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Sample records for facilitative nucleobase transporter

  1. Purine nucleobase transport in human erythrocytes. Reinvestigation with a novel "inhibitor-stop" assay.

    PubMed

    Domin, B A; Mahony, W B; Zimmerman, T P

    1988-07-05

    A novel "inhibitor-stop" method for the determination of initial rates of purine nucleobase transport in human erythrocytes has been developed, based on the addition of seven assay volumes of cold 19 mM papaverine to terminate influx. In view of our finding that the initial velocities of adenine, guanine, and hypoxanthine influx into human erythrocytes were linear for only 4-6 s at 37 degrees C, the present method has been used to reexamine the kinetics of purine nucleobase transport in these cells. Initial influx rates of all three purine nucleobases were shown to be the result of concurrent facilitated and nonfacilitated diffusion. The nonfacilitated influx rates could be estimated either from the linear concentration dependence of nucleobase influx at high concentrations of permeant or from residual influx rates which were not inhibited by the presence of co-permeants. Appropriate corrections for nonfacilitated diffusion were made to the influx rates observed at low nucleobase concentrations. Kinetic analyses indicated that adenine (Km = 13 +/- 1 microM, n = 7), guanine (Km = 37 +/- 2 microM, n = 5), and hypoxanthine (Km = 180 +/- 12 microM, n = 6) were mutually competitive substrates for transport. The Ki values obtained with each nucleobase as an inhibitor of the influx of the other nucleobases were similar to their respective Km values for influx. Furthermore, the transport of the purine nucleobases was not inhibited by nucleosides (uridine, inosine) or by inhibitors of nucleoside transport (6-[(4-nitrobenzyl)thio]-9-beta-D-ribofuranosylpurine, dilazep, dipyridamole). It is concluded that all three purine nucleobases share a common facilitated transport system in human erythrocytes which is functionally distinct from the nucleoside transporter.

  2. Nucleobase and nucleoside transport and integration into plant metabolism.

    PubMed

    Girke, Christopher; Daumann, Manuel; Niopek-Witz, Sandra; Möhlmann, Torsten

    2014-01-01

    Nucleotide metabolism is an essential process in all living organisms. Besides newly synthesized nucleotides, the recycling (salvage) of partially degraded nucleotides, i.e., nucleosides and nucleobases serves to keep the homeostasis of the nucleotide pool. Both types of metabolites are substrates of at least six families of transport proteins in Arabidopsis thaliana (Arabidopsis) with a total of 49 members. In the last years several members of such transport proteins have been analyzed allowing to present a more detailed picture of nucleoside and nucleobase transport and the physiological function of these processes. Besides functioning in nucleotide metabolism it turned out that individual members of the before named transporters exhibit the capacity to transport a wide range of different substrates including vitamins and phytohormones. The aim of this review is to summarize the current knowledge on nucleobase and nucleoside transport processes in plants and integrate this into nucleotide metabolism in general. Thereby, we will focus on those proteins which have been characterized at the biochemical level.

  3. Nucleobase and nucleoside transport and integration into plant metabolism

    PubMed Central

    Girke, Christopher; Daumann, Manuel; Niopek-Witz, Sandra; Möhlmann, Torsten

    2014-01-01

    Nucleotide metabolism is an essential process in all living organisms. Besides newly synthesized nucleotides, the recycling (salvage) of partially degraded nucleotides, i.e., nucleosides and nucleobases serves to keep the homeostasis of the nucleotide pool. Both types of metabolites are substrates of at least six families of transport proteins in Arabidopsis thaliana (Arabidopsis) with a total of 49 members. In the last years several members of such transport proteins have been analyzed allowing to present a more detailed picture of nucleoside and nucleobase transport and the physiological function of these processes. Besides functioning in nucleotide metabolism it turned out that individual members of the before named transporters exhibit the capacity to transport a wide range of different substrates including vitamins and phytohormones. The aim of this review is to summarize the current knowledge on nucleobase and nucleoside transport processes in plants and integrate this into nucleotide metabolism in general. Thereby, we will focus on those proteins which have been characterized at the biochemical level. PMID:25250038

  4. Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.

    PubMed

    Young, J D; Yao, S Y M; Sun, L; Cass, C E; Baldwin, S A

    2008-07-01

    1. The human (h) SLC29 family of integral membrane proteins is represented by four members, designated equilibrative nucleoside transporters (ENTs) because of the properties of the first-characterized family member, hENT1. They belong to the widely distributed eukaryotic ENT family of equilibrative and concentrative nucleoside/nucleobase transporter proteins. 2. A predicted topology of eleven transmembrane helices has been experimentally confirmed for hENT1. The best-characterized members of the family, hENT1 and hENT2, possess similar broad permeant selectivities for purine and pyrimidine nucleosides, but hENT2 also efficiently transports nucleobases. hENT3 has a similar broad permeant selectivity for nucleosides and nucleobases and appears to function in intracellular membranes, including lysosomes. 3. hENT4 is uniquely selective for adenosine, and also transports a variety of organic cations. hENT3 and hENT4 are pH sensitive, and optimally active under acidic conditions. ENTs, including those in parasitic protozoa, function in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis and, in humans, are also responsible for the cellular uptake of nucleoside analogues used in the treatment of cancers and viral diseases. 4. By regulating the concentration of adenosine available to cell surface receptors, mammalian ENTs additionally influence physiological processes ranging from cardiovascular activity to neurotransmission.

  5. Identification and Functional Characterization of the First Nucleobase Transporter in Mammals

    PubMed Central

    Yamamoto, Syunsuke; Inoue, Katsuhisa; Murata, Tomoaki; Kamigaso, Syunsuke; Yasujima, Tomoya; Maeda, Jun-ya; Yoshida, Yukihiro; Ohta, Kin-ya; Yuasa, Hiroaki

    2010-01-01

    Nucleobases are important compounds that constitute nucleosides and nucleic acids. Although it has long been suggested that specific transporters are involved in their intestinal absorption and uptake in other tissues, none of their molecular entities have been identified in mammals to date. Here we describe identification of rat Slc23a4 as the first sodium-dependent nucleobase transporter (rSNBT1). The mRNA of rSNBT1 was expressed highly and only in the small intestine. When transiently expressed in HEK293 cells, rSNBT1 could transport uracil most efficiently. The transport of uracil mediated by rSNBT1 was sodium-dependent and saturable with a Michaelis constant of 21.2 μm. Thymine, guanine, hypoxanthine, and xanthine were also transported, but adenine was not. It was also suggested by studies of the inhibitory effect on rSNBT1-mediated uracil transport that several nucleobase analogs such as 5-fluorouracil are recognized by rSNBT1, but cytosine and nucleosides are not or only poorly recognized. Furthermore, rSNBT1 fused with green fluorescent protein was mainly localized at the apical membrane, when stably expressed in polarized Madin-Darby canine kidney II cells. These characteristics of rSNBT1 were almost fully in agreement with those of the carrier-mediated transport system involved in intestinal uracil uptake. Therefore, it is likely that rSNBT1 is its molecular entity or at least in part responsible for that. It was also found that the gene orthologous to the rSNBT1 gene is genetically defective in humans. This may have a biological and evolutional meaning in the transport and metabolism of nucleobases. The present study provides novel insights into the specific transport and metabolism of nucleobases and their analogs for therapeutic use. PMID:20042597

  6. Mouse equilibrative nucleoside transporter 2 (mENT2) transports nucleosides and purine nucleobases differing from human and rat ENT2.

    PubMed

    Nagai, Katsuhito; Nagasawa, Kazuki; Kyotani, Yoji; Hifumi, Natsuko; Fujimoto, Sadaki

    2007-05-01

    Several mammalian nucleoside transporters have been identified at the molecular level. Human and rat equilibrative nucleoside transporter 2 (hENT2 and rENT2, respectively) was previously reported to have the dual ability of transporting both nucleosides and nucleobases. In the present study, we characterized the transport of a variety of nucleosides and nucleobases via recombinant mouse ENT2 (mENT2). Cloned mENT2 mediated the uptake of nucleosides and purine nucleobases, but not pyrimidine nucleobases. The mENT2-mediated uptake of adenosine was significantly inhibited by nucleosides and nucleobases, irrespective of purine and pyrimidine. The K(m) values for the uptake of nucleosides and purine nucleobases mediated by mENT2 varied between 1.24 and 16.3 microM, and the transport clearances of adenosine and hypoxanthine via the transporter were greater than those of other substrates. Therefore, we concluded that mENT2 is nucleoside and purine nucleobase transporter, and pyrimidine nucleobases are blockers for the transporter, differing from hENT2 and rENT2 that were reported to also transport pyrimidine nucleobases.

  7. FUN26 (Function Unknown Now 26) Protein from Saccharomyces cerevisiae Is a Broad Selectivity, High Affinity, Nucleoside and Nucleobase Transporter*

    PubMed Central

    Boswell-Casteel, Rebba C.; Johnson, Jennifer M.; Duggan, Kelli D.; Roe-Žurž, Zygy; Schmitz, Hannah; Burleson, Carter; Hays, Franklin A.

    2014-01-01

    Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2′)- and C(5′)-positions on the ribose sugar and is not stimulated by a membrane pH differential. [3H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [3H]adenosine or [3H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system. PMID:25035431

  8. A Transition-State Interaction Shifts Nucleobase Ionization Toward Neutrality to Facilitate Small Ribozyme Catalysis

    PubMed Central

    Liberman, Joseph A.; Guo, Man; Jenkins, Jermaine L.; Krucinska, Jolanta; Chen, Yuanyuan; Carey, Paul R.; Wedekind, Joseph E.

    2012-01-01

    One mechanism by which ribozymes can accelerate biological reactions is by adopting folds that favorably perturb nucleobase ionization. Herein we used Raman crystallography to directly measure pKa values for the Ade38 N1-imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pKa value of 6.27 ± 0.05, which agrees strikingly well with values measured by pH-rate analyses. To identify the chemical attributes that contribute to the shifted pKa we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pKa values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pKa >0.8 log units relative to the precatalytic state. The agreement of the microscopic and macroscopic pKa values and the accompanying structural analysis support a mechanism in which Ade38 N1(H)+ functions as a general acid in phosphodiester bond cleavage. Overall the results quantify the contribution of a single electrostatic interaction to base ionization, which has broad relevance for understanding how RNA structure can control chemical reactivity. PMID:22989273

  9. A transition-state interaction shifts nucleobase ionization toward neutrality to facilitate small ribozyme catalysis.

    PubMed

    Liberman, Joseph A; Guo, Man; Jenkins, Jermaine L; Krucinska, Jolanta; Chen, Yuanyuan; Carey, Paul R; Wedekind, Joseph E

    2012-10-17

    One mechanism by which ribozymes can accelerate biological reactions is by adopting folds that favorably perturb nucleobase ionization. Herein we used Raman crystallography to directly measure pK(a) values for the Ade38 N1 imino group of a hairpin ribozyme in distinct conformational states. A transition-state analogue gave a pK(a) value of 6.27 ± 0.05, which agrees strikingly well with values measured by pH-rate analyses. To identify the chemical attributes that contribute to the shifted pK(a), we determined crystal structures of hairpin ribozyme variants containing single-atom substitutions at the active site and measured their respective Ade38 N1 pK(a) values. This approach led to the identification of a single interaction in the transition-state conformation that elevates the base pK(a) > 0.8 log unit relative to the precatalytic state. The agreement of the microscopic and macroscopic pK(a) values and the accompanying structural analysis supports a mechanism in which Ade38 N1(H)+ functions as a general acid in phosphodiester bond cleavage. Overall the results quantify the contribution of a single electrostatic interaction to base ionization, which has broad relevance for understanding how RNA structure can control chemical reactivity.

  10. Carrier facilitated transport through membranes

    SciTech Connect

    Kaper, H.G.; Leaf, G.K.; Matkowsky, B.J.

    1980-06-01

    Facilitated transport is a process whereby the diffusion of a solute across a membrane is chemically enhanced. In this report an analysis is given of a facilitated transport system involving a volatile species A which reacts with a nonvolatile carrier species B to form the nonvolatile product AB. The species A is transported across the membrane by ordinary diffusion, as well as by the diffusion of the product AB. It is assumed that the reaction rates are large, so the reactions are confined mostly to thin boundary layers near the surfaces of the membrane. The method of matched asymptotic expansions is used to derive the asymptotic solution of the nonlinear boundary value problem governing equilibrium. The effect of various parameters on the facilitation factor is analyzed in detail.

  11. SUPERFUND GROUNDWATER ISSUE - FACILITATED TRANSPORT

    EPA Science Inventory

    The Regional Superfund Ground Water Forum is a group of ground-water scientists representing EPA's Regional Superfund Offices, organized to exchange up to date information related to ground-water remediation at Superfund sites. Facilitated transport is an issue identified by the ...

  12. SUPERFUND GROUNDWATER ISSUE - FACILITATED TRANSPORT

    EPA Science Inventory

    The Regional Superfund Ground Water Forum is a group of ground-water scientists representing EPA's Regional Superfund Offices, organized to exchange up to date information related to ground-water remediation at Superfund sites. Facilitated transport is an issue identified by the ...

  13. Identification of the Substrate Recognition and Transport Pathway in a Eukaryotic Member of the Nucleobase-Ascorbate Transporter (NAT) Family

    PubMed Central

    Kosti, Vasiliki; Lambrinidis, George; Myrianthopoulos, Vassilios; Diallinas, George; Mikros, Emmanuel

    2012-01-01

    Using the crystal structure of the uracil transporter UraA of Escherichia coli, we constructed a 3D model of the Aspergillus nidulans uric acid-xanthine/H+ symporter UapA, which is a prototype member of the Nucleobase-Ascorbate Transporter (NAT) family. The model consists of 14 transmembrane segments (TMSs) divided into a core and a gate domain, the later being distinctly different from that of UraA. By implementing Molecular Mechanics (MM) simulations and quantitative structure-activity relationship (SAR) approaches, we propose a model for the xanthine-UapA complex where the substrate binding site is formed by the polar side chains of residues E356 (TMS8) and Q408 (TMS10) and the backbones of A407 (TMS10) and F155 (TMS3). In addition, our model shows several polar interactions between TMS1-TMS10, TMS1-TMS3, TMS8-TMS10, which seem critical for UapA transport activity. Using extensive docking calculations we identify a cytoplasm-facing substrate trajectory (D360, A363, G411, T416, R417, V463 and A469) connecting the proposed substrate binding site with the cytoplasm, as well as, a possible outward-facing gate leading towards the substrate major binding site. Most importantly, re-evaluation of the plethora of available and analysis of a number of herein constructed UapA mutations strongly supports the UapA structural model. Furthermore, modeling and docking approaches with mammalian NAT homologues provided a molecular rationale on how specificity in this family of carriers might be determined, and further support the importance of selectivity gates acting independently from the major central substrate binding site. PMID:22848666

  14. Structural, Dynamical, and Electronic Transport Properties of Modified DNA Duplexes Containing Size-Expanded Nucleobases

    SciTech Connect

    Fuentes-Cabrera, Miguel A; Orozco, Modesto; Luque, Javier; Sumpter, Bobby G; Blas, Jose; Ordejon, Pablo J; Huertas, Oscar; Tabares, Carolina

    2011-01-01

    Among the distinct strategies proposed to expand the genetic alphabet, sizeexpanded nucleobases are promising for the development of modified DNA duplexes with improved biotechnological properties. In particular, duplexes built up by replacing canonical bases with the corresponding benzo-fused counterparts could be valuable as molecular nanowires. In this context, this study reports the results of classical molecular dynamics simulations carried out to examine the structural and dynamical features of size-expanded DNAs, including both hybrid duplexes containing mixed pairs of natural and benzo-fused bases (xDNA) and pure size-expanded (xxDNA) duplexes. Furthermore, the electronic structure of both natural and size-expanded duplexes is examined by means of density functional computations. The results confirm that the structural and flexibility properties of the canonical DNA are globally little affected by the presence of benzo-fused bases. Themost relevant differences are found in the enhanced size of the grooves, and the reduction in the twist. However, the analysis also reveals subtle structural effects related to the nature and sequence of benzo-fused bases in the duplex. On the other hand, electronic structure calculations performed for xxDNAs confirm the reduction in the HOMOLUMO gap predicted from the analysis of the natural bases and their size-expanded counterparts, which suggests that pure size-expanded DNAs can be good conductors. A more complex situation is found for xDNAs, where fluctuations in the electrostatic interaction between base pairs exerts a decisive influence on the modulation of the energy gap.

  15. Proton transfer in nucleobases is mediated by water.

    PubMed

    Khistyaev, Kirill; Golan, Amir; Bravaya, Ksenia B; Orms, Natalie; Krylov, Anna I; Ahmed, Musahid

    2013-08-08

    Water plays a central role in chemistry and biology by mediating the interactions between molecules, altering energy levels of solvated species, modifying potential energy profiles along reaction coordinates, and facilitating efficient proton transport through ion channels and interfaces. This study investigates proton transfer in a model system comprising dry and microhydrated clusters of nucleobases. With mass spectrometry and tunable vacuum ultraviolet synchrotron radiation, we show that water shuts down ionization-induced proton transfer between nucleobases, which is very efficient in dry clusters. Instead, a new pathway opens up in which protonated nucleobases are generated by proton transfer from the ionized water molecule and elimination of a hydroxyl radical. Electronic structure calculations reveal that the shape of the potential energy profile along the proton transfer coordinate depends strongly on the character of the molecular orbital from which the electron is removed; i.e., the proton transfer from water to nucleobases is barrierless when an ionized state localized on water is accessed. The computed energetics of proton transfer is in excellent agreement with the experimental appearance energies. Possible adiabatic passage on the ground electronic state of the ionized system, though energetically accessible at lower energies, is not efficient. Thus, proton transfer is controlled electronically, by the character of the ionized state, rather than statistically, by simple energy considerations.

  16. Characterization of poly(N-isopropylacrylamide)-nucleobase supramolecular complexes featuring bio-multiple hydrogen bonds.

    PubMed

    Yang, Hsiu-Wen; Lee, Ai-Wei; Huang, Chi-Hsien; Chen, Jem-Kun

    2014-11-07

    In this study we employed poly(N-isopropylacrylamide) (PNIPAAm) as a matrix that we hybridized with five different nucleobase units (adenine, thymine, uracil, guanine, cytosine) to generate PNIPAAm-nucleobase supramolecular complexes (PNSCs) stabilized through bio-multiple hydrogen bonds (BMHBs). These nucleobase units interacted with PNIPAAm through BMHBs of various strengths, leading to competition between the BMHBs and the intramolecular hydrogen bonds (HBs) of PNIPAAm. The changes in morphology, crystalline structure, and thermoresponsive behavior of PNIPAAm were related to the strength of its BMHBs with the nucleobases. The strengths of the BMHBs followed the order guanine > adenine > thymine > cytosine > uracil, as verified through analyses of Fourier transform infrared spectra, lower critical solution temperatures, and inter-association equilibrium constants. The PNSCs also exhibited remarkable improvements in conductivity upon the formation of BMHBs, which facilitated proton transport. The neat PNIPAAm film was an insulator, but it transformed into a semiconductor after hybridizing with the nucleobases. In particular, the resistivity of the PNIPAAm-guanine supramolecular complex decreased to 1.35 × 10(5) ohm cm. The resistivity of the PNIPAAm-cytosine supramolecular complex increased significantly from 5.83 × 10(6) to 3 × 10(8) ohm cm upon increasing the temperature from 40 to 50 °C, suggesting that this material might have applicability in thermo-sensing. The ability to significantly improve the conductivity of hydrogels through such a simple approach involving BMHBs might facilitate their use as novel materials in bioelectronics.

  17. Avian and Mammalian Facilitative Glucose Transporters.

    PubMed

    Byers, Mary Shannon; Howard, Christianna; Wang, Xiaofei

    2017-04-05

    The GLUT members belong to a family of glucose transporter proteins that facilitate glucose transport across the cell membrane. The mammalian GLUT family consists of thirteen members (GLUTs 1-12 and H⁺-myo-inositol transporter (HMIT)). Humans have a recently duplicated GLUT member, GLUT14. Avians express the majority of GLUT members. The arrangement of multiple GLUTs across all somatic tissues signifies the important role of glucose across all organisms. Defects in glucose transport have been linked to metabolic disorders, insulin resistance and diabetes. Despite the essential importance of these transporters, our knowledge regarding GLUT members in avians is fragmented. It is clear that there are no chicken orthologs of mammalian GLUT4 and GLUT7. Our examination of GLUT members in the chicken revealed that some chicken GLUT members do not have corresponding orthologs in mammals. We review the information regarding GLUT orthologs and their function and expression in mammals and birds, with emphasis on chickens and humans.

  18. Modeling Facilitated Contaminant Transport by Mobile Bacteria

    NASA Astrophysics Data System (ADS)

    Corapcioglu, M. Yavuz; Kim, Seunghyun

    1995-01-01

    Introduction of exogenous biocolloids such as genetically engineered bacteria in a bioremediation operation can enhance the transport of contaminants in groundwater by reducing the retardation effects. Because of their colloidal size and favorable surface conditions, bacteria are efficient contaminant carriers. In cases where contaminants have a low mobility in porous media because of their high partition with solid matrix, facilitated contaminant transport by mobile bacteria can create high contaminant fluxes. When metabolically active mobile bacteria are present in a subsurface environment, the system can be treated as consisting of three phases: water phase, bacterial phase, and stationary solid matrix phase. In this work a mathematical model based on mass balance equations is developed to describe the facilitated transport and fate of a contaminant and bacteria in a porous medium. Bacterial partition between the bulk solution and the stationary solid matrix and contaminant partition among three phases are represented by expressions in terms of measurable quantities. Solutions were obtained to provide estimates of contaminant and bacterial concentrations. A dimensional analysis of the transport model was utilized to estimate model parameters from the experimental data and to assess the effect of several parameters on model behavior. The model results matched favorably with experimental data of Jenkins and Lion (1993). The presence of mobile bacteria enhances the contaminant transport. However, bacterial consumption of the contaminant, which serves as a bacterial nutrient, can attenuate the contaminant mobility. The work presented in this paper is the first three-phase model to include the effects of substrate metabolism on the fate of groundwater contaminants.

  19. Structure-Function Relationship of a Plant NCS1 Member – Homology Modeling and Mutagenesis Identified Residues Critical for Substrate Specificity of PLUTO, a Nucleobase Transporter from Arabidopsis

    PubMed Central

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M. Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members. PMID:24621654

  20. Structure-function relationship of a plant NCS1 member--homology modeling and mutagenesis identified residues critical for substrate specificity of PLUTO, a nucleobase transporter from Arabidopsis.

    PubMed

    Witz, Sandra; Panwar, Pankaj; Schober, Markus; Deppe, Johannes; Pasha, Farhan Ahmad; Lemieux, M Joanne; Möhlmann, Torsten

    2014-01-01

    Plastidic uracil salvage is essential for plant growth and development. So far, PLUTO, the plastidic nucleobase transporter from Arabidopsis thaliana is the only known uracil importer at the inner plastidic membrane which represents the permeability barrier of this organelle. We present the first homology model of PLUTO, the sole plant NCS1 member from Arabidopsis based on the crystal structure of the benzyl hydantoin transporter MHP1 from Microbacterium liquefaciens and validated by molecular dynamics simulations. Polar side chains of residues Glu-227 and backbones of Val-145, Gly-147 and Thr-425 are proposed to form the binding site for the three PLUTO substrates uracil, adenine and guanine. Mutational analysis and competition studies identified Glu-227 as an important residue for uracil and to a lesser extent for guanine transport. A differential response in substrate transport was apparent with PLUTO double mutants E227Q G147Q and E227Q T425A, both of which most strongly affected adenine transport, and in V145A G147Q, which markedly affected guanine transport. These differences could be explained by docking studies, showing that uracil and guanine exhibit a similar binding mode whereas adenine binds deep into the catalytic pocket of PLUTO. Furthermore, competition studies confirmed these results. The present study defines the molecular determinants for PLUTO substrate binding and demonstrates key differences in structure-function relations between PLUTO and other NCS1 family members.

  1. Proton Transfer in Nucleobases is Mediated by Water

    SciTech Connect

    Khistyaev, Kirill; Golan, Amir; Bravaya, Ksenia B.; Orms, Natalie; Krylov, Anna I.; Ahmed, Musahid

    2013-08-08

    Water plays a central role in chemistry and biology by mediating the interactions between molecules, altering energy levels of solvated species, modifying potential energy proles along reaction coordinates, and facilitating ecient proton transport through ion channels and interfaces. This study investigates proton transfer in a model system comprising dry and microhydrated clusters of nucleobases. With mass spectrometry and tunable vacuum ultraviolet synchrotron radiation, we show that water shuts down ionization-induced proton transfer between nucleobases, which is very ecient in dry clusters. Instead, a new pathway opens up in which protonated nucleo bases are generated by proton transfer from the ionized water molecule and elimination of a hydroxyl radical. Electronic structure calculations reveal that the shape of the potential energy prole along the proton transfer coordinate depends strongly on the character of the molecular orbital from which the electron is removed, i.e., the proton transfer from water to nucleobases is barrierless when an ionized state localized on water is accessed. The computed energetics of proton transfer is in excellent agreement with the experimental appearance energies. Possible adiabatic passage on the ground electronic state of the ionized system, while energetically accessible at lower energies, is not ecient. Thus, proton transfer is controlled electronically, by the character of the ionized state, rather than statistically, by simple energy considerations.

  2. Saturated Zone Colloid-Facilitated Transport

    SciTech Connect

    A. Wolfsberg; P. Reimus

    2001-12-18

    The purpose of the Saturated Zone Colloid-Facilitated Transport Analysis and Modeling Report (AMR), as outlined in its Work Direction and Planning Document (CRWMS M&O 1999a), is to provide retardation factors for colloids with irreversibly-attached radionuclides, such as plutonium, in the saturated zone (SZ) between their point of entrance from the unsaturated zone (UZ) and downgradient compliance points. Although it is not exclusive to any particular radionuclide release scenario, this AMR especially addresses those scenarios pertaining to evidence from waste degradation experiments, which indicate that plutonium and perhaps other radionuclides may be irreversibly attached to colloids. This report establishes the requirements and elements of the design of a methodology for calculating colloid transport in the saturated zone at Yucca Mountain. In previous Total Systems Performance Assessment (TSPA) analyses, radionuclide-bearing colloids were assumed to be unretarded in their migration. Field experiments in fractured tuff at Yucca Mountain and in porous media at other sites indicate that colloids may, in fact, experience retardation relative to the mean pore-water velocity, suggesting that contaminants associated with colloids should also experience some retardation. Therefore, this analysis incorporates field data where available and a theoretical framework when site-specific data are not available for estimating plausible ranges of retardation factors in both saturated fractured tuff and saturated alluvium. The distribution of retardation factors for tuff and alluvium are developed in a form consistent with the Performance Assessment (PA) analysis framework for simulating radionuclide transport in the saturated zone. To improve on the work performed so far for the saturated-zone flow and transport modeling, concerted effort has been made in quantifying colloid retardation factors in both fractured tuff and alluvium. The fractured tuff analysis used recent data

  3. Pyrimidine nucleobase radical reactivity in DNA and RNA

    NASA Astrophysics Data System (ADS)

    Greenberg, Marc M.

    2016-11-01

    Nucleobase radicals are major products of the reactions between nucleic acids and hydroxyl radical, which is produced via the indirect effect of ionizing radiation. The nucleobase radicals also result from hydration of cation radicals that are produced via the direct effect of ionizing radiation. The role that nucleobase radicals play in strand scission has been investigated indirectly using ionizing radiation to generate them. More recently, the reactivity of nucleobase radicals resulting from formal hydrogen atom or hydroxyl radical addition to pyrimidines has been studied by independently generating the reactive intermediates via UV-photolysis of synthetic precursors. This approach has provided control over where the reactive intermediates are produced within biopolymers and facilitated studying their reactivity. The contributions to our understanding of pyrimidine nucleobase radical reactivity by this approach are summarized.

  4. Pyrimidine Nucleobase Radical Reactivity in DNA and RNA.

    PubMed

    Greenberg, Marc M

    2016-11-01

    Nucleobase radicals are major products of the reactions between nucleic acids and hydroxyl radical, which is produced via the indirect effect of ionizing radiation. The nucleobase radicals also result from hydration of cation radicals that are produced via the direct effect of ionizing radiation. The role that nucleobase radicals play in strand scission has been investigated indirectly using ionizing radiation to generate them. More recently, the reactivity of nucleobase radicals resulting from formal hydrogen atom or hydroxyl radical addition to pyrimidines has been studied by independently generating the reactive intermediates via UV-photolysis of synthetic precursors. This approach has provided control over where the reactive intermediates are produced within biopolymers and facilitated studying their reactivity. The contributions to our understanding of pyrimidine nucleobase radical reactivity by this approach are summarized.

  5. Computation Of Facilitated Transport of O2 In Hemoglobin

    NASA Technical Reports Server (NTRS)

    Davis, Sanford

    1991-01-01

    Report describes computations of unsteady facilitated transport of oxygen through liquid membrane of hemoglobin. Used here, "facilitated transport" means diffusion of permeant through membrane in which that diffusion enhanced by reversible chemical reaction between permeant and membrane. In this case, reversible reactions between hemoglobin and oxygen.

  6. Microhydration of Deprotonated Nucleobases

    NASA Astrophysics Data System (ADS)

    Wincel, Henryk

    2016-08-01

    Hydration reactions of deprotonated nucleobases (uracil, thymine, 5-fluorouracil,2-thiouracil, cytosine, adenine, and hypoxanthine) produced by electrospray have been experimentally studied in the gas phase at 10 mbar using a pulsed ion-beam high-pressure mass spectrometer. The thermochemical data, ΔH o , ΔS o , and ΔG o , for the monohydrated systems were determined. The hydration enthalpies were found to be similar for all studied systems and varied between 39.4 and 44.8 kJ/mol. A linear correlation was found between water binding energies in the hydrated complexes and the corresponding acidities of the most acidic site of nucleobases. The structural and energetic aspects of the precursors for the hydrated complexes are discussed in conjunction with available literature data.

  7. Colloid-Facilitated Transport of Radionuclides through the Vadose Zone

    SciTech Connect

    Flury, Markus; Harsh, James B.; Zachara, John M.; McCarthy, John F.; Lichtner, Peter C.

    2006-05-31

    This project seeks to improve the basic understanding of the role of colloids in facilitating the transport of contaminants in the vadose zone. We focus on three major thrusts: (1) thermodynamic stability and mobility of colloids formed by reactions of sediments with highly alkaline tank waste solutions, (2) colloid-contaminant interactions, and (3) in-situ colloid mobilization and colloid facilitated contaminant transport occurring in both contaminated and uncontaminated Hanford sediments.

  8. Facilitated transport of copper with hydroxyapatite nanoparticles in saturated sand

    USDA-ARS?s Scientific Manuscript database

    Saturated packed column experiments were conducted to investigate the facilitated transport of Cu with hydroxyapatite nanoparticles (nHAP) at different pore water velocities (0.22-2.2 cm min–1), solution pH (6.2-9.0), and fraction of Fe oxide coating on grain surfaces (', 0-0.36). The facilitated tr...

  9. Evidence for rotational contribution to protein-facilitated proton transport.

    PubMed Central

    Gros, G; Lavalette, D; Moll, W; Gros, H; Amand, B; Pochon, F

    1984-01-01

    Two modes of molecular motion of carrier molecules can, in principle, lead to a facilitated transport of a substrate: translational and rotational diffusion. In the present study, which deals with the mechanism of the facilitated diffusion of H+ and O2 in solutions of earthworm hemoglobin, examples for both types of facilitation are presented. Only translational, not rotational, diffusion of earthworm hemoglobin appears to lead to a facilitated O2 flux. In contrast, substantial facilitated H+ fluxes of comparable size arise from rotational diffusion as well as from translational diffusion of this large protein. This is derived from measurements of facilitated H+ and O2 fluxes in earthworm hemoglobin solutions and determinations of the rotational and translational diffusion coefficients of earthworm hemoglobin with the help of a theoretical treatment of facilitated diffusion by rotational carrier diffusion. H+ transport by rotational protein diffusion appears to be a case where the often-postulated mechanism of facilitated transport by rotation of a carrier lends itself to experimental verification. Images PMID:6324213

  10. Graphene sculpturene nanopores for DNA nucleobase sensing.

    PubMed

    Sadeghi, Hatef; Algaragholy, L; Pope, T; Bailey, S; Visontai, D; Manrique, D; Ferrer, J; Garcia-Suarez, V; Sangtarash, Sara; Lambert, Colin J

    2014-06-19

    To demonstrate the potential of nanopores in bilayer graphene for DNA sequencing, we computed the current-voltage characteristics of a bilayer graphene junction containing a nanopore and found that they change significantly when nucleobases are transported through the pore. To demonstrate the sensitivity and selectivity of example devices, we computed the probability distribution PX(β) of the quantity β representing the change in the logarithmic current through the pore due to the presence of a nucleobase X (X = adenine, thymine, guanine, or cytosine). We quantified the selectivity of the bilayer-graphene nanopores by showing that PX(β) exhibits distinct peaks for each base X. To demonstrate that such discriminating sensing is a general feature of bilayer nanopores, the well-separated positions of these peaks were shown to be present for different pores, with alternative examples of electrical contacts.

  11. Facilitative plasma membrane transporters function during ER transit

    PubMed Central

    Takanaga, Hitomi; Frommer, Wolf B.

    2010-01-01

    Although biochemical studies suggested a high permeability of the endoplasmic reticulum (ER) membrane for small molecules, proteomics identified few specialized ER transporters. To test functionality of transporters during ER passage, we tested whether glucose transporters (GLUTs, SGLTs) destined for the plasma membrane are active during ER transit. HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEK293T cells to take up glucose across the plasma membrane correlated with low ER transport. Ectopic expression of GLUT1, -2, -4, or -9 induced GLUT isoform-specific ER transport activity in HEK293T cells. In contrast, the Na+-glucose cotransporter SGLT1 mediated efficient plasma membrane glucose transport but no detectable ER uptake, probably because of lack of a sufficient sodium gradient across the ER membrane. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport. Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability.—Takanaga, H., Frommer, W. B. Facilitative plasma membrane transporters function during ER transit. PMID:20354141

  12. Facilitated Ion Transport in Smectic Ordered Ionic Liquid Crystals.

    PubMed

    Lee, Jin Hong; Han, Kee Sung; Lee, Je Seung; Lee, Albert S; Park, Seo Kyung; Hong, Sung Yun; Lee, Jong-Chan; Mueller, Karl T; Hong, Soon Man; Koo, Chong Min

    2016-11-01

    A novel ionic mixture of an imidazolium-based room-temperature ionic liquid containing ethylene-oxide-functionalized phosphite anions is fabricated, which, when doped with lithium salt, self-assembles into a smectic-ordered ionic liquid crystal through Coulombic interactions between the ion species. Interestingly, the smectic order in the ionic-liquid-crystal ionogel facilitates ionic transport.

  13. Facilitated Ion Transport in Smectic Ordered Ionic Liquid Crystals

    SciTech Connect

    Lee, Jin Hong; Han, Kee Sung; Lee, Je Seung; Lee, Albert S.; Park, Seo Kyung; Hong, Sung Yun; Lee, Jong-Chan; Mueller, Karl T.; Hong, Soon Man; Koo, Chong Min

    2016-09-08

    We investigated a novel ionic mixture of an imidazolium-based room temperature IL containing ethylene oxide functionalized phosphite anion and a lithium salt that self-assembles into a smectic-ordered IL crystal. The two key features in this work are the unique origin of the smectic order of the ionic mixtures and the facilitated ion transport behavior in the smectic ordered IL crystal. In fact, the IL crystals are self-assembled through Coulombic interactions between ion species, not through the hydrophilic-phobic interactions between charged ion heads and hydrophobic long alkyl pendants or the steric interaction between mesogenic moieties. Furthermore, the smectic order in the IL crystal ionogel facilitates exceptional and remarkable ionic transport. Large ionic conductivity, viscoelastic robustness, and additional electrochemical stability of the IL crystal ionogels provide promising opportunities for future electrochemical applications.

  14. Liquid transport facilitated by channels in Bacillus subtilis biofilms.

    PubMed

    Wilking, James N; Zaburdaev, Vasily; De Volder, Michael; Losick, Richard; Brenner, Michael P; Weitz, David A

    2013-01-15

    Many bacteria on earth exist in surface-attached communities known as biofilms. These films are responsible for manifold problems, including hospital-acquired infections and biofouling, but they can also be beneficial. Biofilm growth depends on the transport of nutrients and waste, for which diffusion is thought to be the main source of transport. However, diffusion is ineffective for transport over large distances and thus should limit growth. Nevertheless, biofilms can grow to be very large. Here we report the presence of a remarkable network of well-defined channels that form in wild-type Bacillus subtilis biofilms and provide a system for enhanced transport. We observe that these channels have high permeability to liquid flow and facilitate the transport of liquid through the biofilm. In addition, we find that spatial variations in evaporative flux from the surface of these biofilms provide a driving force for the flow of liquid in the channels. These channels offer a remarkably simple system for liquid transport, and their discovery provides insight into the physiology and growth of biofilms.

  15. Colloid-Facilitated Plutonium Transport in Fractured Tuffaceous Rock.

    PubMed

    Wolfsberg, Andrew; Dai, Zhenxue; Zhu, Lin; Reimus, Paul; Xiao, Ting; Ware, Doug

    2017-05-16

    Colloids have the potential to enhance the mobility of strongly sorbing radionuclide contaminants in groundwater at underground nuclear test sites. This study presents an experimental and numerical investigation of colloid-facilitated plutonium transport in fractured porous media to identify plutonium reactive transport processes. The transport parameters for dispersion, diffusion, sorption, and filtration are estimated with inverse modeling by minimizing the least-squares objective function of multicomponent concentration data from multiple transport experiments with the shuffled complex evolution metropolis algorithm. Capitalizing on an unplanned experimental artifact that led to colloid formation, we adopt a stepwise strategy to first interpret the data from each experiment separately and then to incorporate multiple experiments simultaneously to identify a suite of plutonium-colloid transport processes. Nonequilibrium or kinetic attachment and detachment of plutonium-colloid in fractures were clearly demonstrated and captured in the inverted modeling parameters along with estimates of the source plutonium fraction that formed plutonium-colloids. The results from this study provide valuable insights for understanding the transport mechanisms and environmental impacts of plutonium in groundwater aquifers.

  16. Liquid transport facilitated by channels in Bacillus subtilis biofilms

    PubMed Central

    Wilking, James N.; Zaburdaev, Vasily; De Volder, Michael; Losick, Richard; Brenner, Michael P.; Weitz, David A.

    2013-01-01

    Many bacteria on earth exist in surface-attached communities known as biofilms. These films are responsible for manifold problems, including hospital-acquired infections and biofouling, but they can also be beneficial. Biofilm growth depends on the transport of nutrients and waste, for which diffusion is thought to be the main source of transport. However, diffusion is ineffective for transport over large distances and thus should limit growth. Nevertheless, biofilms can grow to be very large. Here we report the presence of a remarkable network of well-defined channels that form in wild-type Bacillus subtilis biofilms and provide a system for enhanced transport. We observe that these channels have high permeability to liquid flow and facilitate the transport of liquid through the biofilm. In addition, we find that spatial variations in evaporative flux from the surface of these biofilms provide a driving force for the flow of liquid in the channels. These channels offer a remarkably simple system for liquid transport, and their discovery provides insight into the physiology and growth of biofilms. PMID:23271809

  17. COLLOID-FACILITATED TRANSPORT OF RADIONUCLIDES THROUGH THE VADOSE ZONE

    SciTech Connect

    Flury, Markus

    2003-09-14

    Contaminants have leaked into the vadose zone at the USDOE Hanford reservation. It is important to understand the fate and transport of these contaminants to design remediation strategies and long-term waste management plans at the Hanford reservation. Colloids may play an important role in fate and transport of strongly sorbing contaminants, such as Cs or Pu. This project seeks to improve the basic understanding of colloid and colloid-facilitated transport of contaminants in the vadose zone. The specific objectives addressed are: (1) Determine the structure, composition, and surface charge characteristics of colloidal particles formed under conditions similar to those occurring during leakage of waste typical of Hanford tank supernatants into soils and sediments surrounding the tanks. (2) Characterize the mutual interactions between colloids, contaminant, and soil matrix in batch experiments under various ionic strength and pH conditions. We will investigate the nature of the solid-liquid interactions and the kinetics of the reactions. (3) Evaluate mobility of colloids through soil under different degrees of water saturation and solution chemistry (ionic strength and pH). (4) Determine the potential of colloids to act as carriers to transport the contaminant through the vadose zone and verify the results through comparison with field samples collected under leaking tanks. (5) Improve conceptual characterization of colloid-contaminant-soil interactions and colloid-facilitated transport for implementation into reactive chemical transport models. This project was in part supported by an NSF-IGERT grant to Washington State University. The IGERT grant provided funding for graduate student research and education, and two graduate students were involved in the EMSP project. The IGERT program also supported undergraduate internships. The project is part of a larger EMSP program to study fate and transport of contaminants under leaking Hanford waste tanks. The project has

  18. Facilitated transport of small molecules and ions for energy-efficient membranes.

    PubMed

    Li, Yifan; Wang, Shaofei; He, Guangwei; Wu, Hong; Pan, Fusheng; Jiang, Zhongyi

    2015-01-07

    In nature, the biological membrane can selectively transport essential small molecules/ions through facilitated diffusion via carrier proteins. Intrigued by this phenomenon and principle, membrane researchers have successfully employed synthetic carriers and carrier-mediated reversible reactions to enhance the separation performance of synthetic membranes. However, the existing facilitated transport membranes as well as the relevant facilitated transport theories have scarcely been comprehensively reviewed in the literature. This tutorial review primarily covers the two aspects of facilitated transport theories: carrier-mediated transport mechanisms and facilitated transport chemistries, including the design and fabrication of facilitated transport membranes. The applications of facilitated transport membranes in energy-intensive membrane processes (gas separation, pervaporation, and proton exchange membrane fuel cells) have also been discussed. Hopefully, this review will provide guidelines for the future research and development of facilitated transport membranes with high energy efficiency.

  19. Integration of a 'proton antenna' facilitates transport activity of the monocarboxylate transporter MCT4.

    PubMed

    Noor, Sina Ibne; Pouyssegur, Jacques; Deitmer, Joachim W; Becker, Holger M

    2017-01-01

    Monocarboxylate transporters (MCTs) mediate the proton-coupled transport of high-energy metabolites like lactate and pyruvate and are expressed in nearly every mammalian tissue. We have shown previously that transport activity of MCT4 is enhanced by carbonic anhydrase II (CAII), which has been suggested to function as a 'proton antenna' for the transporter. In the present study, we tested whether creation of an endogenous proton antenna by introduction of a cluster of histidine residues into the C-terminal tail of MCT4 (MCT4-6xHis) could facilitate MCT4 transport activity when heterologously expressed in Xenopus oocytes. Our results show that integration of six histidines into the C-terminal tail does indeed increase transport activity of MCT4 to the same extent as did coexpression of MCT4-WT with CAII. Transport activity of MCT4-6xHis could be further enhanced by coexpression with extracellular CAIV, but not with intracellular CAII. Injection of an antibody against the histidine cluster into MCT4-expressing oocytes decreased transport activity of MCT4-6xHis, while leaving activity of MCT4-WT unaltered. Taken together, these findings suggest that transport activity of the proton-coupled monocarboxylate transporter MCT4 can be facilitated by integration of an endogenous proton antenna into the transporter's C-terminal tail. © 2016 Federation of European Biochemical Societies.

  20. Evaluation of bacteria-facilitated cadmium transport in gravel columns using the HYDRUS colloid-facilitated solute transport model

    NASA Astrophysics Data System (ADS)

    Pang, Liping; Å Imå¯Nek, Jirka

    2006-12-01

    The colloid-facilitated solute transport model, based on HYDRUS-1D, was evaluated using the column experimental data of Pang et al. (2005) for cadmium (Cd) transport facilitated by B. subtilis spores or E. coli in saturated coarse alluvial gravels. We simulated Cd transport involving convection, dispersion, kinetic adsorption/desorption to/from the aquifer media and to/from mobile/immobile bacteria, and kinetic attachment/detachment of the bacteria to/from the aquifer media. To reduce the number of parameters to be optimized, we independently estimated Cd sorption/desorption rates to mobile bacteria from a batch study. The model described the collected experimental data reasonably well. Extensive sensitivity analysis to various reaction parameters was carried out to obtain an understanding of the relative importance of individual model parameters on model predictions. Our modeling results suggest that the rates of Cd sorption or desorption differ not only between different bacterial species but also between unattached and deposited bacteria. The results of the sensitivity analysis indicated that the Cd sorption rate to unattached bacteria had a significantly greater impact on the model results than its sorption rate to deposited bacteria. For the experimental system investigated here, model results were most sensitive to parameters describing interactions between Cd-aquifer media, bacteria-aquifer media, and Cd-mobile bacteria, and they were less sensitive to interactions between Cd-immobile bacteria and desorption rate from mobile bacteria.

  1. Golgi Localized Barley MTP8 Proteins Facilitate Mn Transport

    PubMed Central

    Pedas, Pai; Schiller Stokholm, Michaela; Hegelund, Josefine Nymark; Ladegård, Anne Hald; Schjoerring, Jan Kofod; Husted, Søren

    2014-01-01

    Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species. PMID:25486417

  2. Golgi localized barley MTP8 proteins facilitate Mn transport.

    PubMed

    Pedas, Pai; Schiller Stokholm, Michaela; Hegelund, Josefine Nymark; Ladegård, Anne Hald; Schjoerring, Jan Kofod; Husted, Søren

    2014-01-01

    Many metabolic processes in plants are regulated by manganese (Mn) but limited information is available on the molecular mechanisms controlling cellular Mn homeostasis. In this study, a yeast assay was used to isolate and characterize two genes, MTP8.1 and MTP8.2, which encode membrane-bound proteins belonging to the cation diffusion facilitator (CDF) family in the cereal species barley (Hordeum vulgare). Transient expression in onion epidermal cells showed that MTP8.1 and MTP8.2 proteins fused to the green fluorescent protein (GFP) are localized to Golgi. When heterologously expressed in yeast, MTP8.1 and MTP8.2 were found to be Mn transporters catalysing Mn efflux in a similar manner as the Golgi localized endogenous yeast protein Pmr1p. The level of MTP8.1 transcripts in barley roots increased with external Mn supply ranging from deficiency to toxicity, while MTP8.2 transcripts decreased under the same conditions, indicating non-overlapping functions for the two genes. In barley leaves, the expression of both MTP8 genes declined in response to toxic Mn additions to the roots suggesting a role in ensuring proper delivery of Mn to Golgi. Based on the above we suggest that barley MTP8 proteins are involved in Mn loading to the Golgi apparatus and play a role in Mn homeostasis by delivering Mn to Mn-dependent enzymes and/or by facilitating Mn efflux via secretory vesicles. This study highlights the importance of MTP transporters in Mn homeostasis and is the first report of Golgi localized Mn2+ transport proteins in a monocot plant species.

  3. Colloid-facilitated radionuclide transport: a regulatory perspective

    NASA Astrophysics Data System (ADS)

    Dam, W. L.; Pickett, D. A.; Codell, R. B.; Nicholson, T. J.

    2001-12-01

    What hydrogeologic-geochemical-microbial conditions and processes affect migration of radionuclides sorbed onto microparticles or native colloid-sized radionuclide particles? The U.S. Nuclear Regulatory Commission (NRC) is responsible for protecting public health, safety, and the environment at numerous nuclear facilities including a potential high-level nuclear waste disposal site. To fulfill these obligations, NRC needs to understand the mechanisms controlling radionuclide release and transport and their importance to performance. The current focus of NRC staff reviews and technical interactions dealing with colloid-facilitated transport relates to the potential nuclear-waste repository at Yucca Mountain, Nevada. NRC staff performed bounding calculations to quantify radionuclide releases available for ground-water transport to potential receptors from a Yucca Mountain repository. Preliminary analyses suggest insignificant doses of plutonium and americium colloids could be derived from spent nuclear fuel. Using surface complexation models, NRC staff found that colloids can potentially lower actinide retardation factors by up to several orders of magnitude. Performance assessment calculations, in which colloidal transport of plutonium and americium was simulated by assuming no sorption or matrix diffusion, indicated no effect of colloids on human dose within the 10,000 year compliance period due largely to long waste-package lifetimes. NRC staff have identified information gaps and developed technical agreements with the U.S. Department of Energy (DOE) to ensure sufficient information will be presented in any potential future Yucca Mountain license application. DOE has agreed to identify which radionuclides could be transported via colloids, incorporate uncertainties in colloid formation, release and transport parameters, and conceptual models, and address the applicability of field data using synthetic microspheres as colloid analogs. NRC is currently

  4. Colloid facilitated transport of lanthanides through discrete fractures in chalk

    NASA Astrophysics Data System (ADS)

    Tran, Emily; Klein Ben-David, Ofra; Teutsch, Nadya; Weisbrod, Noam

    2015-04-01

    Geological disposal of high-level radioactive waste is the internationally agreed-upon, long term solution for the disposal of long lived radionuclides and spent fuel. Eventually, corrosion of the waste canisters may lead to leakage of their hazardous contents, and the radionuclides can ultimately make their way into groundwater and pose a threat to the biosphere. Engineered bentonite barriers placed around nuclear waste repositories are generally considered sufficient to impede the transport of radionuclides from their storage location to the groundwater. However, colloidal-sized mobile bentonite particles eroding from these barriers have come under investigation as a potential transport vector for radionuclides sorbed to them. In addition, the presence of organic matter in groundwater has been shown to additionally facilitate the uptake of radionuclides by the clay colloids. This study aims to evaluate the transport behaviors of radionuclides in colloid-facilitated transport through a fractured chalk matrix and under geochemical conditions representative of the Negev desert, Israel. Lanthanides are considered an acceptable substitute to actinides for research on radionuclide transportation due to their similar chemical behavior. In this study, the migration of Ce both with and without colloidal particles was explored and compared to the migration of a conservative tracer (bromide). Tracer solutions containing known concentrations of Ce, bentonite colloids, humic acid and bromide were prepared in a matrix solution containing salt concentrations representative of that of the average rain water found in the Negev. These solutions were then injected into a flow system constructed around a naturally fractured chalk core. Samples were analyzed for Ce and Br using ICP-MS, and colloid concentrations were determined using spectrophotographic analysis. Breakthrough curves comparing the rates of transportation of each tracer were obtained, allowing for comparison of

  5. Understanding heat facilitated drug transport across human epidermis.

    PubMed

    Wood, D G; Brown, M B; Jones, S A

    2012-08-01

    The application of moderate heat is a safe and effective means to increase drug transport across human skin. However, the cascade of events that follows the exposure of a topical skin formulation to a heating source is not well understood. The aim of this study was to elucidate how three potential rate limiting stages in the drug transport process; formulation release, drug partitioning and epidermal diffusion, responded to changes in local temperature using the model drug lidocaine. Release from the formulation measured using regenerated cellulose membrane was shown to be driven by drug diffusion in the vehicle; it responded linearly when the local temperature was changed (21.6 μg/cm(2)/h for every 1 °C rise) and displayed no measurable partitioning of lidocaine into RCM. Once the drug was within the human epidermis, the structural changes of the barrier controlled its transport. The apparent lidocaine diffusion coefficient through silicone membrane increased from 6.52 to 8.43 × 10(-4) over the 32-45 °C temperature range, but it increased from 7.74 × 10(-5)cm(2)h(-1) to 4.8 × 10(-4)cm(2)h(-1) in the human epidermis. In the absence of large increases in drug partitioning, fluidisation of the lipids in the upper layers of the epidermis at 37-45 °C was shown to facilitate lidocaine diffusion which for human skin transport was the rate limiting process. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Colloid Facilitated Transport of Plutonium in Fractured Volcanic Tuff

    NASA Astrophysics Data System (ADS)

    Kersting, A. B.; Zhao, P.; Walensky, J. R.; Roberts, S. K.; Johnson, M. R.; Zavarin, M.; Ramon, E. C.

    2004-12-01

    The transport of low-solubility radionuclides in a colloidal- or colloidal bound state is frequently suspected or observed. Groundwater contaminated with radionuclides associated with underground nuclear tests was collected from several different well locations at the Nevada Test Site (NTS). In each case, the low-levels of plutonium detected in the groundwater were overwhelmingly (>95percent) associated with the colloidal and not the dissolved fraction of the groundwater. The colloidal fractions consisted of secondary minerals such as clays and zeolites. To better understand the mechanisms controlling the potential colloidal transport of plutonium, colloid-facilitated fracture flow laboratory experiments are being conducted. Pseudocolloids consisting of Pu(IV) sorbed to clinoptilolite were combined with a radionuclide solution cocktail consisting of Np, U, Cs, Sr, Sm and 3H and Re (analog to Tc) tracers in NTS-type synthetic groundwater (4.5mM NaHCO3-). The cocktail was injected into a smooth fracture in a volcanic tuff rock core from the NTS and the effluent analyzed. Autoradiography and secondary ion mass spectrometry will be used to understand the mineral -colloid-radionuclide interactions in the fracture volcanic tuff.

  7. Isoform-selective Inhibition of Facilitative Glucose Transporters

    PubMed Central

    Hresko, Richard C.; Kraft, Thomas E.; Tzekov, Anatoly; Wildman, Scott A.; Hruz, Paul W.

    2014-01-01

    Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design. PMID:24706759

  8. PFLOTRAN: Recent Developments Facilitating Massively-Parallel Reactive Biogeochemical Transport

    NASA Astrophysics Data System (ADS)

    Hammond, G. E.

    2015-12-01

    With the recent shift towards modeling carbon and nitrogen cycling in support of climate-related initiatives, emphasis has been placed on incorporating increasingly mechanistic biogeochemistry within Earth system models to more accurately predict the response of terrestrial processes to natural and anthropogenic climate cycles. PFLOTRAN is an open-source subsurface code that is specialized for simulating multiphase flow and multicomponent biogeochemical transport on supercomputers. The object-oriented code was designed with modularity in mind and has been coupled with several third-party simulators (e.g. CLM to simulate land surface processes and E4D for coupled hydrogeophysical inversion). Central to PFLOTRAN's capabilities is its ability to simulate tightly-coupled reactive transport processes. This presentation focuses on recent enhancements to the code that enable the solution of large parameterized biogeochemical reaction networks with numerous chemical species. PFLOTRAN's "reaction sandbox" is described, which facilitates the implementation of user-defined reaction networks without the need for a comprehensive understanding of PFLOTRAN software infrastructure. The reaction sandbox is written in modern Fortran (2003-2008) and leverages encapsulation, inheritance, and polymorphism to provide the researcher with a flexible workspace for prototyping reactions within a massively parallel flow and transport simulation framework. As these prototypical reactions mature into well-accepted implementations, they can be incorporated into PFLOTRAN as native biogeochemistry capability. Users of the reaction sandbox are encouraged to upload their source code to PFLOTRAN's main source code repository, including the addition of simple regression tests to better ensure the long-term code compatibility and validity of simulation results.

  9. Nucleocytoplasmic transport of ribosomes in a eukaryotic system: Is there a facilitated transport process

    SciTech Connect

    Khanna-Gupta, A.; Ware, V.C. )

    1989-03-01

    The authors have examined the kinetics of the process by which ribosomes are exported from the nucleus to the cytoplasm using Xenopus laevis oocytes microinjected into the germinal vesicle with radiolabeled ribosomes or ribosomal subunits from X. laevis, Tetrahymena thermophila, or Escherichia coli. Microinjected eukaryotic mature ribosomes are redistributed into the oocyte cytoplasm by an apparent carrier-mediated transport process that exhibits saturation kinetics as increasing amounts of ribosomes are injected. T. thermophila ribosomes are competent to traverse the Xenopus nuclear envelope, suggesting that the basic mechanism underlying ribosome transport is evolutionarily conserved. Microinjected E. coli ribosomes are not transported in this system, indicating that prokaryotic ribosomes lack the signals required for transport. Surprisingly, coinjected small (40S) and large (60S) subunits from T. thermophila are transported significantly faster than individual subunits. These observations support a facilitated transport model for the translocation of ribosomal subunits as separate units across the nuclear envelope whereby the transport rate of 60S or 40S subunits is enhanced by the presence of the partner subunit. Although the basic features of the transport mechanism have been preserved through evolution, other aspects of the process may be mediated through species-specific interactions. They hypothesize that a species-specific nuclear 40S-60S subunit association may expedite the transport of individual subunits across the nuclear envelope.

  10. In vitro synthesis of a Major Facilitator Transporter for specific active transport across Droplet Interface Bilayers

    PubMed Central

    Findlay, Heather E.; Harris, Nicola J.; Booth, Paula J.

    2016-01-01

    Nature encapsulates reactions within membrane-bound compartments, affording sequential and spatial control over biochemical reactions. Droplet Interface Bilayers are evolving into a valuable platform to mimic this key biological feature in artificial systems. A major issue is manipulating flow across synthetic bilayers. Droplet Interface Bilayers must be functionalised, with seminal work using membrane-inserting toxins, ion channels and pumps illustrating the potential. Specific transport of biomolecules, and notably transport against a concentration gradient, across these bilayers has yet to be demonstrated. Here, we successfully incorporate the archetypal Major Facilitator Superfamily transporter, lactose permease, into Droplet Interface Bilayers and demonstrate both passive and active, uphill transport. This paves the way for controllable transport of sugars, metabolites and other essential biomolecular substrates of this ubiquitous transporter superfamily in DIB networks. Furthermore, cell-free synthesis of lactose permease during DIB formation also results in active transport across the interface bilayer. This adds a specific disaccharide transporter to the small list of integral membrane proteins that can be synthesised via in vitro transcription/translation for applications of DIB-based artificial cell systems. The introduction of a means to promote specific transport of molecules across Droplet Interface Bilayers against a concentration gradient gives a new facet to droplet networks. PMID:27996025

  11. Silicene-based DNA nucleobase sensing

    NASA Astrophysics Data System (ADS)

    Sadeghi, Hatef; Bailey, S.; Lambert, Colin J.

    2014-03-01

    We propose a DNA sequencing scheme based on silicene nanopores. Using first principles theory, we compute the electrical properties of such pores in the absence and presence of nucleobases. Within a two-terminal geometry, we analyze the current-voltage relation in the presence of nucleobases with various orientations. We demonstrate that when nucleobases pass through a pore, even after sampling over many orientations, changes in the electrical properties of the ribbon can be used to discriminate between bases.

  12. Methanotrophic bacteria and facilitated transport of pollutants in aquifer material

    SciTech Connect

    Jenkins, M.B.; Chen, Jyh-Herng; Kadner, D.J.; Lion, L.W. )

    1994-10-01

    In-situ stimulation of methanotrophic bacteria has been considered for aquifer remediation. Experimental results indicate that both colloidal suspensions containing methanotrophic cells and the soluble extracellular polymers produced by methanotrophic cells have the potential to enhance the transport and removal of other environmental contaminants such as polynuclear aromatic hydrocarbons and transition metals in aquifer material. Three methanotrophic bacteria were used in the experiments: Methylomonas albus BG8, Methylosinus trichosporium OB3b, and Methylocystis parvus OBBP. The distribution coefficients for Cd with extraceullular polymers were of the same order as that obtained with the aquifer sand, indicating polymers from the methanotrophic bacteria could act to increase the transport of Cd in a porous medium. Polymer from BG8 significantly reduced the apparent distribution coefficient for Cd with an aquifer sand. [[sup 14]C]phenanthrene also sorbed to extracellular polymer and to washed, suspended methanotrophic cells. The exopolymer of BG8 and OBBP significantly reduced the apparent distribution coefficient (K[sub d]) for phenanthrene with aquifer sand. The distribution coefficients for phenanthrene with the methanotrophic cells were an order of magnitude greater than those previously reported for other heterotrophic bacteria. Cells of the methanotrophs also significantly reduced the apparent K[sub d] for phenanthrene with an aquifer sand. The three strains of methanotrophs displayed mobility in a column of packed sand, and strain OBBP reduced the retardation coefficient of phenanthrene with an aquifer sand by 27%. These data indicate that both extracellular polymer and mobile cells of methanotrophic bacteria display a capacity to facilitate the mobility of pollutant metals and polynuclear aromatic hydrocarbons in aquifer material. 48 refs., 3 figs., 4 tabs.

  13. Sugar transporter genes of the brown planthopper, Nilaparvata lugens: A facilitated glucose/fructose transporter.

    PubMed

    Kikuta, Shingo; Kikawada, Takahiro; Hagiwara-Komoda, Yuka; Nakashima, Nobuhiko; Noda, Hiroaki

    2010-11-01

    The brown planthopper (BPH), Nilaparvata lugens, attacks rice plants and feeds on their phloem sap, which contains large amounts of sugars. The main sugar component of phloem sap is sucrose, a disaccharide composed of glucose and fructose. Sugars appear to be incorporated into the planthopper body by sugar transporters in the midgut. A total of 93 expressed sequence tags (ESTs) for putative sugar transporters were obtained from a BPH EST database, and 18 putative sugar transporter genes (Nlst1-18) were identified. The most abundantly expressed of these genes was Nlst1. This gene has previously been identified in the BPH as the glucose transporter gene NlHT1, which belongs to the major facilitator superfamily. Nlst1, 4, 6, 9, 12, 16, and 18 were highly expressed in the midgut, and Nlst2, 7, 8, 10, 15, 17, and 18 were highly expressed during the embryonic stages. Functional analyses were performed using Xenopus oocytes expressing NlST1 or 6. This showed that NlST6 is a facilitative glucose/fructose transporter that mediates sugar uptake from rice phloem sap in the BPH midgut in a manner similar to NlST1. Copyright © 2010 Elsevier Ltd. All rights reserved.

  14. Extraterrestrial Nucleobases in Carbonaceous Chondrites

    NASA Astrophysics Data System (ADS)

    Martins, Z.; Botta, O.; Fogel, M.; Sephton, M.; Glavin, D.; Watson, J.; Dworkin, J.; Schwartz, A.; Ehrenfreund, P.

    Nucleobases in Carbonaceous Chondrites Z. Martins (1), O. Botta (2), M. L. Fogel (3), M. A. Sephton (4), D. P. Glavin (2), J. S. Watson (5), J. P. Dworkin (2), A. W. Schwartz (6) and P. Ehrenfreund (1,6). (1) Astrobiology Laboratory, Leiden Institute of Chemistry, Leiden, The Netherlands, (2) NASA Goddard Space Flight Center, Goddard Center for Astrobiology, Greenbelt, MD, USA, (3) GL, Carnegie Institution of Washington, Washington DC, USA, (4) Impacts and Astromaterials Research Centre, Department of Earth Science and Engineering, South Kensington Campus, Imperial College, London, UK, (5) Planetary and Space Sciences Research Institute, The Open University, Walton Hall, Milton Keynes, UK, (6) Radboud University Nijmegen, Nijmegen, The Netherlands. E-mail: z.martins@chem.leidenuniv.nl/Phone:+31715274440 Nucleobases are crucial compounds in terrestrial biochemistry, because they are key components of DNA and RNA. Carbonaceous meteorites have been analyzed for nucleobases by different research groups [1-5]. However, significant quantitative and qualitative differences were observed, leading to the controversial about the origin of these nucleobases. In order to establish the origin of these compounds in carbonaceous chondrites and to assess the plausibility of their exogenous delivery to the early Earth, we have performed formic acid extraction of samples of the Murchison meteorite [6], followed by an extensive purification procedure, analysis and quantification by high-performance liquid chromatography with UV absorption detection and gas chromatography-mass spectrometry. Our results were qualitatively consistent with previous results [3, 4], but showed significant quantitative differences. Compound specific carbon isotope values were obtained, using gas chromatography-combustion- isotope ratio mass spectrometry. A soil sample collected in the proximity of the Murchison meteorite fall site was subjected to the same extraction, purification and analysis procedure

  15. Facilitated transport membrane hybrid systems for olefin purification

    SciTech Connect

    Davis, J.C.; Valus, R.J.; Eshraghi, R.; Velikoff, A.E.

    1993-01-01

    A new membrane system has been developed by BP for refinery and chemical plant olefin purification and recovery. This facilitated transport system, coupled with distillation, offers lower capital and operating costs than conventional distillation alone. Initial results on lab scale hollow fiber devices indicate membrane flux ranging from 8.75 {times} 10{sup {minus}6} to 8 {times} 10{sup {minus}5} m{sup 3}/m{sup 2}/sec (2.5 to 23 scfd/ft{sub 2}) and selectivities from 150 to 300. Pilot plant experiments on propylene/propane and ethylene purge gas recovery over three to six months duration show membrane stability and product purity of 98.5% or greater using refinery grade propylene feed. Hybrid system optimization data for membranes and distillation indicate that using a side draw from the distillation tower provides advantages in terms of membrane area, purity of feed to the membrane, and low per-pass recovery coupled with high overall propylene recovery. Membrane performance data under various conditions are also presented. In addition to performance data, economic evaluation and energy savings are discussed.

  16. Genetic and molecular characterization reveals a unique nucleobase cation symporter 1 in Arabidopsis.

    PubMed

    Mourad, George S; Tippmann-Crosby, Julie; Hunt, Kevin A; Gicheru, Yvonne; Bade, Kaely; Mansfield, Tyler A; Schultes, Neil P

    2012-05-07

    Locus At5g03555 encodes a nucleobase cation symporter 1 (AtNCS1) in the Arabidopsis genome. Arabidopsis insertion mutants, AtNcs1-1 and AtNcs1-3, were used for in planta toxic nucleobase analog growth studies and radio-labeled nucleobase uptake assays to characterize solute transport specificities. These results correlate with similar growth and uptake studies of AtNCS1 expressed in Saccharomyces cerevisiae. Both in planta and heterologous expression studies in yeast revealed a unique solute transport profile for AtNCS1 in moving adenine, guanine and uracil. This is in stark contrast to the canonical transport profiles determined for the well-characterized S. cerevisiae NCS1 proteins FUR4 (uracil transport) or FCY2 (adenine, guanine, and cytosine transport).

  17. Functionalized Solid Electrodes for Electrochemical Biosensing of Purine Nucleobases and Their Analogues: A Review

    PubMed Central

    Sharma, Vimal Kumar; Jelen, Frantisek; Trnkova, Libuse

    2015-01-01

    Interest in electrochemical analysis of purine nucleobases and few other important purine derivatives has been growing rapidly. Over the period of the past decade, the design of electrochemical biosensors has been focused on achieving high sensitivity and efficiency. The range of existing electrochemical methods with carbon electrode displays the highest rate in the development of biosensors. Moreover, modification of electrode surfaces based on nanomaterials is frequently used due to their extraordinary conductivity and surface to volume ratio. Different strategies for modifying electrode surfaces facilitate electron transport between the electrode surface and biomolecules, including DNA, oligonucleotides and their components. This review aims to summarize recent developments in the electrochemical analysis of purine derivatives, as well as discuss different applications. PMID:25594595

  18. Facilitative plasma membrane transporters function during ER transit.

    PubMed

    Takanaga, Hitomi; Frommer, Wolf B

    2010-08-01

    Although biochemical studies suggested a high permeability of the endoplasmic reticulum (ER) membrane for small molecules, proteomics identified few specialized ER transporters. To test functionality of transporters during ER passage, we tested whether glucose transporters (GLUTs, SGLTs) destined for the plasma membrane are active during ER transit. HepG2 cells were characterized by low-affinity ER transport activity, suggesting that ER uptake is protein mediated. The much-reduced capacity of HEK293T cells to take up glucose across the plasma membrane correlated with low ER transport. Ectopic expression of GLUT1, -2, -4, or -9 induced GLUT isoform-specific ER transport activity in HEK293T cells. In contrast, the Na(+)-glucose cotransporter SGLT1 mediated efficient plasma membrane glucose transport but no detectable ER uptake, probably because of lack of a sufficient sodium gradient across the ER membrane. In conclusion, we demonstrate that GLUTs are sufficient for mediating ER glucose transport en route to the plasma membrane. Because of the low volume of the ER, trace amounts of these uniporters contribute to ER solute import during ER transit, while uniporters and cation-coupled transporters carry out export from the ER, together potentially explaining the low selectivity of ER transport. Expression levels and residence time of transporters in the ER, as well as their coupling mechanisms, could be key determinants of ER permeability.

  19. SWEET17, a facilitative transporter, mediates fructose transport across the tonoplast of Arabidopsis roots and leaves.

    PubMed

    Guo, Woei-Jiun; Nagy, Reka; Chen, Hsin-Yi; Pfrunder, Stefanie; Yu, Ya-Chi; Santelia, Diana; Frommer, Wolf B; Martinoia, Enrico

    2014-02-01

    Fructose (Fru) is a major storage form of sugars found in vacuoles, yet the molecular regulation of vacuolar Fru transport is poorly studied. Although SWEET17 (for SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS17) has been characterized as a vacuolar Fru exporter in leaves, its expression in leaves is low. Here, RNA analysis and SWEET17-β-glucuronidase/-GREEN FLUORESCENT PROTEIN fusions expressed in Arabidopsis (Arabidopsis thaliana) reveal that SWEET17 is highly expressed in the cortex of roots and localizes to the tonoplast of root cells. Expression of SWEET17 in roots was inducible by Fru and darkness, treatments that activate accumulation and release of vacuolar Fru, respectively. Mutation and ectopic expression of SWEET17 led to increased and decreased root growth in the presence of Fru, respectively. Overexpression of SWEET17 specifically reduced the Fru content in leaves by 80% during cold stress. These results intimate that SWEET17 functions as a Fru-specific uniporter on the root tonoplast. Vacuoles overexpressing SWEET17 showed increased [14C]Fru uptake compared with the wild type. SWEET17-mediated Fru uptake was insensitive to ATP or treatment with NH4Cl or carbonyl cyanide m-chlorophenyl hydrazone, indicating that SWEET17 functions as an energy-independent facilitative carrier. The Arabidopsis genome contains a close paralog of SWEET17 in clade IV, SWEET16. The predominant expression of SWEET16 in root vacuoles and reduced root growth of mutants under Fru excess indicate that SWEET16 also functions as a vacuolar transporter in roots. We propose that in addition to a role in leaves, SWEET17 plays a key role in facilitating bidirectional Fru transport across the tonoplast of roots in response to metabolic demand to maintain cytosolic Fru homeostasis.

  20. Colloid Facilitated Transport of Radioactive Cations in the Vadose Zone: Field Experiments Oak Ridge

    SciTech Connect

    James E. Saiers

    2012-09-20

    The overarching goal of this study was to improve understanding of colloid-facilitated transport of radioactive cations through unsaturated soils and sediments. We conducted a suite of laboratory experiments and field experiments on the vadose-zone transport of colloids, organic matter, and associated contaminants of interest to the U.S. Department of Energy (DOE). The laboratory and field experiments, together with transport modeling, were designed to accomplish the following detailed objectives: 1. Evaluation of the relative importance of inorganic colloids and organic matter to the facilitation of radioactive cation transport in the vadose zone; 2. Assessment of the role of adsorption and desorption kinetics in the facilitated transport of radioactive cations in the vadose zone; 3. Examination of the effects of rainfall and infiltration dynamics and in the facilitated transport of radioactive cations through the vadose zone; 4. Exploration of the role of soil heterogeneity and preferential flow paths (e.g., macropores) on the facilitated transport of radioactive cations in the vadose zone; 5. Development of a mathematical model of facilitated transport of contaminants in the vadose zone that accurately incorporates pore-scale and column-scale processes with the practicality of predicting transport with readily available parameters.

  1. Spectroscopy of Isolated Prebiotic Nucleobases

    NASA Technical Reports Server (NTRS)

    Svadlenak, Nathan; Callahan, Michael P.; Ligare, Marshall; Gulian, Lisa; Gengeliczki, Zsolt; Nachtigallova, Dana; Hobza, Pavel; deVries, Mattanjah

    2011-01-01

    We use multiphoton ionization and double resonance spectroscopy to study the excited state dynamics of biologically relevant molecules as well as prebiotic nucleobases, isolated in the gas phase. Molecules that are biologically relevant to life today tend to exhibit short excited state lifetimes compared to similar but non-biologically relevant analogs. The mechanism is internal conversion, which may help protect the biologically active molecules from UV damage. This process is governed by conical intersections that depend very strongly on molecular structure. Therefore we have studied purines and pyrimidines with systematic variations of structure, including substitutions, tautomeric forms, and cluster structures that represent different base pair binding motifs. These structural variations also include possible alternate base pairs that may shed light on prebiotic chemistry. With this in mind we have begun to probe the ultrafast dynamics of molecules that exhibit very short excited states and search for evidence of internal conversions.

  2. Plasmodesmata: intercellular tunnels facilitating transport of macromolecules in plants.

    PubMed

    Kragler, Friedrich

    2013-04-01

    In plants, intercellular structures named plasmodesmata (PD) form a continuous cytoplasmic network between neighboring cells. PD pores provide channels for intercellular symplasmic (cell-to-cell) transport throughout most tissues of the plant body. Cell-defining proteins, such as transcription factors, and regulatory non-coding sequences, such as short interfering RNA, micro RNA, protein-encoding messenger RNAs, viroids, and viral RNA/DNA genomes move via PD channels to adjacent cells. PD-mediated intercellular transport of macromolecules is a regulated process depending on the tissue, developmental stage, and nature of the transported macromolecule. In this review, PD channels and their similarity to tunneling nanotubes present in animals are highlighted. In addition, homeodomain protein movement and cellular components regulating transport are discussed.

  3. Understanding transport by the major facilitator superfamily (MFS): structures pave the way.

    PubMed

    Quistgaard, Esben M; Löw, Christian; Guettou, Fatma; Nordlund, Pär

    2016-02-01

    Members of the major facilitator superfamily (MFS) of transport proteins are essential for the movement of a wide range of substrates across biomembranes. As this transport requires a series of conformational changes, structures of MFS transporters captured in different conformational states are needed to decipher the transport mechanism. Recently, a large number of MFS transporter structures have been determined, which has provided us with an unprecedented opportunity to understand general aspects of the transport mechanism. We propose an updated model for the conformational cycle of MFS transporters, the 'clamp-and-switch model', and discuss the role of so-called 'gating residues' and the substrate in modulating these conformational changes.

  4. An artificial transport metabolon facilitates improved substrate utilization in yeast.

    PubMed

    Thomik, Thomas; Wittig, Ilka; Choe, Jun-Yong; Boles, Eckhard; Oreb, Mislav

    2017-09-04

    Efficient substrate utilization is the first and most important prerequisite for economically viable production of biofuels and chemicals by microbial cell factories. However, production rates and yields are often compromised by low transport rates of substrates across biological membranes and their diversion to competing pathways. This is especially true when common chassis organisms are engineered to utilize nonphysiological feedstocks. Here, we addressed this problem by constructing an artificial complex between an endogenous sugar transporter and a heterologous xylose isomerase in Saccharomyces cerevisiae. Direct feeding of the enzyme through the transporter resulted in acceleration of xylose consumption and substantially diminished production of xylitol as an undesired side product, with a concomitant increase in the production of ethanol. This underlying principle could also likely be implemented in other biotechnological applications.

