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Sample records for affinity uptake system

  1. Characterization of the low affinity transport system for NO(3)(-) uptake by Citrus roots.

    PubMed

    Cerezo, M; Flors, V; Legaz, F; García-Agustín, P

    2000-12-07

    Three-month old citrange Troyer (hybrid of Citrus sinensis x Poncirus trifoliata) seedlings were grown hydroponically and, after a period of NO(3)(-) starvation, plants were transferred to solutions enriched with K(15)NO(3) (96% atoms 15N excess) to measure 15NO(3)(-) uptake rates as a function of external 15NO(3)(-) concentrations. Two different NO(3)(-) uptake systems were found. Between 1 and 50 mM 15NO(3)(-) in the uptake solution medium, the uptake rate increased linearly due to the low affinity transport system (LATS). Nitrate reductase activity showed the same response to external [NO(3)(-)], and also appears to be regulated by the rate of nitrate uptake. Nitrate pre-treatments had a represive effect on NO(3)(-) uptake rate measured at 5 or 30 mM external [15NO(3)(-)]. The extent of the inhibition depended on the [NO(3)(-)] during the pre-treatment and in the uptake solution. These results suggest that the LATS of Citrus seedlings is under feedback control by the N status of the plant. Accordingly, addition of amino acids (Glu, Asp, Asn, Gln) to the uptake solution resulted in a decrease in 15NO(3)(-) uptake rate. However, the inactivation of nitrate reductase activity after treatment of the seedlings with either 100 or 500 µM WO(4)(2-) did not affect the activity of the LATS. Metabolic uncouplers, 2,4-DNP and KCN, reduced the uptake rate by 43.3% and 41.4% respectively at 5mM external [15NO(3)(-)]. However, these compounds had little effect when 15NO(3)(-) uptake was assayed at 30 mM external concentration. The ATPase inhibitors DCCD and DES reduced 15NO(3)(-) uptake by 68.8%-35.6%, at both external [15NO(3)(-)]. Nitrate uptake by the LATS declined with the increase of the solution pH beyond pH 4. The data presented are discussed in the context of the kinetics, energy dependence and regulation of NO(3)(-) uptake.

  2. A molecular recognizing system of serotonin in rat fetal axonal growth cones: uptake and high affinity binding.

    PubMed

    Mercado, R; Hernández, J

    1992-09-18

    Axonal growth cone particles (AGCP) isolated from prenatal and postnatal rat brain had different high-affinity 5-HT uptake characteristics. In postnatal AGCP the uptake behaves as in the adult rat brain, while in the prenatal AGCP the uptake characteristics seem to be in a transitional stage. Also in prenatal AGCP we observed specific, high-affinity 5-HT binding sites. These results support the idea of an important role for 5-HT during axogenesis.

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

    PubMed

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

    2003-06-01

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

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

    PubMed Central

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

    2001-01-01

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

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

    PubMed

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

    2001-08-01

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

  6. High-affinity K+ uptake in pepper plants.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2005-06-01

    High-affinity K+ uptake is an essential process for plant nutrition under K+-limiting conditions. The results presented here demonstrate that pepper (Capsicum annuum) plants grown in the absence of NH4+ and starved of K+ show an NH4+-sensitive high-affinity K+ uptake that allows plant roots to deplete external K+ to values below 1 microM. When plants are grown in the presence of NH4+, high-affinity K+ uptake is not inhibited by NH4+. Although NH4+-grown plants deplete external K+ below 1 microM in the absence of NH4+, when 1 mM NH4+ is present they do not deplete external K+ below 10 microM. A K+ transporter of the HAK family, CaHAK1, is very likely mediating the NH4+-sensitive component of the high-affinity K+ uptake in pepper roots. CaHAK1 is strongly induced in the roots that show the NH4+-sensitive high-affinity K+ uptake and its induction is reduced in K+-starved plants grown in the presence of NH4+. The NH4+-insensitive K+ uptake may be mediated by an AKT1-like K+ channel.

