegenerative diseases. PMID:19120692

  10. Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

    PubMed Central

    Nilvebrant, Johan; Åstrand, Mikael; Georgieva-Kotseva, Maria; Björnmalm, Mattias; Löfblom, John; Hober, Sophia

    2014-01-01

    The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein. PMID:25089830

  11. Monoubiquitin-dependent endocytosis of the iron-regulated transporter 1 (IRT1) transporter controls iron uptake in plants.

    PubMed

    Barberon, Marie; Zelazny, Enric; Robert, Stéphanie; Conéjéro, Geneviève; Curie, Cathy; Friml, Jìrí; Vert, Grégory

    2011-08-09

    Plants take up iron from the soil using the iron-regulated transporter 1 (IRT1) high-affinity iron transporter at the root surface. Sophisticated regulatory mechanisms allow plants to tightly control the levels of IRT1, ensuring optimal absorption of essential but toxic iron. Here, we demonstrate that overexpression of Arabidopsis thaliana IRT1 leads to constitutive IRT1 protein accumulation, metal overload, and oxidative stress. IRT1 is unexpectedly found in trans-Golgi network/early endosomes of root hair cells, and its levels and localization are unaffected by iron nutrition. Using pharmacological approaches, we show that IRT1 cycles to the plasma membrane to perform iron and metal uptake at the cell surface and is sent to the vacuole for proper turnover. We also prove that IRT1 is monoubiquitinated on several cytosol-exposed residues in vivo and that mutation of two putative monoubiquitination target residues in IRT1 triggers stabilization at the plasma membrane and leads to extreme lethality. Together, these data suggest a model in which monoubiquitin-dependent internalization/sorting and turnover keep the plasma membrane pool of IRT1 low to ensure proper iron uptake and to prevent metal toxicity. More generally, our work demonstrates the existence of monoubiquitin-dependent trafficking to lytic vacuoles in plants and points to proteasome-independent turnover of plasma membrane proteins.

  12. Preparation of iron-impregnated granular activated carbon for arsenic removal from drinking water.

    PubMed

    Chang, Qigang; Lin, Wei; Ying, Wei-chi

    2010-12-15

    Granular activated carbon (GAC) was impregnated with iron through a new multi-step procedure using ferrous chloride as the precursor for removing arsenic from drinking water. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) analysis demonstrated that the impregnated iron was distributed evenly on the internal surface of the GAC. Impregnated iron formed nano-size particles, and existed in both crystalline (akaganeite) and amorphous iron forms. Iron-impregnated GACs (Fe-GACs) were treated with sodium hydroxide to stabilize iron in GAC and impregnated iron was found very stable at the common pH range in water treatments. Synthetic arsenate-contaminated drinking water was used in isotherm tests to evaluate arsenic adsorption capacities and iron use efficiencies of Fe-GACs with iron contents ranging from 1.64% to 12.13% (by weight). Nonlinear regression was used to obtain unbiased estimates of Langmuir model parameters. The arsenic adsorption capacity of Fe-GAC increased significantly with impregnated iron up to 4.22% and then decreased with more impregnated iron. Fe-GACs synthesized in this study exhibited higher affinity for arsenate as compared with references in literature and shows great potential for real implementations.

  13. New rat models of iron sucrose-induced iron overload.

    PubMed

    Vu'o'ng Lê, Bá; Khorsi-Cauet, Hafida; Villegier, Anne-Sophie; Bach, Véronique; Gay-Quéheillard, Jérôme

    2011-07-01

    The majority of murine models of iron sucrose-induced iron overload were carried out in adult subjects. This cannot reflect the high risk of iron overload in children who have an increased need for iron. In this study, we developed four experimental iron overload models in young rats using iron sucrose and evaluated different markers of iron overload, tissue oxidative stress and inflammation as its consequences. Iron overload was observed in all iron-treated rats, as evidenced by significant increases in serum iron indices, expression of liver hepcidin gene and total tissue iron content compared with control rats. We also showed that total tissue iron content was mainly associated with the dose of iron whereas serum iron indices depended essentially on the duration of iron administration. However, no differences in tissue inflammatory and antioxidant parameters from controls were observed. Furthermore, only rats exposed to daily iron injection at a dose of 75 mg/kg body weight for one week revealed a significant increase in lipid peroxidation in iron-treated rats compared with their controls. The present results suggest a correlation between iron overload levels and the dose of iron, as well as the duration and frequency of iron injection and confirm that iron sucrose may not play a crucial role in inflammation and oxidative stress. This study provides important information about iron sucrose-induced iron overload in rats and may be useful for iron sucrose therapy for iron deficiency anemia as well as for the prevention and diagnosis of iron sucrose-induced iron overload in pediatric patients.

  14. Fructose Uptake in Sinorhizobium meliloti Is Mediated by a High-Affinity ATP-Binding Cassette Transport System

    PubMed Central

    Lambert, Annie; Østerås, Magne; Mandon, Karine; Poggi, Marie-Christine; Le Rudulier, Daniel

    2001-01-01

    By transposon mutagenesis, we have isolated a mutant of Sinorhizobium meliloti which is totally unable to grow on fructose as sole carbon source as a consequence of its inability to transport this sugar. The cloning and sequencing analysis of the chromosomal DNA region flanking the TnphoA insertion revealed the presence of six open reading frames (ORFs) organized in two loci, frcRS and frcBCAK, transcribed divergently. The frcBCA genes encode the characteristic components of an ATP-binding cassette transporter (FrcB, a periplasmic substrate binding protein, FrcC, an integral membrane permease, and FrcA, an ATP-binding cytoplasmic protein), which is the unique high-affinity (Km of 6 μM) fructose uptake system in S. meliloti. The FrcK protein shows homology with some kinases, while FrcR is probably a transcriptional regulator of the repressor-ORF-kinase family. The expression of S. meliloti frcBCAK in Escherichia coli, which transports fructose only via the phosphotransferase system, resulted in the detection of a periplasmic fructose binding activity, demonstrating that FrcB is the binding protein of the Frc transporter. The analysis of substrate specificities revealed that the Frc system is also a high-affinity transporter for ribose and mannose, which are both fructose competitors for the binding to the periplasmic FrcB protein. However, the Frc mutant was still able to grow on these sugars as sole carbon source, demonstrating the presence of at least one other uptake system for mannose and ribose in S. meliloti. The expression of the frcBC genes as determined by measurements of alkaline phosphatase activity was shown to be induced by mannitol and fructose, but not by mannose, ribose, glucose, or succinate, suggesting that the Frc system is primarily targeted towards fructose. Neither Nod nor Fix phenotypes were impared in the TnphoA mutant, demonstrating that fructose uptake is not essential for nodulation and nitrogen fixation, although FrcB protein is

  15. Fructose uptake in Sinorhizobium meliloti is mediated by a high-affinity ATP-binding cassette transport system.

    PubMed

    Lambert, A; Østerås, M; Mandon, K; Poggi, M C; Le Rudulier, D

    2001-08-01

    By transposon mutagenesis, we have isolated a mutant of Sinorhizobium meliloti which is totally unable to grow on fructose as sole carbon source as a consequence of its inability to transport this sugar. The cloning and sequencing analysis of the chromosomal DNA region flanking the TnphoA insertion revealed the presence of six open reading frames (ORFs) organized in two loci, frcRS and frcBCAK, transcribed divergently. The frcBCA genes encode the characteristic components of an ATP-binding cassette transporter (FrcB, a periplasmic substrate binding protein, FrcC, an integral membrane permease, and FrcA, an ATP-binding cytoplasmic protein), which is the unique high-affinity (K(m) of 6 microM) fructose uptake system in S. meliloti. The FrcK protein shows homology with some kinases, while FrcR is probably a transcriptional regulator of the repressor-ORF-kinase family. The expression of S. meliloti frcBCAK in Escherichia coli, which transports fructose only via the phosphotransferase system, resulted in the detection of a periplasmic fructose binding activity, demonstrating that FrcB is the binding protein of the Frc transporter. The analysis of substrate specificities revealed that the Frc system is also a high-affinity transporter for ribose and mannose, which are both fructose competitors for the binding to the periplasmic FrcB protein. However, the Frc mutant was still able to grow on these sugars as sole carbon source, demonstrating the presence of at least one other uptake system for mannose and ribose in S. meliloti. The expression of the frcBC genes as determined by measurements of alkaline phosphatase activity was shown to be induced by mannitol and fructose, but not by mannose, ribose, glucose, or succinate, suggesting that the Frc system is primarily targeted towards fructose. Neither Nod nor Fix phenotypes were impared in the TnphoA mutant, demonstrating that fructose uptake is not essential for nodulation and nitrogen fixation, although FrcB protein is

  16. Integrin avidity regulation: are changes in affinity and conformation underemphasized?

    PubMed

    Carman, Christopher V; Springer, Timothy A

    2003-10-01

    Integrins play critical roles in development, wound healing, immunity and cancer. Central to their function is their unique ability to modulate dynamically their adhesiveness through both affinity- and valency-based mechanisms. Recent advances have shed light on the structural basis for affinity regulation and on the signaling mechanisms responsible for both affinity and valency modes of regulation.

  17. Iron deficiency anaemia.

    PubMed

    Lopez, Anthony; Cacoub, Patrice; Macdougall, Iain C; Peyrin-Biroulet, Laurent

    2016-02-27

    Anaemia affects roughly a third of the world's population; half the cases are due to iron deficiency. It is a major and global public health problem that affects maternal and child mortality, physical performance, and referral to health-care professionals. Children aged 0-5 years, women of childbearing age, and pregnant women are particularly at risk. Several chronic diseases are frequently associated with iron deficiency anaemia--notably chronic kidney disease, chronic heart failure, cancer, and inflammatory bowel disease. Measurement of serum ferritin, transferrin saturation, serum soluble transferrin receptors, and the serum soluble transferrin receptors-ferritin index are more accurate than classic red cell indices in the diagnosis of iron deficiency anaemia. In addition to the search for and treatment of the cause of iron deficiency, treatment strategies encompass prevention, including food fortification and iron supplementation. Oral iron is usually recommended as first-line therapy, but the most recent intravenous iron formulations, which have been available for nearly a decade, seem to replenish iron stores safely and effectively. Hepcidin has a key role in iron homoeostasis and could be a future diagnostic and therapeutic target. In this Seminar, we discuss the clinical presentation, epidemiology, pathophysiology, diagnosis, and acute management of iron deficiency anaemia, and outstanding research questions for treatment.

  18. The ubiquity of iron.

    PubMed

    Frey, Perry A; Reed, George H

    2012-09-21

    The importance of iron in living systems can be traced to the many complexes within which it is found, to its chemical mobility in undergoing oxidation-reduction reactions, and to the abundance of iron in Earth's crust. Iron is the most abundant element, by mass, in the Earth, constituting about 80% of the inner and outer cores of Earth. The molten outer core is about 8000 km in diameter, and the solid inner core is about 2400 km in diameter. Iron is the fourth most abundant element in Earth's crust. It is the chemically functional component of mononuclear iron complexes, dinuclear iron complexes, [2Fe-2S] and [4Fe-4S] clusters, [Fe-Ni-S] clusters, iron protophorphyrin IX, and many other complexes in protein biochemistry. Metals such as nickel, cobalt, copper, and manganese are present in the crust and could in principle function chemically in place of iron, but they are scarce in Earth's crust. Iron is plentiful because of its nuclear stability in stellar nuclear fusion reactions. It seems likely that other solid planets, formed by the same processes as Earth, would also foster the evolution of life and that iron would be similarly important to life on those planets as it is on Earth.

  19. High Affinity Binding of Indium and Ruthenium Ions by Gastrins.

    PubMed

    Baldwin, Graham S; George, Graham N; Pushie, M Jake

    2015-01-01

    The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga3+ were fitted by a 2 site model with dissociation constants (Kd) of 3.3 x 10-7 and 1.1 x 10-6 M. Although the absorption of gastrin did not change upon the addition of In3+ ions, the changes in absorbance on Fe3+ ion binding in the presence of indium ions were fitted by a 2 site model with Kd values for In3+ of 6.5 x 10-15 and 1.7 x 10-7 M. Similar results were obtained with Ru3+ ions, although the Kd values for Ru3+ of 2.6 x 10-13 and 1.2 x 10-5 M were slightly larger than observed for In3+. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0-3.3 Å, the Ru complex clearly demonstrated a short range Ru-Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In3+ or Ru3+ ions, and that the affinity of the first site for In3+ or Ru3+ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.

  20. Electrolytic iron or ferrous sulfate increase body iron in women with moderate to low iron stores.

    PubMed

    Swain, James H; Johnson, LuAnn K; Hunt, Janet R

    2007-03-01

    Commercial elemental iron powders (electrolytic and reduced iron), as well as heme iron supplements, were tested for efficacy in improving the iron status of women. In a randomized, double-blind trial, 51 women with moderate to low iron stores received daily for 12 wk: 1) placebo, 2) 5 mg iron as heme iron or 50 mg iron as 3) electrolytic iron, 4) reduced iron, or 5) FeSO(4). Treatments were provided in 2 capsules (heme carrier) and 3 wheat rolls (other iron sources). Differences in iron status, food nonheme iron absorption, and fecal properties were evaluated. Body iron, assessed from the serum transferrin receptor:ferritin ratio, increased significantly more in subjects administered FeSO(4) (127 +/- 29 mg; mean +/- SEM) and electrolytic (115 +/- 37 mg), but not the reduced (74 +/- 32 mg) or heme (65 +/- 26 mg) iron forms, compared with those given placebo (2 +/- 19 mg). Based on body iron determinations, retention of the added iron was estimated as 3.0, 2.7, 1.8, and 15.5%, in the 4 iron-treated groups, respectively. Iron treatments did not affect food iron absorption. The 50 mg/d iron treatments increased fecal iron and free radical-generating capacity in vitro, but did not affect fecal water cytotoxicity. In subjects administered FeSO(4), fecal water content was increased slightly but significantly more than in the placebo group. In conclusion, electrolytic iron was approximately 86% as efficacious as FeSO(4) for improving body iron, but the power of this study was insufficient to detect any efficacy of the reduced or heme iron within 12 wk. With modification, this methodology of testing higher levels of food fortification for several weeks in healthy women with low iron stores has the potential for economically assessing the efficiency of iron compounds to improve iron status.

  1. Ceruloplasmin ferroxidase activity stimulates cellular iron uptake by a trivalent cation-specific transport mechanism

    NASA Technical Reports Server (NTRS)

    Attieh, Z. K.; Mukhopadhyay, C. K.; Seshadri, V.; Tripoulas, N. A.; Fox, P. L.

    1999-01-01

    The balance required to maintain appropriate cellular and tissue iron levels has led to the evolution of multiple mechanisms to precisely regulate iron uptake from transferrin and low molecular weight iron chelates. A role for ceruloplasmin (Cp) in vertebrate iron metabolism is suggested by its potent ferroxidase activity catalyzing conversion of Fe2+ to Fe3+, by identification of yeast copper oxidases homologous to Cp that facilitate high affinity iron uptake, and by studies of "aceruloplasminemic" patients who have extensive iron deposits in multiple tissues. We have recently shown that Cp increases iron uptake by cultured HepG2 cells. In this report, we investigated the mechanism by which Cp stimulates cellular iron uptake. Cp stimulated the rate of non-transferrin 55Fe uptake by iron-deficient K562 cells by 2-3-fold, using a transferrin receptor-independent pathway. Induction of Cp-stimulated iron uptake by iron deficiency was blocked by actinomycin D and cycloheximide, consistent with a transcriptionally induced or regulated transporter. Cp-stimulated iron uptake was completely blocked by unlabeled Fe3+ and by other trivalent cations including Al3+, Ga3+, and Cr3+, but not by divalent cations. These results indicate that Cp utilizes a trivalent cation-specific transporter. Cp ferroxidase activity was required for iron uptake as shown by the ineffectiveness of two ferroxidase-deficient Cp preparations, copper-deficient Cp and thiomolybdate-treated Cp. We propose a model in which iron reduction and subsequent re-oxidation by Cp are essential for an iron uptake pathway with high ion specificity.

  2. Brain iron homeostasis.

    PubMed

    Moos, Torben

    2002-11-01

    Iron is essential for virtually all types of cells and organisms. The significance of the iron for brain function is reflected by the presence of receptors for transferrin on brain capillary endothelial cells. The transport of iron into the brain from the circulation is regulated so that the extraction of iron by brain capillary endothelial cells is low in iron-replete conditions and the reverse when the iron need of the brain is high as in conditions with iron deficiency and during development of the brain. Whereas there is good agreement that iron is taken up by means of receptor-mediated uptake of iron-transferrin at the brain barriers, there are contradictory views on how iron is transported further on from the brain barriers and into the brain extracellular space. The prevailing hypothesis for transport of iron across the BBB suggests a mechanism that involves detachment of iron from transferrin within barrier cells followed by recycling of apo-transferrin to blood plasma and release of iron as non-transferrin-bound iron into the brain interstitium from where the iron is taken up by neurons and glial cells. Another hypothesis claims that iron-transferrin is transported into the brain by means of transcytosis through the BBB. This thesis deals with the topic "brain iron homeostasis" defined as the attempts to maintain constant concentrations of iron in the brain internal environment via regulation of iron transport through brain barriers, cellular iron uptake by neurons and glia, and export of iron from brain to blood. The first part deals with transport of iron-transferrin complexes from blood to brain either by transport across the brain barriers or by uptake and retrograde axonal transport in motor neurons projecting beyond the blood-brain barrier. The transport of iron and transport into the brain was examined using radiolabeled iron-transferrin. Intravenous injection of [59Fe-125]transferrin led to an almost two-fold higher accumulation of 59Fe than of

  3. Latest European coelacanth shows Gondwanan affinities.

    PubMed

    Cavin, Lionel; Forey, Peter L; Buffetaut, Eric; Tong, Haiyan

    2005-06-22

    The last European fossil occurrence of a coelacanth is from the Mid-Cretaceous of the English Chalk (Turonian, 90 million years ago). Here, we report the discovery of a coelacanth from Late Cretaceous non-marine rocks in southern France. It consists of a left angular bone showing structures that imply close phylogenetic affinities with some extinct Mawsoniidae. The closest relatives are otherwise known from Cretaceous continental deposits of southern continents and suggest that the dispersal of freshwater organisms from Africa to Europe occurred in the Late Cretaceous.

  4. On the electron affinity of Be2

    NASA Technical Reports Server (NTRS)

    Bauschlicher, C. W., Jr.; Partridge, H.

    1984-01-01

    Calculations of the electron affinity (EA) of Be2 using a large Slater-type orbital basis set and extensive correlation based upon a CASSCF reference are reported. The adiabatic EAs are estimated to be 0.44 eV for the 2Sigma sub g(+) state and 0.56 eV for the 2Pi sub u state. The extra electron attaches into an empty bonding orbital, causing a shortening of the bond length and an increase in omega(e). The D(e) of the 2Pi sub u state of Be2 is six times as large as the D(e) of Be2.

  5. Erythropoietin and iron.

    PubMed

    Kaltwasser, J P; Gottschalk, R

    1999-03-01

    Serum ferritin concentration is most informative in estimating the amount of storage iron available for a particular individual. The serum transferrin receptor concentration, in contrast to serum ferritin, provides direct information about any deficit in the adequacy of iron supply to the erythropoiesis. The combination of serum transferrin receptor and serum ferritin provides complete information about storage and functional iron compartments. Using this combination along with the hemoglobin concentration, it is possible to define the iron nutritional status completely. Inflammatory conditions as well as parenteral iron administration interfere, however, with the direct and quantitative ferritin to storage iron relationship and, therefore, have to be considered carefully with respect to diagnostic purposes. The diagnostic use of the serum transferrin receptor is presently limited because of limitations in methodology and definition (standardization) of reference ranges.

  6. On the structure of self-affine convex bodies

    SciTech Connect

    Voynov, A S

    2013-08-31

    We study the structure of convex bodies in R{sup d} that can be represented as a union of their affine images with no common interior points. Such bodies are called self-affine. Vallet's conjecture on the structure of self-affine bodies was proved for d = 2 by Richter in 2011. In the present paper we disprove the conjecture for all d≥3 and derive a detailed description of self-affine bodies in R{sup 3}. Also we consider the relation between properties of self-affine bodies and functional equations with a contraction of an argument. Bibliography: 10 titles.

  7. Iron sensors and signals in response to iron deficiency.

    PubMed

    Kobayashi, Takanori; Nishizawa, Naoko K

    2014-07-01

    The transcription of genes involved in iron acquisition in plants is induced under iron deficiency, but our understanding of iron sensors and signals remains limited. Iron Deficiency-responsive Element-binding Factor 1 (IDEF1) and Hemerythrin motif-containing Really Interesting New Gene- and Zinc-finger proteins (HRZs)/BRUTUS (BTS) have recently emerged as candidate iron sensors because of their functions as potent regulators of iron deficiency responses and their iron-binding properties. IDEF1 is a central transcriptional regulator of graminaceous genes involved in iron uptake and utilization, predominantly during the early stages of iron deficiency. HRZs/BTS are E3 ubiquitin ligases and negative regulators of iron deficiency responses in both graminaceous and non-graminaceous plants. Rice OsHRZ1 and OsHRZ2 are also potent regulators of iron accumulation. Characterizing these putative iron sensors also provides clues to understanding the nature of iron signals, which may involve ionized iron itself, other metals, oxygen, redox status, heme and iron-sulfur clusters, in addition to metabolites affected by iron deficiency. Systemic iron responses may also be regulated by phloem-mobile iron and its chelators such as nicotianamine. Iron sensors and signals will be identified by demonstration of signal transmission by IDEF1, HRZs/BTS, or unknown factors.

  8. Affinity filtration coupled with capillary-based affinity purification for the isolation of protein complexes.

    PubMed

    Qureshi, M S; Sheikh, Q I; Hill, R; Brown, P E; Dickman, M J; Tzokov, S B; Rice, D W; Gjerde, D T; Hornby, D P

    2013-08-01

    The isolation of complex macromolecular assemblies at the concentrations required for structural analysis represents a major experimental challenge. Here we present a method that combines the genetic power of site-specific recombination in order to selectively "tag" one or more components of a protein complex with affinity-based rapid filtration and a final step of capillary-based enrichment. This modified form of tandem affinity purification produces highly purified protein complexes at high concentrations in a highly efficient manner. The application of the method is demonstrated for the yeast Arp2/3 heptameric protein complex involved in mediating reorganization of the actin cytoskeleton.

  9. Development of Iron Aluminides.

    DTIC Science & Technology

    1986-03-01

    IRON ALUMINIDES G. Culbertson C. S. Kortovich TRW Inc. Materials & Manufacturing Center 23555 Euclid Avenue Euclid, Ohio 44117 March 1986 Final Report...NO. N I 1 Ti TILE Inciuav Securty ltassificafton, 621 02F 2420 02 1 flevelonment of Iron Aluminides 12 PERSONAL AUJTHOR(S) rl Cul bertson, C~r...rnumber) nrceqrarl w.as conducted to develop improved iron- aluminide alloys with higher qlevated ernerature strength and room temperature ductility

  10. Iron and the athlete.

    PubMed

    Suedekum, Natalie A; Dimeff, Robert J

    2005-08-01

    Iron is an important mineral necessary for many biologic pathways. Different levels of deficiency can occur in the athlete, resulting in symptoms that range from none to severe fatigue. Iron deficiency without anemia may adversely affect athletic performance. Causes of iron deficiency include poor intake, menstrual losses, gastrointestinal and genitourinary losses due to exercise-induced ischemia or organ movement, foot strike hemolysis, thermohemolysis, and sweat losses. A higher incidence of deficiency occurs in female athletes compared with males.

  11. 35. GREY IRON TUMBLERS, IN THE GREY IRON FOUNDRY ROTATE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    35. GREY IRON TUMBLERS, IN THE GREY IRON FOUNDRY ROTATE CASTINGS WITH SHOT TO REMOVE AND SURFACE OXIDES AND REMAINING EXCESS METALS. - Stockham Pipe & Fittings Company, Grey Iron Foundry, 4000 Tenth Avenue North, Birmingham, Jefferson County, AL

  12. Physics of iron

    NASA Astrophysics Data System (ADS)

    Anderson, O.

    1993-10-01

    This volume comprises papers presented at the AIRAPT Conference, 28 June - 2 July 1993. The iron sessions at the meeting were identified as the Second Ironworkers Convention. The renewal of interest stems from advances in technologies in both diamond-anvil cell (DAC) and shock wave studies as well as from controversies arising from a lack of consensus among both experimentalists and theoreticians. These advances have produced new data on iron in the pressure-temperature regime of interest for phase diagrams and for temperatures of the core/mantle and inner-core/outer-core boundaries. Particularly interesting is the iron phase diagram inferred from DAC studies. A new phase, (beta), with a (gamma)-(beta)-(epsilon) triple point at about 30 GPa and 1190 K, and possible sixth phase, (omega), with an (epsilon)-(Theta)-melt triple point at about 190 GPa and 4000 K are deemed possible. The importance of the equation of state of iron in consideration of Earth's heat budget and the origin of its magnetic field invoke the interest of theoreticians who argue on the basis of molecular dynamics and other first principles methods. While the major thrust of both meetings was on the physics of pure iron, there were notable contributions on iron alloys. Hydrogen-iron alloys, iron-sulfur liquids, and the comparability to rhenium in phase diagram studies are discussed. The knowledge of the physical properties of iron were increased by several contributions.

  13. Physiology of Iron Metabolism

    PubMed Central

    Waldvogel-Abramowski, Sophie; Waeber, Gérard; Gassner, Christoph; Buser, Andreas; Frey, Beat M.; Favrat, Bernard; Tissot, Jean-Daniel

    2014-01-01

    Summary A revolution occurred during the last decade in the comprehension of the physiology as well as in the physiopathology of iron metabolism. The purpose of this review is to summarize the recent knowledge that has accumulated, allowing a better comprehension of the mechanisms implicated in iron homeostasis. Iron metabolism is very fine tuned. The free molecule is very toxic; therefore, complex regulatory mechanisms have been developed in mammalian to insure adequate intestinal absorption, transportation, utilization, and elimination. ‘Ironomics’ certainly will be the future of the understanding of genes as well as of the protein-protein interactions involved in iron metabolism. PMID:25053935

  14. Physics of iron

    SciTech Connect

    Anderson, O.

    1993-10-01

    This volume comprises papers presented at the AIRAPT Conference, June 28 to July 1993. The iron sessions at the meeting were identified as the Second Ironworkers Convention. The renewal of interest stems from advances in technologies in both diamond-anvil cell (DAC) and shock wave studies as well as from controversies arising from a lack of consensus among both experimentalists and theoreticians. These advances have produced new data on iron in the pressure-temperature regime of interest for phase diagrams and for temperatures of the core/mantle and inner-core/outer-core boundaries. Particularly interesting is the iron phase diagram inferred from DAC studies. A new phase, {beta}, with a {gamma}-{beta}-{epsilon} triple point at about 30 GPa and 1190 K, and possible sixth phase, {omega}, with an {epsilon}-{Theta}-melt triple point at about 190 GPa and 4000 K are deemed possible. The importance of the equation of state of iron in consideration of Earth`s heat budget and the origin of its magnetic field invoke the interest of theoreticians who argue on the basis of molecular dynamics and other first principles methods. While the major thrust of both meetings was on the physics of pure iron, there was notable contributions on iron alloys. Hydrogen-iron alloys, iron-sulfur liquids, and the comparability to rhenium in phase diagram studies are discussed. The knowledge of the physical properties of iron were increased by several contributions.