  5. Bubble-Facilitated VOC Transport from LNAPL Smear Zones and Its Potential Effect on Vapor Intrusion.

    PubMed

    Soucy, Nicole C; Mumford, Kevin G

    2017-03-07

    Most conceptual and mathematical models of soil vapor intrusion assume that the transport of volatile organic compounds (VOCs) from a source toward a building is limited by diffusion through the soil gas. Under conditions where advection occurs, transport rates are higher and can lead to higher indoor air concentrations. Advection-dominated conditions can be created by gas bubble flow in the saturated zone. A series of laboratory column experiments were conducted to measure mass flux due to bubble-facilitated VOC transport from light nonaqueous phase liquid (LNAPL) smear zones. Smear zones that contained both LNAPL residual and trapped gas, as well as those that contained only LNAPL residual, were investigated. Results showed that the VOC mass flux due to bubble-facilitated transport was orders-of-magnitude higher than under diffusion-limited conditions. Results also showed that the mass flux due to bubble-facilitated transport was intermittent, and increased with an increased supply of dissolved gases.

  6. Computation of the unsteady facilitated transport of oxygen in hemoglobin

    NASA Technical Reports Server (NTRS)

    Davis, Sanford

    1990-01-01

    The transport of a reacting permeant diffusing through a thin membrane is extended to more realistic dissociation models. A new nonlinear analysis of the reaction-diffusion equations, using implicit finite-difference methods and direct block solvers, is used to study the limits of linearized and equilibrium theories. Computed curves of molecular oxygen permeating through hemoglobin solution are used to illustrate higher-order reaction models, the effect of concentration boundary layers at the membrane interfaces, and the transient buildup of oxygen flux.

  7. Transportation, integration, facilitation: Prehistoric trail networks of the Western Papagueria

    NASA Astrophysics Data System (ADS)

    White, Devin Alan

    This project used many sources of remotely sensed data, digital image analysis methods, advanced geospatial predictive modeling, and traditional archaeological survey to locate, document, contextualize, and interpret over one hundred prehistoric trails and associated networks in the Western Papagueria region of the North American Southwest in order to gain insight into how several landscapes within the region were used in the past. While a traditional culture history of the region suggests that the landscapes would have collectively been used primarily as a corridor for the transportation of exotic materials such as marine shell and salt from the Gulf of California to the Phoenix Basin and other parts of the Southwest, research has shown that the landscapes cannot be treated as a functional monolith. They exhibit a high degree of sub-regional variation motivated by strong local intra-community ties and sacred/ritual concerns in addition to energy-efficient, long distance travel motivated by exchange. This finding further strengthens current thinking related to the settlement of and regional interactions within the Western Papagueria during prehistory, one that continues to highlight its importance as a trade and transportation corridor, but also supports the idea of an indigenous semi-sedentary population that was focused inwards on fulfilling its own unique set of social, economic, and ideological needs.

  8. Defect Facilitated Phonon Transport through Kinks in Boron Carbide Nanowires

    DOE PAGES

    Zhang, Qian; Cui, Zhiguang; Wei, Zhiyong; ...

    2017-05-08

    Nanowires of complex morphologies, such as kinked wires, have been recently synthesized and demonstrated for novel devices and applications. However, the effects of these morphologies on thermal transport have not been well studied. Through systematic experimental measurements, we show in this paper that single-crystalline, defect-free kinks in boron carbide nanowires can pose a thermal resistance up to ~30 times larger than that of a straight wire segment of equivalent length. Analysis suggests that this pronounced resistance can be attributed to the combined effects of backscattering of highly focused phonons and required mode conversion at the kink. Interestingly, it is alsomore » found that instead of posing resistance, structural defects in the kink can actually assist phonon transport through the kink and reduce its resistance. Finally, given the common kink-like wire morphology in nanoelectronic devices and required low thermal conductivity for thermoelectric devices, these findings have important implications in precise thermal management of electronic devices and thermoelectrics.« less

  9. Transport by Collective Flagellar Beating Facilitates Evolutionary Transitions to Multicellularity

    NASA Astrophysics Data System (ADS)

    Short, Martin; Powers, Thomas

    2005-11-01

    A central problem underlying the evolution from single cells to multicellular organisms is the relationship between metabolic requirements and environmental metabolite exchange with increasing size. For organisms that form spherical colonies such as the volvocalean green algae, there is a bottleneck if diffusion alone governs nutrient uptake as they increase in size, for the diffusive flux is linear in the radius while the requirements of surface somatic cells grow quadratically. Using Volvox as a model organism, we examine experimentally and theoretically the role that advection of fluid by surface flagella plays in enhancing nutrient uptake. We show that the fluid flow driven by the coordinated beating of those flagella produces a boundary layer in the concentration of a diffusing solute which renders the metabolite exchange rate quadratic in the colony radius. This bypasses the diffusive bottleneck, facilitating evolutionary transitions to multicellularity which may be driven by other environmental factors. These results suggest that flagella may have evolved not only for motility, but also to enhance metabolite exchange.

  10. Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE.

    PubMed

    Wisedchaisri, Goragot; Park, Min-Sun; Iadanza, Matthew G; Zheng, Hongjin; Gonen, Tamir

    2014-08-04

    The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.

  11. Distribution of Nucleobases in CM and CR Carbonaceous Chondrites

    NASA Astrophysics Data System (ADS)

    Callahan, M. P.; Stern, J. C.; Glavin, D. P.; Whelley, K. E.; Martin, M. G.; Dworkin, J. P.

    2010-04-01

    We have developed an analytical method to target nucleobases in meteorites using HPLC with UV detection and tandem mass spectrometry. The distribution of nucleobases appears to correlate with the degree of aqueous alteration in these meteorites.

  12. Numerical modeling of colloid facilitated virus transport in porus media

    NASA Astrophysics Data System (ADS)

    Katzourakis, Vasileios E.; Chrysikopoulos, Constantinos V.

    2013-04-01

    A conceptual mathematical model was developed to describethe simultaneous transport (cotransport) of viruses and colloids in three-dimensional, water saturated, homogeneous porous media with uniform flow. The model accounts for the migration of individual virus and colloid particles as well as viruses attached onto colloids. Viruses can be suspended in the aqueous phase, attached onto suspended colloids and the solid matrix, and attached onto colloids previously attached on the solid matrix. Colloids can be suspended in the aqueous phase or attached on the solid matrix. Viruses in all four phases (suspended in the aqueous phase, attached onto suspended colloid particles, attached onto the solid matrix, and attached onto colloids previously attached on the solid matrix) may undergo inactivation with different inactivation coefficients. The governing coupled partial differential equations were solved numerically by employing finite difference methods, which were implemented explicitly or implicitly so that both stability and accuracy factors were satisfied. Furthermore, available experimental data were used to test the newly developed cotransport model. The model was shown to simulate quite accurately the available experimental data.

  13. Photoelectron Spectroscopy of Hexachloroplatinate-Nucleobase Complexes: Nucleobase Excited State Decay Observed via Delayed Electron Emission

    SciTech Connect

    Sen, Ananya; Matthews, Edward M.; Hou, Gao-Lei; Wang, Xue B.; Dessent, Caroline

    2015-11-14

    We report low-temperature photoelectron spectra of isolated gas-phase complexes of the hexachloroplatinate dianion bound to the nucleobases uracil, thymine, cytosine and adenine. The spectra display well-resolved, distinct peaks that are consistent with complexes where the hexachloroplatinate dianion is largely intact. Adiabatic electron detachment energies for the hexachloroplatinate-nucleobase complexes are measured as 2.26-2.36 eV. The magnitudes of the repulsive Coulomb barriers (RCBs) of the complexes are all ~1.7 eV, values that are lower than the RCB of the uncomplexed PtCl6 2- dianion as a result of charge solvation by the nucleobases. In addition to the resolved spectral features, broad featureless bands indicative of delayed electron detachment are observed in the 193 nm photoelectron spectra of the four clusters. The 266 nm spectra of the PtCl6 2-∙thymine and PtCl6 2-∙adenine complexes also display very prominent delayed electron emission bands. These results mirror recent results on the related Pt(CN)4 2-∙nucleobase complexes [Sen et al, J. Phys. Chem. B, 119, 11626, 2015]. The observation of delayed electron emission bands in the PtCl6 2-∙nucleobase spectra obtained in this work, as for the previously studied Pt(CN)4 2-∙nucleobase complexes, is attributed to onephoton excitation of nucleobase-centred excited states that can effectively couple to the electron detachment continuum, producing strong electron detachment. Moreover, the selective, strong excitation of the delayed emission bands in the 266 nm spectra is linked to fundamental differences in the individual nucleobase photophysics at this excitation energy. This strongly supports our previous suggestion that the dianion within these clusters can be viewed as a “dynamic tag” which has the propensity to emit electrons when the attached nucleobase decays over a timescale long enough to allow autodetachment.

  14. Photoelectron spectroscopy of hexachloroplatinate-nucleobase complexes: Nucleobase excited state decay observed via delayed electron emission.

    PubMed

    Sen, Ananya; Matthews, Edward M; Hou, Gao-Lei; Wang, Xue-Bin; Dessent, Caroline E H

    2015-11-14

    We report low-temperature photoelectron spectra of isolated gas-phase complexes of the hexachloroplatinate dianion bound to the nucleobases uracil, thymine, cytosine, and adenine. The spectra display well-resolved, distinct peaks that are consistent with complexes where the hexachloroplatinate dianion is largely intact. Adiabatic electron detachment energies for the hexachloroplatinate-nucleobase complexes are measured as 2.26-2.36 eV. The magnitudes of the repulsive Coulomb barriers (RCBs) of the complexes are all ∼1.7 eV, values that are lower than the RCB of the uncomplexed PtCl6 (2-) dianion as a result of charge solvation by the nucleobases. In addition to the resolved spectral features, broad featureless bands indicative of delayed electron detachment are observed in the 193 nm photoelectron spectra of the four clusters. The 266 nm spectra of the PtCl6 (2-) ⋅ thymine and PtCl6 (2-) ⋅ adenine complexes also display very prominent delayed electron emission bands. These results mirror recent results on the related Pt(CN)4 (2-) ⋅ nucleobase complexes [A. Sen et al., J. Phys. Chem. B 119, 11626 (2015)]. The observation of delayed electron emission bands in the PtCl6 (2-) ⋅ nucleobase spectra obtained in this work, as for the previously studied Pt(CN)4 (2-) ⋅ nucleobase complexes, is attributed to one-photon excitation of nucleobase-centred excited states that can effectively couple to the electron detachment continuum, producing strong electron detachment. Moreover, the selective, strong excitation of the delayed emission bands in the 266 nm spectra is linked to fundamental differences in the individual nucleobase photophysics at this excitation energy. This strongly supports our previous suggestion that the dianion within these clusters can be viewed as a "dynamic tag" which has the propensity to emit electrons when the attached nucleobase decays over a time scale long enough to allow autodetachment.

  15. Facilitators for travelling with local public transport among people with mild cognitive limitations after stroke.

    PubMed

    Ståhl, Agneta; Månsson Lexell, Eva

    2017-01-24

    Previous research of how people with stroke manage public transport has mainly focused on barriers due to physical limitations whereas the influence of cognitive limitations is scarce. There is also a lack of knowledge of facilitators that can help to overcome these barriers. The aim of this study was to describe facilitators for travelling with public transport, e.g. local buses, among people with mild cognitive limitations after stroke. A multiple case study research design was used, where quantitative and qualitative data were utilized, and analysed according to a mixed methods design. The case descriptions reveal how people with mild cognitive limitations after stroke manage their trips but constantly have to be prepared to solve problems to unexpected events. Personal characteristics and other individual strategies together with support and solutions from society were important facilitators for travelling with bus. This study takes a new approach by specifically describing facilitators for travelling with public transport among people with mild cognitive limitations after stroke. To facilitate participation in society for this particular traveller group, occupational therapists have an important role when new technology and interventions that target bus travels, and other modes of transport are developed.

  16. Decreased Salinity and Actinide Mobility: Colloid-Facilitated Transport or pH Change?

    PubMed

    Haliena, Brian; Zheng, Hangping; Melson, Nathan; Kaplan, Daniel I; Barnett, Mark O

    2016-01-19

    Colloids have been implicated in influencing the transport of actinides and other adsorbed contaminants in the subsurface, significantly increasing their mobility. Such colloid-facilitated transport can be induced by changes in groundwater chemistry that occur, for example, when high ionic strength contaminant plumes are displaced by infiltrating rainwater. We studied the transport and mobility of Th(IV), as an analogue for Pu(IV) and other tetravalent actinides [An(IV)], in saturated columns packed with a natural heterogeneous subsurface sandy sediment. As expected, decreases in ionic strength both promoted the mobilization of natural colloids and enhanced the transport of previously adsorbed Th(IV). However, colloid-facilitated transport played only a minor role in enhancing the transport of Th(IV). Instead, the enhanced transport of Th(IV) was primarily due to the pH-dependent desorption of Th(IV) caused by the change in ionic strength. In contrast, the adsorption of Th(IV) had a marked impact on the surface charge of the sandy sediment, significantly affecting the mobility of the colloids. In the absence of Th(IV), changes in ionic strength were ineffective at releasing colloids while in the presence of Th(IV), decreases in ionic strength liberated significant concentrations of colloids. Therefore, under the conditions of our experiments which mimicked acidic, high ionic strength groundwater contaminant plumes, Th(IV) had a much greater effect on colloid transport than colloids had on Th(IV) transport.

  17. Carbonaceous meteorites contain a wide range of extraterrestrial nucleobases.

    PubMed

    Callahan, Michael P; Smith, Karen E; Cleaves, H James; Ruzicka, Josef; Stern, Jennifer C; Glavin, Daniel P; House, Christopher H; Dworkin, Jason P

    2011-08-23

    All terrestrial organisms depend on nucleic acids (RNA and DNA), which use pyrimidine and purine nucleobases to encode genetic information. Carbon-rich meteorites may have been important sources of organic compounds required for the emergence of life on the early Earth; however, the origin and formation of nucleobases in meteorites has been debated for over 50 y. So far, the few nucleobases reported in meteorites are biologically common and lacked the structural diversity typical of other indigenous meteoritic organics. Here, we investigated the abundance and distribution of nucleobases and nucleobase analogs in formic acid extracts of 12 different meteorites by liquid chromatography-mass spectrometry. The Murchison and Lonewolf Nunataks 94102 meteorites contained a diverse suite of nucleobases, which included three unusual and terrestrially rare nucleobase analogs: purine, 2,6-diaminopurine, and 6,8-diaminopurine. In a parallel experiment, we found an identical suite of nucleobases and nucleobase analogs generated in reactions of ammonium cyanide. Additionally, these nucleobase analogs were not detected above our parts-per-billion detection limits in any of the procedural blanks, control samples, a terrestrial soil sample, and an Antarctic ice sample. Our results demonstrate that the purines detected in meteorites are consistent with products of ammonium cyanide chemistry, which provides a plausible mechanism for their synthesis in the asteroid parent bodies, and strongly supports an extraterrestrial origin. The discovery of new nucleobase analogs in meteorites also expands the prebiotic molecular inventory available for constructing the first genetic molecules.

  18. Carbonaceous meteorites contain a wide range of extraterrestrial nucleobases

    PubMed Central

    Callahan, Michael P.; Smith, Karen E.; Cleaves, H. James; Ruzicka, Josef; Stern, Jennifer C.; Glavin, Daniel P.; House, Christopher H.; Dworkin, Jason P.

    2011-01-01

    All terrestrial organisms depend on nucleic acids (RNA and DNA), which use pyrimidine and purine nucleobases to encode genetic information. Carbon-rich meteorites may have been important sources of organic compounds required for the emergence of life on the early Earth; however, the origin and formation of nucleobases in meteorites has been debated for over 50 y. So far, the few nucleobases reported in meteorites are biologically common and lacked the structural diversity typical of other indigenous meteoritic organics. Here, we investigated the abundance and distribution of nucleobases and nucleobase analogs in formic acid extracts of 12 different meteorites by liquid chromatography–mass spectrometry. The Murchison and Lonewolf Nunataks 94102 meteorites contained a diverse suite of nucleobases, which included three unusual and terrestrially rare nucleobase analogs: purine, 2,6-diaminopurine, and 6,8-diaminopurine. In a parallel experiment, we found an identical suite of nucleobases and nucleobase analogs generated in reactions of ammonium cyanide. Additionally, these nucleobase analogs were not detected above our parts-per-billion detection limits in any of the procedural blanks, control samples, a terrestrial soil sample, and an Antarctic ice sample. Our results demonstrate that the purines detected in meteorites are consistent with products of ammonium cyanide chemistry, which provides a plausible mechanism for their synthesis in the asteroid parent bodies, and strongly supports an extraterrestrial origin. The discovery of new nucleobase analogs in meteorites also expands the prebiotic molecular inventory available for constructing the first genetic molecules. PMID:21836052

  19. The Ca(2+)-ATPase pump facilitates bidirectional proton transport across the sarco/endoplasmic reticulum.

    PubMed

    Espinoza-Fonseca, L Michel

    2017-03-28

    Ca(2+) transport across the sarco/endoplasmic reticulum (SR) plays an essential role in intracellular Ca(2+) homeostasis, signalling, cell differentiation and muscle contractility. During SR Ca(2+) uptake and release, proton fluxes are required to balance the charge deficit generated by the exchange of Ca(2+) and other ions across the SR. During Ca(2+) uptake by the SR Ca(2+)-ATPase (SERCA), two protons are countertransported from the SR lumen to the cytosol, thus partially compensating for the charge moved by Ca(2+) transport. Studies have shown that protons are also transported from the cytosol to the lumen during Ca(2+) release, but a transporter that facilitates proton transport into the SR lumen has not been described. In this article we propose that SERCA forms pores that facilitate bidirectional proton transport across the SR. We describe the location and structure of water-filled pores in SERCA that form cytosolic and luminal pathways for protons to cross the SR membrane. Based on this structural information, we suggest mechanistic models for proton translocation to the cytosol during active Ca(2+) transport, and into the SR lumen during SERCA inhibition by endogenous regulatory proteins. Finally, we discuss the physiological consequences of SERCA-mediated bidirectional proton transport across the SR membrane of muscle and non-muscle cells.

  20. Computational modelling of amino acid exchange and facilitated transport in placental membrane vesicles.

    PubMed

    Panitchob, N; Widdows, K L; Crocker, I P; Hanson, M A; Johnstone, E D; Please, C P; Sibley, C P; Glazier, J D; Lewis, R M; Sengers, B G

    2015-01-21

    Placental amino acid transport is required for fetal development and impaired transport has been associated with poor fetal growth. It is well known that placental amino acid transport is mediated by a broad array of specific membrane transporters with overlapping substrate specificity. However, it is not fully understood how these transporters function, both individually and as an integrated system. We propose that mathematical modelling could help in further elucidating the underlying mechanisms of how these transporters mediate placental amino acid transport. The aim of this work is to model the sodium independent transport of serine, which has been assumed to follow an obligatory exchange mechanism. However, previous amino acid uptake experiments in human placental microvillous plasma membrane vesicles have persistently produced results that are seemingly incompatible with such a mechanism; i.e. transport has been observed under zero-trans conditions, in the absence of internal substrates inside the vesicles to drive exchange. This observation raises two alternative hypotheses; (i) either exchange is not fully obligatory, or (ii) exchange is indeed obligatory, but an unforeseen initial concentration of amino acid substrate is present within the vesicle which could drive exchange. To investigate these possibilities, a mathematical model for tracer uptake was developed based on carrier mediated transport, which can represent either facilitated diffusion or obligatory exchange (also referred to as uniport and antiport mechanisms, respectively). In vitro measurements of serine uptake by placental microvillous membrane vesicles were carried out and the model applied to interpret the results based on the measured apparent Michaelis-Menten parameters Km and Vmax. In addition, based on model predictions, a new time series experiment was implemented to distinguish the hypothesised transporter mechanisms. Analysis of the results indicated the presence of a facilitated

  1. Arabidopsis WAT1 is a vacuolar auxin transport facilitator required for auxin homoeostasis

    PubMed Central

    Ranocha, Philippe; Dima, Oana; Nagy, Réka; Felten, Judith; Corratgé-Faillie, Claire; Novák, Ondřej; Morreel, Kris; Lacombe, Benoît; Martinez, Yves; Pfrunder, Stephanie; Jin, Xu; Renou, Jean-Pierre; Thibaud, Jean-Baptiste; Ljung, Karin; Fischer, Urs; Martinoia, Enrico; Boerjan, Wout; Goffner, Deborah

    2013-01-01

    The plant hormone auxin (indole-3-acetic acid, IAA) has a crucial role in plant development. Its spatiotemporal distribution is controlled by a combination of biosynthetic, metabolic and transport mechanisms. Four families of auxin transporters have been identified that mediate transport across the plasma or endoplasmic reticulum membrane. Here we report the discovery and the functional characterization of the first vacuolar auxin transporter. We demonstrate that WALLS ARE THIN1 (WAT1), a plant-specific protein that dictates secondary cell wall thickness of wood fibres, facilitates auxin export from isolated Arabidopsis vacuoles in yeast and in Xenopus oocytes. We unambiguously identify IAA and related metabolites in isolated Arabidopsis vacuoles, suggesting a key role for the vacuole in intracellular auxin homoeostasis. Moreover, local auxin application onto wat1 mutant stems restores fibre cell wall thickness. Our study provides new insight into the complexity of auxin transport in plants and a means to dissect auxin function during fibre differentiation. PMID:24129639

  2. Arabidopsis WAT1 is a vacuolar auxin transport facilitator required for auxin homoeostasis.

    PubMed

    Ranocha, Philippe; Dima, Oana; Nagy, Réka; Felten, Judith; Corratgé-Faillie, Claire; Novák, Ondřej; Morreel, Kris; Lacombe, Benoît; Martinez, Yves; Pfrunder, Stephanie; Jin, Xu; Renou, Jean-Pierre; Thibaud, Jean-Baptiste; Ljung, Karin; Fischer, Urs; Martinoia, Enrico; Boerjan, Wout; Goffner, Deborah

    2013-01-01

    The plant hormone auxin (indole-3-acetic acid, IAA) has a crucial role in plant development. Its spatiotemporal distribution is controlled by a combination of biosynthetic, metabolic and transport mechanisms. Four families of auxin transporters have been identified that mediate transport across the plasma or endoplasmic reticulum membrane. Here we report the discovery and the functional characterization of the first vacuolar auxin transporter. We demonstrate that WALLS ARE THIN1 (WAT1), a plant-specific protein that dictates secondary cell wall thickness of wood fibres, facilitates auxin export from isolated Arabidopsis vacuoles in yeast and in Xenopus oocytes. We unambiguously identify IAA and related metabolites in isolated Arabidopsis vacuoles, suggesting a key role for the vacuole in intracellular auxin homoeostasis. Moreover, local auxin application onto wat1 mutant stems restores fibre cell wall thickness. Our study provides new insight into the complexity of auxin transport in plants and a means to dissect auxin function during fibre differentiation.

  3. The multidrug transporters belonging to major facilitator superfamily in Mycobacterium tuberculosis.

    PubMed Central

    De Rossi, Edda; Arrigo, Patrizio; Bellinzoni, Marco; Silva, Pedro A. E.; Martín, Carlos; Aínsa, José A.; Guglierame, Paola; Riccardi, Giovanna

    2002-01-01

    BACKGROUND: Both intrinsic and acquired multidrug resistance play an important role in the insurgence of tuberculosis. Detailed knowledge of the molecular basis of drug recognition and transport by multidrug transport systems is required for the development of new antibiotics that are not extruded or of inhibitors that block the multidrug transporter and allow traditional antibiotics to be effective. MATERIALS AND METHODS: We have undertaken the inventory of the drug transporters subfamily, included in the major facilitator superfamily (MFS), encoded by the complete genome of Mycobacterium tuberculosis (MTB). These proteins were identified on the basis of their characteristic stretches of amino acids and transmembrane segments (TMS) number. CONCLUSIONS: Genome analysis and searches of homology between the identified transporters and proteins characterized in other organisms revealed 16 open reading frames encoding putative drug efflux pumps belonging to MFS. In the case of two of them, we also have demonstrated that they function as drug efflux proteins. PMID:12520088

  4. Sensitivity analyses of a colloid-facilitated contaminant transport model for unsaturated heterogeneous soil conditions.

    NASA Astrophysics Data System (ADS)

    Périard, Yann; José Gumiere, Silvio; Rousseau, Alain N.; Caron, Jean

    2013-04-01

    Certain contaminants may travel faster through soils when they are sorbed to subsurface colloidal particles. Indeed, subsurface colloids may act as carriers of some contaminants accelerating their translocation through the soil into the water table. This phenomenon is known as colloid-facilitated contaminant transport. It plays a significant role in contaminant transport in soils and has been recognized as a source of groundwater contamination. From a mechanistic point of view, the attachment/detachment of the colloidal particles from the soil matrix or from the air-water interface and the straining process may modify the hydraulic properties of the porous media. Šimůnek et al. (2006) developed a model that can simulate the colloid-facilitated contaminant transport in variably saturated porous media. The model is based on the solution of a modified advection-dispersion equation that accounts for several processes, namely: straining, exclusion and attachement/detachement kinetics of colloids through the soil matrix. The solutions of these governing, partial differential equations are obtained using a standard Galerkin-type, linear finite element scheme, implemented in the HYDRUS-2D/3D software (Šimůnek et al., 2012). Modeling colloid transport through the soil and the interaction of colloids with the soil matrix and other contaminants is complex and requires the characterization of many model parameters. In practice, it is very difficult to assess actual transport parameter values, so they are often calibrated. However, before calibration, one needs to know which parameters have the greatest impact on output variables. This kind of information can be obtained through a sensitivity analysis of the model. The main objective of this work is to perform local and global sensitivity analyses of the colloid-facilitated contaminant transport module of HYDRUS. Sensitivity analysis was performed in two steps: (i) we applied a screening method based on Morris' elementary

  5. FACILITATED TRANSPORT OF INORGANIC CONTAMINANTS IN GROUNDWATER: PART II. COLLOIDAL TRANSPORT

    EPA Science Inventory

    This project consisted of both field and laboratory components. Field studies evaluated routine sampling procedures for determination of aqueous inorganicgeochemistry and assessment of contaminant transport by colloidal mobility. Research at three different metal-contaminated sit...

  6. FACILITATED TRANSPORT OF INORGANIC CONTAMINANTS IN GROUNDWATER: PART II. COLLOIDAL TRANSPORT

    EPA Science Inventory

    This project consisted of both field and laboratory components. Field studies evaluated routine sampling procedures for determination of aqueous inorganicgeochemistry and assessment of contaminant transport by colloidal mobility. Research at three different metal-contaminated sit...

  7. Photoelectron spectroscopy of hexachloroplatinate-nucleobase complexes: Nucleobase excited state decay observed via delayed electron emission

    SciTech Connect

    Sen, Ananya; Matthews, Edward M.; Dessent, Caroline E. H. E-mail: xuebin.wang@pnnl.gov; Hou, Gao-Lei; Wang, Xue-Bin E-mail: xuebin.wang@pnnl.gov

    2015-11-14

    We report low-temperature photoelectron spectra of isolated gas-phase complexes of the hexachloroplatinate dianion bound to the nucleobases uracil, thymine, cytosine, and adenine. The spectra display well-resolved, distinct peaks that are consistent with complexes where the hexachloroplatinate dianion is largely intact. Adiabatic electron detachment energies for the hexachloroplatinate-nucleobase complexes are measured as 2.26-2.36 eV. The magnitudes of the repulsive Coulomb barriers (RCBs) of the complexes are all ∼1.7 eV, values that are lower than the RCB of the uncomplexed PtCl{sub 6}{sup 2−} dianion as a result of charge solvation by the nucleobases. In addition to the resolved spectral features, broad featureless bands indicative of delayed electron detachment are observed in the 193 nm photoelectron spectra of the four clusters. The 266 nm spectra of the PtCl{sub 6}{sup 2−} ⋅ thymine and PtCl{sub 6}{sup 2−} ⋅ adenine complexes also display very prominent delayed electron emission bands. These results mirror recent results on the related Pt(CN){sub 4}{sup 2−} ⋅ nucleobase complexes [A. Sen et al., J. Phys. Chem. B 119, 11626 (2015)]. The observation of delayed electron emission bands in the PtCl{sub 6}{sup 2−} ⋅ nucleobase spectra obtained in this work, as for the previously studied Pt(CN){sub 4}{sup 2−} ⋅ nucleobase complexes, is attributed to one-photon excitation of nucleobase-centred excited states that can effectively couple to the electron detachment continuum, producing strong electron detachment. Moreover, the selective, strong excitation of the delayed emission bands in the 266 nm spectra is linked to fundamental differences in the individual nucleobase photophysics at this excitation energy. This strongly supports our previous suggestion that the dianion within these clusters can be viewed as a “dynamic tag” which has the propensity to emit electrons when the attached nucleobase decays over a time scale long enough to

  8. Trehalose transporter 1, a facilitated and high-capacity trehalose transporter, allows exogenous trehalose uptake into cells

    PubMed Central

    Kikawada, Takahiro; Saito, Ayako; Kanamori, Yasushi; Nakahara, Yuichi; Iwata, Ken-ichi; Tanaka, Daisuke; Watanabe, Masahiko; Okuda, Takashi

    2007-01-01

    Trehalose is potentially a useful cryo- or anhydroprotectant molecule for cells and biomolecules such as proteins and nucleotides. A major obstacle to application is that cellular membranes are impermeable to trehalose. In this study, we isolated and characterized the functions of a facilitated trehalose transporter [trehalose transporter 1 (TRET1)] from an anhydrobiotic insect, Polypedilum vanderplanki. Tret1 cDNA encodes a 504-aa protein with 12 predicted transmembrane structures. Tret1 expression was induced by either desiccation or salinity stress. Expression was predominant in the fat body and occurred concomitantly with the accumulation of trehalose, indicating that TRET1 is involved in transporting trehalose synthesized in the fat body into the hemolymph. Functional expression of TRET1 in Xenopus oocytes showed that transport activity was stereochemically specific for trehalose and independent of extracellular pH (between 4.0 and 9.0) and electrochemical membrane potential. These results indicate that TRET1 is a trehalose-specific facilitated transporter and that the direction of transport is reversible depending on the concentration gradient of trehalose. The extraordinarily high values for apparent Km (≥100 mM) and Vmax (≥500 pmol/min per oocyte) for trehalose both indicate that TRET1 is a high-capacity transporter of trehalose. Furthermore, TRET1 was found to function in mammalian cells, suggesting that it confers trehalose permeability on cells, including those of vertebrates as well as insects. These characteristic features imply that TRET1 in combination with trehalose has high potential for basic and practical applications in vivo. PMID:17606922

  9. Transportation Facilitation Education Program: A Handbook for Transportation and Distribution. Part III. Final Report.

    ERIC Educational Resources Information Center

    Oregon Univ., Eugene. Coll. of Business Administration.

    The handbook accents the nature of transportation and related domestic and international business activities. Its objective is to provide basic information for the newcomer to the field. Chapters 2 and 3 describe assistance available from public and private agencies, as well as regulatory requirements for foreign traders and a resume of the…

  10. Synthetic ion transporters can induce apoptosis by facilitating chloride anion transport into cells.

    PubMed

    Ko, Sung-Kyun; Kim, Sung Kuk; Share, Andrew; Lynch, Vincent M; Park, Jinhong; Namkung, Wan; Van Rossom, Wim; Busschaert, Nathalie; Gale, Philip A; Sessler, Jonathan L; Shin, Injae

    2014-10-01

    Anion transporters based on small molecules have received attention as therapeutic agents because of their potential to disrupt cellular ion homeostasis. However, a direct correlation between a change in cellular chloride anion concentration and cytotoxicity has not been established for synthetic ion carriers. Here we show that two pyridine diamide-strapped calix[4]pyrroles induce coupled chloride anion and sodium cation transport in both liposomal models and cells, and promote cell death by increasing intracellular chloride and sodium ion concentrations. Removing either ion from the extracellular media or blocking natural sodium channels with amiloride prevents this effect. Cell experiments show that the ion transporters induce the sodium chloride influx, which leads to an increased concentration of reactive oxygen species, release of cytochrome c from the mitochondria and apoptosis via caspase activation. However, they do not activate the caspase-independent apoptotic pathway associated with the apoptosis-inducing factor. Ion transporters, therefore, represent an attractive approach for regulating cellular processes that are normally controlled tightly by homeostasis.

  11. Synthetic ion transporters can induce apoptosis by facilitating chloride anion transport into cells

    NASA Astrophysics Data System (ADS)

    Ko, Sung-Kyun; Kim, Sung Kuk; Share, Andrew; Lynch, Vincent M.; Park, Jinhong; Namkung, Wan; van Rossom, Wim; Busschaert, Nathalie; Gale, Philip A.; Sessler, Jonathan L.; Shin, Injae

    2014-10-01

    Anion transporters based on small molecules have received attention as therapeutic agents because of their potential to disrupt cellular ion homeostasis. However, a direct correlation between a change in cellular chloride anion concentration and cytotoxicity has not been established for synthetic ion carriers. Here we show that two pyridine diamide-strapped calix[4]pyrroles induce coupled chloride anion and sodium cation transport in both liposomal models and cells, and promote cell death by increasing intracellular chloride and sodium ion concentrations. Removing either ion from the extracellular media or blocking natural sodium channels with amiloride prevents this effect. Cell experiments show that the ion transporters induce the sodium chloride influx, which leads to an increased concentration of reactive oxygen species, release of cytochrome c from the mitochondria and apoptosis via caspase activation. However, they do not activate the caspase-independent apoptotic pathway associated with the apoptosis-inducing factor. Ion transporters, therefore, represent an attractive approach for regulating cellular processes that are normally controlled tightly by homeostasis.

  12. Biology of the major facilitative folate transporters SLC19A1 and SLC46A1.

    PubMed

    Hou, Zhanjun; Matherly, Larry H

    2014-01-01

    This chapter focuses on the biology of the major facilitative membrane folate transporters, the reduced folate carrier (RFC), and the proton-coupled folate transporter (PCFT). Folates are essential vitamins, and folate deficiency contributes to a variety of heath disorders. RFC is ubiquitously expressed and is the major folate transporter in mammalian cells and tissues. PCFT mediates intestinal absorption of dietary folates. Clinically relevant antifolates such as methotrexate (MTX) are transported by RFC, and the loss of RFC transport is an important mechanism of MTX resistance. PCFT is abundantly expressed in human tumors and is active under pH conditions associated with the tumor microenvironment. Pemetrexed (PMX) is an excellent substrate for PCFT as well as for RFC. Novel tumor-targeted antifolates related to PMX with selective membrane transport by PCFT over RFC are being developed. The molecular picture of RFC and PCFT continues to evolve relating to membrane topology, N-glycosylation, energetics, and identification of structurally and functionally important domains and amino acids. The molecular bases for MTX resistance associated with loss of RFC function, and for the rare autosomal recessive condition, hereditary folate malabsorption (HFM), attributable to mutant PCFT, have been established. From structural homologies to the bacterial transporters GlpT and LacY, homology models were developed for RFC and PCFT, enabling new mechanistic insights and experimentally testable hypotheses. RFC and PCFT exist as homo-oligomers, and evidence suggests that homo-oligomerization of RFC and PCFT monomeric proteins may be important for intracellular trafficking and/or transport function. Better understanding of the structure and function of RFC and PCFT should facilitate the rational development of new therapeutic strategies for cancer as well as for HFM.

  13. Facilitated transport of dioxins in soil following unintentional release of pesticide-surfactant formulations.

    PubMed

    Grant, Sharon; Mortimer, Munro; Stevenson, Gavin; Malcolm, Don; Gaus, Caroline

    2011-01-15

    Colloids such as surfactant micelles can act as transport facilitators for highly lipophilic, generally immobile contaminants in soil. Following a fire at a pesticide facility, this study investigated vertical and lateral migration of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in heterogeneous soil beneath bunded ponds, where contaminated wastewater containing high surfactant loads was stored until remediation. Initially, surface and subsurface soil was obtained during excavation, and subsequently intact cores to 5.7 m were collected. ΣPCDD/F concentrations were elevated in the wastewater (15-81 ng/L) and correspondingly in pond surface soils (6.1-61 ng/g). Maximum ΣPCDD/F concentrations were, however, observed at 2-2.5 m depth (68-130 ng/g), far below their expected mobility range based on physicochemical properties. Congener specific analysis further indicated that PCDD/F mobility was reversed, with the least water-soluble congener migrating to the greatest extent. The presence of higher chlorinated PCDD/Fs throughout a core collected in the direction of groundwater flow indicated subsequent lateral transport. These results provide field evidence for rapid vertical migration (2.4 m in <4 months) of highly lipophilic PCDD/Fs and suggest surfactant facilitated transport as the dominant transport mechanism. Quantification and evaluation of such fundamental changes in contaminant transport and fate in the presence of surfactants is required to identify areas at risk of groundwater contamination.

  14. Carbonaceous Meteorites Contain a Wide Range of Extraterrestrial Nucleobases

    NASA Technical Reports Server (NTRS)

    Callahan, Michael P.; Smith, Karen E.; Cleaves, H. James, II; Ruzicka, Josef; Stern, Jennifer C.; Glavin, Daniel P.; House, Christopher H.; Dworkin, Jason P.

    2011-01-01

    All terrestrial organisms depend on nucleic acids (RNA and DNA), which use pyrimidine and purine nucleobases to encode genetic information. Carbon-rich meteorites may have been important sources of organic compounds required for the emergence of life on the early Earth; however, the origin and formation of nuc1eobases in meteorites has been debated for over 50 y. So far, the few nuc1eobases reported in meteorites are biologically common and lacked the structural diversity typical of other indigenous meteoritic organics. Here, we investigated the abundance and distribution of nucleobases and nucleobase analogs in formic acid extracts of 12 different meteorites by liquid chromatography-mass spectrometry. The Murchison and Lonewolf Nunataks 94102 meteorites contained a diverse suite of nucleobases, which included three unusual and terrestrially rare nucleobase analogs; purine, 2,6-diminopurine, and 6,8-diaminopurine. In a parallel experiment, we found an identical suite of nucleobases and nucleobase analogs generated in reactions of ammonium cyanide. Additionally, these nucleobase analoge were not detected above our parts-per-billion detection limits in any of the procedural blanks, control samples, a terrestrial soil sample, and an Antarctic ice sample. Our results demonstrate that the purines detected in meteorites are consistent with products of ammonium cyanide chemistry, which provides a plausible mechanism for their synthesis in the asteroid parent bodies, and strongly supports an extraterrestrial origin. The discovery of new nucleobase analogs in meteorites also expands the prebiotic molecular inventory available for constructing the first genetic molecules.

  15. The effect of myoglobin-facilitated oxygen transport on the basal metabolism of papillary muscle.

    PubMed Central

    Loiselle, D S

    1987-01-01

    A mathematical model of oxygen diffusion into cylindrical papillary muscles is presented. The model partitions total oxygen flux into its simple and myoglobin-facilitated components. The model includes variable sigmoidal, exponential, or hyperbolic functions relating oxygen partial pressure to both fractional myoglobin saturation and rate of oxygen consumption. The behavior of the model was explored for a variety of saturation- and consumption-concentration relations. Facilitation of oxygen transport by myoglobin was considerable as indexed both by the elevation of oxygen partial pressure on the longitudinal axis of the muscle and by the fraction of total oxygen flux at the muscle center contributed by oxymyoglobin. Despite its facilitation of oxygen flux at the muscle center, myoglobin made only a negligible contribution to the total oxygen consumption averaged over the muscle cross-section. Hence the presence of myoglobin fails to explain either the experimentally determined basal metabolism-muscle radius relation or the stretch effect observed in isolated papillary muscle. PMID:3607211

  16. Colloid-Facilitated Transport of Low-Solubility Radionuclides: A Field, Experimental, and Modeling Investigation

    SciTech Connect

    Kersting, A B; Reimus, P W; Abdel-Fattah, A; Allen, P G; Anghel, I; Benedict, F C; Esser, B K; Lu, N; Kung, K S; Nelson, J; Neu, M P; Reilly, S D; Smith, D K; Sylwester, E R; Wang, L; Ware, S D; Warren, RG; Williams, R W; Zavarin, M; Zhao, P

    2003-02-01

    For the last several years, the Underground Test Area (UGTA) program has funded a series of studies carried out by scientists to investigate the role of colloids in facilitating the transport of low-solubility radionuclides in groundwater, specifically plutonium (Pu). Although the studies were carried out independently, the overarching goals of these studies has been to determine if colloids in groundwater at the NTS can and will transport low-solubility radionuclides such as Pu, define the geochemical mechanisms under which this may or may not occur, determine the hydrologic parameters that may or may not enhance transport through fractures and provide recommendations for incorporating this information into future modeling efforts. The initial motivation for this work came from the observation in 1997 and 1998 by scientists from Lawrence Livermore National Laboratory (LLNL) and Los Alamos National Laboratory (LANL) that low levels of Pu originally from the Benham underground nuclear test were detected in groundwater from two different aquifers collected from wells 1.3 km downgradient (Kersting et al., 1999). Greater than 90% of the Pu and other radionuclides were associated with the naturally occurring colloidal fraction (< 1 micron particles) in the groundwater. The colloids consisted mainly of zeolite (mordenite, clinoptilolite/heulandite), clays (illite, smectite) and cristobalite (SiO{sub 2}). These minerals were also identified as alteration mineral components in the host rock aquifer, a rhyolitic tuff. The observation that Pu can and has migrated in the subsurface at the NTS has forced a rethinking of our basic assumptions regarding the mechanical and geochemical transport pathways of low-solubility radionuclides. If colloid-facilitated transport is the primary mechanism for transporting low-solubility radionuclides in the subsurface, then current transport models based solely on solubility arguments and retardation estimates may underestimate the flux and

  17. The major facilitator superfamily transporter ZIFL2 modulates cesium and potassium homeostasis in Arabidopsis.

    PubMed

    Remy, Estelle; Cabrito, Tânia R; Batista, Rita A; Teixeira, Miguel C; Sá-Correia, Isabel; Duque, Paula

    2015-01-01

    Potassium (K(+)) is an essential mineral nutrient for plant growth and development, with numerous membrane transporters and channels having been implicated in the maintenance and regulation of its homeostasis. The cation cesium (Cs(+)) is toxic for plants but shares similar chemical properties to the K(+) ion and hence competes with its transport. Here, we report that K(+) and Cs(+) homeostasis in Arabidopsis thaliana also requires the action of ZIFL2 (Zinc-Induced Facilitator-Like 2), a member of the Major Facilitator Superfamily (MFS) of membrane transporters. We show that the Arabidopsis ZIFL2 is a functional transporter able to mediate K(+) and Cs(+) influx when heterologously expressed in yeast. Promoter-reporter, reverse transcription-PCR and fluorescent protein fusion experiments indicate that the predominant ZIFL2.1 isoform is targeted to the plasma membrane of endodermal and pericyle root cells. ZIFL2 loss of function and overexpression exacerbate and alleviate plant sensitivity, respectively, upon Cs(+) and excess K(+) supply, also influencing Cs(+) whole-plant partitioning. We propose that the activity of this Arabidopsis MFS carrier promotes cellular K(+) efflux in the root, thereby restricting Cs(+)/K(+) xylem loading and subsequent root to shoot translocation under conditions of Cs(+) or high K(+) external supply.

  18. [The role of facilitated diffusion in glucose transport across the apical membrane of enterocytes].

    PubMed

    Gromova, L V; Grefner, N M; Gruzdkov, A A; Komissarchik, Ia Iu

    2006-03-01

    In chronic experiments on Wistar rats, glucose and galactose absorption in the isolated loop of the small intestine considerably decreased in presence of both phloridzine am phloritine (inhibitors of the glucose transporters SGLT1 and GLUT2). The load of the isolated loop with glucose or galactose solutions scarcely influenced the absorption of 2-deoxi-D-glucose (substrate for GLUT2). According to the immunocytochemical analysis by means of confocal microscopy, after the load of the isolated loop with glucose (75 mM) the labels to GLUT2 and proteinkinase C (PKC betalI) were concentrated mainly in the apical part of the enterocytes, whereas after the load with the Ringer solution--in the basal part of the enterocytes. It was shown on the mathematical model that the part of the facilitated diffusion in the total glucose absorption was considerably lesser in comparison with the active transport mediated by SGLT1. Thus the findings support the hypothesis about a recruitment of the transporter GLUT2 into the apical membrane of the enterocytes and its involvement in glucose transfer across this membrane. However, under natural conditions, the active transport is the main mechanism of glucose absorption, whereas the facilitated diffusion plays a certain role only at high carbohydrate loads.

  19. Transport of Glucose by Bifidobacterium animalis subsp. lactis Occurs via Facilitated Diffusion▿

    PubMed Central

    Briczinski, E. P.; Phillips, A. T.; Roberts, R. F.

    2008-01-01

    Two strains of Bifidobacterium animalis subsp. lactis were indistinguishable by several nucleic acid-based techniques; however, the type strain DSMZ 10140 was glucose utilization positive, while RB 4825, an industrially employed strain, was unable to grow rapidly on glucose as the principal carbon source. This difference was attributed to the presence of a low-affinity facilitated-diffusion glucose transporter identified in DSMZ 10140 but lacking in RB 4825. Uptake of d-[U-14C]glucose in DSMZ 10140 was stimulated by monovalent cations (ammonium, sodium, potassium, and lithium) and inhibited by divalent cations (calcium and magnesium). When competitor carbohydrates were included in the uptake assays, stereospecific inhibition was exhibited, with greater competition by methyl-β-glucoside than methyl-α-glucoside. Significant inhibition (>30%) was observed with phloretin, an inhibitor of facilitated diffusion of glucose, whereas there was no inhibition by sodium fluoride, iodoacetate, sodium arsenate, sodium azide, 2,4-dinitrophenol, monensin, or valinomycin, which typically reduce energy-driven transport. Based on kinetic analyses, the mean values for Kt and Vmax were 14.8 ± 3.4 mM d-glucose and 0.13 ± 0.03 μmol glucose/min/mg cell protein, respectively. Glucose uptake by several glucose-utilizing commercial strains of B. animalis subsp. lactis was also inhibited by phloretin, indicating the presence of facilitated diffusion glucose transporters in those strains. Since DSMZ 10140 has been previously reported to lack a functional glucose phosphoenolpyruvate phosphotransferase system, the glucose transporter identified here is responsible for much of the organism's glucose uptake. PMID:18791026

  20. Transport of glucose by Bifidobacterium animalis subsp. lactis occurs via facilitated diffusion.

    PubMed

    Briczinski, E P; Phillips, A T; Roberts, R F

    2008-11-01

    Two strains of Bifidobacterium animalis subsp. lactis were indistinguishable by several nucleic acid-based techniques; however, the type strain DSMZ 10140 was glucose utilization positive, while RB 4825, an industrially employed strain, was unable to grow rapidly on glucose as the principal carbon source. This difference was attributed to the presence of a low-affinity facilitated-diffusion glucose transporter identified in DSMZ 10140 but lacking in RB 4825. Uptake of D-[U-(14)C]glucose in DSMZ 10140 was stimulated by monovalent cations (ammonium, sodium, potassium, and lithium) and inhibited by divalent cations (calcium and magnesium). When competitor carbohydrates were included in the uptake assays, stereospecific inhibition was exhibited, with greater competition by methyl-beta-glucoside than methyl-alpha-glucoside. Significant inhibition (>30%) was observed with phloretin, an inhibitor of facilitated diffusion of glucose, whereas there was no inhibition by sodium fluoride, iodoacetate, sodium arsenate, sodium azide, 2,4-dinitrophenol, monensin, or valinomycin, which typically reduce energy-driven transport. Based on kinetic analyses, the mean values for K(t) and V(max) were 14.8 +/- 3.4 mM D-glucose and 0.13 +/- 0.03 micromol glucose/min/mg cell protein, respectively. Glucose uptake by several glucose-utilizing commercial strains of B. animalis subsp. lactis was also inhibited by phloretin, indicating the presence of facilitated diffusion glucose transporters in those strains. Since DSMZ 10140 has been previously reported to lack a functional glucose phosphoenolpyruvate phosphotransferase system, the glucose transporter identified here is responsible for much of the organism's glucose uptake.

  1. SWEET17, a Facilitative Transporter, Mediates Fructose Transport across the Tonoplast of Arabidopsis Roots and Leaves1[W][OPEN

    PubMed Central

    Guo, Woei-Jiun; Nagy, Reka; Chen, Hsin-Yi; Pfrunder, Stefanie; Yu, Ya-Chi; Santelia, Diana; Frommer, Wolf B.; Martinoia, Enrico

    2014-01-01

    Fructose (Fru) is a major storage form of sugars found in vacuoles, yet the molecular regulation of vacuolar Fru transport is poorly studied. Although SWEET17 (for SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTERS17) has been characterized as a vacuolar Fru exporter in leaves, its expression in leaves is low. Here, RNA analysis and SWEET17-β-glucuronidase/-GREEN FLUORESCENT PROTEIN fusions expressed in Arabidopsis (Arabidopsis thaliana) reveal that SWEET17 is highly expressed in the cortex of roots and localizes to the tonoplast of root cells. Expression of SWEET17 in roots was inducible by Fru and darkness, treatments that activate accumulation and release of vacuolar Fru, respectively. Mutation and ectopic expression of SWEET17 led to increased and decreased root growth in the presence of Fru, respectively. Overexpression of SWEET17 specifically reduced the Fru content in leaves by 80% during cold stress. These results intimate that SWEET17 functions as a Fru-specific uniporter on the root tonoplast. Vacuoles overexpressing SWEET17 showed increased [14C]Fru uptake compared with the wild type. SWEET17-mediated Fru uptake was insensitive to ATP or treatment with NH4Cl or carbonyl cyanide m-chlorophenyl hydrazone, indicating that SWEET17 functions as an energy-independent facilitative carrier. The Arabidopsis genome contains a close paralog of SWEET17 in clade IV, SWEET16. The predominant expression of SWEET16 in root vacuoles and reduced root growth of mutants under Fru excess indicate that SWEET16 also functions as a vacuolar transporter in roots. We propose that in addition to a role in leaves, SWEET17 plays a key role in facilitating bidirectional Fru transport across the tonoplast of roots in response to metabolic demand to maintain cytosolic Fru homeostasis. PMID:24381066

  2. A facilitative urea transporter is localized in the renal collecting tubule of the dogfish Triakis scyllia.

    PubMed

    Hyodo, Susumu; Katoh, Fumi; Kaneko, Toyoji; Takei, Yoshio

    2004-01-01

    Reabsorption of filtered urea by the kidney tubule is essential for retaining high levels of urea in body fluids of marine elasmobranchs. To elucidate the mechanisms of urea reabsorption, we examined the distribution of a facilitative urea transporter (UT) in the kidney of the dogfish Triakis scyllia. We isolated a cDNA encoding a UT that is homologous to the facilitative UT cloned from another dogfish species, Squalus acanthias. The Triakis UT mRNA is abundantly expressed in the kidney, while low levels of expression were detected in the brain and liver. In the dogfish kidney, each nephron makes four turns and traverses repeatedly between bundle zone and sinus zone. In the bundle zone, the resulting five tubular segments are arranged in a countercurrent loop fashion. Immunohistochemistry using specific antibodies raised against the cloned UT revealed that, among the nephron segments, the UT is expressed exclusively in the final segment of the bundle zone, i.e. in the collecting tubule of the Triakis kidney. In contrast to the limited localization of UT, the transport enzyme Na+/K+-ATPase is distributed in the basolateral membrane of numerous tubular segments both in the sinus zone and the bundle zone. However, in the collecting tubule, Na+/K+-ATPase immunoreactivity was not detected. The present study suggests that the collecting tubule is responsible for the reabsorption of urea in the marine elasmobranch kidney. Other countercurrent segments may contribute to production of a driving force for facilitative diffusion of urea through the UT.

  3. Facilitated transport ceramic membranes for high-temperature gas cleanup. Final report, February 1990--April 1994

    SciTech Connect

    Quinn, R.; Minford, E.; Damle, A.S.; Gangwal, S.K.; Hart, B.A.

    1994-04-01

    The objective of this program was to demonstrate the feasibility of developing high temperature, high pressure, facilitated transport ceramic membranes to control gaseous contaminants in Integrated Gasification Combined Cycle (IGCC) power generation systems. Meeting this objective requires that the contaminant gas H{sub 2}S be removed from an IGCC gas mixture without a substantial loss of the other gaseous components, specifically H{sub 2} and CH{sub 4}. As described above this requires consideration of other, nonconventional types of membranes. The solution evaluated in this program involved the use of facilitated transport membranes consisting of molten mixtures of alkali and alkaline earth carbonate salts immobilized in a microporous ceramic support. To accomplish this objective, Air Products and Chemicals, Inc., Golden Technologies Company Inc., and Research Triangle Institute worked together to develop and test high temperature facilitated membranes for the removal of H{sub 2}S from IGCC gas mixtures. Three basic experimental activities were pursued: (1) evaluation of the H{sub 2}S chemistry of a variety of alkali and alkaline earth carbonate salt mixtures; (2) development of microporous ceramic materials which were chemically and physically compatible with molten carbonate salt mixtures under IGCC conditions and which could function as a host to support a molten carbonate mixture and; (3) fabrication of molten carbonate/ceramic immobilized liquid membranes and evaluation of these membranes under conditions approximating those found in the intended application. Results of these activities are presented.

  4. De Novo Pyrimidine Nucleotide Synthesis Mainly Occurs outside of Plastids, but a Previously Undiscovered Nucleobase Importer Provides Substrates for the Essential Salvage Pathway in Arabidopsis[W

    PubMed Central

    Witz, Sandra; Jung, Benjamin; Fürst, Sarah; Möhlmann, Torsten

    2012-01-01

    Nucleotide de novo synthesis is highly conserved among organisms and represents an essential biochemical pathway. In plants, the two initial enzymatic reactions of de novo pyrimidine synthesis occur in the plastids. By use of green fluorescent protein fusions, clear support is provided for a localization of the remaining reactions in the cytosol and mitochondria. This implies that carbamoyl aspartate, an intermediate of this pathway, must be exported and precursors of pyrimidine salvage (i.e., nucleobases or nucleosides) are imported into plastids. A corresponding uracil transport activity could be measured in intact plastids isolated from cauliflower (Brassica oleracea) buds. PLUTO (for plastidic nucleobase transporter) was identified as a member of the Nucleobase:Cation-Symporter1 protein family from Arabidopsis thaliana, capable of transporting purine and pyrimidine nucleobases. A PLUTO green fluorescent protein fusion was shown to reside in the plastid envelope after expression in Arabidopsis protoplasts. Heterologous expression of PLUTO in an Escherichia coli mutant lacking the bacterial uracil permease uraA allowed a detailed biochemical characterization. PLUTO transports uracil, adenine, and guanine with apparent affinities of 16.4, 0.4, and 6.3 μM, respectively. Transport was markedly inhibited by low concentrations of a proton uncoupler, indicating that PLUTO functions as a proton-substrate symporter. Thus, a protein for the absolutely required import of pyrimidine nucleobases into plastids was identified. PMID:22474184

  5. Protonation of Glu135 Facilitates the Outward-to-Inward Structural Transition of Fucose Transporter

    PubMed Central

    Liu, Yufeng; Ke, Meng; Gong, Haipeng

    2015-01-01

    Major facilitator superfamily (MFS) transporters typically need to alternatingly sample the outward-facing and inward-facing conformations, in order to transport the substrate across membrane. To understand the mechanism, in this work, we focused on one MFS member, the L-fucose/H+ symporter (FucP), whose crystal structure exhibits an outward-open conformation. Previous experiments imply several residues critical to the substrate/proton binding and structural transition of FucP, among which Glu135, located in the periplasm-accessible vestibule, is supposed as being involved in both proton translocation and conformational change of the protein. Here, the structural transition of FucP in presence of substrate was investigated using molecular-dynamics simulations. By combining the equilibrium and accelerated simulations as well as thermodynamic calculations, not only was the large-scale conformational change from the outward-facing to inward-facing state directly observed, but also the free energy change during the structural transition was calculated. The simulations confirm the critical role of Glu135, whose protonation facilitates the outward-to-inward structural transition both by energetically favoring the inward-facing conformation in thermodynamics and by reducing the free energy barrier along the reaction pathway in kinetics. Our results may help the mechanistic studies of both FucP and other MFS transporters. PMID:26244736

  6. Facilitation of axon regeneration by enhancing mitochondrial transport and rescuing energy deficits

    PubMed Central

    Yu, Panpan; Lin, Mei-Yao; Chen, Yanmin

    2016-01-01

    Although neuronal regeneration is a highly energy-demanding process, axonal mitochondrial transport progressively declines with maturation. Mature neurons typically fail to regenerate after injury, thus raising a fundamental question as to whether mitochondrial transport is necessary to meet enhanced metabolic requirements during regeneration. Here, we reveal that reduced mitochondrial motility and energy deficits in injured axons are intrinsic mechanisms controlling regrowth in mature neurons. Axotomy induces acute mitochondrial depolarization and ATP depletion in injured axons. Thus, mature neuron-associated increases in mitochondria-anchoring protein syntaphilin (SNPH) and decreases in mitochondrial transport cause local energy deficits. Strikingly, enhancing mitochondrial transport via genetic manipulation facilitates regenerative capacity by replenishing healthy mitochondria in injured axons, thereby rescuing energy deficits. An in vivo sciatic nerve crush study further shows that enhanced mitochondrial transport in snph knockout mice accelerates axon regeneration. Understanding deficits in mitochondrial trafficking and energy supply in injured axons of mature neurons benefits development of new strategies to stimulate axon regeneration. PMID:27268498

  7. Modeling particle-facilitated solute transport using the C-Ride module of HYDRUS

    NASA Astrophysics Data System (ADS)

    Simunek, Jiri; Bradford, Scott A.

    2017-04-01

    Strongly sorbing chemicals (e.g., heavy metals, radionuclides, pharmaceuticals, and/or explosives) in soils are associated predominantly with the solid phase, which is commonly assumed to be stationary. However, recent field- and laboratory-scale observations have shown that, in the presence of mobile colloidal particles (e.g., microbes, humic substances, clays and metal oxides), the colloids could act as pollutant carriers and thus provide a rapid transport pathway for strongly sorbing contaminants. Such transport can be further accelerated since these colloidal particles may travel through interconnected larger pores where the water velocity is relatively high. Additionally, colloidal particles have a considerable adsorption capacity for other species present in water because of their large specific surface areas and their high concentrations in soil-water and groundwater. As a result, the transport of contaminants can be significantly, sometimes dramatically, enhanced when they are adsorbed to mobile colloids. To address this problem, we have developed the C-Ride module for HYDRUS-1D. This one-dimensional numerical module is based on the HYDRUS-1D software package and incorporates mechanisms associated with colloid and colloid-facilitated solute transport in variably saturated porous media. This numerical model accounts for both colloid and solute movement due to convection, diffusion, and dispersion in variably-saturated soils, as well as for solute movement facilitated by colloid transport. The colloids transport module additionally considers processes of attachment/detachment to/from the solid phase, straining, and/or size exclusion. Various blocking and depth dependent functions can be used to modify the attachment and straining coefficients. The module additionally considers the effects of changes in the water content on colloid/bacteria transport and attachment/detachment to/from solid-water and air-water interfaces. For example, when the air

  8. Human transporters, PEPT1/2, facilitate melatonin transportation into mitochondria of cancer cells: an implication of the therapeutic potential.

    PubMed

    Huo, Xiaokui; Wang, Chao; Yu, Zhenlong; Peng, Yulin; Wang, Shumei; Feng, Shengnan; Zhang, Shouji; Tian, Xiangge; Sun, Chengpeng; Liu, Kexin; Deng, Sa; Ma, Xiaochi

    2017-01-18

    Melatonin is present in virtually all organisms from bacteria to mammals, and it exhibits a broad spectrum of biological functions, including synchronization of circadian rhythms and oncostatic activity. Several functions of melatonin are mediated by its membrane receptors but others are receptor-independent. For the latter, melatonin is required to penetrate membrane and enters intracellular compartments. However, the mechanism by which melatonin enters cells remains debatable. In the current study, it was identified that melatonin and its sulfation metabolites were the substrates of oligopeptide transporter (PEPT) 1/2 and organic anion transporter (OAT) 3, respectively. The docking analysis showed that the binding of melatonin to PEPT1/2 was attributed to their low binding energy and suitable binding conformation in which melatonin was embedded in the active site of PEPT1/2 and fitted well with the cavity in three-dimensional space. PEPT1/2 transporters play a pivotal role in melatonin uptake in cells. Melatonin's membrane transportation via PEPT1/2 renders its oncostatic effect in malignant cells. For the first time, PEPT1/2 were identified to localize in the mitochondrial membrane of human cancer cell lines of PC3 and U118. PEPT1/2 facilitated the transportation of melatonin into mitochondria. Melatonin accumulation in mitochondria induced apoptosis of PC3 and U118 cells. Thus, PEPT1/2 can potentially be used as a cancer cell-targeted melatonin delivery system to improve the therapeutic effects of melatonin in cancer treatment. This article is protected by copyright. All rights reserved.

  9. Nucleobase modification by an RNA enzyme.

    PubMed

    Poudyal, Raghav R; Nguyen, Phuong D M; Lokugamage, Melissa P; Callaway, Mackenzie K; Gavette, Jesse V; Krishnamurthy, Ramanarayanan; Burke, Donald H

    2017-02-17

    Ribozymes can catalyze phosphoryl or nucleotidyl transfer onto ribose hydroxyls of RNA chains. We report a single ribozyme that performs both reactions, with a nucleobase serving as initial acceptor moiety. This unprecedented combined reaction was revealed while investigating potential contributions of ribose hydroxyls to catalysis by kinase ribozyme K28. For a 58nt, cis-acting form of K28, each nucleotide could be replaced with the corresponding 2΄F analog without loss of activity, indicating that no particular 2΄OH is specifically required. Reactivities of two-stranded K28 variants with oligodeoxynucleotide acceptor strands devoid of any 2΄OH moieties implicate modification on an internal guanosine N-2, rather than a ribose hydroxyl. Product mass suggests formation of a GDP(S) adduct along with a second thiophosphorylation, implying that the ribozyme catalyzes both phosphoryl and nucleotidyl transfers. This is further supported by transfer of radiolabels into product from both α and γ phosphates of donor molecules. Furthermore, periodate reactivity of the final product signifies acquisition of a ribose sugar with an intact 2΄-3΄ vicinal diol. Neither nucleobase modification nor nucleotidyl transfer has previously been reported for a kinase ribozyme, making this a first-in-class ribozyme. Base-modifying ribozymes may have played important roles in early RNA world evolution by enhancing nucleic acid functions.

  10. Matrix Diffusion and Colloid-Facilitated Transport in Fractured Rocks: Model and Parameter Validation

    SciTech Connect

    Zavarin, M

    2002-08-02

    In this report, we review the results of Reimus et al. (2000a; 2000b) regarding matrix diffusion and colloid-facilitated transport in fractured rock and evaluate the implications of these results on modeling fracture flow at the Nevada Test Site (NTS). In particular, we examine these data in the context of the recent Cheshire hydrologic source term (HST) model results (Pawloski et al., 2001). This report is divided into several sections. In the first, we evaluate the effective diffusion coefficient (D{sub e}) data reported in Reimus et al. (2000a) for conservative tracer species ({sup 3}H, {sup 14}C, and {sup 99}Tc) and fit a simple effective diffusion model to these data. In the second, we use the fitted effective diffusion model, in conjunction with a surface complexation model, to simulate plutonium-colloid transport and compare model results to data reported in Reimus et al. (2000b). In the third, we evaluate the implications of these data with regards to radionuclide transport through fractures at the field scale and, in particular, with regards to the Cheshire HST model (Pawloski et al., 2001). Finally, we make recommendations regarding future radionuclide transport modeling efforts at the NTS.

  11. Channel-facilitated molecular transport: The role of strength and spatial distribution of interactions

    NASA Astrophysics Data System (ADS)

    Uppulury, Karthik; Kolomeisky, Anatoly B.

    2016-12-01

    Molecular transport across channels and pores is critically important for multiple natural and industrial processes. Recent advances in single-molecule techniques have allowed researchers to probe translocation through nanopores with unprecedented spatial and temporal resolution. However, our understanding of the mechanisms of channel-facilitated molecular transport is still not complete. We present a theoretical approach that investigates the role of molecular interactions in the transport through channels. It is based on the discrete-state stochastic analysis that provides a fully analytical description of this complex process. It is found that a spatial distribution of the interactions strongly influences the translocation dynamics. We predict that there is the optimal distribution that leads to the maximal flux through the channel. It is also argued that the channel transport depends on the strength of the molecule-pore interactions, on the shape of interaction potentials and on the relative contributions of entrance and diffusion processes in the system. These observations are discussed using simple physical-chemical arguments.

  12. Cytoplasmic transport of ribosomal subunits microinjected into the Xenopus laevis oocyte nucleus: a generalized, facilitated process

    PubMed Central

    1990-01-01

    To study the biochemistry of ribonucleoprotein export from the nucleus, we characterized an in vivo assay in which the cytoplasmic appearance of radiolabeled ribosomal subunits was monitored after their microinjection into Xenopus oocyte nuclei. Denaturing gel electrophoresis and sucrose density gradient sedimentation demonstrated that injected subunits were transported intact. Consistent with the usual subcellular distribution of ribosomes, transport was unidirectional, as subunits injected into the cytoplasm did not enter the nucleus. Transport displayed properties characteristic of a facilitated, energy-dependent process; the rate of export was saturable and transport was completely inhibited either by lowering the temperature or by depleting nuclei of ATP; the effect of lowered temperature was completely reversible. Transport of injected subunits was likely a process associated with the nuclear pore complex, since export was also inhibited by prior or simultaneous injection of wheat germ agglutinin, a lectin known to inhibit active nuclear transport by binding to N-acetyl glucosamine-containing glycoproteins present in the NPC (Hart, G. W., R. S. Haltiwanger, G. D. Holt, and W. G. Kelly. 1989. Annu. Rev. Biochem. 58:841-874). Although GlcNAc modified proteins exist on both the nuclear and cytoplasmic sides of the nuclear pore complex, ribosomal subunit export was inhibited only when wheat germ agglutinin was injected into the nucleus. Finally, we found that ribosomal subunits from yeast and Escherichia coli were efficiently exported from Xenopus oocyte nuclei, suggesting that export of some RNP complexes may be directed by a collective biochemical property rather than by specific macromolecular primary sequences or structures. PMID:2211825

  13. The Sodium Glucose Cotransporter SGLT1 Is an Extremely Efficient Facilitator of Passive Water Transport.

    PubMed

    Erokhova, Liudmila; Horner, Andreas; Ollinger, Nicole; Siligan, Christine; Pohl, Peter

    2016-04-29

    The small intestine is void of aquaporins adept at facilitating vectorial water transport, and yet it reabsorbs ∼8 liters of fluid daily. Implications of the sodium glucose cotransporter SGLT1 in either pumping water or passively channeling water contrast with its reported water transporting capacity, which lags behind that of aquaporin-1 by 3 orders of magnitude. Here we overexpressed SGLT1 in MDCK cell monolayers and reconstituted the purified transporter into proteoliposomes. We observed the rate of osmotic proteoliposome deflation by light scattering. Fluorescence correlation spectroscopy served to assess (i) SGLT1 abundance in both vesicles and plasma membranes and (ii) flow-mediated dilution of an aqueous dye adjacent to the cell monolayer. Calculation of the unitary water channel permeability, pf, yielded similar values for cell and proteoliposome experiments. Neither the absence of glucose or Na(+), nor the lack of membrane voltage in vesicles, nor the directionality of water flow grossly altered pf Such weak dependence on protein conformation indicates that a water-impermeable occluded state (glucose and Na(+) in their binding pockets) lasts for only a minor fraction of the transport cycle or, alternatively, that occlusion of the substrate does not render the transporter water-impermeable as was suggested by computational studies of the bacterial homologue vSGLT. Although the similarity between the pf values of SGLT1 and aquaporin-1 makes a transcellular pathway plausible, it renders water pumping physiologically negligible because the passive flux would be orders of magnitude larger. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Intrinsic and Carrier Colloid-facilitated transport of lanthanides through discrete fractures in chalk

    NASA Astrophysics Data System (ADS)

    Weisbrod, N.; Tran, E. L.; Klein-BenDavid, O.; Teutsch, N.

    2015-12-01

    Geological disposal of high-level radioactive waste is the long term solution for the disposal of long lived radionuclides and spent fuel. However, some radionuclides might be released from these repositories into the subsurface as a result of leakage, which ultimately make their way into groundwater. Engineered bentonite barriers around nuclear waste repositories are generally considered sufficient to impede the transport of radionuclides from their source to the groundwater. However, colloidal-sized mobile bentonite particles ("carrier" colloids) originating from these barriers have come under investigation as a potential transport vector for radionuclides sorbed to them. As lanthanides are generally accepted to have the same chemical behaviors as their more toxic actinide counterparts, lanthanides are considered an acceptable substitute for research on radionuclide transportation. This study aims to evaluate the transport behaviors of lanthanides in colloid-facilitated transport through a fractured chalk matrix and under geochemical conditions representative the Negev desert, Israel. The migration of Ce both with and without colloidal particles was explored and compared to the migration of a conservative tracer (bromide) using a flow system constructed around a naturally fractured chalk core. Results suggest that mobility of Ce as a solute is negligible. In experiments conducted without bentonite colloids, the 1% of the Ce that was recovered migrated as "intrinsic" colloids in the form of carbonate precipitates. However, the total recovery of the Ce increased to 9% when it was injected into the core in the presence of bentonite colloids and 13% when both bentonite and precipitate colloids were injected. This indicates that lanthanides are essentially immobile in chalk as a solute but may be mobile as carbonate precipitates. Bentonite colloids, however, markedly increase the mobility of lanthanides through fractured chalk matrices.

  15. Functional characterization of multidrug and toxin extrusion protein 1 as a facilitative transporter for fluoroquinolones.

    PubMed

    Ohta, Kin-ya; Imamura, Yuichiro; Okudaira, Noriko; Atsumi, Ryou; Inoue, Katsuhisa; Yuasa, Hiroaki

    2009-02-01

    Many fluoroquinolones are mainly eliminated by urinary excretion, in which tubular secretion by carrier-mediated transport systems has been suggested to be involved. In the present study, we examined the possibility that multidrug and toxin extrusion protein (MATE) 1, which is abundantly expressed in the kidney, might be involved in that, using rat MATE (rMATE) 1 expressed in MDCKII cells. It was found that rMATE1 can transport fluoroquinolones such as ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, norfloxacin (NFX), pazufloxacin, and tosufloxacin. Although rMATE1 has been known as an apical organic cation/H(+) antiporter, detailed investigation of rMATE1-mediated uptake of NFX has revealed that it is not sensitive to intracellular acidification by treatments using NH(4)Cl or nigericin, suggesting that the transmembrane proton gradient is not involved in its transport as a driving force. However, it was dependent on extracellular pH, being greatest at pH 7.0 and smaller at both acidic and basic pH in agreement with the profile of zwitterionization of NFX. The basal-to-apical transcellular transport of NFX in rMATE1-expressing MDCKII cells was greater than that in mock cells and insensitive to acidification of the apical medium, demonstrating proton gradient-independent functionality of rMATE1 in NFX efflux. Finally, rMATE1-mediated NFX uptake at pH 7.4 was saturable with the Michaelis constant of 55.3 microM and inhibited by cationic compounds, such as TEA and cimetidine. These results suggest that rMATE1 mediates the transport of NFX by a facilitative manner. MATE1 may play a key role in the renal tubular secretion of fluoroquinolones.

  16. Shear lines facilitate transport of reactive trace gases from East Asia into the deep tropics.

    NASA Astrophysics Data System (ADS)

    Donets, V.; Atlas, E. L.; Pan, L.; Schauffler, S.; Honomichl, S.; Hornbrook, R. S.; Apel, E. C.; Weinheimer, A. J.; Campos, T. L.; Hall, S. R.; Ullmann, K.; Navarro, M. A.

    2016-12-01

    Rapid industrialization and economic development in Eastern Asia (EA) has in recent decades led to increases in anthropogenic pollutants in the regional atmosphere. This pollutant burden is transported from local and regional sources into the global atmosphere and has been shown to be a significant source of intercontinental and cross-border anthropogenic pollution. This presentation focuses on one significant mechanism of such pollutant transport from EA into the deep tropics. Meteorology of the EA is dominated by the monsoonal low-level wind reversal, which in its winter phase facilitates episodic incursions of cold air masses from Siberia into the Philippine and South China Seas and to the Western Pacific (WP). In that phase cold surface temperatures give rise to stable low-level inversions, resulting in the capping of the boundary layer. In this flow regime wet deposition is almost completely absent, allowing regional pollution to build-up in the lower troposphere. As the cold air surges are carried eastward across EA and into the WP, sweeping off low-level pollution in the process, they also advance equatorward. As the cold air advances southward, heating from the ocean surface reduces the thermal gradient across the frontal boundary, and by the time that air reaches tropical latitudes all that remains of the front is a wind shift referred to as the shear line. North of the shear line, an air mass still contains the diluted pollutants picked up over the source regions in EA. Here we present measurements of chemically reactive trace gases collected during the CONTRAST mission, an airborne research study conducted during January-February, 2014, over the tropical WP Ocean. This presentation describes shear line facilitated transport of pollution into the tropics during boreal winter and its effects on chemical composition of the troposphere in the deep tropics. This is the first analysis that links the phenomenon of shear lines to pollution transport out of EA

  17. Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi.

    PubMed

    Shaheen, Aqsa; Ismat, Fouzia; Iqbal, Mazhar; Haque, Abdul; De Zorzi, Rita; Mirza, Osman; Walz, Thomas; Rahman, Moazur

    2015-05-01

    Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874 conferred resistance to at least ten of the tested antimicrobials: ciprofloxacin, norfloxacin, levofloxacin, kanamycin, streptomycin, gentamycin, nalidixic acid, chloramphenicol, ethidium bromide, and acriflavine, including fluoroquinolone antibiotics, which were drugs of choice to treat S. Typhi infections. Cell-based functional studies using ethidium bromide and acriflavine showed that STY4874 functions as a H(+)-dependent exporter. These results suggest that STY4874 may be an important drug target, which can now be tested by studying the susceptibility of a STY4874-deficient S. Typhi strain to antimicrobials. Copyright © 2015 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. All rights reserved.

  18. Colloid-facilitated transport of cesium in vadose-zone sediments: the importance of flow transients.

    PubMed

    Cheng, Tao; Saiers, James E

    2010-10-01

    Colloid-sized particles are commonly detected in vadose-zone pore waters and are capable of binding chemicals with sorptive affinities for geologic materials. Published research demonstrates that colloids are capable of facilitating the transport of sorptive contaminants under conditions of steady pore water flow, when volumetric moisture content and pore water velocity are constant. Less is known about the role of colloids in governing contaminant mobility under transient-flow conditions, which are characteristic of natural vadose-zone environments. The objective of this study is to elucidate the influences of flow transients on the mobilization and transport of in situ colloids and colloid-associated contaminants. We conducted column experiments in which the mobilization of in situ colloids and (137)Cs was induced by transients associated with the drainage and imbibition of (137)Cs contaminated-sediments. Our results demonstrate that substantial quantities of in situ colloids and colloid-associated (137)Cs are mobilized as volumetric moisture content declines during porous-medium drainage and as volumetric moisture content increases during porous-medium imbibition. We also find that the colloid-effect on (137)Cs transport is sensitive to changes in pore water ionic strength. That is, the quantities of colloids mobilized and the capacity of the these colloids to bind (137)Cs decrease with increasing ionic strength, leading to a decrease of the mass of (137)Cs eluted from the columns during porous-medium drainage and imbibition.

  19. Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates.