  7. Two Plant Bacteria, S. meliloti and Ca. Liberibacter asiaticus, Share Functional znuABC Homologues That Encode for a High Affinity Zinc Uptake System

    PubMed Central

    Vahling-Armstrong, Cheryl M.; Zhou, Huasong; Benyon, Lesley; Morgan, J. Kent; Duan, Yongping

    2012-01-01

    The Znu system, encoded for by znuABC, can be found in multiple genera of bacteria and has been shown to be responsible for the import of zinc under low zinc conditions. Although this high-affinity uptake system is known to be important for both growth and/or pathogenesis in bacteria, it has not been functionally characterized in a plant-associated bacterium. A single homologue of this system has been identified in the plant endosymbiont, Sinorhizobium meliloti, while two homologous systems were found in the destructive citrus pathogen, Candidatus Liberibacter asiaticus. To understand the role of these protein homologues, a complementation assay was devised allowing the individual genes that comprise the system to be assayed independently for their ability to reinstate a partially-inactivated Znu system. Results from the assays have demonstrated that although all of the genes from S. meliloti were able to restore activity, only one of the two Ca. Liberibacter asiaticus encoded gene clusters contained genes that were able to functionally complement the system. Additional analysis of the gene clusters reveals that distinct modes of regulation may also exist between the Ca. Liberibacter asiaticus and S. meliloti import systems despite the intracellular-plant niche common to both of these bacteria. PMID:22655039

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

    PubMed

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

    2009-05-01

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

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

    PubMed Central

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

    2003-01-01

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

  10. Cadmium inhibits the induction of high-affinity nitrate uptake in maize (Zea mays L.) roots.

    PubMed

    Rizzardo, Cecilia; Tomasi, Nicola; Monte, Rossella; Varanini, Zeno; Nocito, Fabio F; Cesco, Stefano; Pinton, Roberto

    2012-12-01

    Cadmium (Cd) detoxification involves glutathione and phytochelatins biosynthesis: the higher need of nitrogen should require increased nitrate (NO(3)(-)) uptake and metabolism. We investigated inducible high-affinity NO(3)(-) uptake across the plasma membrane (PM) in maize seedlings roots upon short exposure (10 min to 24 h) to low Cd concentrations (0, 1 or 10 μM): the activity and gene transcript abundance of high-affinity NO(3)(-) transporters, NO(3)(-) reductases and PM H(+)-ATPases were analyzed. Exposure to 1 mM NO(3)(-) led to a peak in high-affinity (0.2 mM) NO(3)(-) uptake rate (induction), which was markedly lowered in Cd-treated roots. Plasma membrane H(+)-ATPase activity was also strongly limited, while internal NO(3)(-) accumulation and NO(3)(-) reductase activity in extracts of Cd treated roots were only slightly lowered. Kinetics of high- and low-affinity NO(3)(-) uptake showed that Cd rapidly (10 min) blocked the inducible high-affinity transport system; the constitutive high-affinity transport system appeared not vulnerable to Cd and the low-affinity transport system appeared to be less affected and only after a prolonged exposure (12 h). Cd-treatment also modified transcript levels of genes encoding high-affinity NO(3)(-) transporters (ZmNTR2.1, ZmNRT2.2), PM H(+)-ATPases (ZmMHA3, ZmMHA4) and NO(3)(-) reductases (ZmNR1, ZmNADH:NR). Despite an expectable increase in NO(3)(-) demand, a negative effect of Cd on NO(3)(-) nutrition is reported. Cd effect results in alterations at the physiological and transcriptional levels of NO(3)(-) uptake from the external solution and it is particularly severe on the inducible high-affinity anion transport system. Furthermore, Cd would limit the capacity of the plant to respond to changes in NO(3) (-) availability.

  11. High affinity choline uptake: an early index of cholinergic innervation in rat brain.

    PubMed

    Sorimachi, M; Kataoka, K

    1975-08-29

    The uptake of [3H]choline was investigated in nuclei-free homogenates or crude synaptosomal fractions (P2) from rat brain under various stages of development. A comparable sensitivity of uptake to treatment by hyposmotic shock suggested the involvement of synaptosomal populations in choline uptake in immature as well as in adult brains. However, significant changes in the "apparent" Km for the high affinity transport system and quantitative differences in the Na ion requirement for maximal uptake at 0.43 muM choline concentration were found during development; facts which suggested a greater contribution of the low affinity system in the more immature brains. Assuming that the uptake with high and low sensitivity to Na+ reduction reflected that via the high and low affinity system reslectively, we have attempted to obtain real Km values for the high affinity system. These Km values changed less than those measured directly, suggesting that the affinity constant for the high affinity system does not change during development. On these assumptions, the developmental changes of cholinergic synaptogenesis were examined in 5 distinct regions of the brain. It was found that the synaptogenesis begins several days earlier than the increase of choline acetyltransferase (ChAc) level in the frontal cortex, the hippocampus, the superior colliculus and the cerebellum. These regions may be included among the terminal-rich regions according to available evidence related to cholinergic systems. On the other hand, synaptogenesis accompanied the concomitant ChAc increase in the striatum, where the cholinergic interneurons are present. It is concluded that the increase of ChAc in the terminal-rich regions is delayed by the axoplasmic flow; therefore, the earlier index of cholinergic synaptogenesis in these regions is the high affinity uptake activity rather than the enzyme activity.