  15. Iron, Meat and Health

    PubMed Central

    Geissler, Catherine; Singh, Mamta

    2011-01-01

    This article is a summary of the publication “Iron and Health” by the Scientific Advisory Committee on Nutrition (SACN) to the U.K. Government (2010), which reviews the dietary intake of iron and the impact of different dietary patterns on the nutritional and health status of the U.K. population. It concludes that several uncertainties make it difficult to determine dose-response relationships or to confidently characterize the risks associated with iron deficiency or excess. The publication makes several recommendations concerning iron intakes from food, including meat, and from supplements, as well as recommendations for further research. PMID:22254098

  16. Extraction of haemoglobin from human blood by affinity precipitation using a haptoglobin-based stimuli-responsive affinity macroligand.

    PubMed

    Stocker-Majd, Gisela; Hilbrig, Frank; Freitag, Ruth

    2008-06-13

    Affinity precipitation was compared to affinity chromatography and batch adsorption as the final purification step in a protocol for the isolation of haemoglobin from human blood. Haptoglobin was the affinity ligand. The first steps on the process were realized by traditional methods (lyses of red blood cells followed by ammonium sulphate precipitation). For affinity chromatography (and batch adsorption) the ligand was linked to Sepharose, for affinity precipitation to a thermoresponsive polymer, namely poly(N-isopropylacrylamide). Five haptoglobin-poly(N-isopropylacrylamide) bioconjugates (affinity macroligands) were constructed with different polymer: haptoglobin-coupling ratios. Conjugation of haptoglobin to the soluble poly(N-isopropylacrylamide) apparently does not change the interaction thermodynamics with haemoglobin, as the haemoglobin binding constants calculated by a Scatchard analysis for the affinity macroligand were of the same order of magnitude as those described in the literature for the haemoglobin-haptoglobin complex in solution. Two elution protocols were used for haemoglobin release from the various affinity materials, one at pH 2, the other with 5 M urea at pH 11. Both affinity chromatography and affinity precipitation yielded a pure haemoglobin of high quality. Compared to the affinity chromatography, affinity precipitation showed a significantly higher ligand efficiency (ratio of the experimental capacity to the theoretical one). The method thus makes better use of the expensive affinity ligands. As affinity precipitation only requires small temperature changes to bring about precipitation/redissolution of the affinity complexes and a centrifugation step for recovery of the precipitate, the method in addition has advantages in term of scalability and simplicity.

  17. Quantification of hydrophobic interaction affinity of colloids

    NASA Astrophysics Data System (ADS)

    Saini, G.; Nasholm, N.; Wood, B. D.

    2009-12-01

    Colloids play an important role in a wide variety of disciplines, including water and wastewater treatment, subsurface transport of metals and organic contaminants, migration of fines in oil reservoirs, biocolloid (virus and bacteria) transport in subsurface, and are integral to laboratory transport studies. Although the role of hydrophobicity in adhesion and transport of colloids, particularly bacteria, is well known; there is scarcity of literature regarding hydrophobicity measurement of non-bacterial colloids and other micron-sized particles. Here we detail an experimental approach based on differential partitioning of colloids between two liquid phases (hydrocarbon and buffer) as a measure of the hydrophobic interaction affinity of colloids. This assay, known as Microbial adhesion to hydrocarbons or MATH, is frequently used in microbiology and bacteriology for quantifying the hydrophobicity of microbes. Monodispersed colloids and particles, with sizes ranging from 1 micron to 33 micron, were used for the experiments. A range of hydrophobicity values were observed for different particles. The hydrophobicity results are also verified against water contact angle measurements of these particles. This liquid-liquid partitioning assay is quick, easy-to-perform and requires minimal instrumentation. Estimation of the hydrophobic interaction affinity of colloids would lead to a better understanding of their adhesion to different surfaces and subsequent transport in porous media.

  18. Fatigue damage prognosis using affine arithmetic

    NASA Astrophysics Data System (ADS)

    Gbaguidi, Audrey; Kim, Daewon

    2014-02-01

    Among the essential steps to be taken in structural health monitoring systems, damage prognosis would be the field that is least investigated due to the complexity of the uncertainties. This paper presents the possibility of using Affine Arithmetic for uncertainty propagation of crack damage in damage prognosis. The structures examined are thin rectangular plates made of titanium alloys with central mode I cracks and a composite plate with an internal delamination caused by mixed mode I and II fracture modes, under a harmonic uniaxial loading condition. The model-based method for crack growth rates are considered using the Paris Erdogan law model for the isotropic plates and the delamination growth law model proposed by Kardomateas for the composite plate. The parameters for both models are randomly taken and their uncertainties are considered as defined by an interval instead of a probability distribution. A Monte Carlo method is also applied to check whether Affine Arithmetic (AA) leads to tight bounds on the lifetime of the structure.

  19. Affinity-based target deconvolution of safranal

    PubMed Central

    2013-01-01

    Background and the purpose of the study Affinity-based target deconvolution is an emerging method for the identification of interactions between drugs/drug candidates and cellular proteins, and helps to predict potential activities and side effects of a given compound. In the present study, we hypothesized that a part of safranal pharmacological effects, one of the major constituent of Crocus sativus L., relies on its physical interaction with target proteins. Methods Affinity chromatography solid support was prepared by covalent attachment of safranal to agarose beads. After passing tissue lysate through the column, safranal-bound proteins were isolated and separated on SDS-PAGE or two-dimensional gel electrophoresis. Proteins were identified using MALDI-TOF/TOF mass spectrometry and Mascot software. Results and major conclusion Data showed that safranal physically binds to beta actin, cytochrome b-c1 complex sub-unit 1, trifunctional enzyme sub-unit beta and ATP synthase sub-unit alpha and beta. These interactions may explain part of safranal’s pharmacological effects. However, phenotypic and/or biological relevance of these interactions remains to be elucidated by future pharmacological studies. PMID:23514587

  20. Affine conformal vectors in space-time

    NASA Astrophysics Data System (ADS)

    Coley, A. A.; Tupper, B. O. J.

    1992-05-01

    All space-times admitting a proper affine conformal vector (ACV) are found. By using a theorem of Hall and da Costa, it is shown that such space-times either (i) admit a covariantly constant vector (timelike, spacelike, or null) and the ACV is the sum of a proper affine vector and a conformal Killing vector or (ii) the space-time is 2+2 decomposable, in which case it is shown that no ACV can exist (unless the space-time decomposes further). Furthermore, it is proved that all space-times admitting an ACV and a null covariantly constant vector (which are necessarily generalized pp-wave space-times) must have Ricci tensor of Segré type {2,(1,1)}. It follows that, among space-times admitting proper ACV, the Einstein static universe is the only perfect fluid space-time, there are no non-null Einstein-Maxwell space-times, and only the pp-wave space-times are representative of null Einstein-Maxwell solutions. Otherwise, the space-times can represent anisotropic fluids and viscous heat-conducting fluids, but only with restricted equations of state in each case.

  1. Impacts of amount of impregnated iron in granular activated carbon on arsenate adsorption capacities and kinetics.

    PubMed

    Chang, Qigang; Lin, Wei; Ying, Wei-Chi

    2012-06-01

    Iron-impregnated granular activated carbons (Fe-GAC) can remove arsenic effectively from water. In this study, Fe-GACs with iron content of 1.64 to 28.90% were synthesized using a new multi-step procedure for the investigation of effects of iron amount on arsenic adsorption capacities and kinetics. Langmuir model satisfactorily fit arsenic adsorption on Fe-GACs. The maximum arsenic adsorption capacity (q(m)) increased significantly with iron impregnation and reached 1,867 to 1,912 microg/g with iron content of 9.96 to 13.59%. Further increase of iron content (> 13.59%) caused gradual decrease of q(m). It was found that the amount of impregnated iron showed little impact on the affinity for arsenate. Kinetic study showed that the amount of impregnated iron affected the arsenic intraparticle diffusion rate greatly. The pseudo-second-order kinetic model fit arsenic adsorption kinetics on Fe-GACs better than the pseudo-first-order model. The arsenic adsorption rate increased with increasing of iron content from 1.64% to 13.59%, and then decreased with more impregnated iron (13.59 to 28.90%).

  2. Iron-impregnated granular activated carbon for arsenic removal from drinking water

    NASA Astrophysics Data System (ADS)

    Chang, Qigang

    A new multi-step iron impregnation method was developed in this study to impregnate GAC with a high amount of iron that possesses desired characteristics: stable, even distribution, and high arsenic adsorption capacity. Research was carried out to investigate the impact of the amount of impregnated iron on arsenic adsorption properties: capacity, affinity, and kinetics. Fe-GACs were characterized in terms of the amount, stability, distribution, morphology, and species of impregnated iron. It was found that a high amount of iron was stably impregnated in GAC. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) analysis demonstrated that the impregnated iron was evenly distributed on the internal surface of GAC. Impregnated iron formed nano-size particles and existed in both crystalline (akaganeite) and amorphous iron. Arsenic adsorption tests were conducted using Fe-GACs with iron content of 1.64--28.90% in a low arsenic concentration that is typical for drinking water treatment. The amount of impregnated iron affects arsenic maximum adsorption capacity (qm) but has little impact on the Langmuir constant h (the affinity of adsorbent for adsorbate). The qm for both As(V) and As(III) adsorptions increased significantly with increase of the amount of impregnated iron up to 13.59%. Further increase of iron amounts caused a gradual decrease of qm for As(V). BET analysis indicated impregnated iron possesses the highest surface area at iron content of 13.59%. A new second-order kinetic model was developed to investigate the impact of the amounts of impregnated iron on arsenic adsorption kinetics. With iron content increased from 1.64% to 28.90%, the intrinsic adsorption rate constants reduced from 4.6x10-2 1/hr to 1.18x10 -3 1/hr, which indicates that impregnated iron slows arsenic intraparticle diffusion rate in Fe-GAC. The decreased arsenic intraparticle diffusion rate was most likely caused by reduced pore size of Fe-GACs. Column tests were

  3. Mechanism of iron uptake by the pathogenic yeast, Candida albicans

    SciTech Connect

    Ismail, A.

    1986-01-01

    C. albicans requires iron for growth and phenotypic development. When deprived of iron, mycelium and bud formation was suppressed. Survival of the organism was also reduced under iron-limiting conditions. The combination of elevated temperature and iron-deprivation further reduced phenotypic development and survival of the yeast. The combination of elevated temperature and iron starvation resulted in a decrease in both the growth rate and siderophore production. However, with time, the cells were able to show partial recovery in the growth rate which occurred concomitantly with an increase in siderophore production. In order for siderophores to be utilized, ferri-siderophore receptors must be produced. The receptor was shown to be located in the plasma membrane of the yeast. Scatchard analysis of the binding of ferri-siderophores to plasma membrane receptors showed an increase in receptor affinity and number of binding sites in iron-starved cells when compared to control cells. Autoradiograms of the /sup 58/Fe-siderophore-protein complex following SDS-PAGE separation of candidal proteins revealed the presence of a ferri-siderophore receptor of approximately 10,000 daltons. C. albicans strains which lacked the ability to synthesize phenolate siderophore maintained a phenolate receptor and bound candidal phenolate siderophore better than non-candidal phenolate siderophores.

  4. Involvement of NRAMP1 from Arabidopsis thaliana in iron transport.

    PubMed

    Curie, C; Alonso, J M; Le Jean, M; Ecker, J R; Briat, J F

    2000-05-01

    Nramp genes code for a widely distributed class of proteins involved in a variety of processes, ranging from the control of susceptibility to bacterial infection in mammalian cells and taste behaviour in Drosophila to manganese uptake in yeast. Some of the NRAMP proteins in mammals and in yeast are capable of transporting metal ions, including iron. In plants, iron transport was shown to require a reduction/Fe(II) transport system. In Arabidopsis thaliana this process involves the IRT1 and Fro2 genes. Here we report the sequence of five NRAMP proteins from A. thaliana. Sequence comparison suggests that there are two classes of NRAMP proteins in plants: A. thaliana (At) NRAMP1 and Oriza sativa (Os) NRAMP1 and 3 (two rice isologues) represent one class, and AtNRAMP2-5 and OsNRAMP2 the other. AtNramp1 and OsNramp1 are able to complement the fet3fet4 yeast mutant defective both in low- and high-affinity iron transports, whereas AtNramp2 and OsNramp2 fail to do so. In addition, AtNramp1 transcript, but not AtNramp2 transcript, accumulates in response to iron deficiency in roots but not in leaves. Finally, overexpression of AtNramp1 in transgenic A. thaliana plants leads to an increase in plant resistance to toxic iron concentration. Taken together, these results demonstrate that AtNramp1 participates in the control of iron homoeostasis in plants.

  5. Microbial siderophore-based iron assimilation and therapeutic applications.

    PubMed

    Li, Kunhua; Chen, Wei-Hung; Bruner, Steven D

    2016-06-01

    Siderophores are structurally diverse, complex natural products that bind metals with extraordinary specificity and affinity. The acquisition of iron is critical for the survival and virulence of many pathogenic microbes and diverse strategies have evolved to synthesize, import and utilize iron. There has been a substantial increase of known siderophore scaffolds isolated and characterized in the past decade and the corresponding biosynthetic gene clusters have provided insight into the varied pathways involved in siderophore biosynthesis, delivery and utilization. Additionally, therapeutic applications of siderophores and related compounds are actively being developed. The study of biosynthetic pathways to natural siderophores augments the understanding of the complex mechanisms of bacterial iron acquisition, and enables a complimentary approach to address virulence through the interruption of siderophore biosynthesis or utilization by targeting the key enzymes to the siderophore pathways.

  6. Perspectives on nutritional iron deficiency.

    PubMed

    Hallberg, L

    2001-01-01

    Nutritional iron deficiency (ID) is caused by an intake of dietary iron insufficient to cover physiological iron requirements. Studies on iron absorption from whole diets have examined relationships between dietary iron bioavailability/absorption, iron losses, and amounts of stored iron. New insights have been obtained into regulation of iron absorption and expected rates of changes of iron stores or hemoglobin iron deficits when bioavailability or iron content of the diet has been modified and when losses of iron occur. Negative effects of ID are probably related to age, up to about 20 years, explaining some of earlier controversies. Difficulties in establishing the prevalence of mild ID are outlined. The degree of underestimation of the prevalence of mild ID when using multiple diagnostic criteria is discussed. It is suggested that current low-energy lifestyles are a common denominator for the current high prevalence not only of ID but also of obesity, diabetes, and osteoporosis.

  7. Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

    PubMed

    Aguda, Adeleke H; Lavallee, Vincent; Cheng, Ping; Bott, Tina M; Meimetis, Labros G; Law, Simon; Nguyen, Nham T; Williams, David E; Kaleta, Jadwiga; Villanueva, Ivan; Davies, Julian; Andersen, Raymond J; Brayer, Gary D; Brömme, Dieter

    2016-08-26

    Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

  8. [Iron deficiency and digestive disorders].

    PubMed

    Cozon, G J N

    2014-11-01

    Iron deficiency anemia still remains problematic worldwide. Iron deficiency without anemia is often undiagnosed. We reviewed, in this study, symptoms and syndromes associated with iron deficiency with or without anemia: fatigue, cognitive functions, restless legs syndrome, hair loss, and chronic heart failure. Iron is absorbed through the digestive tract. Hepcidin and ferroportin are the main proteins of iron regulation. Pathogenic micro-organisms or intestinal dysbiosis are suspected to influence iron absorption.

  9. Intravenous Iron Sucrose for Children with Iron Deficiency Failing to Respond to Oral Iron Therapy

    PubMed Central

    Crary, Shelley E.; Hall, Katherine; Buchanan, George R.

    2010-01-01

    Background For decades parenteral iron has been used in patients with iron deficiency unresponsive to oral iron therapy and in hemodialysis-dependent patients receiving erythropoietin. Newer intravenous (IV) iron formulations such as iron sucrose have replaced high molecular weight iron dextran in dialysis patients; however, the use of parenteral iron in children without renal disease has not been well defined. Procedure Pharmacy records were reviewed on children (≤ 18 yrs of age) who received IV iron sucrose at Children's Medical Center Dallas between January 1, 2004 and June 30, 2009. Patients who received iron sucrose for chronic renal disease were excluded from analysis. Results Thirty-eight children received iron sucrose for non-renal indications, 13 with iron deficiency refractory to oral iron therapy, 13 with iron malabsorption or dependence on parenteral nutrition, 7 for chronic gastrointestinal blood loss, and 5 for miscellaneous indications. Among these 38 children, who received a total of 510 doses of IV iron sucrose, there were only 6 adverse reactions. Patients in all categories had a good response to the iron sucrose, with a median hemoglobin rise of 1.9 – 3.1 g/dl depending on the indication. Conclusions Parenteral iron is a safe and effective means to treat iron deficiency in children who cannot receive or do not respond to oral iron due to intolerance, poor adherence or iron malabsorption. PMID:21298748

  10. Structure of a High-Affinity

    SciTech Connect

    Saphire, E.O.; Montero, M.; Menendez, A.; Houten, N.E.van; Irving, M.B.; Pantophlet, R.; Swick, M.B.; Parren, P.W.H.I.; Burton, D.R.; Scott, J.K.; Wilson, I.A.; /Scripps Res. Inst. /Simon Fraser U. /British Columbia U.

    2007-07-13

    The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 Angstrom resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining

  11. Taking iron supplements

    MedlinePlus

    ... looking as well as black If they have red streaks Cramps, sharp pains, or soreness in the stomach occur Liquid forms of iron may stain your teeth. Try mixing the iron with water or other liquids (such as fruit juice or ...

  12. Thin Wall Iron Castings

    SciTech Connect

    J.F. Cuttino; D.M. Stefanescu; T.S. Piwonka

    2001-10-31

    Results of an investigation made to develop methods of making iron castings having wall thicknesses as small as 2.5 mm in green sand molds are presented. It was found that thin wall ductile and compacted graphite iron castings can be made and have properties consistent with heavier castings. Green sand molding variables that affect casting dimensions were also identified.

  13. Iron nutrition in adolescence.

    PubMed

    Mesías, Marta; Seiquer, Isabel; Navarro, M Pilar

    2013-01-01

    Adolescence is an important period of nutritional vulnerability due to increased dietary requirements for growth and development. Iron needs are elevated as a result of intensive growth and muscular development, which implies an increase in blood volume; thus, it is extremely important for the adolescent's iron requirements to be met. Diet, therefore, must provide enough iron and, moreover, nutrients producing adequate iron bioavailability to favor element utilization and thus be sufficient for needs at this stage of life. Currently, many adolescents consume monotonous and unbalanced diets which may limit mineral intake and/or bioavailability, leading to iron deficiency and, consequently, to ferropenic anemia, a nutritional deficit of worldwide prevalence. Iron deficiency, apart from provoking important physiological repercussions, can adversely affect adolescents' cognitive ability and behavior. Accordingly, promoting the consumption of a varied, adjusted, and balanced diet by adolescents will facilitate iron utilization, benefiting their health both at present and in adulthood. This review discusses how physiological changes during adolescence can cause iron requirements to increase. Consequently, it is important that diet should contribute an appropriate amount of this mineral and, moreover, with an adequate bioavailability to satisfy needs during this special period of life.

  14. Iron studies in hemophilia

    SciTech Connect

    Lottenberg, R.; Kitchens, C.S.; Roessler, G.S.; Noyes, W.D.

    1981-12-01

    Although iron deficiency is not recognized as a usual complication of hemophilia, we questioned whether intermittent occult loss of blood in urine or stool might predispose hemophiliacs to chronic iron deficiency. Seven men with factor VII and one with factor IX deficiency were studied. Blood studied, bone marrow aspirates, urine and stool samples, and ferrokinetics with total-body counting up to five months were examined. These data showed no excessive loss of blood during the study period; however, marrow iron stores were decidedly decreased, being absent in four subjects. We suggest that in some hemophiliacs, iron deposits in tissues such as synovial membranes may form a high proportion of the body's total iron stores.

  15. The affine cohomology spaces and its applications

    NASA Astrophysics Data System (ADS)

    Fraj, Nizar Ben; Laraiedh, Ismail

    2016-12-01

    We compute the nth cohomology space of the affine Lie superalgebra 𝔞𝔣𝔣(1) on the (1,1)-dimensional real superspace with coefficient in a large class of 𝔞𝔣𝔣(1)-modules M. We apply our results to the module of weight densities and the module of linear differential operators acting on a superspace of weighted densities. This work is the generalization of a result by Basdouri et al. [The linear 𝔞𝔣𝔣(n|1)-invariant differential operators on weighted densities on the superspace ℝ1|n and 𝔞𝔣𝔣(n|1)-relative cohomology, Int. J. Geom. Meth. Mod. Phys. 10 (2013), Article ID: 1320004, 9 pp.

  16. Automatic gesture analysis using constant affine velocity.

    PubMed

    Cifuentes, Jenny; Boulanger, Pierre; Pham, Minh Tu; Moreau, Richard; Prieto, Flavio

    2014-01-01

    Hand human gesture recognition has been an important research topic widely studied around the world, as this field offers the ability to identify, recognize, and analyze human gestures in order to control devices or to interact with computer interfaces. In particular, in medical training, this approach is an important tool that can be used to obtain an objective evaluation of a procedure performance. In this paper, some obstetrical gestures, acquired by a forceps, were studied with the hypothesis that, as the scribbling and drawing movements, they obey the one-sixth power law, an empirical relationship which connects path curvature, torsion, and euclidean velocity. Our results show that obstetrical gestures have a constant affine velocity, which is different for each type of gesture and based on this idea this quantity is proposed as an appropriate classification feature in the hand human gesture recognition field.

  17. Dynamic friction of self-affine surfaces

    NASA Astrophysics Data System (ADS)

    Schmittbuhl, Jean; Vilotte, Jean-Pierre; Roux, Stéphane

    1994-02-01

    We investigate the velocity dependence of the friction between two rigid blocks limited by a self-affine surface such as the one generated by a crack. The upper solid is subjected either to gravity or to an external elastic stiffness, and is driven horizontally at constant velocity, V, while the lower solid is fixed. For low velocities, the apparent friction coefficient is constant. For high velocities, the apparent friction is shown to display a velocity weakening. The weakening can be related to the variation of the mean contact time due to the occurrence of jumps during the motions. The cross-over between these two regimes corresponds to a characteristic velocity which depends on the geometry of the surfaces and on the mean normal force. In the case of simple gravity loading, the velocity dependence of the apparent friction at high velocities is proportional to 1/V^2 where V is the imposed tangential velocity. In the case of external elastic stiffness, two velocity weakening regimes can be identified, the first is identical to the gravity case with a 1/V^2 dependence, the second appears at higher velocities and is characterized by a 1/V variation. The characteristic velocity of this second cross-over depends on the roughness and the elastic stiffness. The statistical distribution of ballistic flight distances is analysed, and is shown to reveal in all cases the self-affinity of the contacting surfaces. Nous analysons la dépendence en vitesse du frottement entre deux solides limités par une surface rugueuse auto-affine comme celle d'une surface de fracture. Le solide supérieur est soumis soit à la gravité, soit à une raideur élastique externe, et est entraîné à vitesse horizontale constante V sur le solide inférieur fixe. A faible vitesse, le coefficient de friction apparent, est constant. A forte vitesse, le coefficient de friction apparent devient inversement proportionnel à la vitesse. Cette dépendance peut être reliée à la variation du temps

  18. Iron and the endurance athlete.

    PubMed

    Hinton, Pamela S

    2014-09-01

    Iron is a trace mineral that is highly significant to endurance athletes. Iron is critical to optimal athletic performance because of its role in energy metabolism, oxygen transport, and acid-base balance. Endurance athletes are at increased risk for suboptimal iron status, with potential negative consequences on performance, because of the combination of increased iron needs and inadequate dietary intake. This review paper summarizes the role of iron in maximal and submaximal exercise and describes the effects of iron deficiency on exercise performance. Mechanisms that explain the increased risk of iron deficiency in endurance athletes, including exercise-associated inflammation and hepcidin release on iron sequestration, are described. Information on screening athletes for iron deficiency is presented, and suggestions to increase iron intake through diet modification or supplemental iron are provided.

  19. Iron acquisition by Cryptococcus neoformans.

    PubMed

    Vartivarian, S E; Cowart, R E; Anaissie, E J; Tashiro, T; Sprigg, H A

    1995-01-01

    Iron is an essential element for the growth and metabolism of microbial cells. Most pathogenic microbes elaborate powerful iron chelating agents (siderophores) to mobilize iron from ferric ligands. The pathogenic yeast, Cryptococcus neoformans has not been found to produce siderophores and its mechanism of iron acquisition is unknown. This investigation explored an alternative pathway for iron acquisition by examining the interactions of iron with the cell surface. Iron uptake experiments were conducted utilizing radiolabelled ferrous iron and ferric iron chelates, with evidence for the presence of iron(II) receptors and the generation of ferrous iron by surface reduction. Hyperbolic kinetics were found when 59FeII was presented to the organism and uptake was blocked with bathophenanthroline sulphonate, an Fe2+ chelator. The yeast also acquired iron as [59Fe3+]-citrate and [59Fe3+]-pyrophosphate while bathophenanthroline sulphonate reduced the acquisition of these ferric ligands by 48% and 52% respectively. Pre-incubation with either ferric ligand also reduced iron acquisition by 50%. KCN inhibited uptake of iron(II) by 90% and uptake of [59Fe3+]-pyrophosphate and [59Fe3+]-citrate by 46% and 56% respectively; dinitrophenol had no effect on these processes. The data suggest that C. neoformans can (i) generate ferrous iron at the cell surface via a reduction of ferric chelates, with the subsequent acquisition of the ferrous iron, and (ii) acquire iron through the interaction of ferric chelates with a surface component.

  20. 21 CFR 310.518 - Drug products containing iron or iron salts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 5 2010-04-01 2010-04-01 false Drug products containing iron or iron salts. 310... Drug products containing iron or iron salts. Drug products containing elemental iron or iron salts as...) that contains iron or iron salts for use as an iron source shall bear the following statement:...

  1. 21 CFR 310.518 - Drug products containing iron or iron salts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 5 2011-04-01 2011-04-01 false Drug products containing iron or iron salts. 310... Drug products containing iron or iron salts. Drug products containing elemental iron or iron salts as...) that contains iron or iron salts for use as an iron source shall bear the following statement:...

  2. 21 CFR 310.518 - Drug products containing iron or iron salts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 5 2014-04-01 2014-04-01 false Drug products containing iron or iron salts. 310... Drug products containing iron or iron salts. Drug products containing elemental iron or iron salts as...) that contains iron or iron salts for use as an iron source shall bear the following statement:...

  3. 21 CFR 310.518 - Drug products containing iron or iron salts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 5 2012-04-01 2012-04-01 false Drug products containing iron or iron salts. 310... Drug products containing iron or iron salts. Drug products containing elemental iron or iron salts as...) that contains iron or iron salts for use as an iron source shall bear the following statement:...

  4. Smooth affine shear tight frames: digitization and applications

    NASA Astrophysics Data System (ADS)

    Zhuang, Xiaosheng

    2015-08-01

    In this paper, we mainly discuss one of the recent developed directional multiscale representation systems: smooth affine shear tight frames. A directional wavelet tight frame is generated by isotropic dilations and translations of directional wavelet generators, while an affine shear tight frame is generated by anisotropic dilations, shears, and translations of shearlet generators. These two tight frames are actually connected in the sense that the affine shear tight frame can be obtained from a directional wavelet tight frame through subsampling. Consequently, an affine shear tight frame indeed has an underlying filter bank from the MRA structure of its associated directional wavelet tight frame. We call such filter banks affine shear filter banks, which can be designed completely in the frequency domain. We discuss the digitization of affine shear filter banks and their implementations: the forward and backward digital affine shear transforms. Redundancy rate and computational complexity of digital affine shear transforms are also investigated in this paper. Numerical experiments and comparisons in image/video processing show the advantages of digital affine shear transforms over many other state-of-art directional multiscale representation systems.