    PubMed

    Bienert, Gerd Patrick; Bienert, Manuela Désirée; Jahn, Thomas Paul; Boutry, Marc; Chaumont, François

    2011-04-01

    Major intrinsic proteins (MIPs) transport water and uncharged solutes across membranes in all kingdoms of life. Recently, an uncharacterized MIP subfamily was identified in the genomes of plants and fungi and named X Intrinsic Proteins (XIPs). Here, we describe the genetic features, localization, expression, and functions of a group of Solanaceae XIPs. XIP cDNA and gDNA were cloned from tobacco, potato, tomato, and morning glory. A conserved sequence motif in the first intron of Solanaceae XIPs initiates an RNA-processing mechanism that results in two splice variants (α and β). When transiently or stably expressed in tobacco plants, yellow fluorescent protein-tagged NtXIP1;1α and NtXIP1;1β were both localized in the plasma membrane. Transgenic tobacco lines expressing NtXIP1;1-promoter-GUS constructs and RT-PCR studies showed that NtXIP1;1 was expressed in all organs. The NtXIP1;1 promoter was mainly active in cell layers facing the environment in all above-ground tissues. Heterologous expression of Solanaceae XIPs in Xenopus laevis oocytes and various Saccharomyces cerevisiae mutants demonstrated that these isoforms facilitate the transport of bulky solutes, such as glycerol, urea, and boric acid. In contrast, permeability for water was undetectable. These data suggest that XIPs function in the transport of uncharged solutes across the cell plasma membrane in specific plant tissues, including at the interface between the environment and external cell layers.

  20. Colloid-facilitated transport of cesium in variably saturated Hanford sediments.

    PubMed

    Chen, Gang; Flury, Markus; Harsh, James B; Lichtner, Peter C

    2005-05-15

    Radioactive 137Cs has leaked from underground waste tanks into the vadose zone at the Hanford Reservation in south-central Washington State. There is concern that 137Cs, currently located in the vadose zone, can reach the groundwater. In this study, we investigated whether, and to what extent, colloidal particles can facilitate the transport of 137Cs at Hanford. We used colloidal materials isolated from Hanford sediments. Transport experiments were conducted under variably saturated, steady-state flow conditions in repacked, 20 cm long Hanford sediment columns, with effective water saturations ranging from 0.2 to 1.0. Cesium, pre-associated with colloids, was stripped off during transport through the sediments. The higher the flow rates, the less Cs was stripped off, indicating in part that Cs desorption from carrying colloids was a residence-time-dependent process. Depending on the flow rate, up to 70% of the initially sorbed Cs desorbed from colloidal carriers and was captured in the stationary sediments. Less Cs was stripped off colloids under unsaturated than under saturated flow conditions at similar flow rates. This phenomenon was likely due to the reduced availability of sorption sites for Cs on the sediments as the water content decreased and water flow was divided between mobile and immobile regions.

  1. Do anthropogenic transports facilitate stored-product pest moth dispersal? A molecular approach

    NASA Astrophysics Data System (ADS)

    Ryne, Camilla; Bensch, Staffan

    2008-02-01

    Stored-product moths cause large economic damage in food processing industries and storage facilities. Control of indoor pests is currently dealt with locally, and control strategies seldom include different mills or cooperative industries in joint efforts to reduce infestations. In colder climates where conditions hinder flight dispersal of stored-product moths, we hypothesize that human transport between mills will facilitate dispersal. Albeit considered intuitive, this hypothesis has so far never been tested. Male moths from three mills (populations) in southern Sweden and Denmark were collected and by using amplified fragment length polymorphism (AFLP) pair-wise F st values were calculated. Cluster (population) origins of the genotypes were computed by using a model-based method, structure. The results suggest that known transportation of flour between two mills generate genetically more similar populations of the economically important stored-product moth, Ephestia kuehniella (Zell.) (Lepidoptera; Pyralidae), compared to the third mill, with another distribution area, but situated geographically in between the other mills. The structure model placed the sampled genotypes to belong to either two or five original populations, with a higher probability of two original populations. The third mill was consistently different from the other two mills independent of the models’ calculated number of populations. Although the study was restricted to three mills and one transportation route, it highlights the possibility that transportation of food products promotes genetic mixing (i.e. dispersal) of insect pest populations. Including cooperating mills in control (or monitor) strategy schemes against stored-product pest insects would therefore be a more effective action, rather than to treat each mill separately.

  2. Functional analysis of an Arabidopsis thaliana abiotic stress-inducible facilitated diffusion transporter for monosaccharides.

    PubMed

    Yamada, Kohji; Osakabe, Yuriko; Mizoi, Junya; Nakashima, Kazuo; Fujita, Yasunari; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2010-01-08

    Sugars play indispensable roles in biological reactions and are distributed into various tissues or organelles via transporters in plants. Under abiotic stress conditions, plants accumulate sugars as a means to increase stress tolerance. Here, we report an abiotic stress-inducible transporter for monosaccharides from Arabidopsis thaliana that is termed ESL1 (ERD six-like 1). Expression of ESL1 was induced under drought and high salinity conditions and with exogenous application of abscisic acid. Promoter analyses using beta-glucuronidase and green fluorescent protein reporters revealed that ESL1 is mainly expressed in pericycle and xylem parenchyma cells. The fluorescence of ESL1-green fluorescent protein-fused protein was detected at tonoplast in transgenic Arabidopsis plants and tobacco BY-2 cells. Furthermore, alanine-scanning mutagenesis revealed that an N-terminal LXXXLL motif in ESL1 was essential for its localization at the tonoplast. Transgenic BY-2 cells expressing mutated ESL1, which was localized at the plasma membrane, showed an uptake ability for monosaccharides. Moreover, the value of K(m) for glucose uptake activity of mutated ESL1 in the transgenic BY-2 cells was extraordinarily high, and the transport activity was independent from a proton gradient. These results indicate that ESL1 is a low affinity facilitated diffusion transporter. Finally, we detected that vacuolar invertase activity was increased under abiotic stress conditions, and the expression patterns of vacuolar invertase genes were similar to that of ESL1. Under abiotic stress conditions, ESL1 might function coordinately with the vacuolar invertase to regulate osmotic pressure by affecting the accumulation of sugar in plant cells.

  3. Do anthropogenic transports facilitate stored-product pest moth dispersal? A molecular approach.

    PubMed

    Ryne, Camilla; Bensch, Staffan

    2008-02-01

    Stored-product moths cause large economic damage in food processing industries and storage facilities. Control of indoor pests is currently dealt with locally, and control strategies seldom include different mills or cooperative industries in joint efforts to reduce infestations. In colder climates where conditions hinder flight dispersal of stored-product moths, we hypothesize that human transport between mills will facilitate dispersal. Albeit considered intuitive, this hypothesis has so far never been tested. Male moths from three mills (populations) in southern Sweden and Denmark were collected and by using amplified fragment length polymorphism (AFLP) pair-wise F(st) values were calculated. Cluster (population) origins of the genotypes were computed by using a model-based method, structure. The results suggest that known transportation of flour between two mills generate genetically more similar populations of the economically important stored-product moth, Ephestia kuehniella (Zell.) (Lepidoptera; Pyralidae), compared to the third mill, with another distribution area, but situated geographically in between the other mills. The structure model placed the sampled genotypes to belong to either two or five original populations, with a higher probability of two original populations. The third mill was consistently different from the other two mills independent of the models' calculated number of populations. Although the study was restricted to three mills and one transportation route, it highlights the possibility that transportation of food products promotes genetic mixing (i.e. dispersal) of insect pest populations. Including cooperating mills in control (or monitor) strategy schemes against stored-product pest insects would therefore be a more effective action, rather than to treat each mill separately.

  4. Silver- and gold-mediated nucleobase bonding.

    PubMed

    Acioli, Paulo H; Srinivas, Sudha

    2014-08-01

    We report the results of a density functional theory investigation of the bonding of nucleobases mediated by silver and gold atoms in the gas phase. Our calculations use the Becke exchange and Perdew-Wang correlation functional (BPW91) combined with the Stuttgart effective core potentials to represent the valence electrons of gold, silver, and platinum, and the all-electron DGTZVP basis set for C, H, N, and O. This combination was chosen based on tests on the metal atoms and tautomers of adenine, cytosine, and guanine. To establish a benchmark to understand the metal-mediated bonding, we calculated the binding energy of each of the base pairs in their canonical forms. Our calculations show rather strong bonds between the Watson-Crick base pairs when compared with typical values for N-H-N and N-H-O hydrogen bonds. The neutral metal atoms tend to bond near the nitrogen atoms. The effect of the metal atoms on the bonding of nucleobases differs depending on whether or not the metal atoms bond to one of the hydrogen-bonding sites. When the silver or gold atoms bond to a non-hydrogen-bonding site, the effect is a slight enhancement of the cytosine-guanine bonding, but there is almost no effect on the adenine-thymine pairing. The metal atoms can block one of the hydrogen-bonding sites, thus preventing the normal cytosine-guanine and adenine-thymine pairings. We also find that both silver and gold can bond to consecutive guanines in a similar fashion to platinum, albeit with a significantly lower binding energy.

  5. Colloid facilitated transport in fractured rock : parameter estimation and comparison with experimental data

    SciTech Connect

    Viswanathan, H. S.; Wolfsberg, A. V.

    2002-01-01

    Many contaminants in groundwater strongly interact with the immobile porous matrix, which retards their movement relative to groundwater flow. Colloidal particles, which are often present in groundwater, have a relatively small size and large specific surface area which makes it possible for them to also adsorb pollutants. The sorption of tracers to colloids may enhance their mobility in groundwater, relative to the case where colloids are not present. A class of pollutants for which colloid-facilitated transport may be of particular significance are radioactive isotopes. A major reason for why geologic repositories are considered suitable for the disposal of spent nuclear fuel is the strong affinity of many radionuclides to adsorb onto the porous matrix. Therefore, radionuclides accidentally released, would be contained in the geological media by adsorption or filtration until sufficient decay takes place. However, the presence of colloids may enhance radionuclide mobility in the groundwater, and reduce the efficiency of geologic media to act as a natural barrier.

  6. Facilitated transport of carbon dioxide through supported liquid membranes of aqueous amine solutions

    SciTech Connect

    Teramoto, Masaaki; Nakai, Katsuya; Ohnishi, Nobuaki; Huang, Q.; Watari, Takashi; Matsuyama, Hideto

    1996-02-01

    A series of experiments on the facilitated transport of CO{sub 2} through supported liquid membranes containing monoethanolamine (MEA) and diethanolamine (DEA) was performed. The feed gas was a mixture of CO{sub 2} and CH{sub 4}, and the CO{sub 2} partial pressure p{sub CO{sub 2},F} was in the range from 0.05 to 0.97 atm. Compared to the MEA membranes, the DEA membranes showed a little higher permeation rate of CO{sub 2} since the equilibrium constant of the reaction between CO{sub 2} and MEA is too large for CO{sub 2} to be released to the receiving phase rapidly. When p{sub CO{sub 2},F} and the MEA concentration were 0.05 atm and 4 mol/dm{sup 3}, respectively, the separation factor {alpha}(CO{sub 2}/CH{sub 4}) was about 2,000. It was found that if the membrane thickness multiplied by the square root of the tortuosity factor of the microporous support membrane is used as the effective pore length, the experimentally observed permeation rates of CO{sub 2} can be satisfactorily simulated by the theory of facilitated transport of CO{sub 2} through aqueous amine membranes. A method for estimating the solubilities of CO{sub 2} in the membrane solutions from the permeation rates of CH{sub 4} was also proposed. It was also found that permeation rates of CO{sub 2} through aqueous DEA membranes reported by Guha et al. were quantitatively explained by the proposed theory.

  7. Colloid-Facilitated Transport of Cations in an Unsaturated Fractured Soil Under Transient Conditions

    SciTech Connect

    Ryan, Joseph

    2015-01-31

    Rainfall experiments were conducted using intact soil cores and an instrumented soil pedon to examine the effect of physical heterogeneity and rainfall characteristics on the mobilization of colloids, organic matter, cesium, and strontium in a fractured soil. To measure the spatial variability of infiltration of colloids and contaminants, samples were collected through a 19-port grid placed below the soil core in laboratory study and in 27 samplers at multiple depths in the soil pedon in the field study. Cesium and strontium were applied to the soil cores and the soil pedon prior to mobilization experiments. Rainwater solutions of multiple ionic strengths and organic matter concentrations were applied to the soil cores and soil pedon to mobilize in situ colloids, cesium, and strontium. The mobilization of colloids and metal cations occurred through preferential flow paths in the soil cores. Compared to steady rainfall, greater amounts of colloids were mobilized during rainfall interrupted by pauses, which indicates that the supply of colloids to be mobilized was replenished during the pauses. A maximum in the amount of mobilized colloids were mobilized during a rainfall following a pause of 2.5 d. Pauses of shorter or longer duration resulted in less colloid mobilization. Freeze-thaw cycles, a transient condition in winter, enhanced colloid mobilization and colloid-facilitated transport of cesium and strontium in the soil cores. The exchange of solutes between the soil matrix and macropores caused a hysteretic mobilization of colloids, cesium, and strontium during changes in ionic strength. Colloid-facilitated mobilization of cesium and strontium was important at low ionic strength in fractures where slow flow allowed greater exchange of flow between the fractures and the surrounding matrix. The release of cesium and strontium by cation exchange occurred at high ionic strength in fractures where there is a little exchange of pore water with the surrounding matrix

  8. Facilitated transport and diffusion take distinct spatial routes through the nuclear pore complex

    PubMed Central

    Fiserova, Jindriska; Richards, Shane A.; Wente, Susan R.; Goldberg, Martin W.

    2010-01-01

    Transport across the nuclear envelope is regulated by nuclear pore complexes (NPCs). Much is understood about the factors that shuttle and control the movement of cargos through the NPC, but less has been resolved about the translocation process itself. Various models predict how cargos move through the channel; however, direct observation of the process is missing. Therefore, we have developed methods to accurately determine cargo positions within the NPC. Cargos were instantly trapped in transit by high-pressure freezing, optimally preserved by low-temperature fixation and then localized by immunoelectron microscopy. A statistical modelling approach was used to identify cargo distribution. We found import cargos localized surprisingly close to the edge of the channel, whereas mRNA export factors were at the very centre of the NPC. On the other hand, diffusion of GFP was randomly distributed. Thus, we suggest that spatially distinguished pathways exist within the NPC. Deletion of specific FG domains of particular NPC proteins resulted in collapse of the peripheral localization and transport defects specific to a certain karyopherin pathway. This further confirms that constraints on the route of travel are biochemical rather than structural and that the peripheral route of travel is essential for facilitated import. PMID:20647373

  9. Facilitated transport and diffusion take distinct spatial routes through the nuclear pore complex.

    PubMed

    Fiserova, Jindriska; Richards, Shane A; Wente, Susan R; Goldberg, Martin W

    2010-08-15

    Transport across the nuclear envelope is regulated by nuclear pore complexes (NPCs). Much is understood about the factors that shuttle and control the movement of cargos through the NPC, but less has been resolved about the translocation process itself. Various models predict how cargos move through the channel; however, direct observation of the process is missing. Therefore, we have developed methods to accurately determine cargo positions within the NPC. Cargos were instantly trapped in transit by high-pressure freezing, optimally preserved by low-temperature fixation and then localized by immunoelectron microscopy. A statistical modelling approach was used to identify cargo distribution. We found import cargos localized surprisingly close to the edge of the channel, whereas mRNA export factors were at the very centre of the NPC. On the other hand, diffusion of GFP was randomly distributed. Thus, we suggest that spatially distinguished pathways exist within the NPC. Deletion of specific FG domains of particular NPC proteins resulted in collapse of the peripheral localization and transport defects specific to a certain karyopherin pathway. This further confirms that constraints on the route of travel are biochemical rather than structural and that the peripheral route of travel is essential for facilitated import.

  10. Self-organized transient facilitated atomic transport in Pt/Al(111).

    PubMed

    Süle, P

    2008-04-07

    During the course of atomic transport in a host material, impurity atoms need to surmount an energy barrier driven by thermodynamic bias or at ultralow temperatures by quantum tunneling. In the present article, we demonstrate using atomistic simulations that at ultralow temperature, transient interlayer atomic transport is also possible without tunneling when the Pt/Al(111) impurity/host system self-organizes itself spontaneously into an intermixed configuration. No such extremely fast athermal concerted process has been reported before at ultralow temperatures. The outlined novel transient atomic exchange mechanism could be of general validity. We find that the source of ultralow temperature heavy particle barrier crossing is intrinsic and no external bias is necessary for atomic intermixing and surface alloying in Pt/Al, although the dynamic barrier height is a few eV. The mechanism is driven by the local thermalization of the Al(111) surface in a self-organized manner arranged spontaneously by the system without any external stimulus. The core of the short lived thermalized region reaches the local temperature of approximately 1000 K (including a few tens of Al atoms), while the average temperature of the simulation cell is approximately 3 K. The transient facilitated intermixing process also takes place with repulsive impurity-host interaction potential leading to negative atomic mobility; hence, the atomic injection is largely independent of the strength of the impurity-surface interaction. We predict that similar exotic behavior is possible in other materials as well.

  11. Proposed structure of putative glucose channel in GLUT1 facilitative glucose transporter.

    PubMed Central

    Zeng, H; Parthasarathy, R; Rampal, A L; Jung, C Y

    1996-01-01

    A family of structurally related intrinsic membrane proteins (facilitative glucose transporters) catalyzes the movement of glucose across the plasma membrane of animal cells. Evidence indicates that these proteins show a common structural motif where approximately 50% of the mass is embedded in lipid bilayer (transmembrane domain) in 12 alpha-helices (transmembrane helices; TMHs) and accommodates a water-filled channel for substrate passage (glucose channel) whose tertiary structure is currently unknown. Using recent advances in protein structure prediction algorithms we proposed here two three-dimensional structural models for the transmembrane glucose channel of GLUT1 glucose transporter. Our models emphasize the physical dimension and water accessibility of the channel, loop lengths between TMHs, the macrodipole orientation in four-helix bundle motif, and helix packing energy. Our models predict that five TMHs, either TMHs 3, 4, 7, 8, 11 (Model 1) or TMHs 2, 5, 11, 8, 7 (Model 2), line the channel, and the remaining TMHs surround these channel-lining TMHs. We discuss how our models are compatible with the experimental data obtained with this protein, and how they can be used in designing new biochemical and molecular biological experiments in elucidation of the structural basis of this important protein function. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 PMID:8770183

  12. Atrazine adsorption and colloid-facilitated transport through the unsaturated zone

    USGS Publications Warehouse

    Sprague, L.A.; Herman, J.S.; Hornberger, G.M.; Mills, A.L.

    2000-01-01

    One explanation for unexpectedly widespread ground water contamination from atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) may be the occurence of colloid-facilitated transport, whereby the dissolved herbicide becomes adsorbed to mobile colloids that migrate through preferential flow-paths in the soil zone and into the ground water. The objectives of this study were to determine the extent of adsorpton of atrazine to bulk soil and to soil colloids and to determine the extent of colloid-facilitated transport of atrazine at a field site in Virginia during simulated rainfall events. Equilibrium batch adsorption experiments were performed over a concentration range of 0.05 to 10.0 mg atrazine L-1 on bulk soil samples and on colloidal suspensions of 75 mg L-1, a concentration comparable with those observed at the field site. Linear partition coefficients ranged from 0.496 to 2.48 L kg-1 for the bulk soil and from 70.8 to 832 L kg-1 for the soil colloids. In the field, gravity lysimeters were insured at a depth of 25 cm below the surface of six 0.25-m2 undisturbed plots. Mass recovery of surface-applied atrazine in the lysimeters was not significantly affected by rainfall rate and was, on average, 2.7% for plots receiving 25 mm h-1 simulated rainfall and 3.6% for plots receiving 50 mm h-1 simulated rainfall. Of the total atrazine collected in the lysimeters, the fraction that was colloid-associated ranged from 4.9 to 30% (mean of 15%), indicating that a measurable portion of mobile atrazine is transported via association with colloids.One explanation for unexpectedly widespread ground water contamination from atrazine (2-chloro-4-ethylamino-6-isopropylamino-s-triazine) may be the occurrence of colloid-facilitated transport, whereby the dissolved herbicide becomes adsorbed to mobile colloids that migrate through preferential flow-paths in the soil zone and into the ground water. The objectives of this study were to determine the extent of adsorption of

  13. Facilitated transport of titanium dioxide nanoparticles by humic substances in saturated porous media under acidic conditions

    NASA Astrophysics Data System (ADS)

    Zhang, Ruichang; Zhang, Haibo; Tu, Chen; Hu, Xuefeng; Li, Lianzhen; Luo, Yongming; Christie, Peter

    2015-04-01

    The transport behavior of titanium dioxide nanoparticles (TiO2 NPs, 30 nm in diameter) was studied in well-defined porous media composed of clean quartz sand over a range of solution chemistry under acidic conditions. Transport of TiO2 NPs was dramatically enhanced by humic substances (HS) at acidic pH (4.0, 5.0 and 6.0), even at a low HS concentration of 0.5 mg L-1. Facilitated transport of TiO2 NPs was likely attributable to the increased stability of TiO2 NPs and repulsive interaction between TiO2 NPs and quartz sands due to the adsorbed HS. The mobility of TiO2 NPs was also increased with increasing pH from 4.0 to 6.0. Although transport of TiO2 NPs was insensitive to low ionic strength, it was significantly inhibited by high concentrations of NaCl and CaCl2. In addition, calculated Derjaguin-Landau-Verwey-Overbeek (DLVO) interaction energy indicated that high energy barriers were responsible for the high mobility of TiO2 NPs, while the secondary energy minimum could play an important role in the retention of TiO2 NPs at 100 mmol L-1 NaCl. Straining and gravitational settlement of larger TiO2 NPs aggregates at 1 mg L-1 HS, pH 5.0, and 2 mmol L-1 CaCl2 could be responsible for the significant retention even in the presence of high energy barriers. Moreover, more favorable interaction between approaching TiO2 NPs and TiO2 NPs that had been already deposited on the collector resulted in a ripening-shape breakthrough curve at 2 mmol L-1 CaCl2. Overall, a combination of mechanisms including DLVO-type force, straining, and physical filtration was involved in the retention of TiO2 NPs over the range of solution chemistry examined in this study.

  14. Modeling of facilitated transport of phenylalanine by emulsion liquid membranes with di(2-ethylhexyl)phosphoric acid as a carrier

    SciTech Connect

    Liu, X.; Liu, D.

    1998-12-01

    A mathematical model is developed in this paper to simulate the facilitated transport of phenylalanine (Phe) in emulsion liquid membrane (ELM) systems with di(2-ethylhexyl)phosphoric acid as a carrier. The model takes into account the mass transfer in both the external aqueous phase and the organic membrane phase interfacial reaction as well as membrane breakage during agitation. The model is tested by comparing theoretical predications with experimental results using Phe extraction by ELM processes. It is found that the model is valid for simulating the facilitated transport of Phe with ELM under various experimental conditions.

  15. Mass transfer model of nanoparticle-facilitated contaminant transport in saturated porous media.

    PubMed

    Johari, Wan Lutfi Wan; Diamessis, Peter J; Lion, Leonard W

    2010-02-01

    A one-dimensional model has been evaluated for transport of hydrophobic contaminants, such as polycyclic aromatic hydrocarbon (PAH) compounds, facilitated by synthetic amphiphilic polyurethane (APU) nanoparticles in porous media. APU particles synthesized from poly(ethylene glycol)-modified urethane acrylate (PMUA) precursor chains have been shown to enhance the desorption rate and mobility of phenanthrene (PHEN) in soil. A reversible process governed by attachment and detachment rates was considered to describe the PMUA binding in soil in addition to PMUA transport through advection and dispersion. Ultimately, an irreversible second-order PMUA attachment rate in which the fractional soil saturation capacity with PMUA was a rate control was found to be adequate to describe the retention of PMUA particles. A gamma-distributed site model (GS) was used to describe the spectrum of physical/chemical constraints for PHEN transfer from solid to aqueous phases. Instantaneous equilibrium was assumed for PMUA-PHEN interactions. The coupled model for PMUA and PHEN behavior successfully described the enhanced elution profile of PHEN by PMUA. Sensitivity analysis was performed to analyze the significance of model parameters on model predictions. The adjustable parameter alpha in the gamma-distribution shapes the contaminant desorption distribution profile as well as elution and breakthrough curves. Model simulations show the use of PMUA can be also expected to improve the release rate of PHEN in soils with higher organic carbon content. The percentage removal of PHEN mass over time is shown to be influenced by the concentration of PMUA added and this information can be used to optimize cost and time require to accomplish a desired remediation goal. Copyright 2009 Elsevier Ltd. All rights reserved.

  16. Expression, purification, and functional characterization of the insulin-responsive facilitative glucose transporter GLUT4.

    PubMed

    Kraft, Thomas E; Hresko, Richard C; Hruz, Paul W

    2015-12-01

    The insulin-responsive facilitative glucose transporter GLUT4 is of fundamental importance for maintenance of glucose homeostasis. Despite intensive effort, the ability to express and purify sufficient quantities of structurally and functionally intact protein for biophysical analysis has previously been exceedingly difficult. We report here the development of novel methods to express, purify, and functionally reconstitute GLUT4 into detergent micelles and proteoliposomes. Rat GLUT4 containing FLAG and His tags at the amino and carboxy termini, respectively, was engineered and stably transfected into HEK-293 cells. Overexpression in suspension culture yielded over 1.5 mg of protein per liter of culture. Systematic screening of detergent solubilized GLUT4-GFP fusion protein via fluorescent-detection size exclusion chromatography identified lauryl maltose neopentyl glycol (LMNG) as highly effective for isolating monomeric GLUT4 micelles. Preservation of structural integrity and ligand binding was demonstrated via quenching of tryptophan fluorescence and competition of ATB-BMPA photolabeling by cytochalasin B. GLUT4 was reconstituted into lipid nanodiscs and proper folding was confirmed. Reconstitution of purified GLUT4 with amphipol A8-35 stabilized the transporter at elevated temperatures for extended periods of time. Functional activity of purified GLUT4 was confirmed by reconstitution of LMNG-purified GLUT4 into proteoliposomes and measurement of saturable uptake of D-glucose over L-glucose. Taken together, these data validate the development of an efficient means to generate milligram quantities of stable and functionally intact GLUT4 that is suitable for a wide array of biochemical and biophysical analyses.

  17. The Hsp90 machinery facilitates the transport of diphtheria toxin into human cells.

    PubMed

    Schuster, Manuel; Schnell, Leonie; Feigl, Peter; Birkhofer, Carina; Mohr, Katharina; Roeder, Maurice; Carle, Stefan; Langer, Simon; Tippel, Franziska; Buchner, Johannes; Fischer, Gunter; Hausch, Felix; Frick, Manfred; Schwan, Carsten; Aktories, Klaus; Schiene-Fischer, Cordelia; Barth, Holger

    2017-04-04

    Diphtheria toxin kills human cells because it delivers its enzyme domain DTA into their cytosol where it inhibits protein synthesis. After receptor-mediated uptake of the toxin, DTA translocates from acidic endosomes into the cytosol, which might be assisted by host cell factors. Here we investigated the role of Hsp90 and its co-chaperones during the uptake of native diphtheria toxin into human cells and identified the components of the Hsp90 machinery including Hsp90, Hsp70, Cyp40 and the FK506 binding proteins FKBP51 and FKBP52 as DTA binding partners. Moreover, pharmacological inhibition of the chaperone activity of Hsp90 and Hsp70 and of the peptidyl-prolyl cis/trans isomerase (PPIase) activity of Cyps and FKBPs protected cells from intoxication with diphtheria toxin and inhibited the pH-dependent trans-membrane transport of DTA into the cytosol. In conclusion, these host cell factors facilitate toxin uptake into human cells, which might lead to development of novel therapeutic strategies against diphtheria.

  18. An inducible ER–Golgi tether facilitates ceramide transport to alleviate lipotoxicity

    PubMed Central

    Choudhary, Vineet

    2017-01-01

    Ceramides are key intermediates in sphingolipid biosynthesis and potent signaling molecules. However, excess ceramide is toxic, causing growth arrest and apoptosis. In this study, we identify a novel mechanism by which cells prevent the toxic accumulation of ceramides; they facilitate nonvesicular ceramide transfer from the endoplasmic reticulum (ER) to the Golgi complex, where ceramides are converted to complex sphingolipids. We find that the yeast protein Nvj2p promotes the nonvesicular transfer of ceramides from the ER to the Golgi complex. The protein is a tether that generates close contacts between these compartments and may directly transport ceramide. Nvj2p normally resides at contacts between the ER and other organelles, but during ER stress, it relocalizes to and increases ER–Golgi contacts. ER–Golgi contacts fail to form during ER stress in cells lacking Nvj2p. Our findings demonstrate that cells regulate ER–Golgi contacts in response to stress and reveal that nonvesicular ceramide transfer out of the ER prevents the buildup of toxic amounts of ceramides. PMID:28011845

  19. Role of LiBF4 in Ionic Liquid Membranes for Facilitated CO2 Transport.

    PubMed

    Choi, Yeji; Hong, Gil Hwan; Kang, Sang Wook

    2016-03-01

    The ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM BF4)/LiBF4 electrolyte was prepared for highly selective facilitated CO2 transport membranes. When LiBF4 was incorporated into BMIM BF4, synergy effects by free Li+ ion and imidazolium cations is expected to enhance the separation performance for CO2/N2 and CO2/CH4. The free state of BF4- ions in BMIM BF4/LiBF4 solutions was investigated by FT-Raman spectroscopy. For the coordination of LiBF4 with BMIMBF4, thermal gravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS) was utilized. Electrolyte membranes consisting of BMIM BF4 and LiBF4 showed selectivities of 8.40 and 8.25 for CO2/N2 and CO2/CH4, respectively. Neat BMIM BF4 membrane showed selectivities of 5.0 and 4.8, respectively. Enhanced separation performance was attributed to increased free Li+ and abundant free imidazolium cations.

  20. Protist-facilitated transport of soil bacteria in an artificial soil micromodel

    NASA Astrophysics Data System (ADS)

    Rubinstein, R. L.; Cousens, V.; Gage, D. J.; Shor, L. M.

    2013-12-01

    Soil bacteria within the rhizosphere benefit plants by protecting roots from pathogens, producing growth factors, and improving nutrient availability. These effects can greatly improve overall plant health and increase crop yield, but as roots grow out from the tips they quickly outpace their bacterial partners. Some soil bacteria are motile and can chemotact towards root tips, but bacterial mobility in unsaturated soils is limited to interconnected hydrated pores. Mobility is further reduced by the tendency of soil bacteria to form biofilms. The introduction of protists to the rhizosphere has been shown to benefit plants, purportedly by selective grazing on harmful bacteria or release of nutrients otherwise sequestered in bacteria. We propose that an additional benefit to the presence of protists is the facilitated transport of beneficial bacteria along root systems. Using microfluidic devices designed to imitate narrow, fluid-filled channels in soil, we have shown that the distribution of bacteria through micro-channels is accelerated in the presence of protists. Furthermore, we have observed that even with predation effects, the bacteria remain viable and continue to reproduce for the duration of our experiments. These results expand upon our understanding of complex bio-physical interactions in the rhizosphere system, and may have important implications for agricultural practices.

  1. Carbon dioxide capture using a CO{sub 2}-selective facilitated transport membrane

    SciTech Connect

    Huang, J.; Zou, J.; Ho, W.S.W.

    2008-02-15

    A novel CO{sub 2}-selective membrane with the facilitated transport mechanism has been synthesized to capture CO{sub 2} from the industrial gas mixtures, including flue gas. Both mobile and fixed amine carriers were incorporated into the cross-linked poly(vinyl alcohol) (PVA) during the membrane synthesis. The membrane showed desirable CO{sub 2} permeability (with a suitable effective thickness) and CO{sub 2}/N{sub 2} selectivity up to 170{sup o} C. In the CO{sub 2} capture experiments from a gas mixture with N{sub 2} and H{sub 2}, a permeate CO{sub 2} dry concentration of {gt}98% was obtained, using steam as the sweep gas. The effects of the feed flow rate and the sweep:feed molar ratio on the membrane separation performance were investigated. A one-dimensional isothermal model was established to examine the performance of a hollow-fiber membrane module composed of the described CO{sub 2}-selective membrane. The modeling results show that a CO{sub 2} recovery of {gt}95% and a permeate CO{sub 2} dry concentration of {gt}98% are achievable from a 1000 standard cubic feet per minute (SCFM) (or 21.06 mol/s) flue gas stream with a 2 ft (0.61 m) hollow-fiber module that contained 980 000 fibers.

  2. Fatty Acid-Binding Protein 5 Facilitates the Blood-Brain Barrier Transport of Docosahexaenoic Acid.

    PubMed

    Pan, Yijun; Scanlon, Martin J; Owada, Yuji; Yamamoto, Yui; Porter, Christopher J H; Nicolazzo, Joseph A

    2015-12-07

    The brain has a limited ability to synthesize the essential polyunsaturated fatty acid (PUFA) docosahexaenoic acid (DHA) from its omega-3 fatty acid precursors. Therefore, to maintain brain concentrations of this PUFA at physiological levels, plasma-derived DHA must be transported across the blood-brain barrier (BBB). While DHA is able to partition into the luminal membrane of brain endothelial cells, its low aqueous solubility likely limits its cytosolic transfer to the abluminal membrane, necessitating the requirement of an intracellular carrier protein to facilitate trafficking of this PUFA across the BBB. As the intracellular carrier protein fatty acid-binding protein 5 (FABP5) is expressed at the human BBB, the current study assessed the putative role of FABP5 in the brain endothelial cell uptake and BBB transport of DHA in vitro and in vivo, respectively. hFAPB5 was recombinantly expressed and purified from Escherichia coli C41(DE3) cells and the binding affinity of DHA to hFABP5 assessed using isothermal titration calorimetry. The impact of FABP5 siRNA on uptake of (14)C-DHA into immortalized human brain microvascular endothelial (hCMEC/D3) cells was assessed. An in situ transcardiac perfusion method was optimized in C57BL/6 mice and subsequently used to compare the BBB influx rate (Kin) of (14)C-DHA between FABP5-deficient (FABP5(-/-)) and wild-type (FABP5(+/+)) C57BL/6 mice. DHA bound to hFABP5 with an equilibrium dissociation constant of 155 ± 8 nM (mean ± SEM). FABP5 siRNA transfection decreased hCMEC/D3 mRNA and protein expression of FABP5 by 53.2 ± 5.5% and 44.8 ± 13.7%, respectively, which was associated with a 14.1 ± 2.7% reduction in (14)C-DHA cellular uptake. By using optimized conditions for the in situ transcardiac perfusion (a 1 min preperfusion (10 mL/min) followed by perfusion of (14)C-DHA (1 min)), the Kin of (14)C-DHA was 0.04 ± 0.01 mL/g/s. Relative to FABP5(+/+) mice, the Kin of (14)C-DHA decreased 36.7 ± 12.4% in FABP5(-/-) mice

  3. Facilitated transport of Cu with hydroxyapatite nanoparticles in saturated sand: Effects of solution ionic strength and composition

    USDA-ARS?s Scientific Manuscript database

    Column experiments were conducted to investigate the facilitated transport of Cu in association with hydroxyapatite nanoparticles (nHAP) in water-saturated quartz sand at different solution concentrations of NaCl (0 to 100 mM) or CaCl2 (0.1 to 1.0 mM). The experimental breakthrough curves and retent...

  4. Role of Desorption Kinetics and Porous Medium Heterogeneity in Colloid-Facilitated Transport of Cesium and Strontium: Preliminary Results

    NASA Astrophysics Data System (ADS)

    Dittrich, T. M.; Ryan, J. N.

    2008-12-01

    The presence of mobile colloids (particles between 1 nm and 1 μm in size) in natural soil and groundwater systems has been well established. Colloids generally have a high sorptive capacity resulting from their high surface area to mass ratio, which makes them effective sorbents of low solubility, strongly sorbing contaminants. Mobile colloids that sorb contaminants can increase the apparent solubility and rate of transport of the contaminants when desorption from the colloids is slow relative to the rate of flow. This process is known as colloid-facilitated transport (CFT). The additional transport of contaminants associated with mobile colloids should be accounted for to accurately predict transport rates of strongly-sorbing contaminants in the environment. Some examples of contaminants that have the potential for CFT are hydrophobic pesticides, polycyclic aromatic hydrocarbons (PAHs), actinide cations (e.g., Th, U, Pu, Am), and many metals (e.g, Pb, Cu, Hg). Many low solubility contaminants that have the potential for CFT are also harmful or toxic to humans, underscoring the importance of accurate modeling techniques to protect water sources from contamination. Contaminated Department of Energy (DOE) sites have been particularly valuable research opportunities for studying the transport of radionuclides in the natural environment. The DOE has conducted energy and weapons research and development in thirty-one states and Puerto Rico and has introduced many toxic and radioactive chemicals into surface waters, soils, and groundwater. Field experiments on DOE sites including the Nevada Test Site, the Hanford 200 Area tank farm, Rocky Flats CO, and Oak Ridge TN, have confirmed that metals and radionuclides have moved further than expected due to colloid-facilitated transport. The major goal of this research project is to identify and quantify the effects of sorption kinetics on colloid- facilitated transport in unsaturated porous media. This information will be used

  5. Evidence for expression of the facilitated glucose transporter in rat hepatocytes.

    PubMed Central

    Rhoads, D B; Takano, M; Gattoni-Celli, S; Chen, C C; Isselbacher, K J

    1988-01-01

    The eukaryotic facilitated glucose transporter (GT) is expressed by many cell types, with the notable exception of hepatocytes; however, GT is expressed by several hepatoma cell lines, including the well-differentiated lines Fao, Hep3B, and HepG2. We report on studies carried out to determine the aspect(s) of the transformed phenotype that might be responsible for activating GT expression. Using RNA blot analysis with probes derived from rat GT cDNA, we found that GT was expressed by rat hepatocytes under two conditions (i) in vitro, when isolated hepatocytes were placed in cell culture, and (ii) in vivo, when rats were subjected to starvation for greater than or equal to 2 days. However, GT expression was not an obligatory feature of hepatomas, since two primary hepatocellular carcinomas did not express any GT mRNA. GT expression in hepatocytes was reduced by addition of dimethyl sulfoxide or sodium butyrate to the culture medium. Since these reagents are known to promote differentiation in some cell culture systems, their effect on hepatocytes may be to maintain the GT repression normally observed in vivo. Inclusion or exclusion in the culture medium of several other agents that enhance hepatocyte viability (serum, insulin, corticosteroids, epidermal growth factor, or triiodothyronine) did not affect GT expression. It is unclear whether the two conditions that led to GT expression in hepatocytes are related by a common signaling mechanism. Possibly, both cases involve a "stress" response: in vivo, a normal physiological response to starvation; in vitro, a response to a major alteration in the cellular environment. Images PMID:3194405

  6. An L-Glutamine Transporter Isoform for Neurogenesis Facilitated by L-Theanine.

    PubMed

    Yoneda, Yukio

    2017-06-09

    L-Theanine (=γ-glutamylethylamide) is an amino acid ingredient in green tea with a structural analogy to L-glutamine (L-GLN) rather than L-glutamic acid (L-GLU), with regards to the absence of a free carboxylic acid moiety from the gamma carbon position. L-theanine markedly inhibits [(3)H]L-GLN uptake without affecting [(3)H]L-GLU uptake in cultured neurons and astroglia. In neural progenitor cells with sustained exposure to L-theanine, upregulation of the L-GLN transporter isoform Slc38a1 expression and promotion of both proliferation and neuronal commitment are seen along with marked acceleration of the phosphorylation of mammalian target of rapamycin (mTOR) and relevant downstream proteins. Stable overexpression of Slc38a1 leads to promotion of cellular growth with facilitated neuronal commitment in pluripotent embryonic carcinoma P19 cells. In P19 cells stably overexpressing Slc38a1, marked phosphorylation is seen with mTOR and downstream proteins in a fashion insensitive to the additional stimulation by L-theanine. The green tea amino acid L-theanine could thus elicit pharmacological actions to up-regulate Slc38a1 expression for activation of the mTOR signaling pathway required for cell growth together with accelerated neurogenesis after sustained exposure in undifferentiated neural progenitor cells. In this review, I summarize a novel pharmacological property of the green tea amino acid L-theanine for embryonic and adult neurogenesis with a focus on the endogenous amino acid analog L-GLN. A possible translational strategy is also discussed on the development of dietary supplements and nutraceuticals enriched of L-theanine for the prophylaxis of a variety of untoward impairments and malfunctions seen in patients with different neurodegenerative and/or neuropsychiatric disorders.

  7. Association of Facilitated Glucose Transporter 2 gene variants with the Myelomeningocele phenotype

    PubMed Central

    Ruggiero, Jaclyn E; Northrup, Hope; Au, Kit Sing

    2015-01-01

    BACKGROUND Neural tube defects (NTDs) remain the second most common cause of congenital malformations. Myelomeningocele (MM), the most common NTD compatible with survival, results from genetic and environmental factors. Epidemiologic studies and murine models support the hypotheses that obesity, diabetes and hyperglycemia confer increased risk of NTDs. Presence of wildtype facilitated glucose transporter, Glut2, in mouse embryos has been shown to increase risk for NTDs in hyperglycemic pregnancy. METHODS The GLUT2 gene of 96 MM patients was amplified, sequenced and compared to the reference sequence (NM_000340). Variants previously unreported in the single nucleotide polymorphisms database (dbSNP) were considered novel. Allele frequencies of reported SNPs were compared to reference populations using Fisher's exact test. RESULTS Analysis revealed three novel variants: a substitution in the core promoter region (c.-331c>t), a substitution (c.-182g>a) in the 5′-untranslated region (UTR), and a single base pair deletion (c.1441delT) in the coding sequences. Polymorphic alleles for 10 SNPs were also identified. Seven SNPs are significantly associated with MM in the Mexican American patients tested (p<0.05) and two of the seven remained significant after Bonferroni correction. CONCLUSION We identified three novel variants and seven SNPs associated with MM. The novel variants in the core promoter and in the 5′-UTR could affect GLUT2 mRNA transcription and stability and translation efficiency. The c.1441delT variant is predicted to alter the reading frame and prematurely terminate translation of the GLUT2 protein at the C-terminus, affecting GLUT2 protein function. Presence of GLUT2 variants may disrupt GLUT2 activity and influence MM susceptibility. PMID:25776730

  8. Nucleobases Undergo Dynamic Rearrangements during RNA Tertiary Folding.

    PubMed

    Welty, Robb; Hall, Kathleen B

    2016-11-06

    The tertiary structure of the GTPase center (GAC) of 23S ribosomal RNA (rRNA) as seen in cocrystals is extremely compact. It is stabilized by long-range hydrogen bonds and nucleobase stacking and by a triloop that forms within its three-way junction. Its folding pathway from secondary structure to tertiary structure has not been previously observed, but it was shown to require Mg(2+) ions in equilibrium experiments. The fluorescent nucleotide 2-aminopurine was substituted at selected sites within the 60-nt GAC. Fluorescence intensity changes upon addition of MgCl2 were monitored over a time-course from 1ms to 100s as the RNA folds. The folding pathway is revealed here to be hierarchical through several intermediates. Observation of the nucleobases during folding provides a new perspective on the process and the pathway, revealing the dynamics of nucleobase conformational exchange during the folding transitions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Recognition of DNA sequencing through binding of nucleobases to graphene

    NASA Astrophysics Data System (ADS)

    Zaffino, Valentina

    Graphene is one of the most promising materials in nanotechnology. Its large surface to volume ratio, high conductivity and electron mobility at room temperature are outstanding properties for use in DNA sensors. For this study, we used Density Functional Theory (DFT), ?with and without the inclusion of van der Waals (vdW) interactions, ?to investigate the adsorption of nucleobases (cytosine, guanine, adenine, thymine, and uracil) on pristine graphene and graphene with defects (Divacancy and Stone-Wales). We investigated the performance of two types of vdW-DF functional (optB86b-vdW and rPW86-vdW), as well as the PBE functional, and their description of the adsorption geometry and electronic structure of the nucleobase-graphene systems.The inclusion of defects results in an increase in binding energy, closer adsorption of the molecule to graphene and greater buckling in both the graphene structure and nucleobase.

  10. MATE transporters facilitate vacuolar uptake of epicatechin 3'-O-glucoside for proanthocyanidin biosynthesis in Medicago truncatula and Arabidopsis.

    PubMed

    Zhao, Jian; Dixon, Richard A

    2009-08-01

    Expression of the Arabidopsis thaliana MYB transcription factor TRANSPARENT TESTA 2 (TT2) in Medicago trunculata hairy roots induces both proanthocyanidin accumulation and the ATP-dependent vacuolar/vesicular uptake of epicatechin 3'-O-glucoside; neither process is active in control roots that do, however, possess anthocyanidin 3-O-glucoside vacuolar uptake activity. A vacuolar membrane-localized multidrug and toxic compound extrusion (MATE) transporter, Medicago MATE1, was identified at the molecular level and shown to preferentially transport epicatechin 3'-O-glucoside. Genetic evidence has implicated TT12, a tonoplastic MATE transporter from Arabidopsis, in the transport of precursors for proanthocyanidin biosynthesis in the seed coat. However, although Arabidopsis TT12 facilitates the transport of cyanidin 3-O-glucoside into membrane vesicles when expressed in yeast, there is no evidence that cyanidin 3-O-glucoside is converted to proanthocyanidins after transport into the vacuole. Here, we show that Arabidopsis TT12, like Medicago MATE1, functions to transport epicatechin 3'-O-glucoside as a precursor for proanthocyanidin biosynthesis, and Medicago MATE1 complements the seed proanthocyanidin phenotype of the Arabidopsis tt12 mutant both quantitatively and qualitatively. On the basis of biochemical properties, tissue-specific expression pattern, and genetic loss-of-function analysis, we conclude that MATE1 is an essential membrane transporter for proanthocyanidin biosynthesis in the Medicago seed coat. Implications of these findings for the assembly of oligomeric proanthocyanidins are discussed.

  11. LONG-TERM COLLOID MOBILIZATION AND COLLOID-FACILITATED TRANSPORT OF RADIONUCLIDES IN A SEMI-ARID VADOSE ZONE

    SciTech Connect

    Markus Flury; James B. Harsh; Fred Zhang; Glendon W. Gee; Earl D. Mattson; Peter C. L

    2012-08-01

    The main purpose of this project was to improve the fundamental mechanistic understanding and quantification of long-term colloid mobilization and colloid-facilitated transport of radionuclides in the vadose zone, with special emphasis on the semi-arid Hanford site. While we focused some of the experiments on hydrogeological and geochemical conditions of the Hanford site, many of our results apply to colloid and colloid-facilitated transport in general. Specific objectives were (1) to determine the mechanisms of colloid mobilization and colloid-facilitated radionuclide transport in undisturbed Hanford sediments under unsaturated flow, (2) to quantify in situ colloid mobilization and colloid-facilitated radionuclidetransport from Hanford sediments under field conditions, and (3) to develop a field-scale conceptual and numerical model for colloid mobilization and transport at the Hanford vadose zone, and use that model to predict long-term colloid and colloid- facilitated radionuclide transport. To achieve these goals and objectives, we have used a combination of experimental, theoretical, and numerical methods at different spatial scales, ranging from microscopic investigationsof single particle attachment and detachment to larger-scale field experiments using outdoor lysimeters at the Hanford site. Microscopic and single particle investigations provided fundamental insight into mechanisms of colloid interactions with the air-water interface. We could show that a moving air water interface (such as a moving water front during infiltration and drainage) is very effective in removing and mobilizing particles from a stationary surface. We further demonstrated that it is particularly the advancing air-water interface which is mainly responsible for colloid mobilization. Forces acting on the colloids calculated from theory corroborated our experimental results, and confirm that the detachment forces (surface tension forces) during the advancing air-water interface

  12. The solute specificity profiles of nucleobase cation symporter 1 (NCS1) from Zea mays and Setaria viridis illustrate functional flexibility.

    PubMed

    Rapp, Micah; Schein, Jessica; Hunt, Kevin A; Nalam, Vamsi; Mourad, George S; Schultes, Neil P

    2016-03-01

    The solute specificity profiles (transport and binding) for the nucleobase cation symporter 1 (NCS1) proteins, from the closely related C4 grasses Zea mays and Setaria viridis, differ from that of Arabidopsis thaliana and Chlamydomonas reinhardtii NCS1. Solute specificity profiles for NCS1 from Z. mays (ZmNCS1) and S. viridis (SvNCS1) were determined through heterologous complementation studies in NCS1-deficient Saccharomyces cerevisiae strains. The four Viridiplantae NCS1 proteins transport the purines adenine and guanine, but unlike the dicot and algal NCS1, grass NCS1 proteins fail to transport the pyrimidine uracil. Despite the high level of amino acid sequence similarity, ZmNCS1 and SvNCS1 display distinct solute transport and recognition profiles. SvNCS1 transports adenine, guanine, hypoxanthine, cytosine, and allantoin and competitively binds xanthine and uric acid. ZmNCS1 transports adenine, guanine, and cytosine and competitively binds, 5-fluorocytosine, hypoxanthine, xanthine, and uric acid. The differences in grass NCS1 profiles are due to a limited number of amino acid alterations. These amino acid residues do not correspond to amino acids essential for overall solute and cation binding or solute transport, as previously identified in bacterial and fungal NCS1, but rather may represent residues involved in subtle solute discrimination. The data presented here reveal that within Viridiplantae, NCS1 proteins transport a broad range of nucleobase compounds and that the solute specificity profile varies with species.

  13. Microbially Produced Organic Matter and Its Role in Facilitating Pu Transport in the Deep Vadose Zone

    NASA Astrophysics Data System (ADS)

    Fisher, J. C.; Tinnacher, R. M.; Zavarin, M.; Kersting, A. B.; Czerwinski, K.; Moser, D. P.

    2010-12-01

    Microorganisms have the potential to affect the fate and mobility of actinides in the deep vadose zone (DVZ) by metabolism (direct oxidation/reduction and changes to ecosystem redox potential), production of colloids and ligands, or by sorption (biofilms). The role of microbial communities in colloid-facilitated Pu transport is currently under investigation at the Nevada Test Site (NTS). Our experimental objective is to obtain both qualitative and quantitative data on the in situ role of biological organic material (DOM, POC, and EPS) on the (de)sorption of Pu at environmentally relevant concentrations. Groundwater samples were collected through vertical ventilation holes from a flooded post-test tunnel at the (NTS), where SSU rRNA gene libraries revealed a range of potential microbial physiotypes. Microbial enrichments were set up with the aim of isolating numerically significant representatives of major relevant physiotypes (e.g. aerobic heterotrophs, Mn/Fe reducers, EPS producers). NTS isolates, a well-characterized Shewanella sp.(str. CN-32), and an EPS-mutant of this strain were screened for their reactivity with Pu(IV). Organisms with both high and low (relative) Kd’s were used in sorption and cell lysis experiments. Viability experiments were conducted for all isolates in NaCl or NaCl/NaHCO3 solutions (I=0.01) for pH = 3, 5, 7, and 9. Products from cell lysis were filtered (0.22 um) or dialyzed (MW cutoff = 20,000 kD). These fractions were normalized by TOC and equilibrated with Pu to determine if Pu sorbs more strongly to either viable cells, EPS, cell membranes, or cell exudates. In our experiments, Pu(IV) sorbed most strongly to cells or cell fractions with EPS (expolysaccharide, the major biofilm component). However, cell fractions and exudates, which may become mobile when released from lysed or senescing cells, also strongly sorbed to Pu(IV). Therefore, changes in groundwater chemistry (e.g., pH or ionic strength) may have both direct chemical

  14. A suite of sucrose transporters expressed in coats of developing legume seeds includes novel pH-independent facilitators.

    PubMed

    Zhou, Yuchan; Qu, Hongxia; Dibley, Katherine E; Offler, Christina E; Patrick, John W

    2007-02-01

    A suite of newly discovered sucrose transporter genes, PsSUF1, PsSUF4, PvSUT1 and PvSUF1, were isolated from the coats of developing pea (Pisum sativum L.) and bean (Phaseolus vulgaris L.) seeds. Sequence analysis indicated that deduced proteins encoded by PsSUF1, PvSUT1 and PvSUF1 clustered in a separate sub-group under sucrose transporter Clade I, whereas the deduced protein encoded by PsSUF4 clustered in Clade II. When expressed in yeast, these genes were shown to encode sucrose transporters with apparent Michaelis Menten constant (Km) values ranging from 8.9 to 99.8 mm. PvSUT1 exhibited functional characteristics of a sucrose/H+ symporter. In contrast, PsSUF1, PvSUF1 and PsSUF4 supported the pH- and energy independent transport of sucrose that was shown to be bi-directional. These transport properties, together with that of counter transport, indicated that PsSUF1, PvSUF1 and PsSUF4 function as carriers that support the facilitated diffusion of sucrose. Carrier function was unaffected by diethylpyrocarbonate and by maltose competition, suggesting that the sucrose binding sites of these transporters differed from those of known sucrose/H+ symporters. All sucrose transporters were expressed throughout the plant and, of greatest interest, were co-expressed in cells considered responsible for sucrose efflux from seed coats. The possible roles played by the novel facilitators in sucrose efflux from seed coats are discussed.

  15. Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

    PubMed

    Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

    2017-06-02

    Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

  16. On the structural regularity in nucleobases and amino acids and relationship to the origin and evolution of the genetic code.

    PubMed

    Yang, Chi Ming

    2005-06-01

    To explore how chemical structures of both nucleobases and amino acids may have played a role in shaping the genetic code, numbers of sp2 hybrid nitrogen atoms in nucleobases were taken as a determinative measure for empirical stereo-electronic property to analyze the genetic code. Results revealed that amino acid hydropathy correlates strongly with the sp2 nitrogen atom numbers in nucleobases rather than with the overall electronic property such as redox potentials of the bases, reflecting that stereo-electronic property of bases may play a role. In the rearranged code, five simple but stereo-structurally distinctive amino acids (Gly, Pro, Val, Thr and Ala) and their codon quartets form a crossed intersection "core". Secondly, a re-categorization of the amino acids according to their beta-carbon stereochemistry, verified by charge density (at beta-carbon) calculation, results in five groups of stereo-structurally distinctive amino acids, the group leaders of which are Gly, Pro, Val, Thr and Ala, remarkably overlapping the above "core". These two lines of independent observations provide empirical arguments for a contention that a seemingly "frozen" "core" could have formed at a certain evolutionary stage. The possible existence of this codon "core" is in conformity with a previous evolutionary model whereby stereochemical interactions may have shaped the code. Moreover, the genetic code listed in UCGA succession together with this codon "core" has recently facilitated an identification of the unprecedented icosikaioctagon symmetry and bi-pyramidal nature of the genetic code.

  17. Facilitated Anion Transport Induces Hyperpolarization of the Cell Membrane That Triggers Differentiation and Cell Death in Cancer Stem Cells.

    PubMed

    Soto-Cerrato, Vanessa; Manuel-Manresa, Pilar; Hernando, Elsa; Calabuig-Fariñas, Silvia; Martínez-Romero, Alicia; Fernández-Dueñas, Víctor; Sahlholm, Kristoffer; Knöpfel, Thomas; García-Valverde, María; Rodilla, Ananda M; Jantus-Lewintre, Eloisa; Farràs, Rosa; Ciruela, Francisco; Pérez-Tomás, Ricardo; Quesada, Roberto

    2015-12-23

    Facilitated anion transport potentially represents a powerful tool to modulate various cellular functions. However, research into the biological effects of small molecule anionophores is still at an early stage. Here we have used two potent anionophore molecules inspired in the structure of marine metabolites tambjamines to gain insight into the effect induced by these compounds at the cellular level. We show how active anionophores, capable of facilitating the transmembrane transport of chloride and bicarbonate in model phospholipid liposomes, induce acidification of the cytosol and hyperpolarization of plasma cell membranes. We demonstrate how this combined effect can be used against cancer stem cells (CSCs). Hyperpolarization of cell membrane induces cell differentiation and loss of stemness of CSCs leading to effective elimination of this cancer cell subpopulation.

  18. Facile thiol-ene thermal crosslinking reaction facilitated hole-transporting layer for highly efficient and stable perovskite solar cells

    DOE PAGES

    Li, Zhong'an; Zhu, Zonglong; Chueh, Chu -Chen; ...

    2016-08-08

    A crosslinked organic hole-transporting layer (HTL) is developed to realize highly efficient and stable perovskite solar cells via a facile thiol-ene thermal reaction. This crosslinked HTL not only facilitates hole extraction from perovskites, but also functions as an effective protective barrier. Lastly, a high-performance (power conversion efficiency: 18.3%) device is demonstrated to show respectable photo and thermal stability without encapsulation.

  19. Facile thiol-ene thermal crosslinking reaction facilitated hole-transporting layer for highly efficient and stable perovskite solar cells

    SciTech Connect

    Li, Zhong'an; Zhu, Zonglong; Chueh, Chu -Chen; Luo, Jingdong; Jen, Alex K. -Y.

    2016-08-08

    A crosslinked organic hole-transporting layer (HTL) is developed to realize highly efficient and stable perovskite solar cells via a facile thiol-ene thermal reaction. This crosslinked HTL not only facilitates hole extraction from perovskites, but also functions as an effective protective barrier. Lastly, a high-performance (power conversion efficiency: 18.3%) device is demonstrated to show respectable photo and thermal stability without encapsulation.

  20. Single Nucleobase Identification Using Biophysical Signatures from Nanoelectronic Quantum Tunneling.

    PubMed

    Korshoj, Lee E; Afsari, Sepideh; Khan, Sajida; Chatterjee, Anushree; Nagpal, Prashant

    2017-03-01

    Nanoelectronic DNA sequencing can provide an important alternative to sequencing-by-synthesis by reducing sample preparation time, cost, and complexity as a high-throughput next-generation technique with accurate single-molecule identification. However, sample noise and signature overlap continue to prevent high-resolution and accurate sequencing results. Probing the molecular orbitals of chemically distinct DNA nucleobases offers a path for facile sequence identification, but molecular entropy (from nucleotide conformations) makes such identification difficult when relying only on the energies of lowest-unoccupied and highest-occupied molecular orbitals (LUMO and HOMO). Here, nine biophysical parameters are developed to better characterize molecular orbitals of individual nucleobases, intended for single-molecule DNA sequencing using quantum tunneling of charges. For this analysis, theoretical models for quantum tunneling are combined with transition voltage spectroscopy to obtain measurable parameters unique to the molecule within an electronic junction. Scanning tunneling spectroscopy is then used to measure these nine biophysical parameters for DNA nucleotides, and a modified machine learning algorithm identified nucleobases. The new parameters significantly improve base calling over merely using LUMO and HOMO frontier orbital energies. Furthermore, high accuracies for identifying DNA nucleobases were observed at different pH conditions. These results have significant implications for developing a robust and accurate high-throughput nanoelectronic DNA sequencing technique. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Identification of an Axonal Kinesin-3 Motor for Fast Anterograde Vesicle Transport that Facilitates Retrograde Transport of Neuropeptides

    PubMed Central

    Barkus, Rosemarie V.; Klyachko, Olga; Horiuchi, Dai; Dickson, Barry J.

    2008-01-01

    A screen for genes required in Drosophila eye development identified an UNC-104/Kif1 related kinesin-3 microtubule motor. Analysis of mutants suggested that Drosophila Unc-104 has neuronal functions that are distinct from those of the classic anterograde axonal motor, kinesin-1. In particular, unc-104 mutations did not cause the distal paralysis and focal axonal swellings characteristic of kinesin-1 (Khc) mutations. However, like Khc mutations, unc-104 mutations caused motoneuron terminal atrophy. The distributions and transport behaviors of green fluorescent protein-tagged organelles in motor axons indicate that Unc-104 is a major contributor to the anterograde fast transport of neuropeptide-filled vesicles, that it also contributes to anterograde transport of synaptotagmin-bearing vesicles, and that it contributes little or nothing to anterograde transport of mitochondria, which are transported primarily by Khc. Remarkably, unc-104 mutations inhibited retrograde runs by neurosecretory vesicles but not by the other two organelles. This suggests that Unc-104, a member of an anterograde kinesin subfamily, contributes to an organelle-specific dynein-driven retrograde transport mechanism. PMID:17989365

  2. The Renaissance of Metal-Pyrimidine Nucleobase Coordination Chemistry.

    PubMed

    Lippert, Bernhard; Sanz Miguel, Pablo J

    2016-08-16

    The significance of metal ions for the function and properties of DNA and RNA, long seen primarily under biological aspects and medicinal uses, has recently gained a renewed momentum. This is a consequence of the advent of novel applications in the fields of materials science, biotechnology, and analytical sensor chemistry that relate to the designed incorporation of transition metal ions into nucleic acid base pairs. Ag(+) and Hg(2+) ions, binding to pyrimidine (pym) nucleobases, represent major players in this development. Interestingly, these metal ions were the ones that some 60 years ago started the field! At the same time, the mentioned metal ions had demonstrated a "special relationship" with the pym nucleobases cytosine, thymine, and uracil! Parallel work conducted with oligonucleotides and model nucleobases fostered numerous significant details of these interactions, in particular when X-ray crystallography was involved, correcting earlier views occasionally. Our own activities during the past three to four decades have focused on, among others, the coordination chemistry of transition and main-group metal ions with pym model nucleobases, with an emphasis on Pt(II) and Pd(II). It has always been our goal to deduce, if possible, the potential relevance of our findings for biological processes. It is interesting to put our data, in particular for trans-a2Pt(II) (a = NH3 or amine), into perspective with those of other metal ions, notably Ag(+) and Hg(2+). Irrespective of major differences in kinetics and lability/inertness between d(8) and d(10) metal ions, there is also a lot of similarity in structural aspects as a result of the preferred linear coordination geometry of these species. Moreover, the apparent clustering of metal ions to the pym nucleobases, which is presumably essential for the formation of nanoclusters on oligonucleotide scaffolds, is impressively reflected in model systems, as are reasons for inter-nucleobase cross-links containing more

  3. Bubble-facilitated VOC transport from LNAPL smear zones and its potential effect on vapor intrusion: Laboratory experiments

    NASA Astrophysics Data System (ADS)

    Soucy, N. C.; Mumford, K. G.

    2016-12-01

    Light non-aqueous phase liquid (LNAPL) sources can pose a significant threat to indoor air through the volatilization of hydrocarbons from the source and the subsequent transport of vapor through the soil. If subjected to the rise and fall of a water table, an LNAPL source can become a smear zone that consists of trapped discontinuous LNAPL blobs (residual) and has a higher aqueous permeability and higher surface area-to-volume ratio than pool sources. The rise and fall of a water table can also trap atmospheric air bubbles alongside the LNAPL. If these bubbles expand and become mobile, either through partitioning of volatile organic compounds (VOCs) or the production of biogenic gases, bubble-facilitated vertical vapor transport can occur. It is important to understand the bubble-facilitated transport of VOCs as it is a mechanism that could lead to faster transport. The transport of VOCs from smear zones was investigated using laboratory column and visualization experiments. In the column experiments, pentane LNAPL was emplaced in a 5 cm sand-packed source zone and the water level was raised and lowered to trap residual LNAPL and air bubbles. Each column also contained a 10 cm-high zone of clean saturated sand, and a 10 cm vadose zone of 4 mm-diameter glass beads. Water was pumped through the source and occlusion zones, and air flowed across the top of the column, where vapor samples were collected and analyzed immediately by gas chromatography. In the visualization experiments, pentane LNAPL was emplaced in a two-dimensional cell designed to allow visualization of mobilized LNAPL and gas through glass walls. Results of the column experiments showed VOC mass fluxes in test columns were 1-2 orders of magnitude greater than in the control columns. In addition, the flux signal was intermittent, consistent with expectations of bubble-facilitated transport. The results from the visualization experiments showed gas fingers growing and mobilizing over time, and supports

  4. Two major facilitator superfamily sugar transporters from Trichoderma reesei and their roles in induction of cellulase biosynthesis.

    PubMed

    Zhang, Weixin; Kou, Yanbo; Xu, Jintao; Cao, Yanli; Zhao, Guolei; Shao, Jing; Wang, Hai; Wang, Zhixing; Bao, Xiaoming; Chen, Guanjun; Liu, Weifeng

    2013-11-15

    Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes. Two other putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement.

  5. Connective Auxin Transport in the Shoot Facilitates Communication between Shoot Apices.

    PubMed

    Bennett, Tom; Hines, Geneviève; van Rongen, Martin; Waldie, Tanya; Sawchuk, Megan G; Scarpella, Enrico; Ljung, Karin; Leyser, Ottoline

    2016-04-01

    The bulk polar movement of the plant signaling molecule auxin through the stem is a long-recognized but poorly understood phenomenon. Here we show that the highly polar, high conductance polar auxin transport stream (PATS) is only part of a multimodal auxin transport network in the stem. The dynamics of auxin movement through stems are inconsistent with a single polar transport regime and instead suggest widespread low conductance, less polar auxin transport in the stem, which we term connective auxin transport (CAT). The bidirectional movement of auxin between the PATS and the surrounding tissues, mediated by CAT, can explain the complex auxin transport kinetics we observe. We show that the auxin efflux carriers PIN3, PIN4, and PIN7 are major contributors to this auxin transport connectivity and that their activity is important for communication between shoot apices in the regulation of shoot branching. We propose that the PATS provides a long-range, consolidated stream of information throughout the plant, while CAT acts locally, allowing tissues to modulate and be modulated by information in the PATS.

  6. Connective Auxin Transport in the Shoot Facilitates Communication between Shoot Apices

    PubMed Central

    Bennett, Tom; Hines, Geneviève; van Rongen, Martin; Sawchuk, Megan G.; Scarpella, Enrico; Ljung, Karin

    2016-01-01

    The bulk polar movement of the plant signaling molecule auxin through the stem is a long-recognized but poorly understood phenomenon. Here we show that the highly polar, high conductance polar auxin transport stream (PATS) is only part of a multimodal auxin transport network in the stem. The dynamics of auxin movement through stems are inconsistent with a single polar transport regime and instead suggest widespread low conductance, less polar auxin transport in the stem, which we term connective auxin transport (CAT). The bidirectional movement of auxin between the PATS and the surrounding tissues, mediated by CAT, can explain the complex auxin transport kinetics we observe. We show that the auxin efflux carriers PIN3, PIN4, and PIN7 are major contributors to this auxin transport connectivity and that their activity is important for communication between shoot apices in the regulation of shoot branching. We propose that the PATS provides a long-range, consolidated stream of information throughout the plant, while CAT acts locally, allowing tissues to modulate and be modulated by information in the PATS. PMID:27119525

  7. Zero-tension lysimeters: An improved design to monitor colloid-facilitated contaminant transport in the vadose zone

    SciTech Connect

    Thompson, M.L.; Scharf, R.L.; Shang, C.

    1995-04-24

    There is increasing evidence that mobile colloids facilitate the long-distance transport of contaminants. The mobility of fine particles and macromolecules has been linked to the movement of actinides, organic contaminants, and heavy metals through soil. Direct evidence for colloid mobility includes the presence of humic materials in deep aquifers as well as coatings of accumulated clay, organic matter, or sesquioxides on particle or aggregate surfaces in subsoil horizons of many soils. The potential for colloid-facilitated transport of contaminants from hazardous-waste sites requires adequate monitoring before, during, and after in-situ remediation treatments. Zero-tension lysimeters (ZTLs) are especially appropriate for sampling water as it moves through saturated soil, although some unsaturated flow events may be sampled as well. Because no ceramic barrier or fiberglass wick is involved to maintain tension on the water (as is the case with other lysimeters), particles suspended in the water as well as dissolved species may be sampled with ZTLs. In this report, a ZTL design is proposed that is more suitable for monitoring colloid-facilitated contaminant migration. The improved design consists of a cylinder made of polycarbonate or polytetrafluoroethylene (PTFE) that is placed below undisturbed soil material. In many soils, a hydraulically powered tube may be used to extract an undisturbed core of soil before placement of the lysimeter. In those cases, the design has significant advantages over conventional designs with respect to simplicity and speed of installation. Therefore, it will allow colloid-facilitated transport of contaminants to be monitored at more locations at a given site.

  8. [Hopping and superexchange mechanisms of charge transport to DNA].

    PubMed

    Lakhno, V D; Sultanov, V B

    2003-01-01

    A theory for charge transport in nucleobase sequences was constructed in which the hole migration proceeds via hopping between guanines. Each hop over the adenine-thymine (A-T) bridge connecting neighboring guanines occurs by means of the superexchange mechanism. The experimental data and theoretical results for various types of nucleobase sequences are compared.

  9. Communication: Photoactivation of nucleobase bound platinum{sup II} metal complexes: Probing the influence of the nucleobase

    SciTech Connect

    Sen, Ananya; Dessent, Caroline E. H.

    2014-12-28

    We present UV laser action spectra (220-300 nm) of isolated nucleobase-bound Pt{sup II}(CN){sub 4}{sup 2−} complexes, i.e., Pt(CN){sub 4}{sup 2−}⋅M, where M = uracil, thymine, cytosine, and adenine. These metal complex-nucleobase clusters represent model systems for identifying the fundamental photophysical and photochemical processes occurring in photodynamic platinum (II) drug therapies that target DNA. This is the first study to explore the specific role of the nucleobase in the photophysics of the aggregate complex. Each of the complexes studied displays a broadly similar absorption spectra, with a strong λ{sub max} ∼ 4.7 eV absorption band (nucleobase localized chromophore) and a subsequent increase in the absorption intensity towards higher spectral-energy (Pt(CN){sub 4}{sup 2−} localized chromophore). However, strikingly different band widths are observed across the series of complexes, decreasing in the order Pt(CN){sub 4}{sup 2−}⋅Thymine > Pt(CN){sub 4}{sup 2−}⋅Uracil > Pt(CN){sub 4}{sup 2−}⋅Adenine > Pt(CN){sub 4}{sup 2−}⋅Cytosine. Changes in the bandwidth of the ∼4.7 eV band are accompanied by distinctive changes in the photofragment product ions observed following photoexcitation, with the narrower-bandwidth complexes showing a greater propensity to decay via electron detachment decay. We discuss these observations in the context of the distinctive nucleobase-dependent excited state lifetimes.

  10. The Effect of a Simulated Macropore on the Colloid-Facilitated Transport of Cesium and Strontium: Experiment and Model Results

    NASA Astrophysics Data System (ADS)

    Dittrich, T. M.; Ryan, J. N.; Saiers, J. E.

    2011-12-01

    The sorption of contaminants to mobile colloids has been shown to increase the transport of the contaminants in a process known as colloid-facilitated transport. Many laboratory experiments and computer model simulations have shown that enhanced transport can occur when a contaminant strongly associates with mobile colloids and release kinetics are slow relative to the rate of flow. Knowing when colloid-facilitated transport will affect field-scale situations and risk assessment decisions has been difficult. The three parts of our research were (1) conduct a set of isotherms and breakthrough curves for a well-characterized system (illite colloids, homogeneous quartz sand, saturated and unsaturated conditions), (2) conduct breakthrough experiments with the addition of a central macropore and, (3) model the results to identify and quantify the effects of desorption kinetics and unsaturated conditions on colloid-facilitated transport with a macropore. Breakthrough experiments used a 12.7 cm diameter and 33.5 cm long column packed with cleaned and sieved quartz sand. The homogeneous experiments used sand with a median grain size of 0.325 mm. For macropore experiments, a 2 cm diameter tube of 1.6 mm sand (about 5× the size of the matrix sand) was packed in the center of the column. A rainfall simulator was suspended over the column and a relative saturation of 1.0, 0.80, or 0.33 was established. Three moisture sensors and three tensiometers monitored the flow conditions. Effluent was collected with a peristaltic pump and a fraction collector and measured for total and dissolved ions, pH, and colloid concentration. Cesium and strontium were used as model contaminants because they are common contaminants found on Department of Energy sites in the United States and because they have contrasting sorption kinetics with illite. A previously developed model for saturated colloid-facilitated transport of cesium and strontium was extended to accommodate unsaturated conditions

  11. Nephrocystins and MKS proteins interact with IFT particle and facilitate transport of selected ciliary cargos.

    PubMed

    Zhao, Chengtian; Malicki, Jarema

    2011-05-20

    Cilia are required for the development and function of many organs. Efficient transport of protein cargo along ciliary axoneme is necessary to sustain these processes. Despite its importance, the mode of interaction between the intraflagellar ciliary transport (IFT) mechanism and its cargo proteins remains poorly understood. Our studies demonstrate that IFT particle components, and a Meckel-Gruber syndrome 1 (MKS1)-related, B9 domain protein, B9d2, bind each other and contribute to the ciliary localization of Inversin (Nephrocystin 2). B9d2, Inversin, and Nephrocystin 5 support, in turn, the transport of a cargo protein, Opsin, but not another photoreceptor ciliary transmembrane protein, Peripherin. Interestingly, the components of this mechanism also contribute to the formation of planar cell polarity in mechanosensory epithelia. These studies reveal a molecular mechanism that mediates the transport of selected ciliary cargos and is of fundamental importance for the differentiation and survival of sensory cells.

  12. COLLOIDAL-FACILITATED TRANSPORT OF INORGANIC CONTAMINANTS IN GROUND WATER: PART I. SAMPLING CONSIDERATIONS

    EPA Science Inventory

    Investigations at Pinal Creek, Arizona, evaluated routine sampling procedures for determination of aqueous inorganic geochemistry and assessment of contaminant transport by colloidal mobility. Sampling variables included pump type and flow rate, collection under air or nitrogen,...

  13. Functional Determinants of Metal Ion Transport and Selectivity in Paralogous Cation Diffusion Facilitator Transporters CzcD and MntE in Streptococcus pneumoniae.

    PubMed

    Martin, Julia E; Giedroc, David P

    2016-01-19

    Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters that collectively possess broad metal specificity and contribute to intracellular metal homeostasis and virulence in bacterial pathogens. Streptococcus pneumoniae expresses two homologous CDF efflux transporters, MntE and CzcD. Cells lacking mntE or czcD are sensitive to manganese (Mn) or zinc (Zn) toxicity, respectively, and specifically accumulate Mn or Zn, respectively, thus suggesting that MntE selectively transports Mn, while CzcD transports Zn. Here, we probe the origin of this metal specificity using a phenotypic growth analysis of pneumococcal variants. Structural homology to Escherichia coli YiiP predicts that both MntE and CzcD are dimeric and each protomer harbors four pairs of conserved metal-binding sites, termed the A site, the B site, and the C1/C2 binuclear site. We find that single amino acid mutations within both the transmembrane domain A site and the B site in both CDFs result in a cellular metal sensitivity similar to that of the corresponding null mutants. However, multiple mutations in the predicted cytoplasmic C1/C2 cluster of MntE have no impact on cellular Mn resistance, in contrast to the analogous substitutions in CzcD, which do have on impact on cellular Zn resistance. Deletion of the MntE-specific C-terminal tail, present only in Mn-specific bacterial CDFs, resulted in only a modest growth phenotype. Further analysis of MntE-CzcD functional chimeric transporters showed that Asn and Asp in the ND-DD A-site motif of MntE and the most N-terminal His in the HD-HD site A of CzcD (the specified amino acids are underlined) play key roles in transporter metal selectivity. Cation diffusion facilitator (CDF) proteins are divalent metal ion transporters that are conserved in organisms ranging from bacteria to humans and that play important roles in cellular physiology, from metal homeostasis and resistance to type I diabetes in vertebrates. The respiratory

  14. Functional Determinants of Metal Ion Transport and Selectivity in Paralogous Cation Diffusion Facilitator Transporters CzcD and MntE in Streptococcus pneumoniae

    PubMed Central

    Martin, Julia E.