  12. Low-affinity Na+ uptake in the halophyte Suaeda maritima.

    PubMed

    Wang, Suo-Min; Zhang, Jin-Lin; Flowers, Timothy J

    2007-10-01

    Na(+) uptake by plant roots has largely been explored using species that accumulate little Na(+) into their shoots. By way of contrast, the halophyte Suaeda maritima accumulates, without injury, concentrations of the order of 400 mM NaCl in its leaves. Here we report that cAMP and Ca(2+) (blockers of nonselective cation channels) and Li(+) (a competitive inhibitor of Na(+) uptake) did not have any significant effect on the uptake of Na(+) by the halophyte S. maritima when plants were in 25 or 150 mM NaCl (150 mM NaCl is near optimal for growth). However, the inhibitors of K(+) channels, TEA(+) (10 mM), Cs(+) (3 mM), and Ba(2+) (5 mM), significantly reduced the net uptake of Na(+) from 150 mM NaCl over 48 h, by 54%, 24%, and 29%, respectively. TEA(+) (10 mM), Cs(+) (3 mM), and Ba(2+) (1 mm) also significantly reduced (22)Na(+) influx (measured over 2 min in 150 mM external NaCl) by 47%, 30%, and 31%, respectively. In contrast to the situation in 150 mm NaCl, neither TEA(+) (1-10 mM) nor Cs(+) (0.5-10 mM) significantly reduced net Na(+) uptake or (22)Na(+) influx in 25 mM NaCl. Ba(2+) (at 5 mm) did significantly decrease net Na(+) uptake (by 47%) and (22)Na(+) influx (by 36% with 1 mM Ba(2+)) in 25 mM NaCl. K(+) (10 or 50 mM) had no effect on (22)Na(+) influx at concentrations below 75 mM NaCl, but the influx of (22)Na(+) was inhibited by 50 mM K(+) when the external concentration of NaCl was above 75 mM. The data suggest that neither nonselective cation channels nor a low-affinity cation transporter are major pathways for Na(+) entry into root cells. We propose that two distinct low-affinity Na(+) uptake pathways exist in S. maritima: Pathway 1 is insensitive to TEA(+) or Cs(+), but sensitive to Ba(2+) and mediates Na(+) uptake under low salinities (25 mM NaCl); pathway 2 is sensitive to TEA(+), Cs(+), and Ba(2+) and mediates Na(+) uptake under higher external salt concentrations (150 mM NaCl). Pathway 1 might be mediated by a high-affinity K transporter

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

    PubMed

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

    2014-10-01

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

  14. Kinetics and autoradiography of high affinity uptake of serotonin by primary astrocyte cultures

    SciTech Connect

    Katz, D.M.; Kimelberg, H.K.

    1985-07-01

    Primary astrocyte cultures prepared from the cerebral cortices of neonatal rats showed significant accumulation of serotonin (5-hydroxytryptamine; (/sup 3/H)-5-HT). At concentrations in the range of 0.01 to 0.7 microM (/sup 3/H)-5-HT, this uptake was 50 to 85% Na+ dependent and gave a Km of 0.40 +/- 0.11 microM (/sup 3/H)-5-HT and a Vmax of 6.42 +/- 0.85 (+/- SEM) pmol of (/sup 3/H)-5-HT/mg of protein/4 min for the Na+-dependent component. In the absence of Na+ the uptake was nonsaturable. Omission of the monoamine oxidase inhibitor pargyline markedly reduced the Na+-dependent component of (/sup 3/H)-5-HT uptake but had a negligible effect on the Na+-independent component. This suggest significant oxidative deamination of serotonin after it has been taken up by the high affinity system, followed by release of its metabolite. The authors estimated that this system enabled the cells to concentrate (/sup 3/H)-5-HT up to 44-fold at an external (/sup 3/H)-5-HT concentration of 10(-7) M. Inhibition of (/sup 3/H)-5-HT uptake by a number of clinically effective antidepressants was also consistent with a specific high affinity uptake mechanism for 5-HT, the order of effectiveness of inhibition being chlorimipramine greater than fluoxetine greater than imipramine = amitriptyline greater than desmethylimipramine greater than iprindole greater than mianserin. Uptake of (/sup 3/H)-5-HT was dependent on the presence of Cl- as well as Na+ in the medium, and the effect of omission of both ions was nonadditive. Varying the concentration of K+ in the media from 1 to 50 mM had a limited effect on (/sup 3/H)-5-HT uptake.