  5. Noncompetitive affinity assays of glucagon and amylin using mirror-image aptamers as affinity probes.

    PubMed

    Yi, Lian; Wang, Xue; Bethge, Lucas; Klussmann, Sven; Roper, Michael G

    2016-03-21

    The ability to detect picomolar concentrations of glucagon and amylin using fluorescently labeled mirror-image aptamers, so-called Spiegelmers, is demonstrated. Spiegelmers rival the specificity of antibodies and overcome the problem of biostability of natural aptamers in a biological matrix. Using Spiegelmers as affinity probes, noncompetitive capillary electrophoresis affinity assays of glucagon and murine amylin were developed and optimized. The detection limit for glucagon was 6 pM and for amylin was 40 pM. Glucagon-like peptide-1 and -2 did not interfere with the glucagon assay, while the amylin assay showed cross-reactivity to calcitonin gene related peptide. The developed assays were combined with a competitive immunoassay for insulin to measure glucagon, amylin, and insulin secretion from batches of islets after incubation with different glucose concentrations. The development of these assays is an important step towards incorporation into an online measurement system for monitoring dynamic secretion from single islets.

  6. IRT1, an Arabidopsis transporter essential for iron uptake from the soil and for plant growth.

    PubMed

    Vert, Grégory; Grotz, Natasha; Dédaldéchamp, Fabienne; Gaymard, Frédéric; Guerinot, Mary Lou; Briat, Jean-François; Curie, Catherine

    2002-06-01

    Plants are the principal source of iron in most diets, yet iron availability often limits plant growth. In response to iron deficiency, Arabidopsis roots induce the expression of the divalent cation transporter IRT1. Here, we present genetic evidence that IRT1 is essential for the uptake of iron from the soil. An Arabidopsis knockout mutant in IRT1 is chlorotic and has a severe growth defect in soil, leading to death. This defect is rescued by the exogenous application of iron. The mutant plants do not take up iron and fail to accumulate other divalent cations in low-iron conditions. IRT1-green fluorescent protein fusion, transiently expressed in culture cells, localized to the plasma membrane. We also show, through promoter::beta-glucuronidase analysis and in situ hybridization, that IRT1 is expressed in the external cell layers of the root, specifically in response to iron starvation. These results clearly demonstrate that IRT1 is the major transporter responsible for high-affinity metal uptake under iron deficiency.

  7. Nonhematological benefits of iron.

    PubMed

    Agarwal, Rajiv

    2007-01-01

    Iron deficiency anemia is common in people with chronic kidney disease (CKD) and its importance in supporting erythropoiesis is unquestioned especially in those patients treated with erythropoietin. Clinical symptomatology such as fatigability, cold intolerance, failure to concentrate and poor effort intolerance is often attributed to anemia or uremia. That iron deficiency, per se, can cause these symptoms is poorly recognized. Clinical and animal studies that support the benefits of iron supplementation, independent of increasing hemoglobin, such as those on immune function, physical performance, thermoregulation, cognition, and restless leg syndrome and aluminum absorption is the subject of this narrative review.

  8. Coal desulfurization. [using iron pentacarbonyl

    NASA Technical Reports Server (NTRS)

    Hsu, G. C. (Inventor)

    1979-01-01

    Organic sulfur is removed from coal by treatment with an organic solution of iron pentacarbonyl. Organic sulfur compounds can be removed by reaction of the iron pentacarbonyl with coal to generate CO and COS off-gases. The CO gas separated from COS can be passed over hot iron fillings to generate iron pentacarbonyl.

  9. Reactive iron in marine sediments

    NASA Technical Reports Server (NTRS)

    Canfield, Donald E.

    1989-01-01

    The influence of reactive iron oxides on sediment pore-water chemistry is considered in detail. A carefully calibrated extraction scheme is used to determine the depth distributions of reactive iron phases at two very different localities: the relatively iron-rich Mississippi Delta and the relatively iron-poor FOAM site in Long Island Sound. Closed system incubations are used to characterize the rates of reaction between sulfide and both naturally occurring and pure iron mineral phases. Rates of iron liberation to pore solution are measured in the presence and absence of sulfate reduction, and the origin of dissolved iron in organic-rich sediments is speculated upon.

  10. In Vivo Analysis of HPr Reveals a Fructose-Specific Phosphotransferase System That Confers High-Affinity Uptake in Streptomyces coelicolor

    PubMed Central

    Nothaft, Harald; Parche, Stephan; Kamionka, Annette; Titgemeyer, Fritz

    2003-01-01

    HPr, the histidine-containing phosphocarrier protein of the bacterial phosphotransferase system (PTS), serves multiple functions in carbohydrate uptake and carbon source regulation in low-G+C-content gram-positive bacteria and in gram-negative bacteria. To assess the role of HPr in the high-G+C-content gram-positive organism Streptomyces coelicolor, the encoding gene, ptsH, was deleted. The ptsH mutant BAP1 was impaired in fructose utilization, while growth on other carbon sources was not affected. Uptake assays revealed that BAP1 could not transport appreciable amounts of fructose, while the wild type showed inducible high-affinity fructose transport with an apparent Km of 2 μM. Complementation and reconstitution experiments demonstrated that HPr is indispensable for a fructose-specific PTS activity. Investigation of the putative fruKA gene locus led to identification of the fructose-specific enzyme II permease encoded by the fruA gene. Synthesis of HPr was not specifically enhanced in fructose-grown cells and occurred also in the presence of non-PTS carbon sources. Transcriptional analysis of ptsH revealed two promoters that are carbon source regulated. In contrast to what happens in other bacteria, glucose repression of glycerol kinase was still operative in a ptsH background, which suggests that HPr is not involved in general carbon regulation. However, fructose repression of glycerol kinase was lost in BAP1, indicating that the fructose-PTS is required for transduction of the signal. This study provides the first molecular genetic evidence of a physiological role of the PTS in S. coelicolor. PMID:12533468

  11. Overexpression of EAR1 and SSH4 that encode PPxY proteins in the multivesicular body provides stability to tryptophan permease Tat2, allowing yeast cells to grow under high hydrostatic pressure

    NASA Astrophysics Data System (ADS)

    Hiraki, Toshiki; Usui, Keiko; Abe, Fumiyoshi

    2010-12-01

    Tryptophan uptake in yeast Saccharomyces cerevisiae is susceptible to high hydrostatic pressure and it limits the growth of tryptophan auxotrophic (Trp-) strains under pressures of 15-25 MPa. The susceptibility of tryptophan uptake is accounted for by the pressure-induced degradation of tryptophan permease Tat2 occurring in a Rsp5 ubiquitin ligase-dependent manner. Ear1 and Ssh4 are multivesicular body proteins that physically interact with Rsp5. We found that overexpression of either of the EAR1 or SSH4 genes enabled the Trp- cells to grow at 15-25 MPa. EAR1 and SSH4 appeared to provide stability to the Tat2 protein when overexpressed. The result suggests that Ear1 and Ssh4 negatively regulate Rsp5 on ubiquitination of Tat2. Currently, high hydrostatic pressure is widely used in bioscience and biotechnology for structurally perturbing macromolecules such as proteins and lipids or in food processing and sterilizing microbes. We suggest that hydrostatic pressure is an operative experimental parameter to screen yeast genes specifically for regulation of Tat2 through the function of Rsp5 ubiquitin ligase.

  12. Overexpression of the FRO2 ferric chelate reductase confers tolerance to growth on low iron and uncovers posttranscriptional control.

    PubMed

    Connolly, Erin L; Campbell, Nathan H; Grotz, Natasha; Prichard, Charis L; Guerinot, Mary Lou

    2003-11-01

    The Arabidopsis FRO2 gene encodes the low-iron-inducible ferric chelate reductase responsible for reduction of iron at the root surface. Here, we report that FRO2 and IRT1, the major transporter responsible for high-affinity iron uptake from the soil, are coordinately regulated at both the transcriptional and posttranscriptional levels. FRO2 and IRT1 are induced together following the imposition of iron starvation and are coordinately repressed following iron resupply. Steady-state mRNA levels of FRO2 and IRT1 are also coordinately regulated by zinc and cadmium. Like IRT1, FRO2 mRNA is detected in the epidermal cells of roots, consistent with its proposed role in iron uptake from the soil. FRO2 mRNA is detected at high levels in the roots and shoots of 35S-FRO2 transgenic plants. However, ferric chelate reductase activity is only elevated in the 35S-FRO2 plants under conditions of iron deficiency, indicating that FRO2 is subject to posttranscriptional regulation, as shown previously for IRT1. Finally, the 35S-FRO2 plants grow better on low iron as compared with wild-type plants, supporting the idea that reduction of ferric iron to ferrous iron is the rate-limiting step in iron uptake.

  13. Ferrous Sulfate (Iron)

    MedlinePlus

    Ferrous sulfate provides the iron needed by the body to produce red blood cells. It is used to ... Ferrous sulfate comes as regular, coated, and extended-release (long-acting) tablets; regular and extended-release capsules; and ...

  14. Iron Partitioning in Ferropericlase

    NASA Astrophysics Data System (ADS)

    Braithwaite, J. W. H.; Stixrude, L. P.; Pinilla, C.; Holmstrom, E.

    2015-12-01

    Ferropericlase, (Mg,Fe)O, is the second most abundant mineral in the Earth's lower mantle. Whether iron favours the liquid or solid phase of (Mg,Fe)O has important implications for the Earth's mantle, both chemically and dynamically. As iron is much heavier than magnesium, the partitioning of iron between liquid and solid will lead to a contrast in densities. This difference in density will lead one phase to be more buoyant than the other and would help, in part, to explain how the mantle crystallised from the magma ocean of the Hadean eon to its current state. The partitioning of iron between the two phases is characterized by partition coefficients. Using ab-initio methods, thermodynamic integration and adiabatic switching these coefficients have been determined. Results are presented for pressures encompassing the region between the upper mantle and the core-mantle boundary (10-140GPa).

  15. Iron-Air Rechargeable Battery

    NASA Technical Reports Server (NTRS)

    Narayan, Sri R. (Inventor); Prakash, G.K. Surya (Inventor); Kindler, Andrew (Inventor)

    2014-01-01

    Embodiments include an iron-air rechargeable battery having a composite electrode including an iron electrode and a hydrogen electrode integrated therewith. An air electrode is spaced from the iron electrode and an electrolyte is provided in contact with the air electrode and the iron electrodes. Various additives and catalysts are disclosed with respect to the iron electrode, air electrode, and electrolyte for increasing battery efficiency and cycle life.

  16. Affine connection form of Regge calculus

    NASA Astrophysics Data System (ADS)

    Khatsymovsky, V. M.

    2016-12-01

    Regge action is represented analogously to how the Palatini action for general relativity (GR) as some functional of the metric and a general connection as independent variables represents the Einstein-Hilbert action. The piecewise flat (or simplicial) spacetime of Regge calculus is equipped with some world coordinates and some piecewise affine metric which is completely defined by the set of edge lengths and the world coordinates of the vertices. The conjugate variables are the general nondegenerate matrices on the three-simplices which play the role of a general discrete connection. Our previous result on some representation of the Regge calculus action in terms of the local Euclidean (Minkowsky) frame vectors and orthogonal connection matrices as independent variables is somewhat modified for the considered case of the general linear group GL(4, R) of the connection matrices. As a result, we have some action invariant w.r.t. arbitrary change of coordinates of the vertices (and related GL(4, R) transformations in the four-simplices). Excluding GL(4, R) connection from this action via the equations of motion we have exactly the Regge action for the considered spacetime.

  17. [Separation of osteoclasts by lectin affinity chromatography].

    PubMed

    Itokazu, M; Tan, A; Tanaka, S

    1991-09-01

    Newborn rat calvaria bone cells obtained by digestion were fractionated on columns of wheat-germ agglutinin (WGA) sepharose 6MB for osteoclast isolation. The initial nonspecific binding cells which were passed through the WGA sepharose column by a buffer acquired a high enzyme activity of alkaline phosphatase, but not that of acid phosphatase. However, elution of cells using a buffer with the addition of N-acetyl-D-glucosamine resulted in a high acid phosphatase activity but no alkaline phosphatase activity. The former WGA binding negative fraction enriched osteoblasts averaging 30 microns in size. The latter WGA binding positive fraction enriched osteoclasts ranging from 20 microns to 60 microns in size. The electron-microscope clearly demonstrated the cellular details of osteoclasts. Isolated cell counts showed a ratio of six to four. These results indicate that our method of osteoclast isolation is simple and useful in lectin affinity chromatography because all cells have sugar moieties on their surface and the binding of osteoclasts can be reversed by the addition of specific lectin-binding sugars to the eluting buffer.

  18. Prostate Cancer and Bone: The Elective Affinities

    PubMed Central

    2014-01-01

    The onset of metastases dramatically changes the prognosis of prostate cancer patients, determining increased morbidity and a drastic fall in survival expectancy. Bone is a common site of metastases in few types of cancer, and it represents the most frequent metastatic site in prostate cancer. Of note, the prevalence of tumor relapse to the bone appears to be increasing over the years, likely due to a longer overall survival of prostate cancer patients. Bone tropism represents an intriguing challenge for researchers also because the preference of prostate cancer cells for the bone is the result of a sequential series of targetable molecular events. Many factors have been associated with the peculiar ability of prostate cancer cells to migrate in bone marrow and to determine mixed osteoblastic/osteolytic lesions. As anticipated by the success of current targeted therapy aimed to block bone resorption, a better understanding of molecular affinity between prostate cancer and bone microenvironment will permit us to cure bone metastasis and to improve prognosis of prostate cancer patients. PMID:24971315

  19. Multiplexed protein profiling by sequential affinity capture

    PubMed Central

    Ayoglu, Burcu; Birgersson, Elin; Mezger, Anja; Nilsson, Mats; Uhlén, Mathias; Nilsson, Peter

    2016-01-01

    Antibody microarrays enable parallelized and miniaturized analysis of clinical samples, and have proven to provide novel insights for the analysis of different proteomes. However, there are concerns that the performance of such direct labeling and single antibody assays are prone to off‐target binding due to the sample context. To improve selectivity and sensitivity while maintaining the possibility to conduct multiplexed protein profiling, we developed a multiplexed and semi‐automated sequential capture assay. This novel bead‐based procedure encompasses a first antigen capture, labeling of captured protein targets on magnetic particles, combinatorial target elution and a read‐out by a secondary capture bead array. We demonstrate in a proof‐of‐concept setting that target detection via two sequential affinity interactions reduced off‐target contribution, while lowered background and noise levels, improved correlation to clinical values compared to single binder assays. We also compared sensitivity levels with single binder and classical sandwich assays, explored the possibility for DNA‐based signal amplification, and demonstrate the applicability of the dual capture bead‐based antibody microarray for biomarker analysis. Hence, the described concept enhances the possibilities for antibody array assays to be utilized for protein profiling in body fluids and beyond. PMID:26935855

  20. Cambrian trilobites with Siberian affinities, southwestern Alaska

    SciTech Connect

    Palmer, A.R.; Egbert, R.M.; Sullivan, R.; Knoth, J.S.

    1985-02-01

    Cambrian trilobites occur in two levels (about 7 m apart) in the core of a large, complex anticlinal structure in the area between the Taylor Mountains and the Hoholitna River in southwestern Alaska. The lower collection contains Erbia, Macannaia (a species close to Soviet forms described as Pagetia ferox Lermontova), two species of Kootenia (including one perhaps cospecific with forms from the central Brooks range), and several species of ptychoparioid trilobites. It is clear that biogeographic affinities are with the transitional facies of the eastern Siberian platform and the south Siberian foldbelt. In Soviet terms, the age of the collection falls in a disputed interval called latest Early Cambrian (Tojonian) by some authors, and earliest Middle Cambrian (Amgan) by others. In North American terms, Macannaia is known only from early Middle Cambrian beds. The younger collection contains abundant agnostids, a variety of conocoryphids, Paradoxides, and several species of ptychoparioid trilobites. This is an assemblage of undoubted late Middle Cambrian age, comparable to faunas described from the Maya State of the Siberian platform and the Paradoxides paradoxissimus Stage of the Baltic region. Both faunas are from ocean-facing or outer shelf environments. None of the key non-agnostid or non-pagetiid elements have been seen previously in deposits of Cambrian North America.

  1. Affinity of guanosine derivatives for polycytidylate revisited

    NASA Technical Reports Server (NTRS)

    Kanavarioti, A.; Hurley, T. B.; Baird, E. E.

    1995-01-01

    Evidence is presented for complexation of guanosine 5'-monophosphate 2-methylimidazolide (2-MeImpG) with polycytidylate (poly(C)) at pH 8.0 and 23 degrees C in the presence of 1.0 M NaCl2 and 0.2 M MgCl2 in water. The association of 2-MeImpG with poly(C) was investigated using UV-vis spectroscopy as well as by monitoring the kinetics of the nucleophilic substitution reaction of the imidazole moiety by amines. The results of both methods are consistent with moderately strong poly(C) 2-MeImpG complexation and the spectrophotometric measurements allowed the construction of a binding isotherm with a concentration of 2-MeImpG equal to 5.55 +/- 0.15 mM at half occupancy. UV spectroscopy was employed to establish the binding of other guanosine derivatives on poly(C). These derivatives are guanosine 5'-monophosphate (5'GMP), guanosine 5'-monophosphate imidazolide (ImpG), and guanosine 5'-monophosphate morpholidate (morpG). Within experimental error these guanosine derivatives exhibit the same affinity for poly(C) as 2-MeImpG.

  2. Macroporous chitin affinity membranes for lysozyme separation.

    PubMed

    Ruckenstein, E; Zeng, X

    1997-12-20

    Macroporous chitin membranes with high, controlled porosity and good mechanical properties have been prepared using a technique developed in this laboratory based on silica particles as porogen. They were employed for the affinity separation of lysozyme. Chitin membranes (1 mm thickness) can be operated at high fluxes (>/=1.1 mL/min/cm(2)) corresponding to pressure drops >/=2 psi. Their adsorption capacity for lysozyme ( approximately 50 mg/mL membrane) is by an order of magnitude higher than that of the chitin beads employed in column separation. In a binary mixture of lysozyme and ovalbumin, the membranes showed very high selectivity towards lysozyme. The effect of some important operation parameters, such as the flow rates during loading and elution were investigated. Lysozyme of very high purity (>98%) was obtained from a mixture of lysozyme and ovalbumin, and from egg white. The results indicate that the macroporous chitin membranes can be used for the separation, purification, and recovery of lysozyme at large scale. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 610-617, 1997.

  3. The extrahepatic role of TFR2 in iron homeostasis

    PubMed Central

    Silvestri, Laura; Nai, Antonella; Pagani, Alessia; Camaschella, Clara

    2014-01-01

    Transferrin receptor 2 (TFR2), a protein homologous to the cell iron importer TFR1, is expressed in the liver and erythroid cells and is reported to bind diferric transferrin, although at lower affinity than TFR1. TFR2 gene is mutated in type 3 hemochromatosis, a disorder characterized by iron overload and inability to upregulate hepcidin in response to iron. Liver TFR2 is considered a sensor of diferric transferrin, possibly in a complex with hemochromatosis protein. In erythroid cells TFR2 is a partner of erythropoietin receptor (EPOR) and stabilizes the receptor on the cell surface. However, Tfr2 null mice as well as TFR2 hemochromatosis patients do not show defective erythropoiesis and tolerate repeated phlebotomy. The iron deficient Tfr2-Tmprss6 double knock out mice have higher red cells count and more severe microcytosis than the liver-specific Tfr2 and Tmprss6 double knock out mice. TFR2 in the bone marrow might be a sensor of iron deficiency that protects against excessive microcytosis in a way that involves EPOR, although the mechanisms remain to be worked out. PMID:24847265

  4. Germinal center reaction: antigen affinity and presentation explain it all.

    PubMed

    Oropallo, Michael A; Cerutti, Andrea

    2014-07-01

    The selection and expansion of B cells undergoing affinity maturation in the germinal center is a hallmark of humoral immunity. A recent paper in Nature provides new insights into the relationships between the affinity of the immunoglobulin receptor for antigen, the ability of B cells to present antigen to T cells, and the processes of selection, mutation, and clonal expansion in the germinal center.

  5. Striving for Empathy: Affinities, Alliances and Peer Sexuality Educators

    ERIC Educational Resources Information Center

    Fields, Jessica; Copp, Martha

    2015-01-01

    Peer sexuality educators' accounts of their work reveal two approaches to empathy with their students: affinity and alliance. "Affinity-based empathy" rests on the idea that the more commonalities sexuality educators and students share (or perceive they share), the more they will be able to empathise with one another, while…

  6. Conformational kinetics reveals affinities of protein conformational states.

    PubMed

    Daniels, Kyle G; Suo, Yang; Oas, Terrence G

    2015-07-28

    Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein's affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.

  7. Affine group formulation of the Standard Model coupled to gravity

    SciTech Connect

    Chou, Ching-Yi; Ita, Eyo; Soo, Chopin

    2014-04-15

    In this work we apply the affine group formalism for four dimensional gravity of Lorentzian signature, which is based on Klauder’s affine algebraic program, to the formulation of the Hamiltonian constraint of the interaction of matter and all forces, including gravity with non-vanishing cosmological constant Λ, as an affine Lie algebra. We use the hermitian action of fermions coupled to gravitation and Yang–Mills theory to find the density weight one fermionic super-Hamiltonian constraint. This term, combined with the Yang–Mills and Higgs energy densities, are composed with York’s integrated time functional. The result, when combined with the imaginary part of the Chern–Simons functional Q, forms the affine commutation relation with the volume element V(x). Affine algebraic quantization of gravitation and matter on equal footing implies a fundamental uncertainty relation which is predicated upon a non-vanishing cosmological constant. -- Highlights: •Wheeler–DeWitt equation (WDW) quantized as affine algebra, realizing Klauder’s program. •WDW formulated for interaction of matter and all forces, including gravity, as affine algebra. •WDW features Hermitian generators in spite of fermionic content: Standard Model addressed. •Constructed a family of physical states for the full, coupled theory via affine coherent states. •Fundamental uncertainty relation, predicated on non-vanishing cosmological constant.

  8. Tending to Change: Toward a Situated Model of Affinity Spaces

    ERIC Educational Resources Information Center

    Bommarito, Dan

    2014-01-01

    The concept of affinity spaces, a theoretical construct used to analyze literate activity from a spatial perspective, has gained popularity among scholars of literacy studies and, particularly, video-game studies. This article seeks to expand current notions of affinity spaces by identifying key assumptions that have limited researchers'…

  9. Affinity based information diffusion model in social networks

    NASA Astrophysics Data System (ADS)

    Liu, Hongli; Xie, Yun; Hu, Haibo; Chen, Zhigao

    2014-12-01

    There is a widespread intuitive sense that people prefer participating in spreading the information in which they are interested. The affinity of people with information disseminated can affect the information propagation in social networks. In this paper, we propose an information diffusion model incorporating the mechanism of affinity of people with information which considers the fitness of affinity values of people with affinity threshold of the information. We find that the final size of information diffusion is affected by affinity threshold of the information, average degree of the network and the probability of people's losing their interest in the information. We also explore the effects of other factors on information spreading by numerical simulations and find that the probabilities of people's questioning and confirming the information can affect the propagation speed, but not the final scope.

  10. Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

    PubMed Central

    Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

    2016-01-01

    Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

  11. Affinity Monolith-Integrated Microchips for Protein Purification and Concentration.

    PubMed

    Gao, Changlu; Sun, Xiuhua; Wang, Huaixin; Qiao, Wei; Hu, Bo

    2016-01-01

    Affinity chromatography is a valuable method to purify and concentrate minute amount of proteins. Monoliths with epoxy groups for affinity immobilization were prepared by direct in-situ photopolymerization of glycidyl methacrylate and ethylene glycol dimethacrylate in porogenic solvents consisting of 1-dodecanol and cyclohexanol. By integrating affinity monoliths onto a microfluidic system, targeted biomolecules can be captured and retained on affinity column, while other biomolecules having no specific interactions toward the immobilized ligands flow through the microchannel. Therefore, proteins which remain on the affinity column are purified and concentrated, and then eluted by appropriate solutions and finally, separated by microchip capillary electrophoresis. This integrated microfluidic device has been applied to the purification and separation of specific proteins (FITC-labeled human serum albumin and IgG) in a mixture.

  12. Optimal T-cell receptor affinity for inducing autoimmunity

    PubMed Central

    Koehli, Sabrina; Naeher, Dieter; Galati-Fournier, Virginie; Zehn, Dietmar; Palmer, Ed

    2014-01-01

    T-cell receptor affinity for self-antigen has an important role in establishing self-tolerance. Three transgenic mouse strains expressing antigens of variable affinity for the OVA transgenic-I T-cell receptor were generated to address how TCR affinity affects the efficiency of negative selection, the ability to prime an autoimmune response, and the elimination of the relevant target cell. Mice expressing antigens with an affinity just above the negative selection threshold exhibited the highest risk of developing experimental autoimmune diabetes. The data demonstrate that close to the affinity threshold for negative selection, sufficient numbers of self-reactive T cells escape deletion and create an increased risk for the development of autoimmunity. PMID:25411315

  13. Detection of protein-protein interactions using tandem affinity purification.

    PubMed

    Goodfellow, Ian; Bailey, Dalan

    2014-01-01

    Tandem affinity purification (TAP) is an invaluable technique for identifying interaction partners for an affinity tagged bait protein. The approach relies on the fusion of dual tags to the bait before separate rounds of affinity purification and precipitation. Frequently two specific elution steps are also performed to increase the specificity of the overall technique. In the method detailed here, the two tags used are protein G and a short streptavidin binding peptide; however, many variations can be employed. In our example the tags are separated by a cleavable tobacco etch virus protease target sequence, allowing for specific elution after the first round of affinity purification. Proteins isolated after the final elution step in this process are concentrated before being identified by mass spectrometry. The use of dual affinity tags and specific elution in this technique dramatically increases both the specificity and stringency of the pull-downs, ensuring a low level of background nonspecific interactions.

  14. Affinity Regulates Spatial Range of EGF Receptor Autocrine Ligand Binding

    SciTech Connect

    Dewitt, Ann; Iida, Tomoko; Lam, Ho-Yan; Hill, Virginia; Wiley, H S.; Lauffenburger, Douglas A.

    2002-08-08

    Proper spatial localization of EGFR signaling activated by autocrine ligands represents a critical factor in embryonic development as well as tissue organization and function, and ligand/receptor binding affinity is among the molecular and cellular properties suggested to play a role in governing this localization. The authors employ a computational model to predict how receptor-binding affinity affects local capture of autocrine ligand vis-a-vis escape to distal regions, and provide experimental test by constructing cell lines expressing EGFR along with either wild-type EGF or a low-affinity mutant, EGF{sup L47M}. The model predicts local capture of a lower affinity autocrine ligand to be less efficient when the ligand production rate is small relative to receptor appearance rate. The experimental data confirm this prediction, demonstrating that cells can use ligand/receptor binding affinity to regulate ligand spatial distribution when autocrine ligand production is limiting for receptor signaling.

  15. Persistence of iron limitation in the western subarctic Pacific SEEDS II mesoscale fertilization experiment

    NASA Astrophysics Data System (ADS)

    Wells, Mark L.; Trick, Charles G.; Cochlan, William P.; Beall, Ben

    2009-12-01

    The cumulative evidence from more than a dozen mesoscale iron-enrichment studies in high nitrate low chlorophyll (HNLC) waters demonstrates that iron limitation is widespread and very likely affects atmospheric carbon dioxide and thus global climate. However, the responses of microphytoplankton (>20 μm), predominantly diatoms, vary greatly among these mesoscale experiments even though similar amounts of iron were added, making it difficult to quantitatively incorporate iron effects into global climate models. Nowhere is this difference more dramatic than between the massive bloom observed during Subarctic Pacific Iron Experiment for Ecosystem Dynamics Study (SEEDS) I and the order of magnitude smaller ecosystem response in SEEDS II; two mesocale experiments performed in the same HNLC region of the western subarctic Pacific in different years. Deckboard incubation experiments initiated during the early, middle, and late stages of the 32-day SEEDS II experiment show that while the two iron infusions increased phytoplankton growth, diatoms remained significantly limited by iron availability, despite total dissolved Fe concentrations in the patch being well above the diffusion-limited threshold for rapid diatom growth. This iron limitation was apparent <6 days after the initial iron infusion and was not alleviated by the second, smaller iron infusion. In contrast, smaller phytoplankton (<20 μm) showed a more restricted response to further iron amendments, indicating that their iron nutrition was near optimal. Iron complexed to desferrioximine B, a commonly available siderophore produced by at least one marine bacterium, was poorly available to diatoms throughout the patch evolution, indicating that these diatoms lacked the ability to induce high-affinity iron uptake systems. These results suggest that the strong organic complexation of Fe(III) observed in the SEEDS II-fertilized patch was not compatible with rapid diatom growth. In contrast, iron associated with

  16. Chasing polys: Interdisciplinary affinity and its connection to physics identity

    NASA Astrophysics Data System (ADS)

    Scott, Tyler D.