    2016-01-01

    ABSTRACT Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters that collectively possess broad metal specificity and contribute to intracellular metal homeostasis and virulence in bacterial pathogens. Streptococcus pneumoniae expresses two homologous CDF efflux transporters, MntE and CzcD. Cells lacking mntE or czcD are sensitive to manganese (Mn) or zinc (Zn) toxicity, respectively, and specifically accumulate Mn or Zn, respectively, thus suggesting that MntE selectively transports Mn, while CzcD transports Zn. Here, we probe the origin of this metal specificity using a phenotypic growth analysis of pneumococcal variants. Structural homology to Escherichia coli YiiP predicts that both MntE and CzcD are dimeric and each protomer harbors four pairs of conserved metal-binding sites, termed the A site, the B site, and the C1/C2 binuclear site. We find that single amino acid mutations within both the transmembrane domain A site and the B site in both CDFs result in a cellular metal sensitivity similar to that of the corresponding null mutants. However, multiple mutations in the predicted cytoplasmic C1/C2 cluster of MntE have no impact on cellular Mn resistance, in contrast to the analogous substitutions in CzcD, which do have on impact on cellular Zn resistance. Deletion of the MntE-specific C-terminal tail, present only in Mn-specific bacterial CDFs, resulted in only a modest growth phenotype. Further analysis of MntE-CzcD functional chimeric transporters showed that Asn and Asp in the ND-DD A-site motif of MntE and the most N-terminal His in the HD-HD site A of CzcD (the specified amino acids are underlined) play key roles in transporter metal selectivity. IMPORTANCE Cation diffusion facilitator (CDF) proteins are divalent metal ion transporters that are conserved in organisms ranging from bacteria to humans and that play important roles in cellular physiology, from metal homeostasis and resistance to type I diabetes in vertebrates

  15. Convective transport of highly plasma protein bound drugs facilitates direct penetration into deep tissues after topical application.

    PubMed

    Dancik, Yuri; Anissimov, Yuri G; Jepps, Owen G; Roberts, Michael S

    2012-04-01

    Many products are applied to human skin for local effects in deeper tissues. Animal studies suggest that deep dermal and/or subcutaneous delivery may be facilitated by both dermal diffusion and transport via the cutaneous vasculature. However, the relationship between the extent and pathways of penetration, drug physicochemical properties and deeper tissue physiology is not well understood. We have used a physiologically based pharmacokinetic model to analyze published human cutaneous microdialysis data, complemented by our own in vitro skin penetration studies. We found that convective blood, lymphatic and interstitial flow led to significant deep tissue concentrations for drugs that are highly plasma protein bound. In such cases, deeper tissue concentrations will occur earlier and may be several orders of magnitude greater than predicted by passive dermal diffusion alone. To relate the varying dermal, subcutaneous and muscle microdialysate concentrations found in man after topical application to the nature of the drug applied and to the underlying physiology. We developed a physiologically based pharmacokinetic model in which transport to deeper tissues was determined by tissue diffusion, blood, lymphatic and intersitial flow transport and drug properties. The model was applied to interpret published human microdialysis data, estimated in vitro dermal diffusion and protein binding affinity of drugs that have been previously applied topically in vivo and measured in deep cutaneous tissues over time. Deeper tissue microdialysis concentrations for various drugs in vivo vary widely. Here, we show that carriage by the blood to the deeper tissues below topical application sites facilitates the transport of highly plasma protein bound drugs that penetrate the skin, leading to rapid and significant concentrations in those tissues. Hence, the fractional concentration for the highly plasma protein bound diclofenac in deeper tissues is 0.79 times that in a probe 4.5

  16. Structural basis of transport function in major facilitator superfamily protein from Trichoderma harzianum.

    PubMed

    Chaudhary, Nitika; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

    2017-02-01

    Trichothecenes are the sesquiterpenes secreted by Trichoderma spp. residing in the rhizosphere. These compounds have been reported to act as plant growth promoters and bio-control agents. The structural knowledge for the transporter proteins of their efflux remained limited. In this study, three-dimensional structure of Thmfs1 protein, a trichothecene transporter from Trichoderma harzianum, was homology modelled and further Molecular Dynamics (MD) simulations were used to decipher its mechanism. Fourteen transmembrane helices of Thmfs1 protein are observed contributing to an inward-open conformation. The transport channel and ligand binding sites in Thmfs1 are identified based on heuristic, iterative algorithm and structural alignment with homologous proteins. MD simulations were performed to reveal the differential structural behaviour occurring in the ligand free and ligand bound forms. We found that two discrete trichothecene binding sites are located on either side of the central transport tunnel running from the cytoplasmic side to the extracellular side across the Thmfs1 protein. Detailed analysis of the MD trajectories showed an alternative access mechanism between N and C-terminal domains contributing to its function. These results also demonstrate that the transport of trichodermin occurs via hopping mechanism in which the substrate molecule jumps from one binding site to another lining the transport tunnel.

  17. Content variations of triterpenic acid, nucleoside, nucleobase, and sugar in jujube (Ziziphus jujuba) fruit during ripening.

    PubMed

    Guo, Sheng; Duan, Jin-Ao; Qian, Dawei; Tang, Yuping; Wu, Dawei; Su, Shulan; Wang, Hanqing; Zhao, Yunan

    2015-01-15

    Jujube (Ziziphus jujuba) fruit is widely consumed as food and traditional Chinese medicine in Asian countries due to its potential effects for human health. To facilitate selection of the maturity stage providing optimum health benefits, jujube fruits were analysed at six stages of growth (S1-6) for triterpenic acids, nucleosides, nucleobases, and sugars by UHPLC-MS/MS or HPLC-ELSD methods. The content levels of most triterpenic acids and sugars increased with ripening, and reached the highest at S5 and S6, respectively. The accumulation of the cyclic nucleotides (cAMP and cGMP) was mainly in the later stage of ripening (S5-6). Therefore, if taking triterpenic acids as the major quality indicator, S5 should be the ideal time to harvest jujube fruit, and the full ripen stage (S6) maybe the best choice when taking sugars and cyclic nucleotides as the most important components.

  18. Ghrelin Facilitates GLUT2-, SGLT1- and SGLT2-mediated Intestinal Glucose Transport in Goldfish (Carassius auratus)

    PubMed Central

    Blanco, Ayelén Melisa; Bertucci, Juan Ignacio; Ramesh, Naresh; Delgado, María Jesús; Valenciano, Ana Isabel; Unniappan, Suraj

    2017-01-01

    Glucose homeostasis is an important biological process that involves a variety of regulatory mechanisms. This study aimed to determine whether ghrelin, a multifunctional gut-brain hormone, modulates intestinal glucose transport in goldfish (Carassius auratus). Three intestinal glucose transporters, the facilitative glucose transporter 2 (GLUT2), and the sodium/glucose co-transporters 1 (SGLT1) and 2 (SGLT2), were studied. Immunostaining of intestinal sections found colocalization of ghrelin and GLUT2 and SGLT2 in mucosal cells. Some cells containing GLUT2, SGLT1 and SGLT2 coexpressed the ghrelin/growth hormone secretagogue receptor 1a (GHS-R1a). Intraperitoneal glucose administration led to a significant increase in serum ghrelin levels, as well as an upregulation of intestinal preproghrelin, ghrelin O-acyltransferase and ghs-r1 expression. In vivo and in vitro ghrelin treatment caused a concentration- and time-dependent modulation (mainly stimulatory) of GLUT2, SGLT1 and SGLT2. These effects were abolished by the GHS-R1a antagonist [D-Lys3]-GHRP-6 and the phospholipase C inhibitor U73122, suggesting that ghrelin actions on glucose transporters are mediated by GHS-R1a via the PLC/PKC signaling pathway. Finally, ghrelin stimulated the translocation of GLUT2 into the plasma membrane of goldfish primary intestinal cells. Overall, data reported here indicate an important role for ghrelin in the modulation of glucoregulatory machinery and glucose homeostasis in fish. PMID:28338019

  19. Proteome scale census of major facilitator superfamily transporters in Trichoderma reesei using protein sequence and structure based classification enhanced ranking.

    PubMed

    Chaudhary, Nitika; Kumari, Indu; Sandhu, Padmani; Ahmed, Mushtaq; Akhter, Yusuf

    2016-07-01

    Trichoderma spp. have been acknowledged as potent bio-control agents against microbial pathogens and also as plant growth promoters. Various secondary metabolites are attributed for these beneficial activities. Major facilitator superfamily (MFS) includes the large proportion of efflux-pumps which are linked with membrane transport of these secondary metabolites. We have carried out a proteome-wide identification of MFS transporters using protein sequence and structure based hierarchical method in Trichoderma reesei. 448 proteins out of 9115 were detected to carry transmembrane helices. MFS specific intragenic gene duplication and its context with transport function have been presented. Finally, using homology based techniques, domains and motifs of MFS families have been identified and utilized to classify them. From query dataset of 448 transmembrane proteins, 148 proteins are identified as potential MFS transporters. Sugar porter, drug: H(+) antiporter-1, monocarboxylate porter and anion: cation symporter emerged as major MFS families with 51, 35, 17 and 11 members respectively. Representative protein tertiary structures of these families are homology modeled for structure-function analysis. This study may help to understand the molecular basis of secretion and transport of agriculturally valuable secondary metabolites produced by these bio-control fungal agents which may be exploited in future for enhancing its biotechnological applications in eco-friendly sustainable development.

  20. Ghrelin Facilitates GLUT2-, SGLT1- and SGLT2-mediated Intestinal Glucose Transport in Goldfish (Carassius auratus).

    PubMed

    Blanco, Ayelén Melisa; Bertucci, Juan Ignacio; Ramesh, Naresh; Delgado, María Jesús; Valenciano, Ana Isabel; Unniappan, Suraj

    2017-03-24

    Glucose homeostasis is an important biological process that involves a variety of regulatory mechanisms. This study aimed to determine whether ghrelin, a multifunctional gut-brain hormone, modulates intestinal glucose transport in goldfish (Carassius auratus). Three intestinal glucose transporters, the facilitative glucose transporter 2 (GLUT2), and the sodium/glucose co-transporters 1 (SGLT1) and 2 (SGLT2), were studied. Immunostaining of intestinal sections found colocalization of ghrelin and GLUT2 and SGLT2 in mucosal cells. Some cells containing GLUT2, SGLT1 and SGLT2 coexpressed the ghrelin/growth hormone secretagogue receptor 1a (GHS-R1a). Intraperitoneal glucose administration led to a significant increase in serum ghrelin levels, as well as an upregulation of intestinal preproghrelin, ghrelin O-acyltransferase and ghs-r1 expression. In vivo and in vitro ghrelin treatment caused a concentration- and time-dependent modulation (mainly stimulatory) of GLUT2, SGLT1 and SGLT2. These effects were abolished by the GHS-R1a antagonist [D-Lys3]-GHRP-6 and the phospholipase C inhibitor U73122, suggesting that ghrelin actions on glucose transporters are mediated by GHS-R1a via the PLC/PKC signaling pathway. Finally, ghrelin stimulated the translocation of GLUT2 into the plasma membrane of goldfish primary intestinal cells. Overall, data reported here indicate an important role for ghrelin in the modulation of glucoregulatory machinery and glucose homeostasis in fish.

  1. Structural basis for the facilitative diffusion mechanism by SemiSWEET transporter

    NASA Astrophysics Data System (ADS)

    Lee, Yongchan; Nishizawa, Tomohiro; Yamashita, Keitaro; Ishitani, Ryuichiro; Nureki, Osamu

    2015-01-01

    SWEET family proteins mediate sugar transport across biological membranes and play crucial roles in plants and animals. The SWEETs and their bacterial homologues, the SemiSWEETs, are related to the PQ-loop family, which is characterized by highly conserved proline and glutamine residues (PQ-loop motif). Although the structures of the bacterial SemiSWEETs were recently reported, the conformational transition and the significance of the conserved motif in the transport cycle have remained elusive. Here we report crystal structures of SemiSWEET from Escherichia coli, in the both inward-open and outward-open states. A structural comparison revealed that SemiSWEET undergoes an intramolecular conformational change in each protomer. The conserved PQ-loop motif serves as a molecular hinge that enables the ‘binder clip-like’ motion of SemiSWEET. The present work provides the framework for understanding the overall transport cycles of SWEET and PQ-loop family proteins.

  2. Perfluorocarbon facilitated O(2) transport in a hepatic hollow fiber bioreactor.

    PubMed

    Chen, Guo; Palmer, Andre F

    2009-01-01

    A mathematical model describing O(2) transport in a hepatic hollow fiber (HF) bioreactor supplemented with perfluorocarbons (PFCs) in the circulating cell culture media was developed to explore the potential of PFCs in properly oxygenating a bioartificial liver assist device (BLAD). The 2-dimensional model is based on the geometry of a commercial HF bioreactor operated under steady-state conditions. The O(2) transport model considers fluid motion of a homogeneous mixture of cell culture media and PFCs, and mass transport of dissolved O(2) in a single HF. Each HF consists of three distinct regions: (1) the lumen (conducts the homogeneous mixture of cell culture media and PFCs), (2) the membrane (physically separates the lumen from the extracapillary space (ECS), and (3) the ECS (hepatic cells reside in this compartment). In a single HF, dissolved O(2) is predominantly transported in the lumen via convection in the axial direction and via diffusion in the radial direction through the membrane and ECS. The resulting transport equations are solved using the finite element method. The calculated O(2) transfer flux showed that supplementation of the cell culture media with PFCs can significantly enhance O(2) transport to the ECS of the HF when compared with a control with no PFC supplementation. Moreover, the O(2) distribution and subsequent analysis of ECS zonation demonstrate that limited in vivo-like O(2) gradients can be recapitulated with proper selection of the operational settings of the HF bioreactor. Taken together, this model can also be used to optimize the operating conditions for future BLAD development that aim to fully recapitulate the liver's varied functions. 2009 American Institute of Chemical Engineers Biotechnol.

  3. Facilitated transport of copper in bulk liquid membranes containing LIX 984N

    SciTech Connect

    Yang, X.J.; Fane, A.G.

    1999-06-01

    The liquid-liquid extraction of copper(II) using a novel extractant LIX 984N (a mixture of 5-nonylsalicylaldoxime and 2-hydroxy-5-nonyl-acetophenone oxime) was carried out. Following the liquid-liquid extraction, coupled uphill transport of copper(II) across a bulk liquid membrane containing LIX 984N as carrier was performed. The effects of aqueous stirring speed, carrier concentration, and inventory of membrane liquid were investigated. The rate-limiting step for the coupled transport of copper across a bulk liquid membrane was identified.

  4. New HYDRUS Modules for Simulating Preferential Flow, Colloid-Facilitated Contaminant Transport, and Various Biogeochemical Processes in Soils

    NASA Astrophysics Data System (ADS)

    Simunek, J.; Sejna, M.; Jacques, D.; Langergraber, G.; Bradford, S. A.; van Genuchten, M. Th.

    2012-04-01

    We have dramatically expanded the capabilities of the HYDRUS (2D/3D) software package by developing new modules to account for processes not available in the standard HYDRUS version. These new modules include the DualPerm, C-Hitch, HP2/3, Wetland, and Unsatchem modules. The dual-permeability modeling approach of Gerke and van Genuchten [1993] simulating preferential flow and transport is implemented into the DualPerm module. Colloid transport and colloid-facilitated solute transport, the latter often observed for many contaminants, such as heavy metals, radionuclides, pharmaceuticals, pesticides, and explosives [Šimůnek et al., 2006] are implemented into the C-Hitch module. HP2 and HP3 are the two and three-dimensional alternatives of the HP1 module, currently available with HYDRUS-1D [Jacques and Šimůnek, 2005], that couple HYDRUS flow and transport routines with the generic geochemical model PHREEQC of Parkhurst and Appelo [1999]. The Wetland module includes two alternative approaches (CW2D of Langergraber and Šimůnek [2005] and CWM1 of Langergraber et al. [2009]) for modeling aerobic, anaerobic, and anoxic biogeochemical processes in natural and constructed wetlands. Finally, the Unsatchem module simulates the transport and reactions of major ions in a soil profile. Brief descriptions and an application of each module will be presented. Except for HP3, all modules simulate flow and transport processes in two-dimensional transport domains. All modules are fully supported by the HYDRUS graphical user interface. Further development of these modules, as well as of several other new modules (such as Overland), is still envisioned. Continued feedback from the research community is encouraged.

  5. Undergraduate Laboratory Experiment Facilitating Active Learning of Concepts in Transport Phenomena: Experiment with a Subliming Solid

    ERIC Educational Resources Information Center

    Utgikar, Vivek P.

    2015-01-01

    An experiment based on the sublimation of a solid was introduced in the undergraduate Transport Phenomena course. The experiment required the students to devise their own apparatus and measurement techniques. The theoretical basis, assignment of the experiment, experimental results, and student/instructor observations are described in this paper.…

  6. An Active Learning Exercise to Facilitate Understanding of Nephron Function: Anatomy and Physiology of Renal Transporters

    ERIC Educational Resources Information Center

    Dirks-Naylor, Amie J.

    2016-01-01

    Renal transport is a central mechanism underlying electrolyte homeostasis, acid base balance and other essential functions of the kidneys in human physiology. Thus, knowledge of the anatomy and physiology of the nephron is essential for the understanding of kidney function in health and disease. However, students find this content difficult to…

  7. Undergraduate Laboratory Experiment Facilitating Active Learning of Concepts in Transport Phenomena: Experiment with a Subliming Solid

    ERIC Educational Resources Information Center

    Utgikar, Vivek P.

    2015-01-01

    An experiment based on the sublimation of a solid was introduced in the undergraduate Transport Phenomena course. The experiment required the students to devise their own apparatus and measurement techniques. The theoretical basis, assignment of the experiment, experimental results, and student/instructor observations are described in this paper.…

  8. An Active Learning Exercise to Facilitate Understanding of Nephron Function: Anatomy and Physiology of Renal Transporters

    ERIC Educational Resources Information Center

    Dirks-Naylor, Amie J.

    2016-01-01

    Renal transport is a central mechanism underlying electrolyte homeostasis, acid base balance and other essential functions of the kidneys in human physiology. Thus, knowledge of the anatomy and physiology of the nephron is essential for the understanding of kidney function in health and disease. However, students find this content difficult to…

  9. Local piezoresponse and polarization switching in nucleobase thymine microcrystals

    NASA Astrophysics Data System (ADS)

    Bdikin, Igor; Heredia, Alejandro; Neumayer, Sabine M.; Bystrov, Vladimir S.; Gracio, José; Rodriguez, Brian J.; Kholkin, Andrei L.

    2015-08-01

    Thymine (2-oxy-4-oxy-5 methyl pyrimidine) is one of the four nucleobases of deoxyribonucleic acid (DNA). In the DNA molecule, thymine binds to adenine via two hydrogen bonds, thus stabilizing the nucleic acid structure and is involved in pairing and replication. Here, we show that synthetic thymine microcrystals grown from the solution exhibit local piezoelectricity and apparent ferroelectricity, as evidenced by nanoscale electromechanical measurements via Piezoresponse Force Microscopy. Our experimental results demonstrate significant electromechanical activity and polarization switchability of thymine, thus opening a pathway for piezoelectric and ferroelectric-based applications of thymine and, perhaps, of other DNA nucleobase materials. The results are supported by molecular modeling of polarization switching under an external electric field.

  10. Toxin-Mediated Paracellular Transport of Antitoxin Antibodies Facilitates Protection against Clostridium difficile Infection

    PubMed Central

    Zhang, Z.; Chen, X.; Hernandez, L. D.; Lipari, P.; Flattery, A.; Chen, S.-C.; Kramer, S.; Polishook, J. D.; Racine, F.; Cape, H.; Kelly, C. P.

    2014-01-01

    The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile. Circulating neutralizing antitoxin antibodies are protective in C. difficile infection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralize toxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in active antibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevant human IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in hamsters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In an in vitro two-dimensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increased upon addition of toxin to the apical side. Similar data were obtained with F(ab′)2 fragments, which lack an Fc domain, consistent with FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damage caused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mechanism of humoral response-mediated protection against enteric pathogens. PMID:25385797

  11. Mucolipin 1 positively regulates TLR7 responses in dendritic cells by facilitating RNA transportation to lysosomes.

    PubMed

    Li, Xiaobing; Saitoh, Shin-Ichiroh; Shibata, Takuma; Tanimura, Natsuko; Fukui, Ryutaro; Miyake, Kensuke

    2015-02-01

    Toll-like receptor 7 (TLR7) and TLR9 sense microbial single-stranded RNA (ssRNA) and ssDNA in endolysosomes. Nucleic acid (NA)-sensing in endolysosomes is thought to be important for avoiding TLR7/9 responses to self-derived NAs. Aberrant self-derived NA transportation to endolysosomes predisposes to autoimmune diseases. To restrict NA-sensing in endolysosomes, TLR7/9 trafficking is tightly controlled by a multiple transmembrane protein Unc93B1. In contrast to TLR7/9 trafficking, little is known about a mechanism underlying NA transportation. We here show that Mucolipin 1 (Mcoln1), a member of the transient receptor potential (TRP) cation channel gene family, has an important role in ssRNA trafficking into lysosomes. Mcoln1(-/-) dendritic cells (DCs) showed impaired TLR7 responses to ssRNA. A mucolipin agonist specifically enhanced TLR7 responses to ssRNAs. The channel activity of Mcoln1 is activated by a phospholipid phosphatidylinositol (3,5) bisphosphate (PtdIns(3,5)P2), which is generated by a class III lipid kinase PIKfyve. A PIKfyve inhibitor completely inhibited TLR7 responses to ssRNA in DCs. Confocal analyses showed that ssRNA transportation to lysosomes in DCs was impaired by PIKfyve inhibitor as well as by the lack of Mcoln1. Transportation of TLR9 ligands was also impaired by the PIKfyve inhibitor. These results demonstrate that the PtdIns(3,5)P2-Mcoln1 axis has an important role in ssRNA transportation into lysosomes in DCs.

  12. Isolation and functional analysis of Thmfs1, the first major facilitator superfamily transporter from the biocontrol fungus Trichoderma harzianum.

    PubMed

    Liu, Mu; Liu, Jun; Wang, Wei Min

    2012-10-01

    A novel major facilitator superfamily (MFS) transporter gene, Thmfs1, was isolated from Trichoderma harzianum (T. harzianum). A Thmfs1 over-expressing mutant displayed enhanced antifungal activity and fungicide tolerance, while the Thmfs1 disruption mutant showed the opposite trend. Trichodermin production in Thmfs1 disruption group (185 mg l(-1)) was decreased by less than 17 % compared to the parental strain, suggesting that Thmfs1 is not mainly responsible for trichodermin secretion. Real-time PCR showed that Thmfs1 transcript level could be induced by a certain range of trichodermin concentrations, while expression of Tri5, encoding a trichodiene synthase, was strongly inhibited under these conditions. To our knowledge, Thmfs1 is the first MFS transporter gene identified in T. harzianum.

  13. Replacing the Nucleobases in DNA with Designer Molecules

    DTIC Science & Technology

    2007-11-02

    electrophiles or especially strong nucleophiles, and they generally do not change protonation near neutral pH. They offer (to the first approximation) only four...DNA bases can impart important biological activity, such as replacing the methyl group of thymine with fluorine , or the oxygen of guanine with sulfur...nucleobases that lack hydrogen- bonding functionality. The design involves replacing oxygen with fluorine and nitrogen with carbon, and keeping aromaticity

  14. T tubules and surface membranes provide equally effective pathways of carbonic anhydrase-facilitated lactic acid transport in skeletal muscle.

    PubMed

    Hallerdei, Janine; Scheibe, Renate J; Parkkila, Seppo; Waheed, Abdul; Sly, William S; Gros, Gerolf; Wetzel, Petra; Endeward, Volker

    2010-12-13

    We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV-CAIX-CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli.

  15. Two Major Facilitator Superfamily Sugar Transporters from Trichoderma reesei and Their Roles in Induction of Cellulase Biosynthesis*

    PubMed Central

    Zhang, Weixin; Kou, Yanbo; Xu, Jintao; Cao, Yanli; Zhao, Guolei; Shao, Jing; Wang, Hai; Wang, Zhixing; Bao, Xiaoming; Chen, Guanjun; Liu, Weifeng

    2013-01-01

    Proper perception of the extracellular insoluble cellulose is key to initiating the rapid synthesis of cellulases by cellulolytic Trichoderma reesei. Uptake of soluble oligosaccharides derived from cellulose hydrolysis represents a potential point of control in the induced cascade. In this study, we identified a major facilitator superfamily sugar transporter Stp1 capable of transporting cellobiose by reconstructing a cellobiose assimilation system in Saccharomyces cerevisiae. The absence of Stp1 in T. reesei resulted in differential cellulolytic response to Avicel versus cellobiose. Transcriptional profiling revealed a different expression profile in the Δstp1 strain from that of wild-type strain in response to Avicel and demonstrated that Stp1 somehow repressed induction of the bulk of major cellulase and hemicellulose genes. Two other putative major facilitator superfamily sugar transporters were, however, up-regulated in the profiling. Deletion of one of them identified Crt1 that was required for growth and enzymatic activity on cellulose or lactose, but was not required for growth or hemicellulase activity on xylan. The essential role of Crt1 in cellulase induction did not seem to rely on its transporting activity because the overall uptake of cellobiose or sophorose by T. reesei was not compromised in the absence of Crt1. Phylogenetic analysis revealed that orthologs of Crt1 exist in the genomes of many filamentous ascomycete fungi capable of degrading cellulose. These data thus shed new light on the mechanism by which T. reesei senses and transmits the cellulose signal and offers potential strategies for strain improvement. PMID:24085297

  16. T Tubules and Surface Membranes Provide Equally Effective Pathways of Carbonic Anhydrase-Facilitated Lactic Acid Transport in Skeletal Muscle

    PubMed Central

    Hallerdei, Janine; Scheibe, Renate J.; Parkkila, Seppo; Waheed, Abdul; Sly, William S.; Gros, Gerolf; Wetzel, Petra; Endeward, Volker

    2010-01-01

    We have studied lactic acid transport in the fast mouse extensor digitorum longus muscles (EDL) by intracellular and cell surface pH microelectrodes. The role of membrane-bound carbonic anhydrases (CA) of EDL in lactic acid transport was investigated by measuring lactate flux in muscles from wildtype, CAIV-, CAIX- and CAXIV-single ko, CAIV-CAXIV double ko and CAIV–CAIX–CAXIV-triple ko mice. This was complemented by immunocytochemical studies of the subcellular localization of CAIV, CAIX and CAXIV in mouse EDL. We find that CAXIV and CAIX single ko EDL exhibit markedly but not maximally reduced lactate fluxes, whereas triple ko and double ko EDL show maximal or near-maximal inhibition of CA-dependent lactate flux. Interpretation of the flux measurements in the light of the immunocytochemical results leads to the following conclusions. CAXIV, which is homogeneously distributed across the surface membrane of EDL fibers, facilitates lactic acid transport across this membrane. CAIX, which is associated only with T tubular membranes, facilitates lactic acid transport across the T tubule membrane. The removal of lactic acid from the lumen of T tubuli towards the interstitial space involves a CO2-HCO3- diffusional shuttle that is maintained cooperatively by CAIX within the T tubule and, besides CAXIV, by the CAIV, which is strategically located at the opening of the T tubules. The data suggest that about half the CA-dependent muscular lactate flux occurs across the surface membrane, while the other half occurs across the membranes of the T tubuli. PMID:21179203

  17. Three tapasin docking sites in TAP cooperate to facilitate transporter stabilization and heterodimerization

    PubMed Central

    Leonhardt, Ralf M.; Abrahimi, Parwiz; Mitchell, Susan M.; Cresswell, Peter

    2014-01-01

    The transporter associated with antigen processing (TAP) translocates peptide antigens into the lumen of the endoplasmic reticulum (ER) for loading onto major histocompatibility complex (MHC) class I molecules. MHC class I acquires its peptide cargo in the peptide loading complex (PLC), an oligomeric complex that the chaperone tapasin organizes by bridging TAP to MHC class I and recruiting accessory molecules such as ERp57 and calreticulin. Three tapasin binding sites on TAP have been described, two of which are located in the N-terminal domains (N domains) of TAP1 and TAP2. The third binding site is present in the core transmembrane domain (coreTMD) of TAP1 and is only used by the unassembled subunits. Tapasin is required to promote TAP stability, but through which binding site(s) it is acting is unknown. In particular the role of tapasin binding to the coreTMD of TAP1 single chains is mysterious as this interaction is lost upon TAP2 association. In this study, we map the respective binding site in TAP1 to the polar face of the amphipathic transmembrane helix TM9 and identify key residues that are essential to establish the interaction. We find that this interaction is dispensable for the peptide transport function but essential to achieve full stability of human TAP1. The interaction is also required for proper heterodimerization of the transporter. Based on similar results obtained using TAP mutants lacking tapasin binding to either N domain, we conclude that all three tapasin-binding sites in TAP cooperate to achieve high transporter stability and efficient heterodimerization. PMID:24501197

  18. Facilitated transporters mediate net efflux of amino acids to the fetus across the basal membrane of the placental syncytiotrophoblast

    PubMed Central

    Cleal, J K; Glazier, J D; Ntani, G; Crozier, S R; Day, P E; Harvey, N C; Robinson, S M; Cooper, C; Godfrey, K M; Hanson, M A; Lewis, R M

    2011-01-01

    Fetal growth depends on placental transfer of amino acids from maternal to fetal blood. The mechanisms of net amino acid efflux across the basal membrane (BM) of the placental syncytiotrophoblast to the fetus, although vital for amino acid transport, are poorly understood. We examined the hypothesis that facilitated diffusion by the amino acid transporters TAT1, LAT3 and LAT4 plays an important role in this process, with possible effects on fetal growth. Amino acid transfer was measured in isolated perfused human placental cotyledons (n= 5 per experiment) using techniques which distinguish between different transport processes. Placental TAT1, LAT3 and LAT4 proteins were measured, and mRNA expression levels (measured using real-time quantitative-PCR) were related to fetal and neonatal anthropometry and dual-energy X-ray absorptiometry measurements of neonatal lean mass in 102 Southampton Women's Survey (SWS) infants. Under conditions preventing transport by amino acid exchangers, all amino acids appearing in the fetal circulation were substrates of TAT1, LAT3 or LAT4. Western blots demonstrated the presence of TAT1, LAT3 and LAT4 in placental BM preparations. Placental TAT1 and LAT3 mRNA expression were positively associated with measures of fetal growth in SWS infants (P < 0.05). We provide evidence that the efflux transporters TAT1, LAT3 and LAT4 are present in the human placental BM, and may play an important role in the net efflux of amino acids to the fetus. Unlike other transporters they can increase fetal amino acid concentrations. Consistent with a role in placental amino acid transfer capacity and fetal growth TAT1 and LAT3 mRNA expression showed positive associations with infant size at birth. PMID:21224231

  19. Colloid-Facilitated Transport of Contaminants in Groundwater: Mobilization of Transuranic Radionuclides from Disposal Trenches by Natural Organic Matter

    NASA Astrophysics Data System (ADS)

    McCarthy, J. F.

    1998-03-01

    The role of natural organic matter (NOM) in enhancing the field-scale transport of transuranic radionuclides (TRU) in groundwater is used as a case study to illustrate the value of integrated laboratory and field approaches to understanding colloid-facilitated transport. Field observations provide evidence that TRU are mobilized and co-transported by NOM when hydrologic processes bring the groundwater in contact with waste buried in shallow trenches. This hypothesis receives further support from laboratory speciation studies and geochemical modeling. However, laboratory sorption studies indicate that the groundwater NOM should sorb to, and thus be retarded by the mineral surfaces of the formation. This issue is resolved through field studies of NOM transport. Discrepancies between laboratory predictions and field results reveal that the key process in NOM transport in natural-as opposed to model laboratory-systems is competitive adsorption among NOM subcomponents. Unlike laboratory studies of adsorption of NOM to clean mineral surfaces, surfaces in natural systems are coated with groundwater NOM, and binding sites are “passivated” with respect to further binding of the same NOM. The hypothesis that highly mobile NOM enhances TRU migration was tested by using lanthanides as field tracers to determine the extent of retardation of the TRU-NOM complex. The lanthanides, which have sorption and transport properties similar to TRU, migrated as NOM complexes without significant retardation over flow paths of 75-m. It is evident that assumptions inherent in many risk assessments for existing waste facilities, and performance assessments for future repositories, must begin to account for the role that even typically low levels of groundwater NOM plays in contaminant mobility.

  20. Tunneling holes in microparticles to facilitate the transport of lithium ions for high volumetric density batteries.

    PubMed

    Zhu, Jian; Ng, K Y Simon; Deng, Da

    2015-09-14

    Microscale materials generally have a higher tap density than that of random nanoparticles. Therefore, microparticles have been attracting much attention for application as high volumetric density electrodes for lithium ion batteries. However, microparticles have much longer electrolyte diffusion and Li-ion migration length and less accessibility to the electrolyte than that of nanoparticles. Therefore, it will be interesting to tunnel-holes in the high volumetric density microparticles to facilitate the reversible storage of lithium ions. Here, tunnel-like holes were generated in microparticles to dramatically increase the accessibility of the active materials to facilitate the lithium ion transfer. A plausible formation mechanism to explain the generation of tunnel-like holes was proposed based on time-course experiments and intensive characterization. Impressively, the as-prepared microbeads with tunnels demonstrated dramatically improved performance compared to the solid microbeads without tunnels in lithium ion storage. The microparticles with tunnels could achieve comparable electrochemical performances to those nanoparticles reported in the literature, suggesting that microparticles, properly tuned, could be promising candidates as negative electrodes for lithium-ion batteries and worthy of further studies. We also directly measured the volumetric density of the microparticles. We would like to highlight that a superior volumetric capacity of 514 mA h cm(-3) has been achieved. We hope to promote more frequent use of the unit mA h cm(-3) in addition to the conventional unit mA h g(-1) in the battery community.

  1. Chemical factors influencing colloid-facilitated transport of contaminants in porous media

    USGS Publications Warehouse

    Roy, Sujoy B.; Dzombak, David A.

    1997-01-01

    The effects of colloids on the transport of two strongly sorbing solutesa hydrophobic organic compound, phenanthrene, and a metal ion, Ni2+were studied in sand-packed laboratory columns under different pH and ionic strength conditions. Two types of column experiments were performed as follows:  (i) sorption/mobilization experiments where the contaminant was first sorbed in the column under conditions where no colloids were released and mobilized under conditions where colloids were released as a result of ionic strength reduction in the influent; and (ii) transport experiments where the contaminant, dissolved or sorbed on colloids, was injected into columns packed with a strongly sorbing porous medium. In the first type of experiment, contaminant mobilization was significant only when all releasable colloids were flushed from the column. In all other cases, although high colloid particle concentrations were encountered, there was no marked effect on total contaminant concentrations. In the second type of experiment, colloid deposition efficiencies were shown to control the enhancement of transport. The deposition efficiency was a function of the pH (for a high organic content sand) and of the contaminant concentration (for a charged species such as Ni2+).

  2. Structural basis for the facilitative diffusion mechanism by SemiSWEET transporter

    PubMed Central

    Lee, Yongchan; Nishizawa, Tomohiro; Yamashita, Keitaro; Ishitani, Ryuichiro; Nureki, Osamu

    2015-01-01

    SWEET family proteins mediate sugar transport across biological membranes and play crucial roles in plants and animals. The SWEETs and their bacterial homologues, the SemiSWEETs, are related to the PQ-loop family, which is characterized by highly conserved proline and glutamine residues (PQ-loop motif). Although the structures of the bacterial SemiSWEETs were recently reported, the conformational transition and the significance of the conserved motif in the transport cycle have remained elusive. Here we report crystal structures of SemiSWEET from Escherichia coli, in the both inward-open and outward-open states. A structural comparison revealed that SemiSWEET undergoes an intramolecular conformational change in each protomer. The conserved PQ-loop motif serves as a molecular hinge that enables the ‘binder clip-like’ motion of SemiSWEET. The present work provides the framework for understanding the overall transport cycles of SWEET and PQ-loop family proteins. PMID:25598322

  3. Mammalian glucose permease GLUT1 facilitates transport of arsenic trioxide and methylarsonous acid†

    PubMed Central

    Liu, Zijuan; Sanchez, Marco A.; Jiang, Xuan; Boles, Eckhard; Landfear, Scott M.; Rosen, Barry P.

    2006-01-01

    Arsenic exposure is associated with hypertension, diabetes and cancer. Some mammals methylate arsenic. Saccharomyces cerevisiae hexose permeases catalyze As(OH)3 uptake. Here we report that mammalian glucose transporter GLUT1 catalyzes As(OH)3 and CH3As(OH)2 uptake in yeast or in Xenopus laevis öocytes. Expression of GLUT1 in a yeast lacking other glucose transporters allows for growth on glucose. Yeast expressing yeast HXT1 or rat GLUT1 transport As(OH)3 and CH3As(OH)2. The Km of GLUT1 is to 1.2 mM for CH3As(OH)2, compared to a Km of 3 mM for glucose. Inhibition between glucose and CH3As(OH)2 is noncompetitive, suggesting differences between the translocation pathways of hexoses and arsenicals. Both human and rat GLUT1 catalyze uptake of both As(OH)3 and CH3As(OH)2 in öocytes. Thus GLUT1 may be a major pathway uptake of both inorganic and methylated arsenicals in erythrocytes or the epithelial cells of the blood-brain barrier, contributing to arsenic-related cardiovascular problems and neurotoxicity. PMID:17064664

  4. An efficient strategy for small-scale screening and production of archaeal membrane transport proteins in Escherichia coli.

    PubMed

    Ma, Pikyee; Varela, Filipa; Magoch, Malgorzata; Silva, Ana Rita; Rosário, Ana Lúcia; Brito, José; Oliveira, Tânia Filipa; Nogly, Przemyslaw; Pessanha, Miguel; Stelter, Meike; Kletzin, Arnulf; Henderson, Peter J F; Archer, Margarida

    2013-01-01

    Membrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional characterisation of membrane proteins still remains a challenge, partially due to the difficulties in recombinant expression and purification. Therefore the need for development of efficient methods for heterologous production is essential. Fifteen integral membrane transport proteins from Archaea were selected as test targets, chosen to represent two superfamilies widespread in all organisms known as the Major Facilitator Superfamily (MFS) and the 5-Helix Inverted Repeat Transporter superfamily (5HIRT). These proteins typically have eleven to twelve predicted transmembrane helices and are putative transporters for sugar, metabolite, nucleobase, vitamin or neurotransmitter. They include a wide range of examples from the following families: Metabolite-H(+)-symporter; Sugar Porter; Nucleobase-Cation-Symporter-1; Nucleobase-Cation-Symporter-2; and neurotransmitter-sodium-symporter. Overproduction of transporters was evaluated with three vectors (pTTQ18, pET52b, pWarf) and two Escherichia coli strains (BL21 Star and C43 (DE3)). Thirteen transporter genes were successfully expressed; only two did not express in any of the tested vector-strain combinations. Initial trials showed that seven transporters could be purified and six of these yielded quantities of ≥ 0.4 mg per litre suitable for functional and structural studies. Size-exclusion chromatography confirmed that two purified transporters were almost homogeneous while four others were shown to be non-aggregating, indicating that they are ready for up-scale production and crystallisation trials. Here, we describe an efficient strategy for heterologous production of membrane transport proteins in E. coli. Small-volume cultures (10 mL) produced sufficient

  5. Effects of oil dispersants on settling of marine sediment particles and particle-facilitated distribution and transport of oil components.

    PubMed

    Cai, Zhengqing; Fu, Jie; Liu, Wen; Fu, Kunming; O'Reilly, S E; Zhao, Dongye

    2017-01-15

    This work investigated effects of three model oil dispersants (Corexit EC9527A, Corexit EC9500A and SPC1000) on settling of fine sediment particles and particle-facilitated distribution and transport of oil components in sediment-seawater systems. All three dispersants enhanced settling of sediment particles. The nonionic surfactants (Tween 80 and Tween 85) play key roles in promoting particle aggregation. Yet, the effects varied with environmental factors (pH, salinity, DOM, and temperature). Strongest dispersant effect was observed at neutral or alkaline pH and in salinity range of 0-3.5wt%. The presence of water accommodated oil and dispersed oil accelerated settling of the particles. Total petroleum hydrocarbons in the sediment phase were increased from 6.9% to 90.1% in the presence of Corexit EC9527A, and from 11.4% to 86.7% for PAHs. The information is useful for understanding roles of oil dispersants in formation of oil-sediment aggregates and in sediment-facilitated transport of oil and PAHs in marine eco-systems.

  6. Bacillus cereus efflux protein BC3310 - a multidrug transporter of the unknown major facilitator family, UMF-2.

    PubMed

    Kroeger, Jasmin K; Hassan, Karl; Vörös, Aniko; Simm, Roger; Saidijam, Massoud; Bettaney, Kim E; Bechthold, Andreas; Paulsen, Ian T; Henderson, Peter J F; Kolstø, Anne-Brit

    2015-01-01

    Phylogenetic classification divides the major facilitator superfamily (MFS) into 82 families, including 25 families that are comprised of transporters with no characterized functions. This study describes functional data for BC3310 from Bacillus cereus ATCC 14579, a member of the "unknown major facilitator family-2" (UMF-2). BC3310 was shown to be a multidrug efflux pump conferring resistance to ethidium bromide, SDS and silver nitrate when heterologously expressed in Escherichia coli DH5α ΔacrAB. A conserved aspartate residue (D105) in putative transmembrane helix 4 was identified, which was essential for the energy dependent ethidium bromide efflux by BC3310. Transport proteins of the MFS comprise specific sequence motifs. Sequence analysis of UMF-2 proteins revealed that they carry a variant of the MFS motif A, which may be used as a marker to distinguish easily between this family and other MFS proteins. Genes orthologous to bc3310 are highly conserved within the B. cereus group of organisms and thus belong to the core genome, suggesting an important conserved functional role in the normal physiology of these bacteria.

  7. Bacillus cereus efflux protein BC3310 – a multidrug transporter of the unknown major facilitator family, UMF-2

    PubMed Central

    Kroeger, Jasmin K.; Hassan, Karl; Vörös, Aniko; Simm, Roger; Saidijam, Massoud; Bettaney, Kim E.; Bechthold, Andreas; Paulsen, Ian T.; Henderson, Peter J. F.; Kolstø, Anne-Brit

    2015-01-01

    Phylogenetic classification divides the major facilitator superfamily (MFS) into 82 families, including 25 families that are comprised of transporters with no characterized functions. This study describes functional data for BC3310 from Bacillus cereus ATCC 14579, a member of the “unknown major facilitator family-2” (UMF-2). BC3310 was shown to be a multidrug efflux pump conferring resistance to ethidium bromide, SDS and silver nitrate when heterologously expressed in Escherichia coli DH5α ΔacrAB. A conserved aspartate residue (D105) in putative transmembrane helix 4 was identified, which was essential for the energy dependent ethidium bromide efflux by BC3310. Transport proteins of the MFS comprise specific sequence motifs. Sequence analysis of UMF-2 proteins revealed that they carry a variant of the MFS motif A, which may be used as a marker to distinguish easily between this family and other MFS proteins. Genes orthologous to bc3310 are highly conserved within the B. cereus group of organisms and thus belong to the core genome, suggesting an important conserved functional role in the normal physiology of these bacteria. PMID:26528249

  8. Tunneling holes in microparticles to facilitate the transport of lithium ions for high volumetric density batteries

    NASA Astrophysics Data System (ADS)

    Zhu, Jian; Ng, K. Y. Simon; Deng, Da

    2015-08-01

    Microscale materials generally have a higher tap density than that of random nanoparticles. Therefore, microparticles have been attracting much attention for application as high volumetric density electrodes for lithium ion batteries. However, microparticles have much longer electrolyte diffusion and Li-ion migration length and less accessibility to the electrolyte than that of nanoparticles. Therefore, it will be interesting to tunnel-holes in the high volumetric density microparticles to facilitate the reversible storage of lithium ions. Here, tunnel-like holes were generated in microparticles to dramatically increase the accessibility of the active materials to facilitate the lithium ion transfer. A plausible formation mechanism to explain the generation of tunnel-like holes was proposed based on time-course experiments and intensive characterization. Impressively, the as-prepared microbeads with tunnels demonstrated dramatically improved performance compared to the solid microbeads without tunnels in lithium ion storage. The microparticles with tunnels could achieve comparable electrochemical performances to those nanoparticles reported in the literature, suggesting that microparticles, properly tuned, could be promising candidates as negative electrodes for lithium-ion batteries and worthy of further studies. We also directly measured the volumetric density of the microparticles. We would like to highlight that a superior volumetric capacity of 514 mA h cm-3 has been achieved. We hope to promote more frequent use of the unit mA h cm-3 in addition to the conventional unit mA h g-1 in the battery community.Microscale materials generally have a higher tap density than that of random nanoparticles. Therefore, microparticles have been attracting much attention for application as high volumetric density electrodes for lithium ion batteries. However, microparticles have much longer electrolyte diffusion and Li-ion migration length and less accessibility to the

  9. Diffusion or bulk flow: how plasmodesmata facilitate pre-phloem transport of assimilates.

    PubMed

    Schulz, Alexander

    2015-01-01

    Assimilates synthesized in the mesophyll of mature leaves move along the pre-phloem transport pathway to the bundle sheath of the minor veins from which they are loaded into the phloem. The present review discusses the most probable driving force(s) for the pre-phloem pathway, diffusion down the concentration gradient or bulk flow along a pressure gradient. The driving force seems to depend on the mode of phloem loading. In a majority of plant species phloem loading is a thermodynamically active process, involving the activity of membrane transporters in the sieve-element companion cell complex. Since assimilate movement includes an apoplasmic step, this mode is called apoplasmic loading. Well established is also the polymer-trap loading mode, where the phloem-transport sugars are raffinose-family oligomers in herbaceous plants. Also this mode depends on the investment of energy, here for sugar oligomerization, and leads to a high sugar accumulation in the phloem, even though the phloem is not symplasmically isolated, but well coupled by plasmodesmata (PD). Hence the mode polymer-trap mode is also designated active symplasmic loading. For woody angiosperms and gymnosperms an alternate loading mode is currently matter of discussion, called passive symplasmic loading. Based on the limited material available, this review compares the different loading modes and suggests that diffusion is the driving force in apoplasmic loaders, while bulk flow plays an increasing role in plants having a continuous symplasmic pathway from mesophyll to sieve elements. Crucial for the driving force is the question where water enters the pre-phloem pathway. Surprisingly, the role of PD in water movement has not been addressed so far appropriately. Modeling of assimilate and water fluxes indicates that in symplasmic loaders a considerable part of water flux happens through the PD between bundle sheath and phloem.

  10. Nitrate facilitates cadmium uptake, transport and accumulation in the hyperaccumulator Sedum plumbizincicola.

    PubMed

    Hu, Pengjie; Yin, Yong-Gen; Ishikawa, Satoru; Suzui, Nobuo; Kawachi, Naoki; Fujimaki, Shu; Igura, Masato; Yuan, Cheng; Huang, Jiexue; Li, Zhu; Makino, Tomoyuki; Luo, Yongming; Christie, Peter; Wu, Longhua

    2013-09-01

    The aims of this study are to investigate whether and how the nitrogen form (nitrate (NO3 (-)) versus ammonium (NH4 (+))) influences cadmium (Cd) uptake and translocation and subsequent Cd phytoextraction by the hyperaccumulator species Sedum plumbizincicola. Plants were grown hydroponically with N supplied as either NO3 (-) or NH4 (+). Short-term (36 h) Cd uptake and translocation were determined innovatively and quantitatively using a positron-emitting (107)Cd tracer and positron-emitting tracer imaging system. The results show that the rates of Cd uptake by roots and transport to the shoots in the NO3 (-) treatment were more rapid than in the NH4 (+) treatment. After uptake for 36 h, 5.6 (0.056 μM) and 29.0 % (0.290 μM) of total Cd in the solution was non-absorbable in the NO3 (-) and NH4 (+) treatments, respectively. The local velocity of Cd transport was approximately 1.5-fold higher in roots (3.30 cm h(-1)) and 3.7-fold higher in shoots (10.10 cm h(-1)) of NO3 (-)- than NH4 (+)-fed plants. Autoradiographic analysis of (109)Cd reveals that NO3 (-) nutrition enhanced Cd transportation from the main stem to branches and young leaves. Moreover, NO3 (-) treatment increased Cd, Ca and K concentrations but inhibited Fe and P in the xylem sap. In a 21-day hydroponic culture, shoot biomass and Cd concentration were 1.51 and 2.63 times higher in NO3 (-)- than in NH4 (+)-fed plants. We conclude that compared with NH4 (+), NO3 (-) promoted the major steps in the transport route followed by Cd from solution to shoots in S. plumbizincicola, namely its uptake by roots, xylem loading, root-to-shoot translocation in the xylem and uploading to the leaves. S. plumbizincicola prefers NO3 (-) nutrition to NH4 (+) for Cd phytoextraction.

  11. Paraoxonase 2 Facilitates Pancreatic Cancer Growth and Metastasis by Stimulating GLUT1-Mediated Glucose Transport.

    PubMed

    Nagarajan, Arvindhan; Dogra, Shaillay Kumar; Sun, Lisha; Gandotra, Neeru; Ho, Thuy; Cai, Guoping; Cline, Gary; Kumar, Priti; Cowles, Robert A; Wajapeyee, Narendra

    2017-08-17

    Metabolic deregulation is a hallmark of human cancers, and the glycolytic and glutamine metabolism pathways were shown to be deregulated in pancreatic ductal adenocarcinoma (PDAC). To identify new metabolic regulators of PDAC tumor growth and metastasis, we systematically knocked down metabolic genes that were overexpressed in human PDAC tumor samples using short hairpin RNAs. We found that p53 transcriptionally represses paraoxonase 2 (PON2), which regulates GLUT1-mediated glucose transport via stomatin. The loss of PON2 initiates the cellular starvation response and activates AMP-activated protein kinase (AMPK). In turn, AMPK activates FOXO3A and its transcriptional target, PUMA, which induces anoikis to suppress PDAC tumor growth and metastasis. Pharmacological or genetic activation of AMPK, similar to PON2 inhibition, blocks PDAC tumor growth. Collectively, our results identify PON2 as a new modulator of glucose transport that regulates a pharmacologically tractable pathway necessary for PDAC tumor growth and metastasis. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Cloning, expression, and localization of a chloride-facilitated, cocaine-sensitive serotonin transporter from Drosophila melanogaster.

    PubMed

    Demchyshyn, L L; Pristupa, Z B; Sugamori, K S; Barker, E L; Blakely, R D; Wolfgang, W J; Forte, M A; Niznik, H B

    1994-05-24

    We report here on the isolation and characterization of a serotonin (5HT) transporter from Drosophila melanogaster. A 3.1-kb complementary DNA clone (dSERT) was found to encode a protein of 622 amino acid residues with a predicted molecular mass of approximately 69 kDa and a putative transmembrane topology characteristic of cloned members of the mammalian Na+/Cl- neurotransmitter cotransporter gene family. dSERT displays highest overall amino acid sequence identity with the mammalian 5HT (51%), norepinephrine (47%), and dopamine (47%) transporters and shares with all transporters 104 absolutely conserved amino acid residues. Upon transient expression in HeLa cells, dSERT exhibited saturable, high-affinity, and sodium-dependent [3H]5HT uptake with estimated Km and Vmax values of approximately 500 nM and 5.2 x 10(-18) mol per cell per min, respectively. In marked contrast to the human SERT (hSERT), 5HT-mediated transport by dSERT was not absolutely dependent on extracellular Cl-, while the sodium-dependent uptake of 5HT was facilitated by increased extracellular Cl- concentrations. dSERT displays a pharmacological profile and rank order of potency consistent with, but not identical to, mammalian 5HT transporters. Comparison of the affinities of various compounds for the inhibition of 5HT transport by both dSERT and hSERT revealed that antidepressants were 3- to 300-fold less potent on dSERT than on hSERT, while mazindol displayed approximately 30-fold greater potency for dSERT. Both cocaine and RTI-55 inhibited 5HT uptake by dSERT with estimated inhibition constants of approximately 500 nM, while high concentrations (> 10 microM) of dopamine, norepinephrine, octopamine, tyramine, and histamine failed to inhibit transport. In situ hybridization reveals the selective expression of dSERT mRNA to specific cell bodies in the ventral ganglion of the embryonic and larval Drosophila nervous system with a distribution pattern virtually identical to that of 5HT

  13. Mrt, a gene unique to fungi, encodes an oligosaccharide transporter and facilitates rhizosphere competency in Metarhizium robertsii.

    PubMed

    Fang, Weiguo; St Leger, Raymond J

    2010-11-01

    The symbiotic associations between rhizospheric fungi and plants have enormous environmental impact. Fungi are crucial to plant health as antagonists of pathogens and herbivores and facilitate the uptake of soil nutrients. However, little is known about the plant products obtained by fungi in exchange or how they are transported through the symbiotic interface. Here, we demonstrate that sucrose and raffinose family oligosaccharides in root exudates are important for rhizosphere competence in the insect pathogen Metarhizium robertsii (formerly known as Metarhizium anisopliae). We identified mutants in the Metarhizium raffinose transporter (Mrt) gene of M. robertsii that grew poorly in root exudate and were greatly reduced in rhizosphere competence on grass roots. Studies on sugar uptake, including competition assays, revealed that MRT was a sucrose and galactoside transporter. Disrupting MRT resulted in greatly reduced or no growth on sucrose and galactosides but did not affect growth on monosaccharides or oligosaccharides composed entirely of glucose subunits. Consistent with this, expression of Mrt is exclusively up-regulated by galactosides and sucrose. Expressing a green fluorescent protein gene under the control of the Mrt promoter confirmed that MRT was expressed by germlings in the vicinity of grass roots but not in surrounding bulk soil. Disrupting Mrt did not reduce virulence to insects, demonstrating that Mrt is exclusively involved in M. robertsii's interactions with plants. To our knowledge, MRT is the first oligosaccharide transporter identified and characterized in a fungus and is unique to filamentous fungi, but homologous genes in Magnaporthe, Ustilago, Aspergillus, Fusarium, Epichloe, and Penicillium species indicate that oligosaccharide transport is of widespread significance.

  14. Facilitating guest transport in clathrate hydrates by tuning guest-host interactions

    SciTech Connect

    Moudrakovski, Igor L.; Udachin, Konstantin A.; Ratcliffe, Christopher I.; Alavi, Saman; Ripmeester, John A.

    2015-02-21

    The understanding and eventual control of guest molecule transport in gas hydrates is of central importance for the efficient synthesis and processing of these materials for applications in the storage, separation, and sequestration of gases and natural gas production. Previously, some links have been established between dynamics of the host water molecules and guest-host hydrogen bonding interactions, but direct observation of transport in the form of cage-to-cage guest diffusion is still lacking. Recent calculations have suggested that pairs of different guest molecules in neighboring cages can affect guest-host hydrogen bonding and, therefore, defect injection and water lattice motions. We have chosen two sets of hydrate guest pairs, tetrahydrofuran (THF)-CO{sub 2} and isobutane-CO{sub 2}, that are predicted to enhance or to diminish guest–host hydrogen bonding interactions as compared to those in pure CO{sub 2} hydrate and we have studied guest dynamics in each using {sup 13}C nuclear magnetic resonance (NMR) methods. In addition, we have obtained the crystal structure of the THF-CO{sub 2} sII hydrate using the combined single crystal X-ray diffraction and {sup 13}C NMR powder pattern data and have performed molecular dynamics-simulation of the CO{sub 2} dynamics. The NMR powder line shape studies confirm the enhanced and delayed dynamics for the THF and isobutane containing hydrates, respectively, as compared to those in the CO{sub 2} hydrate. In addition, from line shape studies and 2D exchange spectroscopy NMR, we observe cage-to-cage exchange of CO{sub 2} molecules in the THF-CO{sub 2} hydrate, but not in the other hydrates studied. We conclude that the relatively rapid intercage guest dynamics are the result of synergistic guest A–host water–guest B interactions, thus allowing tuning of the guest transport properties in the hydrates by choice of the appropriate guest molecules. Our experimental value for inter-cage hopping is slower by a factor of 10

  15. Facilitating guest transport in clathrate hydrates by tuning guest-host interactions.

    PubMed

    Moudrakovski, Igor L; Udachin, Konstantin A; Alavi, Saman; Ratcliffe, Christopher I; Ripmeester, John A

    2015-02-21

    The understanding and eventual control of guest molecule transport in gas hydrates is of central importance for the efficient synthesis and processing of these materials for applications in the storage, separation, and sequestration of gases and natural gas production. Previously, some links have been established between dynamics of the host water molecules and guest-host hydrogen bonding interactions, but direct observation of transport in the form of cage-to-cage guest diffusion is still lacking. Recent calculations have suggested that pairs of different guest molecules in neighboring cages can affect guest-host hydrogen bonding and, therefore, defect injection and water lattice motions. We have chosen two sets of hydrate guest pairs, tetrahydrofuran (THF)-CO2 and isobutane-CO2, that are predicted to enhance or to diminish guest-host hydrogen bonding interactions as compared to those in pure CO2 hydrate and we have studied guest dynamics in each using (13)C nuclear magnetic resonance (NMR) methods. In addition, we have obtained the crystal structure of the THF-CO2 sII hydrate using the combined single crystal X-ray diffraction and (13)C NMR powder pattern data and have performed molecular dynamics-simulation of the CO2 dynamics. The NMR powder line shape studies confirm the enhanced and delayed dynamics for the THF and isobutane containing hydrates, respectively, as compared to those in the CO2 hydrate. In addition, from line shape studies and 2D exchange spectroscopy NMR, we observe cage-to-cage exchange of CO2 molecules in the THF-CO2 hydrate, but not in the other hydrates studied. We conclude that the relatively rapid intercage guest dynamics are the result of synergistic guest A-host water-guest B interactions, thus allowing tuning of the guest transport properties in the hydrates by choice of the appropriate guest molecules. Our experimental value for inter-cage hopping is slower by a factor of 10(6) than a published calculated value.

  16. The role of organic complexants and microparticulates in the facilitated transport of radionuclides

    SciTech Connect

    Schilk, A.J.; Robertson, D.E.; Abel, K.H.; Thomas, C.W.

    1996-12-01

    This progress report describes the results of ongoing radiological and geochemical investigations of the mechanisms of radionuclide transport in groundwater at two low-level waste (LLW) disposal sites within the waste management area of the Chalk River Laboratories (CRL), Ontario, Canada. These sites, the Chemical Pit liquid disposal facility and the Waste Management Area C solid LLW disposal site, have provided valuable 30- to 40-year-old field locations for characterizing the migration of radionuclides and evaluating a number of recent site performance objectives for LLW disposal facilities. This information will aid the NRC and other federal, state, and local regulators, as well as LLW disposal site developers and waste generators, in maximizing the effectiveness of existing or projected LLW disposal facilities for isolating radionuclides from the general public and thereby improving the health and safety aspects of LLW disposal.

  17. Specific aquaporins facilitate Nox-produced hydrogen peroxide transport through plasma membrane in leukaemia cells.

    PubMed

    Vieceli Dalla Sega, Francesco; Zambonin, Laura; Fiorentini, Diana; Rizzo, Benedetta; Caliceti, Cristiana; Landi, Laura; Hrelia, Silvana; Prata, Cecilia

    2014-04-01

    In the last decade, the generation and the role of reactive oxygen species (ROS), particularly hydrogen peroxide, in cell signalling transduction pathways have been intensively studied, and it is now clear that an increase of ROS level affects cellular growth and proliferation pathways related to cancer development. Hydrogen peroxide (H2O2) has been long thought to permeate biological membranes by simple diffusion since recent evidence challenged this notion disclosing the role of aquaporin water channels (AQP) in mediating H2O2 transport across plasma membranes. We previously demonstrated that NAD(P)H oxidase (Nox)-generated ROS sustain glucose uptake and cellular proliferation in leukaemia cells. The aim of this study was to assess whether specific AQP isoforms can channel Nox-produced H2O2 across the plasma membrane of leukaemia cells affecting downstream pathways linked to cell proliferation. In this work, we demonstrate that AQP inhibition caused a decrease in intracellular ROS accumulation in leukaemia cells both when H2O2 was produced by Nox enzymes and when it was exogenously added. Furthermore, AQP8 overexpression or silencing resulted to modulate VEGF capacity of triggering an H2O2 intracellular level increase or decrease, respectively. Finally, we report that AQP8 is capable of increasing H2O2-induced phosphorylation of both PI3K and p38 MAPK and that AQP8 expression affected positively cell proliferation. Taken together, the results here reported indicate that AQP8 is able to modulate H2O2 transport through the plasma membrane affecting redox signalling linked to leukaemia cell proliferation. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Uranium facilitated transport by water-dispersible colloids in field and soil columns.

    PubMed

    Crançon, P; Pili, E; Charlet, L

    2010-04-01

    The transport of uranium through a sandy podzolic soil has been investigated in the field and in column experiments. Field monitoring, numerous years after surface contamination by depleted uranium deposits, revealed a 20 cm deep uranium migration in soil. Uranium retention in soil is controlled by the <50 microm mixed humic and clayey coatings in the first 40 cm i.e. in the E horizon. Column experiments of uranium transport under various conditions were run using isotopic spiking. After 100 pore volumes elution, 60% of the total input uranium is retained in the first 2 cm of the column. Retardation factor of uranium on E horizon material ranges from 1300 (column) to 3000 (batch). In parallel to this slow uranium migration, we experimentally observed a fast elution related to humic colloids of about 1-5% of the total-uranium input, transferred at the mean porewater velocity through the soil column. In order to understand the effect of rain events, ionic strength of the input solution was sharply changed. Humic colloids are retarded when ionic strength increases, while a major mobilization of humic colloids and colloid-borne uranium occurs as ionic strength decreases. Isotopic spiking shows that both (238)U initially present in the soil column and (233)U brought by input solution are desorbed. The mobilization process observed experimentally after a drop of ionic strength may account for a rapid uranium migration in the field after a rainfall event, and for the significant uranium concentrations found in deep soil horizons and in groundwater, 1 km downstream from the pollution source.

  19. Long-range transport of hydrothermal iron facilitated by dissolved-particulate exchange

    NASA Astrophysics Data System (ADS)

    Forsch, K.; Fitzsimmons, J. N.; German, C. R.; Sherrell, R. M.

    2016-02-01

    Deep-sea hydrothermal venting has a strong impact on ocean chemistry close to vent sites; however, effects of these systems on basin- and global-scale ocean biogeochemistry have only started to be observed and modeled. The U.S. GEOTRACES Eastern Pacific Zonal Transect investigated potential long-range hydrothermal influences by sampling the length of the 15°S Southern East Pacific Rise (EPR) plume as far as 150°W, following a plume extending >4000 km from the EPR based on He-3 distributions and the distribution of metal-rich sediments. It was recently reported that dissolved iron (dFe) in this plume is apparently transported conservatively >4000km across the South Pacific, with potential for ultimate upwelling in the Fe-limited Southern Ocean. The mechanism by which this dissolved Fe is maintained in solution, however, remains a topic of active debate. Here we show that hydrothermal Fe in the particulate phase, which exceeds the dissolved Fe concentration over much of the plume, is also enriched relative to background across the same 4300 km transect but that maximum pFe concentrations both decrease non-conservatively and deepen progressively relative to dissolved 3He anomalies across the section. Stokesian settling would require an FeOOH particle size of 0.5 µm in order to explain the 500 m "sag" observed over the 50 y of plume transport. A striking feature of the dissolved Fe plume is that it, too, descends in the water column over the length of the plume, following the particulate Fe plume. This suggests an active exchange between dissolved and particulate Fe species on timescales that are short relative to plume advection across the South Pacific. Thus, the large-scale distribution of particulate Fe appears to influence the preservation of the dFe pool, suggesting that the basin-scale impact of hydrothermal vent systems needs to consider both Fe phases and their long-term interactions.

  20. Facilitation of Drug Transport across the Blood–Brain Barrier with Ultrasound and Microbubbles

    PubMed Central

    Meairs, Stephen

    2015-01-01

    Medical treatment options for central nervous system (CNS) diseases are limited due to the inability of most therapeutic agents to penetrate the blood–brain barrier (BBB). Although a variety of approaches have been investigated to open the BBB for facilitation of drug delivery, none has achieved clinical applicability. Mounting evidence suggests that ultrasound in combination with microbubbles might be useful for delivery of drugs to the brain through transient opening of the BBB. This technique offers a unique non-invasive avenue to deliver a wide range of drugs to the brain and promises to provide treatments for CNS disorders with the advantage of being able to target specific brain regions without unnecessary drug exposure. If this method could be applied for a range of different drugs, new CNS therapeutic strategies could emerge at an accelerated pace that is not currently possible in the field of drug discovery and development. This article reviews both the merits and potential risks of this new approach. It assesses methods used to verify disruption of the BBB with MRI and examines the results of studies aimed at elucidating the mechanisms of opening the BBB with ultrasound and microbubbles. Possible interactions of this novel delivery method with brain disease, as well as safety aspects of BBB disruption with ultrasound and microbubbles are addressed. Initial translational research for treatment of brain tumors and Alzheimer’s disease is presented. PMID:26404357

  1. Osteopontin facilitates West Nile virus neuroinvasion via neutrophil "Trojan horse" transport.

    PubMed

    Paul, Amber M; Acharya, Dhiraj; Duty, Laurel; Thompson, E Ashley; Le, Linda; Stokic, Dobrivoje S; Leis, A Arturo; Bai, Fengwei

    2017-07-05

    West Nile virus (WNV) can cause severe human neurological diseases including encephalitis and meningitis. The mechanisms by which WNV enters the central nervous system (CNS) and host-factors that are involved in WNV neuroinvasion are not completely understood. The proinflammatory chemokine osteopontin (OPN) is induced in multiple neuroinflammatory diseases and is responsible for leukocyte recruitment to sites of its expression. In this study, we found that WNV infection induced OPN expression in both human and mouse cells. Interestingly, WNV-infected OPN deficient (Opn (-/-)) mice exhibited a higher survival rate (70%) than wild type (WT) control mice (30%), suggesting OPN plays a deleterious role in WNV infection. Despite comparable levels of viral load in circulating blood cells and peripheral organs in the two groups, WNV-infected polymorphonuclear neutrophil (PMN) infiltration and viral burden in brain of Opn (-/-) mice were significantly lower than in WT mice. Importantly, intracerebral administration of recombinant OPN into the brains of Opn (-/-) mice resulted in increased WNV-infected PMN infiltration and viral burden in the brain, which was coupled to increased mortality. The overall results suggest that OPN facilitates WNV neuroinvasion by recruiting WNV-infected PMNs into the brain.

  2. The major facilitator superfamily transporter Knq1p modulates boron homeostasis in Kluyveromyces lactis.

    PubMed

    Svrbicka, Alexandra; Toth Hervay, Nora; Gbelska, Yvetta

    2016-03-01

    Boron is an essential micronutrient for living cells, yet its excess causes toxicity. To date, the mechanisms of boron toxicity are poorly understood. Recently, the ScATR1 gene has been identified encoding the main boron efflux pump in Saccharomyces cerevisiae. In this study, we analyzed the ScATR1 ortholog in Kluyveromyces lactis--the KNQ1 gene, to understand whether it participates in boron stress tolerance. We found that the KNQ1 gene, encoding a permease belonging to the major facilitator superfamily, is required for K. lactis boron tolerance. Deletion of the KNQ1 gene led to boron sensitivity and its overexpression increased K. lactis boron tolerance. The KNQ1 expression was induced by boron and the intracellular boron concentration was controlled by Knq1p. The KNQ1 promoter contains two putative binding motifs for the AP-1-like transcription factor KlYap1p playing a central role in oxidative stress defense. Our results indicate that the induction of the KNQ1 expression requires the presence of KlYap1p and that Knq1p like its ortholog ScAtr1p in S. cerevisiae functions as a boron efflux pump providing boron resistance in K. lactis.

  3. Particle-facilitated lead and arsenic transport in abandoned mine sites soil influenced by simulated acid rain.

    PubMed

    Shaoping, Hu; Xincai, Chen; Jiyan, Shi; Yingxu, Chen; Qi, Lin

    2008-05-01

    The role of acid rain in affecting Pb and As transport from mine tailings was investigated by pumping simulated acid rain at a infiltration rate of 10.2 cm/h through soil columns. Simulated acid rain with pH of 3.0, 4.5 and 5.6 were used as leaching solutions. Results showed that 86.9-95.9% of Pb and 90-91.8% of As eluted from the columns were adsorbed by particles in the leachates. Scanning electron microscopy (SEM) analysis showed that particles released from the columns were mainly composed of flocculated aggregates and plate or rod shaped discrete grains. Transmission electron microscopy (TEM) coupled with energy dispersive X-ray analysis (EDX) showed that these particles were predominantly silicate minerals. Results from our experiments demonstrated that when rapid infiltration conditions or a rainstorm exist, particle-facilitated transport of contaminants is likely to the dominant metal transport pathway influenced by acid rain.

  4. Role of a major facilitator superfamily transporter in adaptation capacity of Penicillium funiculosum under extreme acidic stress.

    PubMed

    Xu, Xiaoxue; Chen, Jinyin; Xu, Houjuan; Li, Duochuan

    2014-08-01

    Fungal species present in extreme low pH environments are expected to have adapted for tolerance to high H(+) concentrations. However, their adaptability mechanism is unclear. In this study, we isolated an acid-tolerant strain of Penicillium funiculosum, which can grow actively at pH 1.0 and thrived in pH 0.6. A major facilitator superfamily transporter (PfMFS) was isolated from an acid-sensitive random insertional mutant (M4) of the fungus. It encodes a putative protein of 551 residues and contains 14 transmembrane-spanning segments. A targeted mutant (M7) carrying an inactivated copy of PfMFS showed an obvious reduction of growth compared with the wild type (WT) and complementation of M7 with PfMFS restored the wild-type level of growth at pH 1.0. Further data showed that the wild-type showed higher intracellular pH than M7 in response to pH 1. Subcellular localization showed that PfMFS was a cell membrane protein. Homology modeling showed structural similarity with an MFS transporter EmrD from Escherichiacoli. These results demonstrate that the PfMFS transporter is involved in the acid resistance and intracellular pH homeostasis of P. funiculosum.

  5. Relocation of active site carboxylates in major facilitator superfamily multidrug transporter LmrP reveals plasticity in proton interactions

    PubMed Central

    Nair, Asha V.; Singh, Himansha; Raturi, Sagar; Neuberger, Arthur; Tong, Zhen; Ding, Ning; Agboh, Kelvin; van Veen, Hendrik W.

    2016-01-01

    The expression of polyspecific membrane transporters is one important mechanism by which cells can obtain resistance to structurally different antibiotics and cytotoxic agents. These transporters reduce intracellular drug concentrations to subtoxic levels by mediating drug efflux across the cell envelope. The major facilitator superfamily multidrug transporter LmrP from Lactococcus lactis catalyses drug efflux in a membrane potential and chemical proton gradient-dependent fashion. To enable the interaction with protons and cationic substrates, LmrP contains catalytic carboxyl residues on the surface of a large interior chamber that is formed by transmembrane helices. These residues co-localise together with polar and aromatic residues, and are predicted to be present in two clusters. To investigate the functional role of the catalytic carboxylates, we generated mutant proteins catalysing membrane potential-independent dye efflux by removing one of the carboxyl residues in Cluster 1. We then relocated this carboxyl residue to six positions on the surface of the interior chamber, and tested for restoration of wildtype energetics. The reinsertion at positions towards Cluster 2 reinstated the membrane potential dependence of dye efflux. Our data uncover a remarkable plasticity in proton interactions in LmrP, which is a consequence of the flexibility in the location of key residues that are responsible for proton/multidrug antiport. PMID:27917857

  6. The human concentrative and equilibrative nucleoside transporter families, SLC28 and SLC29.

    PubMed

    Young, James D; Yao, Sylvia Y M; Baldwin, Jocelyn M; Cass, Carol E; Baldwin, Stephen A

    2013-01-01

    Nucleoside transport in humans is mediated by members of two unrelated protein families, the SLC28 family of cation-linked concentrative nucleoside transporters (CNTs) and the SLC29 family of energy-independent, equilibrative nucleoside transporters (ENTs). These families contain three and four members, respectively, which differ both in the stoichiometry of cation coupling and in permeant selectivity. Together, they play key roles in nucleoside and nucleobase uptake for salvage pathways of nucleotide synthesis. Moreover, they facilitate cellular uptake of several nucleoside and nucleobase drugs used in cancer chemotherapy and treatment of viral infections. Thus, the transporter content of target cells can represent a key determinant of the response to treatment. In addition, by regulating the concentration of adenosine available to cell surface receptors, nucleoside transporters modulate many physiological processes ranging from neurotransmission to cardiovascular activity. This review describes the molecular and functional properties of the two transporter families, with a particular focus on their physiological roles in humans and relevance to disease treatment.

  7. Facilitated Strontium Transport by Remobilization of Strontium-Containing Secondary Precipitates in Hanford Site Subsurface

    SciTech Connect

    Wang, Guohui; Um, Wooyong

    2013-03-15

    Significantly enhanced immobilization of radionuclides (such as 90Sr and 137Cs) due to adsorption and coprecipitation with neo-formed colloid-sized secondary precipitates has been reported at the U.S. Department of Energy’s Hanford Site. However, the stability of these secondary precipitates containing radionuclides in the subsurface under changeable field conditions is not clear. Here, the authors tested the remobilization possibility of Sr containing secondary precipitates (nitrate-cancrinite) in the subsurface using saturated column experiments under different geochemical and flow conditions. The columns were packed with quartz sand that contained secondary precipitates (nitrate-cancrinite containing Sr), and leached using colloid-free solutions under different flow rates, varying pH, and ionic strength conditions. The results indicate remobilization of the neo-formed secondary precipitates could be possible given a change of background conditions. The remobility of the neo formed precipitates increased with the rise in the leaching solution flow rate and pH (in a range of pH 4 to 11), as well as with decreasing solution ionic strength. The increased mobility of Sr-containing secondary precipitates with changing background conditions can be a potential source for additional radionuclide transport in Hanford Site subsurface environments.

  8. Transport of surfactant-facilitated multiwalled carbon nanotube suspensions in columns packed with sized soil particles.

    PubMed

    Lu, Yinying; Yang, Kun; Lin, Daohui

    2014-09-01

    Transport of carbon nanotubes (CNTs) in soil/sediment matrixes can regulate their potential eco-effects and has been however rarely studied. Herein, column experiments were conducted to investigate mobility of CNT suspensions stabilized by dodecylbenzenesulfonic acid sodium salt (SDBS), octyl-phenol-ethoxylate (TX-100) and cetylpyridinium chloride (CPC) in four soil samples with certain particle sizes. Humic acid was extracted from a soil sample and was coated on quartz sands to explore the effect of soil organic matter (SOM) on the mobility. Results showed that the positively-charged CPC-CNT was entirely retained in the columns while the negatively-charged SDBS-CNT and TX-100-CNT more or less broke through the columns. Pearson correlation analyses revealed that soil texture rather than SOM controlled the mobility. Electrostatic attraction to and/or precipitation on the grain surfaces together with the straining effect could explain the CNT retention. These novel results will help to understand the eco-effects of CNTs.