    This research is based on two motivations that merge by means of the frameworks of interdisciplinary affinity and physics identity. First, a goal of education is to develop interdisciplinary abilities in students' thinking and work. But an often ignored factor is students interests and beliefs about being interdisciplinary. Thus, this work develops and uses a framework called interdisciplinary affinity. It encompasses students interests in making connections across disciplines and their beliefs about their abilities to make those connections. The second motivation of this research is to better understand how to engage more students with physics. Physics identity describes how a student sees themselves in relation to physics. By understanding how physics identity is developed, researchers and educators can identify factors that increase interest and engagement in physics classrooms. Therefore, physics identity was used in conjunction with interdisciplinary affinity. Using a mixed methods approach, this research used quantitative data to identify the relationships interdisciplinary affinity has with physics identity and the physics classroom. These connections were explored in more detail using a case study of three students in a high school physics class. Results showed significant and positive relationships between interdisciplinary affinity and physics identity, including the individual interest and recognition components of identity. It also identified characteristics of physics classrooms that had a significant, positive relationship with interdisciplinary affinity. The qualitative case study highlighted the importance of student interest to the relationship between interdisciplinary affinity and physics identity. It also identified interest and mastery orientation as key to understanding the link between interdisciplinary affinity and the physics classroom. These results are a positive sign that by understanding interdisciplinary affinity and physics identity

  17. Adsorption of natural organic matter from waters by iron coated pumice.

    PubMed

    Kitis, M; Kaplan, S S; Karakaya, E; Yigit, N O; Civelekoglu, G

    2007-01-01

    Natural pumice particles were used as granular support media and coated with iron oxides to investigate their adsorptive natural organic matter (NOM) removal from waters. The impacts of natural pumice source, particle size fraction, pumice dose, pumice surface chemistry and specific surface area, and NOM source on the ultimate extent and rate of NOM removal were studied. All adsorption isotherm experiments were conducted employing the variable-dose completely mixed batch reactor bottle-point method. Iron oxide coating overwhelmed the surface electrical properties of the underlying pumice particles. Surface areas as high as 20.6m(2)g(-1) were achieved after iron coating of pumice samples, which are above than those of iron coated sand samples reported in the literature. For all particle size fractions, iron coating of natural pumices significantly increased their NOM uptakes both on an adsorbent mass- and surface area-basis. The smallest size fractions (<63 microm) of coated pumices generally exhibited the highest NOM uptakes. A strong linear correlation between the iron contents of coated pumices and their Freundlich affinity parameters (K(F)) indicated that the enhanced NOM uptake is due to iron oxides bound on pumice surfaces. Iron oxide coated pumice surfaces preferentially removed high UV-absorbing fractions of NOM, with UV absorbance reductions up to 90%. Control experiments indicated that iron oxide species bound on pumice surfaces are stable, and potential iron release to the solution is not a concern at pH values of typical natural waters. Based on high NOM adsorption capacities, iron oxide coated pumice may be a promising novel adsorbent in removing NOM from waters. Furthermore, due to preferential removal of high UV-absorbing NOM fractions, iron oxide coated pumice may also be effective in controlling the formation of disinfection by-products in drinking water treatment.

  18. Affinity capillary electrophoresis: the theory of electromigration.

    PubMed

    Dubský, Pavel; Dvořák, Martin; Ansorge, Martin

    2016-12-01

    We focus on the state-of-the-art theory of electromigration under single and multiple complexation equilibrium. Only 1:1 complexation stoichiometry is discussed because of its unique status in the field of affinity capillary electrophoresis (ACE). First, we summarize the formulas for the effective mobility in various ACE systems as they appeared since the pioneering days in 1992 up to the most recent theories till 2015. Disturbing phenomena that do not alter the mobility of the analyte directly but cause an unexpected peak broadening have been studied only recently and are also discussed in this paper. Second, we turn our attention to the viscosity effects in ACE. Change in the background electrolyte viscosity is unavoidable in ACE but numerous observations scattered throughout the literature have not been reviewed previously. This leads to an uncritical employment of correction factors that may or may not be appropriate in practice. Finally, we consider the ionic strength effects in ACE, too. Limitations of the current theories are also discussed and the tasks identified where open problems still prevail. Graphical Abstract A weak base (A) undergoes an acidic-basic equilibria (in blue) and migrates with an electrophoretic mobility of [Formula: see text]. Simultaneously, it interacts with a selector (sel) while the analyte-selector complex migrates with an electrophoretic mobility of [Formula: see text]. The strength of the interaction (in orange) is governed by the binding constant, K A , and the concentration of the selector, c sel . This all gives the analyte an effective mobility of [Formula: see text] and moves it out of the zero position (EOF; right top insert). The interaction of the positively charged analyte with the neutral selector slows down the analyte with increasing selector concentration (right bottom insert).

  19. Affinity purification of copper-chelating peptides from sunflower protein hydrolysates.

    PubMed

    Megías, Cristina; Pedroche, Justo; Yust, Maria M; Girón-Calle, Julio; Alaiz, Manuel; Millan, Francisco; Vioque, Javier

    2007-08-08

    Copper-chelating peptides were purified from sunflower protein hydrolysates by affinity chromatography using immobilized copper. A variety of protein hydrolysates were obtained by incubation with the proteases Alcalase and Flavourzyme for different periods of time. Chelating activity was indirectly determined by measuring the inhibitory effect of hydrolysates on the oxidation of beta-carotene by copper. Copper-binding peptides purified from the two hydrolysates that inhibited oxidation by copper the most contained 25.4 and 42.0% histidine and inhibited beta-carotene oxidation 8 and 3 times more than the original hydrolysates, which had 2.4 and 2.6% histidine, respectively. Thus, histidine content is not the only factor involved in antioxidant activity, and probably other factors such as peptide size and amino acid sequence are also important. This work shows that affinity chromatography can be used for the purification of copper-chelating peptides and probably other metals of nutritional interest such as calcium, iron, and zinc. In addition to their antioxidant potential, chelating peptides are of nutritional interest because they increase bioavailability of minerals.

  20. 46 CFR 148.275 - Iron oxide, spent; iron sponge, spent.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 5 2013-10-01 2013-10-01 false Iron oxide, spent; iron sponge, spent. 148.275 Section... § 148.275 Iron oxide, spent; iron sponge, spent. (a) Before spent iron oxide or spent iron sponge is... been cooled and weathered for at least eight weeks. (b) Both spent iron oxide and spent iron sponge...

  1. 46 CFR 148.275 - Iron oxide, spent; iron sponge, spent.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 5 2014-10-01 2014-10-01 false Iron oxide, spent; iron sponge, spent. 148.275 Section... § 148.275 Iron oxide, spent; iron sponge, spent. (a) Before spent iron oxide or spent iron sponge is... been cooled and weathered for at least eight weeks. (b) Both spent iron oxide and spent iron sponge...

  2. 46 CFR 148.275 - Iron oxide, spent; iron sponge, spent.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 5 2011-10-01 2011-10-01 false Iron oxide, spent; iron sponge, spent. 148.275 Section... § 148.275 Iron oxide, spent; iron sponge, spent. (a) Before spent iron oxide or spent iron sponge is... been cooled and weathered for at least eight weeks. (b) Both spent iron oxide and spent iron sponge...

  3. 46 CFR 148.275 - Iron oxide, spent; iron sponge, spent.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 5 2012-10-01 2012-10-01 false Iron oxide, spent; iron sponge, spent. 148.275 Section... § 148.275 Iron oxide, spent; iron sponge, spent. (a) Before spent iron oxide or spent iron sponge is... been cooled and weathered for at least eight weeks. (b) Both spent iron oxide and spent iron sponge...

  4. Iron Absorption in Drosophila melanogaster

    PubMed Central

    Mandilaras, Konstantinos; Pathmanathan, Tharse; Missirlis, Fanis

    2013-01-01

    The way in which Drosophila melanogaster acquires iron from the diet remains poorly understood despite iron absorption being of vital significance for larval growth. To describe the process of organismal iron absorption, consideration needs to be given to cellular iron import, storage, export and how intestinal epithelial cells sense and respond to iron availability. Here we review studies on the Divalent Metal Transporter-1 homolog Malvolio (iron import), the recent discovery that Multicopper Oxidase-1 has ferroxidase activity (iron export) and the role of ferritin in the process of iron acquisition (iron storage). We also describe what is known about iron regulation in insect cells. We then draw upon knowledge from mammalian iron homeostasis to identify candidate genes in flies. Questions arise from the lack of conservation in Drosophila for key mammalian players, such as ferroportin, hepcidin and all the components of the hemochromatosis-related pathway. Drosophila and other insects also lack erythropoiesis. Thus, systemic iron regulation is likely to be conveyed by different signaling pathways and tissue requirements. The significance of regulating intestinal iron uptake is inferred from reports linking Drosophila developmental, immune, heat-shock and behavioral responses to iron sequestration. PMID:23686013

  5. Iron absorption in Drosophila melanogaster.

    PubMed

    Mandilaras, Konstantinos; Pathmanathan, Tharse; Missirlis, Fanis

    2013-05-17

    The way in which Drosophila melanogaster acquires iron from the diet remains poorly understood despite iron absorption being of vital significance for larval growth. To describe the process of organismal iron absorption, consideration needs to be given to cellular iron import, storage, export and how intestinal epithelial cells sense and respond to iron availability. Here we review studies on the Divalent Metal Transporter-1 homolog Malvolio (iron import), the recent discovery that Multicopper Oxidase-1 has ferroxidase activity (iron export) and the role of ferritin in the process of iron acquisition (iron storage). We also describe what is known about iron regulation in insect cells. We then draw upon knowledge from mammalian iron homeostasis to identify candidate genes in flies. Questions arise from the lack of conservation in Drosophila for key mammalian players, such as ferroportin, hepcidin and all the components of the hemochromatosis-related pathway. Drosophila and other insects also lack erythropoiesis. Thus, systemic iron regulation is likely to be conveyed by different signaling pathways and tissue requirements. The significance of regulating intestinal iron uptake is inferred from reports linking Drosophila developmental, immune, heat-shock and behavioral responses to iron sequestration.

  6. A theoretical study on the electronic structures and equilibrium constants evaluation of Deferasirox iron complexes.

    PubMed

    Salehi, Samie; Saljooghi, Amir Shokooh; Izadyar, Mohammad

    2016-10-01

    Elemental iron is essential for cellular growth and homeostasis but it is potentially toxic to the cells and tissues. Excess iron can contribute in tumor initiation and tumor growth. Obviously, in iron overload issues using an iron chelator in order to reduce iron concentration seems to be vital. This study presents the density functional theory calculations of the electronic structure and equilibrium constant for iron-deferasirox (Fe-DFX) complexes in the gas phase, water and DMSO. A comprehensive study was performed to investigate the Deferasirox-iron complexes in chelation therapy. Calculation was performed in CAMB3LYP/6-31G(d,p) to get the optimized structures for iron complexes in high and low spin states. Natural bond orbital and quantum theory of atoms in molecules analyses was carried out with B3LYP/6-311G(d,p) to understand the nature of complex bond character and electronic transition in complexes. Electrostatic potential effects on the complexes were evaluated using the CHelpG calculations. The results indicated that higher affinity for Fe(III) is not strictly a function of bond length but also the degree of Fe-X (X=O,N) covalent bonding. Based on the quantum reactivity parameters which have been investigated here, it is possible reasonable design of the new chelators to improve the chelator abilities.

  7. Siderophore uptake in bacteria and the battle for iron with the host; a bird's eye view.

    PubMed

    Chu, Byron C; Garcia-Herrero, Alicia; Johanson, Ted H; Krewulak, Karla D; Lau, Cheryl K; Peacock, R Sean; Slavinskaya, Zoya; Vogel, Hans J

    2010-08-01

    Siderophores are biosynthetically produced and secreted by many bacteria, yeasts, fungi and plants, to scavenge for ferric iron (Fe(3+)). They are selective iron-chelators that have an extremely high affinity for binding this trivalent metal ion. The ferric ion is poorly soluble but it is the form of iron that is predominantly found in oxygenated environments. Siderophore uptake in bacteria has been extensively studied and over the last decade, detailed structural information for many of the proteins that are involved in their transport has become available. Specifically, numerous crystal structures for outer membrane siderophore transporters, as well as for soluble periplasmic siderophore-binding proteins, have been reported. Moreover, unique siderophore-binding proteins have recently been serendipitously discovered in humans, and the structures of some of their siderophore-complexes have been characterized. The binding pockets for different ferric-siderophores in these proteins have been described in great molecular detail. In addition to highlighting this structural information, in this review paper we will also briefly discuss the relevant chemical properties of iron, and provide a perspective on our current understanding of the human and bacterial iron uptake pathways. Potential clinical uses of siderophores will also be discussed. The emerging overall picture is that iron metabolism plays an extremely important role during bacterial infections. Because levels of free ferric iron in biological systems are always extremely low, there is serious competition for iron and for ferric-siderophores between pathogenic bacteria and the human or animal host.

  8. The hazards of iron loading.

    PubMed

    Weinberg, Eugene D

    2010-11-01

    Excessive or misplaced tissue iron now is recognized to pose a substantial health risk for an extensive array of endocrinological, gastrointestinal, infectious, neoplasmic, neurodegenerative, obstetric, ophthalmic, orthopedic, pulmonary and vascular diseases. Ingested, injected, inhaled and decompartmentalized iron contributes not only to disease, but also to aging and mortality. Iron is dangerous by catalyzing free radical formation and by serving as an essential nutrient for microbial and neoplasmic cell invaders. Our body cells exhibit wide variation in sensitivity to iron toxicity. Efficacy of our iron withholding defense system is modulated by numerous environmental, behavioral and genetic factors. A notable variety of methods for prevention and therapy of iron toxicity are now becoming available.

  9. Analysis of biomolecular interactions using affinity microcolumns: A review

    PubMed Central

    Zheng, Xiwei; Li, Zhao; Beeram, Sandya; Podariu, Maria; Matsuda, Ryan; Pfaunmiller, Erika L.; White, Christopher J.; Carter, NaTasha; Hage, David S.

    2014-01-01

    Affinity chromatography has become an important tool for characterizing biomolecular interactions. The use of affinity microcolumns, which contain immobilized binding agents and have volumes in the mid-to-low microliter range, has received particular attention in recent years. Potential advantages of affinity microcolumns include the many analysis and detection formats that can be used with these columns, as well as the need for only small amounts of supports and immobilized binding agents. This review examines how affinity microcolumns have been used to examine biomolecular interactions. Both capillary-based microcolumns and short microcolumns are considered. The use of affinity microcolumns with zonal elution and frontal analysis methods are discussed. The techniques of peak decay analysis, ultrafast affinity extraction, split-peak analysis, and band-broadening studies are also explored. The principles of these methods are examined and various applications are provided to illustrate the use of these methods with affinity microcolumns. It is shown how these techniques can be utilized to provide information on the binding strength and kinetics of an interaction, as well as on the number and types of binding sites. It is further demonstrated how information on competition or displacement effects can be obtained by these methods. PMID:24572459

  10. Hepcidin response to acute iron intake and chronic iron loading in dysmetabolic iron overload syndrome

    PubMed Central

    Trombini, Paola; Paolini, Valentina; Pelucchi, Sara; Mariani, Raffaella; Nemeth, Elizabeta; Ganz, Tomas; Piperno, Alberto

    2014-01-01

    Background The pathogenesis of dysmetabolic iron overload syndrome (DIOS) is still unclear. Hepcidin is the key regulator of iron homeostasis controlling iron absorption and macrophage release. Aim To investigate hepcidin regulation by iron in DIOS. Methods We analysed urinary hepcidin at baseline and 24 h after a 65 mg oral iron dose in 24 patients at diagnosis and after iron depletion (n=13) and compared data with those previously observed in 23 healthy controls. Serum iron indices, liver histology and metabolic data were available for all patients. Results At diagnosis, hepcidin values were significantly higher than in controls (P<0.001). After iron depletion, hepcidin levels decreased to normal values in all patients. At baseline, a significant response of hepcidin to iron challenge was observed only in the subgroup with lower basal hepcidin concentration (P=0.007). In iron-depleted patients, urinary hepcidin significantly increased after oral iron test (P=0.006). Conclusions Ours findings suggest that in DIOS, the progression of iron accumulation is counteracted by the increase in hepcidin production and progressive reduction of iron absorption, explaining why these patients develop a mild–moderate iron overload that tends to a plateau. PMID:21733088

  11. Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria.

    PubMed Central

    Crosa, J H

    1997-01-01

    Iron is an essential element for nearly all living cells. Thus, the ability of bacteria to utilize iron is a crucial survival mechanism independent of the ecological niche in which the microorganism lives, because iron is scarce both in potential biological hosts, where it is bound by high-affinity iron-binding proteins, and in the environment, where it is present as part of insoluble complex hydroxides. Therefore, pathogens attempting to establish an infection and environmental microorganisms must all be able to utilize the otherwise unavailable iron. One of the strategies to perform this task is the possession of siderophore-mediated iron uptake systems that are capable of scavenging the hoarded iron. This metal is, however, a double-edged sword for the cell because it can catalyze the production of deadly free hydroxyl radicals, which are harmful to the cells. It is therefore imperative for the cell to control the concentration of iron at levels that permit key metabolic steps to occur without becoming a messenger of cell death. Early work identified a repressor, Fur, which as a complex with iron repressed the expression of most iron uptake systems as well as other iron-regulated genes when the iron concentration reached a certain level. However, later work demonstrated that this regulation by Fur was not the only answer under low-iron conditions, there was a need for activation of iron uptake genes as well as siderophore biosynthetic genes. Furthermore, it was also realized that in some instances the actual ferric iron-siderophore complex induced the transcription of the cognate receptor and transport genes. It became evident that control of the expression of iron-regulated genes was more complex than originally envisioned. In this review, I analyze the processes of signal transduction, transcriptional control, and posttranscriptional control of iron-regulated genes as reported for the ferric dicitrate system in Escherichia coli; the pyochelin, pyoverdin, and

  12. Fur-regulated iron uptake system of Edwardsiella ictaluri and its influence on pathogenesis and immunogenicity in the catfish host.

    PubMed

    Santander, Javier; Golden, Greg; Wanda, Soo-Young; Curtiss, Roy

    2012-08-01

    The ability of bacterial pathogens to take up iron from the host during infection is necessary for their multiplication within the host. However, host high-affinity iron binding proteins limit levels of free iron in fluids and tissues. To overcome this deficiency of iron during infection, bacterial pathogens have developed iron uptake systems that are upregulated in the absence of iron, typically tightly controlled by the ferric uptake regulator (Fur) protein. The iron uptake system of Edwardsiella ictaluri, a host-restricted pathogen of channel catfish (Ictalurus punctatus) and the main pathogen of this fish in aquaculture, is unknown. Here we describe the E. ictaluri Fur protein, the iron uptake machinery controlled by Fur, and the effects of fur gene deletion on virulence and immunogenicity in the fish host. Analysis of the E. ictaluri Fur protein shows that it lacks the N-terminal region found in the majority of pathogen-encoded Fur proteins. However, it is fully functional in regulated genes encoding iron uptake proteins. E. ictaluri grown under iron-limited conditions upregulates an outer membrane protein (HemR) that shows heme-hemoglobin transport activity and is tightly regulated by Fur. In vivo studies showed that an E. ictaluri Δfur mutant is attenuated and immune protective in zebrafish (Danio rerio) and catfish (Ictalurus punctatus), triggering systemic immunity. We conclude that an E. ictaluri Δfur mutant could be an effective component of an immersion-oral vaccine for the catfish industry.

  13. Fur-Regulated Iron Uptake System of Edwardsiella ictaluri and Its Influence on Pathogenesis and Immunogenicity in the Catfish Host

    PubMed Central

    Golden, Greg; Wanda, Soo-Young; Curtiss, Roy

    2012-01-01

    The ability of bacterial pathogens to take up iron from the host during infection is necessary for their multiplication within the host. However, host high-affinity iron binding proteins limit levels of free iron in fluids and tissues. To overcome this deficiency of iron during infection, bacterial pathogens have developed iron uptake systems that are upregulated in the absence of iron, typically tightly controlled by the ferric uptake regulator (Fur) protein. The iron uptake system of Edwardsiella ictaluri, a host-restricted pathogen of channel catfish (Ictalurus punctatus) and the main pathogen of this fish in aquaculture, is unknown. Here we describe the E. ictaluri Fur protein, the iron uptake machinery controlled by Fur, and the effects of fur gene deletion on virulence and immunogenicity in the fish host. Analysis of the E. ictaluri Fur protein shows that it lacks the N-terminal region found in the majority of pathogen-encoded Fur proteins. However, it is fully functional in regulated genes encoding iron uptake proteins. E. ictaluri grown under iron-limited conditions upregulates an outer membrane protein (HemR) that shows heme-hemoglobin transport activity and is tightly regulated by Fur. In vivo studies showed that an E. ictaluri Δfur mutant is attenuated and immune protective in zebrafish (Danio rerio) and catfish (Ictalurus punctatus), triggering systemic immunity. We conclude that an E. ictaluri Δfur mutant could be an effective component of an immersion-oral vaccine for the catfish industry. PMID:22615248

  14. A randomized trial of iron isomaltoside versus iron sucrose in patients with iron deficiency anemia

    PubMed Central

    Roman, Eloy; Modiano, Manuel R.; Achebe, Maureen M.; Thomsen, Lars L.; Auerbach, Michael

    2017-01-01

    Abstract Iron deficiency anemia (IDA) is common in many chronic diseases, and intravenous (IV) iron offers a rapid and efficient iron correction. This trial compared the efficacy and safety of iron isomaltoside and iron sucrose in patients with IDA who were intolerant of, or unresponsive to, oral iron. The trial was an open‐label, comparative, multi‐center trial. Five hundred and eleven patients with IDA from different causes were randomized 2:1 to iron isomaltoside or iron sucrose and followed for 5 weeks. The cumulative dose of iron isomaltoside was based on body weight and hemoglobin (Hb), administered as either a 1000 mg infusion over more than 15 minutes or 500 mg injection over 2 minutes. The cumulative dose of iron sucrose was calculated according to Ganzoni and administered as repeated 200 mg infusions over 30 minutes. The mean cumulative dose of iron isomaltoside was 1640.2 (standard deviation (SD): 357.6) mg and of iron sucrose 1127.9 (SD: 343.3) mg. The primary endpoint was the proportion of patients with a Hb increase ≥2 g/dL from baseline at any time between weeks 1‐5. Both non‐inferiority and superiority were confirmed for the primary endpoint, and a shorter time to Hb increase ≥2 g/dL was observed with iron isomaltoside. For all biochemical efficacy parameters, faster and/or greater improvements were found with iron isomaltoside. Both treatments were well tolerated; 0.6% experienced a serious adverse drug reaction. Iron isomaltoside was more effective than iron sucrose in achieving a rapid improvement in Hb. Furthermore, iron isomaltoside has an advantage over iron sucrose in allowing higher cumulative dosing in fewer administrations. Both treatments were well tolerated in a broad population with IDA. PMID:28052413

  15. A randomized trial of iron isomaltoside versus iron sucrose in patients with iron deficiency anemia.

    PubMed

    Derman, Richard; Roman, Eloy; Modiano, Manuel R; Achebe, Maureen M; Thomsen, Lars L; Auerbach, Michael

    2017-03-01

    Iron deficiency anemia (IDA) is common in many chronic diseases, and intravenous (IV) iron offers a rapid and efficient iron correction. This trial compared the efficacy and safety of iron isomaltoside and iron sucrose in patients with IDA who were intolerant of, or unresponsive to, oral iron. The trial was an open-label, comparative, multi-center trial. Five hundred and eleven patients with IDA from different causes were randomized 2:1 to iron isomaltoside or iron sucrose and followed for 5 weeks. The cumulative dose of iron isomaltoside was based on body weight and hemoglobin (Hb), administered as either a 1000 mg infusion over more than 15 minutes or 500 mg injection over 2 minutes. The cumulative dose of iron sucrose was calculated according to Ganzoni and administered as repeated 200 mg infusions over 30 minutes. The mean cumulative dose of iron isomaltoside was 1640.2 (standard deviation (SD): 357.6) mg and of iron sucrose 1127.9 (SD: 343.3) mg. The primary endpoint was the proportion of patients with a Hb increase ≥2 g/dL from baseline at any time between weeks 1-5. Both non-inferiority and superiority were confirmed for the primary endpoint, and a shorter time to Hb increase ≥2 g/dL was observed with iron isomaltoside. For all biochemical efficacy parameters, faster and/or greater improvements were found with iron isomaltoside. Both treatments were well tolerated; 0.6% experienced a serious adverse drug reaction. Iron isomaltoside was more effective than iron sucrose in achieving a rapid improvement in Hb. Furthermore, iron isomaltoside has an advantage over iron sucrose in allowing higher cumulative dosing in fewer administrations. Both treatments were well tolerated in a broad population with IDA.

  16. Affinity+: Semi-Structured Brainstorming on Large Displays

    SciTech Connect

    Burtner, Edwin R.; May, Richard A.; Scarberry, Randall E.; LaMothe, Ryan R.; Endert, Alexander

    2013-04-27

    Affinity diagraming is a powerful method for encouraging and capturing lateral thinking in a group environment. The Affinity+ Concept was designed to improve the collaborative brainstorm process through the use of large display surfaces in conjunction with mobile devices like smart phones and tablets. The system works by capturing the ideas digitally and allowing users to sort and group them on a large touch screen manually. Additionally, Affinity+ incorporates theme detection, topic clustering, and other processing algorithms that help bring structured analytic techniques to the process without requiring explicit leadership roles and other overhead typically involved in these activities.

  17. Binding Affinities Controlled by Shifting Conformational Equilibria: Opportunities and Limitations

    PubMed Central

    Michielssens, Servaas; de Groot, Bert L.; Grubmüller, Helmut

    2015-01-01

    Conformational selection is an established mechanism in molecular recognition. Despite its power to explain binding events, it is hardly used in protein/ligand design to modulate molecular recognition. Here, we explore the opportunities and limitations of design by conformational selection. Using appropriate thermodynamic cycles, our approach predicts the effects of a conformational shift on binding affinity and also allows one to disentangle the effects induced by a conformational shift from other effects influencing the binding affinity. The method is assessed and applied to explain the contribution of a conformational shift on the binding affinity of six ubiquitin mutants showing different conformational shifts in six different complexes. PMID:25992736

  18. Regulation of cellular iron metabolism

    PubMed Central

    Wang, Jian; Pantopoulos, Kostas

    2011-01-01

    Iron is an essential but potentially hazardous biometal. Mammalian cells require sufficient amounts of iron to satisfy metabolic needs or to accomplish specialized functions. Iron is delivered to tissues by circulating transferrin, a transporter that captures iron released into the plasma mainly from intestinal enterocytes or reticuloendothelial macrophages. The binding of iron-laden transferrin to the cell-surface transferrin receptor 1 results in endocytosis and uptake of the metal cargo. Internalized iron is transported to mitochondria for the synthesis of haem or iron–sulfur clusters, which are integral parts of several metalloproteins, and excess iron is stored and detoxified in cytosolic ferritin. Iron metabolism is controlled at different levels and by diverse mechanisms. The present review summarizes basic concepts of iron transport, use and storage and focuses on the IRE (iron-responsive element)/IRP (iron-regulatory protein) system, a well known post-transcriptional regulatory circuit that not only maintains iron homoeostasis in various cell types, but also contributes to systemic iron balance. PMID:21348856

  19. Forging the anthropogenic iron cycle.

    PubMed

    Wang, Tao; Müller, Daniel B; Graedel, T E

    2007-07-15

    Metallurgical iron cycles are characterized for four anthropogenic life stages: production, fabrication and manufacturing, use, and waste management and recycling. This analysis is conducted for year 2000 and at three spatial levels: 68 countries and territories, nine world regions, and the planet. Findings include the following: (1) contemporary iron cycles are basically open and substantially dependent on environmental sources and sinks; (2) Asia leads the world regions in iron production and use; Oceania, Latin America and the Caribbean, Africa, and the Commonwealth of Independent States present a highly production-biased iron cycle; (3) purchased scrap contributes a quarter of the global iron and steel production; (4) iron exiting use is three times less than that entering use; (5) about 45% of global iron entering use is devoted to construction, 24% is devoted to transport equipment, and 20% goes to industrial machinery; (6) with respect to international trade of iron ore, iron and steel products, and scrap, 54 out of the 68 countries are net iron importers, while only 14 are net exporters; (7) global iron discharges in tailings, slag, and landfill approximate one-third of the iron mined. Overall, these results provide a foundation for studies of iron-related resource policy, industrial development, and waste and environmental management.

  20. Iron regulatory proteins and their role in controlling iron metabolism.

    PubMed

    Kühn, Lukas C

    2015-02-01

    Cellular iron homeostasis is regulated by post-transcriptional feedback mechanisms, which control the expression of proteins involved in iron uptake, release and storage. Two cytoplasmic proteins with mRNA-binding properties, iron regulatory proteins 1 and 2 (IRP1 and IRP2) play a central role in this regulation. Foremost, IRPs regulate ferritin H and ferritin L translation and thus iron storage, as well as transferrin receptor 1 (TfR1) mRNA stability, thereby adjusting receptor expression and iron uptake via receptor-mediated endocytosis of iron-loaded transferrin. In addition splice variants of iron transporters for import and export at the plasma-membrane, divalent metal transporter 1 (DMT1) and ferroportin are regulated by IRPs. These mechanisms have probably evolved to maintain the cytoplasmic labile iron pool (LIP) at an appropriate level. In certain tissues, the regulation exerted by IRPs influences iron homeostasis and utilization of the entire organism. In intestine, the control of ferritin expression limits intestinal iron absorption and, thus, whole body iron levels. In bone marrow, erythroid heme biosynthesis is coordinated with iron availability through IRP-mediated translational control of erythroid 5-aminolevulinate synthase mRNA. Moreover, the translational control of HIF2α mRNA in kidney by IRP1 coordinates erythropoietin synthesis with iron and oxygen supply. Besides IRPs, body iron absorption is negatively regulated by hepcidin. This peptide hormone, synthesized and secreted by the liver in response to high serum iron, downregulates ferroportin at the protein level and thereby limits iron absorption from the diet. Hepcidin will not be discussed in further detail here.

  1. Identification of an Iron-Regulated, Hemin-Binding Outer Membrane Protein in Sinorhizobium meliloti

    PubMed Central

    Battistoni, Federico; Platero, Raúl; Duran, Rosario; Cerveñansky, Carlos; Battistoni, Julio; Arias, Alicia; Fabiano, Elena

    2002-01-01

    Rhizobia are soil bacteria that are able to establish symbiotic associations with leguminous hosts. In iron-limited environments these bacteria can use iron present in heme or heme compounds (hemoglobin, leghemoglobin). Here we report the presence in Sinorhizobium meliloti of an iron-regulated outer membrane protein that is able to bind hemin but not hemoglobin. Protein assignment was done by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Tryptic peptides correlated with the mass measurements obtained accounted for 54% of the translated sequence of a putative heme receptor gene present in the chromosome of S. meliloti 1021. The results which we obtained suggest that this protein (designated ShmR for Sinorhizobium heme receptor) is involved in high-affinity heme-mediated iron transport. PMID:12450806

  2. How Is Iron-Deficiency Anemia Treated?

    MedlinePlus

    ... the NHLBI on Twitter. How Is Iron-Deficiency Anemia Treated? Treatment for iron-deficiency anemia will depend ... may be advised. Treatments for Severe Iron-Deficiency Anemia Blood Transfusion If your iron-deficiency anemia is ...

  3. Earth's core iron

    NASA Astrophysics Data System (ADS)

    Geophysicist J. Michael Brown of Texas A & M University noted recently at the Spring AGU Meeting in Baltimore that the structure and phase of metallic iron at pressures of the earth's inner core (approximately 3.3 Mbar) could have great significance in defining geometrical aspects of the core itself. Brown worked at the Los Alamos Scientific Laboratory with R.B. McQueen to redetermine the phase relations of metallic iron in a series of new shock-wave experiments. They found the melting point of iron at conditions equal to those at the boundary of the earth's outer (liquid) and inner (solid) cores to be 6000°±500°C (Geophysical Research Letters, 7, 533-536, 1980).

  4. Iron-induced virulence of Salmonella enterica serovar typhimurium at the intestinal epithelial interface can be suppressed by carvacrol.

    PubMed

    Kortman, Guus A M; Roelofs, Rian W H M; Swinkels, Dorine W; de Jonge, Marien I; Burt, Sara A; Tjalsma, Harold

    2014-01-01

    Oral iron therapy can increase the abundance of bacterial pathogens, e.g., Salmonella spp., in the large intestine of African children. Carvacrol is a natural compound with antimicrobial activity against various intestinal bacterial pathogens, among which is the highly prevalent Salmonella enterica serovar Typhimurium. This study aimed to explore a presumed interaction between carvacrol and bacterial iron handling and to assess the potential of carvacrol in preventing the increase of bacterial pathogenicity during high iron availability. S. Typhimurium was cultured with increasing concentrations of iron and carvacrol to study the effects of these combined interventions on growth, adhesion to intestinal epithelial cells, and iron uptake/influx in both bacterial and epithelial cells. In addition, the ability of carvacrol to remove iron from the high-affinity ligand transferrin and an Fe-dye complex was examined. Carvacrol retarded growth of S. Typhimurium at all iron conditions. Furthermore, iron-induced epithelial adhesion was effectively reduced by carvacrol at high iron concentrations. The reduction of growth and virulence by carvacrol was not paralleled by a change in iron uptake or influx into S. Typhimurium. In contrast, bioavailability of iron for epithelial cells was moderately decreased under these conditions. Further, carvacrol was shown to lack the properties of an iron binding molecule; however, it was able to weaken iron-ligand interactions by which it may possibly interfere with bacterial virulence. In conclusion, our in vitro data suggest that carvacrol has the potential to serve as a novel dietary supplement to prevent pathogenic overgrowth and colonization in the large intestine during oral iron therapy.

  5. Iron-Induced Virulence of Salmonella enterica Serovar Typhimurium at the Intestinal Epithelial Interface Can Be Suppressed by Carvacrol

    PubMed Central

    Kortman, Guus A. M.; Roelofs, Rian W. H. M.; Swinkels, Dorine W.; de Jonge, Marien I.; Burt, Sara A.

    2014-01-01

    Oral iron therapy can increase the abundance of bacterial pathogens, e.g., Salmonella spp., in the large intestine of African children. Carvacrol is a natural compound with antimicrobial activity against various intestinal bacterial pathogens, among which is the highly prevalent Salmonella enterica serovar Typhimurium. This study aimed to explore a presumed interaction between carvacrol and bacterial iron handling and to assess the potential of carvacrol in preventing the increase of bacterial pathogenicity during high iron availability. S. Typhimurium was cultured with increasing concentrations of iron and carvacrol to study the effects of these combined interventions on growth, adhesion to intestinal epithelial cells, and iron uptake/influx in both bacterial and epithelial cells. In addition, the ability of carvacrol to remove iron from the high-affinity ligand transferrin and an Fe-dye complex was examined. Carvacrol retarded growth of S. Typhimurium at all iron conditions. Furthermore, iron-induced epithelial adhesion was effectively reduced by carvacrol at high iron concentrations. The reduction of growth and virulence by carvacrol was not paralleled by a change in iron uptake or influx into S. Typhimurium. In contrast, bioavailability of iron for epithelial cells was moderately decreased under these conditions. Further, carvacrol was shown to lack the properties of an iron binding molecule; however, it was able to weaken iron-ligand interactions by which it may possibly interfere with bacterial virulence. In conclusion, our in vitro data suggest that carvacrol has the potential to serve as a novel dietary supplement to prevent pathogenic overgrowth and colonization in the large intestine during oral iron therapy. PMID:24379194

  6. INTERIOR VIEW OF IRON TREATMENT (DESULPHURIZATION) AREA. MOLTEN IRON PROCEEDS ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    INTERIOR VIEW OF IRON TREATMENT (DESULPHURIZATION) AREA. MOLTEN IRON PROCEEDS FROM CUPOLA TO IRON TREATMENT AREAS BEFORE BEING TRANSFERRED TO PIPE CASTING MACHINES. - United States Pipe & Foundry Company Plant, Melting & Treatment Areas, 2023 St. Louis Avenue at I-20/59, Bessemer, Jefferson County, AL

  7. Hexadecylamine Adsorption at the Iron Oxide–Oil Interface

    PubMed Central

    2013-01-01

    The adsorption behavior of a model additive, hexadecylamine, onto an iron surface from hexadecane oil has been characterized using polarized neutron reflectometry, sum-frequency generation spectroscopy, solution depletion isotherm, and X-ray photoelectron spectroscopy (XPS). The amine showed a strong affinity for the metal surface, forming a dense monolayer at relatively low concentrations; a layer thickness of 16 (±3) Å at low concentrations, increasing to 20 (±3) Å at greater amine concentrations, was determined from the neutron data. These thicknesses suggest that the molecules in the layer are tilted. Adsorption was also indicated by sum-frequency generation spectroscopy and XPS, the latter indicating that the most dominant amine–surface interaction was via electron donation from the nitrogen lone pair to the positively charged iron ions. Sum-frequency generation spectroscopy was used to determine the alkyl chain conformation order and orientation on the surface. PMID:24106786

  8. Iron Meteorite on Mars

    NASA Technical Reports Server (NTRS)

    2005-01-01

    NASA's Mars Exploration Rover Opportunity has found an iron meteorite on Mars, the first meteorite of any type ever identified on another planet. The pitted, basketball-size object is mostly made of iron and nickel. Readings from spectrometers on the rover determined that composition. Opportunity used its panoramic camera to take the images used in this approximately true-color composite on the rover's 339th martian day, or sol (Jan. 6, 2005). This composite combines images taken through the panoramic camera's 600-nanometer (red), 530-nanometer (green), and 480-nanometer (blue) filters.

  9. Iron deficiency: definition and diagnosis.

    PubMed

    Cook, J D; Skikne, B S

    1989-11-01

    There has been a continuous refinement over the past several decades of methods to detect iron deficiency and assess its magnitude. The optimal combination of measurements differs for clinical and epidemiological assessment. Clinically, the major problem is to distinguish true iron deficiency from other causes of iron-deficient erythropoiesis, such as the anaemia of chronic disease. Epidemiologically, techniques that provide quantified estimates of body iron are preferable. For both purposes, the serum ferritin is the focal point of the laboratory detection of iron deficiency. Serum ferritin measurements provide a reliable index of body iron stores in healthy individuals, a cost-effective method of screening for iron deficiency, and a useful alternative to bone marrow examinations in the evaluation of anaemic patients. Preliminary studies indicate that measurement of the serum transferrin receptor may be the most reliable way to assess deficits in tissue iron supply.

  10. Hepcidin Suppresses Brain Iron Accumulation by Downregulating Iron Transport Proteins in Iron-Overloaded Rats.

    PubMed

    Du, Fang; Qian, Zhong-Ming; Luo, Qianqian; Yung, Wing-Ho; Ke, Ya

    2015-08-01

    Iron accumulates progressively in the brain with age, and iron-induced oxidative stress has been considered as one of the initial causes for Alzheimer's disease (AD) and Parkinson's disease (PD). Based on the role of hepcidin in peripheral organs and its expression in the brain, we hypothesized that this peptide has a role to reduce iron in the brain and hence has the potential to prevent or delay brain iron accumulation in iron-associated neurodegenerative disorders. Here, we investigated the effects of hepcidin expression adenovirus (ad-hepcidin) and hepcidin peptide on brain iron contents, iron transport across the brain-blood barrier, iron uptake and release, and also the expression of transferrin receptor-1 (TfR1), divalent metal transporter 1 (DMT1), and ferroportin 1 (Fpn1) in cultured microvascular endothelial cells and neurons. We demonstrated that hepcidin significantly reduced brain iron in iron-overloaded rats and suppressed transport of transferrin-bound iron (Tf-Fe) from the periphery into the brain. Also, the peptide significantly inhibited expression of TfR1, DMT1, and Fpn1 as well as reduced Tf-Fe and non-transferrin-bound iron uptake and iron release in cultured microvascular endothelial cells and neurons, while downregulation of hepcidin with hepcidin siRNA retrovirus generated opposite results. We concluded that, under iron-overload, hepcidin functions to reduce iron in the brain by downregulating iron transport proteins. Upregulation of brain hepcidin by ad-hepcidin emerges as a new pharmacological treatment and prevention for iron-associated neurodegenerative disorders.

  11. Toughness Properties of Nodular Iron

    NASA Astrophysics Data System (ADS)

    Bradley, Walter L.

    1985-01-01

    The German government recently certified ductile iron for construction of nuclear waste transport containers. This approved use of ductile iron for such a critical application represents the culmination of ten years worth of research bringing to light the surprising toughness of ductile iron. This article explains how modern fracture mechanics and microstructure/property relationships have altered the stereotype of ductile iron as a low toughness material.

  12. Bidirectional elastic image registration using B-spline affine transformation.

    PubMed

    Gu, Suicheng; Meng, Xin; Sciurba, Frank C; Ma, Hongxia; Leader, Joseph; Kaminski, Naftali; Gur, David; Pu, Jiantao

    2014-06-01

    A registration scheme termed as B-spline affine transformation (BSAT) is presented in this study to elastically align two images. We define an affine transformation instead of the traditional translation at each control point. Mathematically, BSAT is a generalized form of the affine transformation and the traditional B-spline transformation (BST). In order to improve the performance of the iterative closest point (ICP) method in registering two homologous shapes but with large deformation, a bidirectional instead of the traditional unidirectional objective/cost function is proposed. In implementation, the objective function is formulated as a sparse linear equation problem, and a sub-division strategy is used to achieve a reasonable efficiency in registration. The performance of the developed scheme was assessed using both two-dimensional (2D) synthesized dataset and three-dimensional (3D) volumetric computed tomography (CT) data. Our experiments showed that the proposed B-spline affine model could obtain reasonable registration accuracy.

  13. Bidirectional Elastic Image Registration Using B-Spline Affine Transformation

    PubMed Central

    Gu, Suicheng; Meng, Xin; Sciurba, Frank C.; Wang, Chen; Kaminski, Naftali; Pu, Jiantao

    2014-01-01

    A registration scheme termed as B-spline affine transformation (BSAT) is presented in this study to elastically align two images. We define an affine transformation instead of the traditional translation at each control point. Mathematically, BSAT is a generalized form of the affine transformation and the traditional B-Spline transformation (BST). In order to improve the performance of the iterative closest point (ICP) method in registering two homologous shapes but with large deformation, a bi-directional instead of the traditional unidirectional objective / cost function is proposed. In implementation, the objective function is formulated as a sparse linear equation problem, and a sub-division strategy is used to achieve a reasonable efficiency in registration. The performance of the developed scheme was assessed using both two-dimensional (2D) synthesized dataset and three-dimensional (3D) volumetric computed tomography (CT) data. Our experiments showed that the proposed B-spline affine model could obtain reasonable registration accuracy. PMID:24530210

  14. A thermodynamic approach to the affinity optimization of drug candidates.

    PubMed

    Freire, Ernesto

    2009-11-01

    High throughput screening and other techniques commonly used to identify lead candidates for drug development usually yield compounds with binding affinities to their intended targets in the mid-micromolar range. The affinity of these molecules needs to be improved by several orders of magnitude before they become viable drug candidates. Traditionally, this task has been accomplished by establishing structure activity relationships to guide chemical modifications and improve the binding affinity of the compounds. As the binding affinity is a function of two quantities, the binding enthalpy and the binding entropy, it is evident that a more efficient optimization would be accomplished if both quantities were considered and improved simultaneously. Here, an optimization algorithm based upon enthalpic and entropic information generated by Isothermal Titration Calorimetry is presented.

  15. Antibody Affinity Maturation in Fishes—Our Current Understanding

    PubMed Central

    Magor, Brad G.

    2015-01-01

    It has long been believed that fish lack antibody affinity maturation, in part because they were thought to lack germinal centers. Recent research done on sharks and bony fishes indicates that these early vertebrates are able to affinity mature their antibodies. This article reviews the functionality of the fish homologue of the immunoglobulin (Ig) mutator enzyme activation-induced cytidine deaminase (AID). We also consider the protein and molecular evidence for Ig somatic hypermutation and antibody affinity maturation. In the context of recent evidence for a putative proto-germinal center in fishes we propose some possible reasons that observed affinity maturation in fishes often seems lacking and propose future work that might shed further light on this process in fishes. PMID:26264036

  16. Structure of the nucleotide-binding domain of a dipeptide ABC transporter reveals a novel iron-sulfur cluster-binding domain.

    PubMed

    Li, Xiaolu; Zhuo, Wei; Yu, Jie; Ge, Jingpeng; Gu, Jinke; Feng, Yue; Yang, Maojun; Wang, Linfang; Wang, Na

    2013-02-01

    Dipeptide permease (Dpp), which belongs to an ABC transport system, imports peptides consisting of two or three L-amino acids from the matrix to the cytoplasm in microbes. Previous studies have indicated that haem competes with dipeptides to bind DppA in vitro and in vivo and that the Dpp system can also translocate haem. Here, the crystal structure of DppD, the nucleotide-binding domain (NBD) of the ABC-type dipeptide/oligopeptide/nickel-transport system from Thermoanaerobacter tengcongensis, bound with ATP, Mg(2+) and a [4Fe-4S] iron-sulfur cluster is reported. The N-terminal domain of DppD shares a similar structural fold with the NBDs of other ABC transporters. Interestingly, the C-terminal domain of DppD contains a [4Fe-4S] cluster. The UV-visible absorbance spectrum of DppD was consistent with the presence of a [4Fe-4S] cluster. A search with DALI revealed that the [4Fe-4S] cluster-binding domain is a novel structural fold. Structural analysis and comparisons with other ABC transporters revealed that this iron-sulfur cluster may act as a mediator in substrate (dipeptide or haem) binding by electron transfer and may regulate the transport process in Dpp ABC transport systems. The crystal structure provides a basis for understanding the properties of ABC transporters and will be helpful in investigating the functions of NBDs in the regulation of ABC transporter activity.

  17. 21 CFR 184.1375 - Iron, elemental.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Iron, elemental. 184.1375 Section 184.1375 Food and... Substances Affirmed as GRAS § 184.1375 Iron, elemental. (a) Iron, elemental (CAS Reg. No. 7439-89-6) is metallic iron obtained by any of the following processes: reduced iron, electrolytic iron, and...

  18. Electrospun polyethersulfone affinity membrane: membrane preparation and performance evaluation.

    PubMed

    Ma, Zuwei; Lan, Zhengwei; Matsuura, Takeshi; Ramakrishna, Seeram

    2009-11-01

    Non-woven polyethersulfone (PES) membranes were prepared by electrospinning. After heat treatment and surface activation, the membranes were covalently functionalized with ligands to be used as affinity membranes. The membranes were characterized in terms of fiber diameter, porosity, specific area, pore size, ligand density and binding capacities. To evaluate the binding efficiency of the membrane, dynamic adsorption of bovine serum albumin (BSA) on the Cibacron blue F3GA (CB) functionalized PES membrane was studied. Experimental breakthrough curves were fitted with the theoretical curves based on the plate model to estimate plate height (H(p)) of the affinity membrane. The high value of H(p) (1.6-8 cm) of the affinity membrane implied a poor dynamic binding efficiency, which can be explained by the intrinsic microstructures of the material. Although the electrospun membrane might not be an ideal candidate for the preparative affinity membrane chromatography for large-scale production, it still can be used for fast small-scale protein purification in which a highly efficient binding is not required. Spin columns packed with protein A/G immobilized PES membranes were demonstrated to be capable of binding IgG specifically. SDS-PAGE results demonstrated that the PES affinity membrane had high specific binding selectivity for IgG molecules and low non-specific protein adsorption. Compared with other reported affinity membranes, the PES affinity membrane had a comparable IgG binding capacity of 4.5 mg/ml, and had a lower flow through pressure drop due to its larger pore size. In conclusion, the novel PES affinity membrane is an ideal spin column packing material for fast protein purification.

  19. Considering affinity: an ethereal conversation (part two of three).

    PubMed

    Winsor, Mary P

    2015-06-01

    In 1840 Hugh Strickland published a diagram showing the relationships of genera of birds in the kingfisher family. Three years later he applied this mapping idea to genera of birds of prey and songbirds, creating a large wall chart that he displayed to colleagues but never published. Both of his diagrams featured a scale of degrees of affinity. The meaning of taxonomic affinity was something Darwin thought about deeply. Details in the chart undermine Strickland's claim that his method was purely inductive.

  20. Proton affinity of methyl nitrate - Less than proton affinity of nitric acid

    NASA Technical Reports Server (NTRS)

    Lee, Timothy J.; Rice, Julia E.

    1992-01-01

    Several state-of-the-art ab initio quantum mechanical methods were used to investigate the equilibrium structure, dipole moments, harmonic vibrational frequencies, and IR intensities of methyl nitrate, methanol, and several structures of protonated methyl nitrate, using the same theoretical methods as in an earlier study (Lee and Rice, 1992) of nitric acid. The ab initio results for methyl nitrate and methanol were found to be in good agreement with available experimental data. The proton affinity (PA) of methyl nitrate was calculated to be 176.9 +/-5 kcal/mol, in excellent agreement with the experimental value 176 kcal/mol obtained by Attina et al. (1987) and less than the PA value of nitric acid. An explanation of the discrepancy of the present results with those of an earlier study on protonated nitric acid is proposed.

  1. Direct measurement of equilibrium constants for high-affinity hemoglobins.

    PubMed

    Kundu, Suman; Premer, Scott A; Hoy, Julie A; Trent, James T; Hargrove, Mark S

    2003-06-01

    The biological functions of heme proteins are linked to their rate and affinity constants for ligand binding. Kinetic experiments are commonly used to measure equilibrium constants for traditional hemoglobins comprised of pentacoordinate ligand binding sites and simple bimolecular reaction schemes. However, kinetic methods do not always yield reliable equilibrium constants with more complex hemoglobins for which reaction mechanisms are not clearly understood. Furthermore, even where reaction mechanisms are clearly understood, it is very difficult to directly measure equilibrium constants for oxygen and carbon monoxide binding to high-affinity (K(D) < 1 micro M) hemoglobins. This work presents a method for direct measurement of equilibrium constants for high-affinity hemoglobins that utilizes a competition for ligands between the "target" protein and an array of "scavenger" hemoglobins with known affinities. This method is described for oxygen and carbon monoxide binding to two hexacoordinate hemoglobins: rice nonsymbiotic hemoglobin and Synechocystis hemoglobin. Our results demonstrate that although these proteins have different mechanisms for ligand binding, their affinities for oxygen and carbon monoxide are similar. Their large affinity constants for oxygen, 285 and approximately 100 micro M(-1) respectively, indicate that they are not capable of facilitating oxygen transport.

  2. Functionalized multi-walled carbon nanotubes as affinity ligands

    NASA Astrophysics Data System (ADS)

    Yu, L.; Li, C. M.; Zhou, Q.; Gan, Y.; Bao, Q. L.

    2007-03-01

    Functionalization of carbon nanotubes is very challenging for their applications. The paper here describes a new method to functionalize multi-walled carbon nanotubes (MWCNTs) as specific affinity adsorbents. MWCNTs were acid purified and pretreated with (3-aminopropyl)-triethoxysilane (APTES) in order to introduce abundant amino groups on the surface of MWCNTs. After the conversion of amino groups to carboxyl groups by succinic acid anhydride, MWCNTs were attached to protein A or aminodextran using 1-ethyl-3,3' (dimethylamion)-propylcarbodiimide as a biofunctional crosslinker. The incorporation of aminodextran as a spacer arm noticeably increased the binding capacity of the APTES-modified MWCNTs for protein A. The application of affinity MWCNTs for purification of immunoglobulin G was then evaluated. The affinity of MWCNTs with AMD spacer exhibited a high adsorption capacity of ~361 µg IgG/mg MWCNT (wet basis). About 75% of bound IgG was eluted from affinity MWCNTs (ANT-I and ANT-II) and ELISA confirmed that the biological activity of IgG was well preserved during the course of affinity separation. The functionalized MWCNTs could be potentially used in affinity chromatography.

  3. Extracting Iron from Cereal.

    ERIC Educational Resources Information Center

    Katz, David A.

    1992-01-01

    Describes an activity in which students can investigate and evaluate the amount of iron found in most fortified breakfast cereals or cream of wheat. Includes a list of necessary materials, safety precautions, experimental procedure, disposal protocol, and nutritional explanation, utilization, and variations. (JJK)

  4. Development of iron aluminides

    SciTech Connect

    Viswanathan, S.; Sikka, V.K.; Andleigh, V.K.

    1995-06-01

    The primary reason for the poor room-temperature ductility of Fe{sub 3}Al-based alloys is generally accepted to be environmental embrittlement due to hydrogen produced by the reaction of aluminum with water vapor present in the test atmosphere. In the as-cast condition, another possible reason for the low room-temperature ductility is the large grain size (0.5 to 3 mm) of the cast material. While recent studies on iron aluminides in the wrought condition have led to higher room-temperature ductility and increased high-temperature strength, limited studies have been conducted on iron aluminides in the as-cast condition. The purpose of this study was to induce grain refinement of the as-cast alloy through alloying additions to the melt and study the effect on room-temperature ductility as measured by the strain corresponding to the maximum stress obtained in a three-point bend test. A base charge of Fe-28% Al-5% Cr alloy was used; as in previous studies this ternary alloy exhibited the highest tensile ductility of several alloys tested. Iron aluminide alloys are being considered for many structural uses, especially for applications where their excellent corrosion resistance is needed. Several alloy compositions developed at ORNL have been licensed to commercial vendors for development of scale-up procedures. With the licensees and other vendors, several applications for iron aluminides are being pursued.

  5. The Iron Project

    NASA Technical Reports Server (NTRS)

    Pradhan, Anil K.

    2000-01-01

    Recent advances in theoretical atomic physics have enabled large-scale calculation of atomic parameters for a variety of atomic processes with high degree of precision. The development and application of these methods is the aim of the Iron Project. At present the primary focus is on collisional processes for all ions of iron, Fe I - FeXXVI, and other iron-peak elements; new work on radiative processes has also been initiated. Varied applications of the Iron Project work to X-ray astronomy are discussed, and more general applications to other spectral ranges are pointed out. The IP work forms the basis for more specialized projects such as the RmaX Project, and the work on photoionization/recombination, and aims to provide a comprehensive and self-consistent set of accurate collisional and radiative cross sections, and transition probabilities, within the framework of relativistic close coupling formulation using the Breit-Pauli R-Matrix method. An illustrative example is presented of how the IP data may be utilized in the formation of X-ray spectra of the K alpha complex at 6.7 keV from He-like Fe XXV.