  9. Glucose transporter recycling in response to insulin is facilitated by myosin Myo1c.

    PubMed

    Bose, Avirup; Guilherme, Adilson; Robida, Stacey I; Nicoloro, Sarah M C; Zhou, Qiong L; Jiang, Zhen Y; Pomerleau, Darcy P; Czech, Michael P

    Insulin stimulates glucose uptake in muscle and adipocytes by signalling the translocation of GLUT4 glucose transporters from intracellular membranes to the cell surface. The translocation of GLUT4 may involve signalling pathways that are both independent of and dependent on phosphatidylinositol-3-OH kinase (PI(3)K). This translocation also requires the actin cytoskeleton, and the rapid movement of GLUT4 along linear tracks may be mediated by molecular motors. Here we report that the unconventional myosin Myo1c is present in GLUT4-containing vesicles purified from 3T3-L1 adipocytes. Myo1c, which contains a motor domain, three IQ motifs and a carboxy-terminal cargo domain, is highly expressed in primary and cultured adipocytes. Insulin enhances the localization of Myo1c with GLUT4 in cortical tubulovesicular structures associated with actin filaments, and this colocalization is insensitive to wortmannin. Insulin-stimulated translocation of GLUT4 to the adipocyte plasma membrane is augmented by the expression of wild-type Myo1c and inhibited by a dominant-negative cargo domain of Myo1c. A decrease in the expression of endogenous Myo1c mediated by small interfering RNAs inhibits insulin-stimulated uptake of 2-deoxyglucose. Thus, myosin Myo1c functions in a PI(3)K-independent insulin signalling pathway that controls the movement of intracellular GLUT4-containing vesicles to the plasma membrane.

  10. Colloid facilitated transport of humic substances in soil: laboratory experiment and modeling calculation.

    NASA Astrophysics Data System (ADS)

    Dinu, Marina; Moiseenko, Tatyana

    2016-04-01

    An understanding of ability to predict the fate and transport of colloids in soil systems are of great importance in many environmental and industrial applications. Especially, in the case study sizes and zeta potentials of lignin and humus components (as a parameter reflecting the mobility and tread of organic substances). The objects of investigation were water extracts of gleepodzolic soil of European territory of Russia and Western Siberia, as well as humus substances extracted from this soil. In this study, evaluation of size, molecular weight distribution and zeta potential were used to predict the mobility of the organic component fractions of the soil. Fractionation was performed using multistage filtration plant (100 Da) and measuring physic-chemical parameters measured with the Malvern Zetasizer Nano ZSP. Significant differences in the distribution of organic matter on the molecular weight, charge (sign) of the zeta potential and the size of the sample of European Russia in comparison with samples of Western Siberia have been found. Also, laboratory studies have demonstrated of any differences in physicochemical parameters as infrared spectra, ultraviolet spectra, complexing ability of samples of the same soil type but different areas of Russia. The results can be used in the prediction of the migration ability of fractions humus substances and their stability at change physic-chemical conditions (the coefficient of mobility of the organic components by calculated in MathCad). This work was supported by the grant № 14-17-00460 RSF from 07.11.2014

  11. A Physical Interaction between the Dopamine Transporter and DJ-1 Facilitates Increased Dopamine Reuptake.

    PubMed

    Luk, Beryl; Mohammed, Mohinuddin; Liu, Fang; Lee, Frank J S

    2015-01-01

    The regulation of the dopamine transporter (DAT) impacts extracellular dopamine levels after release from dopaminergic neurons. Furthermore, a variety of protein partners have been identified that can interact with and modulate DAT function. In this study we show that DJ-1 can potentially modulate DAT function. Co-expression of DAT and DJ-1 in HEK-293T cells leads to an increase in [3H] dopamine uptake that does not appear to be mediated by increased total DAT expression but rather through an increase in DAT cell surface localization. In addition, through a series of GST affinity purifications and co-immunoprecipitations, we provide evidence that the DAT can be found in a complex with DJ-1, which involve distinct regions within both DAT and DJ-1. Using in vitro binding experiments we also show that this complex can be formed in part by a direct interaction between DAT and DJ-1. Co-expression of a mini-gene that can disrupt the DAT/DJ-1 complex appears to block the increase in [3H] dopamine uptake by DJ-1. Mutations in DJ-1 have been linked to familial forms of Parkinson's disease, yet the normal physiological function of DJ-1 remains unclear. Our study suggests that DJ-1 may also play a role in regulating dopamine levels by modifying DAT activity.

  12. Colloidal-facilitated transport of inorganic contaminants in ground water: part 1, sampling considerations

    USGS Publications Warehouse

    Puls, Robert W.; Eychaner, James H.; Powell, Robert M.

    1996-01-01

    Investigations at Pinal Creek, Arizona, evaluated routine sampling procedures for determination of aqueous inorganic geochemistry and assessment of contaminant transport by colloidal mobility. Sampling variables included pump type and flow rate, collection under air or nitrogen, and filter pore diameter. During well purging and sample collection, suspended particle size and number as well as dissolved oxygen, temperature, specific conductance, pH, and redox potential were monitored. Laboratory analyses of both unfiltered samples and the filtrates were performed by inductively coupled argon plasma, atomic absorption with graphite furnace, and ion chromatography. Scanning electron microscopy with Energy Dispersive X-ray was also used for analysis of filter particulates. Suspended particle counts consistently required approximately twice as long as the other field-monitored indicators to stabilize. High-flow-rate pumps entrained normally nonmobile particles. Difference in elemental concentrations using different filter-pore sizes were generally not large with only two wells having differences greater than 10 percent in most wells. Similar differences (>10%) were observed for some wells when samples were collected under nitrogen rather than in air. Fe2+/Fe3+ ratios for air-collected samples were smaller than for samples collected under a nitrogen atmosphere, reflecting sampling-induced oxidation.

  13. Facilitated strontium transport by remobilization of strontium-containing secondary precipitates in Hanford Site subsurface.

    PubMed

    Wang, Guohui; Um, Wooyong

    2013-03-15

    Significantly enhanced immobilization of radionuclides (such as (90)Sr and (137)Cs) due to adsorption and coprecipitation with neo-formed colloid-sized secondary precipitates has been reported at the U.S. Department of Energy's Hanford Site. However, the stability of these secondary precipitates containing radionuclides in the subsurface under changeable field conditions is not clear. Here, the authors tested the remobilization possibility of Sr-containing secondary precipitates (nitrate-cancrinite) in the subsurface using saturated column experiments under different geochemical and flow conditions. The columns were packed with quartz sand that contained secondary precipitates (nitrate-cancrinite containing Sr), and leached using colloid-free solutions under different flow rates, varying pH, and ionic strength conditions. The results indicate remobilization of the neo-formed secondary precipitates could be possible given a change of pH of ionic strength and flow rate conditions. The remobility of the neo-formed precipitates increased with the rise in the leaching solution flow rate and pH (in a range of pH 4-11), as well as with decreasing solution ionic strength. The increased mobility of Sr-containing secondary precipitates with changing background conditions can be a potential source for additional radionuclide transport in Hanford Site subsurface environments.

  14. A Physical Interaction between the Dopamine Transporter and DJ-1 Facilitates Increased Dopamine Reuptake

    PubMed Central

    Luk, Beryl; Mohammed, Mohinuddin; Liu, Fang; Lee, Frank J. S.

    2015-01-01

    The regulation of the dopamine transporter (DAT) impacts extracellular dopamine levels after release from dopaminergic neurons. Furthermore, a variety of protein partners have been identified that can interact with and modulate DAT function. In this study we show that DJ-1 can potentially modulate DAT function. Co-expression of DAT and DJ-1 in HEK-293T cells leads to an increase in [3H] dopamine uptake that does not appear to be mediated by increased total DAT expression but rather through an increase in DAT cell surface localization. In addition, through a series of GST affinity purifications and co-immunoprecipitations, we provide evidence that the DAT can be found in a complex with DJ-1, which involve distinct regions within both DAT and DJ-1. Using in vitro binding experiments we also show that this complex can be formed in part by a direct interaction between DAT and DJ-1. Co-expression of a mini-gene that can disrupt the DAT/DJ-1 complex appears to block the increase in [3H] dopamine uptake by DJ-1. Mutations in DJ-1 have been linked to familial forms of Parkinson’s disease, yet the normal physiological function of DJ-1 remains unclear. Our study suggests that DJ-1 may also play a role in regulating dopamine levels by modifying DAT activity. PMID:26305376

  15. Facilitated charge transport in ternary interconnected electrodes for flexible supercapacitors with excellent power characteristics.

    PubMed

    Chen, Wanjun; He, Yongmin; Li, Xiaodong; Zhou, Jinyuan; Zhang, Zhenxing; Zhao, Changhui; Gong, Chengshi; Li, Shuankui; Pan, Xiaojun; Xie, Erqing

    2013-12-07

    Flexible and high performance supercapacitors are very critical in modern society. In order to develop the flexible supercapacitors with high power density, free-standing and flexible three-dimensional graphene/carbon nanotubes/MnO2 (3DG/CNTs/MnO2) composite electrodes with interconnected ternary 3D structures were fabricated, and the fast electron and ion transport channels were effectively constructed in the rationally designed electrodes. Consequently, the obtained 3DG/CNTs/MnO2 composite electrodes exhibit superior specific capacitance and rate capability compared to 3DG/MnO2 electrodes. Furthermore, the 3DG/CNTs/MnO2 based asymmetric supercapacitor demonstrates the maximum energy and power densities of 33.71 W h kg(-1) and up to 22,727.3 W kg(-1), respectively. Moreover, the asymmetric supercapacitor exhibits excellent cycling stability with 95.3% of the specific capacitance maintained after 1000 cycle tests. Our proposed synthesis strategy to construct the novel ternary 3D structured electrodes can be efficiently applied to other high performance energy storage/conversion systems.

  16. Facilitated transport of uranium(VI) across supported liquid membranes containing T2EHDGA as the carrier extractant.

    PubMed

    Panja, S; Mohapatra, P K; Tripathi, S C; Manchanda, V K

    2011-04-15

    Facilitated transport of uranyl ion from nitric acid feed solutions was investigated across PTFE supported liquid membranes using N,N,N',N'-tetra-2-ethylhexyl-3-pentane-diamide (T2EHDGA) in n-dodecane as the carrier extractant containing 30% iso-decanol as the phase modifier. Solvent extraction studies indicated extraction of species of the type, UO(2)(NO(3))(2)·T2EHDGA. The distribution coefficients increased in the presence of NaNO(3) as compared to equivalent concentration of HNO(3) which was exactly the opposite of what was reported for Am(III)-TODGA extraction system. Supported liquid membrane studies indicated about 11h were required for quantitative transport of U(VI) from a feed of 3M HNO(3) using 0.2M T2EHDGA in n-dodecane containing 30% iso-decanol as the carrier extractant. Effect of various parameters such as feed acidity, T2EHDGA concentration, and nature of the strippant on the transport rate was investigated. The transport was found to be diffusion controlled in the membrane phase and the permeability coefficient was calculated to be (3.20 ± 0.13)× 10(-4)cm/s for the feed composition of 3M HNO(3), receiver phase composition of 0.01 M HNO(3) and membrane carrier phase of 0.2M T2EHDGA in n-dodecane containing 30% iso-decanol. The present results may be useful for the separation of U from lean solutions or radioactive wastes considered hazardous due to the presence of alpha-particle emitting radionuclides.

  17. Biosorption of dichlorodiphenyltrichloroethane and hexachlorobenzene in groundwater and its implications for facilitated transport.

    PubMed Central

    Lindqvist, R; Enfield, C G

    1992-01-01

    The potential for enhanced mobility of hydrophobic pollutants by cotransport with bacteria in saturated soils was evaluated from measurements of biosorption of 14C-labeled hexachlorobenzene and dichlorodiphenyltrichloroethane (DDT) to five strains of soil and sewage bacteria. The sorption process could be described by a linear partition equation and appeared to be reversible, but desorption kinetics were slow and/or partly irreversible. The DDT partition coefficients varied with equilibration time, possibly reflecting DDT-induced changes in the physiology of the bacteria. The partition coefficients, normalized to the masses of the bacteria, ranged from 250 to 14,000 for live cells, but the largest coefficients were associated with autoclaved cells of a Pseudomonas sp. The sorptive capacity of the bacterial biomass was greater for DDT than for hexachlorobenzene but was not correlated to overall bacterial hydrophobicity, measured by hydrophobic interaction chromatography. In a column study, 1.2 x 10(9) cells of a Bacillus sp. strain per ml enhanced DDT transport about 8-fold, whereas an advective-dispersive-sorptive equilibrium model for two mobile phases, water and free-living bacteria, suggested a 14-fold enhancement, based on the DDT partition coefficient. The disagreement was in part due to a retarded nonequilibrium movement of the bacteria. Model calculations based on literature data covering a wide range of organisms and compounds suggested that 10(6) cells ml-1 would increase the mobility of very hydrophobic compounds (log octanol-water partition coefficient [K(ow) of greater than or equal to 6), whereas higher densities of bacteria (10(8) cells ml-1) would have a significant impact on compounds with a log K(ow) of greater than or equal to 4.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1637158

  18. Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport.

    PubMed

    McCauliff, Leslie A; Xu, Zhi; Li, Ran; Kodukula, Sarala; Ko, Dennis C; Scott, Matthew P; Kahn, Peter C; Storch, Judith

    2015-11-06

    The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2(-/-) fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment.

  19. Long-lived Species Facilitate Substantial Summertime Transpacific Transport of Ozone

    NASA Astrophysics Data System (ADS)

    Guo, Y.; Liu, J.; Mauzerall, D. L.; Li, X.; Horowitz, L. W.; Tao, W.; Tao, S.

    2016-12-01

    Ground-level ozone (O3), harmful to most living things, is produced from both domestic and foreign emissions of anthropogenic precursors. For the U.S., upwind emissions affect compliance with tightening national ambient ozone standards. Previous estimates of contributions from distant sources rely on the sensitivity approach (i.e., modeling the change of O3 concentrations that result from modifying precursor emissions), and O3 tagging approach (i.e., tracking O3 produced within one region or from nitrogen oxides emitted from one region). However, these approaches either perturb the non-linear O3 chemistry or exclude important O3 precursors, making accurate estimation of the total contribution of distant emissions challenging. Here, for the first time, we quantify the contribution of trans-Pacific transport of O3 precursors to U.S. ground-level O3 by tracking all O3 precursors (i.e. nitrogen oxides, NOx; carbon monoxide, CO; volatile organic compounds, VOCs) without perturbing the state of the atmosphere. We show that even in summer nearly 3 ppbv (parts per billion by volume) of surface ozone over North American can be attributed to East Asian anthropogenic emissions. This is 4 times larger than obtained using the sensitivity approach (0.7 ppbv) and 6 times larger than obtained by tagging reactive nitrogen oxides (0.5 ppbv). Longer lived species (e.g. VOCs and CO), largely ignored by previous studies, play an important role in extending long-distance photochemical production of O3. We conclude that East Asian CO and VOCs have substantial impact on the U.S. boundary layer photochemistry and influence O3 production and surface concentrations especially in summer.

  20. Effect of stochastic gating on channel-facilitated transport of non-interacting and strongly repelling solutes

    NASA Astrophysics Data System (ADS)

    Berezhkovskii, Alexander M.; Bezrukov, Sergey M.

    2017-08-01

    Ligand- or voltage-driven stochastic gating—the structural rearrangements by which the channel switches between its open and closed states—is a fundamental property of biological membrane channels. Gating underlies the channel's ability to respond to different stimuli and, therefore, to be functionally regulated by the changing environment. The accepted understanding of the gating effect on the solute flux through the channel is that the mean flux is the product of the flux through the open channel and the probability of finding the channel in the open state. Here, using a diffusion model of channel-facilitated transport, we show that this is true only when the gating is much slower than the dynamics of solute translocation through the channel. If this condition breaks, the mean flux could differ from this simple estimate by orders of magnitude.

  1. Stomatal Spacing Safeguards Stomatal Dynamics by Facilitating Guard Cell Ion Transport Independent of the Epidermal Solute Reservoir.

    PubMed

    Papanatsiou, Maria; Amtmann, Anna; Blatt, Michael R

    2016-09-01

    Stomata enable gaseous exchange between the interior of the leaf and the atmosphere through the stomatal pore. Control of the pore aperture depends on osmotic solute accumulation by, and its loss from the guard cells surrounding the pore. Stomata in most plants are separated by at least one epidermal cell, and this spacing is thought to enhance stomatal function, although there are several genera that exhibit stomata in clusters. We made use of Arabidopsis (Arabidopsis thaliana) stomatal patterning mutants to explore the impact of clustering on guard cell dynamics, gas exchange, and ion transport of guard cells. These studies showed that stomatal clustering in the Arabidopsis too many mouths (tmm1) mutant suppressed stomatal movements and affected CO2 assimilation and transpiration differentially between dark and light conditions and were associated with alterations in K(+) channel gating. These changes were consistent with the impaired dynamics of tmm1 stomata and were accompanied by a reduced accumulation of K(+) ions in the guard cells. Our findings underline the significance of spacing for stomatal dynamics. While stomatal spacing may be important as a reservoir for K(+) and other ions to facilitate stomatal movements, the effects on channel gating, and by inference on K(+) accumulation, cannot be explained on the basis of a reduced number of epidermal cells facilitating ion supply to the guard cells.

  2. Stereoselective Actions of Methylenedioxypyrovalerone (MDPV) To Inhibit Dopamine and Norepinephrine Transporters and Facilitate Intracranial Self-Stimulation in Rats.

    PubMed

    Kolanos, R; Partilla, J S; Baumann, M H; Hutsell, B A; Banks, M L; Negus, S S; Glennon, R A

    2015-05-20

    The designer stimulant methylenedioxypyrovalerone (MDPV) is a potent reuptake inhibitor at transporters for dopamine (DAT) and norepinephrine (NET) that produces a constellation of abuse-related behavioral effects. MDPV possesses a chiral center, and the abused formulation of the drug is a racemic mixture, but no data are available on the pharmacology of its isomers. Here, the individual optical isomers of MDPV were prepared and examined with respect to their neurochemical actions on neurotransmitter reuptake and behavioral effects in an assay of intracranial self-stimulation (ICSS) in rats. In assays of DAT uptake inhibition, S(+)MDPV (EC50 = 2.13 nM) was more potent than either (±)MDPV (EC50 = 4.85 nM) or R(-)MDPV (EC50 = 382.80 nM); the three drugs were less potent at NET uptake inhibition, with the same rank order of potency. Neither racemic MDPV nor its optical isomers inhibited the reuptake of serotonin at concentrations up to 10 μM. S(+)MDPV produced an abuse-related and dose-dependent facilitation of ICSS, and the potency of S(+)MDPV (significant facilitation at doses ≥ 0.1 mg/kg) was greater than that of the racemate (significant facilitation at doses ≥ 0.32 mg/kg). R(-)MDPV failed to alter ICSS at doses up to 100 times greater than the lowest effective dose of S(+)MDPV. The results indicate that abuse-related neurochemical and behavioral effects of racemic MDPV reside primarily with its S(+) isomer.

  3. A Re-Examination of Nucleobases in Carbonaceous Chondrites

    NASA Astrophysics Data System (ADS)

    Martins, Z.; Botta, O.; de Vries, M.; Becker, L.; Ehrenfreund, E.

    The biomolecular building blocks of life, as we know it, are amino acids, purines and pyrimidines. The latter two form the bases of DNA and RNA, molecules that are used in the storage, transcription and translation of genetic information in all terrestrial organisms. A dedicated search for these compounds in meteorites can shed light on the origins of life in two ways: (i) Results can help assess the plausibility of extraterrestrial formation of prebiotic molecules followed by their meteoritic delivery to the early Earth. (ii) Such studies can also provide insights into possible prebiotic synthetic routes. We will search for these compounds in selected carbonaceous chondrites using formic acid extraction and reverse phase high performance liquid chromatography (HPLC) to isolate specific nucleobases from the bulk meteorite material as previously reported [1,2,3]. We will also use a new technique, resonant two-photon ionization mass spectrometry (R2PI) that can, not only identify organic compounds by their mass, but at the same time by their vibronic spectroscopy [4]. R2PI dramatically enhances the specificity for certain compounds (e.g. amino acids, nucleobases) and allows for distinction of structural isomers, tautomers and enantiomers as well as providing additional information due to isotope shifts. The optical spectroscopy can thus help us to further discriminate between terrestrial and extraterrestrial nucleobases. References: [1] Van Der Velden, W. and Schwarts, A. W. (1977) Geochim. Cosmochim. Acta, 41, 961-968. [2] Stoks, P. G. and Schwartz, A. W. (1979a) Nature, 282, 709-10. [3] Glavin, D. P. and Bada, J. L. (2004) In Lunar and Planetary Science XXXV, Abstract # 1022, Houston. [4] Nir, E., Grace, L. I., Brauer, B. and de Vries, M. S. (1999) Journal of the American Chemical Society, 121, 4896-4897.

  4. Human copper transporter 2 is localized in late endosomes and lysosomes and facilitates cellular copper uptake

    PubMed Central

    vandenBerghe, Peter V. E.; Folmer, Dineke E.; Malingré, Helga E. M.; vanBeurden, Ellen; Klomp, Adriana E. M.; vandeSluis, Bart; Merkx, Maarten; Berger, Ruud; Klomp, Leo W. J.

    2007-01-01

    High-affinity cellular copper uptake is mediated by the CTR (copper transporter) 1 family of proteins. The highly homologous hCTR (human CTR) 2 protein has been identified, but its function in copper uptake is currently unknown. To characterize the role of hCTR2 in copper homoeostasis, epitope-tagged hCTR2 was transiently expressed in different cell lines. hCTR2–vsvG (vesicular-stomatitis-virus glycoprotein) predominantly migrated as a 17 kDa protein after imunoblot analysis, consistent with its predicted molecular mass. Chemical cross-linking resulted in the detection of higher-molecular-mass complexes containing hCTR2–vsvG. Furthermore, hCTR2–vsvG was co-immunoprecipitated with hCTR2–FLAG, suggesting that hCTR2 can form multimers, like hCTR1. Transiently transfected hCTR2–eGFP (enhanced green fluorescent protein) was localized exclusively to late endosomes and lysosomes, and was not detected at the plasma membrane. To functionally address the role of hCTR2 in copper metabolism, a novel transcription-based copper sensor was developed. This MRE (metal-responsive element)–luciferase reporter contained four MREs from the mouse metallothionein 1A promoter upstream of the firefly luciferase open reading frame. Thus the MRE–luciferase reporter measured bioavailable cytosolic copper. Expression of hCTR1 resulted in strong activation of the reporter, with maximal induction at 1 μM CuCl2, consistent with the Km of hCTR1. Interestingly, expression of hCTR2 significantly induced MRE–luciferase reporter activation in a copper-dependent manner at 40 and 100 μM CuCl2. Taken together, these results identify hCTR2 as an oligomeric membrane protein localized in lysosomes, which stimulates copper delivery to the cytosol of human cells at relatively high copper concentrations. This work suggests a role for endosomal and lysosomal copper pools in the maintenance of cellular copper homoeostasis. PMID:17617060

  5. Probing nucleobase photo protection with soft x-rays

    NASA Astrophysics Data System (ADS)

    Gühr, Markus

    2013-05-01

    We [1] present a new method for ultrafast spectroscopy of molecular photoexcited dynamics. The technique uses a pair of femtosecond pulses: a photoexcitation pulse initiating excited state dynamics followed by a soft x-ray (SXR) probe pulse that core ionizes certain atoms inside the molecule. We observe the Auger decay of the core hole as a function of delay between the photoexcitation and SXR pulses. The core hole decay is particularly sensitive to the local valence electrons near the core and shows new types of propensity rules, compared to dipole selection rules in SXR absorption or emission spectroscopy. We apply the delayed ultrafast x-ray Auger probing (DUXAP) method to the specific problem of nucleobase photoprotection to demonstrate its potential. The ultraviolet photoexcited ππ * states of nucleobases are prone to chemical reactions with neighboring bases. To avoid this, the single molecules funnel the ππ * population to lower lying electronic states on an ultrafast timescale under violation of the Born-Oppenheimer approximation. The new type of propensity rule, which is confirmed by Auger decay simulations, allows us to have increased sensitivity on the direct relaxation from the ππ * state to the vibrationally hot electronic ground state. For the nucleobase thymine, we measure a decay of the ππ * state and a subsequent filling of the vibrationally hot ground state in 300 fs. This work was supported by the AMOS program within the Chemical Sciences, Geosciences, and Biosciences Division of the Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy.Portions of this research were carried out at the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. LCLS is an Office of Science User Facility operated for the U.S. Department of Energy Office of Science by Stanford University. Other portions of this research were carried out at the Advanced Light Source, which is supported by the Director, Office of

  6. A DTX/MATE-type transporter facilitates abscisic acid efflux and modulates ABA sensitivity and drought tolerance in Arabidopsis.

    PubMed

    Zhang, Haiwen; Zhu, Huifen; Pan, Yajun; Yu, Yuexuan; Luan, Sheng; Li, Legong

    2014-10-01

    Abscisic acid (ABA) regulates numerous physiological and developmental processes in plants. Recent studies identify intracellular ABA receptors, implicating the transport of ABA across cell membranes as crucial for ABA sensing and response. Here, we report that a DTX/Multidrug and Toxic Compound Extrusion (MATE) family member in Arabidopsis thaliana, AtDTX50, functions as an ABA efflux transporter. When expressed heterologously in both an Escherichia coli strain and Xenopus oocyte cells, AtDTX50 was found to facilitate ABA efflux. Furthermore, dtx50 mutant mesophyll cells preloaded with ABA released less ABA compared with the wild-type (WT). The AtDTX50 gene was expressed mainly in the vascular tissues and guard cells and its expression was strongly up-regulated by exogenous ABA. The AtDTX50::GFP fusion protein was localized predominantly to the plasma membrane. The dtx50 mutant plants were observed to be more sensitive to ABA in growth inhibition. In addition, compared with the WT, dtx50 mutant plants were more tolerant to drought with lower stomatal conductance, consistent with its function as an ABA efflux carrier in guard cells. © The Author 2014. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.

  7. Colloid-Facilitated Transport of 137Cs in Fracture-Fill Material. Experiments and Modeling

    SciTech Connect

    Dittrich, Timothy M.; Reimus, Paul William

    2015-10-29

    In this study, we demonstrate how a combination of batch sorption/desorption experiments and column transport experiments were used to effectively parameterize a model describing the colloid-facilitated transport of Cs in the Grimsel granodiorite/FFM system. Cs partition coefficient estimates onto both the colloids and the stationary media obtained from the batch experiments were used as initial estimates of partition coefficients in the column experiments, and then the column experiment results were used to obtain refined estimates of the number of different sorption sites and the adsorption and desorption rate constants of the sites. The desorption portion of the column breakthrough curves highlighted the importance of accounting for adsorption-desorption hysteresis (or a very nonlinear adsorption isotherm) of the Cs on the FFM in the model, and this portion of the breakthrough curves also dictated that there be at least two different types of sorption sites on the FFM. In the end, the two-site model parameters estimated from the column experiments provided excellent matches to the batch adsorption/desorption data, which provided a measure of assurance in the validity of the model.

  8. Facilitating mass transport in gas diffusion layer of PEMFC by fabricating micro-porous layer with dry layer preparation

    NASA Astrophysics Data System (ADS)

    Chen, Jian; Xu, Haifeng; Zhang, Huamin; Yi, Baolian

    For a proton exchange membrane fuel cell (PEMFC), dry layer preparation was optimized and applied to fabricate a micro-porous layer (MPL) for a gas diffusion layer (GDL). The MPLs fabricated by dry layer preparation and the conventional wet layer preparation were compared by physical and electrochemical methods. The PEMFC using dry layer MPLs showed better performance than that using wet layer MPLs, especially when the cells were operated under conditions of high oxygen utilization rate and high humidification temperature of air. The mass transport properties of the GDLs with the dry layer MPLs were also better than with the wet layer MPLs, and were found to be related to the pore size distribution in GDLs. The differences in surface morphology and pore size distribution for the GDLs with the dry layer and wet layer MPLs were investigated and analyzed. The dry layer preparation for MPLs was found to be more beneficial for forming meso-pores (pore size in the range of 0.5-15 μm), which are important and advantageous for facilitating gas transport in the GDLs. Moreover, the GDLs with the dry layer MPLs exhibited better electronic conductivity and more stable hydrophobicity than those with the wet layer MPLs. The reproducibility of the dry layer preparation for MPLs was also satisfying.

  9. Isoform-selective inhibition of facilitative glucose transporters: elucidation of the molecular mechanism of HIV protease inhibitor binding.

    PubMed

    Hresko, Richard C; Kraft, Thomas E; Tzekov, Anatoly; Wildman, Scott A; Hruz, Paul W

    2014-06-06

    Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Two-Photon-Induced Fluorescence of Isomorphic Nucleobase Analogs

    PubMed Central

    Lane, Richard S. K.; Jones, Rosemary; Sinkeldam, Renatus W.

    2014-01-01

    Five isomorphic fluorescent uridine mimics have been subjected to two-photon (2P) excitation analysis to investigate their potential applicability as non-perturbing probes for the single-molecule detection of nucleic acids. We find that small structural differences can cause major changes in the two-photon excitation probability, with the 2P cross sections varying by over one order of magnitude. Two of the probes, both furan-modified uridine analogs, have the highest 2P cross sections (3.8 GM and 7.6 GM) reported for nucleobase analogs, using a conventional Ti:sapphire laser for excitation at 690 nm; they also have the lowest emission quantum yields. In contrast, the analogs with the highest reported quantum yields have the lowest 2P cross sections. The structure-photophysical property relationship presented here is a first step towards the rational design of emissive nucleobase analogs with controlled 2P characteristics. The results demonstrate the potential for major improvements through judicious structural modifications. PMID:24604669

  11. Distinction of nucleobases – a tip-enhanced Raman approach

    PubMed Central

    Treffer, Regina; Lin, Xiumei; Bailo, Elena; Deckert-Gaudig, Tanja

    2011-01-01

    Summary The development of novel DNA sequencing methods is one of the ongoing challenges in various fields of research seeking to address the demand for sequence information. However, many of these techniques rely on some kind of labeling or amplification steps. Here we investigate the intrinsic properties of tip-enhanced Raman scattering (TERS) towards the development of a novel, label-free, direct sequencing method. It is known that TERS allows the acquisition of spectral information with high lateral resolution and single-molecule sensitivity. In the presented experiments, single stranded adenine and uracil homopolymers were immobilized on different kinds of substrates (mica and gold nanoplates) and TERS experiments were conducted, which demonstrated the reproducibility of the technique. To elucidate the signal contributions from the specific nucleobases, TERS spectra were collected on single stranded calf thymus DNA with arbitrary sequence. The results show that, while the Raman signals with respect to the four nucleobases differ remarkably, specific markers can be determined for each respective base. The combination of sensitivity and reproducibility shows that the crucial demands for a sequencing procedure are met. PMID:22003468

  12. Mg2+ ions: do they bind to nucleobase nitrogens?

    PubMed Central

    Leonarski, Filip; D'Ascenzo, Luigi; Auffinger, Pascal

    2017-01-01

    Given the many roles proposed for Mg2+ in nucleic acids, it is essential to accurately determine their binding modes. Here, we surveyed the PDB to classify Mg2+ inner-sphere binding patterns to nucleobase imine N1/N3/N7 atoms. Among those, purine N7 atoms are considered to be the best nucleobase binding sites for divalent metals. Further, Mg2+ coordination to N7 has been implied in several ribozyme catalytic mechanisms. We report that Mg2+ assigned near imine nitrogens derive mostly from poor interpretations of electron density patterns and are most often misidentified Na+, K+, NH4+ ions, water molecules or spurious density peaks. Consequently, apart from few documented exceptions, Mg2+ ions do not bind to N7 atoms. Without much of a surprise, Mn2+, Zn2+ and Cd2+, which have a higher affinity for nitrogens, may contact N7 atoms when present in crystallization buffers. In this respect, we describe for the first time a potential Zn2+ ribosomal binding site involving two purine N7 atoms. Further, we provide a set of guidelines to help in the assignment of Mg2+ in crystallographic, cryo-EM, NMR and model building practices and discuss implications of our findings related to ion substitution experiments. PMID:27923930

  13. Inhibiting Glycine Transporter-1 Facilitates Cocaine-Cue Extinction and Attenuates Reacquisition of Cocaine-Seeking Behavior*

    PubMed Central

    Nic Dhonnchadha, Bríd Á; Pinard, Emmanuel; Alberati, Daniela; Wettstein, Joseph G.; Spealman, Roger D.; Kantak, Kathleen M.

    2011-01-01

    Background Combining extinction training with cognitive-enhancing pharmacotherapy represents a novel strategy for improving the efficacy of exposure therapy for drug relapse prevention. We investigated if the selective glycine transporter-1 (GlyT-1) inhibitor RO4543338 could facilitate extinction of cocaine-conditioned responses and attenuate reacquisition of cocaine-seeking behavior. Methods Rats were trained to self-administer cocaine (0.3 mg/kg), which was associated with a 2-sec light cue under a second-order schedule of i.v. drug injection. Rats received vehicle, 30 or 45 mg/kg of RO4543338 prior to three 1-hr extinction-training sessions spaced at weekly intervals. Responses were extinguished by substituting saline for cocaine while maintaining response-contingent cue presentations. Reacquisition of cocaine-seeking behavior during self-administration sessions began one week after the last extinction session. Control experiments were conducted under conditions that precluded explicit extinction of cocaine-conditioned responses. Results Compared to vehicle, 30 and 45 mg/kg RO4543338 significantly decreased responding early in extinction training and during subsequent reacquisition sessions. The latter effect persisted for at least five sessions. In control studies, reacquisition of cocaine-seeking behavior was not altered when RO4543338 was administered either prior to weekly self-administration control sessions or prior to weekly control sessions in which cocaine and cues were omitted and the levers retracted. Conclusions As the GlyT-1 inhibitor facilitated cocaine-cue extinction learning and attenuated subsequent reacquisition of cocaine-seeking behavior, this class of compounds may have utility as a pharmacological adjunct to cocaine-cue exposure therapy in addicts. PMID:21992874

  14. Intracellular cholesterol transport proteins enhance hydrolysis of HDL-CEs and facilitate elimination of cholesterol into bile

    PubMed Central

    Wang, Jing; Bie, Jinghua; Ghosh, Shobha

    2016-01-01

    While HDL-associated unesterified or free cholesterol (FC) is thought to be rapidly secreted into the bile, the fate of HDL-associated cholesteryl esters (HDL-CEs) that represent >80% of HDL-cholesterol, is only beginning to be understood. In the present study, we examined the hypothesis that intracellular cholesterol transport proteins [sterol carrier protein 2 (SCP2) and fatty acid binding protein-1 (FABP1)] not only facilitate CE hydrolase-mediated hydrolysis of HDL-CEs, but also enhance elimination of cholesterol into bile. Adenovirus-mediated overexpression of FABP1 or SCP2 in primary hepatocytes significantly increased hydrolysis of HDL-[3H]CE, reduced resecretion of HDL-CE-derived FC as nascent HDL, and increased its secretion as bile acids. Consistently, the flux of [3H]cholesterol from HDL-[3H]CE to biliary bile acids was increased by overexpression of SCP2 or FABP1 in vivo and reduced in SCP2−/− mice. Increased flux of HDL-[3H]CE to biliary FC was noted with FABP1 overexpression and in SCP2−/− mice that have increased FABP1 expression. Lack of a significant decrease in the flux of HDL-[3H]CE to biliary FC or bile acids in FABP1−/− mice indicates the likely compensation of its function by an as yet unidentified mechanism. Taken together, these studies demonstrate that FABP1 and SCP2 facilitate the preferential movement of HDL-CEs to bile for final elimination. PMID:27381048

  15. Prevalence of syn nucleobases in the active sites of functional RNAs

    PubMed Central

    Sokoloski, Joshua E.; Godfrey, Stephanie A.; Dombrowski, Sarah E.; Bevilacqua, Philip C.

    2011-01-01

    Biological RNAs, like their DNA counterparts, contain helical stretches, which have standard Watson-Crick base pairs in the anti conformation. Most functional RNAs also adopt geometries with far greater complexity such as bulges, loops, and multihelical junctions. Occasionally, nucleobases in these regions populate the syn conformation wherein the base resides close to or over the ribose sugar, which leads to a more compact state. The importance of the syn conformation to RNA function is largely unknown. In this study, we analyze 51 RNAs with tertiary structure, including aptamers, riboswitches, ribozymes, and ribosomal RNAs, for number, location, and properties of syn nucleobases. These RNAs represent the set of nonoverlapping, moderate- to high-resolution structures available at present. We find that syn nucleobases are much more common among purines than pyrimidines, and that they favor C2′-endo-like conformations especially among those nucleobases in the intermediate syn conformation. Strikingly, most syn nucleobases participate in tertiary stacking and base-pairing interactions: Inspection of RNA structures revealed that the majority of the syn nucleobases are in regions assigned to function, with many syn nucleobases interacting directly with a ligand or ribozyme active site. These observations suggest that judicious placement of conformationally restricted nucleotides biased into the syn conformation could enhance RNA folding and catalysis. Such changes could also be useful for locking RNAs into functionally competent folds for use in X-ray crystallography and NMR. PMID:21873463

  16. Dual Functional Polymer Interlayer for Facilitating Ion Transport and Reducing Charge Recombination in Dye-Sensitized Solar Cells.

    PubMed

    Wang, Ying-Chiao; Li, Shao-Sian; Wen, Cheng-Yen; Chen, Liang-Yih; Ho, Kuo-Chuan; Chen, Chun-Wei

    2016-12-14

    Dye-sensitized solar cells (DSSCs) present low-cost alternatives to conventional wafer-based inorganic solar cells and have remarkable power conversion efficiency. To further enhance performance, we propose a new DSSC architecture with a novel dual-functional polymer interlayer that prevents charge recombination and facilitates ionic conduction, as well as maintaining dye loading and regeneration. Poly(vinylidene fluoride-trifluoroethylene) (p(VDF-TrFE)) was coated on the outside of a dye-sensitized TiO2 photoanode by a simple solution process that did not sacrifice the amount of adsorbed dye molecules in the DSSC device. Light-intensity-modulated photocurrent and photovoltage spectroscopy revealed that the proposed p(VDF-TrFE)-coated anode yielded longer electron lifetime and improved the injection of photogenerated electrons into TiO2, thereby reducing the electron transport time. Comparative cyclic voltammetry and UV-visible absorption spectroscopy based on a ferrocene-ferrocenium external standard material demonstrated that p(VDF-TrFE) enhanced the power conversion efficiency from 7.67% to 9.11%. This dual functional p(VDF-TrFE) interlayer is a promising candidate for improving the performance of DSSCs and can also be employed in other electrochemical devices.

  17. A New Natural Product Analog of Blasticidin S Reveals Cellular Uptake Facilitated by the NorA Multidrug Transporter.

    PubMed

    Davison, Jack R; Lohith, Katheryn M; Wang, Xiaoning; Bobyk, Kostyantyn; Mandadapu, Sivakoteswara R; Lee, Su-Lin; Cencic, Regina; Nelson, Justin; Simpkins, Scott; Frank, Karen M; Pelletier, Jerry; Myers, Chad L; Piotrowski, Jeff; Smith, Harold E; Bewley, Carole A

    2017-04-03

    The permeation of antibiotics through bacterial membranes to their target site is a crucial determinant of drug activity, but in many cases remains poorly understood. During screening efforts to discover new broad-spectrum antibiotic compounds from marine sponge samples, we identified a new analog of the peptidyl nucleoside antibiotic blasticidin S that exhibited up to 16-fold improved potency against a range of laboratory and clinical bacterial strains, which we named P10. Whole genome sequencing of laboratory-evolved strains of Staphylococcus aureus resistant to blasticidin S and P10, combined with genome-wide assessment of the fitness of barcoded Escherichia coli knockout strains in the presence of the antibiotics, revealed that the restriction of cellular access was a key feature in the development of resistance to this class of drug. In particular, the gene encoding the well-characterized multidrug efflux pump NorA was found to be mutated in 69% of all S. aureus isolates resistant to blasticidin S or P10. Unexpectedly, resistance was associated with inactivation of norA, suggesting that the NorA transporter facilitates cellular entry of peptidyl nucleosides in addition to its known role in the efflux of diverse compounds including fluoroquinolone antibiotics.

  18. Progress report on colloid-facilitated transport at Yucca Mountain: Yucca Mountain site characterization program milestone 3383

    SciTech Connect

    Triay, I.R.; Degueldre, C.; Wistrom, A.O.; Cotter, C.R.; Lemons, W.W.

    1996-06-01

    To assess colloid-facilitated radionuclide transport in groundwaters at the potential nuclear waste repository at Yucca Mountain, it is very important to understand the generation and stability of colloids, including naturally occurring colloids. To this end, we measured the colloid concentration in waters from Well J-13, which is on the order of 106 particles per milliliter (for particle sizes larger than 100 manometers). At this low particle loading, the sorption of radionuclides to colloids would have to be extremely high before the colloids could carry a significant amount of radionuclides from the repository to the accessible environment. We also performed aggregation experiments to evaluate the stability of silica (particle diameter: 85 nm) and clay colloids (particle diameter: 140 nm) as a function of ionic strength in a carbonate-rich synthetic groundwater. When the concentration of electrolyte is increased to induce aggregation, the aggregation is irreversible and the rate of aggregation increases with increasing electrolyte strength. We used autocorrelation photon spectroscopy to estimate the rate of particle aggregation for both types of colloids. By relating the measured aggregation rate to the Smoluchowski rate expression, we determined the stability ratio, W. Aggregation of silica particles and kaolinite clay particles decreased dramatically for an electrolyte concentration, C{sub NaCl}, below 300 mM and 200 mM, respectively.

  19. Experimental evidence for ternary colloid-facilitated transport of Th(IV) with hematite (α-Fe2O3) colloids and Suwannee River fulvic acid.

    PubMed

    Emerson, Hilary P; Hickok, Katherine A; Powell, Brian A

    2016-12-01

    Previous field experiments have suggested colloid-facilitated transport via inorganic and organic colloids as the primary mechanism of enhanced actinide transport in the subsurface at former nuclear weapons facilities. In this work, research was guided by the hypothesis that humic substances can enhance tetravalent actinide (An(IV)) migration by coating and mobilizing natural colloids in environmental systems and increasing An(IV) sorption to colloids. This mechanism is expected to occur under relatively acidic conditions where organic matter can sorb and coat colloid surfaces and facilitate formation of ternary colloid-ligand-actinide complexes. The objective of this work was to examine Th transport through packed columns in the presence of hematite colloids and/or Suwannee River fulvic acid (SRFA). In the presence of SRFA, with or without hematite colloids, significant transport (>60% recovery within the effluent) of thorium occurred through quartz columns. It is notable that the SRFA contributed to increased transport of both Th and hematite colloids, while insignificant transport occurred in the absence of fulvic acid. Further, in the presence of a natural sandy sediment (as opposed to pure quartz), transport is negligible in the presence of SRFA due to interactions with natural, clay-sized sediment coatings. Moreover, this data shows that the transport of Th through quartz columns is enhanced in ternary Th-colloid-SRFA and binary Th-SRFA systems as compared to a system containing only Th. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. PNA containing isocytidine nucleobase: synthesis and recognition of double helical RNA.

    PubMed

    Zengeya, Thomas; Li, Ming; Rozners, Eriks

    2011-04-01

    Peptide nucleic acid (PNA1) containing a 5-methylisocytidine (iC) nucleobase has been synthesized. Triple helix formation between PNA1 and RNA hairpins having variable base pairs interacting with iC was studied using isothermal titration calorimetry. The iC nucleobase recognized the proposed target, C-G inversion in polypurine tract of RNA, with slightly higher affinity than the natural nucleobases, though the sequence selectivity of recognition was low. Compared to non-modified PNA, PNA1 had lower affinity for its RNA target. Copyright © 2011 Elsevier Ltd. All rights reserved.

  1. PNA containing isocytidine nucleobase: synthesis and recognition of double helical RNA

    PubMed Central

    Zengeya, Thomas; Li, Ming; Rozners, Eriks

    2011-01-01

    Peptide nucleic acid (PNA1) containing a 5-methylisocytidine (iC) nucleobase has been synthesized. Triple helix formation between PNA1 and RNA hairpins having variable base pairs interacting with iC was studied using isothermal titration calorimetry. The iC nucleobase recognized the proposed target, C-G inversion in polypurine tract of RNA, with slightly higher affinity than the natural nucleobases, though the sequence selectivity of recognition was low. Compared to non-modified PNA, PNA1 had lower affinity for its RNA target. PMID:21333533

  2. Structural evolution of nucleobase clusters using force field models and density functional theory

    NASA Astrophysics Data System (ADS)

    Chiriki, Siva; Dagar, Anuradha; Bulusu, Satya S.

    2015-08-01

    We report global minima for all nucleobase clusters (nucleobase)n, with 2 ≤ n ≤ 4. The global minima are predicted using force field based global optimization methods followed by local optimizations using the dispersion corrected DFT method. In this study, we use both non-polarizable (OPLS-AA) and polarizable (AMOEBA) force fields for global optimization. Here we emphasize on the reliability of AMOEBA force field used for predicting accurate global minima of nucleobase clusters. The average deviation in binding energies using AMOEBA is 3 kcal/mol from the DFT while the average deviation using OPLS-AA is 8 kcal/mol from DFT.

  3. Click Reaction on Solid Phase Enables High Fidelity Synthesis of Nucleobase-Modified DNA.

    PubMed

    Tolle, Fabian; Rosenthal, Malte; Pfeiffer, Franziska; Mayer, Günter

    2016-03-16

    The post-synthetic functionalization of nucleic acids via click chemistry (CuAAC) has seen tremendous implementation, extending the applicability of nucleobase-modified nucleic acids in fields like fluorescent labeling, nanotechnology, and in vitro selection. However, the production of large quantities of high-density functionalized material via solid phase synthesis has been hampered by oxidative by-product formation associated with the alkaline workup conditions. Herein, we describe a rapid and cost-effective protocol for the high fidelity large-scale production of nucleobase-modified nucleic acids, exemplified with a recently described nucleobase-modified aptamer.

  4. Colloid and Colloid-Facilitated Contaminant Transport Experiments and Models to Support Assessments of Radionuclide Migration at Yucca Mountain and the Nevada Test Site

    SciTech Connect

    P. Reimus

    2004-06-01

    In recent years, numerous laboratory and field experiments have been conducted to assess and parameterize colloid and colloid-facilitated radionuclide transport for the Yucca Mountain Project and the Nevada Test Site (NTS) Environmental Restoration Project. Radionuclide contamination of ground water currently exists within or near underground nuclear test cavities at the NTS, and the proposed Yucca Mountain high-level nuclear waste repository represents a potential future source of radionuclide contamination of ground water at the NTS. Furthermore, recent field observations have indicated that small amounts of Plutonium, which normally adsorbs very strongly to mineral surfaces in aquifers, can transport quite rapidly and over significant distances in ground water when associated with inorganic colloids (Kersting et al., 1999). Groundwater samples from all over the Nevada Test Site have been analyzed for colloid concentrations and size distributions, and it is clear that there are significant mass loadings of colloids in the ground water at some locations. These colloids represent mobile surface area for potentially transporting strongly-adsorbed radionuclides. Field transport experiments have involved the use of fluorescent-dyed carboxylate-modified latex (CML) microspheres in the 250- to 650-nm diameter size range as surrogates for natural colloids in forced-gradient tracer tests. These experiments have indicated that effective colloid filtration coefficients appear to decrease as time and length scales increase. They suggest that a small fraction of colloids may be able to transport significant distances in groundwater systems. Laboratory experiments have been conducted to determine radionuclide sorption and desorption parameters onto inorganic colloids present in the groundwater systems and also to determine transport parameters for inorganic colloids in both fractured and porous media present at the Nevada Test Site. More recent laboratory experiments have

  5. Assaying RNA Localization in Situ with Spatially Restricted Nucleobase Oxidation.

    PubMed

    Li, Ying; Aggarwal, Mahima B; Nguyen, Kim; Ke, Ke; Spitale, Robert C

    2017-10-09

    We report herein a novel chemical-genetic method for assaying RNA localization within living cells. RNA localization is critical for normal physiology as well as the onset of cancer and neurodegenerative disorders. Despite its importance, there is a real lack of chemical methods to directly assay RNA localization with high resolution in living cells. Our novel approach relies on in situ nucleobase oxidation by singlet oxygen generated from spatially confined fluorophores. We demonstrate that our novel method can identify RNA molecules localized within specific cellular compartments. We anticipate that this platform will provide the community with a much-needed methodology for tracking RNA localization within living cells, and set the stage for systematic large scale analysis of RNA localization in living systems.

  6. Formation of nucleobases in a Miller-Urey reducing atmosphere.

    PubMed

    Ferus, Martin; Pietrucci, Fabio; Saitta, Antonino Marco; Knížek, Antonín; Kubelík, Petr; Ivanek, Ondřej; Shestivska, Violetta; Civiš, Svatopluk

    2017-04-25

    The Miller-Urey experiments pioneered modern research on the molecular origins of life, but their actual relevance in this field was later questioned because the gas mixture used in their research is considered too reducing with respect to the most accepted hypotheses for the conditions on primordial Earth. In particular, the production of only amino acids has been taken as evidence of the limited relevance of the results. Here, we report an experimental work, combined with state-of-the-art computational methods, in which both electric discharge and laser-driven plasma impact simulations were carried out in a reducing atmosphere containing NH3 + CO. We show that RNA nucleobases are synthesized in these experiments, strongly supporting the possibility of the emergence of biologically relevant molecules in a reducing atmosphere. The reconstructed synthetic pathways indicate that small radicals and formamide play a crucial role, in agreement with a number of recent experimental and theoretical results.

  7. Carbonic anhydrase II is found in the placenta of a viviparous, matrotrophic lizard and likely facilitates embryo-maternal CO2 transport.

    PubMed

    Van Dyke, James U; Lindsay, Laura A; Murphy, Christopher R; Thompson, Michael B

    2015-11-01

    The evolution of viviparity requires the development of mechanisms that facilitate transport of respiratory gases between mother and developing embryo. Of particular importance is maternal excretion of embryonic carbon dioxide (CO2 ), which increases as the embryo grows in size during development. The carbonic anhydrases are a family of enzymes that convert CO2 to bicarbonate for transport throughout the cardiovascular system and which may also be important for CO2 transport from embryo to mother. We used immunohistochemistry to localize carbonic anhydrase II in the placental tissues of a viviparous and highly placentotrophic lizard, Pseudemoia entrecasteauxii. Carbonic anhydrase II is localized in the uterine component of the paraplacentome, presumably to facilitate transport of embryonic CO2 to the mother. Carbonic anhydrase II is also localized in both the uterine and embryonic components of the placentome, a region heavily involved in placental nutrient transport rather than respiratory gas exchange. In contrast, carbonic anhydrase II is not present in the uterine or embryonic components of the omphaloplacenta, another region responsible for nutrient transport. While carbonic anhydrase II in the paraplacentomal uterus is likely to be responsible for embryo-maternal CO2 transport, the distribution of carbonic anhydrase II throughout the placentome indicates a different function. Instead of transporting embryonic CO2 , placentomal carbonic anhydrase II appears to be responsible for transporting CO2 produced by energetically expensive nutrient transport mechanisms in both the uterus and the embryo, which implies that the mechanisms of nutrient transport in the omphaloplacenta may not be as energetically expensive. © 2015 Wiley Periodicals, Inc.

  8. The Formation of Nucleobases from the Ultraviolet Photoirradiation of Purine in Simple Astrophysical Ice Analogues

    NASA Astrophysics Data System (ADS)

    Materese, Christopher K.; Nuevo, Michel; Sandford, Scott A.

    2017-08-01

    Nucleobases are the informational subunits of RNA and DNA and are essential to all known forms of life. The nucleobases can be divided into two groups of molecules: the pyrimidine-based compounds that include uracil, cytosine, and thymine, and the purine-based compounds that include adenine and guanine. Previous work in our laboratory has demonstrated that uracil, cytosine, thymine, and other nonbiological, less common nucleobases can form abiotically from the UV photoirradiation of pyrimidine in simple astrophysical ice analogues containing combinations of H2O, NH3, and CH4. In this work, we focused on the UV photoirradiation of purine mixed with combinations of H2O and NH3 ices to determine whether or not the full complement of biological nucleobases can be formed abiotically under astrophysical conditions. Room-temperature analyses of the resulting photoproducts resulted in the detection of adenine, guanine, and numerous other functionalized purine derivatives.

  9. First Principles Study of Nuclear Quadrupole Interactions in Single and Double Chain DNA and Solid Nucleobases

    NASA Astrophysics Data System (ADS)

    Das, T. P.; Pink, R. H.; Badu, S. R.; Dubey, Archana; Scheicher, R. H.; Saha, H. P.; Chow, Lee; Huang, M. B.

    2009-03-01

    Nuclear Quadrupole Interactions (NQI) of ^17O, ^14N and ^2H nuclei have been studied for free nucleobases and nucleobases in single strand and double strand DNA and in solid state. Our first-principles investigations were carried out using the Gaussian 2003 set of programs to implement the Hartree-Fock procedure combined with many-body effects included using many-body perturbation theory. As expected for NQI in general, many-body effects are found to be small. Results will be presented for the quadrupole coupling constants (e^2qQ) and asymmetry parameters (η) for the nucleobases in the various environments. Trends in e^2qQ and η in the different environments will be discussed. In the case of the solid nucleobases, comparisons will be made with available experimental data [1] for ^17O nuclei.[3pt] [1] Gang Wu et al., J. Am. Chem. Soc. 124, 1768 (2002)

  10. Contents Changes of Triterpenic Acids, Nucleosides, Nucleobases, and Saccharides in Jujube (Ziziphus jujuba) Fruit During the Drying and Steaming Process.

    PubMed

    Guo, Sheng; Duan, Jin-Ao; Zhang, Ying; Qian, Dawei; Tang, Yuping; Zhu, Zhenhua; Wang, Hanqing

    2015-12-12

    Chinese jujube (Ziziphus jujuba), a medicinal and edible plant, is widely consumed in Asian countries owing to the remarkable health activities of its fruits. To facilitate selection of the suitable processing method for jujube fruits, in this study their contents of triterpenic acids, nucleosides, nucleobases and saccharides after drying and steaming treatment were determined using ultra-high performance liquid chromatography and high performance liquid chromatography coupled with evaporative light scattering detector methods. The results showed that except for sucrose, the content levels of most analytes were increasing in the jujube fruits during drying treatment at 45 °C. The levels of cyclic nucleotides such as adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, were significantly decreased after the fruits were steamed. Therefore, owing to the bioactivities of these components for human health, the dried fruits would be the better choice as medicinal material or functional food, and dried jujube fruit should not be further steamed.

  11. DNA-nucleobases: Gate Dielectric/Passivation Layer for Flexible GFET-based Sensor Applications (Postprint)

    DTIC Science & Technology

    2015-09-24

    AFRL-RX-WP-JA-2016-0271 DNA -NUCLEOBASES: GATE DIELECTRIC/ PASSIVATION LAYER FOR FLEXIBLE GFET-BASED SENSOR APPLICATIONS (POSTPRINT...TITLE AND SUBTITLE DNA -NUCLEOBASES: GATE DIELECTRIC/ PASSIVATION LAYER FOR FLEXIBLE GFET-BASED SENSOR APPLICATIONS (POSTPRINT) 5a. CONTRACT...deposition of the gate dielectric layer used for making transistor devices. The approach was introducing a thin film of deoxyribonucleic acid ( DNA

  12. Which Electronic and Structural Factors Control the Photostability of DNA and RNA Purine Nucleobases?

    NASA Astrophysics Data System (ADS)

    Pollum, Marvin; Reichardt, Christian; Crespo-Hernández, Carlos E.; Martínez-Fernández, Lara; Corral, Inés; Rauer, Clemens; Mai, Sebastian; Marquetand, Philipp; González, Leticia

    2015-06-01

    Following ultraviolet excitation, the canonical purine nucleobases, guanine and adenine, are able to efficiently dissipate the absorbed energy within hundreds of femtoseconds. This property affords these nucleobases with great photostability. Conversely, non-canonical purine nucleobases exhibit high fluorescence quantum yields or efficiently populate long-lived triplet excited states from which chemistry can occur. Using femtosecond broadband transient absorption spectroscopy in combination with ab initio static and surface hopping dynamics simulations we have determined the electronic and structural factors that regulate the excited state dynamics of the purine nucleobase derivatives. Importantly, we have uncovered that the photostability of the guanine and adenine nucleobases is not due to the structure of the purine core itself and that the substituent at the C6 position of the purine nucleobase plays a more important role than that at the C2 position in the ultrafast relaxation of deleterious electronic energy. [The authors acknowledge the CAREER program of the National Science Foundation (Grant No. CHE-1255084) for financial support.

  13. Synthesis, structural, solubility and anticancer activity studies of salts using nucleobases and sulfonic acids coformer

    NASA Astrophysics Data System (ADS)

    Singh, Neetu; Singh, Udai P.; Nikhil, Kumar; Roy, Partha; Singh, Hariji

    2017-10-01

    The reactions of natural and unnatural nucleobases (cytosine (Cyt), adenine (Ade), 5-aminouracil (AU) and caffeine (Caff)) with sulfonic acids coformer (1,5-naphthalenedisulfonic acid, NDSA; 5-sulfosalicylic acid, SSA) resulted in the formation of salts viz. [NDSA.Cyt] (1), [NDSA.Ade] (2), [NDSA.AU] (3), [NDSA.Caff] (4), [SSA.Cyt] (5), [SSA.Ade] (6), [SSA.AU] (7), and [SSA.Caff] (8). The structural analysis revealed that salts 1, 4, 6 and 7 have intermolecular interactions between adjacent nucleobases which form two different homodimer shown in R22 (8) motif and assembled via complementary Nsbnd H⋯O and Nsbnd H⋯N interactions. However, in all other salts an intermediate supramolecular synthon pattern was observed between nucleobases and sulfonic acids. The lattice energy was also calculated by DFT to investigate whether salts were thermodynamically more stable than its coformer. The same was further confirmed by differential scanning calorimetry-thermogravimetric (DSC-TG) analysis. The anticancer activity study of individual nucleobases and their NDSA salts were also performed on human breast (MCF-7) and lung (A 549) cancer cell. The salts formation of nucleobases with sulfonic acids improved their solubility, thereby demonstrating up to 8-fold increase in solubility of nucleobases.

  14. Synthesis of alanyl nucleobase amino acids and their incorporation into proteins.

    PubMed

    Talukder, Poulami; Dedkova, Larisa M; Ellington, Andrew D; Yakovchuk, Petro; Lim, Jaebum; Anslyn, Eric V; Hecht, Sidney M

    2016-09-15

    Proteins which bind to nucleic acids and regulate their structure and functions are numerous and exceptionally important. Such proteins employ a variety of strategies for recognition of the relevant structural elements in their nucleic acid substrates, some of which have been shown to involve rather subtle interactions which might have been difficult to design from first principles. In the present study, we have explored the preparation of proteins containing unnatural amino acids having nucleobase side chains. In principle, the introduction of multiple nucleobase amino acids into the nucleic acid binding domain of a protein should enable these modified proteins to interact with their nucleic acid substrates using Watson-Crick and other base pairing interactions. We describe the synthesis of five alanyl nucleobase amino acids protected in a fashion which enabled their attachment to a suppressor tRNA, and their incorporation into each of two proteins with acceptable efficiencies. The nucleobases studied included cytosine, uracil, thymine, adenine and guanine, i.e. the major nucleobase constituents of DNA and RNA. Dihydrofolate reductase was chosen as one model protein to enable direct comparison of the facility of incorporation of the nucleobase amino acids with numerous other unnatural amino acids studied previously. The Klenow fragment of DNA polymerase I was chosen as a representative DNA binding protein whose mode of action has been studied in detail. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Canonical and unconventional pairing schemes between bis(nucleobase) complexes of trans-a2PtII: Artificial nucleobase quartets and C—H…N bonds

    PubMed Central

    Freisinger, Eva; Rother, Irene B.; Lüth, Marc Sven; Lippert, Bernhard

    2003-01-01

    If two nucleobases are crosslinked by trans-a2PtII, self-association via H bonding may take place either through individual bases or jointly through both bases. Due to the blockage of an acceptor site by the metal, the number of feasible pairing patterns can be reduced, and the preferred ones altered. If the metalated base pair as a whole undergoes association, base quartets can form. Various scenarios resulting from the application of guanine, hypoxanthine, and cytosine model nucleobases are discussed. Unconventional C—H…N hydrogen bonding has been observed in several instances. PMID:12651957

  16. Inhibition of Mycoplasma pneumoniae growth by FDA-approved anticancer and antiviral nucleoside and nucleobase analogs

    PubMed Central

    2013-01-01

    Background Mycoplasma pneumoniae (Mpn) is a human pathogen that causes acute and chronic respiratory diseases and has been linked to many extrapulmonary diseases. Due to the lack of cell wall, Mpn is resistant to antibiotics targeting cell wall synthesis such as penicillin. During the last 10 years macrolide-resistant Mpn strains have been frequently reported in Asian countries and have been spreading to Europe and the United States. Therefore, new antibiotics are needed. In this study, 30 FDA-approved anticancer or antiviral drugs were screened for inhibitory effects on Mpn growth and selected analogs were further characterized by inhibition of target enzymes and metabolism of radiolabeled substrates. Results Sixteen drugs showed varying inhibitory effects and seven showed strong inhibition of Mpn growth. The anticancer drug 6-thioguanine had a MIC (minimum inhibitory concentration required to cause 90% of growth inhibition) value of 0.20 μg ml-1, whereas trifluorothymidine, gemcitabine and dipyridamole had MIC values of approximately 2 μg ml-1. In wild type Mpn culture the presence of 6-thioguanine and dipyridamole strongly inhibited the uptake and metabolism of hypoxanthine and guanine while gemcitabine inhibited the uptake and metabolism of all nucleobases and thymidine. Trifluorothymidine and 5-fluorodeoxyuridine, however, stimulated the uptake and incorporation of radiolabeled thymidine and this stimulation was due to induction of thymidine kinase activity. Furthermore, Mpn hypoxanthine guanine phosphoribosyl transferase (HPRT) was cloned, expressed, and characterized. The 6-thioguanine, but not other purine analogs, strongly inhibited HPRT, which may in part explain the observed growth inhibition. Trifluorothymidine and 5-fluorodeoxyuridine were shown to be good substrates and inhibitors for thymidine kinase from human and Mycoplasma sources. Conclusion We have shown that several anticancer and antiviral nucleoside and nucleobase analogs are potent

  17. The Major Facilitative Folate Transporters Solute Carrier 19A1 and Solute Carrier 46A1: Biology and Role in Antifolate Chemotherapy of Cancer

    PubMed Central

    Wilson, Mike R.; Hou, Zhanjun

    2014-01-01

    This review summarizes the biology of the major facilitative membrane transporters, the reduced folate carrier (RFC) (Solute Carrier 19A1) and the proton-coupled folate transporter (PCFT) (Solute Carrier 46A1). Folates are essential vitamins, and folate deficiency contributes to a variety of health disorders. RFC is ubiquitously expressed and is the major folate transporter in mammalian cells and tissues. PCFT mediates the intestinal absorption of dietary folates and appears to be important for transport of folates into the central nervous system. Clinically relevant antifolates for cancer, such as methotrexate and pralatrexate, are transported by RFC, and loss of RFC transport is an important mechanism of methotrexate resistance in cancer cell lines and in patients. PCFT is expressed in human tumors, and is active at pH conditions associated with the tumor microenvironment. Pemetrexed is an excellent substrate for both RFC and PCFT. Novel tumor-targeted antifolates related to pemetrexed with selective membrane transport by PCFT over RFC are being developed. In recent years, there have been major advances in understanding the structural and functional properties and the regulation of RFC and PCFT. The molecular bases for methotrexate resistance associated with loss of RFC transport and for hereditary folate malabsorption, attributable to mutant PCFT, were determined. Future studies should continue to translate molecular insights from basic studies of RFC and PCFT biology into new therapeutic strategies for cancer and other diseases. PMID:24396145

  18. MATE Transporters Facilitate Vacuolar Uptake of Epicatechin 3′-O-Glucoside for Proanthocyanidin Biosynthesis in Medicago truncatula and Arabidopsis[C][W

    PubMed Central

    Zhao, Jian; Dixon, Richard A.

    2009-01-01

    Expression of the Arabidopsis thaliana MYB transcription factor TRANSPARENT TESTA 2 (TT2) in Medicago trunculata hairy roots induces both proanthocyanidin accumulation and the ATP-dependent vacuolar/vesicular uptake of epicatechin 3′-O-glucoside; neither process is active in control roots that do, however, possess anthocyanidin 3-O-glucoside vacuolar uptake activity. A vacuolar membrane-localized multidrug and toxic compound extrusion (MATE) transporter, Medicago MATE1, was identified at the molecular level and shown to preferentially transport epicatechin 3′-O-glucoside. Genetic evidence has implicated TT12, a tonoplastic MATE transporter from Arabidopsis, in the transport of precursors for proanthocyanidin biosynthesis in the seed coat. However, although Arabidopsis TT12 facilitates the transport of cyanidin 3-O-glucoside into membrane vesicles when expressed in yeast, there is no evidence that cyanidin 3-O-glucoside is converted to proanthocyanidins after transport into the vacuole. Here, we show that Arabidopsis TT12, like Medicago MATE1, functions to transport epicatechin 3′-O-glucoside as a precursor for proanthocyanidin biosynthesis, and Medicago MATE1 complements the seed proanthocyanidin phenotype of the Arabidopsis tt12 mutant both quantitatively and qualitatively. On the basis of biochemical properties, tissue-specific expression pattern, and genetic loss-of-function analysis, we conclude that MATE1 is an essential membrane transporter for proanthocyanidin biosynthesis in the Medicago seed coat. Implications of these findings for the assembly of oligomeric proanthocyanidins are discussed. PMID:19684242

  19. The major facilitative folate transporters solute carrier 19A1 and solute carrier 46A1: biology and role in antifolate chemotherapy of cancer.

    PubMed

    Matherly, Larry H; Wilson, Mike R; Hou, Zhanjun

    2014-04-01

    This review summarizes the biology of the major facilitative membrane transporters, the reduced folate carrier (RFC) (Solute Carrier 19A1) and the proton-coupled folate transporter (PCFT) (Solute Carrier 46A1). Folates are essential vitamins, and folate deficiency contributes to a variety of health disorders. RFC is ubiquitously expressed and is the major folate transporter in mammalian cells and tissues. PCFT mediates the intestinal absorption of dietary folates and appears to be important for transport of folates into the central nervous system. Clinically relevant antifolates for cancer, such as methotrexate and pralatrexate, are transported by RFC, and loss of RFC transport is an important mechanism of methotrexate resistance in cancer cell lines and in patients. PCFT is expressed in human tumors, and is active at pH conditions associated with the tumor microenvironment. Pemetrexed is an excellent substrate for both RFC and PCFT. Novel tumor-targeted antifolates related to pemetrexed with selective membrane transport by PCFT over RFC are being developed. In recent years, there have been major advances in understanding the structural and functional properties and the regulation of RFC and PCFT. The molecular bases for methotrexate resistance associated with loss of RFC transport and for hereditary folate malabsorption, attributable to mutant PCFT, were determined. Future studies should continue to translate molecular insights from basic studies of RFC and PCFT biology into new therapeutic strategies for cancer and other diseases.

  20. Biologically-validated HIV integrase inhibitors with nucleobase scaffolds: structure, synthesis, chemical biology, molecular modeling, and antiviral activity.

    PubMed

    Nair, Vasu; Uchil, Vinod; Chi, Guochen; Dams, Iwona; Cox, Arthur; Seo, Byung

    2007-01-01

    Integrase, an enzyme of the pol gene of HIV, is a significant viral target for the discovery of anti-HIV agents. In this presentation, we report on the continuation of our work on the discovery of diketo acids, constructed on nucleobase scaffolds, that are inhibitors of HIV integrase. An example of our synthetic approach to inhibitors with purine nucleobase scaffolds is given. Comparison is made between integrase inhibition data arising from compounds with pyrimidine versus purine nucleobase scaffold. Antiviral results are cited.

  1. Life Stage-Specific Cargo Receptors Facilitate Glycosylphosphatidylinositol-Anchored Surface Coat Protein Transport in Trypanosoma brucei.

    PubMed

    Kruzel, Emilia K; Zimmett, George P; Bangs, James D

    2017-01-01

    The critical virulence factor of bloodstream-form Trypanosoma brucei is the glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG). Endoplasmic reticulum (ER) exit of VSG is GPI dependent and relies on a discrete subset of COPII machinery (TbSec23.2/TbSec24.1). In other systems, p24 transmembrane adaptor proteins selectively recruit GPI-anchored cargo into nascent COPII vesicles. Trypanosomes have eight putative p24s (TbERP1 to TbERP8) that are constitutively expressed at the mRNA level. However, only four TbERP proteins (TbERP1, -2, -3, and -8) are detectable in bloodstream-form parasites. All four colocalize to ER exit sites, are required for efficient GPI-dependent ER exit, and are interdependent for steady-state stability. These results suggest shared function as an oligomeric ER GPI-cargo receptor. This cohort also mediates rapid forward trafficking of the soluble lysosomal hydrolase TbCatL. Procyclic insect-stage trypanosomes have a distinct surface protein, procyclin, bearing a different GPI anchor structure. A separate cohort of TbERP proteins (TbERP1, -2, -4, and -8) are expressed in procyclic parasites and also function in GPI-dependent ER exit. Collectively, these results suggest developmentally regulated TbERP cohorts, likely in obligate assemblies, that may recognize stage-specific GPI anchors to facilitate GPI-cargo trafficking throughout the parasite life cycle. IMPORTANCE African trypanosomes are protozoan parasites that cause African sleeping sickness. Critical to the success of the parasite is the variant surface glycoprotein (VSG), which covers the parasite cell surface and which is essential for evasion of the host immune system. VSG is membrane bound by a glycolipid (GPI) anchor that is attached in the earliest compartment of the secretory pathway, the endoplasmic reticulum (ER). We have previously shown that the anchor acts as a positive forward trafficking signal for ER exit, implying a cognate receptor mechanism for

  2. Seeding the Pregenetic Earth: Meteoritic Abundances of Nucleobases and Potential Reaction Pathways

    NASA Astrophysics Data System (ADS)

    Pearce, Ben K. D.; Pudritz, Ralph E.

    2015-07-01

    Carbonaceous chondrites are a class of meteorite known for having high contents of water and organics. In this study, the abundances of the nucleobases, i.e., the building blocks of RNA and DNA, found in carbonaceous chondrites are collated from a variety of published data and compared across various meteorite classes. An extensive review of abiotic chemical reactions producing nucleobases is then performed. These reactions are then reduced to a list of 15 individual reaction pathways that could potentially occur within meteorite parent bodies. The nucleobases guanine, adenine, and uracil are found in carbonaceous chondrites in amounts of 1-500 ppb. It is currently unknown which reaction is responsible for their synthesis within the meteorite parent bodies. One class of carbonaceous meteorite dominates the abundances of both amino acids and nucleobases—the so-called CM2 (e.g., Murchison meteorite). CR2 meteorites (e.g., Graves Nunataks) also dominate the abundances of amino acids, but are the least abundant in nucleobases. The abundances of total nucleobases in these two classes are 330 ± 250 and 16 ± 13 ppb, respectively. Guanine most often has the greatest abundances in carbonaceous chondrites with respect to the other nucleobases, but is 1-2 orders of magnitude less abundant in CM2 meteorites than glycine (the most abundant amino acid). Our survey of the reaction mechanisms for nucleobase formation suggests that Fischer-Tropsch synthesis (i.e., CO, H2, and NH3 gases reacting in the presence of a catalyst such as alumina or silica) is the most likely candidate for conditions that characterize the early states of planetesimals.

  3. Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site.

    PubMed

    Abe, Yukiko; Nakagawa, Osamu; Yamaguchi, Rie; Sasaki, Shigeki

    2012-06-01

    DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnostic purposes. In this study, we have developed nucleobase-polyamine conjugates as the AP site binding ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleobase opposite the AP site by the Watson-Crick base pair formation and that the polyamine part should contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate backbone. The nucleobase conjugated with 3,3'-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that the specific stabilization is due to specific binding of the ligands to the complementary AP site. These results have suggested that the complementary base pairs of the Watson-Crick type are formed at the AP site.

  4. Nucleobases in Space: Laboratory Studies of Polycyclic Aromatic Nitrogen Heterocycles

    NASA Technical Reports Server (NTRS)

    Elsila, Jamie; Mattioda, Andy; Bernstein, Max; Sandford, Scott; Hudgins, Doug

    2005-01-01

    Polycyclic Aromatic Nitrogen Heterocycles (PANHs) are heterocyclic aromatics Le., PAHs with carbon atoms replaced by a nitrogen atom. These molecules have been detected in meteorite extracts, and in general these nitrogen heterocycles are of astrobiological interest since this class of molecules include nucleobases, basic components of our nucleic acids. These compounds are predicted to be present in the interstellar medium and in Titan tholin, but have received relatively little attention. We will present spectra and reactions of PANHs, frozen in solid H2O at 12 K, conditions germane to astronomical observations. In contrast to simple PAHs, that do not interact strongly with solid H2O, the nitrogen atoms in PANHs are potentially capable of hydrogen bonding with H20 changing their spectra, complicating their remote detection on the surfaces of icy bodies. Moreover, we have studied the photo-chemistry of these interesting compounds under astrophysical conditions and will use our lab studies to assess a potential interstellar heritage of these compounds in carbonaceous chondrites.

  5. Accumulation of formamide in hydrothermal pores to form prebiotic nucleobases.

    PubMed

    Niether, Doreen; Afanasenkau, Dzmitry; Dhont, Jan K G; Wiegand, Simone

    2016-04-19

    Formamide is one of the important compounds from which prebiotic molecules can be synthesized, provided that its concentration is sufficiently high. For nucleotides and short DNA strands, it has been shown that a high degree of accumulation in hydrothermal pores occurs, so that temperature gradients might play a role in the origin of life [Baaske P, et al. (2007)Proc Natl Acad Sci USA104(22):9346-9351]. We show that the same combination of thermophoresis and convection in hydrothermal pores leads to accumulation of formamide up to concentrations where nucleobases are formed. The thermophoretic properties of aqueous formamide solutions are studied by means of Infrared Thermal Diffusion Forced Rayleigh Scattering. These data are used in numerical finite element calculations in hydrothermal pores for various initial concentrations, ambient temperatures, and pore sizes. The high degree of formamide accumulation is due to an unusual temperature and concentration dependence of the thermophoretic behavior of formamide. The accumulation fold in part of the pores increases strongly with increasing aspect ratio of the pores, and saturates to highly concentrated aqueous formamide solutions of ∼85 wt% at large aspect ratios. Time-dependent studies show that these high concentrations are reached after 45-90 d, starting with an initial formamide weight fraction of[Formula: see text]wt % that is typical for concentrations in shallow lakes on early Earth.

  6. Accumulation of formamide in hydrothermal pores to form prebiotic nucleobases

    PubMed Central

    Niether, Doreen; Afanasenkau, Dzmitry; Dhont, Jan K. G.

    2016-01-01

    Formamide is one of the important compounds from which prebiotic molecules can be synthesized, provided that its concentration is sufficiently high. For nucleotides and short DNA strands, it has been shown that a high degree of accumulation in hydrothermal pores occurs, so that temperature gradients might play a role in the origin of life [Baaske P, et al. (2007) Proc Natl Acad Sci USA 104(22):9346−9351]. We show that the same combination of thermophoresis and convection in hydrothermal pores leads to accumulation of formamide up to concentrations where nucleobases are formed. The thermophoretic properties of aqueous formamide solutions are studied by means of Infrared Thermal Diffusion Forced Rayleigh Scattering. These data are used in numerical finite element calculations in hydrothermal pores for various initial concentrations, ambient temperatures, and pore sizes. The high degree of formamide accumulation is due to an unusual temperature and concentration dependence of the thermophoretic behavior of formamide. The accumulation fold in part of the pores increases strongly with increasing aspect ratio of the pores, and saturates to highly concentrated aqueous formamide solutions of ∼85 wt% at large aspect ratios. Time-dependent studies show that these high concentrations are reached after 45–90 d, starting with an initial formamide weight fraction of 10−3 wt % that is typical for concentrations in shallow lakes on early Earth. PMID:27044100

  7. The role of nucleobase interactions in RNA structure and dynamics

    PubMed Central

    Bottaro, Sandro; Di Palma, Francesco; Bussi, Giovanni

    2014-01-01

    The intricate network of interactions observed in RNA three-dimensional structures is often described in terms of a multitude of geometrical properties, including helical parameters, base pairing/stacking, hydrogen bonding and backbone conformation. We show that a simple molecular representation consisting in one oriented bead per nucleotide can account for the fundamental structural properties of RNA. In this framework, canonical Watson-Crick, non-Watson-Crick base-pairing and base-stacking interactions can be unambiguously identified within a well-defined interaction shell. We validate this representation by performing two independent, complementary tests. First, we use it to construct a sequence-independent, knowledge-based scoring function for RNA structural prediction, which compares favorably to fully atomistic, state-of-the-art techniques. Second, we define a metric to measure deviation between RNA structures that directly reports on the differences in the base–base interaction network. The effectiveness of this metric is tested with respect to the ability to discriminate between structurally and kinetically distant RNA conformations, performing better compared to standard techniques. Taken together, our results suggest that this minimalist, nucleobase-centric representation captures the main interactions that are relevant for describing RNA structure and dynamics. PMID:25355509

  8. Excitation Energies of Canonical Nucleobases Computed by Multiconfigurational Perturbation Theories.

    PubMed

    Wiebeler, Christian; Borin, Veniamin; Sanchez de Araújo, Adalberto Vasconcelos; Schapiro, Igor; Borin, Antonio Carlos

    2017-05-01

    In this computational work, we assessed the performance of ab initio multireference (MR) methods for the calculation of vertical excitation energies of five nucleobases: adenine, guanine, cytosine, thymine and uracil. In total, we have studied 38 singlet and 30 triplet excited states. Where possible we used the multireference configuration interaction (MRCI) method as a reference for various flavors of multireference perturbation theory to second order. In particular, we have benchmarked CASPT2, NEVPT2 and XMCQDPT2. For CASPT2, we have analyzed the single-state, multistate (MS) and extended MS variants. In addition, we have assessed the effect of the ionization potential electron affinity (IPEA) shift. For NEVPT2, we have used the partially and the strongly contracted variants. Further, we have tested the commonly used RI-CC2, RI-ADC2 and EOM-CCSD methods. Generally, we observe the following trends for singlet excited states: NEVPT2 is the closest MR method to MRCISD+Q, closely followed by CASPT2 with the default IPEA shift. The same trend is observed for triplet states, although NEVPT2 and CASPT2-IPEA are getting closer. Interestingly, the n, π* singlet excited states were described more accurately than π, π* excited states, while for triplet states the trend is inverted except for NEVPT2. This work is an important benchmark for future photochemical investigations. © 2017 The American Society of Photobiology.

  9. Nucleobases in Space: Laboratory Studies of Polycyclic Aromatic Nitrogen Heterocycles

    NASA Technical Reports Server (NTRS)

    Elsila, Jamie; Mattioda, Andy; Bernstein, Max; Sandford, Scott; Hudgins, Doug

    2005-01-01

    Polycyclic Aromatic Nitrogen Heterocycles (PANHs) are heterocyclic aromatics Le., PAHs with carbon atoms replaced by a nitrogen atom. These molecules have been detected in meteorite extracts, and in general these nitrogen heterocycles are of astrobiological interest since this class of molecules include nucleobases, basic components of our nucleic acids. These compounds are predicted to be present in the interstellar medium and in Titan tholin, but have received relatively little attention. We will present spectra and reactions of PANHs, frozen in solid H2O at 12 K, conditions germane to astronomical observations. In contrast to simple PAHs, that do not interact strongly with solid H2O, the nitrogen atoms in PANHs are potentially capable of hydrogen bonding with H20 changing their spectra, complicating their remote detection on the surfaces of icy bodies. Moreover, we have studied the photo-chemistry of these interesting compounds under astrophysical conditions and will use our lab studies to assess a potential interstellar heritage of these compounds in carbonaceous chondrites.

  10. The nucleobase adenine as a signalling molecule in the kidney.

    PubMed

    Thimm, D; Schiedel, A C; Peti-Peterdi, J; Kishore, B K; Müller, C E

    2015-04-01

    In 2002, the first receptor activated by the nucleobase adenine was discovered in rats. In the past years, two adenine receptors (AdeRs) in mice and one in Chinese hamsters, all of which belong to the family of G protein-coupled receptors (GPCRs), were cloned and pharmacologically characterized. Based on the nomenclature for other purinergic receptor families (P1 for adenosine receptors and P2 for nucleotide, e.g. ATP, receptors), AdeRs were designated P0 receptors. Pharmacological data indicate the existence of G protein-coupled AdeRs in pigs and humans as well; however, those have not been cloned so far. Current data suggest a role for adenine and AdeRs in renal proximal tubules. Furthermore, AdeRs are suggested to be functional counterplayers of vasopressin in the collecting duct system, thus exerting diuretic effects. We are only at the beginning of understanding the significance of this new class of purinergic receptors, which might become future drug targets.

  11. DNA photoreacts by nucleobase ring cleavage to form labile isocyanates.

    PubMed

    Buschhaus, Laura; Rolf, Josefin; Kleinermanns, Karl

    2013-11-14

    Differential infrared absorption spectroscopy was used to study the formation of isocyanates and further photo-products in the oligonucleotides dG10, dC10 and dT10 and in their mononucleosides by ultraviolet light at 266 nm. We find that α-cleavage takes place in oligonucleotides and mononucleosides both in films and in solution. The very intense and spectrally isolated isocyanate (N=C=O) asymmetric stretch vibration at 2277 cm(-1) is used as a spectroscopic marker for detection of the photo-product. The band disappears upon reaction with small amounts of water vapour as expected for isocyanates. Quantum yields for isocyanate formation by nucleobase ring cleavage in the α-position to the carbonyl group are ∼5 × 10(-5) in the mononucleosides and up to 5 × 10(-4) in the oligonucleotides. In the mixed oligonucleotides dG10/dC10 and dA10/dT10 the quantum yield of α-cleavage drops by a factor of 10 compared to the single oligonucleotides. Implications for DNA repair and photo-induced DNA-protein cross-linking via isocyanate reaction with NH2 groups of amino acids are discussed.

  12. An engineered cryptic Hxt11 sugar transporter facilitates glucose-xylose co-consumption in Saccharomyces cerevisiae.

    PubMed

    Shin, Hyun Yong; Nijland, Jeroen G; de Waal, Paul P; de Jong, René M; Klaassen, Paul; Driessen, Arnold J M

    2015-01-01

    The yeast Saccharomyces cerevisiae is unable to ferment pentose sugars like d-xylose. Through the introduction of the respective metabolic pathway, S. cerevisiae is able to ferment xylose but first utilizes d-glucose before the d-xylose can be transported and metabolized. Low affinity d-xylose uptake occurs through the endogenous hexose (Hxt) transporters. For a more robust sugar fermentation, co-consumption of d-glucose and d-xylose is desired as d-xylose fermentation is in particular prone to inhibition by compounds present in pretreated lignocellulosic feedstocks. Evolutionary engineering of a d-xylose-fermenting S. cerevisiae strain lacking the major transporter HXT1-7 and GAL2 genes yielded a derivative that shows improved growth on xylose because of the expression of a normally cryptic HXT11 gene. Hxt11 also supported improved growth on d-xylose by the wild-type strain. Further selection for glucose-insensitive growth on d-xylose employing a quadruple hexokinase deletion yielded mutations at N366 of Hxt11 that reversed the transporter specificity for d-glucose into d-xylose while maintaining high d-xylose transport rates. The Hxt11 mutant enabled the efficient co-fermentation of xylose and glucose at industrially relevant sugar concentrations when expressed in a strain lacking the HXT1-7 and GAL2 genes. Hxt11 is a cryptic sugar transporter of S. cerevisiae that previously has not been associated with effective d-xylose transport. Mutagenesis of Hxt11 yielded transporters that show a better affinity for d-xylose as compared to d-glucose while maintaining high transport rates. d-glucose and d-xylose co-consumption is due to a redistribution of the sugar transport flux while maintaining the total sugar conversion rate into ethanol. This method provides a single transporter solution for effective fermentation on lignocellulosic feedstocks.

  13. Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides.

    PubMed

    Hayashi, Keisuke; Schoonbeek, Henk-Jan; De Waard, Maarten A

    2002-10-01

    Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.

  14. N-h and N-C bond activation of pyrimidinic nucleobases and nucleosides promoted by an osmium polyhydride.

    PubMed

    Esteruelas, Miguel A; García-Raboso, Jorge; Oliván, Montserrat; Oñate, Enrique

    2012-05-21

    Complex OsH(6)(P(i)Pr(3))(2) (1) reacts with 1-methylthymine and 1-methyluracil to give OsH(3)(P(i)Pr(3))(2)(nucleobase') (2, 3) containing the deprotonated nucleobases (nucleobase') κ(2)-N,O coordinated by the nitrogen atom at position 3 and the oxygen bonded to the carbon atom of the ring at position 4. Similarly, the reactions of 1 with thymidine, 5-methyluridine, deoxyuridine, and uridine lead to OsH(3)(P(i)Pr(3))(2)(nucleoside') (4-7) with the deprotonated nucleoside (nucleoside') κ(2)-N,O coordinated by the nitrogen atom at position 3 and the oxygen bonded to the carbon atom at position 4 of the nucleobases. Treatment of complexes 5 and 7, containing nucleosides derived from ribose, with OsH(2)Cl(2)(P(i)Pr(3))(2) (8) in the presence of Et(3)N affords dinuclear species OsH(3)(P(i)Pr(3))(2)(nucleobase')-(ribose)(P(i)Pr(3))(2)H(2)Os (9, 10) formed by two different metal fragments. Complex 1 also promotes the cleavage of the N-C bond of 2-7 to give the dinuclear species {OsH(3)(P(i)Pr(3))(2)}(2)(nucleobase'') (11, 12) with the nucleobase skeleton (nucleobase'') κ(2)-N,O coordinated to both metal fragments. These compounds can be also prepared by reaction of 1 with 0.5 equiv of thymine and uracil. The use of 1:1 hexahydride:nucleobase molar ratios gives rise to the preferred formation of the mononuclear complexes OsH(3)(P(i)Pr(3))(2)(nucleobase''') (13, 14; nucleobase''' = monodeprotonated thymine or uracil). The X-ray structures of complexes 6, 11, and 14 are also reported.

  15. The Formation of Nucleobases from the Irradiation of Purine in Astophysical Ices and Comparisons with Meteorites.

    NASA Technical Reports Server (NTRS)

    Sandford, S. A.; Materese, C. K.; Nuevo, M.

    2016-01-01

    N-heterocycles have been identified in meteorites and their extraterrestrial origins are suggested by isotopic ratio measurements. Although small N- heterocycles have not been detected in the interstellar medium (ISM), recent experiments in our lab have shown that the irradiation of the aromatic molecules like benzene (C6H6) and naphthalene (C10H8) in mixed molecular ices leads to the formation of O- and N-heterocyclic molecules. Among the class of N-heterocycles are the nucleobases, which are of astrobiological interest because they are the information bearing units of DNA and RNA. Nucleobases have been detected in meteorites [3-5], with isotopic signatures that are also consistent with an extraterrestrial origin. Three of the biologically relevant nucleobases (uracil, cytosine, and guanine) have a pyrimidine core structure while the remaining two (adenine and guanine) possess a purine core. Previous experiments in our lab have demonstrated that all of the bio-logical nucleobases (and numerous other molecules) with a pyrimidine core structure can be produced by irradiating pyrimidine in mixed molecular ices of several compositions [6-8]. In this work, we study the formation of purine-based molecules, including the nucleobases adenine, and guanine, from the ultraviolet (UV) irradiation of purine in ices consisting mixtures of H2O and NH3 at low temperature. The experiments are designed to simulate the astrophysical conditions under which these species may be formed in dense molecular clouds, protoplanetary disks, or on the surfaces of icy bodies in planetary systems.

  16. Meteorites and the RNA World: A Thermodynamic Model of Nucleobase Synthesis within Planetesimals

    NASA Astrophysics Data System (ADS)

    Pearce, Ben K. D.; Pudritz, Ralph E.

    2016-11-01

    The possible meteorite parent body origin of Earth's pregenetic nucleobases is substantiated by the guanine (G), adenine (A), and uracil (U) measured in various meteorites. Cytosine (C) and thymine (T), however, are absent in meteorites, making the emergence of an RNA and later RNA/DNA/protein world problematic. We investigated the meteorite parent body (planetesimal) origin of all nucleobases by computationally modeling 18 reactions that potentially contribute to nucleobase formation in such environments. Out of this list, we identified the two most important reactions for each nucleobase and found that these involve small molecules such as HCN, CO, NH3, and water that ultimately arise from the protoplanetary disks in which planetesimals are built. The primary result of this study is that cytosine is unlikely to persist within meteorite parent bodies due to aqueous deamination. Thymine has a thermodynamically favorable reaction pathway from uracil, formaldehyde, and formic acid but likely did not persist within planetesimals containing H2O2 due to an oxidation reaction with this molecule. Finally, while Fischer-Tropsch (FT) synthesis is found to be the dominant source of nucleobases within our model planetesimal, non-catalytic (NC) synthesis may still be significant under certain chemical conditions (e.g., within CR2 parent bodies). We discuss several major consequences of our results for the origin of the RNA world.

  17. High resolution mapping of modified DNA nucleobases using excision repair enzymes

    PubMed Central

    Bryan, D. Suzi; Ransom, Monica; Adane, Biniam; York, Kerri

    2014-01-01

    The incorporation and creation of modified nucleobases in DNA have profound effects on genome function. We describe methods for mapping positions and local content of modified DNA nucleobases in genomic DNA. We combined in vitro nucleobase excision with massively parallel DNA sequencing (Excision-seq) to determine the locations of modified nucleobases in genomic DNA. We applied the Excision-seq method to map uracil in E. coli and budding yeast and discovered significant variation in uracil content, wherein uracil is excluded from the earliest and latest replicating regions of the genome, possibly driven by changes in nucleotide pool composition. We also used Excision-seq to identify sites of pyrimidine dimer formation induced by UV light exposure, where the method could distinguish between sites of cyclobutane and 6-4 photoproduct formation. These UV mapping data enabled analysis of local sequence bias around pyrimidine dimers and suggested a preference for an adenosine downstream from 6-4 photoproducts. The Excision-seq method is broadly applicable for high precision, genome-wide mapping of modified nucleobases with cognate repair enzymes. PMID:25015380

  18. The Formation of Nucleobases from the UV Irradiation of Astrophysical Ice Analogs

    NASA Technical Reports Server (NTRS)

    Materese, C. K.; Nuevo, M.; Sandford, S. A.

    2017-01-01

    Nucleobases are the fundamental information bearing components of both RNA and DNA. They are central to all known terrestrial life and they are generally conserved between species. Biological nucleobases can be divided into two groups based on the N-heterocyclic molecules pyrimidine (uracil, cytosine, and thymine) and purine (adenine and guanine) respectively. Do date, no experimental conditions have been determined that could produce both pyrimidines and purines together, abiotically, in a ter-restrial environment or an early terrestrial analog. Organic materials produced in extraterrestrial envi-ronments may have been delivered to the primitive earth by comets and meteorites and may have contrib-uted to the emergence of life. To date, some, but not all nucleobases have been detected in meteorites and their isotopic signatures may be consistent with an extraterrestrial origin. Earlier work in our lab demonstrated that it is possible to produce all of the pyrimidine group nucleobases from the UV-irradiation of pyrimidine in astrophysically relevant ice analogs. Here we report our most recent work, which studied the formation of the purine group nucleobases under similar conditions.

  19. Meteorites and the RNA World: A Thermodynamic Model of Nucleobase Synthesis within Planetesimals.

    PubMed

    Pearce, Ben K D; Pudritz, Ralph E

    2016-11-01

    The possible meteorite parent body origin of Earth's pregenetic nucleobases is substantiated by the guanine (G), adenine (A), and uracil (U) measured in various meteorites. Cytosine (C) and thymine (T), however, are absent in meteorites, making the emergence of an RNA and later RNA/DNA/protein world problematic. We investigated the meteorite parent body (planetesimal) origin of all nucleobases by computationally modeling 18 reactions that potentially contribute to nucleobase formation in such environments. Out of this list, we identified the two most important reactions for each nucleobase and found that these involve small molecules such as HCN, CO, NH3, and water that ultimately arise from the protoplanetary disks in which planetesimals are built. The primary result of this study is that cytosine is unlikely to persist within meteorite parent bodies due to aqueous deamination. Thymine has a thermodynamically favorable reaction pathway from uracil, formaldehyde, and formic acid but likely did not persist within planetesimals containing H2O2 due to an oxidation reaction with this molecule. Finally, while Fischer-Tropsch (FT) synthesis is found to be the dominant source of nucleobases within our model planetesimal, non-catalytic (NC) synthesis may still be significant under certain chemical conditions (e.g., within CR2 parent bodies). We discuss several major consequences of our results for the origin of the RNA world. Key Words: Astrobiology-Cosmochemistry-Meteorites-RNA world-Abiotic organic synthesis. Astrobiology 16, 853-872.

  20. Identification of nucleobases using variable currents through graphene nanopores: A first principles study

    NASA Astrophysics Data System (ADS)

    Haraldsen, J. T.; McFarland, H.; Ahmed, T.; Zhu, J.-X.; Balatsky, A. V.

    2015-03-01

    Nanopore-based technology has the potential to be an efficient method for DNA/RNA base sequencing, as well as an identifier of other biomolecules. However, the thickness of the nanopore substrate is critical for the identification of individual nucleobases due to resulting noise and resolution problems. Recently, graphene has been suggested as a possible nanopore substrate due to its single atomic thickness and robust strength. In this study, we examine a possible device mechanism for the voltage dependence of nucleobases passing through a graphene nanopore. We utilize density functional theory with a generalized gradient approach on a graphene ribbon with a nucleobase in order to calculate the transmission spectra for each base. Transmission spectra for each base allows for the calculation of the ballistic current and differential current as a function of voltage. We show that applying various bias voltages across a graphene ribbon for the general, energy-minimized position of the translocated nucleobase, it is possible to distinguish individual bases using the resulting current. Overall, our goal is to improve nanopore device design by helping to further DNA/RNA nucleobase identification and sequencing.

  1. Simultaneous determination of 10 nucleosides and nucleobases in Antrodia camphorata using QTRAP LC-MS/MS.

    PubMed

    Chen, Fei; Zhang, Fengsu; Yang, Nianyun; Liu, Xunhong

    2014-09-01

    A liquid chromatography-triple-quadrupole linear ion trap mass spectrometry (LC-QTrap-MS) analysis has been developed for the identification and quantification of 10 nucleosides and nucleobases in extracts of Antrodia camphorata. The method was successfully used to qualitatively identify for six nucleosides namely, cytidine, uridine, inosine, guanosine, thymidine, adenosine and four nucleobases namely, uracil, guanine, xanthine, adenine in A. camphorata. Under optimized chromatographic conditions, good separation for 10 target compounds were obtained on an Agilent HC-C18(2) column (4.6 × 250 mm, 5 μm) eluted by a mobile phase of 5 mM ammonium acetate solution-methanol at a flow rate of 0.5 mL/min. Data acquisition was carried out in multiple reaction monitoring transition mode. Additional identification and confirmation of target compounds were performed using the enhanced product ion modus of the linear ion trap. It was the first report about simultaneous analysis of nucleosides and nucleobases in A. camphorata using this method. These results demonstrated that the QTRAP LC-MS/MS was a useful tool for quality evaluation of some medicinal plant products by using nucleosides and nucleobases as chemical markers. This method might also be utilized for the investigation of edible plant materials and agricultural products containing nucleosides and nucleobases. © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  2. A primary fish gill cell culture model to assess pharmaceutical uptake and efflux: Evidence for passive and facilitated transport

    PubMed Central

    Stott, Lucy C.; Schnell, Sabine; Hogstrand, Christer; Owen, Stewart F.; Bury, Nic R.

    2015-01-01

    The gill is the principle site of xenobiotic transfer to and from the aqueous environment. To replace, refine or reduce (3Rs) the large numbers of fish used in in vivo uptake studies an effective in vitro screen is required that mimics the function of the teleost gill. This study uses a rainbow trout (Oncorhynchus mykiss) primary gill cell culture system grown on permeable inserts, which tolerates apical freshwater thus mimicking the intact organ, to assess the uptake and efflux of pharmaceuticals across the gill. Bidirectional transport studies in media of seven pharmaceuticals (propranolol, metoprolol, atenolol, formoterol, terbutaline, ranitidine and imipramine) showed they were transported transcellularly across the epithelium. However, studies conducted in water showed enhanced uptake of propranolol, ranitidine and imipramine. Concentration-equilibrated conditions without a concentration gradient suggested that a proportion of the uptake of propranolol and imipramine is via a carrier-mediated process. Further study using propranolol showed that its transport is pH-dependent and at very low environmentally relevant concentrations (ng L−1), transport deviated from linearity. At higher concentrations, passive uptake dominated. Known inhibitors of drug transport proteins; cimetidine, MK571, cyclosporine A and quinidine inhibited propranolol uptake, whilst amantadine and verapamil were without effect. Together this suggests the involvement of specific members of SLC and ABC drug transporter families in pharmaceutical transport. PMID:25544062

  3. Membrane transport of camptothecin: facilitation by human P-glycoprotein (ABCB1) and multidrug resistance protein 2 (ABCC2)

    PubMed Central

    Lalloo, Anita K; Luo, Feng R; Guo, Ailan; Paranjpe, Pankaj V; Lee, Sung-Hack; Vyas, Viral; Rubin, Eric; Sinko, Patrick J

    2004-01-01

    Background The purpose of the present study was to continue the investigation of the membrane transport mechanisms of 20-(S)-camptothecin (CPT) in order to understand the possible role of membrane transporters on its oral bioavailability and disposition. Methods The intestinal transport kinetics of CPT were characterized using Caco-2 cells, MDCKII wild-type cells and MDCKII cells transfected with human P-glycoprotein (PGP) (ABCB1) or human multidrug resistance protein 2 (MRP2) (ABCC2). The effects of drug concentration, inhibitors and temperature on CPT directional permeability were determined. Results The absorptive (apical to basolateral) and secretory (basolateral to apical) permeabilities of CPT were found to be saturable. Reduced secretory CPT permeabilities with decreasing temperatures suggests the involvement of an active, transporter-mediated secretory pathway. In the presence of etoposide, the CPT secretory permeability decreased 25.6%. However, inhibition was greater in the presence of PGP and of the breast cancer resistant protein inhibitor, GF120918 (52.5%). The involvement of additional secretory transporters was suggested since the basolateral to apical permeability of CPT was not further reduced in the presence of increasing concentrations of GF120918. To investigate the involvement of specific apically-located secretory membrane transporters, CPT transport studies were conducted using MDCKII/PGP cells and MDCKII/MRP2 cells. CPT carrier-mediated permeability was approximately twofold greater in MDCKII/PGP cells and MDCKII/MRP2 cells than in MDCKII/wild-type cells, while the apparent Km values were comparable in all three cell lines. The efflux ratio of CPT in MDCKII/PGP in the presence of 0.2 μM GF120918 was not completely reversed (3.36 to 1.49). However, the decrease in the efflux ratio of CPT in MDCKII/MRP2 cells (2.31 to 1.03) suggests that CPT efflux was completely inhibited by MK571, a potent inhibitor of the Multidrug Resistance Protein

  4. Influence of biofilms on the movement of colloids in porous media. Implications for colloid facilitated transport in subsurface environments.

    PubMed

    Leon Morales, Carlos Felipe; Strathmann, Martin; Flemming, Hans-Curt

    2007-05-01

    Colloid transport through porous media can be influenced by the presence of biofilms. Sterile and non-sterile sand columns were investigated using Laponite RD as model colloid and a highly mucoid strain of Pseudomonas aeruginosa as model biofilm former. Laponite RD was marked specifically by fluorescent complexes with rhodamine 6G. Breakthrough curves (BTCs) were used as parameters for determination of colloid transport characteristics. In the sterile columns, the colloid was mobile (collision efficiencies from 0.05 to 0.08) both after the presence of Na(+) and Ca(2+) ions followed by deionised water influent. In the biofilm-grown column, the same treatment did not result in colloid retention in the case of Na(+) exposure, but in altered or enhanced colloid transport. In the case of Ca(2+) ions exposure, colloid retention increased with biofilm age. After 3 weeks, almost complete retention was observed. Similar observations were made in columns packed with material from slow sand filtration units. These data reveal the complex interactions between biofilms, cations and colloid transport. Changes in the electrolyte composition of water percolating the subsurface can frequently occur and will result in different colloid transport characteristics with regard to the dominating species of ions and the relative abundance of microbial biofilms. This has to be considered when modelling colloid transport through the subsurface.

  5. Photoelectron Spectroscopy of Rare-Gas Solvated Nucleobase Anions

    NASA Astrophysics Data System (ADS)

    Buonaugurio, Angela M.; Chen, Jing; Bowen, Kit H.

    2012-06-01

    Gas-phase polar molecular anions [uracil (U^-), thymine (T^-), 1-3 dimethyluracil (DMU^-)] solvated by rare gas atoms were studied by means of negative ion photoelectron spectroscopy. The photoelectron spectrum (PES) of U^-, T^-, and DMU^- each exhibit a distinctive dipole-bound (DB) spectral signature. The spectra of U^-, U^- (Ar)_1,2 and U^- (Kr)_1 also only displayed the DB anion feature. Upon the solvation of more rare gas atoms, the spectra of U^- (Ar)_3, U^- (Kr)_2, and U^- (Xe)1-3 not only retained the DB signature but also exhibited the valence anion features. Moreover, the DB and the valence features shifted together to higher electron binding energies (EBEs) with increasing numbers of rare gas solvent atoms. Therefore, the co-existing DB and the valence anions appeared to be strongly coupled with each other, i.e. they effectively form a single state that is a superposition of both DB and valence anion states. For both U^- and T^- series, the ``onset size" of the Xe, Kr, and Ar solvents for the co-existing of the two anionic states was 1, 2, and 3 respectively. In addition, a minimum of 2 methane (CH_4) molecules or 1 ethane (C_2H_6) molecule were required to induce the coupling between the two states in the T^- series. Thus, the nucleobase anion interaction with non-polar solvent atoms tracks as the sum of the solvent polarizabilities. However for the DMU- series, the DB and the valence anions of DMU^-(Xe)_1, DMU^-(Kr)_2, and DMU^-(Ar)_3 were completely absent in both the mass spectra and the PES. Beyond these ``holes", their PES displayed the similar behaviors to the U^- and T^- series. Extrapolated EA values for these missing species were at or very close to zero, which may explain why they were not seen. However, why this was the case is not clear. With better Franck-Condon overlap between the origins of the NB^- (Rg)_n valence anion and the neutral NB(Rg)n than between those of the NB^- (H2O)n valence anion and the neutral NB(H2O)n, extrapolation of

  6. Intermolecular interaction in nucleobases and dimethyl sulfoxide/water molecules: A DFT, NBO, AIM and NCI analysis.

    PubMed

    Venkataramanan, Natarajan Sathiyamoorthy; Suvitha, Ambigapathy; Kawazoe, Yoshiyuki

    2017-10-02

    This study aims to cast light on the physico-chemical nature and energetics of interactions between the nucleobases and water/DMSO molecules which occurs through the non-conventional CH⋯O/N-H bonds using a comprehensive quantum-chemical approach. The computed interaction energies do not show any appreciable change for all the nucleobase-solvent complexes, conforming the experimental findings on the hydration enthalpies. Compared to water, DMSO form complexes with high interaction energies. The quantitative molecular electrostatic potentials display a charge transfer during the complexation. NBO analysis shows the nucleobase-DMSO complexes, have higher stabilization energy values than the nucleobase-water complexes. AIM analysis illustrates that the in the nucleobase-DMSO complexes, SO⋯H-N type interaction have strongest hydrogen bond strength with high EHB values. Furthermore, the Laplacian of electron density and total electron density were negative indicating the partial covalent nature of bonding in these systems, while the other bonds are classified as noncovalent interactions. EDA analysis indicates, the electrostatic interaction is more pronounced in the case of nucleobase-water complexes, while the dispersion contribution is more dominant in nucleobase-DMSO complexes. NCI-RDG analysis proves the existence of strong hydrogen bonding in nucleobase-DMSO complex, which supports the AIM results. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. The phage shock protein PspA facilitates divalent metal transport and is required for virulence of Salmonella enterica sv. Typhimurium.

    PubMed

    Karlinsey, Joyce E; Maguire, Michael E; Becker, Lynne A; Crouch, Marie-Laure V; Fang, Ferric C

    2010-11-01

    The phage shock protein (Psp) system is induced by extracytoplasmic stress and thought to be important for the maintenance of proton motive force. We investigated the contribution of PspA to Salmonella virulence. A pspA deletion mutation significantly attenuates the virulence of Salmonella enterica serovar Typhimurium following intraperitoneal inoculation of C3H/HeN (Ity(r) ) mice. PspA was found to be specifically required for virulence in mice expressing the natural resistance-associated macrophage protein 1 (Nramp1) (Slc11a1) divalent metal transporter, which restricts microbial growth by limiting the availability of essential divalent metals within the phagosome. Salmonella competes with Nramp1 by expressing multiple metal uptake systems including the Nramp-homologue MntH, the ABC transporter SitABCD and the ZIP family transporter ZupT. PspA was found to facilitate Mn(2+) transport by MntH and SitABCD, as well as Zn(2+) and Mn(2+) transport by ZupT. In vitro uptake of (54) Mn(2+) by MntH and ZupT was reduced in the absence of PspA. Transport-deficient mutants exhibit reduced viability in the absence of PspA when grown under metal-limited conditions. Moreover, the ZupT transporter is required for Salmonella enterica serovar Typhimurium virulence in Nramp1-expressing mice. We propose that PspA promotes Salmonella virulence by maintaining proton motive force, which is required for the function of multiple transporters mediating bacterial divalent metal acquisition during infection.

  8. Dissociative electron attachment to the gas-phase nucleobase hypoxanthine

    SciTech Connect

    Dawley, M. Michele; Tanzer, Katrin; Denifl, Stephan E-mail: Sylwia.Ptasinska.1@nd.edu; Carmichael, Ian; Ptasińska, Sylwia E-mail: Sylwia.Ptasinska.1@nd.edu

    2015-06-07

    We present high-resolution measurements of the dissociative electron attachment (DEA) to isolated gas-phase hypoxanthine (C{sub 5}H{sub 4}N{sub 4}O, Hyp), a tRNA purine base. The anion mass spectra and individual ion efficiency curves from Hyp were measured as a function of electron energy below 9 eV. The mass spectra at 1 and 6 eV exhibit the highest anion yields, indicating possible common precursor ions that decay into the detectable anionic fragments. The (Hyp − H) anion (C{sub 5}H{sub 3}N{sub 4}O{sup −}) exhibits a sharp resonant peak at 1 eV, which we tentatively assign to a dipole-bound state of the keto-N1H,N9H tautomer in which dehydrogenation occurs at either the N1 or N9 position based upon our quantum chemical computations (B3LYP/6-311+G(d,p) and U(MP2-aug-cc-pVDZ+)) and prior studies with adenine. This closed-shell dehydrogenated anion is the dominant fragment formed upon electron attachment, as with other nucleobases. Seven other anions were also observed including (Hyp − NH){sup −}, C{sub 4}H{sub 3}N{sub 4}{sup −}/C{sub 4}HN{sub 3}O{sup −}, C{sub 4}H{sub 2}N{sub 3}{sup −}, C{sub 3}NO{sup −}/HC(HCN)CN{sup −}, OCN{sup −}, CN{sup −}, and O{sup −}. Most of these anions exhibit broad but weak resonances between 4 and 8 eV similar to many analogous anions from adenine. The DEA to Hyp involves significant fragmentation, which is relevant to understanding radiation damage of biomolecules.

  9. Accumulation of formamide in hydrothermal pores to form prebiotic nucleobases

    NASA Astrophysics Data System (ADS)

    Niether, Doreen; Afanasenkau, Dzmitry; Dhont, Jan K. G.

    2016-04-01

    Formamide is one of the important compounds from which prebiotic molecules can be synthesized, provided that its concentration is sufficiently high. For nucleotides and short DNA strands, it has been shown that a high degree of accumulation in hydrothermal pores occurs, so that temperature gradients might play a role in the origin of life [Baaske P, et al. (2007) Proc Natl Acad Sci USA 104(22):9346-9351]. We show that the same combination of thermophoresis and convection in hydrothermal pores leads to accumulation of formamide up to concentrations where nucleobases are formed. The thermophoretic properties of aqueous formamide solutions are studied by means of Infrared Thermal Diffusion Forced Rayleigh Scattering. These data are used in numerical finite element calculations in hydrothermal pores for various initial concentrations, ambient temperatures, and pore sizes. The high degree of formamide accumulation is due to an unusual temperature and concentration dependence of the thermophoretic behavior of formamide. The accumulation fold in part of the pores increases strongly with increasing aspect ratio of the pores, and saturates to highly concentrated aqueous formamide solutions of ˜85 wt% at large aspect ratios. Time-dependent studies show that these high concentrations are reached after 45-90 d, starting with an initial formamide weight fraction of 10-310-3 wt % that is typical for concentrations in shallow lakes on early Earth.

  10. The origin of efficient triplet state population in sulfur-substituted nucleobases.

    PubMed

    Mai, Sebastian; Pollum, Marvin; Martínez-Fernández, Lara; Dunn, Nicholas; Marquetand, Philipp; Corral, Inés; Crespo-Hernández, Carlos E; González, Leticia

    2016-10-05

    Elucidating the photophysical mechanisms in sulfur-substituted nucleobases (thiobases) is essential for designing prospective drugs for photo- and chemotherapeutic applications. Although it has long been established that the phototherapeutic activity of thiobases is intimately linked to efficient intersystem crossing into reactive triplet states, the molecular factors underlying this efficiency are poorly understood. Herein we combine femtosecond transient absorption experiments with quantum chemistry and nonadiabatic dynamics simulations to investigate 2-thiocytosine as a necessary step to unravel the electronic and structural elements that lead to ultrafast and near-unity triplet-state population in thiobases in general. We show that different parts of the potential energy surfaces are stabilized to different extents via thionation, quenching the intrinsic photostability of canonical DNA and RNA nucleobases. These findings satisfactorily explain why thiobases exhibit the fastest intersystem crossing lifetimes measured to date among bio-organic molecules and have near-unity triplet yields, whereas the triplet yields of canonical nucleobases are nearly zero.

  11. Survival of gas phase amino acids and nucleobases in space radiation conditions

    NASA Astrophysics Data System (ADS)

    Pilling, S.; Andrade, D. P. P.; de Castilho, R. B.; Cavasso-Filho, R. L.; Lago, A. F.; Coutinho, L. H.; de Souza, G. G. B.; Boechat-Roberty, H. M.; de Brito, A. Naves

    2008-10-01

    We present experimental studies on the photoionization and photodissociation processes (photodestruction) of gaseous amino acids and nucleobases in interstellar and interpla-netary radiation analogs conditions. The measurements have been undertaken at the Brazilian Synchrotron Light Laboratory (LNLS), employing vacuum ultraviolet (VUV) and soft X-ray photons. The experimental set up basically consists of a time-of-flight mass spectrometer kept under high vacuum conditions. Mass spectra were obtained using a photoelectron photoion coincidence technique. We have shown that the amino acids are effectively more destroyed (up to 70 80%) by the stellar radiation than the nucleobases, mainly in the VUV. Since polycyclic aromatic hydrocarbons have the same survival capability and seem to be ubiquitous in the ISM, it is not unreasonable to predict that nucleobases could survive in the interstellar medium and/or in comets, even as a stable cation.

  12. The origin of efficient triplet state population in sulfur-substituted nucleobases

    NASA Astrophysics Data System (ADS)

    Mai, Sebastian; Pollum, Marvin; Martínez-Fernández, Lara; Dunn, Nicholas; Marquetand, Philipp; Corral, Inés; Crespo-Hernández, Carlos E.; González, Leticia

    2016-10-01

    Elucidating the photophysical mechanisms in sulfur-substituted nucleobases (thiobases) is essential for designing prospective drugs for photo- and chemotherapeutic applications. Although it has long been established that the phototherapeutic activity of thiobases is intimately linked to efficient intersystem crossing into reactive triplet states, the molecular factors underlying this efficiency are poorly understood. Herein we combine femtosecond transient absorption experiments with quantum chemistry and nonadiabatic dynamics simulations to investigate 2-thiocytosine as a necessary step to unravel the electronic and structural elements that lead to ultrafast and near-unity triplet-state population in thiobases in general. We show that different parts of the potential energy surfaces are stabilized to different extents via thionation, quenching the intrinsic photostability of canonical DNA and RNA nucleobases. These findings satisfactorily explain why thiobases exhibit the fastest intersystem crossing lifetimes measured to date among bio-organic molecules and have near-unity triplet yields, whereas the triplet yields of canonical nucleobases are nearly zero.

  13. Nucleobases and Other Prebiotic Species from the UV Irradiation of Pyrimidine in Astrophysical Ices

    NASA Technical Reports Server (NTRS)

    Sandford, Scott; Materese, Christopher; Nuevo, Michel

    2012-01-01

    Nucleobases are aromatic N-heterocycles that constitute the informational subunits of DNA and RNA and are divided into two families: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites and their extraterrestrial origin confirmed by isotope measurement. Although no N-heterocycles have been individually identified in the ISM, the 6.2-micron interstellar emission feature seen towards many astronomical objects suggests a population of such molecules is likely present. We report on a study of the formation of pyrimidine-based molecules, including nucleobases and other species of prebiotic interest, from the ultraviolet (UV) irradiation of pyrimidine in low temperature ices containing H2O, NH3, C3OH, and CH4, to simulate the astrophysical conditions under which prebiotic species may be formed in the Solar System.

  14. Conformational Change of a Tryptophan Residue in BtuF Facilitates Binding and Transport of Cobinamide by the Vitamin B12 Transporter BtuCD-F

    PubMed Central

    Mireku, S. A.; Ruetz, M.; Zhou, T.; Korkhov, V. M.; Kräutler, B.; Locher, K. P.

    2017-01-01

    BtuCD-F is an ABC transporter that mediates cobalamin uptake into Escherichia coli. Early in vivo data suggested that BtuCD-F might also be involved in the uptake of cobinamide, a cobalamin precursor. However, neither was it demonstrated that BtuCD-F indeed transports cobinamide, nor was the structural basis of its recognition known. We synthesized radiolabeled cyano-cobinamide and demonstrated BtuCD-catalyzed in vitro transport, which was ATP- and BtuF-dependent. The crystal structure of cobinamide-bound BtuF revealed a conformational change of a tryptophan residue (W66) in the substrate binding cleft compared to the structure of cobalamin-bound BtuF. High-affinity binding of cobinamide was dependent on W66, because mutation to most other amino acids substantially reduced binding. The structures of three BtuF W66 mutants revealed that tight packing against bound cobinamide was only provided by tryptophan and phenylalanine, in line with the observed binding affinities. In vitro transport rates of cobinamide and cobalamin were not influenced by the substitutions of BtuF W66 under the experimental conditions, indicating that W66 has no critical role in the transport reaction. Our data present the molecular basis of the cobinamide versus cobalamin specificity of BtuCD-F and provide tools for in vitro cobinamide transport and binding assays. PMID:28128319

  15. M4FT-16LL080303052-State of Knowledge for Colloid Facilitated Radionuclide Transport and Update on Actinide Diffusion in Bentonite Backfill

    SciTech Connect

    Zavarin, Mavrik; Joseph, C.

    2016-08-16

    This progress report (Level 4 Milestone Number M4FT-16LL080303052) summarizes research conducted at Lawrence Livermore National Laboratory (LLNL) within the Crystalline Disposal R&D Activity Number FT-16LL080303051 and Crystalline International Collaborations Activity Number FT-16LL080303061. The focus of this research is the interaction of radionuclides with Engineered Barrier System (EBS) and host rock materials at various physico-chemical conditions relevant to subsurface repository environments. They include both chemical and physical processes such as solubility, sorption, and diffusion. The colloid-facilitated transport effort focused on preparation of a draft manuscript summarizing the state of knowledge and parameterization of colloid-facilitated transport mechanisms in support of reactive transport and performance assessment models for generic crystalline repositories. This draft manuscript is being submitted as a level 3 milestone with LANL as the primary author. LLNL’s contribution to that effort is summarized only briefly in the present report. A manuscript summarizing long-term U(VI) diffusion experiments through bentonite backfill material was recently accepted for publication; the contents of that manuscript are summarized in the present report. The Np(IV) diffusion experiments were started mid-year and are ongoing. The completion of these experiments is planned for early FY17. Our progress in quantifying Np(IV) diffusion in bentonite backfill is summarized in the present report. Our involvement with the NEA TDB project was summarized in a recent Argillite Disposal activity report. It is not included in this report.

  16. AtSWEET4, a hexose facilitator, mediates sugar transport to axial sinks and affects plant development

    PubMed Central

    Liu, Xiaozhu; Zhang, Yan; Yang, Chao; Tian, Zhihong; Li, Jianxiong

    2016-01-01

    Plants transport photoassimilates from source organs to sink tissues through the phloem translocation pathway. In the transport phloem, sugars that escape from the sieve tubes are released into the apoplasmic space between the sieve element/companion cell complex (SE/CC) and phloem parenchyma cells (PPCs) during the process of long-distance transport. The competition for sugar acquisition between SE/CC and adjoining PPCs is mediated by plasma membrane translocators. YFP-tagged AtSWEET4 protein is localized in the plasma membrane, and PromoterAtSWEET4-GUS analysis showed that AtSWEET4 is expressed in the stele of roots and veins of leaves and flowers. Overexpression of AtSWEET4 in Arabidopsis increases plant size and accumulates more glucose and fructose. By contrast, knock-down of AtSWEET4 by RNA-interference leads to small plant size, reduction in glucose and fructose contents, chlorosis in the leaf vein network, and reduction in chlorophyll content in leaves. Yeast assays demonstrated that AtSWEET4 is able to complement both fructose and glucose transport deficiency. Transgenic plants of AtSWEET4 overexpression exhibit higher freezing tolerance and support more growth of bacterium Pseudomonas syringae pv. phaseolicola NPS3121. We conclude that AtSWEET4 plays an important role in mediating sugar transport in axial tissues during plant growth and development. PMID:27102826

  17. A single-component multidrug transporter of the major facilitator superfamily is part of a network that protects E scherichia coli from bile salt stress

    PubMed Central

    Paul, Stephanie; Alegre, Kamela O; Holdsworth, Scarlett R; Rice, Matthew; Brown, James A; McVeigh, Paul; Kelly, Sharon M; Law, Christopher J

    2014-01-01

    Resistance to high concentrations of bile salts in the human intestinal tract is vital for the survival of enteric bacteria such as E scherichia coli. Although the tripartite AcrAB–TolC efflux system plays a significant role in this resistance, it is purported that other efflux pumps must also be involved. We provide evidence from a comprehensive suite of experiments performed at two different pH values (7.2 and 6.0) that reflect pH conditions that E . coli may encounter in human gut that MdtM, a single-component multidrug resistance transporter of the major facilitator superfamily, functions in bile salt resistance in E . coli by catalysing secondary active transport of bile salts out of the cell cytoplasm. Furthermore, assays performed on a chromosomal ΔacrB mutant transformed with multicopy plasmid encoding MdtM suggested a functional synergism between the single-component MdtM transporter and the tripartite AcrAB–TolC system that results in a multiplicative effect on resistance. Substrate binding experiments performed on purified MdtM demonstrated that the transporter binds to cholate and deoxycholate with micromolar affinity, and transport assays performed on inverted vesicles confirmed the capacity of MdtM to catalyse electrogenic bile salt/H+ antiport. PMID:24684269

  18. MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples.

    PubMed

    Hu, Da-Gang; Sun, Cui-Hui; Ma, Qi-Jun; You, Chun-Xiang; Cheng, Lailiang; Hao, Yu-Jin

    2016-03-01

    Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H(+)-pumping activities of vacuolar H(+)-ATPase (VHA) and/or vacuolar H(+)-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H(+)-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants. © 2016 American Society of Plant Biologists. All Rights Reserved.

  19. MdMYB1 Regulates Anthocyanin and Malate Accumulation by Directly Facilitating Their Transport into Vacuoles in Apples1[OPEN

    PubMed Central

    Hu, Da-Gang; Sun, Cui-Hui; Ma, Qi-Jun; You, Chun-Xiang; Hao, Yu-Jin

    2016-01-01

    Tonoplast transporters, including proton pumps and secondary transporters, are essential for plant cell function and for quality formation of fleshy fruits and ornamentals. Vacuolar transport of anthocyanins, malate, and other metabolites is directly or indirectly dependent on the H+-pumping activities of vacuolar H+-ATPase (VHA) and/or vacuolar H+-pyrophosphatase, but how these proton pumps are regulated in modulating vacuolar transport is largely unknown. Here, we report a transcription factor, MdMYB1, in apples that binds to the promoters of two genes encoding the B subunits of VHA, MdVHA-B1 and MdVHA-B2, to transcriptionally activate its expression, thereby enhancing VHA activity. A series of transgenic analyses in apples demonstrates that MdMYB1/10 controls cell pH and anthocyanin accumulation partially by regulating MdVHA-B1 and MdVHA-B2. Furthermore, several other direct target genes of MdMYB10 are identified, including MdVHA-E2, MdVHP1, MdMATE-LIKE1, and MdtDT, which are involved in H+-pumping or in the transport of anthocyanins and malates into vacuoles. Finally, we show that the mechanism by which MYB controls malate and anthocyanin accumulation in apples also operates in Arabidopsis (Arabidopsis thaliana). These findings provide novel insights into how MYB transcription factors directly modulate the vacuolar transport system in addition to anthocyanin biosynthesis, consequently controlling organ coloration and cell pH in plants. PMID:26637549

  20. Computational analysis of stacking interactions between 3-nitropyrrole and natural nucleobases.

    PubMed

    Ukawa, Hisashi; Seio, Kohji; Sekine, Mitsuo

    2002-01-01

    The stacking energies between natural nucleobases and a universal base of 3-nitropyrrole (3-NP) were calculated by use of two theoretically independent quantum chemical methods, namely, molecular orbital (MO) and density function theory (DFT) calculations. The parameters required for molecular mechanics calculation of 3-NP were obtained by use of a software of Direct Force Field and used to evaluate the stacking energy of the complexes formed between 3-NP and canonical four nucleobases. Dependence of the twist angle between the two stacked bases on the stacking energy was studied in great detail.

  1. Infrared spectral investigations of UV irradiated nucleobases adsorbed on mineral surfaces

    NASA Astrophysics Data System (ADS)

    Fornaro, Teresa; Brucato, John Robert; Pace, Emanuele; Guidi, Mariangela Cestelli; Branciamore, Sergio; Pucci, Amaranta

    2013-09-01

    The interaction between electromagnetic radiation and bio-molecules in heterogeneous environments is a prebiotically relevant process. Minerals may have a pivotal role in the prebiotic evolution of complex chemical systems, mediating the effects of electromagnetic radiation, influencing the photostability of bio-molecules, catalyzing important chemical reactions and/or protecting molecules against degradation. In particular, nucleobases are relevant bio-molecules to investigate both in the prebiotic context, because they are coding components of nucleic acids, and from the standpoint of the survival of biological systems in space conditions. Several studies on the photodynamics of nucleobases suggest that their structure could have been naturally selected for the ability to dissipate electronic energy through ultrafast photophysical decay. Considering the putative involvement of minerals in the prebiotic chemistry, it is necessary to study the photostability of nucleobases under space conditions in the presence of mineral matrices, to investigate both the prebiotic processes that might have had a role in the development of the first living entities on Earth and the physical and chemical processes occurring in extraterrestrial environments. We focused our study on the characterization of the nature of the interaction between nucleobases and the surface of the minerals magnesium oxide and forsterite by infrared vibrational spectroscopy. We observed that most of the characteristic bands of pure nucleobases vanished when adsorbed on magnesium oxide. On the contrary, in the case of adenine and uracil adsorbed on forsterite, very intense nucleobase absorption peaks appeared. This phenomenon pertains to the surface selection rules changes related to molecular orientation. Moreover, based on the vibrational shifts, we deduced the molecular interaction sites with the mineral surfaces. Furthermore, we investigated the photostability of nucleobases adsorbed on such minerals

  2. Infrared spectral investigations of UV irradiated nucleobases adsorbed on mineral surfaces

    NASA Astrophysics Data System (ADS)

    Brucato, John Robert; Pace, Emanuele; Pucci, Amaranta; Branciamore, Sergio; Cestelli Guidi, Mariangela; Fornaro, Teresa

    The interaction between electromagnetic radiation and bio-molecules in heterogeneous environments is a prebiotically relevant process. Minerals may have a pivotal role in the prebiotic evolution of complex chemical systems, mediating the effects of electromagnetic radiation, influencing the photostability of bio-molecules, catalyzing important chemical reactions and/or protecting molecules against degradation. In particular, nucleobases are relevant bio-molecules to investigate both in the prebiotic context, because they are coding components of nucleic acids, and from the standpoint of the survival of biological systems in space conditions. In this talk, laboratory results on photostability of nucleobases adsorbed on minerals will be presented.

  3. Assessment of new triplet forming artificial nucleobases as RNA ligands directed towards HCV IRES IIId loop.

    PubMed

    Safir Filho, Mauro; Martin, Anthony R; Benhida, Rachid

    2017-04-15

    We report the synthesis of two new artificial nucleobase scaffolds, 1 and 2, featuring adequate hydrogen bonding donors and acceptors for the molecular recognition of U:A and C:G base pairs, respectively. The tethering of these structures to various amino acids and the assessment of these artificial nucleobase-amino acid conjugates as RNA ligands against a model of HCV IRES IIId domain are also reported. Compound 1e displayed the highest affinity (Kd twice lower than neomycin - control). Moreover, it appears that this interaction is enthalpically and entropically favored.

  4. Humic acid facilitates the transport of ARS-labeled hydroxyapatite nanoparticles in iron oxyhydroxide-coated sand

    USDA-ARS?s Scientific Manuscript database

    Hydroxyapatite nanoparticles (nHAP) have been widely used to remediate soil and wastewater contaminated with metals and radionuclides. However, our understanding of nHAP transport and fate is limited in natural environments that exhibit significant variability in solid and solution chemistry. The tr...

  5. Reptation-induced coalescence of tunnels and cavities in Escherichia Coli XylE transporter conformers accounts for facilitated diffusion.

    PubMed

    Cunningham, Philip; Naftalin, Richard J

    2014-11-01

    Structural changes and xylose docking to eight conformers of Escherichia Coli XylE, a xylose transporter similar to mammalian passive glucose transporters GLUTs, have been examined. Xylose docks to inward and outward facing conformers at a high affinity central site (K(i) 4-20 µM), previously identified by crystallography and additionally consistently docks to lower affinity sites in the external and internal vestibules (K(i) 12-50 µM). All these sites lie within intramolecular tunnels and cavities. Several local regions in the central transmembrane zone have large positional divergences of both skeleton carbon Cα positions and side chains. One such in TM 10 is the destabilizing sequence G388-P389-V390-C391 with an average RMSD (4.5 ± 0.4 Å). Interchange between conformer poses results in coalescence of tunnels with adjacent cavities, thereby producing a transitory channel spanning the entire transporter. A fully open channel exists in one inward-facing apo-conformer, (PDB 4ja4c) as demonstrated by several different tunnel-finding algorithms. The conformer interchanges produce a gated network within a branched central channel that permits staged ligand diffusion across the transporter during the open gate periods. Simulation of this model demonstrates that small-scale conformational changes required for sequentially opening gate with frequencies in the ns-μs time domain accommodate diffusive ligand flow between adjacent sites with association-dissociation rates in the μs-ms domain without imposing delays. This current model helps to unify the apparently opposing concepts of alternate access and multisite models of ligand transport.

  6. Facilitated transport of titanium dioxide nanoparticles via hydrochars in the presence of ammonium in saturated sands: Effects of pH, ionic strength, and ionic composition.

    PubMed

    Xu, Nan; Cheng, Xueying; Zhou, Kairong; Xu, Xiaoting; Li, Zuling; Chen, Jianping; Wang, Dongtian; Li, Duo

    2017-09-08

    The widespread use of nanoparticles (NPs) has led to their inevitable introduction into environmental systems. How the existence of hydrochars in crop soils will affect the mobility of nanoparticle titanium dioxide (nTiO2), especially in the presence of ammonium (NH4(+)), remains unknown. Research is needed to study the effects of hydrochars on the transport and retention of nTiO2 and to uncover the mechanisms of these effects on nTiO2 transport. Column experiments with nTiO2 and hydrochars were performed in various electrolyte (NaCl, NH4Cl, and CaCl2) solutions under a controlled pH (6.0 and 8.0). Additionally, the size distributions and scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the NPs were observed. The experimental results suggested that the mobility of the hydrochars was much better than that of nTiO2. Thus, the mobility of nTiO2 was improved upon their attachment to the hydrochars. The facilitated transport of nTiO2 in the presence of hydrochars was stronger at pH8.0 than at pH6.0, and facilitated transport was nearly independent of the electrolyte cation at pH8.0. However, at pH6.0, the facilitated transport in various electrolytes had the following order: NaCl>NH4Cl>CaCl2. The conversion from a completely reversible to a partially irreversible deposition of nTiO2 in sand was induced by the partially irreversible retention of hydrochars, and this phenomenon was more pronounced in the presence of NH4(+) than in the presence of Na(+). In particular, the irreversible deposition of nTiO2-hydrochars was enhanced as the cation concentration increased. The increased irreversible retention of nTiO2 was related to the greater k2 value (irreversible attachment coefficients) on site 2 for hydrochars based on two-site kinetic retention modeling. Thus, there is a potential risk of contaminating crops, soil, and underground water when nTiO2 exists in a hydrochar-amended environment, especially when associated with NH4-N

  7. Blue blood on ice: modulated blood oxygen transport facilitates cold compensation and eurythermy in an Antarctic octopod.

    PubMed

    Oellermann, Michael; Lieb, Bernhard; Pörtner, Hans-O; Semmens, Jayson M; Mark, Felix C

    2015-01-01

    The Antarctic Ocean hosts a rich and diverse fauna despite inhospitable temperatures close to freezing, which require specialist adaptations to sustain animal activity and various underlying body functions. While oxygen transport has been suggested to be key in setting thermal tolerance in warmer climates, this constraint is relaxed in Antarctic fishes and crustaceans, due to high levels of dissolved oxygen. Less is known about how other Antarctic ectotherms cope with temperatures near zero, particularly the more active invertebrates like the abundant octopods. A continued reliance on the highly specialised blood oxygen transport system of cephalopods may concur with functional constraints at cold temperatures. We therefore analysed the octopod's central oxygen transport component, the blue blood pigment haemocyanin, to unravel strategies that sustain oxygen supply at cold temperatures. To identify adaptive compensation of blood oxygen transport in octopods from different climatic regions, we compared haemocyanin oxygen binding properties, oxygen carrying capacities as well as haemolymph protein and ion composition between the Antarctic octopod Pareledone charcoti, the South-east Australian Octopus pallidus and the Mediterranean Eledone moschata. In the Antarctic Pareledone charcoti at 0°C, oxygen unloading by haemocyanin was poor but supported by high levels of dissolved oxygen. However, lower oxygen affinity and higher oxygen carrying capacity compared to warm water octopods, still enabled significant contribution of haemocyanin to oxygen transport at 0°C. At warmer temperatures, haemocyanin of Pareledone charcoti releases most of the bound oxygen, supporting oxygen supply at 10°C. In warm water octopods, increasing oxygen affinities reduce the ability to release oxygen from haemocyanin at colder temperatures. Though, unlike Eledone moschata, Octopus pallidus attenuated this increase below 15°C. Adjustments of haemocyanin physiological function and

  8. BC4707 Is a Major Facilitator Superfamily Multidrug Resistance Transport Protein from Bacillus cereus Implicated in Fluoroquinolone Tolerance

    PubMed Central

    Simm, Roger; Vörös, Aniko; Ekman, Jaakko V.; Sødring, Marianne; Nes, Ingerid; Kroeger, Jasmin K.; Saidijam, Massoud; Bettaney, Kim E.; Henderson, Peter J. F.; Salkinoja-Salonen, Mirja; Kolstø, Anne-Brit

    2012-01-01

    Transcriptional profiling highlighted a subset of genes encoding putative multidrug transporters in the pathogen Bacillus cereus that were up-regulated during stress produced by bile salts. One of these multidrug transporters (BC4707) was selected for investigation. Functional characterization of the BC4707 protein in Escherichia coli revealed a role in the energized efflux of xenobiotics. Phenotypic analyses after inactivation of the gene bc4707 in Bacillus cereus ATCC14579 suggested a more specific, but modest role in the efflux of norfloxacin. In addition to this, transcriptional analyses showed that BC4707 is also expressed during growth of B. cereus under non-stressful conditions where it may have a role in the normal physiology of the bacteria. Altogether, the results indicate that bc4707, which is part of the core genome of the B. cereus group of bacteria, encodes a multidrug resistance efflux protein that is likely involved in maintaining intracellular homeostasis during growth of the bacteria. PMID:22615800

  9. Cloning and expression pattern of facilitative glucose transporter 1 (GLUT1) in gilthead sea bream Sparus aurata in response to salinity acclimation.

    PubMed

    Balmaceda-Aguilera, C; Martos-Sitcha, J A; Mancera, J M; Martínez-Rodríguez, G

    2012-09-01

    Facilitative glucose transporters (GLUT) are transmembrane transporter proteins involved in glucose transport across the plasma membrane. In fish, several GLUT mRNAs have been cloned, but to date there is no information about these transporters in the marine euryhaline teleost Sparus aurata. In the present study we obtained the complete coding sequence from S. aurata GLUT1 (saGLUT1), composed by 4483 bases, presenting a 79 to 95% identity with respect to other fish GLUT1 mRNAs. The analysis of the 5' and 3' UTRs showed the presence of several post-transcriptional regulatory elements. In addition, the effect on saGLUT1 mRNA expression of the osmotic acclimation to four different environmental salinities (5, 12, 40 and 55 ppt), in gills, kidney, liver and brain, was studied. Changes in mRNA expression levels were detected in gills and brain, indicating that GLUT1 has an important role in these organs for osmotic acclimation in S. aurata.

  10. The activity of anandamide at vanilloid VR1 receptors requires facilitated transport across the cell membrane and is limited by intracellular metabolism.

    PubMed

    De Petrocellis, L; Bisogno, T; Maccarrone, M; Davis, J B; Finazzi-Agro, A; Di Marzo, V

    2001-04-20

    The endogenous ligand of CB(1) cannabinoid receptors, anandamide, is also a full agonist at vanilloid VR1 receptors for capsaicin and resiniferatoxin, thereby causing an increase in cytosolic Ca(2+) concentration in human VR1-overexpressing (hVR1-HEK) cells. Two selective inhibitors of anandamide facilitated transport into cells, VDM11 and VDM13, and two inhibitors of anandamide enzymatic hydrolysis, phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, inhibited and enhanced, respectively, the VR1-mediated effect of anandamide, but not of resiniferatoxin or capsaicin. The nitric oxide donor, sodium nitroprusside, known to stimulate anandamide transport, enhanced anandamide effect on the cytosolic Ca(2+) concentration. Accordingly, hVR1-HEK cells contain an anandamide membrane transporter inhibited by VDM11 and VDM13 and activated by sodium nitroprusside, and an anandamide hydrolase activity sensitive to phenylmethylsulfonyl fluoride and methylarachidonoyl fluorophosphonate, and a fatty acid amide hydrolase transcript. These findings suggest the following. (i) Anandamide activates VR1 receptors by acting at an intracellular site. (ii) Degradation by fatty acid amide hydrolase limits anandamide activity on VR1; and (iii) the anandamide membrane transporter inhibitors can be used to distinguish between CB(1) or VR1 receptor-mediated actions of anandamide. By contrast, the CB(1) receptor antagonist SR141716A inhibited also the VR1-mediated effect of anandamide and capsaicin on cytosolic Ca(2+) concentration, although at concentrations higher than those required for CB(1) antagonism.

  11. Humic acid facilitates the transport of ARS-labeled hydroxyapatite nanoparticles in iron oxyhydroxide-coated sand

    USGS Publications Warehouse

    Wang, Dengjun; Bradford, Scott A.; Harvey, Ronald W.; Gao, Bin; Cang, Long; Zhou, Dongmei

    2012-01-01

    Hydroxyapatite nanoparticles (nHAP) have been widely used to remediate soil and wastewater contaminated with metals and radionuclides. However, our understanding of nHAP transport and fate is limited in natural environments that exhibit significant variability in solid and solution chemistry. The transport and retention kinetics of Alizarin red S (ARS)-labeled nHAP were investigated in water-saturated packed columns that encompassed a range of humic acid concentrations (HA, 0–10 mg L–1), fractional surface coverage of iron oxyhydroxide coatings on sand grains (λ, 0–0.75), and pH (6.0–10.5). HA was found to have a marked effect on the electrokinetic properties of ARS-nHAP, and on the transport and retention of ARS-nHAP in granular media. The transport of ARS-nHAP was found to increase with increasing HA concentration because of enhanced colloidal stability and the reduced aggregate size. When HA = 10 mg L–1, greater ARS-nHAP attachment occurred with increasing λ because of increased electrostatic attraction between negatively charged nanoparticles and positively charged iron oxyhydroxides, although alkaline conditions (pH 8.0 and 10.5) reversed the surface charge of the iron oxyhydroxides and therefore decreased deposition. The retention profiles of ARS-nHAP exhibited a hyperexponential shape for all test conditions, suggesting some unfavorable attachment conditions. Retarded breakthrough curves occurred in sands with iron oxyhydroxide coatings because of time-dependent occupation of favorable deposition sites. Consideration of the above effects is necessary to improve remediation efficiency of nHAP for metals and actinides in soils and subsurface environments.

  12. Humic acid facilitates the transport of ARS-labeled hydroxyapatite nanoparticles in iron oxyhydroxide-coated sand.

    PubMed

    Wang, Dengjun; Bradford, Scott A; Harvey, Ronald W; Gao, Bin; Cang, Long; Zhou, Dongmei

    2012-03-06

    Hydroxyapatite nanoparticles (nHAP) have been widely used to remediate soil and wastewater contaminated with metals and radionuclides. However, our understanding of nHAP transport and fate is limited in natural environments that exhibit significant variability in solid and solution chemistry. The transport and retention kinetics of Alizarin red S (ARS)-labeled nHAP were investigated in water-saturated packed columns that encompassed a range of humic acid concentrations (HA, 0-10 mg L(-1)), fractional surface coverage of iron oxyhydroxide coatings on sand grains (λ, 0-0.75), and pH (6.0-10.5). HA was found to have a marked effect on the electrokinetic properties of ARS-nHAP, and on the transport and retention of ARS-nHAP in granular media. The transport of ARS-nHAP was found to increase with increasing HA concentration because of enhanced colloidal stability and the reduced aggregate size. When HA = 10 mg L(-1), greater ARS-nHAP attachment occurred with increasing λ because of increased electrostatic attraction between negatively charged nanoparticles and positively charged iron oxyhydroxides, although alkaline conditions (pH 8.0 and 10.5) reversed the surface charge of the iron oxyhydroxides and therefore decreased deposition. The retention profiles of ARS-nHAP exhibited a hyperexponential shape for all test conditions, suggesting some unfavorable attachment conditions. Retarded breakthrough curves occurred in sands with iron oxyhydroxide coatings because of time-dependent occupation of favorable deposition sites. Consideration of the above effects is necessary to improve remediation efficiency of nHAP for metals and actinides in soils and subsurface environments.

  13. An Efficient Strategy for Small-Scale Screening and Production of Archaeal Membrane Transport Proteins in Escherichia coli

    PubMed Central

    Ma, Pikyee; Varela, Filipa; Magoch, Malgorzata; Silva, Ana Rita; Rosário, Ana Lúcia; Brito, José; Oliveira, Tânia Filipa; Nogly, Przemyslaw; Pessanha, Miguel; Stelter, Meike; Kletzin, Arnulf; Henderson, Peter J. F.; Archer, Margarida

    2013-01-01

    Background Membrane proteins play a key role in many fundamental cellular processes such as transport of nutrients, sensing of environmental signals and energy transduction, and account for over 50% of all known drug targets. Despite their importance, structural and functional characterisation of membrane proteins still remains a challenge, partially due to the difficulties in recombinant expression and purification. Therefore the need for development of efficient methods for heterologous production is essential. Methodology/Principal Findings Fifteen integral membrane transport proteins from Archaea were selected as test targets, chosen to represent two superfamilies widespread in all organisms known as the Major Facilitator Superfamily (MFS) and the 5-Helix Inverted Repeat Transporter superfamily (5HIRT). These proteins typically have eleven to twelve predicted transmembrane helices and are putative transporters for sugar, metabolite, nucleobase, vitamin or neurotransmitter. They include a wide range of examples from the following families: Metabolite-H+-symporter; Sugar Porter; Nucleobase-Cation-Symporter-1; Nucleobase-Cation-Symporter-2; and neurotransmitter-sodium-symporter. Overproduction of transporters was evaluated with three vectors (pTTQ18, pET52b, pWarf) and two Escherichia coli strains (BL21 Star and C43 (DE3)). Thirteen transporter genes were successfully expressed; only two did not express in any of the tested vector-strain combinations. Initial trials showed that seven transporters could be purified and six of these yielded quantities of ≥ 0.4 mg per litre suitable for functional and structural studies. Size-exclusion chromatography confirmed that two purified transporters were almost homogeneous while four others were shown to be non-aggregating, indicating that they are ready for up-scale production and crystallisation trials. Conclusions/Significance Here, we describe an efficient strategy for heterologous production of membrane transport

  14. Vaccinia Virus Uses Retromer-Independent Cellular Retrograde Transport Pathways To Facilitate the Wrapping of Intracellular Mature Virions during Virus Morphogenesis

    PubMed Central

    Harrison, Kate; Haga, Ismar R.; Pechenick Jowers, Tali; Jasim, Seema; Cintrat, Jean-Christophe; Gillet, Daniel; Schmitt-John, Thomas; Digard, Paul

    2016-01-01

    ABSTRACT Poxviruses, such as vaccinia virus (VACV), undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms and includes a crucial wrapping step whereby intracellular mature virions (IMVs) are wrapped in two additional membranes to form intracellular enveloped virions (IEVs). To determine if cellular retrograde transport pathways are required for this wrapping step, we examined VACV morphogenesis in cells with reduced expression of the tetrameric tethering factor known as the GARP (Golgi-associated retrograde pathway), a central component of retrograde transport. VACV multistep replication was significantly impaired in cells transfected with small interfering RNA targeting the GARP complex and in cells with a mutated GARP complex. Detailed analysis revealed that depletion of the GARP complex resulted in a reduction in the number of IEVs, thereby linking retrograde transport with the wrapping of IMVs. In addition, foci of viral wrapping membrane proteins without an associated internal core accumulated in cells with a mutated GARP complex, suggesting that impaired retrograde transport uncouples nascent IMVs from the IEV membranes at the site of wrapping. Finally, small-molecule inhibitors of retrograde transport strongly suppressed VACV multistep growth in vitro and reduced weight loss and clinical signs in an in vivo murine model of systemic poxviral disease. This work links cellular retrograde transport pathways with the morphogenesis of poxviruses and identifies a panel of novel inhibitors of poxvirus replication. IMPORTANCE Cellular retrograde transport pathways traffic cargo from endosomes to the trans-Golgi network and are a key part of the intracellular membrane network. This work reveals that the prototypic poxvirus vaccinia virus (VACV) exploits cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions and therefore promote the production of extracellular virus

  15. Vaccinia virus uses retromer-independent cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions during viral morphogenesis.

    PubMed

    Harrison, Kate; Haga, Ismar R; Pechenick Jowers, Tali; Jasim, Seema; Cintrat, Jean-Christophe; Gillet, Daniel; Schmitt-John, Thomas; Digard, Paul; Beard, Philippa M

    2016-08-31

    Poxviruses such as Vaccinia virus (VACV) undertake a complex cytoplasmic replication cycle which involves morphogenesis through four distinct virion forms, and includes a crucial "wrapping" step whereby intracellular mature virions (IMVs) are wrapped in two additional membranes to form intracellular enveloped virions (IEVs). To determine if cellular retrograde transport pathways were required for this wrapping step we examined VACV morphogenesis in cells with reduced expression of the tetrameric tethering factor complex GARP (Golgi-associated retrograde pathway complex), a central component of retrograde transport. VACV multi-step replication was significantly impaired in cells transfected with siRNA targeting the GARP complex or in cells with a mutated GARP complex. Detailed analysis revealed that depletion of the GARP complex resulted in a reduction in the number of IEVs, thereby linking retrograde transport with the wrapping of IMVs. In addition foci of viral wrapping membrane proteins without an associated internal core accumulated in cells with a mutated GARP complex, suggesting that impaired retrograde transport uncouples nascent IMVs from the IEV membranes at the site of wrapping. Finally, small molecule inhibitors of retrograde transport strongly suppressed VACV multi-step growth in vitro and reduced weight loss and clinical signs in an in vivo murine model of systemic poxviral disease. This work links cellular retrograde transport pathways with morphogenesis of poxviruses and identifies a panel of novel inhibitors of poxvirus replication. Cellular retrograde transport pathways traffic cargo from endosomes to the trans-Golgi network and are a key part of the intracellular membrane network. This work reveals the prototypic poxvirus Vaccinia virus (VACV) exploits cellular retrograde transport pathways to facilitate the wrapping of intracellular mature virions and therefore promote the production of extracellular virus. Inhibition of retrograde transport by

  16. Characterization of nucleobases and nucleosides in the fruit of Alpinia oxyphylla collected from different cultivation regions.

    PubMed

    Song, Wenjing; Li, Yonghui; Wang, Jianguo; Li, Zeyou; Zhang, Junqing

    2014-03-01

    The fruit of Alpinia oxyphylla, known as Yizhi, Yakuchi and Ikji in Chinese, Japanese, and Korean, respectively, has been utilized as an important drug for the treatment of diarrhea, dyspepsia, spermatorrhea, kidney asthenia and abdominal pain in East Asian traditional medicine for thousands of years. Since the therapeutic effects of A. oxyphylla are attributed to multiple components and nucleobases and nucleosides exhibit various bioactivities, it is necessary to explore the chemical characterization of nucleobases and nucleosides in this herb. Herein, 10 common nucleobases and nucleosides, including cytidine, adenosine, thymidine, inosine, guanosine, 2'-deoxyinosine, guanine, adenine, cytosine, and hypoxanthine, were quantified simultaneously in the fruit of A. oxyphylla collected from different geographical regions. Changes in their contents were discussed, and hierarchical cluster analysis (HCA) was performed to classify all samples on the basis of the contents of the investigated analytes. The results indicated that there was a large variation in the contents of nucleobases and nucleosides among the herbs from different regions, and the samples collected from the same cultivation region were mostly classified in one cluster. The method can be used for comprehensive quality evaluation of A. oxyphylla. Copyright © 2013 John Wiley & Sons, Ltd.

  17. Origin of the RNA world: The fate of nucleobases in warm little ponds.

    PubMed

    Pearce, Ben K D; Pudritz, Ralph E; Semenov, Dmitry A; Henning, Thomas K

    2017-10-02

    Before the origin of simple cellular life, the building blocks of RNA (nucleotides) had to form and polymerize in favorable environments on early Earth. At this time, meteorites and interplanetary dust particles delivered organics such as nucleobases (the characteristic molecules of nucleotides) to warm little ponds whose wet-dry cycles promoted rapid polymerization. We build a comprehensive numerical model for the evolution of nucleobases in warm little ponds leading to the emergence of the first nucleotides and RNA. We couple Earth's early evolution with complex prebiotic chemistry in these environments. We find that RNA polymers must have emerged very quickly after the deposition of meteorites (less than a few years). Their constituent nucleobases were primarily meteoritic in origin and not from interplanetary dust particles. Ponds appeared as continents rose out of the early global ocean, but this increasing availability of "targets" for meteorites was offset by declining meteorite bombardment rates. Moreover, the rapid losses of nucleobases to pond seepage during wet periods, and to UV photodissociation during dry periods, mean that the synthesis of nucleotides and their polymerization into RNA occurred in just one to a few wet-dry cycles. Under these conditions, RNA polymers likely appeared before 4.17 billion years ago.

  18. 6-Pyrazolylpurine as an Artificial Nucleobase for Metal-Mediated Base Pairing in DNA Duplexes

    PubMed Central

    Léon, J. Christian; Sinha, Indranil; Müller, Jens

    2016-01-01

    The artificial nucleobase 6-pyrazol-1-yl-purine (6PP) has been investigated with respect to its usability in metal-mediated base pairing. As was shown by temperature-dependent UV spectroscopy, 6PP may form weakly stabilizing 6PP–Ag(I)–6PP homo base pairs. Interestingly, 6PP can be used to selectively recognize a complementary pyrimidine nucleobase. The addition of Ag(I) to a DNA duplex comprising a central 6PP:C mispair (C = cytosine) leads to a slight destabilization of the duplex. In contrast, a stabilizing 6PP–Ag(I)–T base pair is formed with a complementary thymine (T) residue. It is interesting to note that 6PP is capable of differentiating between the pyrimidine moieties despite the fact that it is not as sterically crowded as 6-(3,5-dimethylpyrazol-1-yl)purine, an artificial nucleobase that had previously been suggested for the recognition of nucleic acid sequences via the formation of a metal-mediated base pair. Hence, the additional methyl groups of 6-(3,5-dimethylpyrazol-1-yl)purine may not be required for the specific recognition of the complementary nucleobase. PMID:27089326

  19. Optical properties of organically functionalized silicon surfaces: Uracil-like nucleobases on Si(001)

    NASA Astrophysics Data System (ADS)

    Molteni, Elena; Cappellini, Giancarlo; Onida, Giovanni; Fratesi, Guido

    2017-02-01

    We predict UV reflectance anisotropy spectra (RAS) of the organically functionalized silicon (001) surface covered by pyrimidinic uracil-like nucleobases. First-principles results based on density functional theory show characteristic spectral features appearing in the UV range between 3 and 7 eV, besides the expected quench in the well-known two-minima RAS signal of clean Si(001). Nucleobase adsorption in the energetically favored "dimer bridge" configuration gives rise to a characteristic RAS line shape, common to thymine, uracil, and 5-fluorouracil. We trace back the origin of such spectral features by singling out RAS structures induced by relaxation and passivation effects on the Si surface, and those directly associated with molecular excitations. The former turn out to be the same for the three nucleobases, and are totally unaffected by molecular tilting. The sign and position of the latter RAS peaks at higher energy exhibit a moderate nucleobase dependence, and can be fully rationalized in terms of the molecular orbitals involved. The present theoretical results call for a RAS experimental study in the UV region extending up to ≃6 -7 eV.