  6. Iron toxicity screening.

    PubMed

    Cheng, C S; Sullivan, T D; Li, P K

    1979-06-01

    Fischer's method for rapid detection of acute iron toxicity is modified to suit pediatric cases. TPTZ (2,4,6-tripyridyl-s-triazine) is the chromogen of choice since in a small volume of serum slight to moderate hemolysis can cause a false positive result bathophenanthroline. Ordinary labware is amenable to this simplified procedure.

  7. Iron dominated magnets

    SciTech Connect

    Fischer, G.E.

    1985-07-01

    These two lectures on iron dominated magnets are meant for the student of accelerator science and contain general treatments of the subjects design and construction. The material is arranged in the categories: General Concepts and Cost Considerations, Profile Configuration and Harmonics, Magnetic Measurements, a few examples of ''special magnets'' and Materials and Practices. Extensive literature is provided.

  8. New insights into iron deficiency and iron deficiency anemia.

    PubMed

    Camaschella, Clara

    2017-02-13

    Recent advances in iron metabolism have stimulated new interest in iron deficiency (ID) and its anemia (IDA), common conditions worldwide. Absolute ID/IDA, i.e. the decrease of total body iron, is easily diagnosed based on decreased levels of serum ferritin and transferrin saturation. Relative lack of iron in specific organs/tissues, and IDA in the context of inflammatory disorders, are diagnosed based on arbitrary cut offs of ferritin and transferrin saturation and/or marker combination (as the soluble transferrin receptor/ferritin index) in an appropriate clinical context. Most ID patients are candidate to traditional treatment with oral iron salts, while high hepcidin levels block their absorption in inflammatory disorders. New iron preparations and new treatment modalities are available: high-dose intravenous iron compounds are becoming popular and indications to their use are increasing, although long-term side effects remain to be evaluated.

  9. Local BLyS production by T follicular cells mediates retention of high affinity B cells during affinity maturation

    PubMed Central

    Goenka, Radhika; Matthews, Andrew H.; Zhang, Bochao; O’Neill, Patrick J.; Scholz, Jean L.; Migone, Thi-Sau; Leonard, Warren J.; Stohl, William; Hershberg, Uri

    2014-01-01

    We have assessed the role of B lymphocyte stimulator (BLyS) and its receptors in the germinal center (GC) reaction and affinity maturation. Despite ample BLyS retention on B cells in follicular (FO) regions, the GC microenvironment lacks substantial BLyS. This reflects IL-21–mediated down-regulation of the BLyS receptor TACI (transmembrane activator and calcium modulator and cyclophilin ligand interactor) on GC B cells, thus limiting their capacity for BLyS binding and retention. Within the GC, FO helper T cells (TFH cells) provide a local source of BLyS. Whereas T cell–derived BLyS is dispensable for normal GC cellularity and somatic hypermutation, it is required for the efficient selection of high affinity GC B cell clones. These findings suggest that during affinity maturation, high affinity clones rely on TFH-derived BLyS for their persistence. PMID:24367004

  10. 49 CFR 192.373 - Service lines: Cast iron and ductile iron.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 3 2013-10-01 2013-10-01 false Service lines: Cast iron and ductile iron. 192.373... Regulators, and Service Lines § 192.373 Service lines: Cast iron and ductile iron. (a) Cast or ductile iron... cast iron pipe or ductile iron pipe is installed for use as a service line, the part of the...

  11. 49 CFR 192.373 - Service lines: Cast iron and ductile iron.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 3 2014-10-01 2014-10-01 false Service lines: Cast iron and ductile iron. 192.373... Regulators, and Service Lines § 192.373 Service lines: Cast iron and ductile iron. (a) Cast or ductile iron... cast iron pipe or ductile iron pipe is installed for use as a service line, the part of the...

  12. 49 CFR 192.373 - Service lines: Cast iron and ductile iron.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 3 2012-10-01 2012-10-01 false Service lines: Cast iron and ductile iron. 192.373... Regulators, and Service Lines § 192.373 Service lines: Cast iron and ductile iron. (a) Cast or ductile iron... cast iron pipe or ductile iron pipe is installed for use as a service line, the part of the...

  13. 49 CFR 192.373 - Service lines: Cast iron and ductile iron.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 3 2011-10-01 2011-10-01 false Service lines: Cast iron and ductile iron. 192.373... Regulators, and Service Lines § 192.373 Service lines: Cast iron and ductile iron. (a) Cast or ductile iron... cast iron pipe or ductile iron pipe is installed for use as a service line, the part of the...

  14. 49 CFR 192.373 - Service lines: Cast iron and ductile iron.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 3 2010-10-01 2010-10-01 false Service lines: Cast iron and ductile iron. 192.373... Regulators, and Service Lines § 192.373 Service lines: Cast iron and ductile iron. (a) Cast or ductile iron... cast iron pipe or ductile iron pipe is installed for use as a service line, the part of the...

  15. Iron aluminide composites

    SciTech Connect

    Schneibel, J.H.

    1999-07-01

    Iron aluminides with the B2 structure are highly oxidation and corrosion resistant. They are thermodynamically compatible with a wide range of ceramics such as TiC, WC, TiB{sub 2}, and ZrB{sub 2}. In addition, liquid iron aluminides wet these ceramics very well. Therefore, FeAl/ceramic composites may be produced by techniques such as liquid phase sintering of powder mixtures, or pressureless melt infiltration of ceramic powders with liquid FeAl. These techniques, the resulting microstructures, and their advantages as well as limitations are described. Iron aluminide composites can be very strong. Room temperature flexure strengths as high as 1.8 GPa have been observed for FeAl/WC. Substantial gains in strength of elevated temperatures (1,073 K) have also been demonstrated. Above 40 vol.% WC the room temperature flexure strength becomes flaw-limited. This is thought to be due to processing flaws and limited interfacial strength. The fracture toughness of FeAl/WC is unexpectedly high and follows a rule of mixtures. Interestingly, sufficiently thin ({lt}1 {micro}m) FeAl ligaments between adjacent WC particles fracture not by cleavage, but in a ductile manner. For these thin ligaments the dislocation pile-ups formed during deformation are not long enough to nucleate cleavage fracture, and their fracture mode is therefore ductile. For several reasons, this brittle-to-ductile size transition does not improve the fracture toughness of the composites significantly. However, since no cleavage cracks are nucleated in sufficiently thin FeAl ligaments, slow crack growth due to ambient water vapor does not occur. Therefore, as compared to monolithic iron aluminides, environmental embrittlement is dramatically reduced in iron aluminide composites.

  16. Iron bacterial phylogeny and their execution towards iron availability in Equatorial Indian Ocean and coastal Arabian Sea.

    PubMed

    Rajasabapathy, Raju; Mohandass, Chellandi; Vijayaraj, Ajakkalamoole Srinivas; Madival, Varsha Vinayak; Meena, Ram Murti

    2013-01-01

    Based on distinct colony morphology, color, size, shape and certain other traits, 92 bacterial isolates were investigated to understand their managerial ability on iron from the Arabian Sea and Equatorial Indian Ocean samples. The ARDRA (amplified rDNA restriction analysis) applied to eliminate the duplication of the bacterial strains, resulted 39 different banding patterns. The 16S rRNA gene sequencing data indicate the dominancy of three phylogenetic groups, alpha-Proteobacteria (10.25%), gamma-Proteobacteria (35.89%) and Bacilli (53.84%) in these waters. Marinobacter and Bacillus were the only common genera from both of the regions. Pseudoalteromonas, Halomonas, Rheinheimera, Staphylococcus and Idiomarina were some of the other genera obtained from the Arabian Sea. Erythrobacter, Roseovarius, Sagittula and Nitratireductor were found mostly in Equatorial Indian Ocean. In addition, 16S rRNA gene sequence data of some of our iron bacterial strains belong to novel species and one isolate ASS2A could form a new genus. Close to 23% of the isolates were able to produce high affinity sets of ligands like siderophores to mediate iron transport into the cell. The current study indicated that the Equatorial Indian Ocean species were well adapted to oxidize iron as an electron acceptor and the Arabian Sea species preferably go through siderophore production.

  17. Carbo-Iron - An Fe/AC composite - As alternative to nano-iron for groundwater treatment.

    PubMed

    Mackenzie, Katrin; Bleyl, Steffen; Georgi, Anett; Kopinke, Frank-Dieter

    2012-08-01

    Carbo-Iron(®)(1) is a novel colloidal composite consisting of activated carbon colloids (ACC) with a d(50) particle size of 0.8 μm and anchored deposits of zero-valent iron clusters. This study discusses the principal material properties of Carbo-Iron colloids (CIC) relevant for groundwater treatment in comparison to commercially available nano-sized zero-valent iron (nZVI). CIC with 10-25 wt% Fe(0) have been developed and tested in laboratory studies for their suitability as dehalogenation reagent and are especially designed to overcome some limitations known from the utilization of nZVI: CIC combine the sorption properties of ACC and the chemical reactivity of nZVI. In column tests, flushed-in CIC showed an enhanced mobility in sediment material compared to nZVI, without the need for colloid stabilizers. However, adding 1-3 wt-% of carboxymethyl cellulose (CMC) related to CIC as colloid stabilizer was found to assure long-lived stable suspensions under laboratory conditions which may additionally support the already improved mobility of the CIC and the homogeneity of particle deposition on the sediment matrix. The hydrophobic character of the ACC carrier provides a high affinity of CIC to non-aqueous phase liquids (NAPL). In undisturbed flow, the reactive particles are collected at the water-NAPL interface. The reagent accumulation at the organic phase is necessary for a successful source attack.

  18. Effect of dietary iron source and iron status on iron bioavailability tests in the rat

    SciTech Connect

    Zhang, D.; Hendricks, D.G.; Mahoney, A.W.

    1986-03-05

    Weanling male rats were made anemic in 7 days by feeding a low iron diet and bleeding. Healthy rats were fed the low iron diet supplemented with ferrous sulfate (29 ppm Fe). Each group was subdivided and fed for 10 days on test diets containing about 29 ppm iron that were formulated with meat:spinach mixtures or meat:soy mixtures to provided 100:0, 75:25, 50:50, 25:75, or 0:100% of the dietary iron from these sources or from a ferrous sulfate diet. After 3 days on the diets all rats were dosed orally with 2 or 5 micro curries of /sup 59/Fe after a 18 hour fast and refeeding for 1.5 hours. Iron status influenced liver iron, carcass iron, liver radio activity and percent of radioactive dose retained. Diet influenced fecal iron and apparent absorption of iron. In iron bioavailability studies assessment methodology and iron status of the test subject greatly influences the estimates of the value of dietary sources of iron.

  19. Identification of iron and heme utilization genes in Aeromonas and their role in the colonization of the leech digestive tract

    PubMed Central

    Maltz, Michele; LeVarge, Barbara L.; Graf, Joerg

    2015-01-01

    It is known that many pathogens produce high-affinity iron uptake systems like siderophores and/or proteins for utilizing iron bound to heme-containing molecules, which facilitate iron-acquisition inside a host. In mutualistic digestive-tract associations, iron uptake systems have not been as well studied. We investigated the importance of two iron utilization systems within the beneficial digestive-tract association Aeromonas veronii and the medicinal leech, Hirudo verbana. Siderophores were detected in A. veronii using chrome azurol S. Using a mini Tn5, a transposon insertion in viuB generated a mutant unable to utilize iron using siderophores. The A. veronii genome was then searched for genes potentially involved in iron utilization bound to heme-containing molecules. A putative outer membrane heme receptor (hgpB) was identified with a transcriptional activator, termed hgpR, downstream. The hgpB gene was interrupted with an antibiotic resistance cassette in both the parent strain and the viuB mutant, yielding an hgpB mutant and a mutant with both iron uptake systems inactivated. In vitro assays indicated that hgpB is involved in utilizing iron bound to heme and that both iron utilization systems are important for A. veronii to grow in blood. In vivo colonization assays revealed that the ability to acquire iron from heme-containing molecules is critical for A. veronii to colonize the leech gut. Since iron and specifically heme utilization is important in this mutualistic relationship and has a potential role in virulence factor of other organisms, genomes from different Aeromonas strains (both clinical and environmental) were queried with iron utilization genes of A. veronii. This analysis revealed that in contrast to the siderophore utilization genes heme utilization genes are widely distributed among aeromonads. The importance of heme utilization in the colonization of the leech further confirms that symbiotic and pathogenic relationships possess similar

  20. Genetic defects of iron transport.

    PubMed

    Bannerman, R M

    1976-09-01

    Five genetic traits in man and laboratory animals have major effects on iron transport. The heterogeneous condition, hemochromatosis, in some families appears to segregate as a Mendelian trait, and is associated with defective control of intestinal iron absorption. In the very rare human autosomal recessive trait, atransferrinemia, there is an almost total lack of transferrin and gross maldistribution of iron through the body. In mice, sex-linked anemia (an X-linked recessive trait) causes iron deficiency through defective iron absorption, at the "exit" step; a similar defect probably exists in placental iron transfer. In microcytic anemia of mice, an autosomal recessive trait, iron absorption is also impaired because of a defect of iron entry into cells, which is probably generalized. Belgrade rat anemia, less understood at present, also may involve a major disorder of iron metabolism. Study of these mutations has provided new knowledge of iron metabolism and its genetic control Their phenotypic interaction with nutritional factors, especially the form and quantity of iron in the diet, may provide new insights for the study of nutrition.

  1. Iron and cancer: recent insights.

    PubMed

    Manz, David H; Blanchette, Nicole L; Paul, Bibbin T; Torti, Frank M; Torti, Suzy V

    2016-03-01

    Iron is an essential dietary element. However, the ability of iron to cycle between oxidized and reduced forms also renders it capable of contributing to free radical formation, which can have deleterious effects, including promutagenic effects that can potentiate tumor formation. Dysregulation of iron metabolism can increase cancer risk and promote tumor growth. Cancer cells exhibit an enhanced dependence on iron relative to their normal counterparts, a phenomenon we have termed iron addiction. Work conducted in the past few years has revealed new cellular processes and mechanisms that deepen our understanding of the link between iron and cancer. Control of iron efflux through the combined action of ferroportin, an iron efflux pump, and its regulator hepcidin appears to play an important role in tumorigenesis. Ferroptosis is a form of iron-dependent cell death involving the production of reactive oxygen species. Specific mechanisms involved in ferroptosis, including depletion of glutathione and inhibition of glutathione peroxidase 4, have been uncovered. Ferritinophagy is a newly identified mechanism for degradation of the iron storage protein ferritin. Perturbations of mechanisms that control transcripts encoding proteins that regulate iron have been observed in cancer cells, including differences in miRNA, methylation, and acetylation. These new insights may ultimately provide new therapeutic opportunities for treating cancer.

  2. Iron status in the elderly

    PubMed Central

    Fairweather-Tait, Susan J.; Wawer, Anna A.; Gillings, Rachel; Jennings, Amy; Myint, Phyo K.

    2014-01-01

    Iron deficiency anaemia is prevalent in older age, particularly after the age of 80. Serum ferritin concentrations also decline, although there is no evidence to suggest that changes in iron stores are an inevitable consequence of ageing. Chronic inflammation is a common condition in older people, making the measurement of iron status difficult, and it is likely that elevated levels of circulating hepcidin are responsible for changes in iron metabolism that result in systemic iron depletion. Other contributory factors are poor diet and some medications, such as aspirin. Anaemia in older age has undesirable health outcomes, including increased susceptibility to falling and depression. However, there are concerns about possible adverse effects of iron supplements, either in relation to pro-inflammatory effects in the gut or inappropriate tissue iron deposition. Brain iron levels are increased with age-related degenerative diseases, but it is not known if this is the cause or a consequence of the disease, and genetic factors are likely to play a role. In order to maintain body iron within the normal range a personalised approach is required, taking into account all of the factors that may affect iron metabolism and the available strategies for preventing iron deficiency or overload. PMID:24275120

  3. Iron status in the elderly.

    PubMed

    Fairweather-Tait, Susan J; Wawer, Anna A; Gillings, Rachel; Jennings, Amy; Myint, Phyo K

    2014-01-01

    Iron deficiency anaemia is prevalent in older age, particularly after the age of 80. Serum ferritin concentrations also decline, although there is no evidence to suggest that changes in iron stores are an inevitable consequence of ageing. Chronic inflammation is a common condition in older people, making the measurement of iron status difficult, and it is likely that elevated levels of circulating hepcidin are responsible for changes in iron metabolism that result in systemic iron depletion. Other contributory factors are poor diet and some medications, such as aspirin. Anaemia in older age has undesirable health outcomes, including increased susceptibility to falling and depression. However, there are concerns about possible adverse effects of iron supplements, either in relation to pro-inflammatory effects in the gut or inappropriate tissue iron deposition. Brain iron levels are increased with age-related degenerative diseases, but it is not known if this is the cause or a consequence of the disease, and genetic factors are likely to play a role. In order to maintain body iron within the normal range a personalised approach is required, taking into account all of the factors that may affect iron metabolism and the available strategies for preventing iron deficiency or overload.

  4. The essential basic helix-loop-helix protein FIT1 is required for the iron deficiency response.

    PubMed

    Colangelo, Elizabeth P; Guerinot, Mary Lou

    2004-12-01

    Regulation of iron uptake is critical for plant survival. Although the activities responsible for reduction and transport of iron at the plant root surface have been described, the genes controlling these activities are largely unknown. We report the identification of the essential gene Fe-deficiency Induced Transcription Factor 1 (FIT1), which encodes a putative transcription factor that regulates iron uptake responses in Arabidopsis thaliana. Like the Fe(III) chelate reductase FRO2 and high affinity Fe(II) transporter IRT1, FIT1 mRNA is detected in the outer cell layers of the root and accumulates in response to iron deficiency. fit1 mutant plants are chlorotic and die as seedlings but can be rescued by the addition of supplemental iron, pointing to a defect in iron uptake. fit1 mutant plants accumulate less iron than wild-type plants in root and shoot tissues. Microarray analysis shows that expression of many (72 of 179) iron-regulated genes is dependent on FIT1. We demonstrate that FIT1 regulates FRO2 at the level of mRNA accumulation and IRT1 at the level of protein accumulation. We propose a new model for iron uptake in Arabidopsis where FRO2 and IRT1 are differentially regulated by FIT1.

  5. Systems and Trans-System Level Analysis Identifies Conserved Iron Deficiency Responses in the Plant Lineage[W][OA

    PubMed Central

    Urzica, Eugen I.; Casero, David; Yamasaki, Hiroaki; Hsieh, Scott I.; Adler, Lital N.; Karpowicz, Steven J.; Blaby-Haas, Crysten E.; Clarke, Steven G.; Loo, Joseph A.; Pellegrini, Matteo; Merchant, Sabeeha S.

    2012-01-01

    We surveyed the iron nutrition-responsive transcriptome of Chlamydomonas reinhardtii using RNA-Seq methodology. Presumed primary targets were identified in comparisons between visually asymptomatic iron-deficient versus iron-replete cells. This includes the known components of high-affinity iron uptake as well as candidates for distributive iron transport in C. reinhardtii. Comparison of growth-inhibited iron-limited versus iron-replete cells revealed changes in the expression of genes in chloroplastic oxidative stress response pathways, among hundreds of other genes. The output from the transcriptome was validated at multiple levels: by quantitative RT-PCR for assessing the data analysis pipeline, by quantitative proteomics for assessing the impact of changes in RNA abundance on the proteome, and by cross-species comparison for identifying conserved or universal response pathways. In addition, we assessed the functional importance of three target genes, VITAMIN C 2 (VTC2), MONODEHYDROASCORBATE REDUCTASE 1 (MDAR1), and CONSERVED IN THE GREEN LINEAGE AND DIATOMS 27 (CGLD27), by biochemistry or reverse genetics. VTC2 and MDAR1, which are key enzymes in de novo ascorbate synthesis and ascorbate recycling, respectively, are likely responsible for the 10-fold increase in ascorbate content of iron-limited cells. CGLD27/At5g67370 is a highly conserved, presumed chloroplast-localized pioneer protein and is important for growth of Arabidopsis thaliana in low iron. PMID:23043051

  6. Arabidopsis copper transport protein COPT2 participates in the cross talk between iron deficiency responses and low-phosphate signaling.

    PubMed

    Perea-García, Ana; Garcia-Molina, Antoni; Andrés-Colás, Nuria; Vera-Sirera, Francisco; Pérez-Amador, Miguel A; Puig, Sergi; Peñarrubia, Lola

    2013-05-01

    Copper and iron are essential micronutrients for most living organisms because they participate as cofactors in biological processes, including respiration, photosynthesis, and oxidative stress protection. In many eukaryotic organisms, including yeast (Saccharomyces cerevisiae) and mammals, copper and iron homeostases are highly interconnected; yet, such interdependence is not well established in higher plants. Here, we propose that COPT2, a high-affinity copper transport protein, functions under copper and iron deficiencies in Arabidopsis (Arabidopsis thaliana). COPT2 is a plasma membrane protein that functions in copper acquisition and distribution. Characterization of the COPT2 expression pattern indicates a synergic response to copper and iron limitation in roots. We characterized a knockout of COPT2, copt2-1, that leads to increased resistance to simultaneous copper and iron deficiencies, measured as reduced leaf chlorosis and improved maintenance of the photosynthetic apparatus. We propose that COPT2 could play a dual role under iron deficiency. First, COPT2 participates in the attenuation of copper deficiency responses driven by iron limitation, possibly to minimize further iron consumption. Second, global expression analyses of copt2-1 versus wild-type Arabidopsis plants indicate that low-phosphate responses increase in the mutant. These results open up new biotechnological approaches to fight iron deficiency in crops.

  7. High efficiency iron electrode and additives for use in rechargeable iron-based batteries

    DOEpatents

    Narayan, Sri R.; Prakash, G. K. Surya; Aniszfeld, Robert; Manohar, Aswin; Malkhandi, Souradip; Yang, Bo

    2017-02-21

    An iron electrode and a method of manufacturing an iron electrode for use in an iron-based rechargeable battery are disclosed. In one embodiment, the iron electrode includes carbonyl iron powder and one of a metal sulfide additive or metal oxide additive selected from the group of metals consisting of bismuth, lead, mercury, indium, gallium, and tin for suppressing hydrogen evolution at the iron electrode during charging of the iron-based rechargeable battery. An iron-air rechargeable battery including an iron electrode comprising carbonyl iron is also disclosed, as is an iron-air battery wherein at least one of the iron electrode and the electrolyte includes an organosulfur additive.

  8. Nanoparticle multivalency counterbalances the ligand affinity loss upon PEGylation.

    PubMed

    Hennig, Robert; Pollinger, Klaus; Veser, Anika; Breunig, Miriam; Goepferich, Achim

    2014-11-28

    The conjugation of receptor ligands to shielded nanoparticles is a widely used strategy to precisely control nanoparticle-cell interactions. However, it is often overlooked that a ligand's affinity can be severely impaired by its attachment to the polyethylene glycol (PEG) chains that are frequently used to protect colloids from serum protein adsorption. Using the model ligand EXP3174, a small-molecule antagonist for the angiotensin II receptor type 1 (AT1R), we investigated the ligand's affinity before and after its PEGylation and when attached to PEGylated nanoparticles. The PEGylated ligand displayed a 580-fold decreased receptor affinity compared to the native ligand. Due to their multivalency, the nanoparticles regained a low nanomolar receptor affinity, which is in the range of the affinity of the native ligand. Moreover, a four orders of magnitude higher concentration of free ligand was required to displace PEGylated nanoparticles carrying EXP3174 from the receptor. On average, one nanoparticle was decorated with 11.2 ligand molecules, which led to a multivalent enhancement factor of 22.5 compared to the monovalent PEGylated ligand. The targeted nanoparticles specifically bound the AT1R and showed no interaction to receptor negative cells. Our study shows that the attachment of a small-molecule ligand to a PEG chain can severely affect its receptor affinity. Concomitantly, when the ligand is tethered to nanoparticles, the immense avidity greatly increases the ligand-receptor interaction. Based on our results, we highly recommend the affinity testing of receptor ligands before and after PEGylation to identify potent molecules for active nanoparticle targeting.

  9. Protein Complex Affinity Capture from Cryomilled Mammalian Cells.

    PubMed

    LaCava, John; Jiang, Hua; Rout, Michael P

    2016-12-09

    Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

  10. Protein Complex Affinity Capture from Cryomilled Mammalian Cells

    PubMed Central

    LaCava, John; Jiang, Hua; Rout, Michael P.

    2016-01-01

    Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets. PMID:28060343

  11. Degradation of chlorofluorocarbons using granular iron and bimetallic irons.

    PubMed

    Jeen, Sung-Wook; Lazar, Snezana; Gui, Lai; Gillham, Robert W

    2014-03-01

    Degradation of trichlorofluoromethane (CFC11) and 1,1,2-trichloro-1,2,2-trifluoroethane (CFC113) by granular iron and bimetallic (nickel- or palladium-enhanced) irons was studied in flow-through column tests. Both compounds were rapidly degraded, following pseudo-first-order kinetics with respect to the parent compounds. The average pseudo-first-order rate constants for CFC11 were similar among different materials, except for palladium-enhanced iron (PdFe), in which the rate of degradation was about two times faster than for the other materials. In the case of CFC113, the rate constants for bimetallic irons were about two to three times greater than for the regular iron material. The smaller than expected differences in degradation rate constants of chlorofluorocarbons (CFCs) between regular iron and bimetallic irons suggested little, if any, catalytic effect of the bimetallic materials in the initial degradation step. Subsequent degradation steps involved catalytic hydrogenation, however, playing a significant role in further degradation of reaction intermediates. The degradation intermediates and final products of CFC11 and CFC113 suggested that degradation proceeded through hydrogenolysis and α/β-elimination in the presence of regular iron (Fe) and nickel-enhanced iron (NiFe). Even though there is only minor benefit in the use of bimetallic iron in terms of degradation kinetics of the parent CFCs, enhanced degradation rates of intermediates such as chlorotriflouroethene (CTFE) in subsequent reaction steps could be beneficial.

  12. Gradient-based habitat affinities predict species vulnerability to drought.

    PubMed

    Debinski, Diane M; Caruthers, Jennet C; Cook, Dianne; Crowley, Jason; Wickham, Hadley

    2013-05-01

    Ecological fingerprints of climate change are becoming increasingly evident at broad geographical scales as measured by species range shifts and changes in phenology. However, finer-scale species-level responses to environmental fluctuations may also provide an important bellwether of impending future community responses. Here we examined changes in abundance of butterfly species along a hydrological gradient of six montane meadow habitat types in response to drought. Our data collection began prior to the drought, and we were able to track changes for 11 years, of which eight were considered mild to extreme drought conditions. We separated the species into those that had an affinity for hydric vs. xeric habitats. We suspected that drought would favor species with xeric habitat affinities, but that there could be variations in species-level responses along the hydrological gradient. We also suspected that mesic meadows would be most sensitive to drought conditions. Temporal trajectories were modeled for both species groups (hydric vs. xeric affinity) and individual species. Abundances of species with affinity for xeric habitats increased in virtually all meadow types. Conversely, abundances of species with affinity for hydric habitats decreased, particularly in mesic and xeric meadows. Mesic meadows showed the most striking temporal abundance trajectory: Increasing abundances of species with xeric habitat affinity were offset by decreasing or stable abundances of species with hydric habitat affinity. The one counterintuitive finding was that, in some hydric meadows, species with affinity for hydric habitats increased. In these cases, we suspect that decreasing moisture conditions in hydric meadows actually increased habitat suitability because sites near the limit of moisture extremes for some species became more acceptable. Thus, species responses were relatively predictable based upon habitat affinity and habitat location along the hydrological gradient, and

  13. Coenzyme-like ligands for affinity isolation of cholesterol oxidase.

    PubMed

    Xin, Yu; Lu, Liushen; Wang, Qing; Zhang, Ling; Tong, Yanjun; Wang, Wu

    2016-05-15

    Two coenzyme-like chemical ligands were designed and synthesized for affinity isolation of cholesterol oxidase (COD). To simulate the structure of natural coenzyme of COD (flavin adenine dinucleotide (FAD)), on Sepharose beads, 5-aminouracil, cyanuric chloride and 1, 4-butanediamine were composed and then modified. The COD gene from Brevibacterium sp. (DQ345780) was expressed in Escherichia coli BL21 (DE3), and then the sorbents were applied to adsorption analysis with the pure enzyme. Subsequently, the captured enzyme was applied to SDS-PAGE and activity analysis. As calculated, the theoretical maximum adsorption (Qmax) of the two affinity sorbents (RL-1 and RL-2) were ∼83.5 and 46.3mg/g wet gel; and the desorption constant Kd of the two sorbents were ∼6.02×10(-4) and 1.19×10(-4)μM. The proteins after cell lysis were applied to affinity isolation, and then after one step of affinity binding on the two sorbents, the protein recoveries of RL-1 and RL-2 were 9.2% and 9.7%; the bioactivity recoveries were 92.7% and 91.3%, respectively. SDS-PAGE analysis revealed that the purities of COD isolated with the two affinity sorbents were approximately 95%.