  20. Macrocyclic Metal Complex-DNA Conjugates for Electrochemical Sensing of Single Nucleobase Changes in DNA.

    PubMed

    Duprey, Jean-Louis H A; Carr-Smith, James; Horswell, Sarah L; Kowalski, Jarosław; Tucker, James H R

    2016-01-27

    The direct incorporation of macrocyclic cyclidene complexes into DNA via automated synthesis results in a new family of metal-functionalized DNA derivatives that readily demonstrate their utility through the ability of one redox-active copper(II)-containing strand to distinguish electrochemically between all four canonical DNA nucleobases at a single site within a target sequence of DNA.

  1. The Photochemistry of Pyrimidine in Realistic Astrophysical Ices and the Production of Nucleobases

    NASA Astrophysics Data System (ADS)

    Nuevo, Michel; Materese, Christopher K.; Sandford, Scott A.

    2014-10-01

    Nucleobases, together with deoxyribose/ribose and phosphoric acid, are the building blocks of DNA and RNA for all known life. The presence of nucleobase-like compounds in carbonaceous chondrites delivered to the Earth raises the question of an extraterrestrial origin for the molecules that triggered life on our planet. Whether these molecules are formed in interstellar/protostellar environments, in small parent bodies in the solar system, or both, is currently unclear. Recent experiments show that the UV irradiation of pyrimidine (C4H4N2) in H2O-rich ice mixtures that contain NH3, CH3OH, or CH4 leads to the formation of the pyrimidine-based nucleobases uracil, cytosine, and thymine. In this work, we discuss the low-temperature UV irradiation of pyrimidine in realistic astrophysical ice mixtures containing H2O, CH3OH, and NH3, with or without CH4, to search for the production of nucleobases and other prebiotic compounds. These experiments show the presence of uracil, urea, glycerol, hexamethylenetetramine, small amino acids, and small carboxylic acids in all samples. Cytosine was only found in one sample produced from ices irradiated with a higher UV dose, while thymine was not found in any sample, even after irradiation with a higher UV dose. Results are discussed to evaluate the role of the photochemistry of pyrimidine in the inventory of organic molecules detected in meteorites and their astrophysical/astrobiological implications.

  2. Hartree-Fock Cluster Study of Electronic Structures and Nuclear Quadrupole Interactions in Solid Nucleobases.

    NASA Astrophysics Data System (ADS)

    Scheicher, R. H.; Dubey, Archana; Badu, S. R.; Saha, H. P.; Pink, R. H.; Nagamine, K.; Torikai, E.; Chow, Lee; Das, T. P.

    2008-03-01

    In recent work [1] we have studied nucleobases attached to a CH3 group to simulate the influence of their binding to the sugar rings and the phosphate groups in DNA and RNA and the effect of this binding on the nuclear quadrupole interactions of ^14N, ^17O and ^2H nuclei. Our results from this work have indicated that for ^17O, the binding to the CH3 group moves our results from the free nucleobases closer to the experimentally observed data [2] in the solid nucleobases. We are now investigating the solid nucleobases by the first --principles Hartree-Fock cluster procedure that we have employed earlier for the halogen molecular solids [3]. Our results for the binding energy of an imidazole molecule in the molecular solid system and the ^14N, ^17O and ^2H nuclear quadrupole interaction parameters will be presented. [1] T.P. Das et al (at this APS meeting), [2] Gang Wu et al, J. Am.Chem. Soc. 124, 1768(2002). [3] M.M. Aryal et al Hyperfine Interactions (to be published).

  3. Dependence of Binding Free Energies between RNA Nucleobases and Protein Side Chains on Local Dielectric Properties.

    PubMed

    de Ruiter, Anita; Polyansky, Anton A; Zagrovic, Bojan

    2017-09-12

    In order to fully understand the microscopic origins of binding specificity between nucleic acids and proteins, it is imperative to study the dependence of the binding preferences between nucleobases and protein side chains on the properties of the environment. Here, we employ molecular dynamics simulations and umbrella sampling to derive the potentials of mean force and the associated absolute binding free energies between the four standard RNA nucleobases and the side chains of aspartic acid and tryptophan in water/methanol mixtures exhibiting a wide range of dielectric constants. In addition to their opposing character when it comes to hydrophobicity, aspartate and tryptophan side chains were chosen because they exhibit the greatest change in binding free energies with nucleobases between pure water and methanol environments. We exploit a strong linear dependence of the derived ΔG values on the mole fraction of methanol to estimate the binding free energies of all possible combinations of different standard RNA nucleobases and side chains at multiple values of dielectric constants. Finally, we critically assess the recently proposed complementarity hypothesis concerning direct, coaligned binding between mRNAs and their cognate proteins in light of the present results.

  4. Human SLC2A9a and SLC2A9b isoforms mediate electrogenic transport of urate with different characteristics in the presence of hexoses.

    PubMed

    Witkowska, Kate; Smith, Kyla M; Yao, Sylvia Y M; Ng, Amy M L; O'Neill, Debbie; Karpinski, Edward; Young, James D; Cheeseman, Christopher I

    2012-08-15

    Human SLC2A9 (GLUT9) is a novel high-capacity urate transporter belonging to the facilitated glucose transporter family. In the present study, heterologous expression in Xenopus oocytes has allowed us to undertake an in-depth radiotracer flux and electrophysiological study of urate transport mediated by both isoforms of SLC2A9 (a and b). Addition of urate to SLC2A9-producing oocytes generated outward currents, indicating electrogenic transport. Urate transport by SLC2A9 was voltage dependent and independent of the Na(+) transmembrane gradient. Urate-induced outward currents were affected by the extracellular concentration of Cl(-), but there was no evidence for exchange of the two anions. [(14)C]urate flux studies under non-voltage-clamped conditions demonstrated symmetry of influx and efflux, suggesting that SLC2A9 functions in urate efflux driven primarily by the electrochemical gradient of the cell. Urate uptake in the presence of intracellular hexoses showed marked differences between the two isoforms, suggesting functional differences between the two splice variants. Finally, the permeant selectivity of SLC2A9 was examined by testing the ability to transport a panel of radiolabeled purine and pyrimidine nucleobases. SLC2A9 mediated the uptake of adenine in addition to urate, but did not function as a generalized nucleobase transporter. The differential expression pattern of the two isoforms of SLC2A9 in the human kidney's proximal convoluted tubule and its electrogenic transport of urate suggest that these transporters play key roles in the regulation of plasma urate levels and are therefore potentially important participants in hyperuricemia and hypouricemia.

  5. Urea Mimics Nucleobases by Preserving the Helical Integrity of B-DNA Duplexes via Hydrogen Bonding and Stacking Interactions.

    PubMed

    Suresh, Gorle; Padhi, Siladitya; Patil, Indrajit; Priyakumar, U Deva

    2016-10-11

    Urea lesions are formed in DNA because of free radical damage of the thymine base, and their occurrence in DNA blocks DNA polymerases, which has deleterious consequences. Recently, it has been shown that urea is capable of forming hydrogen bonding and stacking interactions with nucleobases, which are responsible for the unfolding of RNA in aqueous urea. Base pairing and stacking are inherent properties of nucleobases; because urea is able to form both, this study attempts to investigate if urea can mimic nucleobases in the context of nucleic acid structures by examining the effect of introducing urea lesions complementary to the four different nucleobases on the overall helical integrity of B-DNA duplexes and their thermodynamic stabilities using molecular dynamics (MD) simulations. The MD simulations resulted in stable duplexes without significant changes in the global B-DNA conformation. The urea lesions occupy intrahelical positions by forming hydrogen bonds with nitrogenous nucleobases, in agreement with experimental results. Furthermore, these urea lesions form hydrogen bonding and stacking interactions with other nucleobases of the same and partner strands, analogous to nucleobases in typical B-DNA duplexes. Direct hydrogen bond interactions are observed for the urea-purine pairs within DNA duplexes, whereas two different modes of pairing, namely, direct hydrogen bonds and water-mediated hydrogen bonds, are observed for the urea-pyrimidine pairs. The latter explains the complexities involved in interpreting the experimental nuclear magnetic resonance data reported previously. Binding free energy calculations were further performed to confirm the thermodynamic stability of the urea-incorporated DNA duplexes with respect to pure duplexes. This study suggests that urea mimics nucleobases by pairing opposite all four nucleobases and maintains the overall structure of the B-DNA duplexes.

  6. The Role of Desorption Kinetics on the Colloid-Facilitated Transport of Cesium and Strontium in a Partially-Saturated Quartz Sand Column

    NASA Astrophysics Data System (ADS)

    Dittrich, T. M.; Ryan, J. N.; Saiers, J. E.

    2010-12-01

    Colloid-facilitated transport (CFT) is a mechanism for enhanced transport of contaminants under certain environmental conditions. In order for CFT to be significant, three conditions must be met: (1) colloids must be present at a significant concentration, 2) the contaminant must associate with and remain associated with the colloids, and 3) colloids must be transported faster than the contaminant on its own. Colloids are often present in significant concentrations; therefore, CFT strongly depends on slow desorption kinetics and colloid mobility. The goal of this research was to identify and quantify the effects of desorption kinetics on the transport of cesium and strontium through a quartz sand column at different degrees of saturation (moisture contents). Breakthrough experiments were conducted using a rainfall simulator suspended over a column (12.7 cm diameter, 33.5 cm depth) packed with clean, sieved quartz sand (d50 = 360 µm). The effluent was collected with a fraction collector. Cesium and strontium were used as model contaminants because they are common contaminants found on Department of Energy sites in the US and because they have contrasting sorption kinetics with illite, which was used as the model colloid. The column was instrumented with three tensiometers and three moisture sensors to ensure uniform flow under partially-saturated conditions. Relative saturations of 0.33, 0.80, and 1.0 were established in the column. For aqueous breakthrough experiments, solutions at pH 7.3 containing cesium or strontium at a concentration of 7.5 x 10-6 M were applied to the top of the column until breakthrough (C/C0 > 0.5) was achieved. Following breakthrough, the solution was switched to a cesium- or strontium-free pH 7.3 solution for about 40 pore volumes. These experiments were repeated with the addition of illite colloids at a concentration of 100 mg L-1 in equilibrium with the cesium of strontium solution. Cesium and strontium breakthrough was monitored by

  7. TFG facilitates outer coat disassembly on COPII transport carriers to promote tethering and fusion with ER-Golgi intermediate compartments.

    PubMed

    Hanna, Michael G; Block, Samuel; Frankel, E B; Hou, Feng; Johnson, Adam; Yuan, Lin; Knight, Gavin; Moresco, James J; Yates, John R; Ashton, Randolph; Schekman, Randy; Tong, Yufeng; Audhya, Anjon

    2017-08-29

    The conserved coat protein complex II (COPII) mediates the initial steps of secretory protein trafficking by assembling onto subdomains of the endoplasmic reticulum (ER) in two layers to generate cargo-laden transport carriers that ultimately fuse with an adjacent ER-Golgi intermediate compartment (ERGIC). Here, we demonstrate that Trk-fused gene (TFG) binds directly to the inner layer of the COPII coat. Specifically, the TFG C terminus interacts with Sec23 through a shared interface with the outer COPII coat and the cargo receptor Tango1/cTAGE5. Our findings indicate that TFG binding to Sec23 outcompetes these other associations in a concentration-dependent manner and ultimately promotes outer coat dissociation. Additionally, we demonstrate that TFG tethers vesicles harboring the inner COPII coat, which contributes to their clustering between the ER and ERGIC in cells. Together, our studies define a mechanism by which COPII transport carriers are retained locally at the ER/ERGIC interface after outer coat disassembly, which is a prerequisite for fusion with ERGIC membranes.

  8. Nuclear Factor Erythroid 2-Related Factor 2 Facilitates Neuronal Glutathione Synthesis by Upregulating Neuronal Excitatory Amino Acid Transporter 3 Expression

    PubMed Central

    Escartin, Carole; Won, Seok Joon; Malgorn, Carole; Auregan, Gwennaelle; Berman, Ari E.; Chen, Pei-Chun; Déglon, Nicole; Johnson, Jeffrey A.; Suh, Sang Won; Swanson, Raymond A.

    2012-01-01

    Astrocytes support neuronal antioxidant capacity by releasing glutathione, which is cleaved to cysteine in brain extracellular space. Free cysteine is then taken up by neurons through excitatory amino acid transporter 3 [EAAT3; also termed Slc1a1 (solute carrier family 1 member 1)] to support de novo glutathione synthesis. Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway by oxidative stress promotes astrocyte release of glutathione, but it remains unknown how this release is coupled to neuronal glutathione synthesis. Here we evaluated transcriptional regulation of the neuronal cysteine transporter EAAT3 by the Nrf2-ARE pathway. Nrf2 activators and Nrf2 overexpression both produced EAAT3 transcriptional activation in C6 cells. A conserved ARE-related sequence was found in the EAAT3 promoter of several mammalian species. This ARE-related sequence was bound by Nrf2 in mouse neurons in vivo as observed by chromatin immunoprecipitation. Chemical activation of the Nrf2-ARE pathway in mouse brain increased both neuronal EAAT3 levels and neuronal glutathione content, and these effects were abrogated in mice genetically deficient in either Nrf2 or EAAT3. Selective overexpression of Nrf2 in brain neurons by lentiviral gene transfer was sufficient to upregulate both neuronal EAAT3 protein and glutathione content. These findings identify a mechanism whereby Nrf2 activation can coordinate astrocyte glutathione release with neuronal glutathione synthesis through transcriptional upregulation of neuronal EAAT3 expression. PMID:21593323

  9. Nuclear factor erythroid 2-related factor 2 facilitates neuronal glutathione synthesis by upregulating neuronal excitatory amino acid transporter 3 expression.

    PubMed

    Escartin, Carole; Won, Seok Joon; Malgorn, Carole; Auregan, Gwennaelle; Berman, Ari E; Chen, Pei-Chun; Déglon, Nicole; Johnson, Jeffrey A; Suh, Sang Won; Swanson, Raymond A

    2011-05-18

    Astrocytes support neuronal antioxidant capacity by releasing glutathione, which is cleaved to cysteine in brain extracellular space. Free cysteine is then taken up by neurons through excitatory amino acid transporter 3 [EAAT3; also termed Slc1a1 (solute carrier family 1 member 1)] to support de novo glutathione synthesis. Activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway by oxidative stress promotes astrocyte release of glutathione, but it remains unknown how this release is coupled to neuronal glutathione synthesis. Here we evaluated transcriptional regulation of the neuronal cysteine transporter EAAT3 by the Nrf2-ARE pathway. Nrf2 activators and Nrf2 overexpression both produced EAAT3 transcriptional activation in C6 cells. A conserved ARE-related sequence was found in the EAAT3 promoter of several mammalian species. This ARE-related sequence was bound by Nrf2 in mouse neurons in vivo as observed by chromatin immunoprecipitation. Chemical activation of the Nrf2-ARE pathway in mouse brain increased both neuronal EAAT3 levels and neuronal glutathione content, and these effects were abrogated in mice genetically deficient in either Nrf2 or EAAT3. Selective overexpression of Nrf2 in brain neurons by lentiviral gene transfer was sufficient to upregulate both neuronal EAAT3 protein and glutathione content. These findings identify a mechanism whereby Nrf2 activation can coordinate astrocyte glutathione release with neuronal glutathione synthesis through transcriptional upregulation of neuronal EAAT3 expression.

  10. Monocarboxylate transporter 4 facilitates cell proliferation and migration and is associated with poor prognosis in oral squamous cell carcinoma patients.

    PubMed

    Zhu, Jiang; Wu, Yu-Nong; Zhang, Wei; Zhang, Xiao-Min; Ding, Xu; Li, Huai-Qi; Geng, Meiyu; Xie, Zuo-Quan; Wu, He-Ming

    2014-01-01

    Monocarboxylate transporter 4 (MCT4) is a cell membrane transporter of lactate. Recent studies have shown that MCT4 is over-expressed in various cancers; however, its role in cancer maintenance and aggressiveness has not been fully demonstrated. This study investigated the role of MCT4 in oral squamous cell carcinoma (OSCC), and found that it is highly expressed in OSCC patients by using immunohistochemistry. Moreover, this over-expression of MCT4 was closely associated with tumor size, TNM classification, lymphatic metastasis, distant metastasis and tumor recurrence, and also poor prognosis. To further study mechanisms of MCT4 in vitro, we used small-interfering RNA to silence its expression in OSCC cell lines. The results showed that knock-down of MCT4 decreased cell proliferation, migration, and invasion. The inhibition of proliferation was associated with down-regulation of p-AKT and p-ERK1/2, while decreased cell migration and invasion may be caused by down-regulation of integrin β4-SRC-FAK and MEK-ERK signaling. Together, these findings provide new insight into the critical role of MCT4 in cell proliferation and metastasis in OSCC.

  11. The maltose ABC transporter in Lactococcus lactis facilitates high-level sensitivity to the circular bacteriocin garvicin ML.

    PubMed

    Gabrielsen, Christina; Brede, Dag A; Hernández, Pablo E; Nes, Ingolf F; Diep, Dzung B

    2012-06-01

    We generated and characterized a series of spontaneous mutants of Lactococcus lactis IL1403 with average 6- to 11-fold-lowered sensitivities to the circular bacteriocin garvicin ML (GarML). Carbohydrate fermentation assays highlighted changes in carbohydrate metabolism, specifically loss of the ability to metabolize starch and maltose, in these mutants. PCR and sequencing showed that a 13.5-kb chromosomal deletion encompassing 12 open reading frames, mainly involved in starch and maltose utilization, had spontaneously occurred in the GarML-resistant mutants. Growth experiments revealed a correlation between sensitivity to GarML and carbon catabolite repression (CCR); i.e., sensitivity to GarML increased significantly when wild-type cells were grown on maltose and galactose as sole carbohydrates, an effect which was alleviated by the presence of glucose. Among the genes deleted in the mutants were malEFG, which encode a CCR-regulated membrane-bound maltose ABC transporter. The complementation of mutants with these three genes recovered normal sensitivity to the bacteriocin, suggesting an essential role of the maltose ABC transporter in the antimicrobial activity of GarML. This notion was supported by the fact that the level of sensitivity to GarML was dose dependent, increasing with higher expression levels of malEFG over a 50-fold range. To our knowledge, this is the first time a specific protein complex has been demonstrated to be involved in sensitivity to a circular bacteriocin.

  12. FGT-1 Is a Mammalian GLUT2-Like Facilitative Glucose Transporter in Caenorhabditis elegans Whose Malfunction Induces Fat Accumulation in Intestinal Cells

    PubMed Central

    Kitaoka, Shun; Morielli, Anthony D.; Zhao, Feng-Qi

    2013-01-01

    Caenorhabditis elegans (C. elegans) is an attractive animal model for biological and biomedical research because it permits relatively easy genetic dissection of cellular pathways, including insulin/IGF-like signaling (IIS), that are conserved in mammalian cells. To explore C. elegans as a model system to study the regulation of the facilitative glucose transporter (GLUT), we have characterized the GLUT gene homologues in C. elegans: fgt-1, R09B5.11, C35A11.4, F53H8.3, F48E3.2, F13B12.2, Y61A9LA.1, K08F9.1 and Y37A1A.3. The exogenous expression of these gene products in Xenopus oocytes showed transport activity to unmetabolized glucose analogue 2-deoxy-D-glucose only in FGT-1. The FGT-1-mediated transport activity was inhibited by the specific GLUT inhibitor phloretin and exhibited a Michaelis constant (Km) of 2.8 mM. Mannose, galactose, and fructose were able to inhibit FGT-1-mediated 2-deoxy-D-glucose uptake (P < 0.01), indicating that FGT-1 is also able to transport these hexose sugars. A GFP fusion protein of FGT-1 was observed only on the basolateral membrane of digestive tract epithelia in C. elegans, but not in other tissues. FGT-1::eGFP expression was observed from early embryonic stages. The knockdown or mutation of fgt-1 resulted in increased fat staining in both wild-type and daf-2 (mammalian insulin receptor homologue) mutant animals. Other common phenotypes of IIS mutant animals, including dauer formation and brood size reduction, were not affected by fgt-1 knockdown in wild-type or daf-2 mutants. Our results indicated that in C. elegans, FGT-1 is mainly a mammalian GLUT2-like intestinal glucose transporter and is involved in lipid metabolism. PMID:23826391

  13. Structure determination of a major facilitator peptide transporter: Inward facing PepTSt from Streptococcus thermophilus crystallized in space group P3121

    PubMed Central

    Quistgaard, Esben M.; Martinez Molledo, Maria

    2017-01-01

    Major facilitator superfamily (MFS) peptide transporters (typically referred to as PepT, POT or PTR transporters) mediate the uptake of di- and tripeptides, and so play an important dietary role in many organisms. In recent years, a better understanding of the molecular basis for this process has emerged, which is in large part due to a steep increase in structural information. Yet, the conformational transitions underlying the transport mechanism are still not fully understood, and additional data is therefore needed. Here we report in detail the detergent screening, crystallization, experimental MIRAS phasing, and refinement of the peptide transporter PepTSt from Streptococcus thermophilus. The space group is P3121, and the protein is crystallized in a monomeric inward facing form. The binding site is likely to be somewhat occluded, as the lobe encompassing transmembrane helices 10 and 11 is markedly bent towards the central pore of the protein, but the extent of this potential occlusion could not be determined due to disorder at the apex of the lobe. Based on structural comparisons with the seven previously determined P212121 and C2221 structures of inward facing PepTSt, the structural flexibility as well as the conformational changes mediating transition between the inward open and inward facing occluded states are discussed. In conclusion, this report improves our understanding of the structure and conformational cycle of PepTSt, and can furthermore serve as a case study, which may aid in supporting future structure determinations of additional MFS transporters or other integral membrane proteins. PMID:28264013

  14. Mapping the UV Photophysics of Platinum Metal Complexes Bound to Nucleobases

    NASA Astrophysics Data System (ADS)

    Sen, Ananya; Dessent, Caroline

    2015-03-01

    We report the first UV laser spectroscopic study of isolated gas-phase complexes of Platinum metal complex anions bound to a nucleobase as model systems for exploring at the molecular level the key photophysical processes involved in photodynamic therapy. Spectra of the PtIV CN 6 2 - • Uracil and PtII CN 4 2 - • Uracil complexes were acquired across the 220 -320 nm range using mass-selective photodepletion and photofragment action spectroscopy. The spectra of both complexes reveal prominent UV absorption bands that we assign primarily to excitation of the Uracil π - π * localized chromophore. Distinctive UV photofragments are observed for the complexes, with PtIV CN 6 2 - • Uracil photoexcitation resulting in complex fission, while PtII CN 4 2 - • Uracil photoexcitation initiates a nucleobase proton-transfer reaction across 4.4 -5.2 eV and electron detachment above 5.2 eV. The observed photofragments are consistent with ultrafast decay of a Uracil localized excited state back to the electronic ground state followed by intramolecular vibrational relaxation and ergodic complex fragmentation. In addition, we present recent results to explore how the photophysics of the Platinum complex-nucleobase clusters evolves as a function of nucleobase. Results are presented for PtII CN 4 2 - • Uracil complexed to Cytosine, Thymine and Adenine, reveal distinctive decay dynamics which we attribute to the intrinsic decay dynamics of the nucleobase. JPC. Lett. 2014, 5, 3281 to 3285 and PCCP 2014, 16, 15490 to 15500.

  15. Photochemistry of Pyrimidine in Astrophysical Ices: Formation of Nucleobases and Other Prebiotic Species

    NASA Technical Reports Server (NTRS)

    Nuevo, Michel; Sandford, Scott A.; Materese, Christopher K.; Milam, Stefanie N.

    2012-01-01

    Nucleobases are N-heterocycles that are the informational subunits of DNA and RNA. They are divided into two molecular groups: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites, and their extraterrestrial origin confirmed by isotopic measurements. Although no N-heterocycles have ever been observed in the ISM, the positions of the 6.2- m interstellar emission features suggest a population of such molecules is likely to be present. However, laboratory experiments have shown that the ultraviolet (UV) irradiation of pyrimidine in ices of astrophysical relevance such as H2O, NH3, CH3OH, CH4, CO, or combinations of these at low temperature (less than or equal to 20 K) leads to the formation of several pyrimidine derivatives including the nucleobases uracil and cytosine, as well as precursors such as 4(3H)-pyrimidone and 4-aminopyrimidine. Quantum calculations on the formation of 4(3H)-pyrimidone and uracil from the irradiation of pyrimidine in pure H2O ices are in agreement with their experimental formation pathways.10 In those residues, other species of prebiotic interest such as urea as well as the amino acids glycine and alanine could also be identified. However, only very small amounts of pyrimidine derivatives containing CH3 groups could be detected, suggesting that the addition of methyl groups to pyrimidine is not an efficient process. For this reason, the nucleobase thymine was not observed in any of the samples. In this work, we study the formation of nucleobases and other photo-products of prebiotic interest from the UV irradiation of pyrimidine in ices containing H2O, NH3, CH3OH, and CO, mixed in astrophysical proportions.

  16. Monsoon-facilitated characteristics and transport of atmospheric mercury at a high-altitude background site in southwestern China

    NASA Astrophysics Data System (ADS)

    Zhang, Hui; Fu, Xuewu; Lin, Che-Jen; Shang, Lihai; Zhang, Yiping; Feng, Xinbin; Lin, Cynthia

    2016-10-01

    To better understand the influence of monsoonal climate and transport of atmospheric mercury (Hg) in southwestern China, measurements of total gaseous mercury (TGM, defined as the sum of gaseous elemental mercury, GEM, and gaseous oxidized mercury, GOM), particulate bound mercury (PBM) and GOM were carried out at Ailaoshan Station (ALS, 2450 m a.s.l.) in southwestern China from May 2011 to May 2012. The mean concentrations (± SD) for TGM, GOM and PBM were 2.09 ± 0.63, 2.2 ± 2.3 and 31.3 ± 28.4 pg m-3, respectively. TGM showed a monsoonal distribution pattern with relatively higher concentrations (2.22 ± 0.58 ng m-3, p = 0.021) during the Indian summer monsoon (ISM, from May to September) and the east Asia summer monsoon (EASM, from May to September) periods than that (1.99 ± 0.66 ng m-3) in the non-ISM period. Similarly, GOM and PBM concentrations were higher during the ISM period than during the non-ISM period. This study suggests that the ISM and the EASM have a strong impact on long-range and transboundary transport of Hg between southwestern China and south and southeast Asia. Several high TGM events were accompanied by the occurrence of northern wind during the ISM period, indicating anthropogenic Hg emissions from inland China could rapidly increase TGM levels at ALS due to strengthening of the EASM. Most of the TGM and PBM events occurred at ALS during the non-ISM period. Meanwhile, high CO concentrations were also observed at ALS, indicating that a strong south tributary of westerlies could have transported Hg from south and southeast Asia to southwestern China during the non-ISM period. The biomass burning in southeast Asia and anthropogenic Hg emissions from south Asia are thought to be the source of atmospheric Hg in remote areas of southwestern China during the non-ISM period.

  17. A Major Facilitator Superfamily Transporter-Mediated Resistance to Oxidative Stress and Fungicides Requires Yap1, Skn7, and MAP Kinases in the Citrus Fungal Pathogen Alternaria alternata.

    PubMed

    Chen, Li-Hung; Tsai, Hsieh-Chin; Yu, Pei-Ling; Chung, Kuang-Ren

    2017-01-01

    Major Facilitator Superfamily (MFS) transporters play an important role in multidrug resistance in fungi. We report an AaMFS19 gene encoding a MFS transporter required for cellular resistance to oxidative stress and fungicides in the phytopathogenic fungus Alternaria alternata. AaMFS19, containing 12 transmembrane domains, displays activity toward a broad range of substrates. Fungal mutants lacking AaMFS19 display profound hypersensitivities to cumyl hydroperoxide, potassium superoxide, many singlet oxygen-generating compounds (eosin Y, rose Bengal, hematoporphyrin, methylene blue, and cercosporin), and the cell wall biosynthesis inhibitor, Congo red. AaMFS19 mutants also increase sensitivity to copper ions, clotrimazole, fludioxonil, and kocide fungicides, 2-chloro-5-hydroxypyridine (CHP), and 2,3,5-triiodobenzoic acid (TIBA). AaMFS19 mutants induce smaller necrotic lesions on leaves of a susceptible citrus cultivar. All observed phenotypes in the mutant are restored by introducing and expressing a wild-type copy of AaMFS19. The wild-type strain of A. alternata treated with either CHP or TIBA reduces radial growth and formation and germination of conidia, increases hyphal branching, and results in decreased expression of the AaMFS19 gene. The expression of AaMFS19 is regulated by the Yap1 transcription activator, the Hog1 and Fus3 mitogen-activated protein (MAP) kinases, the 'two component' histidine kinase, and the Skn7 response regulator. Our results demonstrate that A. alternata confers resistance to different chemicals via a membrane-bound MFS transporter.

  18. A novel major facilitator transporter TrSTR1 is essential for pentose utilization and involved in xylanase induction in Trichoderma reesei.

    PubMed

    Huang, Zhen-Bang; Chen, Xiu-Zhen; Qin, Li-Na; Wu, Hong-Qing; Su, Xiao-Yun; Dong, Zhi-Yang

    2015-05-08

    Trichoderma reesei (teleomorph Hypocrea jecorina) is an industrially important filamentous fungus for glycoside hydrolases production, with its xylanolytic enzymes widely applied in many areas. However, the molecular mechanisms underlying xylanase expression are still insufficiently understood. In particular, the effect of sugar transporter on the induction of xylanase expression is unclear. In this work, we identified a novel major facilitator transporter TrSTR1 that is capable of transporting xylose by using a xylose utilization system in Saccharomyces cerevisiae. In T. reesei, TrSTR1 is essential for the utilization of d-xylose, l-arabinose, and even their downstream metabolites D-xylitol and L-arabitol. TrSTR1 is also involved in the induction of xylanase expression since both the xylanase activity and extracellular protein concentration in the Tu6△str1 strain were decreased, which further confirmed by a qRT-PCR analysis of the transcript levels of the key transcriptional regulators. Our observations provide new insights into connections between pentose utilization and xylanase production in T. reesei.

  19. Macroscopic and microscopic observations of particle-facilitated mercury transport from New Idria and Sulphur Bank mercury mine tailings

    USGS Publications Warehouse

    Lowry, G.V.; Shaw, S.; Kim, C.S.; Rytuba, J.J.; Brown, Gordon E.

    2004-01-01

    Mercury (Hg) release from inoperative Hg mines in the California Coast Range has been documented, but little is known about the release and transport mechanisms. In this study, tailings from Hg mines located in different geologic settings-New Idria (NI), a Si-carbonate Hg deposit, and Sulphur Bank (SB), a hot-spring Hg deposit-were characterized, and particle release from these wastes was studied in column experiments to (1) investigate the mechanisms of Hg release from NI and SB mine wastes, (2) determine the speciation of particle-bound Hg released from the mine wastes, and (3) determine the effect of calcinations on Hg release processes. The physical and chemical properties of tailings and the colloids released from them were determined using chemical analyses, selective chemical extractions, XRD, SEM, TEM, and X-ray absorption spectroscopy techniques. The total Hg concentration in tailings increased with decreasing particle size in NI and SB calcines (roasted ore), but reached a maximum at an intermediate particle size in the SB waste rock (unroasted ore). Hg in the tailings exists predominantly as low-solubility HgS (cinnabar and metacinnabar), with NI calcines having >50% HgS, SB calcines having >89% HgS, and SB waste rock having ???100% HgS. Leaching experiments with a high-ionic-strength solution (0.1 M NaCl) resulted in a rapid but brief release of soluble and particulate Hg. Lowering the ionic strength of the leach solution (0.005 M NaCl) resulted in the release of colloidal Hg from two of the three mine wastes studied (NI calcines and SB waste rock). Colloid-associated Hg accounts for as much as 95% of the Hg released during episodic particle release. Colloids generated from the NI calcines are produced by a breakup and release mechanism and consist of hematite, jarosite/alunite, and Al-Si gel with particle sizes of 10-200 nm. ATEM and XAFS analyses indicate that the majority (???78%) of the mercury is present in the form of HgS. SB calcines also

  20. Monitoring of event based mobilization of hydrophobic pollutants in rivers: Calibration of turbidity as a proxy for particle facilitated transport

    NASA Astrophysics Data System (ADS)

    Rügner, Hermann; Schwientek, Marc; Grathwohl, Peter

    2014-05-01

    Transport of many pollutants in rivers is coupled to transport of suspended particles which is typically enhanced during events such as floods, snow melts etc. As the amount of total suspended solids (TSS) in rivers can be monitored by turbidity measurements this may be used as a proxy for the total concentration of particle associated pollutants in rivers such as polycyclic aromatic hydrocarbons (PAHs), PCBs, etc. and several heavy metals. On-line turbidity measurements (e.g. by optical backscattering sensors) then allow for an assessment of particle and pollutant flux dynamics. In this study, pronounced flood and thus turbidity events were sampled at high temporal resolution in three contrasting catchments in Southwest Germany (Rivers Ammer, Goldersbach, Steinlach) as well as in the River Neckar. Samples were analyzed for turbidity, the total amount of PAH and total suspended solids (TSS) in water. Additionally, the grain size distributions of suspended solids were determined. Discharge and turbidity were measured on-line at gauging stations in three of the catchments. Results showed that turbidity and TSS were linearly correlated over an extended turbidity range up to 2000 NTU for the flood samples (i.e. independent on grain size). This also holds for total PAH concentrations which can be reasonably well predicted based on the turbidity measurements and TSS versus PAH relationships - even for very high turbidity or TSS values (> 2000 NTU or mg l-1, respectively). From these linear regressions concentrations of PAHs on suspended particles were obtained which varied by catchment. The values comprise a robust measure of the average sediment quality in a river network and may be correlated to the degree of urbanization represented by the number of inhabitants per total flux of suspended particles. Based on long-term on-line turbidity measurements mass flow rates of particle bound pollutants over time could be calculated. Results showed that by far the largest amount

  1. Enhanced vascular permeability facilitates entry of plasma HDL and promotes macrophage-reverse cholesterol transport from skin in mice

    PubMed Central

    Kareinen, Ilona; Cedó, Lídia; Silvennoinen, Reija; Laurila, Pirkka-Pekka; Jauhiainen, Matti; Julve, Josep; Blanco-Vaca, Francisco; Escola-Gil, Joan Carles; Kovanen, Petri T.; Lee-Rueckert, Miriam

    2015-01-01

    Reverse cholesterol transport (RCT) pathway from macrophage foam cells initiates when HDL particles cross the endothelium, enter the interstitial fluid, and induce cholesterol efflux from these cells. We injected [3H]cholesterol-loaded J774 macrophages into the dorsal skin of mice and measured the transfer of macrophage-derived [3H]cholesterol to feces [macrophage-RCT (m-RCT)]. Injection of histamine to the macrophage injection site increased locally vascular permeability, enhanced influx of intravenously administered HDL, and stimulated m-RCT from the histamine-treated site. The stimulatory effect of histamine on m-RCT was abolished by prior administration of histamine H1 receptor (H1R) antagonist pyrilamine, indicating that the histamine effect was H1R-dependent. Subcutaneous administration of two other vasoactive mediators, serotonin or bradykinin, and activation of skin mast cells to secrete histamine and other vasoactive compounds also stimulated m-RCT. None of the studied vasoactive mediators affected serum HDL levels or the cholesterol-releasing ability of J774 macrophages in culture, indicating that acceleration of m-RCT was solely due to increased availability of cholesterol acceptors in skin. We conclude that disruption of the endothelial barrier by vasoactive compounds enhances the passage of HDL into interstitial fluid and increases the rate of RCT from peripheral macrophage foam cells, which reveals a novel tissue cholesterol-regulating function of these compounds. PMID:25473102

  2. Enhanced vascular permeability facilitates entry of plasma HDL and promotes macrophage-reverse cholesterol transport from skin in mice.

    PubMed

    Kareinen, Ilona; Cedó, Lídia; Silvennoinen, Reija; Laurila, Pirkka-Pekka; Jauhiainen, Matti; Julve, Josep; Blanco-Vaca, Francisco; Escola-Gil, Joan Carles; Kovanen, Petri T; Lee-Rueckert, Miriam

    2015-02-01

    Reverse cholesterol transport (RCT) pathway from macrophage foam cells initiates when HDL particles cross the endothelium, enter the interstitial fluid, and induce cholesterol efflux from these cells. We injected [(3)H]cholesterol-loaded J774 macrophages into the dorsal skin of mice and measured the transfer of macrophage-derived [(3)H]cholesterol to feces [macrophage-RCT (m-RCT)]. Injection of histamine to the macrophage injection site increased locally vascular permeability, enhanced influx of intravenously administered HDL, and stimulated m-RCT from the histamine-treated site. The stimulatory effect of histamine on m-RCT was abolished by prior administration of histamine H1 receptor (H1R) antagonist pyrilamine, indicating that the histamine effect was H1R-dependent. Subcutaneous administration of two other vasoactive mediators, serotonin or bradykinin, and activation of skin mast cells to secrete histamine and other vasoactive compounds also stimulated m-RCT. None of the studied vasoactive mediators affected serum HDL levels or the cholesterol-releasing ability of J774 macrophages in culture, indicating that acceleration of m-RCT was solely due to increased availability of cholesterol acceptors in skin. We conclude that disruption of the endothelial barrier by vasoactive compounds enhances the passage of HDL into interstitial fluid and increases the rate of RCT from peripheral macrophage foam cells, which reveals a novel tissue cholesterol-regulating function of these compounds.

  3. A Salt Bridge Linking the First Intracellular Loop with the C Terminus Facilitates the Folding of the Serotonin Transporter*

    PubMed Central

    Koban, Florian; El-Kasaby, Ali; Häusler, Cornelia; Stockner, Thomas; Simbrunner, Benedikt M.; Sitte, Harald H.; Freissmuth, Michael; Sucic, Sonja

    2015-01-01

    The folding trajectory of solute carrier 6 (SLC6) family members is of interest because point mutations result in misfolding and thus cause clinically relevant phenotypes in people. Here we examined the contribution of the C terminus in supporting folding of the serotonin transporter (SERT; SLC6A4). Our working hypothesis posited that the amphipathic nature of the C-terminal α-helix (Thr603–Thr613) was important for folding of SERT. Accordingly, we disrupted the hydrophobic moment of the α-helix by replacing Phe604, Ile608, or Ile612 by Gln. The bulk of the resulting mutants SERT-F604Q, SERT-I608Q, and SERT-I612Q were retained in the endoplasmic reticulum, but their residual delivery to the cell surface still depended on SEC24C. This indicates that the amphipathic nature of the C-terminal α-helix was dispensable to endoplasmic reticulum export. The folding trajectory of SERT is thought to proceed through the inward facing conformation. Consistent with this conjecture, cell surface expression of the misfolded mutants was restored by (i) introducing second site suppressor mutations, which trap SERT in the inward facing state, or (ii) by the pharmacochaperone noribogaine, which binds to the inward facing conformation. Finally, mutation of Glu615 at the end of the C-terminal α-helix to Lys reduced surface expression of SERT-E615K. A charge reversal mutation in intracellular loop 1 restored surface expression of SERT-R152E/E615K to wild type levels. These observations support a mechanistic model where the C-terminal amphipathic helix is stabilized by an intramolecular salt bridge between residues Glu615 and Arg152. This interaction acts as a pivot in the conformational search associated with folding of SERT. PMID:25869136

  4. The gelatinous extracellular matrix facilitates transport studies in kelp: visualization of pressure-induced flow reversal across sieve plates

    PubMed Central

    Knoblauch, Jan; Peters, Winfried S.; Knoblauch, Michael

    2016-01-01

    Background and Aims In vascular plants, important questions regarding phloem function remain unanswered due to problems with invasive experimental procedures in this highly sensitive tissue. Certain brown algae (kelps; Laminariales) also possess sieve tubes for photoassimilate transport, but these are embedded in large volumes of a gelatinous extracellular matrix which isolates them from neighbouring cells. Therefore, we hypothesized that kelp sieve tubes might tolerate invasive experimentation better than their analogues in higher plants, and sought to establish Nereocystis luetkeana as an experimental system. Methods The predominant localization of cellulose and the gelatinous extracellular matrix in N. luetkeana was verified using specific fluorescent markers and confocal laser scanning microscopy. Sieve tubes in intact specimens were loaded with fluorescent dyes, either passively (carboxyfluorescein diacetate; CFDA) or by microinjection (rhodamine B), and the movement of the dyes was monitored by fluorescence microscopy. Key Results Application of CFDA demonstrated source to sink bulk flow in N. luetkeana sieve tubes, and revealed the complexity of sieve tube structure, with branches, junctions and lateral connections. Microinjection into sieve elements proved comparatively easy. Pulsed rhodamine B injection enabled the determination of flow velocity in individual sieve elements, and the direct visualization of pressure-induced reversals of flow direction across sieve plates. Conclusions The reversal of flow direction across sieve plates by pressurizing the downstream sieve element conclusively demonstrates that a critical requirement of the Münch theory is satisfied in kelp; no such evidence exists for tracheophytes. Because of the high tolerance of its sieve elements to experimental manipulation, N. luetkeana is a promising alternative to vascular plants for studying the fluid mechanics of sieve tube networks. PMID:26929203

  5. Role of Achiral Nucleobases in Multicomponent Chiral Self-Assembly: Purine-Triggered Helix and Chirality Transfer.

    PubMed

    Deng, Ming; Zhang, Li; Jiang, Yuqian; Liu, Minghua

    2016-11-21

    Chiral self-assembly is a basic process in biological systems, where many chiral biomolecules such as amino acids and sugars play important roles. Achiral nucleobases usually covalently bond to saccharides and play a significant role in the formation of the double helix structure. However, it remains unclear how the achiral nucleobases can function in chiral self-assembly without the sugar modification. Herein, we have clarified that purine nucleobases could trigger N-(9-fluorenylmethox-ycarbonyl) (Fmoc)-protected glutamic acid to self-assemble into helical nanostructures. Moreover, the helical nanostructure could serve as a matrix and transfer the chirality to an achiral fluorescence probe, thioflavin T (ThT). Upon chirality transfer, the ThT showed not only supramolecular chirality but also circular polarized fluorescence (CPL). Without the nucleobase, the self-assembly processes cannot happen, thus providing an example where achiral molecules played an essential role in the expression and transfer of the chirality.

  6. [QTRAP LC-MS/MS Analytical Study on Nucleosides and Nucleobases of Pseudostellariae Radix Cultivated in Different Idioplasm Resources].

    PubMed

    Ma, Yang; Hou, Ya; Zou, Li-si; Liu, Xun-hong; Xu, Li; Lan, Cai-wu; Yuan, Ji-duan

    2015-04-01

    To analyze nucleosides and nucleobases of Pseudostellariae Radix cultivated in different idibplasni resources and to compare the differences. QTRAP LC-MS/MS method was applied for the analysis of 13 kinds of nucleosides and nucleobases in Pseudostellariae Radix and the data obtained was analyzed by SPSS 16. 0 software. There were some differences between Pseudostellariae Radix cultivated in different idioplasm resources. The highest amount of nucleosides and nucleobases was ZS2 which came from Zherong in Fujian Province. The total content of nucleosides and nucleobases in the sample from Shibing in Guizhou Province was the lowest. There was little difference between ZS1 (Zherong in Fujian Province) and XC(Xuancheng in Anhui Province). This study provides evidence for the influence of eco-environment on the metabolites of Pseudostellariae Radix.

  7. [Dynamic Changes of Nucleosides and Nucleobases in Different Harvest Periods of Polygoni Multiflori Radix by UPLC-QTRAP-MS/MS].

    PubMed

    Luo, Yi-Yuan; Liu, Juan-xiu; Liu, Xun-hong; Lan, Cai-wu; Hou, Ya; Ma, Yang; Xu, Li

    2015-05-01

    To analyze the dynamic changes of nucleosides and nucleobases in Polygoni Multiflori Radix harvested in different periods. UPLC-QTRAP-MS/MS method was applied for the analysis of nine kinds of nucleosides and nucleobases in Polygoni Multiflori Radix. The content of uridine, adenine, guanosine and cytidine was higher in Polygoni Multiflori Radix harvested in different periods and assumed some difference. The trends of nucleosides and nucleobases from Polygoni Multiflori Radix according to the peak valley shape changed. The highest contents of them were in December. The accumulation of nucleosides and nucleobases in Polygoni Multiflori Radix is closely related to its growth cycle. It is found to be basically the same as that obtained when the herb is collected during the conventional collecting time.

  8. Stomatal Spacing Safeguards Stomatal Dynamics by Facilitating Guard Cell Ion Transport Independent of the Epidermal Solute Reservoir12[CC-BY

    PubMed Central

    Papanatsiou, Maria; Amtmann, Anna

    2016-01-01

    Stomata enable gaseous exchange between the interior of the leaf and the atmosphere through the stomatal pore. Control of the pore aperture depends on osmotic solute accumulation by, and its loss from the guard cells surrounding the pore. Stomata in most plants are separated by at least one epidermal cell, and this spacing is thought to enhance stomatal function, although there are several genera that exhibit stomata in clusters. We made use of Arabidopsis (Arabidopsis thaliana) stomatal patterning mutants to explore the impact of clustering on guard cell dynamics, gas exchange, and ion transport of guard cells. These studies showed that stomatal clustering in the Arabidopsis too many mouths (tmm1) mutant suppressed stomatal movements and affected CO2 assimilation and transpiration differentially between dark and light conditions and were associated with alterations in K+ channel gating. These changes were consistent with the impaired dynamics of tmm1 stomata and were accompanied by a reduced accumulation of K+ ions in the guard cells. Our findings underline the significance of spacing for stomatal dynamics. While stomatal spacing may be important as a reservoir for K+ and other ions to facilitate stomatal movements, the effects on channel gating, and by inference on K+ accumulation, cannot be explained on the basis of a reduced number of epidermal cells facilitating ion supply to the guard cells. PMID:27406168

  9. Charge transfer from 2-aminopurine radical cation and radical anion to nucleobases: A pulse radiolysis study

    NASA Astrophysics Data System (ADS)

    Manoj, P.; Mohan, H.; Mittal, J. P.; Manoj, V. M.; Aravindakumar, C. T.

    2007-01-01

    Pulse radiolysis study has been carried out to investigate the properties of the radical cation of 2-aminopurine (2AP) and the probable charge transfer from the radical cation and radical anion of 2AP to natural nucleobases in aqueous medium. The radical cation of 2AP was produced by the reaction of sulfate radical anion ( SO4rad -). The time resolved absorption spectra obtained by the reaction of SO4rad - with 2AP at neutral pH have two distinct maxima at 380 and 470 nm and is assigned to the formation of a neutral radical of the form 2AP-N 2(-H) rad ( k2 = 4.7 × 10 9 dm 3 mol -1 s -1 at pH 7). This neutral radical is formed from the deprotonation reaction of a very short-lived radical cation of 2AP. The transient absorption spectra recorded at pH 10.2 have two distinct maxima at 400 and 480 nm and is assigned to the formation of a nitrogen centered radical (2AP-N(9) rad ). As the hole transport from 2AP to guanine is a highly probable process, the reaction of SO4rad - is carried out in the presence of guanosine, adenosine and inosine. The spectrum obtained in the presence of guanosine was significantly different from that in the absence and it showed prominent absorption maxima at 380 and 470 nm, and a weak broad maximum centered around 625 nm which match well with the reported spectrum of a neutral guanine radical (G(-H) rad ). The electron transfer reaction from the radical anion of 2AP to thymine (T), cytidine (Cyd) and uridine (Urd) was also investigated at neutral pH. Among the three pyrimidines, only the transient spectrum in the presence of T gave a significant difference from the spectral features of the electron adduct of 2AP, which showed a prominent absorption maximum at 340 nm and this spectrum is similar to the electron adduct spectrum of T. The preferential reduction of thymine by 2AP rad - and the oxidation of guanosine by 2AP rad + clearly follow the oxidation/reduction potentials of the purines and pyrimidines.

  10. High Nucleobase-Solubilizing Ability of Low-Viscous Ionic Liquid/Water Mixtures: Measurements and Mechanism.

    PubMed

    Ghoshdastidar, Debostuti; Ghosh, Dibbendu; Senapati, Sanjib

    2016-01-28

    Research on nucleobases has always been on the forefront owing to their ever-increasing demand in the pharmaceutical, agricultural, and other industries. The applications, however, became limited due to their poor solubility in water. Recently, ionic liquids (ILs) have emerged as promising solvents for nucleobase dissolution, as they exhibit >100-fold increased solubility compared to water. But the high viscosity of ILs remains as a bottleneck in the field. Here, by solubility and viscosity measurements, we show that addition of low-to-moderate quantity of water preserves the high solubilizing capacity of ILs, while reducing the viscosity of the solution by several folds. To understand the mechanism of nucleobase dissolution, molecular dynamics simulations were carried out, which showed that at low concentrations water incorporates into the IL-nucleobase network without much perturbing of the nucleobase-IL interactions. At higher concentrations, increasing numbers of IL anion-water hydrogen bonds replace IL-nucleobase interactions, which have been confirmed by (1)H- and (13)C NMR chemical shifts of the IL ions.

  11. Exciton energy transfer-based quantum dot fluorescence sensing array: "chemical noses" for discrimination of different nucleobases.

    PubMed

    Liu, Jianbo; Li, Gui; Yang, Xiaohai; Wang, Kemin; Li, Li; Liu, Wei; Shi, Xing; Guo, Yali

    2015-01-20

    A novel exciton energy transfer-based fluorescence sensing array for the discrimination of different nucleobases was developed through target nucleobase-triggered self-assembly of quantum dots (QDs). Four QD nanoprobes with different ligand receptors, including mercaptoethylamine, N-acetyl-l-cysteine, 2-dimethyl-aminethanethiol, and thioglycolic acid, were created to detect and identify nucleobase targets. These QDs served as both selective recognition scaffolds and signal transduction elements for a biomolecule target. The extent of particle assembly, induced by the analyte-triggered self-assembly of QDs, led to an exciton energy transfer effect between interparticles that gave a readily detectable fluorescence quenching and distinct fluorescence response patterns. These patterns are characteristic for each nucleobase and can be quantitatively differentiated by linear discriminate analysis. Furthermore, a fingerprint-based barcode was established to conveniently discriminate the nucleobases. This pattern sensing was successfully used to identify nucleobase samples at unknown concentrations and five rare bases. In this "chemical noses" strategy, the robust characteristics of QD nanoprobes, coupled with the diversity of surface functionality that can be readily obtained using nanoparticles, provides a simple and label-free biosensing approach that shows great promise for biomedical applications.

  12. MgMfs1, a major facilitator superfamily transporter from the fungal wheat pathogen Mycosphaerella graminicola, is a strong protectant against natural toxic compounds and fungicides.

    PubMed

    Roohparvar, Ramin; De Waard, Maarten A; Kema, Gert H J; Zwiers, Lute-Harm

    2007-05-01

    MgMfs1, a major facilitator superfamily (MFS) gene from the wheat pathogenic fungus Mycosphaerella graminicola, was identified in expressed sequence tag (EST) libraries. The encoded protein has high homology to members of the drug:H(+) antiporter efflux family of MFS transporters with 14 predicted transmembrane spanners (DHA14), implicated in mycotoxin secretion and multidrug resistance. Heterologous expression of MgMfs1 in a hypersensitive Saccharomyces cerevisiae strain resulted in a strong decrease in sensitivity of this organism to a broad range of unrelated synthetic and natural toxic compounds. The sensitivity of MgMfs1 disruption mutants of M. graminicola to most of these compounds was similar when compared to the wild-type but the sensitivity to strobilurin fungicides and the mycotoxin cercosporin was increased. Virulence of the disruption mutants on wheat seedlings was not affected. The results indicate that MgMfs1 is a true multidrug transporter that can function as a determinant of pathogen sensitivity and resistance to fungal toxins and fungicides.

  13. The role of facilitated diffusion in oxygen transport by cell-free hemoglobins: implications for the design of hemoglobin-based oxygen carriers.

    PubMed

    McCarthy, M R; Vandegriff, K D; Winslow, R M

    2001-08-30

    We compared rates of oxygen transport in an in vitro capillary system using red blood cells (RBCs) and cell-free hemoglobins. The axial PO(2) drop down the capillary was calculated using finite-element analysis. RBCs, unmodified hemoglobin (HbA(0)), cross-linked hemoglobin (alpha alpha-Hb) and hemoglobin conjugated to polyethylene-glycol (PEG-Hb) were evaluated. According to their fractional saturation curves, PEG-Hb showed the least desaturation down the capillary, which most closely matched the RBCs; HbA(0) and alpha alpha-Hb showed much greater desaturation. A lumped diffusion parameter, K*, was calculated based on the Fick diffusion equation with a term for facilitated diffusion. The overall rates of oxygen transfer are consistent with hemoglobin diffusion rates according to the Stokes-Einstein Law and with previously measured blood pressure responses in rats. This study provides a conceptual framework for the design of a 'blood substitute' based on mimicking O(2) transport by RBCs to prevent autoregulatory changes in blood flow and pressure.

  14. Colloid facilitated transport of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) to the groundwater at Ma Da area, Vietnam.

    PubMed

    Hofmann, Thilo; Wendelborn, Anke

    2007-06-01

    PCDD/Fs are hydrophobic organic substances and strongly sorbing to soil particles. Once adsorbed to soil particles they are believed to be virtually immobile. However, research in the last decades confirmed that strong sorbing contaminants may reach the groundwater via colloid-facilitated transport. This pathway has not been investigated before in Vietnam. Ma Da area, 100 km north of Ho Chi Minh City, was repeatedly sprayed during the Vietnam War (1962-1971) with herbicides like Agent Orange containing, beside others, the teratogenic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). 11 surface soil samples and 12 water samples were collected in Ma Da area for analysis of PCDD/Fs in solids. Soil TCDD concentrations ranged from 1-41 ppt with a mean of 8.8 ppt and a mean I-TEQ of 9.7 ppt. Two surface water samples showed colloid bound TCDD (7 and 19 ppt). Groundwater samples showed elevated colloid bound PCDD concentrations (mean 770 ng/kg), mainly octachlorodibenzo-p-dioxin. Groundwater colloids separated by filtration did not show any TCDD. The results support that TCDD/Fs can be relocated from the top soil to the groundwater by colloidal pathway. They did not provide evidence that the dioxins bound to groundwater colloids are leftovers from the Second Indochinese War. However, this study reinforces that the colloidal transport pathway has to be included investigating the relocation of strong sorbing organic contaminants.

  15. The rural Prehospital Acute Stroke Triage (PAST) trial protocol: a controlled trial for rapid facilitated transport of rural acute stroke patients to a regional stroke centre.

    PubMed

    Garnett, Ashley R; Marsden, Dianne L; Parsons, Mark W; Quain, Debbie A; Spratt, Neil J; Loudfoot, Allan R; Middleton, Paul M; Levi, Christopher R

    2010-12-01

    Access to intravenous thrombolysis for acute ischaemic stroke is limited worldwide, particularly in regional and rural areas including in Australia. We are testing the effectiveness of a new rural Prehospital Acute Stroke Triage protocol that includes prehospital assessment and rapid transport of patients from a rural catchment to the major stroke centre in Newcastle, NSW, Australia. The local district hospitals within the rural catchment do not have the capability or infrastructure to deliver acute stroke thrombolysis. The trial has relevance to stroke clinicians, health service managers and planners responsible for rural populations. To implement a system of rapid prehospital assessment and facilitated transport that will significantly increase stroke thrombolysis rates to 10% of ischaemic stroke cases in the rural catchment. Validate an eight-point modified National Institutes of Health Stroke Scale for use by paramedics in the prehospital setting to assess patients' potential eligibility for stroke thrombolysis. The joint project between the John Hunter Hospital Acute Stroke Team and the Ambulance Service of NSW will use a prospective cohort with an historical control group. Tools and protocols have been developed and education undertaken for ambulance field and operations centre personnel. These include a cut-down eight-item National Institutes of Health Stroke Scale (Hunter NIHSS-8) score to be used in the field by paramedics and a transport decision matrix to expedite transport for a suspected stroke patient (road or road plus air transport). The primary outcome measure will be the rate of intravenous tissue plasminogen activator delivery for those who suffer an ischaemic stroke following protocol implementation, in comparison with historical rates over a corresponding period prior to implementation, for residents within the catchment. Sixty cases are required in the postimplementation time epoch to demonstrate a statistically significant absolute increase

  16. A Major Facilitator Superfamily Transporter-Mediated Resistance to Oxidative Stress and Fungicides Requires Yap1, Skn7, and MAP Kinases in the Citrus Fungal Pathogen Alternaria alternata

    PubMed Central

    Chen, Li-Hung; Tsai, Hsieh-Chin; Yu, Pei-Ling

    2017-01-01

    Major Facilitator Superfamily (MFS) transporters play an important role in multidrug resistance in fungi. We report an AaMFS19 gene encoding a MFS transporter required for cellular resistance to oxidative stress and fungicides in the phytopathogenic fungus Alternaria alternata. AaMFS19, containing 12 transmembrane domains, displays activity toward a broad range of substrates. Fungal mutants lacking AaMFS19 display profound hypersensitivities to cumyl hydroperoxide, potassium superoxide, many singlet oxygen-generating compounds (eosin Y, rose Bengal, hematoporphyrin, methylene blue, and cercosporin), and the cell wall biosynthesis inhibitor, Congo red. AaMFS19 mutants also increase sensitivity to copper ions, clotrimazole, fludioxonil, and kocide fungicides, 2-chloro-5-hydroxypyridine (CHP), and 2,3,5-triiodobenzoic acid (TIBA). AaMFS19 mutants induce smaller necrotic lesions on leaves of a susceptible citrus cultivar. All observed phenotypes in the mutant are restored by introducing and expressing a wild-type copy of AaMFS19. The wild-type strain of A. alternata treated with either CHP or TIBA reduces radial growth and formation and germination of conidia, increases hyphal branching, and results in decreased expression of the AaMFS19 gene. The expression of AaMFS19 is regulated by the Yap1 transcription activator, the Hog1 and Fus3 mitogen-activated protein (MAP) kinases, the ‘two component’ histidine kinase, and the Skn7 response regulator. Our results demonstrate that A. alternata confers resistance to different chemicals via a membrane-bound MFS transporter. PMID:28060864

  17. Important factors stabilizing stacking interaction between 3-nitropyrrole and natural nucleobases revealed by ab initio calculations.

    PubMed

    Seio, Kohji; Ukawa, Hisashi; Shohda, Koh-ichiro; Sekine, Mitsuo

    2003-01-01

    Stacking energies between canonical nucleobases and a universal base, 3-nitropyrrole (3-NP), were estimated by use of molecular orbital (MO) and molecular mechanics (MM) calculations. The detailed analysis of the energy profiles revealed the importance of the London dispersion energy to stabilize the stacked dimers and electrostatic interactions to determine the orientation of 3-NP to the nucleobases in the dimers. Although the energy profiles of 3-NP/natural base dimers obtained by the MO and MM calculations were qualitatively correlated with each other, the correlations were poorer than those obtained for the stacking between natural bases. The origin of the difference between 3-NP and natural bases will be discussed to understand the possibility and limitation of the current MM calculations for the simulation and design of other universal bases.

  18. Terahertz spectra of DNA nucleobase crystals: A joint experimental and computational study.

    PubMed

    Wang, Fang; Zhao, Dongbo; Dong, Hao; Jiang, Ling; Liu, Yunfei; Li, Shuhua

    2017-02-21

    Terahertz (THz) spectra of DNA nucleobase crystals were experimentally studied by terahertz time domain spectroscopy (THz-TDS), Fourier transform infrared spectroscopy (FTIR), and computationally studied by the generalized energy-based fragmentation approach under periodic boundary conditions (denoted as PBC-GEBF). We analyzed the vibrational spectra of solid-state DNA nucleobases and assigned the corresponding vibrational modes to the main peaks in the experimental spectra with the PBC-GEBF results. The computational results were verified to be in good accordance with the experimental data. Harmonic vibrational frequency results revealed that all the vibrational modes belong to collective vibrational modes, which involve complicated mixtures of inter- and intramolecular displacements, somewhere in the vicinity of 0.5-9THz.

  19. Terahertz spectra of DNA nucleobase crystals: A joint experimental and computational study

    NASA Astrophysics Data System (ADS)

    Wang, Fang; Zhao, Dongbo; Dong, Hao; Jiang, Ling; Liu, Yunfei; Li, Shuhua

    2017-05-01

    Terahertz (THz) spectra of DNA nucleobase crystals were experimentally studied by terahertz time domain spectroscopy (THz-TDS), Fourier transform infrared spectroscopy (FTIR), and computationally studied by the generalized energy-based fragmentation approach under periodic boundary conditions (denoted as PBC-GEBF). We analyzed the vibrational spectra of solid-state DNA nucleobases and assigned the corresponding vibrational modes to the main peaks in the experimental spectra with the PBC-GEBF results. The computational results were verified to be in good accordance with the experimental data. Harmonic vibrational frequency results revealed that all the vibrational modes belong to collective vibrational modes, which involve complicated mixtures of inter- and intramolecular displacements, somewhere in the vicinity of 0.5-9 THz.

  20. Formation of Nucleobases from the UV Irradiation of Pyrimidine in Interstellar Ice Analogs

    NASA Technical Reports Server (NTRS)

    Milam, Stefanie N.; Nuevo, Michel; Sandford, Scott A.; Elsila, Jamie E.; Dworkin, Jason P.

    2010-01-01

    Previous laboratory simulations showed that complex molecules, including prebiotic compounds/can be formed under interstellar conditions from the vacuum UV irradiation of interstellar ice analogs containing H2O, CO, NH3 etc. Although some complex prebiotic species have not been confirmed In the interstellar medium, they are known to be present in meteorites. Nucleobases, the building blocks of DNA and RNA, have also been detected in meteorites. Here, we present a study of the formation of pyrimidine-based compounds from the UV irradiation of pyrimidine in H2O- and/or NH3-ices at 20-30 K, Our results show that various derivatives, induding the nucleobases uracil and cytosine, are formed under these conditions.

  1. Characterization of the stacking interactions between DNA or RNA nucleobases and the aromatic amino acids

    NASA Astrophysics Data System (ADS)

    Rutledge, Lesley R.; Campbell-Verduyn, Lachlan S.; Wetmore, Stacey D.

    2007-08-01

    MP2/6-31G ∗(0.25) gas-phase potential energy surfaces of stacked dimers between the four aromatic amino acids and the natural (DNA or RNA) nucleobases were considered as a function of three variables (vertical separation, angle of rotation, and horizontal displacement). The maximum stacking interaction was found to increase with the amino acid according to PHE < HIS ≈ TYR < TRP, while the stacking energy is generally largest for the purines compared to the pyrimidines. Most notably, the interaction energies are up to -43 kJ mol -1. Comparison of the magnitude of these interactions with, for example, hydrogen-bonding and stacking interactions that stabilize DNA duplexes suggests that π-π stacking between nucleobases and amino acids likely plays a large role in many fundamental biological processes.

  2. Nucleobase and Ribose Modifications Control Immunostimulation by a MicroRNA-122-mimetic RNA

    PubMed Central

    Peacock, Hayden; Fucini, Raymond V.; Jayalath, Prasanna; Ibarra-Soza, José M.; Haringsma, Henry J.; Flanagan, W. Michael; Willingham, Aarron; Beal, Peter A.

    2011-01-01

    Immune stimulation is a significant hurdle in the development of effective and safe RNA interference therapeutics. Here, we address this problem in the context of a mimic of microRNA-122 by employing novel nucleobase and known 2′-ribose modifications. The nucleobase modifications are analogues of adenosine and guanosine that contain cyclopentyl and propyl minor-groove projections. Via a site-by-site chemical modification analysis, we identify several immunostimulatory ‘hot spots’ within the miRNA guide strand at which single base modifications significantly reduce immune stimulation. A duplex containing one base modification on each strand proved to be most effective in preventing immune stimulation. PMID:21612237

  3. Formation of Nucleobases from the UV Irradiation of Pyrimidine in Interstellar Ice Analogs

    NASA Technical Reports Server (NTRS)

    Milam, Stefanie N.; Nuevo, Michel; Sandford, Scott A.; Elsila, Jamie E.; Dworkin, Jason P.

    2010-01-01

    Previous laboratory simulations showed that complex molecules, including prebiotic compounds/can be formed under interstellar conditions from the vacuum UV irradiation of interstellar ice analogs containing H2O, CO, NH3 etc. Although some complex prebiotic species have not been confirmed In the interstellar medium, they are known to be present in meteorites. Nucleobases, the building blocks of DNA and RNA, have also been detected in meteorites. Here, we present a study of the formation of pyrimidine-based compounds from the UV irradiation of pyrimidine in H2O- and/or NH3-ices at 20-30 K, Our results show that various derivatives, induding the nucleobases uracil and cytosine, are formed under these conditions.

  4. Synthesis of rigid homo- and heteroditopic nucleobase-terminated molecules incorporating adenine and/or thymine.

    PubMed

    Jacobsen, Mikkel F; Andersen, Casper S; Knudsen, Martin M; Gothelf, Kurt V

    2007-07-19

    A series of homo- and heteroditopic thymine- and/or adenine-terminated molecules incorporating rigid aryl or oligo(phenylene ethynylene) linkers has been efficiently synthesized. The key steps involved in the synthesis are the construction of the N-arylated nucleobases using the Chan-Lam-Evans-modified Ullman coupling and their further elaboration using the Sonogashira coupling. Furthermore, the synthesis of a rigid tripodal thymine derivative is reported.

  5. The photochemistry of pyrimidine in realistic astrophysical ices and the production of nucleobases

    SciTech Connect

    Nuevo, Michel; Materese, Christopher K.; Sandford, Scott A.

    2014-10-01

    Nucleobases, together with deoxyribose/ribose and phosphoric acid, are the building blocks of DNA and RNA for all known life. The presence of nucleobase-like compounds in carbonaceous chondrites delivered to the Earth raises the question of an extraterrestrial origin for the molecules that triggered life on our planet. Whether these molecules are formed in interstellar/protostellar environments, in small parent bodies in the solar system, or both, is currently unclear. Recent experiments show that the UV irradiation of pyrimidine (C{sub 4}H{sub 4}N{sub 2}) in H{sub 2}O-rich ice mixtures that contain NH{sub 3}, CH{sub 3}OH, or CH{sub 4} leads to the formation of the pyrimidine-based nucleobases uracil, cytosine, and thymine. In this work, we discuss the low-temperature UV irradiation of pyrimidine in realistic astrophysical ice mixtures containing H{sub 2}O, CH{sub 3}OH, and NH{sub 3}, with or without CH{sub 4}, to search for the production of nucleobases and other prebiotic compounds. These experiments show the presence of uracil, urea, glycerol, hexamethylenetetramine, small amino acids, and small carboxylic acids in all samples. Cytosine was only found in one sample produced from ices irradiated with a higher UV dose, while thymine was not found in any sample, even after irradiation with a higher UV dose. Results are discussed to evaluate the role of the photochemistry of pyrimidine in the inventory of organic molecules detected in meteorites and their astrophysical/astrobiological implications.

  6. Studies on effective atomic numbers and electron densities of nucleobases in DNA

    NASA Astrophysics Data System (ADS)

    Kumar, Ashok

    2016-10-01

    Various parameters of dosimetric importance such as effective atomic numbers (Zeff) and electron densities (Nel) of nucleobases in DNA have been calculated for the total and partial photon interaction processes in the wide energy range of 1 keV-100 GeV. The variations of Zeff and Nel with energy are shown graphically for all partial and total interaction processes and are found to be similar. Up to 10 keV, Zeff and Nel show a sharp increase for cytosine-guanine and thymine-adenine whereas for all the other nucleobases, it is almost constant. Then there is sharp decrease in Zeff and Nel with energy up to 100 keV for all the nucleobases. From 100 keV to 6 MeV, Zeff and Nel are almost independent of energy. From 6 MeV to 100 MeV, there is regular increase in Zeff and Nel with photon energy. Above 400 MeV, Zeff and Nel remain almost constant. The obtained results are due to the dominance of photoelectric absorption, Compton scattering and pair production in different energy regions as respectively stated above and their dependences on the chemical compositions of the interacting media.

  7. Formation of Nucleobases from the UV Irradiation of Pyrimidine in Astrophysical Ice Analogs

    NASA Technical Reports Server (NTRS)

    Sandford, Scott A.; Nuevo, Michel; Materese, Christopher K.

    2014-01-01

    Nucleobases are the informational subunits of DNA and RNA. They consist of Nheterocycles that belong to either the pyrimidine-base group (uracil, cytosine, and thymine) or the purinebase group (adenine and guanine). Several nucleobases, mostly purine bases, have been detected in meteorites [1-3], with isotopic signatures consistent with an extraterrestrial origin [4]. Uracil is the only pyrimidine-base compound formally reported in meteorites [2], though the presence of cytosine cannot be ruled out [5,6]. However, the actual process by which the uracil was made and the reasons for the non-detection of thymine in meteorites have yet to be fully explained. Although no N-heterocycles have ever been observed in the ISM [7,8], the positions of the 6.2-µm interstellar emission features suggest a population of such molecules is likely to be present [9]. In this work we study the formation of pyrimidine-based molecules, including the three nucleobases uracil, cytosine, and thymine from the ultraviolet (UV) irradiation of pyrimidine in ices consisting of several combinations of H(sub2)O, NH(sub3), CH(sub3)OH, and CH(sub4) at low temperature, in order to simulate the astrophysical conditions under which prebiotic species may be formed in the interstellar medium, in the protosolar nebula, and on icy bodies of the Solar System.

  8. Application of the Mars Organic Analyzer to Nucleobase and Amine Biomarker Detection

    NASA Astrophysics Data System (ADS)

    Skelley, Alison M.; Cleaves, H. James; Jayarajah, Christine N.; Bada, Jeffrey L.; Mathies, Richard A.

    2006-12-01

    The Mars Organic Analyzer (MOA), a portable microfabricated capillary electrophoresis instrument being developed for planetary exploration, is used to analyze a wide variety of fluorescamine-labeled amine-containing biomarker compounds, including amino acids, mono and diaminoalkanes, amino sugars, nucleobases, and nucleobase degradation products. The nucleobases cytosine and adenine, which contain an exocyclic primary amine, were effectively labeled, separated, and detected at concentrations <500 nM. To test the general applicability of the MOA for biomarker detection, amino acids and mono- and diamines were extracted from bacterial cells using both hydrolysis and sublimation followed by analysis. The extrapolated limit of detection provided by the valine biomarker was ~4 × 103 cells per sample. Products of an NH4CN polymerization that simulate a prebiotic synthesis were also successfully isolated via sublimation and analyzed. Adenine and alanine/serine were detected with no additional sample cleanup at 120 +/- 13 µM and 4.1 +/- 1 µM, respectively, corresponding to a reaction yield of 0.04% and 0.0003%, respectively. This study demonstrates that the MOA provides sensitive detection and analysis of low levels of a wide variety of amine-containing organic compounds from both biological and abiotic sources.

  9. Binding of Organometallic Ruthenium(II) Anticancer Compounds to Nucleobases: A Computational Study

    NASA Astrophysics Data System (ADS)

    Gossens, Christian; Tavernelli, Ivano; Rothlisberger, Ursula

    2009-09-01

    The reaction of the anticancer compound [(η6-benzene)Ru(en)(OH2)]2+ (1) toward the nucleobases guanine, adenine, and cytosine is studied computationally using DFT/BP86 calculations. The aqua leaving group of such compounds is known to undergo ligand exchange reactions with nucleophilic centers in DNA and preferentially with the N7 atom of guanine, N7(G). Our results show that an H-bonded reactant adduct with nucleobases is formed via either the aqua ligand (cis adduct) or the en (ethylenediamine) ligand (trans adduct) of 1. All studied nucleobases favor an H-bonded cis adduct. Only guanine forms also a trans reactant adduct in the gas phase. The guanine N7 and O6 atoms in this trans adduct are situated in an ideal position to form each a strong H-bond to both amino groups of the en ligand of 1. A docking study shows that this unique recognition pattern is also plausible for the interaction with double stranded DNA. For the reaction of 1 with guanine, we identified three different reaction pathways: (i) A cis (G)N7-Ru-OH2 transition state (TS). (ii) A direct trans reaction pathway. (iii) A 2-step trans mechanism. The activation energies for the cis pathway are smaller than for the trans pathways. The ultimately formed Ru-N7(G) product is characterized by a thermally stable H-bond between the O6(G) and a diamine-NH2 hydrogen.

  10. Determination of HDV ribozyme N(-1) nucleobase and functional group specificity using internal competition kinetics

    PubMed Central

    Kellerman, Daniel L; Simmons, Kandice S; Pedraza, Mayra; Piccirilli, Joseph A; York, Darrin M; Harris, Michael E

    2015-01-01

    Biological catalysis involves interactions distant from the site of chemistry that can position the substrate for reaction. Catalysis of RNA 2′-O-transphosphorylation by the HDV ribozyme is sensitive to the identity of the N(-1) nucleotide flanking the reactive phosphoryl group. However, the interactions that affect the conformation of this position, and in turn the 2′O nucleophile, are unclear. Here, we describe the application of multiple substrate internal competition kinetic analyses to understand how the N(-1) nucleobase contributes to HDV catalysis, and to test the utility of this approach for RNA structure-function studies. Internal competition reactions containing all four substrate sequence variants at the N(-1) position in reactions using ribozyme active site mutations at A77 and A78 were used to test a proposed basepairing interaction. Mutants A78U, A78G and A79G retain significant catalytic activity, but do not alter the specificity for the N(-1) nucleobase. Effects of nucleobase analog substitutions at N(-1) indicate that U is preferred due to the ability to donate an H-bond in the Watson-Crick face and avoid minor groove steric clash. The results provide information essential for evaluating models of the HDV active site, and illustrate multiple-substrate kinetic analyses as a practical approach for characterizing structure-function relationships in RNA reactions. PMID:25937290

  11. Molecularly resolved label-free sensing of single nucleobase mismatches by interfacial LNA probes

    PubMed Central

    Mishra, Sourav; Lahiri, Hiya; Banerjee, Siddhartha; Mukhopadhyay, Rupa

    2016-01-01

    So far, there has been no report on molecularly resolved discrimination of single nucleobase mismatches using surface-confined single stranded locked nucleic acid (ssLNA) probes. Herein, it is exemplified using a label-independent force-sensing approach that an optimal coverage of 12-mer ssLNA sensor probes formed onto gold(111) surface allows recognition of ssDNA targets with twice stronger force sensitivity than 12-mer ssDNA sensor probes. The force distributions are reproducible and the molecule-by-molecule force measurements are largely in agreement with ensemble on-surface melting temperature data. Importantly, the molecularly resolved detection is responsive to the presence of single nucleobase mismatches in target sequences. Since the labelling steps can be eliminated from protocol, and each force-based detection event occurs within milliseconds' time scale, the force-sensing assay is potentially capable of rapid detection. The LNA probe performance is indicative of versatility in terms of substrate choice - be it gold (for basic research and array-based applications) or silicon (for ‘lab-on-a-chip’ type devices). The nucleic acid microarray technologies could therefore be generally benefited by adopting the LNA films, in place of DNA. Since LNA is nuclease-resistant, unlike DNA, and the LNA-based assay is sensitive to single nucleobase mismatches, the possibilities for label-free in vitro rapid diagnostics based on the LNA probes may be explored. PMID:27025649

  12. Nucleobase and amino acid formation through impacts of meteorites on the early ocean

    NASA Astrophysics Data System (ADS)

    Furukawa, Yoshihiro; Nakazawa, Hiromoto; Sekine, Toshimori; Kobayashi, Takamichi; Kakegawa, Takeshi

    2015-11-01

    The emergence of life's building blocks on the prebiotic Earth was the first crucial step for the origins of life. Extraterrestrial delivery of intact amino acids and nucleobases is the prevailing hypothesis for their availability on prebiotic Earth because of the difficulties associated with the production of these organics from terrestrial carbon and nitrogen sources under plausible prebiotic conditions. However, the variety and amounts of these intact organics delivered by meteorites would have been limited. Previous shock-recovery experiments have demonstrated that meteorite impact reactions could have generated organics on the prebiotic Earth. Here, we report on the simultaneous formation of nucleobases (cytosine and uracil) found in DNA and/or RNA, various proteinogenic amino acids (glycine, alanine, serine, aspartic acid, glutamic acid, valine, leucine, isoleucine, and proline), non-proteinogenic amino acids, and aliphatic amines in experiments simulating reactions induced by extraterrestrial objects impacting on the early oceans. To the best of our knowledge, this is the first report of the formation of nucleobases from inorganic materials by shock conditions. In these experiments, bicarbonate was used as the carbon source. Bicarbonate, which is a common dissolved carbon species in CO2-rich atmospheric conditions, was presumably the most abundant carbon species in the early oceans and in post-impact plumes. Thus, the present results expand the possibility that impact-induced reactions generated various building blocks for life on prebiotic Earth in large quantities through the use of terrestrial carbon reservoirs.