  14. The proton affinities of saturated and unsaturated heterocyclic molecules

    NASA Astrophysics Data System (ADS)

    Kabli, Samira; van Beelen, Eric S. E.; Ingemann, Steen; Henriksen, Lars; Hammerum, Steen

    2006-03-01

    The proton affinities derived from G3-calculations of 23 five-membered ring heteroaromatic molecules agree well with the experimentally determined values available in the literature. The calculated local proton affinities show that the principal site of protonation of the heteroaromatic compounds examined is an atom of the ring, carbon when there is only one heteroatom in the ring, and nitrogen where there are two or more heteroatoms. The experimental proton affinities of non-aromatic cyclic ethers, amines and thioethers are also in excellent agreement with the calculated values, with two exceptions (oxetane, N-methylazetidine). The literature proton affinities of the four simple cyclic ethers, oxetane, tetrahydrofuran, tetrahydropyran and oxepane were confirmed by Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry, in order to examine the disagreement between the values predicted by extrapolation or additivity for tetrahydrofuran and tetrahydropyran and those determined by experiment and by calculation. The proton affinity differences between the pairs tetrahydropyran/1,4-dioxane, piperidine/morpholine and related compounds show that introduction of an additional oxygen atom in the ring considerably lowers the basicity.

  15. Improving antibody binding affinity and specificity for therapeutic development.

    PubMed

    Bostrom, Jenny; Lee, Chingwei V; Haber, Lauric; Fuh, Germaine

    2009-01-01

    Affinity maturation is an important part of the therapeutic antibody development process as in vivo activity often requires high binding affinity. Here, we describe a targeted approach for affinity improvement of therapeutic antibodies. Sets of CDR residues that are solvent accessible and relatively diverse in natural antibodies are targeted for diversification. Degenerate oligonucleotides are used to generate combinatorial phage-displayed antibody libraries with varying degree of diversity at randomized positions from which high-affinity antibodies can be selected. An advantage of using antibodies for therapy is their exquisite target specificity, which enables selective antigen binding and reduces off-target effects. However, it can be useful, and often it is necessary, to generate cross-reactive antibodies binding to not only the human antigen but also the corresponding non-human primate or rodent orthologs. Such cross-reactive antibodies can be used to validate the therapeutic targeting and examine the safety profile in preclinical animal models before committing to a costly development track. We show how affinity improvement and cross-species binding can be achieved in a one-step process.

  16. High-affinity K+ uptake in pepper plants.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2005-06-01

    High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

  17. Affine kinematics in planar fibrous connective tissues: an experimental investigation.

    PubMed

    Jayyosi, C; Affagard, J-S; Ducourthial, G; Bonod-Bidaud, C; Lynch, B; Bancelin, S; Ruggiero, F; Schanne-Klein, M-C; Allain, J-M; Bruyère-Garnier, K; Coret, M

    2017-03-29

    The affine transformation hypothesis is usually adopted in order to link the tissue scale with the fibers scale in structural constitutive models of fibrous tissues. Thanks to the recent advances in imaging techniques, such as multiphoton microscopy, the microstructural behavior and kinematics of fibrous tissues can now be monitored at different stretching within the same sample. Therefore, the validity of the affine hypothesis can be investigated. In this paper, the fiber reorientation predicted by the affine assumption is compared to experimental data obtained during mechanical tests on skin and liver capsule coupled with microstructural imaging using multiphoton microscopy. The values of local strains and the collagen fibers orientation measured at increasing loading levels are used to compute a theoretical estimation of the affine reorientation of collagen fibers. The experimentally measured reorientation of collagen fibers during loading could not be successfully reproduced with this simple affine model. It suggests that other phenomena occur in the stretching process of planar fibrous connective tissues, which should be included in structural constitutive modeling approaches.

  18. Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis.

    PubMed

    Deng, Zhenzhen; Dong, Mingming; Wang, Yan; Dong, Jing; Li, Shawn S-C; Zou, Hanfa; Ye, Mingliang

    2017-02-21

    Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.

  19. Protein purification by aminosquarylium cyanine dye-affinity chromatography.

    PubMed

    Silva, M S; Graça, V C; Reis, L V; Santos, P F; Almeida, P; Queiroz, J A; Sousa, F

    2013-12-01

    The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography.

  20. Magnetorotational iron core collapse

    NASA Technical Reports Server (NTRS)

    Symbalisty, E. M. D.

    1984-01-01

    During its final evolutionary stages, a massive star, as considered in current astrophysical theory, undergoes rapid collapse, thereby triggering a sequence of a catastrophic event which results in a Type II supernova explosion. A remnant neutron star or a black hole is left after the explosion. Stellar collapse occurs, when thermonuclear fusion has consumed the lighter elements present. At this stage, the core consists of iron. Difficulties arise regarding an appropriate model with respect to the core collapse. The present investigation is concerned with the evolution of a Type II supernova core including the effects of rotation and magnetic fields. A simple neutrino model is developed which reproduced the spherically symmetric results of Bowers and Wilson (1982). Several two-dimensional computational models of stellar collapse are studied, taking into account a case in which a 15 solar masses iron core was artificially given rotational and magnetic energy.

  1. Development of Iron Aluminides

    DTIC Science & Technology

    1987-05-01

    the precipitation of an ordered perovskite carbide for strength. A second series was modeled after the austenitic iron-based superalloys from the FeNi ... Thinned Foils .. 67 11 Grain Aspect Ratio and TiB. Dispersion Size for Fe.Al + TiB2 Produced With Fine and Coarse Powders...aluminide, the addition of titanium and boron produced a finer microstructure. Transmission electron microscopy (TEM) on prepared thin sections of

  2. Measurement of iron absorption from meals contaminated with iron

    SciTech Connect

    Hallberg, L.; Bjoern-Rasmussen, E.

    1981-12-01

    A method is described to measure in vitro the extent of isotopic exchange between the native nonheme food iron and added inorganic reduction to radioiron tracer. The food is digested with pepsin and trypsin in the presence of radioiron. The exchangeability of food iron is calculated from the specific activity in the food and in an extract of bathophenantroline in isoamyl alcohol obtained after digesting this food. The precision and accuracy of the method is illustrated by two kinds of studies, those in which different amounts of contamination iron are added to a meal and those evaluating contamination iron in natural meals. The present method will make it possible to measure validly iron absorption from meals contaminated with unknown amounts of iron of unknown exchangeability with the extrinsic radioiron tracer.

  3. Iron homoeostasis in rheumatic disease.

    PubMed

    Baker, Joshua F; Ghio, Andrew J

    2009-11-01

    Iron is critical in nearly all cell functions and the ability of a cell, tissue and organism to procure this metal is obligatory for survival. Iron is necessary for normal immune function, and relative iron deficiency is associated with mild immunosuppression. Concentrations of this metal in excess of those required for function can present both an oxidative stress and elevate risks for infection. As a result, the human has evolved to have a complex mechanism of regulating iron and limiting its availability. This homoeostasis can be disrupted. Autoimmune diseases and gout often present with abnormal iron homoeostasis, thus supporting a participation of the metal in these injuries. We review the role of iron in normal immune function and discuss both clinical evidence of altered iron homoeostasis in autoimmune diseases and gout as well as possible implications of both depletion and supplementation of this metal in this patient population. We conclude that altered iron homoeostasis may represent a purposeful response to inflammation that could have theoretical anti-inflammatory benefits. We encourage physicians to avoid routine iron supplementation in those without depleted iron stores.

  4. Current issues in iron deficiency.

    PubMed

    Baynes, R D; Cook, J D

    1996-03-01

    This brief review of developments relating to iron deficiency during the past year covers three main areas: iron supplementation, the regulation of iron absorption, and the use of the serum transferrin receptor for the assessment of iron status. The intermittent administration of iron supplement once or twice weekly rather than daily has been advocated by international health agencies in recent years, but radioiron absorption studies in human subjects have failed to demonstrate any absorptive advantage of the intermittent schedule. The value of prophylactic iron supplementation in elderly blood donors was evaluated and shown to offer limited benefit in maintaining donation frequency. A recent model of the regulation of iron absorption involving erythropoietic and store regulators is discussed and a recent article indicating a potential non-hematopoietic effect of hematopoietic growth factors on iron absorption by the gastrointestinal mucosal cell is reviewed. A new measure of functional iron deficiency, namely the serum transferrin receptor, is discussed, with particular reference to its mechanism of production and its great value in distinguishing iron deficiency anemia from the anemia of chronic disease.

  5. Iron homeostasis and eye disease

    PubMed Central

    Loh, Allison; Hadziahmetovic, Majda; Dunaief, Joshua L.

    2009-01-01

    Summary Iron is necessary for life, but excess iron can be toxic to tissues. Iron is thought to damage tissues primarily by generating oxygen free radicals through the Fenton reaction. We present an overview of the evidence supporting iron's potential contribution to a broad range of eye disease using an anatomical approach. Firstly, iron can be visualized in the cornea as iron lines in the normal aging cornea as well as in diseases like keratoconus and pterygium. In the lens, we present the evidence for the role of oxidative damage in cataractogenesis. Also, we review the evidence that iron may play a role in the pathogenesis of the retinal disease age-related macular degeneration. Although currently there is no direct link between excess iron and development of optic neuropathies, ferrous iron's ability to form highly reactive oxygen species may play a role in optic nerve pathology. Lastly, we discuss recent advances in prevention and therapeutics for eye disease with antioxidants and iron chelators,. PMID:19059309

  6. Replacement of the glucose phosphotransferase transport system by galactose permease reduces acetate accumulation and improves process performance of Escherichia coli for recombinant protein production without impairment of growth rate.

    PubMed

    De Anda, Ramón; Lara, Alvaro R; Hernández, Vanessa; Hernández-Montalvo, Verónica; Gosset, Guillermo; Bolívar, Francisco; Ramírez, Octavio T

    2006-05-01

    Acetate accumulation under aerobic conditions is a common problem in Escherichia coli cultures, as it causes a reduction in both growth rate and recombinant protein productivity. In this study, the effect of replacing the glucose phosphotransferase transport system (PTS) with an alternate glucose transport activity on growth kinetics, acetate accumulation and production of two model recombinant proteins, was determined. Strain VH32 is a W3110 derivative with an inactive PTS. The promoter region of the chromosomal galactose permease gene galP of VH32 was replaced by the strong trc promoter. The resulting strain, VH32GalP+ acquired the capacity to utilize glucose as a carbon source. Strains W3110 and VH32GalP+ were transformed for the production of recombinant TrpLE-proinsulin accumulated as inclusion bodies (W3110-PI and VH32GalP+-PI) and for production of soluble intracellular green fluorescent protein (W3110-pV21 and VH32GalP+-pV21). W3110-pV21 and VH32GalP+-pV21 were grown in batch cultures. Maximum recombinant protein concentration, as determined from fluorescence, was almost four-fold higher in VH32GalP+-pV21, relative to W3110-pV21. Maximum acetate concentration reached 2.8 g/L for W3110-pV21 cultures, whereas a maximum of 0.39 g/L accumulated in VH32GalP+-pV21. W3110-PI and VH32GalP+-PI were grown in batch and fed-batch cultures. Compared to W3110-PI, the engineered strain maintained similar production and growth rate capabilities while reducing acetate accumulation. Specific glucose consumption rate was lower and product yield on glucose was higher in VH32GalP+-PI fed-batch cultures. Altogether, strains with the engineered glucose uptake system showed improved process performance parameters for recombinant protein production over the wild-type strain.

  7. Altered srf expression in Bacillus subtilis resulting from changes in culture pH is dependent on the Spo0K oligopeptide permease and the ComQX system of extracellular control.

    PubMed

    Cosby, W M; Vollenbroich, D; Lee, O H; Zuber, P

    1998-03-01

    The expression of the srf operon of Bacillus subtilis, encoding surfactin synthetase and the competence regulatory protein ComS, was observed to be reduced when cells were grown in a rich glucose- and glutamine-containing medium in which late-growth culture pH was 5.0 or lower. The production of the surfactin synthetase subunits and of surfactin itself was also reduced. Raising the pH to near neutrality resulted in dramatic increases in srf expression and surfactin production. This apparent pH-dependent induction of srf expression required spo0K, which encodes the oligopeptide permease that functions in cell-density-dependent control of sporulation and competence, but not CSF, the competence-inducing pheromone that regulates srf expression in a Spo0K-dependent manner. Both ComP and ComA, the two-component regulatory pair that stimulates cell-density-dependent srf transcription, were required for optimal expression of srf at low and high pHs, but ComP was not required for pH-dependent srf induction. The known negative regulators of srf, RapC and CodY, were found not to function significantly in pH-dependent srf expression. Late-growth culture supernatants at low pH were not active in inducing srf expression in cells of low-density cultures but were rendered active when their pH was raised to near neutrality. ComQ (and very likely the srf-inducing pheromone ComX) and Spo0K were found to be required for the extracellular induction of srf-lacZ at neutral pH. The results suggest that srf expression, in response to changes in culture pH, requires Spo0K and another, as yet unidentified, extracellular factor. The study also provides evidence consistent with the hypothesis that ComP acts both positively and negatively in the regulation of ComA and that both activities are controlled by the ComX pheromone.

  8. Flare Plasma Iron Abundance

    NASA Technical Reports Server (NTRS)

    Dennis, Brian R.; Dan, Chau; Jain, Rajmal; Schwartz, Richard A.; Tolbert, Anne K.

    2008-01-01

    The equivalent width of the iron-line complex at 6.7 keV seen in flare X-ray spectra suggests that the iron abundance of the hottest plasma at temperatures >approx.10 MK may sometimes be significantly lower than the nominal coronal abundance of four times the photospheric value that is commonly assumed. This conclusion is based on X-ray spectral observations of several flares seen in common with the Ramaty High Energy Solar Spectroscopic Imager (RHESSI) and the Solar X-ray Spectrometer (SOXS) on the second Indian geostationary satellite, GSAT-2. The implications of this will be discussed as it relates to the origin of the hot flare plasma - either plasma already in the corona that is directly heated during the flare energy release process or chromospheric plasma that is heated by flare-accelerated particles and driven up into the corona. Other possible explanations of lower-than-expected equivalent widths of the iron-line complex will also be discussed.

  9. The affine structure of gravitational theories: Symplectic groups and geometry

    NASA Astrophysics Data System (ADS)

    Capozziello, Salvatore; Cirilo-Lombardo, D. J.; de Laurentis, Mariafelicia

    2014-09-01

    We give a geometrical description of gravitational theories from the viewpoint of symmetries and affine structure. We show how gravity, considered as a gauge theory, can be consistently achieved by the nonlinear realization of the conformal-affine group in an indirect manner: due to the partial isomorphism between CA(3, 1) and the centrally extended Sp( 8), we perform a nonlinear realization of the centrally extended (CE)Sp( 8) in its semi-simple version. In particular, starting from the bundle structure of gravity, we derive the conformal-affine Lie algebra and then, by the nonlinear realization, we define the coset field transformations, the Cartan forms and the inverse Higgs constraints. Finally, we discuss the geometrical Lagrangians where all the information on matter fields and their interactions can be contained.

  10. Affinity maturation of antibodies requires integrity of the adult thymus.

    PubMed

    AbuAttieh, Mouhammed; Bender, Diane; Liu, Esther; Wettstein, Peter; Platt, Jeffrey L; Cascalho, Marilia

    2012-02-01

    The generation of B-cell responses to proteins requires a functional thymus to produce CD4(+) T cells which helps in the activation and differentiation of B cells. Because the mature T-cell repertoire has abundant cells with the helper phenotype, one might predict that in mature individuals, the generation of B-cell memory would proceed independently of the thymus. Contrary to that prediction, we show here that the removal of the thymus after the establishment of the T-cell compartment or sham surgery without removal of the thymus impairs the affinity maturation of antibodies. Because removal or manipulation of the thymus did not decrease the frequency of mutation of the Ig variable heavy chain exons encoding antigen-specific antibodies, we conclude that the thymus controls affinity maturation of antibodies in the mature individual by facilitating the selection of B cells with high-affinity antibodies.

  11. A fast quantum algorithm for the affine Boolean function identification

    NASA Astrophysics Data System (ADS)

    Younes, Ahmed

    2015-02-01

    Bernstein-Vazirani algorithm (the one-query algorithm) can identify a completely specified linear Boolean function using a single query to the oracle with certainty. The first aim of the paper is to show that if the provided Boolean function is affine, then one more query to the oracle (the two-query algorithm) is required to identify the affinity of the function with certainty. The second aim of the paper is to show that if the provided Boolean function is incompletely defined, then the one-query and the two-query algorithms can be used as bounded-error quantum polynomial algorithms to identify certain classes of incompletely defined linear and affine Boolean functions respectively with probability of success at least 2/3.

  12. Enhancing Community Detection By Affinity-based Edge Weighting Scheme

    SciTech Connect

    Yoo, Andy; Sanders, Geoffrey; Henson, Van; Vassilevski, Panayot

    2015-10-05

    Community detection refers to an important graph analytics problem of finding a set of densely-connected subgraphs in a graph and has gained a great deal of interest recently. The performance of current community detection algorithms is limited by an inherent constraint of unweighted graphs that offer very little information on their internal community structures. In this paper, we propose a new scheme to address this issue that weights the edges in a given graph based on recently proposed vertex affinity. The vertex affinity quantifies the proximity between two vertices in terms of their clustering strength, and therefore, it is ideal for graph analytics applications such as community detection. We also demonstrate that the affinity-based edge weighting scheme can improve the performance of community detection algorithms significantly.

  13. On the electron affinity of the oxygen atom

    NASA Technical Reports Server (NTRS)

    Bauschlicher, C. W., Jr.; Langhoff, S. R.; Partridge, H.; Taylor, P. R.

    1986-01-01

    The electron affinity (EA) of oxygen is computed to be 1.287 eV, using 2p electron full configuration-interaction (CI) wave functions expanded in a 6s5p3d2f Slater-type orbital basis. The best complete active space self-consistent field - multireference CI (CASSCF-MRCI) result including only 2p correlation is 1.263 eV. However, inclusion of 2s intrashell and 2s2p intershell correlation increases the computed EA to 1.290 at the CASSCF-MRCI level. At the full CI basis set limit, the 2s contribution to the electron affinity is estimated to be as large as 0.1 eV. This study clearly establishes the synergistic effect between the higher excitations and basis set completeness on the electron affinity when the 2s electrons are correlated.

  14. On the electron affinity of the oxygen atom

    NASA Technical Reports Server (NTRS)

    Bauschlicher, C. W., Jr.; Langhoff, S. R.; Partridge, H.; Taylor, P. R.

    1986-01-01

    The electron affinity of oxygen is computed to be 1.287 eV, at the full CI level using a 6s5p3d 2f Slater-type orbital basis and correlating only the 2p electrons. The best CASSCF-MRCI result including only 2p correlation is 1.263 eV. However, inclusion of 2s intrashell and 2s2p intershell coorelation increases the computed EA to 1.290 eV at the CASSCF-MRCI level. At the full CI basis set limit, the 2s contribution to the electron affinity is estimated to be as large as 0.1 eV. The higher excitation contribution to the electron affinity is found to increase substantially with basis set completeness, especially when the 2s electrons are correlated. Relativistic effects are shown to make a small (less than 0.01 eV) change in the EA.

  15. AMPK beta subunits display isoform specific affinities for carbohydrates.

    PubMed

    Koay, Ann; Woodcroft, Ben; Petrie, Emma J; Yue, Helen; Emanuelle, Shane; Bieri, Michael; Bailey, Michael F; Hargreaves, Mark; Park, Jong-Tae; Park, Kwan-Hwa; Ralph, Stuart; Neumann, Dietbert; Stapleton, David; Gooley, Paul R

    2010-08-04

    AMP-activated protein kinase (AMPK) is a heterotrimer of catalytic (alpha) and regulatory (beta and gamma) subunits with at least two isoforms for each subunit. AMPK beta1 is widely expressed whilst AMPK beta2 is highly expressed in muscle and both beta isoforms contain a mid-molecule carbohydrate-binding module (beta-CBM). Here we show that beta2-CBM has evolved to contain a Thr insertion and increased affinity for glycogen mimetics with a preference for oligosaccharides containing a single alpha-1,6 branched residue. Deletion of Thr-101 reduces affinity for single alpha-1,6 branched oligosaccharides by 3-fold, while insertion of this residue into the equivalent position in the beta1-CBM sequence increases affinity by 3-fold, confirming the functional importance of this residue.

  16. High-Affinity Manganese Uptake by the Metal Transporter NRAMP1 Is Essential for Arabidopsis Growth in Low Manganese Conditions[C][W

    PubMed Central

    Cailliatte, Rémy; Schikora, Adam; Briat, Jean-François; Mari, Stéphane; Curie, Catherine

    2010-01-01

    In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis. PMID:20228245

  17. Age-associated mitochondrial oxidative decay: Improvement of carnitine acetyltransferase substrate-binding affinity and activity in brain by feeding old rats acetyl-l- carnitine and/or R-α-lipoic acid

    PubMed Central

    Liu, Jiankang; Killilea, David W.; Ames, Bruce N.

    2002-01-01

    We test whether the dysfunction with age of carnitine acetyltransferase (CAT), a key mitochondrial enzyme for fuel utilization, is due to decreased binding affinity for substrate and whether this substrate, fed to old rats, restores CAT activity. The kinetics of CAT were analyzed by using the brains of young and old rats and of old rats supplemented for 7 weeks with the CAT substrate acetyl-l-carnitine (ALCAR) and/or the mitochondrial antioxidant precursor R-α-lipoic acid (LA). Old rats, compared with young rats, showed a decrease in CAT activity and in CAT-binding affinity for both substrates, ALCAR and CoA. Feeding ALCAR or ALCAR plus LA to old rats significantly restored CAT-binding affinity for ALCAR and CoA, and CAT activity. To explore the underlying mechanism, lipid peroxidation and total iron and copper levels were assayed; all increased in old rats. Feeding old rats LA or LA plus ALCAR inhibited lipid peroxidation but did not decrease iron and copper levels. Ex vivo oxidation of young-rat brain with Fe(II) caused loss of CAT activity and binding affinity. In vitro oxidation of purified CAT with Fe(II) inactivated the enzyme but did not alter binding affinity. However, in vitro treatment of CAT with the lipid peroxidation products malondialdehyde or 4-hydroxy-nonenal caused a decrease in CAT-binding affinity and activity, thus mimicking age-related change. Preincubation of CAT with ALCAR or CoA prevented malondialdehyde-induced dysfunction. Thus, feeding old rats high levels of key mitochondrial metabolites can ameliorate oxidative damage, enzyme activity, substrate-binding affinity, and mitochondrial dysfunction. PMID:11854488

  18. Iron and ageing: an introduction to iron regulatory mechanisms.

    PubMed

    Levenson, Cathy W; Tassabehji, Nadine M

    2004-07-01

    While there have been significant advances made in our understanding of the cellular and molecular mechanisms that regulate iron absorption, transport, storage, and utilization, the effect of ageing on these mechanisms and the role of iron in the ageing process is not fully understood. Thus, this review will provide an overview of the iron regulatory mechanisms that may be a factor in the ageing process. Additional reviews in this volume represent an attempt to explore the very latest information on the regulation of iron with a particular emphasis on age-related pathology including mitochondrial function, Parkinson's disease, Alzheimer's disease, stroke, and cardiovascular disease.

  19. Iron homeostasis: new players, newer insights.

    PubMed

    Edison, Eunice S; Bajel, Ashish; Chandy, Mammen

    2008-12-01

    Although iron is a relatively abundant element in the universe, it is estimated that more than 2 billion people worldwide suffer from iron deficiency anemia. Iron deficiency results in impaired production of iron-containing proteins, the most prominent of which is hemoglobin. Cellular iron deficiency inhibits cell growth and subsequently leads to cell death. Hemochromatosis, an inherited disorder results in disproportionate absorption of iron and the extra iron builds up in tissues resulting in organ damage. As both iron deficiency and iron overload have adverse effects, cellular and systemic iron homeostasis is critically important. Recent advances in the field of iron metabolism have led to newer understanding of the pathways involved in iron homeostasis and the diseases which arise from alteration in the regulators. Although insight into this complex regulation of the proteins involved in iron homeostasis has been obtained mainly through animal studies, it is most likely that this knowledge can be directly extrapolated to humans.

  20. Solid support resins and affinity purification mass spectrometry.

    PubMed

    Havis, Spencer; Moree, Wilna J; Mali, Sujina; Bark, Steven J

    2017-02-28

    Co-affinity purification-mass spectrometry (CoAP-MS) is a primary technology for elucidating the protein-protein interactions that form the basis of all biological processes. A critical component of CoAP-MS is the affinity purification (AP) of the bait protein, usually by immobilization of an antibody to a solid-phase resin. This Minireview discusses common resins, reagents, tagging methods, and their consideration for successful AP of tagged proteins. We discuss our experiences with different solid supports, their impact in AP experiments, and propose areas where chemistry can advance this important technology.

  1. Affine generalization of the Komar complex of general relativity

    NASA Astrophysics Data System (ADS)

    Mielke, Eckehard W.

    2001-02-01

    On the basis of the ``on shell'' Noether identities of the metric-affine gauge approach of gravity, an affine superpotential is derived which comprises the energy- and angular-momentum content of exact solutions. In the special case of general relativity (GR) or its teleparallel equivalent, the Komar or Freud complex, respectively, are recovered. Applying this to the spontaneously broken anti-de Sitter gauge model of McDowell and Mansouri with an induced Euler term automatically yields the correct mass and spin of the Kerr-AdS solution of GR with a (induced) cosmological constant without the factor two discrepancy of the Komar formula.

  2. A quantum affine algebra for the deformed Hubbard chain

    NASA Astrophysics Data System (ADS)

    Beisert, Niklas; Galleas, Wellington; Matsumoto, Takuya

    2012-09-01

    The integrable structure of the one-dimensional Hubbard model is based on Shastry's R-matrix and the Yangian of a centrally extended \\mathfrak {sl}(2|2) superalgebra. Alcaraz and Bariev have shown that the model admits an integrable deformation whose R-matrix has recently been found. This R-matrix is of trigonometric type and here we derive its underlying exceptional quantum affine algebra. We also show how the algebra reduces to the above-mentioned Yangian and to the conventional quantum affine \\mathfrak {sl}(2|2) algebra in two special limits.

  3. Iron-Binding Catechols and Virulence in Escherichia coli

    PubMed Central

    Rogers, Henry J.