  13. Determination of nucleosides and nucleobases in the pollen of Typha angustifolia by UPLC-PDA-MS.

    PubMed

    Tao, Wei-Wei; Duan, Jin-Ao; Yang, Nian-Yun; Guo, Sheng; Zhu, Zhen-Hua; Tang, Yu-Ping; Qian, Da-Wei

    2012-01-01

    The pollen of Typha angustifolia L. has been used traditionally for the treatment of dysmenorrhea, stranguria and metrorrhagia in China. Recently, nucleosides and nucleobases have been proven as important bioactive compounds. Exploration of the nucleoside and nucleobase profiles from the pollen of T. angustifolia is important for improving its therapeutic value and could be convenient for its quality evaluation. To establish an UPLC-PDA-MS method for simultaneous determination of nucleosides and nucleobases in the pollen of T. angustifolia. The analysis was performed on an Acuity UPLCHSS T3 column with a gradient elution of 5 mM ammonium acetate and methanol solution at a flow rate of 0.3 mL/min. Satisfactory separation of these compounds was obtained in less than 12 min. All calibration curves showed good linear regression (r²  > 0.9995). The method provided good accuracy, precision, recovery, and sensitivity for the quantification of the 10 compounds analysed. The UPLC method established is very helpful for optimising their content and could be convenient for quality evaluation of the pollen of T. angustifolia, which has not been reported as far as we are aware. Copyright © 2011 John Wiley & Sons, Ltd.

  14. The origin of efficient triplet state population in sulfur-substituted nucleobases

    PubMed Central

    Mai, Sebastian; Pollum, Marvin; Martínez-Fernández, Lara; Dunn, Nicholas; Marquetand, Philipp; Corral, Inés; Crespo-Hernández, Carlos E.; González, Leticia

    2016-01-01

    Elucidating the photophysical mechanisms in sulfur-substituted nucleobases (thiobases) is essential for designing prospective drugs for photo- and chemotherapeutic applications. Although it has long been established that the phototherapeutic activity of thiobases is intimately linked to efficient intersystem crossing into reactive triplet states, the molecular factors underlying this efficiency are poorly understood. Herein we combine femtosecond transient absorption experiments with quantum chemistry and nonadiabatic dynamics simulations to investigate 2-thiocytosine as a necessary step to unravel the electronic and structural elements that lead to ultrafast and near-unity triplet-state population in thiobases in general. We show that different parts of the potential energy surfaces are stabilized to different extents via thionation, quenching the intrinsic photostability of canonical DNA and RNA nucleobases. These findings satisfactorily explain why thiobases exhibit the fastest intersystem crossing lifetimes measured to date among bio-organic molecules and have near-unity triplet yields, whereas the triplet yields of canonical nucleobases are nearly zero. PMID:27703148

  15. A Critical Role for the Putative NCS2 Nucleobase Permease YjcD in the Sensitivity of Escherichia coli to Cytotoxic and Mutagenic Purine Analogs

    PubMed Central

    Kozmin, Stanislav G.; Stepchenkova, Elena I.; Chow, Stephen C.; Schaaper, Roel M.

    2013-01-01

    ABSTRACT The base analogs 6-N-hydroxylaminopurine (HAP) and 2-amino-HAP (AHAP) are potent mutagens in bacteria and eukaryotic organisms. Previously, we demonstrated that a defect in the Escherichia coli ycbX gene, encoding a molybdenum cofactor-dependent oxidoreductase, dramatically enhances sensitivity to the toxic and mutagenic action of these agents. In the present study, we describe the discovery and properties of a novel suppressor locus, yjcD, that strongly reduces the HAP sensitivity of the ycbX strain. Suppressor effects are also observed for other purine analogs, like AHAP, 6-mercaptopurine, 6-thioguanine, and 2-aminopurine. In contrast, the yjcD defect did not affect the sensitivity to the pyrimidine analog 5-fluorouracil. Homology searches have predicted that yjcD encodes a putative permease of the NCS2 family of nucleobase transporters. We further investigated the effects of inactivation of all other members of the NCS2 family, XanQ, XanP, PurP, UacT, UraA, RutG, YgfQ, YicO, and YbbY, and of the NCS1 family nucleobase permeases CodB and YbbW. None of these other defects significantly affected sensitivity to either HAP or AHAP. The combined data strongly suggest that YjcD is the primary importer for modified purine bases. We also present data showing that this protein may, in fact, also be a principal permease involved in transport of the normal purines guanine, hypoxanthine, and/or xanthine. PMID:24169576

  16. Increased gene expression of a facilitated diffusion urea transporter in the skin of the African lungfish (Protopterus annectens) during massively elevated post-terrestrialization urea excretion.

    PubMed

    Hung, Carrie Y C; Galvez, Fernando; Ip, Yuen K; Wood, Chris M

    2009-04-01

    The full-length cDNA sequence of a putative urea transporter (lfUT) of the facilitated diffusion UT-A type has been cloned from the African lungfish Protopterus annectens. The lfUT cDNA is 1990 bp in length and its open reading frame encodes a 409 amino acid long protein, with a calculated molecular mass of 44,723 Da. The sequence is closest to those of amphibians ( approximately 65% amino acid homology), followed by mammals and elasmobranchs ( approximately 60%), and then teleosts ( approximately 50%). lfUT was clearly expressed in gill, kidney, liver, skeletal muscle and skin. Upon re-immersion in water after 33 days of air exposure ('terrestrialization'), lungfish exhibited a massive rise in urea-N excretion which peaked at 12-30 h with rates of 2000-5000 micromol-N kg(-1) h(-1) (versus normal aquatic rates of <130 micromol-N kg(-1) h(-1)) and persisted until 70 h. This appears to occur mainly through the skin. Total 'excess' urea-N excretion amounted to approximately 81,000-91,000 micromol-N kg(-1) over 3 days. By real-time PCR, there was no difference in lfUT expression in the ventral abdominal skin between aquatic ammoniotelic controls and terrestrialized lungfish immediately after return to water (0 h), and no elevation of urea-N excretion at this time. However, skin biopsies revealed a significant 2.55-fold elevation of lfUT expression at 14 h, coincident with peak urea-N excretion. At 48 h, there was no longer any significant difference in lfUT mRNA levels from those at 0 and 14 h, or from aquatic fed controls. In accordance with earlier studies, which identified elevated urea-N excretion via the skin of P. dolloi with pharmacology typical of UT-A carriers, these results argue that transcriptional activation of a facilitated diffusion type urea transporter (lfUT) occurs in the skin during re-immersion. This serves to clear the body burden of urea-N accumulated during terrestrialization.

  17. Nucleobases and other Prebiotic Species from the Ultraviolet Irradiation of Pyrimidine in Astrophysical Ices

    NASA Technical Reports Server (NTRS)

    Sandford, S. A.; Nuevo, M.; Materese, C. K.; Milam, S. N.

    2012-01-01

    Nucleobases are N-heterocycles that are the informational subunits of DNA and RNA, and are divided into two families: pyrimidine bases (uracil, cytosine, and thymine) and purine bases (adenine and guanine). Nucleobases have been detected in meteorites and their extraterrestrial origin confirmed by isotope measurement. Although no Nheterocycles have ever been observed in the ISM, the positions of the 6.2-m interstellar emission features suggest a population of such molecules is likely to be present. In this work we study the formation of pyrimidine-based molecules, including nucleobases, as well as other species of prebiotic interest, from the ultraviolet (UV) irradiation of pyrimidine in combinations of H2O, NH3, CH3OH, and CH4 ices at low temperature, in order to simulate the astrophysical conditions under which prebiotic species may be formed in the interstellar medium and icy bodies of the Solar System. Experimental: Gas mixtures are prepared in a glass mixing line (background pressure approx. 10(exp -6)-10(exp -5) mbar). Relative proportions between mixture components are determined by their partial pressures. Gas mixtures are then deposited on an aluminum foil attached to a cold finger (15-20 K) and simultaneously irradiated with an H2 lamp emitting UV photons (Lyman and a continuum at approx.160 nm). After irradiation samples are warmed to room temperature, at which time the remaining residues are recovered to be analyzed with liquid and gas chromatographies. Results: These experiments showed that the UV irradiation of pyrimidine mixed in these ices at low temperature leads to the formation of several photoproducts derived from pyrimidine, including the nucleobases uracil and cytosine, as well as their precursors 4(3H)-pyrimidone and 4-aminopyrimidine (Fig. 1). Theoretical quantum calculations on the formation of 4(3H)-pyrimidone and uracil from the irradiation of pyrimidine in pure H2O ices are in agreement with their experimental formation pathways. In

  18. Enhanced Binding Affinity for an i-Motif DNA Substrate Exhibited by a Protein Containing Nucleobase Amino Acids.

    PubMed

    Bai, Xiaoguang; Talukder, Poulami; Daskalova, Sasha M; Roy, Basab; Chen, Shengxi; Li, Zhongxian; Dedkova, Larisa M; Hecht, Sidney M

    2017-04-05

    Several variants of a nucleic acid binding motif (RRM1) of putative transcription factor hnRNP LL containing nucleobase amino acids at specific positions have been prepared and used to study binding affinity for the BCL2 i-motif DNA. Molecular modeling suggested a number of amino acids in RRM1 likely to be involved in interaction with the i-motif DNA, and His24 and Arg26 were chosen for modification based on their potential ability to interact with G14 of the i-motif DNA. Four nucleobase amino acids were introduced into RRM1 at one or both of positions 24 and 26. The introduction of cytosine nucleobase 2 into position 24 of RRM1 increased the affinity of the modified protein for the i-motif DNA, consistent with the possible Watson-Crick interaction of 2 and G14. In comparison, the introduction of uracil nucleobase 3 had a minimal effect on DNA affinity. Two structurally simplified nucleobase analogues (1 and 4) lacking both the N-1 and the 2-oxo substituents were also introduced in lieu of His24. Again, the RRM1 analogue containing 1 exhibited enhanced affinity for the i-motif DNA, while the protein analogue containing 4 bound less tightly to the DNA substrate. Finally, the modified protein containing 1 in lieu of Arg26 also bound to the i-motif DNA more strongly than the wild-type protein, but a protein containing 1 both at positions 24 and 26 bound to the DNA less strongly than wild type. The results support the idea of using nucleobase amino acids as protein constituents for controlling and enhancing DNA-protein interaction. Finally, modification of the i-motif DNA at G14 diminished RRM1-DNA interaction, as well as the ability of nucleobase amino acid 1 to stabilize RRM1-DNA interaction.

  19. Functional interplay between the ATP binding cassette Msr(D) protein and the membrane facilitator superfamily Mef(E) transporter for macrolide resistance in Escherichia coli.

    PubMed

    Nunez-Samudio, Virginia; Chesneau, Olivier

    2013-04-01

    Macrolides have wide clinical applications in the treatment of community-acquired respiratory tract infections, among which streptococci are the most frequent causative agents. An active efflux-based mechanism of macrolide resistance, referred to as the M phenotype in streptococcal isolates, has been associated with the presence of mef genes that encode a subset of major facilitator superfamily (MFS) transporters like Mef(E). An msr(D) gene, adjacent to and co-transcribed with mef in the presence of erythromycin, has also been implicated in drug efflux, but its role remains elusive. Msr(D) belongs to the ATP binding cassette (ABC) proteins and harbors two fused nucleotide-binding domains with no membrane-spanning domains. The present work indicates that the major resistance traits of the M phenotype in Escherichia coli may be due to Msr(D) and not to Mef(E). Fluorescence microscopy using Mef(E) tagged with GFP linked low efficacy of the chimera in conferring macrolide resistance with improper subcellular localization. The active role of Msr(D) in directing Mef(E)-GFP to the cell poles was demonstrated, as was synergistic effect in terms of levels of resistance when both proteins were expressed. A trans-dominant negative mutation within ABC Msr(D) affecting MFS Mef(E) strongly suggests that both proteins can interact in vivo, and such a physical interaction was supported in vitro. This is the first reported example of a functional interplay between an ABC component and an MFS transporter. The direct involvement of Msr(D) in the efflux of macrolides remains to be demonstrated.

  20. Cytoplasmic fragment of Alcadein α generated by regulated intramembrane proteolysis enhances amyloid β-protein precursor (APP) transport into the late secretory pathway and facilitates APP cleavage.

    PubMed

    Takei, Norio; Sobu, Yuriko; Kimura, Ayano; Urano, Satomi; Piao, Yi; Araki, Yoichi; Taru, Hidenori; Yamamoto, Tohru; Hata, Saori; Nakaya, Tadashi; Suzuki, Toshiharu

    2015-01-09

    The neural type I membrane protein Alcadein α (Alcα), is primarily cleaved by amyloid β-protein precursor (APP) α-secretase to generate a membrane-associated carboxyl-terminal fragment (Alcα CTF), which is further cleaved by γ-secretase to secrete p3-Alcα peptides and generate an intracellular cytoplasmic domain fragment (Alcα ICD) in the late secretory pathway. By association with the neural adaptor protein X11L (X11-like), Alcα and APP form a ternary complex that suppresses the cleavage of both Alcα and APP by regulating the transport of these membrane proteins into the late secretory pathway where secretases are active. However, it has not been revealed how Alcα and APP are directed from the ternary complex formed largely in the Golgi into the late secretory pathway to reach a nerve terminus. Using a novel transgenic mouse line expressing excess amounts of human Alcα CTF (hAlcα CTF) in neurons, we found that expression of hAlcα CTF induced excess production of hAlcα ICD, which facilitated APP transport into the nerve terminus and enhanced APP metabolism, including Aβ generation. In vitro cell studies also demonstrated that excess expression of Alcα ICD released both APP and Alcα from the ternary complex. These results indicate that regulated intramembrane proteolysis of Alcα by γ-secretase regulates APP trafficking and the production of Aβ in vivo. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. A structural model of the copper ATPase ATP7B to facilitate analysis of Wilson disease-causing mutations and studies of the transport mechanism.

    PubMed

    Schushan, Maya; Bhattacharjee, Ashima; Ben-Tal, Nir; Lutsenko, Svetlana

    2012-07-01

    The copper-transporting ATPase ATP7B has an essential role in human physiology, particularly for the liver and brain function. Inactivation of ATP7B is associated with a severe hepato-neurologic disorder, Wilson disease (WD). Hundreds of WD related mutations have been identified in ATP7B to date. The low frequency and the compound-heterozygous nature of causative mutations complicate the analysis of individual mutants and the establishment of genotype-phenotype correlations. To facilitate studies of disease-causing mutations and mechanistic understanding of WD, we have homology-modelled the ATP7B core (residues 643-1377) using the recent structure of the bacterial copper-ATPase LCopA as a template. The model, supported by evolutionary conservation and hydrophobicity analysis, as well as existing and new mutagenesis data, allows molecular interpretations of experimentally characterized clinical mutations. We also illustrate that structure and conservation can be used to grade potential deleterious effects for many WD mutations, which were clinically detected but have not yet been experimentally characterized. Finally, we compare the structural features of ATP7B and LCopA and discuss specific features of the eukaryotic copper pump.

  2. TRANSPORT

    EPA Science Inventory

    Presentation outline: transport principles, effective solubility; gasoline composition; and field examples (plume diving).
    Presentation conclusions: MTBE transport follows from - phyiscal and chemical properties and hydrology. Field examples show: MTBE plumes > benzene plu...

  3. 49 CFR 38.2 - Equivalent facilitation.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Equivalent facilitation. 38.2 Section 38.2 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES General § 38.2 Equivalent facilitation. Departures from...

  4. 49 CFR 38.2 - Equivalent facilitation.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 1 2011-10-01 2011-10-01 false Equivalent facilitation. 38.2 Section 38.2 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES General § 38.2 Equivalent facilitation. Departures from...

  5. 49 CFR 38.2 - Equivalent facilitation.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 49 Transportation 1 2013-10-01 2013-10-01 false Equivalent facilitation. 38.2 Section 38.2 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES General § 38.2 Equivalent facilitation. Departures from...

  6. 49 CFR 38.2 - Equivalent facilitation.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 49 Transportation 1 2014-10-01 2014-10-01 false Equivalent facilitation. 38.2 Section 38.2 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES General § 38.2 Equivalent facilitation. Departures from...

  7. 49 CFR 38.2 - Equivalent facilitation.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 49 Transportation 1 2012-10-01 2012-10-01 false Equivalent facilitation. 38.2 Section 38.2 Transportation Office of the Secretary of Transportation AMERICANS WITH DISABILITIES ACT (ADA) ACCESSIBILITY SPECIFICATIONS FOR TRANSPORTATION VEHICLES General § 38.2 Equivalent facilitation. Departures from...

  8. Probing Nucleobase Interactions and Predicting Mechanisms of Synthetic Interest Using Computational Chemistry, and Furthering the Development of BVI Education in Chemistry

    ERIC Educational Resources Information Center

    Harrison, Jason Gordon

    2013-01-01

    Quantum mechanical (QM) and molecular docking methods are used to probe systems of biological and synthetic interest. Probing interactions of nucleobases within proteins, and properly modeling said interactions toward novel nucleobase development, is extremely difficult, and of great utility in RNA interference (RNAi) therapeutics. The issues in…

  9. Probing Nucleobase Interactions and Predicting Mechanisms of Synthetic Interest Using Computational Chemistry, and Furthering the Development of BVI Education in Chemistry

    ERIC Educational Resources Information Center

    Harrison, Jason Gordon

    2013-01-01

    Quantum mechanical (QM) and molecular docking methods are used to probe systems of biological and synthetic interest. Probing interactions of nucleobases within proteins, and properly modeling said interactions toward novel nucleobase development, is extremely difficult, and of great utility in RNA interference (RNAi) therapeutics. The issues in…

  10. Mumps Virus Is Released from the Apical Surface of Polarized Epithelial Cells, and the Release Is Facilitated by a Rab11-Mediated Transport System

    PubMed Central

    Nakatsu, Yuichiro; Kubota, Toru; Sakata, Masafumi; Takeda, Makoto; Kidokoro, Minoru

    2015-01-01

    ABSTRACT Mumps virus (MuV) is an airborne virus that causes a systemic infection in patients. In vivo, the epithelium is a major replication site of MuV, and thus, the mode of MuV infection of epithelial cells is a subject of interest. Our data in the present study showed that MuV entered polarized epithelial cells via both the apical and basolateral surfaces, while progeny viruses were predominantly released from the apical surface. In polarized cells, intracellular transport of viral ribonucleoprotein (vRNP) complexes was dependent on Rab11-positive endosomes, and vRNP complexes were transported to the apical membrane. Expression of a dominant negative form of Rab11 (Rab11S25N) reduced the progeny virus release in polarized cells but not in nonpolarized cells. Although in this way these effects were correlated with cell polarity, Rab11S25N did not modulate the direction of virus release from the apical surface. Therefore, our data suggested that Rab11 is not a regulator of selective apical release of MuV, although it acts as an activator of virus release from polarized epithelial cells. In addition, our data and previous studies on Sendai virus, respiratory syncytial virus, and measles virus suggested that selective apical release from epithelial cells is used by many paramyxoviruses, even though they cause either a systemic infection or a local respiratory infection. IMPORTANCE Mumps virus (MuV) is the etiological agent of mumps and causes a systemic infection. However, the precise mechanism by which MuV breaks through the epithelial barriers and achieves a systemic infection remains unclear. In the present study, we show that the entry of MuV is bipolar, while the release is predominantly from the apical surface in polarized epithelial cells. In addition, the release of progeny virus was facilitated by a Rab11-positive recycling endosome and microtubule network. Our data provide important insights into the mechanism of transmission and pathogenesis of MuV. PMID

  11. Ab Initio Inverstagation of the Excited States of Nucleobases and Nucleosides

    NASA Astrophysics Data System (ADS)

    Szalay, Péter G.; Fogarasi, Géza; Watson, Thomas; Perera, Ajith; Lotrich, Victor; Bartlett, Rod J.

    2011-06-01

    Most living bodies are exposed to sunlight, essential life sustaining processes are using this natural radiation. Sunlight has, however, several components (has a broad "spectrum") and in particular the invisible component (UV, ultraviolet) is harmful for living organisms. Scientists around the word are busy to understand what happens in the cell when it is exposed to light: it seems that the building blocks of cells and in particular those carrying the genetic information (DNA and RNA) are highly protected against this exposition. Our research focuses on the spectral properties of the building blocks of DNA and RNA, the so called nucleobases and nucleosides, in order to understand this mechanism. Due to improvement in computer technology both at hardware and software side we are now able to use the most accurate methods of ab initio quantum chemistry to investigate the spectroscopic properties of these building blocks. These calculations provide direct information on the properties of these molecules but also provide important benchmarks for cheaper methods which can be used for even larger systems. We have calculated the excited state properties for the nucleobases (cytosine, guanine and adenine), their complexes with water and with each other (Watson-Crick base pairs and stacks) as well as corresponding nucleosides at the EOM-CCSD(T)/aug-cc-pVDZ level of theory and try to answer the following questions: (1) how the order of excited states varies in different nucleobases; (2) how hydration influences the excitation energy and order of excited states; (3) is there any effect of the sugar substituent; (4) how do close lying other bases change the spectrum. The calculations involve over hundred correlated electrons and up to thousand basis functions. Such calculations are now routinely available with the recently developed ACESIII code and can make use of hundreds or even several thousand of processors. V. Lotrich, N. Flocke, M. Ponton, A. Yau, A. Perera, E. Deumens

  12. Reactions of β-Propiolactone with Nucleobase Analogues, Nucleosides, and Peptides

    PubMed Central

    Uittenbogaard, Joost P.; Zomer, Bert; Hoogerhout, Peter; Metz, Bernard

    2011-01-01

    β-Propiolactone is often applied for inactivation of viruses and preparation of viral vaccines. However, the exact nature of the reactions of β-propiolactone with viral components is largely unknown. The purpose of the current study was to elucidate the chemical modifications occurring on nucleotides and amino acid residues caused by β-propiolactone. Therefore, a set of nucleobase analogues was treated with β-propiolactone, and reaction products were identified and quantified. NMR revealed at least one modification in either deoxyguanosine, deoxyadenosine, or cytidine after treatment with β-propiolactone. However, no reaction products were found from thymidine and uracil. The most reactive sides of the nucleobase analogues and nucleosides were identified by NMR. Furthermore, a series of synthetic peptides was used to determine the conversion of reactive amino acid residues by liquid chromatography-mass spectrometry. β-Propiolactone was shown to react with nine different amino acid residues. The most reactive residues are cysteine, methionine, and histidine and, to a lesser degree, aspartic acid, glutamic acid, tyrosine, lysine, serine, and threonine. Remarkably, cystine residues (disulfide groups) do not react with β-propiolactone. In addition, no reaction was observed for β-propiolactone with asparagine, glutamine, and tryptophan residues. β-Propiolactone modifies proteins to a larger extent than expected from current literature. In conclusion, the study determined the reactivity of β-propiolactone with nucleobase analogues, nucleosides, and amino acid residues and elucidated the chemical structures of the reaction products. The study provides detailed knowledge on the chemistry of β-propiolactone inactivation of viruses. PMID:21868382

  13. The Trehalose Phosphotransferase System (PTS) in E. coli W Can Transport Low Levels of Sucrose that Are Sufficient to Facilitate Induction of the csc Sucrose Catabolism Operon

    PubMed Central

    Steen, Jennifer A.; Bohlke, Nina; Vickers, Claudia E.; Nielsen, Lars K.

    2014-01-01

    Plasticity in substrate acceptance is a well-characterised phenomenon for disaccharide transporters. Sucrose, a non-reducing disaccharide, is usually metabolised via either the permease-mediated chromosomally-encoded sucrose catabolism (csc) regulon or the sucrose phosphotransferase system (PTS). E. coli W is a fast-growing strain which efficiently utilises sucrose at concentrations above 1% via the csc regulon. To examine if sucrose could be metabolised via other routes, a library of transposon mutants was generated and screened on 0.2% sucrose. One mutant identified from this library had an insertion in the repressor for the regulon controlling catabolism of the disaccharide trehalose (treR). A series of mutants was constructed to elucidate the mechanism of sucrose utilization in the treR insertion strain. Analysis of these mutants provided evidence that deletion of TreR enables uptake of sucrose via TreB, an enzyme II protein required for PTS-mediated uptake of trehalose. Once inside the cell, this sucrose is not processed by the TreC hydrolase, nor is it sufficient for growth of the strain. QRT-PCR analysis showed that levels of cscA (invertase) transcript increased in the WΔtreR mutant relative to the wild-type strain when grown under low sucrose conditions. This result suggests that the intracellular sucrose provided by TreB can facilitate de-repression of the csc regulon, leading to increased gene expression, sucrose uptake and sucrose utilization in the treR mutant. PMID:24586369

  14. Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment.

    PubMed

    Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves; Einav, Shirit

    2016-11-01

    Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses.

  15. Engineering Rhodosporidium toruloides with a membrane transporter facilitates production and separation of carotenoids and lipids in a bi-phasic culture.

    PubMed

    Lee, Jaslyn J L; Chen, Liwei; Cao, Bin; Chen, Wei Ning

    2016-01-01

    The oleaginous yeast Rhodosporidium toruloides has great biotechnological potential. It accumulates a high amount of lipids which can be used for biofuels and also produces carotenoids which are valuable in the food and pharmaceutical industry. However, the location of these two hydrophobic products in the cell membrane prohibits its efficient harvesting and separation. Here, the transporter Pdr10 was engineered into R. toruloides and cultured in two-phase media containing oil. This enabled the production and in situ export of carotenoids into the oil and concurrent separation from intracellular lipids in the cells. When Pdr10 strain was cultured in the two-phase media, carotenoids and fatty acids yield increased from 1.9 to 2.9 μg/mg and 0.07 to 0.09 mg/mg, respectively. A total of 1.8 μg/mg carotenoids was exported by Pdr10 strain, as compared to 0.3 μg/mg in the wild type. In the Pdr10 strain, the composition of carotenoids and fatty acid it produced also changed. Torulene became the major carotene produced instead of torularhodin. Also, the unsaturated fatty acid C18:2 became the dominant fatty acid produced instead of the saturated C16:0, which was similar to the grape seed oil used in the two-phase media. This indicated that oil was being consumed by the cells, which was supported by the increased intracellular glycerol levels detected by gas chromatography-mass spectrometry (GC-MS). Our approach represents an easy and greener extraction method which could serve to increase the yield and facilitate separation of carotenoids and fatty acids.

  16. Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment

    PubMed Central

    Barouch-Bentov, Rina; Neveu, Gregory; Xiao, Fei; Beer, Melanie; Bekerman, Elena; Schor, Stanford; Campbell, Joseph; Boonyaratanakornkit, Jim; Lindenbach, Brett; Lu, Albert; Jacob, Yves

    2016-01-01

    ABSTRACT Enveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack the endosomal sorting complex required for transport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses. PMID:27803188

  17. Targeting DNA base pair mismatch with artificial nucleobases. Advances and perspectives in triple helix strategy.

    PubMed

    Malnuit, Vincent; Duca, Maria; Benhida, Rachid

    2011-01-21

    This review, divided into three sections, describes the contribution of the chemists' community to the development and application of triple helix strategy by using artificial nucleic acids, particularly for the recognition of DNA sequences incorporating base pair inversions. Firstly, the development of nucleobases that recognise CG inversion is surveyed followed secondly by specific recognition of TA inverted base pair. Finally, we point out in the last section recent perspectives and applications, driven from knowledge in nucleic acids interactions, in the growing field of nanotechnology and supramolecular chemistry at the border area of physics, chemistry and molecular biology.

  18. Role of pKa of Nucleobases in the Origins of Chemical Evolution

    PubMed Central

    2012-01-01

    The formation of canonical base pairs through Watson–Crick hydrogen bonding sits at the heart of the genetic apparatus. The specificity of the base pairing of adenine with thymine/uracil and guanine with cytosine preserves accurate information for the biochemical blueprint and replicates the instructions necessary for carrying out biological function. The chemical evolution question of how these five canonical nucleobases were selected over various other possibilities remains intriguing. Since these and alternative nucleobases would have been available for chemical evolution, the reasons for the emergence of this system appear to be primarily functional. While investigating the base-pairing properties of structural nucleic acid analogs, we encountered a relationship between the pKa of a series of nonstandard (and canonical) nucleobases and the pH of the aqueous medium. This relationship appeared to correspond with the propensity of these molecules to self-assemble via Watson–Crick-type base-pairing interactions. A simple correlation of the “magnitude of the difference between the pKa and pH” (pKa–pH correlation) enables a general prediction of which types of heterocyclic recognition elements form hydrogen-bonded base pairs in aqueous media. Using the pKa–pH relationship, we can rationalize why nature chose the canonical nucleobases in terms of hydrophobic and hydrophilic interactions, and further extrapolate its significance within the context of chemical evolution. The connection between the physicochemical properties of bioorganic compounds and the interactions with their aqueous environment directly affects structure and function, at both a molecular and a supramolecular level. A general structure–function pattern emerges in biomolecules and biopolymers in aqueous media near neutral pH. A pKa – pH < 2 generally prompts catalytic functions, central to metabolism, but a difference in pKa – pH > 2 seems to result in the emergence of structure

  19. Investigation of DNA nucleobases-thin films for potential application in electronics and photonics

    NASA Astrophysics Data System (ADS)

    Ouchen, Fahima; Gomez, Eliot; Joyce, Donna; Yaney, Perry; Kim, Steve; Williams, Adrienne; Steckl, Andrew; Venkat, Narayanan; Grote, James

    2013-10-01

    In previous research we have demonstrated improvements in device performance with the incorporation of a deoxyribonucleic acid (DNA)-based biopolymer into organic light emitting diodes, organic thin film transistors and other organic photonic and electronic devices. Here, we investigate nucleobases, nitrogen-containing biological compounds found within DNA, ribonucleic acid (RNA), nucleotides and nucleosides, for use in a few of those previously investigated photonic and electronic devices. Used as an electron blocking layer in OLEDs, a gate insulator for grapheme transistors and as a dielectric in organic-based capacitors, we have produced comparable results to those using DNA-based biopolymers.

  20. The role of intramolecular hydrogen bonding on nucleobase acidification following metal coordination: possible implications of an "indirect" role of metals in acid-base catalysis of nucleic acids.

    PubMed

    Roitzsch, Michael; Añorbe, Marta Garijo; Miguel, Pablo J Sanz; Müller, Barbara; Lippert, Bernhard

    2005-11-01

    The acidifying effect of Pt(II) on nucleobase -NH and -NH2 groups depends both on the site of metal coordination and on the efficiency of stabilization of the deprotonated nucleobase via intracomplex hydrogen bonding. Weakly acidic nucleobase protons with pK (a) values between 9 and 17 can be acidified by a single Pt(II) to have pK (a) values which are well within the physiological pH range. This could open the possibility of an acid-base catalysis occurring at pH 7, with the metal-nucleobase entity functioning either as an acid or a base. Examples of Pt(II) complexes studied here include, among others, mixed nucleobase systems of 1-methylcytosine and 1,9-dimethyladenine as well as a complex of the rare iminooxo tautomer of 1-methylcytosine having the metal bonded at N4.

  1. Perceived Barriers and Facilitators of Exercise and Healthy Dietary Choices: A Study of Employees and Managers within a Large Transport Organisation

    ERIC Educational Resources Information Center

    Donaldson-Feilder, Emma; Lewis, Rachel; Pavey, Louisa; Jones, Bethan; Green, Melanie; Webster, Angela

    2017-01-01

    Objective: The objective of this study was to examine employees' perceived barriers and facilitators of physical activity and healthy dietary choices, and managers' perceptions of how best to facilitate physical activity and healthy dietary choices among their team members. Design: Single time-point survey with categorical and open-ended…

  2. Ultraviolet Irradiation of Pyrimidine in Interstellar Ice Analogs: Formation and Photo-Stability of Nucleobases

    NASA Technical Reports Server (NTRS)

    Nuevo, Michel; Milam, Stefanie N.; Sandford, Scott A.; Elsila, Jamie E.; Dworkin, Jason P.

    2010-01-01

    Astrochemistry laboratory experiments recently showed that molecules of prebiotic interest can potentially form in space, as supported by the detection of amino acids in organic residues formed by the UV photolysis of ices simulating interstellar and cometary environments (H2O, CO, CO2, CH3OH, NH3, etc.). Although the presence of amino acids in the interstellar medium (ISM) is still under debate, experiments and the detection of amino acids in meteorites both support a scenario in which prebiotic molecules could be of extraterrestrial origin, before they are delivered to planets by comets, asteroids, and interplanetary dust particles. Nucleobases, the informational subunits of DNA and RNA, have also been detected in meteorites, although they have not yet been observed in the ISM. Thus, these molecules constitute another family of prebiotic compounds that can possibly form via abiotical processes in astrophysical environments. Nucleobases are nitrogen-bearing cyclic aromatic species with various functional groups attached, which are divided into two classes: pyrimidines (uracil, cytosine, and thymine) and purines (adenine and guanine). In this work, we study how UV irradiation affects pyrimidine mixed in interstellar ice analogs (H2O, NH3, CH3OH). In particular, we show that the UV irradiation of H2O:pyrimidine mixtures leads to the production of oxidized compounds including uracil, and show that both uracil and cytosine are formed upon irradiation of H2O:NH3:pyrimidine mixtures. We also study the photostability of pyrimidine and its photoproducts formed during these experiments.

  3. First-principles study of interaction of serine with nucleobases of DNA and RNA.

    PubMed

    Abbas, Haider

    2017-03-01

    The nature of interaction between serine-a vital molecule for cancer cell proliferation and nucleic acid bases-adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U) is investigated within the framework of Møller-Plesset perturbation theory (MP2) and density functional theory (DFT). To quantify the interaction strength between serine and nucleobases, the corresponding binding energies were computed, showing energetic ordering such that G > C > T > A > U. This shows that the interaction energy of serine with guanine is the highest, while with uracil it is the lowest. The amount of charge transferred is the lowest in case of the serine-guanine complex and highest in case of the serine-uracil complex. The results show the serine-guanine complex to be more stable and to have a salt bridge structure involving the -COOH group. Theoretical analysis based on MP2 and DFT shows that the interaction between the serine and nucleobases is mainly determined by hydrogen bonding.

  4. Biochemical retrosynthesis of 2'-deoxyribonucleosides from glucose, acetaldehyde, and a nucleobase.

    PubMed

    Horinouchi, Nobuyuki; Ogawa, Jun; Kawano, Takako; Sakai, Takafumi; Saito, Kyota; Matsumoto, Seiichiro; Sasaki, Mie; Mikami, Yoichi; Shimizu, Sakayu

    2006-08-01

    2'-Deoxyribonucleosides are important as building blocks for the synthesis of antisense drugs, antiviral nucleosides, and 2'-deoxyribonucleotides for polymerase chain reaction. The microbial production of 2'-deoxyribonucleosides from simple materials, glucose, acetaldehyde, and a nucleobase, through the reverse reactions of 2'-deoxyribonucleoside degradation and the glycolytic pathway, was investigated. The glycolytic pathway of baker's yeast yielded fructose 1,6-diphosphate from glucose using the energy of adenosine 5'-triphosphate generated from adenosine 5'-monophosphate through alcoholic fermentation with the yeast. Fructose 1,6-diphosphate was further transformed to 2-deoxyribose 5-phosphate in the presence of acetaldehyde by deoxyriboaldolase-expressing Escherichia coli cells via D-glyceraldehyde 3-phosphate. E. coli transformants expressing phosphopentomutase and nucleoside phosphorylase produced 2'-deoxyribonucleosides from 2-deoxyribose 5-phosphate and a nucleobase via 2-deoxyribose 1-phosphate through the reverse reactions of 2'-deoxyribonucleoside degradation. Coupling of the glycolytic pathway and deoxyriboaldolase-catalyzing reaction efficiently supplied 2-deoxyribose 5-phosphate, which is a key intermediate for 2'-deoxyribonucleoside synthesis. 2'-Deoxyinosine (9.9 mM) was produced from glucose, acetaldehyde, and adenine through three-step reactions via fructose 1,6-diphosphate and then 2-deoxyribose 5-phosphate, the molar yield as to glucose being 17.8%.

  5. Supramolecular copolymer micelles based on the complementary multiple hydrogen bonds of nucleobases for drug delivery.

    PubMed

    Wang, Dali; Su, Yue; Jin, Chengyu; Zhu, Bangshang; Pang, Yan; Zhu, Lijuan; Liu, Jinyao; Tu, Chunlai; Yan, Deyue; Zhu, Xinyuan

    2011-04-11

    Novel supramolecular copolymer micelles with stimuli-responsive abilities were successfully prepared through the complementary multiple hydrogen bonds of nucleobases and then applied for rapid intracellular release of drugs. First, both adenine-terminated poly(ε-caprolactone) (PCL-A) and uracil-terminated poly(ethylene glycol) (PEG-U) were synthesized. The supramolecular amphiphilic block copolymers (PCL-A:U-PEG) were formed based on multiple hydrogen bonding interactions between PCL-A and PEG-U. The micelles self-assembled from PCL-A:U-PEG were sufficiently stable in water but prone to fast aggregation in acidic condition due to the dynamic and sensitive nature of noncovalent interactions. The low cytotoxicity of supramolecular copolymer micelles was confirmed by MTT assay against NIH/3T3 normal cells. As a hydrophobic anticancer model drug, doxorubicin (DOX) was encapsulated into these supramolecular copolymer micelles. In vitro release studies demonstrated that the release of DOX from micelles was significantly faster at mildly acid pH of 5.0 compared to physiological pH. MTT assay against HeLa cancer cells showed DOX-loaded micelles had high anticancer efficacy. Hence, these supramolecular copolymer micelles based on the complementary multiple hydrogen bonds of nucleobases are very promising candidates for rapid controlled release of drugs.

  6. The search for and identification of amino acids, nucleobases and nucleosides in samples returned from Mars

    NASA Technical Reports Server (NTRS)

    Gehrke, Charles W.; Ponnamperuma, Cyril; Kuo, Kenneth C.; Stalling, David L.; Zumwalt, Robert W.

    1989-01-01

    An investigation of the returned Mars samples for biologically important organic compounds, with emphasis on amino acid, the puring and pyrimidine bases, and nucleosides is proposed. These studies would be conducted on subsurface samples obtained by drilling past the surface oxidizing layer with emphasis on samples containing the larges quantities of organic carbon as determined by the rover gas chromatographic mass spectrometer (GCMS). Extraction of these molecules from the returned samples will be performed using the hydrothermal extraction technique described by Cheng and Ponnamperuma. More rigorous extraction methods will be developed and evaluated. For analysis of the extract for free amino acids or amino acids present in a bound or peptidic form, aliquots will be analyzed by capillary GCMS both before and after hydrolysis with 6N hydrochloric acid. Establishment of the presence of amino acids would then lead to the next logical step which would be the use of chiral stationary gas chromatography phases to determine the enatiomeic composition of the amino acids present, and thus potentially establish their biotic or abiotic origin. Confirmational analyses for amino acids would include ion-exchange and reversed-phase liquid chromatographic analysis. For analyses of the returned Mars samples for nucleobases and nucleosides, affinity and reversed-phase liquid chromatography would be utilized. This technology coupled with scanning UV detection for identification, presents a powerful tool for nucleobase and nucleoside analysis. Mass spectrometric analysis of these compounds would confirm their presence in samples returned form Mars.

  7. Meteoritic Input of Amino Acids and Nucleobases: Methodology and Implications for the Origins of Life

    NASA Technical Reports Server (NTRS)

    Burton, Aaron S.; Stern, Jennifer C.; Elsila, Jamie E.; Glavin, Daniel P.; Dworkin, Jason P.

    2012-01-01

    The discoveries of amino acids of extraterrestrial origin in many meteorites over the last 40 years have revolutionized the Astrobiology field. A variety of non-terrestrial amino acids similar to those found in life on Earth have been detected in meteorites. A few amino acids have even been found with chiral excesses, suggesting that meteorites could have contributed to the origin of homochirality in life on Earth. In addition to amino acids, which have been productively studied for years, sugar-like molecules, activated phosphates, and nucleobases have also been determined to be indigenous to numerous meteorites. Because these molecules are essential for life as we know it, and meteorites have been delivering them to the Earth since accretion, it is plausible that the origin(s) of life on Earth were aided by extraterrestrially-synthesized molecules. Understanding the origins of life on Earth guides our search for life elsewhere, helping to answer the question of whether biology is unique to Earth. This tutorial review focuses on meteoritic amino acids and nucleobases, exploring modern analytical methods and possible formation mechanisms. We will also discuss the unique window that meteorites provide into the chemistry that preceded life on Earth, a chemical record we do not have access to on Earth due to geologic recycling of rocks and the pervasiveness of biology across the planet. Finally, we will address the future of meteorite research, including asteroid sample return mIssIons.

  8. New approach for designing single-chain magnets: organization of chains via hydrogen bonding between nucleobases.

    PubMed

    Zhang, Wei-Xiong; Shiga, Takuya; Miyasaka, Hitoshi; Yamashita, Masahiro

    2012-04-25

    Two one-dimensional (1D) manganese complexes, [Mn(2)(naphtmen)(2)(L)](ClO(4))·2Et(2)O·2MeOH·H(2)O (1) and [Mn(2)(naphtmen)(2)(HL)](ClO(4))(2)·MeOH (2), were synthesized by using a bridging ligand with a nucleobase moiety, 6-amino-9-β-carboxyethylpurine, and a salen-type manganese(III) dinuclear complex, [Mn(2)(naphtmen)(2)(H(2)O)(2)](ClO(4))(2) (naphtmen(2-) = N,N'-(1,1,2,2-tetramethylethylene)bis(naphthylideneiminato) dianion). In 1 and 2, the carboxylate-bridged Mn(III) dinuclear units are alternately linked by two kinds of weak Mn···O interactions into 1D chains. As a result, canted antiferromagnetic and ferromagnetic interactions are alternately present along the chains, leading to a 1D chain with non-cancellation of anisotropic spins. Since the chains connected via H-bonds between nucleobase moieties are magnetically isolated, both 1 and 2 act as single-chain magnets (SCMs). More importantly, this result shows the smaller canting angles hinder long-range ordering in favor of SCM dynamics.

  9. Understanding Prebiotic Chemistry Through the Analysis of Extraterrestrial Amino Acids and Nucleobases in Meteorites

    NASA Technical Reports Server (NTRS)

    Burton, Aaron S.; Stern, Jennifer C.; Elsila, Jamie E.; Glavin, Daniel P.; Dworkin, Jason P.

    2012-01-01

    The discoveries of amino acids of extraterrestrial origin in many meteorites over the last 50 years have revolutionized the Astrobiology field. A variety of non-terrestrial amino acids similar to those found in life on Earth have been detected in meteorites. A few amino acids have even been found with chiral excesses, suggesting that meteorites could have contributed to the origin of homochirality in life on Earth. In addition to amino acids, which have been productively studied for years, sugar-like molecules, activated phosphates, and nucleobases have also been determined to be indigenous to numerous meteorites. Because these molecules are essential for life as we know it, and meteorites have been delivering them to the Earth since accretion, it is plausible that the origines) of life on Earth were aided by extrataterrestrially-synthesized molecules. Understanding the origins of life on Earth guides our search for life elsewhere, helping to answer the question of whether biology is unique to Earth. This tutorial focuses on meteoritic amino acids and nucleobases, exploring modern analytical methods and possible formation mechanisms. We will also discuss the unique window that meteorites provide into the chemistry that preceded life on Earth, a chemical record we do not have access to on Earth due to geologic recycling of rocks and the pervasiveness of biology across the planet. Finally. we will address the future of meteorite research, including asteroid sample return missions.

  10. Catalytic Role of Manganese Oxides in Prebiotic Nucleobases Synthesis from Formamide.

    PubMed

    Bhushan, Brij; Nayak, Arunima; Kamaluddin

    2016-06-01

    Origin of life processes might have begun with the formation of important biomonomers, such as amino acids and nucleotides, from simple molecules present in the prebiotic environment and their subsequent condensation to biopolymers. While studying the prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide, the manganese oxides demonstrated not only good binding for formamide but demonstrated novel catalytic activity. A novel one pot manganese oxide catalyzed synthesis of pyrimidine nucleobases like thymine is reported along with the formation of other nucleobases like purine, 9-(hydroxyacetyl) purine, cytosine, 4(3 H)-pyrimidinone and adenine in acceptable amounts. The work reported is significant in the sense that the synthesis of thymine has exhibited difficulties especially under one pot conditions and also such has been reported only under the catalytic activity of TiO2. The lower oxides of manganese were reported to show higher potential as catalysts and their existence were favored by the reducing atmospheric conditions prevalent on early Earth; thereby confirming the hypothesis that mineral having metals in reduced form might have been more active during the course of chemical evolution. Our results further confirm the role of formamide as a probable precursor for the formation of purine and pyrimidine bases during the course of chemical evolution and origin of life.

  11. Nucleobase-functionalized graphene nanoribbons for accurate high-speed DNA sequencing

    NASA Astrophysics Data System (ADS)

    Paulechka, Eugene; Wassenaar, Tsjerk A.; Kroenlein, Kenneth; Kazakov, Andrei; Smolyanitsky, Alex

    2016-01-01

    We propose a water-immersed nucleobase-functionalized suspended graphene nanoribbon as an intrinsically selective device for nucleotide detection. The proposed sensing method combines Watson-Crick selective base pairing with graphene's capacity for converting anisotropic lattice strain to changes in an electrical current at the nanoscale. Using detailed atomistic molecular dynamics (MD) simulations, we study sensor operation at ambient conditions. We combine simulated data with theoretical arguments to estimate the levels of measurable electrical signal variation in response to strains and determine that the proposed sensing mechanism shows significant promise for realistic DNA sensing devices without the need for advanced data processing, or highly restrictive operational conditions.We propose a water-immersed nucleobase-functionalized suspended graphene nanoribbon as an intrinsically selective device for nucleotide detection. The proposed sensing method combines Watson-Crick selective base pairing with graphene's capacity for converting anisotropic lattice strain to changes in an electrical current at the nanoscale. Using detailed atomistic molecular dynamics (MD) simulations, we study sensor operation at ambient conditions. We combine simulated data with theoretical arguments to estimate the levels of measurable electrical signal variation in response to strains and determine that the proposed sensing mechanism shows significant promise for realistic DNA sensing devices without the need for advanced data processing, or highly restrictive operational conditions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07061a

  12. Interaction of nucleobases with silicon doped and defective silicon doped graphene and optical properties.

    PubMed

    Mudedla, Sathish Kumar; Balamurugan, Kanagasabai; Kamaraj, Manoharan; Subramanian, Venkatesan

    2016-01-07

    The interaction of nucleobases (NBs) with the surface of silicon doped graphene (SiGr) and defective silicon doped graphene (dSiGr) has been studied using electronic structure methods. A systematic comparison of the calculated interaction energies (adsorption strength) of NBs with the surface of SiGr and dSiGr with those of pristine graphene (Gr) has also been made. The doping of graphene with silicon increases the adsorption strength of NBs. The introduction of defects in SiGr further enhances the strength of interaction with NBs. The appreciable stability of complexes (SiGr-NBs and dSiGr-NBs) arises due to the partial electrostatic and covalent (Si···O(N)) interaction in addition to π-π stacking. The interaction energy increases with the size of graphene models. The strong interaction between dSiGr-NBs and concomitant charge transfer causes significant changes in the electronic structure of dSiGr in contrast to Gr and SiGr. Further, the calculated optical properties of all the model systems using time dependent density functional theory (TD-DFT) reveal that absorption spectra of SiGr and dSiGr undergo appreciable changes after adsorption of NBs. Thus, the significant variations in the HOMO-LUMO gap and absorption spectra of dSiGr after interaction with the NBs can be exploited for possible applications in the sensing of DNA nucleobases.

  13. Ultraviolet Irradiation of Pyrimidine in Interstellar Ice Analogs: Formation and Stability of Nucleobases

    NASA Astrophysics Data System (ADS)

    Milam, Stefanie; Nuevo, Michel; Sandford, Scott; Elsila, Jamie; Dworkin, Jason

    The detection of amino acids in organic residues formed by the UV photolysis of 10 K ices representative of interstellar and cometary environments (H2 O, CO, CO2 , CH3 OH, NH3 , etc.) show that molecules of prebiotic interest could potentially form in space. The detection of amino acids in meteorites supports a scenario where the organic molecules required for life are of extraterrestrial origin. Nucleobases, the informational units of RNA and DNA, have also been detected in meteorites and constitute another family of prebiotic compounds that can possibly form in interstellar environments. These molecules are functionalized heterocyclic aromatic species. There are two classes of nucleobases: pyrimidines (e.g. thymine, uracil, and cytosine) and purines (e.g. adenine and guanine). The functionalization of PAHs from UV photolysis in mixed molecular ices has been proven effective in the laboratory. This work aims at studying how UV irradiation affects pyrimidine in interstellar ice analogs. In particular, we show how H2 O/ pyrimidine mixtures lead to the production of oxidized compounds and study their photostability.

  14. Molecular hydrogen attenuates radiation-induced nucleobase damage to DNA in aerated aqueous solutions.

    PubMed

    Abou-Hamdan, Mhamad; Gardette, Bernard; Cadet, Jean; Gharib, Bouchra; De Reggi, Max; Douki, Thierry; Triantaphylides, Christian

    2016-09-01

    The main aim of the present study is to gain mechanistic insights into the modulating effect of molecular hydrogen on the γ-radiation-induced alteration pathways of DNA nucleobases. Aerated aqueous solutions of calf thymus DNA were exposed to a (60)Co source at doses ranging from 0 to 55 Gy under normoxic conditions, in the presence or not of 0.7 MPa hydrogen or helium. The measurement of several modified bases was performed using HPLC associated with electrospray ionization tandem pass spectrometry (HPLC-ESI-MS/MS). Bleaching of aqueous solutions of p-nitrosodimethylaniline (p-NDA) solutions was also used to allow the quantification of hydroxyl radical (•OH) formation. pNDA bleaching was significantly reduced in the presence of hyperbaric hydrogen. This is undoubtedly due to (•)OH scavenging by H2 since, under the same conditions, He had no effect. Similarly, base alterations were significantly reduced in the presence of hydrogen, as compared to controls under normal atmosphere or in the presence of helium. The relative proportions of modified nucleobases were not changed, showing that the only effect of H2 is to scavenge (•)OH without exhibiting reducing properties. Our findings demonstrate that H2 exerts a significant protection against radiation-induced DNA base damage in aqueous solutions, (•)OH scavenging being the only mechanism involved.

  15. Understanding prebiotic chemistry through the analysis of extraterrestrial amino acids and nucleobases in meteorites.

    PubMed

    Burton, Aaron S; Stern, Jennifer C; Elsila, Jamie E; Glavin, Daniel P; Dworkin, Jason P

    2012-08-21

    The discoveries of amino acids of extraterrestrial origin in many meteorites over the last 50 years have revolutionized the Astrobiology field. A variety of non-terrestrial amino acids similar to those found in life on Earth have been detected in meteorites. A few amino acids have even been found with chiral excesses, suggesting that meteorites could have contributed to the origin of homochirality in life on Earth. In addition to amino acids, which have been productively studied for years, sugar-like molecules, activated phosphates, and nucleobases have also been determined to be indigenous to numerous meteorites. Because these molecules are essential for life as we know it, and meteorites have been delivering them to the Earth since accretion, it is plausible that the origin(s) of life on Earth were aided by extraterrestrially-synthesized molecules. Understanding the origins of life on Earth guides our search for life elsewhere, helping to answer the question of whether biology is unique to Earth. This tutorial review focuses on meteoritic amino acids and nucleobases, exploring modern analytical methods and possible formation mechanisms. We will also discuss the unique window that meteorites provide into the chemistry that preceded life on Earth, a chemical record we do not have access to on Earth due to geologic recycling of rocks and the pervasiveness of biology across the planet. Finally, we will address the future of meteorite research, including asteroid sample return missions.

  16. Understanding Prebiotic Chemistry Through the Analysis of Extraterrestrial Amino Acids and Nucleobases in Meteorites

    NASA Technical Reports Server (NTRS)

    Burton, Aaron S.; Stern, Jennifer C.; Elsila, Jamie E.; Glavin, Daniel P.; Dworkin, Jason P.

    2012-01-01

    The discoveries of amino acids of extraterrestrial origin in many meteorites over the last 50 years have revolutionized the Astrobiology field. A variety of non-terrestrial amino acids similar to those found in life on Earth have been detected in meteorites. A few amino acids have even been found with chiral excesses, suggesting that meteorites could have contributed to the origin of homochirality in life on Earth. In addition to amino acids, which have been productively studied for years, sugar-like molecules, activated phosphates, and nucleobases have also been determined to be indigenous to numerous meteorites. Because these molecules are essential for life as we know it, and meteorites have been delivering them to the Earth since accretion, it is plausible that the origines) of life on Earth were aided by extrataterrestrially-synthesized molecules. Understanding the origins of life on Earth guides our search for life elsewhere, helping to answer the question of whether biology is unique to Earth. This tutorial focuses on meteoritic amino acids and nucleobases, exploring modern analytical methods and possible formation mechanisms. We will also discuss the unique window that meteorites provide into the chemistry that preceded life on Earth, a chemical record we do not have access to on Earth due to geologic recycling of rocks and the pervasiveness of biology across the planet. Finally. we will address the future of meteorite research, including asteroid sample return missions.

  17. Recent discovery of non-nucleobase thymidine phosphorylase inhibitors targeting cancer.

    PubMed

    Bera, Hriday; Chigurupati, Sridevi

    2016-11-29

    Thymidine phosphorylase (TP, EC 2.4.2.4), an enzyme involved in pyrimidine salvage pathway, is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and gliostatin. It is extremely upregulated in a variety of solid tumours. The TP amplification is associated with concomitant overexpression of many angiogenic factors such as matrix metalloproteases (MMPs), interleukins (ILs), vascular endothelial growth factor (VEGF) etc., resulting in promotion of angiogenesis and cancer metastasis. In addition, overshooting TP level protects tumour cells from apoptosis and helps cell survival. Thus, TP is identified as a prime target for developing novel anticancer therapies. Pioneering research activities investigated a large number of TP inhibitors, most of which are pyrimidine or purine analogues. Recently, an array of structurally diverse non-nucleobase derivatives was designed, synthesized and established as promising TP inhibitors. This review, following an outline on the TP structure and functions, gives an overview of the recent advancement of various non-nucleobase TP inhibitors as novel anti-cancer agents.

  18. Infrared spectral investigations of UV irradiated nucleobases adsorbed on mineral surfaces

    NASA Astrophysics Data System (ADS)

    Brucato, J. R.; Fornaro, T.

    2014-04-01

    Spectroscopic studies of the effects of UV radiation on biomolecules such as nucleobases in heterogeneous environments are particularly relevant in prebiotic chemistry to unravel the role of minerals in the transformation/preservation of biomolecules in abiotic environments. Minerals may have a pivotal role in the prebiotic evolution of complex chemical systems, mediating the effects of electromagnetic radiation, influencing the photostability of biomolecules, catalyzing important chemical reactions and/or protecting molecules against degradation. Studies on the photodegradation of biomolecules adsorbed on minerals have applications also in the life detection context to identify potential biomarkers for future space mission and hence to develop suitable sample-extraction protocols for bioanalytical instruments [1]. Moreover, the characterization of the spectroscopic features of biomolecules-mineral complexes provides a support in remote sensing spectroscopy for detecting organic compounds on planetary surfaces or cometary grains and asteroid surfaces. In this context we will present laboratory results on UV photostability of nucleobases adsorbed on magnesium oxide and forsterite minerals and analysed with infrared spectroscopic [2,3].

  19. Enthalpy-Entropy Tuning in the Adsorption of Nucleobases at the Au(111) Surface.

    PubMed

    Rosa, Marta; Corni, Stefano; Di Felice, Rosa

    2014-04-08

    The interaction of DNA molecules with hard substrates is of paramount importance both for the study of DNA itself and for the variety of possible technological applications. Interaction with inorganic surfaces strongly modifies the helical shape of DNA. Hence, an accurate understanding of DNA structure and function at interfaces is a fundamental question with enormous impact in science and society. This work sets the fundamentals for the simulation of entire DNA oligomers on gold surfaces in dry and wet conditions. Thanks to the new GolDNA-AMBER force field, which was derived from first principles and includes dispersion interactions and polarization effects, we simulated self-assembled guanine and adenine monolayers on Au(111) in vacuo and the adsorption of all nucleobases on the same substrate in aqueous conditions. The periodic monolayers obtained from classical simulations match very well those from first principle calculations and experiments, assessing the robustness of the force field and motivating the application to more complex systems for which quantum calculations are not affordable and experiments are elusive. The energetics of nucleobases on Au(111) in solution reveal fundamental physicochemical effects: we find that the adsorption paradigm shifts from purely enthalpic to dominantly entropic by changing the environment and aggregation phase.

  20. Catalytic Role of Manganese Oxides in Prebiotic Nucleobases Synthesis from Formamide

    NASA Astrophysics Data System (ADS)

    Bhushan, Brij; Nayak, Arunima; Kamaluddin

    2016-06-01

    Origin of life processes might have begun with the formation of important biomonomers, such as amino acids and nucleotides, from simple molecules present in the prebiotic environment and their subsequent condensation to biopolymers. While studying the prebiotic synthesis of naturally occurring purine and pyrimidine derivatives from formamide, the manganese oxides demonstrated not only good binding for formamide but demonstrated novel catalytic activity. A novel one pot manganese oxide catalyzed synthesis of pyrimidine nucleobases like thymine is reported along with the formation of other nucleobases like purine, 9-(hydroxyacetyl) purine, cytosine, 4(3 H)-pyrimidinone and adenine in acceptable amounts. The work reported is significant in the sense that the synthesis of thymine has exhibited difficulties especially under one pot conditions and also such has been reported only under the catalytic activity of TiO2. The lower oxides of manganese were reported to show higher potential as catalysts and their existence were favored by the reducing atmospheric conditions prevalent on early Earth; thereby confirming the hypothesis that mineral having metals in reduced form might have been more active during the course of chemical evolution. Our results further confirm the role of formamide as a probable precursor for the formation of purine and pyrimidine bases during the course of chemical evolution and origin of life.

  1. Synthesis and degradation of nucleobases and nucleic acids by formamide in the presence of montmorillonites.

    PubMed

    Saladino, Raffaele; Crestini, Claudia; Ciambecchini, Umberto; Ciciriello, Fabiana; Costanzo, Giovanna; Di Mauro, Ernesto

    2004-11-05

    We describe the role of formamide, a product of the hydrolysis of hydrogen cyanide, as precursor of several components of nucleic acids under prebiotic conditions. When formamide is heated in the presence of montmorillonites, the efficient one-pot synthesis of purine, adenine, cytosine, and uracil is obtained. Along with these nucleobases, several components of the inosine pathway are obtained: 5-aminoimidazole-4-carboxamide, 5-formamidoimidazole-4-carboxamide and hypoxanthine. This almost complete catalogue of nucleic acid precursors is accompanied by N(9)-formylpurine, which, containing a masked glycosidic bond in its formyl moiety, is a plausible precursor of purine acyclonucleosides. In addition, montmorillonites differentially affect the rate of degradation of nucleobases when embedded in 2'-deoxyoligonucleotides; namely, montmorillonites protect adenine and guanine from the degradative action of formamide, while thymine degradation is enhanced. The oligonucleotide backbone reactivity to formamide is also affected; this shows that the interaction with montmorillonites modifies the rate of abstraction of the Halpha and Hbeta protons on the sugar moieties.

  2. Identification of the distribution of adenosine phosphates, nucleosides and nucleobases in royal jelly.

    PubMed

    Wu, Liming; Chen, Lanzhen; Selvaraj, Jonathan Nimal; Wei, Yue; Wang, Yong; Li, Yi; Zhao, Jing; Xue, Xiaofeng

    2015-04-15

    Nucleotides, nucleosides and nucleobases play a greater role in the physiological activity of organisms which are highly present in royal jelly (RJ). The objective of the present study is to develop a HPLC method to simultaneous determine nucleotides, nucleosides and nucleobases in RJ and access them in fresh and commercial RJ samples. The LOD and LOQ were 12.2-99.6 μg/L and 40.8-289.4 μg/L, respectively with nearly 100.9% recoveries. Except uric acid, all other compounds were found in RJ samples. Significant difference in the average content of compounds in fresh (2682.93 mg/kg) and commercial samples (3152.78 mg/kg) were observed. AMP, adenosine and adenine were found predominant in all the samples. Significant higher levels of ATP, ADP and AMP was seen in fresh RJ samples, and IMP, uridine, guanosine, and thymidine was seen in commercial RJ samples. The investigated compounds can be used as indexes for assessment RJ freshness and quality. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. kmerPyramid: an interactive visualization tool for nucleobase and k-mer frequencies.

    PubMed

    Kruppa, Jochen; van der Vries, Erhard; Jo, Wendy K; Postel, Alexander; Becher, Paul; Osterhaus, Albert; Jung, Klaus

    2017-10-01

    Bioinformatics methods often incorporate the frequency distribution of nulecobases or k-mers in DNA or RNA sequences, for example as part of metagenomic or phylogenetic analysis. Because the frequency matrix with sequences in the rows and nucleobases in the columns is multi-dimensional it is hard to visualize. We present the R-package 'kmerPyramid' that allows to display each sequence, based on its nucleobase or k-mer distribution projected to the space of principal components, as a point within a 3-dimensional, interactive pyramid. Using the computer mouse, the user can turn the pyramid's axes, zoom in and out and identify individual points. Additionally, the package provides the k-mer frequency matrices of about 2000 bacteria and 5000 virus reference sequences calculated from the NCBI RefSeq genbank. The 'kmerPyramid' can particularly be used for visualization of intra- and inter species differences. The R-package 'kmerPyramid' is available from the GitHub website at https://github.com/jkruppa/kmerPyramid. klaus.jung@tiho-hannover.de. Supplementary data are available at Bioinformatics online.

  4. Intrinsic flexibility of snRNA hairpin loops facilitates protein binding

    PubMed Central

    Rau, Michael; Stump, W. Tom; Hall, Kathleen B.

    2012-01-01

    Stem–loop II of U1 snRNA and Stem–loop IV of U2 snRNA typically have 10 or 11 nucleotides in their loops. The fluorescent nucleobase 2-aminopurine was used as a substitute for the adenines in each loop to probe the local and global structures and dynamics of these unusually long loops. Using steady-state and time-resolved fluorescence, we find that, while the bases in the loops are stacked, they are able to undergo significant local motion on the picosecond/nanosecond timescale. In addition, the loops have a global conformational change at low temperatures that occurs on the microsecond timescale, as determined using laser T-jump experiments. Nucleobase and loop motions are present at temperatures far below the melting temperature of the hairpin stem, which may facilitate the conformational change required for specific protein binding to these RNA loops. PMID:23012481

  5. Construction of peptides with nucleobase amino acids: design and synthesis of the nucleobase-conjugated peptides derived from HIV-1 Rev and their binding properties to HIV-1 RRE RNA.

    PubMed

    Takahashi, T; Hamasaki, K; Ueno, A; Mihara, H

    2001-04-01

    In order to develop a novel molecule that recognizes a specific structure of RNA, we have attempted to design peptides having L-alpha-amino acids with a nucleobase at the side chain (nucleobase amino acid (NBA)), expecting that the function of a nucleobase which can specifically recognize a base in RNA is regulated in a peptide conformation. In this study, to demonstrate the applicability of the NBA units in the peptide to RNA recognition, we designed and synthesized a variety of NBA-conjugated peptides, derived from HIV-1 Rev. Circular dichroism study revealed that the conjugation of the Rev peptide with an NBA unit did not disturb the peptide conformation. RNA-binding affinities of the designed peptides with RRE IIB RNA were dependent on the structure of the nucleobase moieties in the peptides. The peptide having the cytosine NBA at the position of the Asn40 site in the Rev showed a higher binding ability for RRE IIB RNA, despite the diminishing the Asn40 function. Furthermore, the peptide having the guanine NBA at the position of the Arg44 site, which is the most important residue for the RNA binding in the Rev, bound to RRE IIB RNA in an ability similar to Rev34-50 with native sequence. These results demonstrate that an appropriate NBA unit in the peptide plays an important role in the RNA binding with a specific contact such as hydrogen bonding, and the interaction between the nucleobase in the peptide and the base in the RNA can enhance the RNA-binding affinity and specificity.

  6. Absolute binding-free energies between standard RNA/DNA nucleobases and amino-acid sidechain analogs in different environments.

    PubMed

    de Ruiter, Anita; Zagrovic, Bojan

    2015-01-01

    Despite the great importance of nucleic acid-protein interactions in the cell, our understanding of their physico-chemical basis remains incomplete. In order to address this challenge, we have for the first time determined potentials of mean force and the associated absolute binding free energies between all standard RNA/DNA nucleobases and amino-acid sidechain analogs in high- and low-dielectric environments using molecular dynamics simulations and umbrella sampling. A comparison against a limited set of available experimental values for analogous systems attests to the quality of the computational approach and the force field used. Overall, our analysis provides a microscopic picture behind nucleobase/sidechain interaction preferences and creates a unified framework for understanding and sculpting nucleic acid-protein interactions in different contexts. Here, we use this framework to demonstrate a strong relationship between nucleobase density profiles of mRNAs and nucleobase affinity profiles of their cognate proteins and critically analyze a recent hypothesis that the two may be capable of direct, complementary interactions.

  7. On the Origin of the Canonical Nucleobases: An Assessment of Selection Pressures across Chemical and Early Biological Evolution.

    PubMed

    Rios, Andro C; Tor, Yitzhak

    2013-06-01

    The native bases of RNA and DNA are prominent examples of the narrow selection of organic molecules upon which life is based. How did nature "decide" upon these specific heterocycles? Evidence suggests that many types of heterocycles could have been present on the early Earth. It is therefore likely that the contemporary composition of nucleobases is a result of multiple selection pressures that operated during early chemical and biological evolution. The persistence of the fittest heterocycles in the prebiotic environment towards, for example, hydrolytic and photochemical assaults, may have given some nucleobases a selective advantage for incorporation into the first informational polymers. The prebiotic formation of polymeric nucleic acids employing the native bases remains, however, a challenging problem to reconcile. Hypotheses have proposed that the emerging RNA world may have included many types of nucleobases. This is supported by the extensive utilization of non-canonical nucleobases in extant RNA and the resemblance of many of the modified bases to heterocycles generated in simulated prebiotic chemistry experiments. Selection pressures in the RNA world could have therefore narrowed the composition of the nucleic acid bases. Two such selection pressures may have been related to genetic fidelity and duplex stability. Considering these possible selection criteria, the native bases along with other related heterocycles seem to exhibit a certain level of fitness. We end by discussing the strength of the N-glycosidic bond as a potential fitness parameter in the early DNA world, which may have played a part in the refinement of the alphabetic bases.

  8. Microwave-promoted facile and efficient preparation of N-(alkoxycarbonylmethyl) nucleobases--building blocks for peptide nucleic acids.

    PubMed

    Qu, Guirong; Zhang, Zhiguang; Guo, Haiming; Geng, Mingwei; Xia, Ran

    2007-03-19

    A simple, rapid, and regioselective approach for the synthesis of N-(methoxy-carbonylmethyl)- and N-(n-propoxycarbonylmethyl) nucleobases was developed. By using DMF as the solvent and in the presence of K2CO3 as the base, all the desired products were obtained in moderate yields within 8 min under microwave irradiation.

  9. Synthesis of oligonucleotides containing N,N-disubstituted 3-deazacytosine nucleobases by post-elongation modification and their triplex-forming ability with double-stranded DNA.

    PubMed

    Akabane-Nakata, Masaaki; Obika, Satoshi; Hari, Yoshiyuki

    2014-11-28

    A phosphoramidite of a 2'-O,4'-C-methylene-bridged nucleoside, bearing 4-(2,4,6-triisopropylbenzenesulfonyloxy)pyridin-2-one as a nucleobase precursor, was synthesized and introduced into an oligonucleotide. Treatment with various secondary amines after elongating the oligonucleotide on an automated DNA synthesizer enabled facile and mild conversion of the precursor into the corresponding N,N-disubstituted 3-deazacytosine nucleobases. The evaluation of the triplex-forming ability of the synthesized oligonucleotides with double-stranded DNA showed that the nucleobase possessing the (3S)-3-guanidinopyrrolidine moiety can recognize a CG base pair with high sequence-selectivity and binding-affinity.

  10. Alpha-beta chimeric oligo-DNA bearing intercalator-conjugated nucleobase inside the linker sequence remarkably improves thermal stability of an alternate-stranded triple helix.

    PubMed

    Zafrul Azam, A T M; Hasegawa, Minoru; Moriguchi, Tomohisa; Shinozuka, Kazuo

    2004-12-06

    Novel alpha-beta chimeric oligodeoxynucleotides bearing an intercalator-conjugated nucleobase located at the internal 4-nt linker region were synthesized, and their triplex-stabilizing property was examined. The triple helical DNA formed between the modified chimera DNA and double-stranded DNA exhibited remarkable thermal stability; however, the position of the intercalator-conjugated nucleobase had little influence on the stability. Among the examined, modified chimera DNA bearing the two intercalator-conjugated nucleobases at adjacent positions exhibited the highest stability.

  11. Transportation

    NASA Technical Reports Server (NTRS)

    Vontiesenhausen, G.

    1986-01-01

    A summary of tether transportation is given. Four steps were used over a period of time. First, theoretical engineering feasibility and technology requirements were determined. Then the survivors of that effort went into step two in the analysis of promising candidates. Those survivors went into the third phase which is engineering design and cost benefits. Survivors entered into the demonstration mission definition phase. Transportation studies have covered two kinds of deployments. First, steady state deployment was studied. Like the TSS, it's nearly vertical. It takes a long time to deploy and involves relatively high tether tension. Secondly, dynamic deployment was studied. Deployment started in an almost horizontal direction under a very shallow angle which allows a high deployment rate under very low tension. Momentum transfer here occurs by libration. Specific payloads were used to study tethered transportation benefits. Four transportation concepts were studied with regard to cost benefits. A tethered orbiter deboost from the space station, an OTV boost up from the Space Station, a science platform on a tether with a possible micro-g lab moving in between platform and station, and a tethered boost of payloads fromthe orbiter are the four concepts. These benefits are examined in detail.

  12. New size-expanded RNA nucleobase analogs: a detailed theoretical study.

    PubMed

    Zhang, Laibin; Zhang, Zhenwei; Ren, Tingqi; Tian, Jianxiang; Wang, Mei

    2015-04-05

    Fluorescent nucleobase analogs have attracted much attention in recent years due to their potential applications in nucleic acids research. In this work, four new size-expanded RNA base analogs were computationally designed and their structural, electronic, and optical properties are investigated by means of DFT calculations. The results indicate that these analogs can form stable Watson-Crick base pairs with natural counterparts and they have smaller ionization potentials and HOMO-LUMO gaps than natural ones. Particularly, the electronic absorption spectra and fluorescent emission spectra are calculated. The calculated excitation maxima are greatly red-shifted compared with their parental and natural bases, allowing them to be selectively excited. In gas phase, the fluorescence from them would be expected to occur around 526, 489, 510, and 462 nm, respectively. The influences of water solution and base pairing on the relevant absorption spectra of these base analogs are also examined. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. New size-expanded RNA nucleobase analogs: A detailed theoretical study

    NASA Astrophysics Data System (ADS)

    Zhang, Laibin; Zhang, Zhenwei; Ren, Tingqi; Tian, Jianxiang; Wang, Mei

    2015-04-01

    Fluorescent nucleobase analogs have attracted much attention in recent years due to their potential applications in nucleic acids research. In this work, four new size-expanded RNA base analogs were computationally designed and their structural, electronic, and optical properties are investigated by means of DFT calculations. The results indicate that these analogs can form stable Watson-Crick base pairs with natural counterparts and they have smaller ionization potentials and HOMO-LUMO gaps than natural ones. Particularly, the electronic absorption spectra and fluorescent emission spectra are calculated. The calculated excitation maxima are greatly red-shifted compared with their parental and natural bases, allowing them to be selectively excited. In gas phase, the fluorescence from them would be expected to occur around 526, 489, 510, and 462 nm, respectively. The influences of water solution and base pairing on the relevant absorption spectra of these base analogs are also examined.

  14. Direct Oxidative Damage of Naked DNA Generated upon Absorption of UV Radiation by Nucleobases.

    PubMed

    Gomez-Mendoza, Miguel; Banyasz, Akos; Douki, Thierry; Markovitsi, Dimitra; Ravanat, Jean-Luc

    2016-10-06

    It has been shown that in addition to formation of pyrimidine dimers, UV irradiation of DNA in the absence of photosensitizer also induces formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, but the mechanism of formation of that oxidized base has not been clearly established. In the present study, we provide an unambiguous demonstration that absorption of UVC and UVB radiation by the nucleobases induces DNA oxidation via a direct process (one-electron oxidation) and not singlet oxygen. Evidence arose from the fact that polyamine-guanine adducts that are specifically produced through the transient formation of guanine radical cation are generated following UV irradiation of DNA in the presence of a polyamine even in the absence of any photosensitizer.

  15. Synthesis, structure and imaging of oligodeoxyribonucleotides with tellurium-nucleobase derivatization

    SciTech Connect

    Sheng, J.; Soares, A.; Hassan, A. E. A.; Zhang, W.; Zhou, J.; Xu, B.; Huang, Z.

    2011-05-01

    We report here the first synthesis of 5-phenyl-telluride-thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases (thymine) derivatized with 5-phenyl-telluride functionality (5-Te). Our results show that the 5-Te-DNA is stable, and that the Te-DNA duplex has the thermo-stability similar to the corresponding native duplex. The crystal structure indicates that the 5-Te-DNA duplex structure is virtually identical to the native one, and that the Te-modified T and native A interact similarly to the native T and A pair. Furthermore, while the corresponding native showed weak signals, the DNA duplex modified with electron-rich tellurium functionality showed strong topographic and current peaks by STM imaging, suggesting a potential strategy to directly image DNA without structural perturbation.

  16. Synthesis Structure and Imaging of Oligodeoxyribonucleotides with Tellurium-nucleobase Derivatization

    SciTech Connect

    J Sheng; A Hassan; W Zhang; J Zhou; B Xu; A Soares; Z Huang

    2011-12-31

    We report here the first synthesis of 5-phenyl-telluride-thymidine derivatives and the Te-phosphoramidite. We also report here the synthesis, structure and STM current-imaging studies of DNA oligonucleotides containing the nucleobases