    1973-01-01

    Previous work suggested that virulent bacteria, which can grow rapidly in serum, must possess a specific mechanism for removing iron from its transferrin complex. Two strains of Escherichia coli were examined with this in mind. Strain O141, which showed inoculum-dependent growth in serum and multiplied in the mouse peritoneum, secreted iron-binding catechols into both synthetic medium and serum. One of these compounds has an association constant for iron similar to that of transferrin. Both transferrin and ethylenediamine-di-o-hydroxyphenyl acetic acid (EDDA), which have very high affinities for ferric iron, induced catechol synthesis in growing cultures of strain O111. This organism was inhibited by normal horse serum. Further work showed that traces of specific antibody inhibited catechol synthesis by O111 exposed to EDDA; therefore, the existence of this inhibitory process means that the organism can no longer obtain Fe3+, which all remains bound to transferrin in serum. In vivo, the inhibition of O111 is similar to that produced by serum in vitro. Neither phagocytosis nor killing by complement appeared to be of any significance during the first 4 h of the infections. Significantly, the purified catechol was capable of abolishing bacteriostasis in vivo. Since these results show that the production of iron-binding catechols is essential for rapid bacterial growth both in vitro and in vivo, these compounds should therefore be considered as true virulence factors. Conversely, any interference by the host with the production or activity of these compounds would constitute an important aspect of antibacterial defense. Images PMID:16558077

  4. Microbial reduction of iron ore

    DOEpatents

    Hoffmann, M.R.; Arnold, R.G.; Stephanopoulos, G.

    1989-11-14

    A process is provided for reducing iron ore by treatment with microorganisms which comprises forming an aqueous mixture of iron ore, microorganisms operable for reducing the ferric iron of the iron ore to ferrous iron, and a substrate operable as an energy source for the microbial reduction; and maintaining the aqueous mixture for a period of time and under conditions operable to effect the reduction of the ore. Preferably the microorganism is Pseudomonas sp. 200 and the reduction conducted anaerobically with a domestic wastewater as the substrate. An aqueous solution containing soluble ferrous iron can be separated from the reacted mixture, treated with a base to precipitate ferrous hydroxide which can then be recovered as a concentrated slurry. 11 figs.

  5. Microbial reduction of iron ore

    DOEpatents

    Hoffmann, Michael R.; Arnold, Robert G.; Stephanopoulos, Gregory

    1989-01-01

    A process is provided for reducing iron ore by treatment with microorganisms which comprises forming an aqueous mixture of iron ore, microorganisms operable for reducing the ferric iron of the iron ore to ferrous iron, and a substrate operable as an energy source for the microbial reduction; and maintaining the aqueous mixture for a period of time and under conditions operable to effect the reduction of the ore. Preferably the microorganism is Pseudomonas sp. 200 and the reduction conducted anaerobically with a domestic wastewater as the substrate. An aqueous solution containing soluble ferrous iron can be separated from the reacted mixture, treated with a base to precipitate ferrous hydroxide which can then be recovered as a concentrated slurry.

  6. Iron deficiency and brain development.

    PubMed

    Lozoff, Betsy; Georgieff, Michael K

    2006-09-01

    Iron deficiency (ID) is common in pregnant women and infants worldwide. Rodent models show that ID during gestation/lactation alters neurometabolism, neurotransmitters, myelination, and gene/protein profiles before and after iron repletion at weaning. Human infants with iron deficiency anemia test lower in cognitive, motor, social-emotional, and neurophysiologic development than comparison group infants. Iron therapy does not consistently improve developmental outcome, with long-term differences observed. Poorer outcome has also been shown in human and monkey infants with fetal/neonatal ID. Recent randomized trials of infant iron supplementation show benefits, indicating that adverse effects can be prevented and/or reversed with iron earlier in development or before ID becomes severe or chronic. This body of research emphasizes the importance of protecting the developing brain from ID.

  7. The Irony of Iron – Biogenic Iron Oxides as an Iron Source to the Ocean

    PubMed Central

    Emerson, David

    2016-01-01

    Primary productivity in at least a third of the sunlit open ocean is thought to be iron-limited. Primary sources of dissolved iron (dFe) to the ocean are hydrothermal venting, flux from the sediments along continental margins, and airborne dust. This article provides a general review of sources of hydrothermal and sedimentary iron to the ocean, and speculates upon the role that iron-cycling microbes play in controlling iron dynamics from these sources. Special attention is paid to iron-oxidizing bacteria (FeOB) that live by oxidizing iron and producing biogenic iron oxides as waste products. The presence and ubiquity of FeOB both at hydrothermal systems and in sediments is only beginning to be appreciated. The biogenic oxides they produce have unique properties that could contribute significantly to the dynamics of dFe in the ocean. Changes in the physical and chemical characteristics of the ocean due to climate change and ocean acidification will undoubtedly impact the microbial iron cycle. A better understanding of the contemporary role of microbes in the iron cycle will help in predicting how these changes could ultimately influence marine primary productivity. PMID:26779157

  8. The Irony of Iron - Biogenic Iron Oxides as an Iron Source to the Ocean.

    PubMed

    Emerson, David

    2015-01-01

    Primary productivity in at least a third of the sunlit open ocean is thought to be iron-limited. Primary sources of dissolved iron (dFe) to the ocean are hydrothermal venting, flux from the sediments along continental margins, and airborne dust. This article provides a general review of sources of hydrothermal and sedimentary iron to the ocean, and speculates upon the role that iron-cycling microbes play in controlling iron dynamics from these sources. Special attention is paid to iron-oxidizing bacteria (FeOB) that live by oxidizing iron and producing biogenic iron oxides as waste products. The presence and ubiquity of FeOB both at hydrothermal systems and in sediments is only beginning to be appreciated. The biogenic oxides they produce have unique properties that could contribute significantly to the dynamics of dFe in the ocean. Changes in the physical and chemical characteristics of the ocean due to climate change and ocean acidification will undoubtedly impact the microbial iron cycle. A better understanding of the contemporary role of microbes in the iron cycle will help in predicting how these changes could ultimately influence marine primary productivity.

  9. Iron deficiency in the young athlete.

    PubMed

    Rowland, T W

    1990-10-01

    Although overt anemia is uncommon, depletion of body iron stores is common among adolescent female athletes. Poor dietary iron intake, menstruation, and increased iron losses associated with physical training all appear to be important factors. Whether nonanemic iron deficiency can impair exercise performance is uncertain. Nonetheless, athletes with low ferritin levels are at risk for impaired erythropoiesis and should receive therapeutic iron supplementation.

  10. 21 CFR 184.1375 - Iron, elemental.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... by any of the following processes: reduced iron, electrolytic iron, and carbonyl iron. (1) Reduced... temperature. The process results in a grayish-black powder, all of which should pass through a 100-mesh sieve... stable in dry air. (3) Carbonyl iron is prepared by the decomposition of iron pentacarbonyl. It occurs...

  11. Four new iron meteorite finds

    NASA Technical Reports Server (NTRS)

    Scott, E. R. D.; Wasson, J. T.; Bild, R. W.

    1977-01-01

    Four new iron meteorites are described: Buenaventura (IIIB) from Chihuahua, Mexico: mass 114 kg; Denver City (anomalous) from Texas, USA: mass 26.1 kg; Kinsella (IIIB) from Alberta, Canada: mass 3.7 kg; and Tacoma (IA) from Washington, USA: mass 17 g. Denver City is unique - i.e., not related to any other known iron. Tacoma is the smallest iron meteorite recorded. The meteorites were initially discovered in 1969, 1975, 1946, and between 1925 and 1932, respectively.

  12. Gum Arabic coated magnetic nanoparticles with affinity ligands specific for antibodies.

    PubMed

    Batalha, Iris L; Hussain, Abid; Roque, A C A

    2010-01-01

    A novel magnetic support based on gum Arabic (GA) coated iron oxide magnetic nanoparticles (MNP) has been endowed with affinity properties towards immunoglobulin G (IgG) molecules. The success of the in situ triazine ligand synthesis was confirmed by fluorescence assays. Two synthetic ligands previously developed for binding to IgG, named as ligand 22/8 (artificial Protein A) and ligand 8/7 (artificial Protein L) were immobilized on to MNPs coated with GA (MNP_GA). The dimension of the particles core was not affected by the surface functionalization with GA and triazine ligands. The hydrodynamic diameters of the magnetic supports indicate that the coupling of GA leads to the formation of larger agglomerates of particles with about 1 microm, but the introduction of the triazine ligands leads to a decrease on MNPs size. The non-functionalized MNP_GA bound 28 mg IgG/g, two times less than bare MNP (60 mg IgG/g). MNP_GA modified with ligand 22/8 bound 133 mg IgG/g support, twice higher than the value obtained for ligand 8/7 magnetic adsorbents (65 mg/g). Supports modified with ligand 22/8 were selected to study the adsorption and the elution of IgG. The adsorption of human IgG on this support followed a Langmuir behavior with a Q(máx) of 344 mg IgG/g support and K(a) of 1.5 x 10(5) M. The studies on different elution conditions indicated that although the 0.05 M citrate buffer (pH 3) presented good recovery yields (elution 64% of bound protein), there was occurrence of iron leaching at this acidic pH. Therefore, a potential alternative would be to elute bound protein with a 0.05 M glycine-NaOH (pH 11) buffer.

  13. Aerosol Iron/Phytoplankton Interactions

    DTIC Science & Technology

    1994-08-16

    effect on the photocycling of able and particulate iron in the euphotic zone at iron. The TRFe levels in carboys to which catalase O0N, 140’W that...experiments without catalase (Figs. 4 filter averaged 0.05 nM in the upper 100 m and and 5). The effects of adding catalase to a carboy particulate iron... Effects on dimethyl sulfide and dimethylsulphoniopropionate production. Nightingale, P. D., Turner, S. M. Malin, G. and L1ss, P. S. Iron

  14. Iron Deficiency Anemia in Pregnancy.

    PubMed

    Breymann, Christian

    2015-10-01

    Anemia is a common problem in obstetrics and perinatal care. Any hemoglobin below 10.5 g/dL can be regarded as true anemia regardless of gestational age. Reasons for anemia in pregnancy are mainly nutritional deficiencies, parasitic and bacterial diseases, and inborn red blood cell disorders such as thalassemias. The main cause of anemia in obstetrics is iron deficiency, which has a worldwide prevalence between estimated 20%-80% and consists of a primarily female population. Stages of iron deficiency are depletion of iron stores, iron-deficient erythropoiesis without anemia, and iron deficiency anemia, the most pronounced form of iron deficiency. Pregnancy anemia can be aggravated by various conditions such as uterine or placental bleedings, gastrointestinal bleedings, and peripartum blood loss. In addition to the general consequences of anemia, there are specific risks during pregnancy for the mother and the fetus such as intrauterine growth retardation, prematurity, feto-placental miss ratio, and higher risk for peripartum blood transfusion. Besides the importance of prophylaxis of iron deficiency, the main therapy options for the treatment of pregnancy anemia are oral iron and intravenous iron preparations.

  15. Chemiluminescence of iron-chlorophyllin.

    PubMed

    Nagoshi, Toshimasa; Ohno, Osamu; Kotake, Tomohiko; Igarashi, Shukuro

    2005-01-01

    The iron-chlorophyllin complex was found to be chemiluminescent (CL) in an acetonitrile (22%)/water mixed solvent. In the presence of hydrogen peroxide, when iron-chlorophyllin was added to the mixed solvent, a sharp CL signal immediately appeared. Also, analysis of the absorption spectra revealed decomposition of iron-chlorophyllin (based on decrease in absorbance at 396 nm), hence iron-chlorophyllin is the CL substance. Moreover, the CL intensity decreased in the presence of potassium thiocyanate (KSCN), indicating that the axial coordinative position of iron-chlorophyllin acts as a point of catalytic activation. In addition, when fluorophores were present with iron-chlorophyllin CL, their CL intensity values were similar to or greater than that of the well-known trichlorophenylperoxalate ester (TCPO) CL. Thus, during the decomposition reaction of iron-chlorophyllin, the latter transfers its energy to the coexisting fluorophores. Moreover, since the decomposed compound in this CL reaction had a fluorescence, it was found that the iron-chlorophyllin also functions as an energy donor. Therefore, the iron-chlorophyllin complex acts not only as a CL substance, but also as a catalyst and energy donor in the reaction.

  16. Iron, microbiota and colorectal cancer.

    PubMed

    Ng, Oliver

    2016-10-01

    Iron deficiency and anaemia are common in colorectal cancer. Replacement with oral or intravenous iron effectively treats this deficiency. However, mechanistic and population studies suggest that excess iron promotes colorectal carcinogenesis. Growing research into gut microbiota and dysbiosis suggests one explanation for this association. Iron is growth limiting for many pathogenic bacteria and may promote a shift in the ratio of pathogenic to protective bacteria. This may increase the toxic bacterial metabolites, promoting inflammation and carcinogenesis. This has important implications as we seek to correct anaemia in our patients.

  17. 46 CFR 56.60-10 - Cast iron and malleable iron.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 46 Shipping 2 2011-10-01 2011-10-01 false Cast iron and malleable iron. 56.60-10 Section 56.60-10... APPURTENANCES Materials § 56.60-10 Cast iron and malleable iron. (a) The low ductility of cast iron and malleable iron should be recognized and the use of these metals where shock loading may occur should...

  18. 46 CFR 56.60-10 - Cast iron and malleable iron.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 46 Shipping 2 2014-10-01 2014-10-01 false Cast iron and malleable iron. 56.60-10 Section 56.60-10... APPURTENANCES Materials § 56.60-10 Cast iron and malleable iron. (a) The low ductility of cast iron and malleable iron should be recognized and the use of these metals where shock loading may occur should...

  19. 46 CFR 56.60-10 - Cast iron and malleable iron.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 46 Shipping 2 2012-10-01 2012-10-01 false Cast iron and malleable iron. 56.60-10 Section 56.60-10... APPURTENANCES Materials § 56.60-10 Cast iron and malleable iron. (a) The low ductility of cast iron and malleable iron should be recognized and the use of these metals where shock loading may occur should...

  20. 49 CFR 192.489 - Remedial measures: Cast iron and ductile iron pipelines.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 3 2012-10-01 2012-10-01 false Remedial measures: Cast iron and ductile iron... for Corrosion Control § 192.489 Remedial measures: Cast iron and ductile iron pipelines. (a) General graphitization. Each segment of cast iron or ductile iron pipe on which general graphitization is found to...

  1. 49 CFR 192.489 - Remedial measures: Cast iron and ductile iron pipelines.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 3 2011-10-01 2011-10-01 false Remedial measures: Cast iron and ductile iron... for Corrosion Control § 192.489 Remedial measures: Cast iron and ductile iron pipelines. (a) General graphitization. Each segment of cast iron or ductile iron pipe on which general graphitization is found to...

  2. 46 CFR 56.60-10 - Cast iron and malleable iron.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 46 Shipping 2 2013-10-01 2013-10-01 false Cast iron and malleable iron. 56.60-10 Section 56.60-10... APPURTENANCES Materials § 56.60-10 Cast iron and malleable iron. (a) The low ductility of cast iron and malleable iron should be recognized and the use of these metals where shock loading may occur should...

  3. 49 CFR 192.489 - Remedial measures: Cast iron and ductile iron pipelines.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 3 2013-10-01 2013-10-01 false Remedial measures: Cast iron and ductile iron... for Corrosion Control § 192.489 Remedial measures: Cast iron and ductile iron pipelines. (a) General graphitization. Each segment of cast iron or ductile iron pipe on which general graphitization is found to...

  4. 49 CFR 192.489 - Remedial measures: Cast iron and ductile iron pipelines.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 3 2014-10-01 2014-10-01 false Remedial measures: Cast iron and ductile iron... for Corrosion Control § 192.489 Remedial measures: Cast iron and ductile iron pipelines. (a) General graphitization. Each segment of cast iron or ductile iron pipe on which general graphitization is found to...

  5. 46 CFR 56.60-10 - Cast iron and malleable iron.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 46 Shipping 2 2010-10-01 2010-10-01 false Cast iron and malleable iron. 56.60-10 Section 56.60-10... APPURTENANCES Materials § 56.60-10 Cast iron and malleable iron. (a) The low ductility of cast iron and malleable iron should be recognized and the use of these metals where shock loading may occur should...

  6. 49 CFR 192.489 - Remedial measures: Cast iron and ductile iron pipelines.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 3 2010-10-01 2010-10-01 false Remedial measures: Cast iron and ductile iron... for Corrosion Control § 192.489 Remedial measures: Cast iron and ductile iron pipelines. (a) General graphitization. Each segment of cast iron or ductile iron pipe on which general graphitization is found to...

  7. Synthesis of hierarchical iron hydrogen phosphate crystal as a robust peroxidase mimic for stable H₂O₂ detection.

    PubMed

    Zhang, Tongbao; Lu, Yangcheng; Luo, Guangsheng

    2014-08-27

    To develop a green, cost-efficient and robust peroxidase mimic, micro/nano hierarchical morphology (for ease of separation and reuse), relative chemically stable composition (for ease of storage) and stable crystal structure (for long-term stability) are highly desired. Herein, using phosphoric acid as a chelating ligand to control the release of iron ions, hierarchical iron(III) hydrogen phosphate hydrate crystals are successfully prepared by nanosheets formation and following self-assembling in a facile low-temperature hydrothermal process. They are first found to have peroxidase-like activity and showed higher affinity for H2O2 and lower affinity for 3,3',5,5'-tetramethylbenzidine compared with horseradish peroxidase. The affinity feature is used for quantitative detection of H2O2 and shows a wide linear detection range from 57.4 to 525.8 μM (R(2) = 0.994) with a low detection limit of 1 μM. Benefited from chemical stability of hierarchical iron(III) salt crystals, they own good reproducibility (relative standard deviation = 1.95% for 10 independent measurements), long-term stability (no activity loss after 10 cycles), and ease of recovery (by simple centrifugation). Because the method is easily accessible, iron hydrogen phosphate hierarchical crystals have great potential for practical use of H2O2 sensing and detection under harsh conditions.

  8. Effects of Iron Limitation on the Degradation of Toluene by Pseudomonas Strains Carrying the TOL (pWWO) Plasmid

    PubMed Central

    Dinkla, Inez J. T.; Gabor, Esther M.; Janssen, Dick B.

    2001-01-01

    Most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases. In iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants. We investigated the effects of iron limitation on the degradation of toluene by Pseudomonas putida mt2 and the transconjugant rhizosphere bacterium P. putida WCS358(pWWO), both of which contain the pWWO (TOL) plasmid that harbors the genes for toluene degradation. The results of continuous-culture experiments showed that the activity of the upper-pathway toluene monooxygenase decreased but that the activity of benzyl alcohol dehydrogenase was not affected under iron-limited conditions. In contrast, the activities of three meta-pathway (lower-pathway) enzymes were all found to be reduced when iron concentrations were decreased. Additional experiments in which citrate was used as a growth substrate and the pathways were induced with the gratuitous inducer o-xylene showed that expression of the TOL genes increased the iron requirement in both strains. Growth yields were reduced and substrate affinities decreased under iron-limited conditions, suggesting that iron availability can be an important parameter in the oxidative breakdown of hydrocarbons. PMID:11472911

  9. Evidence for a protein-protein complex during iron loading into ferritin by ceruloplasmin.

    PubMed

    Reilly, C A; Sorlie, M; Aust, S D

    1998-06-01

    The formation of a protein-protein complex for the loading of iron into ferritin by ceruloplasmin was investigated. Ferritin stimulated the ferroxidase activity of ceruloplasmin unless the ferritin was fully loaded, in which case it inhibited the ferroxidase activity of ceruloplasmin. The apparent association constant for the interaction of ferritin and ceruloplasmin was 24 nM. Isothermal titration calorimetry indicated that the interaction of ceruloplasmin and ferritin was endothermic, driven by positive changes in entropy. The association constants for complex formation between ferritin and ceruloplasmin were 4.5 +/- 0.7 x 10(5) and 9.5 +/- 0.3 x 10(4) M-1 for the reduced and oxidized forms of ceruloplasmin, respectively. The oxidized form of ceruloplasmin was retained on an affinity column with ferritin immobilized as the ligand and remained bound to the column with mobile phases of increased hydrophobicity, but was eluted with increased ionic strength. The ability of ceruloplasmin to remain bound to the affinity resin was affected by the species from which ceruloplasmin was isolated. Gradient ultracentrifugation also provided evidence that the two proteins were associated, since ferritin promoted migration of ceruloplasmin through the gradient. Including ferrous iron in the gradient resulted in reduction of ceruloplasmin and increased the mobility of ceruloplasmin with ferritin. These data provide evidence that ferritin and ceruloplasmin form a protein-protein complex during iron loading into ferritin, which may limit redox cycling of iron in vivo.

  10. Iron oxide surfaces

    NASA Astrophysics Data System (ADS)

    Parkinson, Gareth S.

    2016-03-01

    The current status of knowledge regarding the surfaces of the iron oxides, magnetite (Fe3O4), maghemite (γ-Fe2O3), haematite (α-Fe2O3), and wüstite (Fe1-xO) is reviewed. The paper starts with a summary of applications where iron oxide surfaces play a major role, including corrosion, catalysis, spintronics, magnetic nanoparticles (MNPs), biomedicine, photoelectrochemical water splitting and groundwater remediation. The bulk structure and properties are then briefly presented; each compound is based on a close-packed anion lattice, with a different distribution and oxidation state of the Fe cations in interstitial sites. The bulk defect chemistry is dominated by cation vacancies and interstitials (not oxygen vacancies) and this provides the context to understand iron oxide surfaces, which represent the front line in reduction and oxidation processes. Fe diffuses in and out from the bulk in response to the O2 chemical potential, forming sometimes complex intermediate phases at the surface. For example, α-Fe2O3 adopts Fe3O4-like surfaces in reducing conditions, and Fe3O4 adopts Fe1-xO-like structures in further reducing conditions still. It is argued that known bulk defect structures are an excellent starting point in building models for iron oxide surfaces. The atomic-scale structure of the low-index surfaces of iron oxides is the major focus of this review. Fe3O4 is the most studied iron oxide in surface science, primarily because its stability range corresponds nicely to the ultra-high vacuum environment. It is also an electrical conductor, which makes it straightforward to study with the most commonly used surface science methods such as photoemission spectroscopies (XPS, UPS) and scanning tunneling microscopy (STM). The impact of the surfaces on the measurement of bulk properties such as magnetism, the Verwey transition and the (predicted) half-metallicity is discussed. The best understood iron oxide surface at present is probably Fe3O4(100); the structure is

  11. Student Engagement and Neoliberalism: Mapping an Elective Affinity

    ERIC Educational Resources Information Center

    Zepke, Nick

    2015-01-01

    The purpose of this article is to argue that student engagement, an important area for research about learning and teaching in formal higher education, has an elective affinity with neoliberalism, a hegemonic ideology in many countries of the developed world. The paper first surveys an extensive research literature examining student engagement and…

  12. "The Hunger Games": Literature, Literacy, and Online Affinity Spaces

    ERIC Educational Resources Information Center

    Curwood, Jen Scott

    2013-01-01

    This article examines adolescent literacy practices related to "The Hunger Games," a young adult novel and the first of a trilogy. By focusing on the interaction of social identities, discourses, and media paratexts within an online affinity space, this ethnographic study offers insight into how young adults engage with contemporary…

  13. Affinities and beyond! Developing Ways of Seeing in Online Spaces

    ERIC Educational Resources Information Center

    Davies, Julia

    2006-01-01

    This article presents an insider view of an online community of adults involved in sharing digital photography through a host website, Flickr. It describes how reciprocal teaching and learning partnerships in a dynamic multimodal environment are achieved through the creation of a "Third Space" or "Affinity Space", where "Funds of Knowledge" are…

  14. Electrochemical affinity biosensors for detection of mycotoxins: A review.

    PubMed

    Vidal, Juan C; Bonel, Laura; Ezquerra, Alba; Hernández, Susana; Bertolín, Juan R; Cubel, Carlota; Castillo, Juan R

    2013-11-15

    This review discusses the current state of electrochemical biosensors in the determination of mycotoxins in foods. Mycotoxins are highly toxic secondary metabolites produced by molds. The acute toxicity of these results in serious human and animal health problems, although it has been only since early 1960s when the first studied aflatoxins were found to be carcinogenic. Mycotoxins affect a broad range of agricultural products, most important cereals and cereal-based foods. A majority of countries, mentioning especially the European Union, have established preventive programs to control contamination and strict laws of the permitted levels in foods. Official methods of analysis of mycotoxins normally requires sophisticated instrumentation, e.g. liquid chromatography with fluorescence or mass detectors, combined with extraction procedures for sample preparation. For about sixteen years, the use of simpler and faster analytical procedures based on affinity biosensors has emerged in scientific literature as a very promising alternative, particularly electrochemical (i.e., amperometric, impedance, potentiometric or conductimetric) affinity biosensors due to their simplicity and sensitivity. Typically, electrochemical biosensors for mycotoxins use specific antibodies or aptamers as affinity ligands, although recombinant antibodies, artificial receptors and molecular imprinted polymers show potential utility. This article deals with recent advances in electrochemical affinity biosensors for mycotoxins and covers complete literature from the first reports about sixteen years ago.

  15. Toward an Affinity Space Methodology: Considerations for Literacy Research

    ERIC Educational Resources Information Center

    Lammers, Jayne C.; Curwood, Jen Scott; Magnifico, Alecia Marie

    2012-01-01

    As researchers seek to make sense of young people's online literacy practices and participation, questions of methodology are important to consider. In our work to understand the culture of physical, virtual and blended spheres that adolescents inhabit, we find it necessary to expand Gee's (2004) notion of affinity spaces. In this article, we draw…

  16. A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

    PubMed Central

    Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A.; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

    2016-01-01

    Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6. PMID:27291418

  17. Kinetic Studies of Biological Interactions By Affinity Chromatography

    PubMed Central

    Schiel, John E.; Hage, David S.

    2009-01-01

    The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This review will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this review and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. PMID:19391173

  18. Affinity of cefoperazone for penicillin-binding proteins.

    PubMed Central

    Matsubara, N; Minami, S; Matsuhashi, M; Takaoka, M; Mitsuhashi, S

    1980-01-01

    Cefoperazone (T-1551, CFP) a new semisynthetic cephalosporin, has a broad spectrum of antibacterial activity. We investigated the affinity of CFP to penicillin-binding proteins (PBPs) and the inhibition of peptidoglycan synthesis by CFP. CFP had high affinities for Escherichia coli PBP-3, -1Bs, -2, and -1A, in descending order, and low affinities for PBP-4, -5, and -6. Similarly, CFP showed high affinity for Pseudomonas aeruginosa PBP-3, -1A, -1B, -2, and -4, in descending order. It is known that E. coli PBP-3 and P. aeruginosa PBP-3 participate in cell division. These results are in good agreement with the formation of filamentous cells of E. coli and P. aeruginosa treated with CFP. CFP had lower inhibitory activities on D-alanine carboxypeptidase IA and IB of E. coli than that of penicillin G, but its inhibitory activities on the cross-link formation in peptidoglycan synthesis were the same as those of penicillin G and higher than those of ampicillin. Images PMID:6448021

  19. A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

    NASA Astrophysics Data System (ADS)

    Velisetty, Phanindra; Borbiro, Istvan; Kasimova, Marina A.; Liu, Luyu; Badheka, Doreen; Carnevale, Vincenzo; Rohacs, Tibor

    2016-06-01

    Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6.

  20. Limit measures for affine cellular automata on topological Markov subgroups

    NASA Astrophysics Data System (ADS)

    Maass, Alejandro; Martínez, Servet; Sobottka, Marcelo

    2006-09-01

    Consider a topological Markov subgroup which is ps-torsion (with p prime) and an affine cellular automaton defined on it. We show that the Cesàro mean of the iterates, by the automaton of a probability measure with complete connections and summable memory decay that is compatible with the topological Markov subgroup, converges to the Haar measure.