  15. Side-by-Side Comparison of Commonly Used Biomolecules That Differ in Size and Affinity on Tumor Uptake and Internalization.

    PubMed

    Leelawattanachai, Jeerapond; Kwon, Keon-Woo; Michael, Praveesuda; Ting, Richard; Kim, Ju-Young; Jin, Moonsoo M

    2015-01-01

    The ability to use a systemically injected agent to image tumor is influenced by tumor characteristics such as permeability and vascularity, and the size, shape, and affinity of the imaging agent. In this study, six different imaging biomolecules, with or without specificity to tumor, were examined for tumor uptake and internalization at the whole body, ex-vivo tissue, and cellular levels: antibodies, antibody fragments (Fab), serum albumin, and streptavidin. The time of peak tumor uptake was dependent solely on the size of molecules, suggesting that molecular size is the major factor that influences tumor uptake by its effect on systemic clearance and diffusion into tumor. Affinity to tumor antigen failed to augment tumor uptake of Fab above non-specific accumulation, which suggests that Fab fragments of typical monoclonal antibodies may fall below an affinity threshold for use as molecular imaging agents. Despite abundant localization into the tumor, albumin and streptavidin were not found on cell surface or inside cells. By comparing biomolecules differing in size and affinity, our study highlights that while pharmacokinetics are a dominant factor in tumor uptake for biomolecules, affinity to tumor antigen is required for tumor binding and internalization.

  16. Agrobacterium tumefaciens Zur Regulates the High-Affinity Zinc Uptake System TroCBA and the Putative Metal Chaperone YciC, along with ZinT and ZnuABC, for Survival under Zinc-Limiting Conditions

    PubMed Central

    Chaoprasid, Paweena; Dokpikul, Thanittra; Johnrod, Jaruwan; Sirirakphaisarn, Sirin; Nookabkaew, Sumontha; Mongkolsuk, Skorn

    2016-01-01

    ABSTRACT Agrobacterium tumefaciens has a cluster of genes (Atu3178, Atu3179, and Atu3180) encoding an ABC-type transporter, here named troA, troB, and troC, respectively, which is shown here to be a zinc-specific uptake system. Reverse transcription (RT)-PCR analysis confirmed that troA, troB, and troC are cotranscribed, with troC as the first gene of the operon. The yciC (Atu3181) gene is transcribed in the opposite orientation to that of the troCBA operon and belongs to a metal-binding GTPase family. Expression of troCBA and yciC was inducible under zinc-limiting conditions and was controlled by the zinc uptake regulator, Zur. Compared to the wild type, the mutant strain lacking troC was hypersensitive to a metal chelator, EDTA, and the phenotype could be rescued by the addition of zinc, while the strain with a single yciC mutation showed no phenotype. However, yciC was important for survival under zinc limitation when either troC or zinT was inactivated. The periplasmic zinc-binding protein, ZinT, could not function when TroC was inactivated, suggesting that ZinT may interact with TroCBA in zinc uptake. Unlike many other bacteria, the ABC-type transporter ZnuABC was not the major zinc uptake system in A. tumefaciens. However, the important role of A. tumefaciens ZnuABC was revealed when TroCBA was impaired. The strain containing double mutations in the znuA and troC genes exhibited a growth defect in minimal medium. A. tumefaciens requires cooperation of zinc uptake systems and zinc chaperones, including TroCBA, ZnuABC, ZinT, and YciC, for survival under a wide range of zinc-limiting conditions. IMPORTANCE Both host and pathogen battle over access to essential metals, including zinc. In low-zinc environments, physiological responses that make it possible to acquire enough zinc are important for bacterial survival and could determine the outcome of host-pathogen interactions. A. tumefaciens was found to operate a novel pathway for zinc uptake in which Zin

  17. High-affinity uptake of gamma-aminobutyric acid in cultured glial and neuronal cells.

    PubMed

    Balcar, V J; Mark, J; Borg, J; Mandel, P

    1979-06-01

    Both glial and neuronal cells maintained in primary culture were found to accumulate [3H]GABA by an efficient "high-affinity" uptake system (apparent Km = 9 muM, Vmax = 0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1mM) also strongly inhibited uptake of [3H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, beta-alanine, and 2,4-diaminobutyrate) inhibited uptake of [3H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine, L-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (Km = 92 muM, Vmax = 0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [3H]GABA against a concentration gradient. Apparent Km of this uptake was relatively high (819 muM), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, non of the nontransformed continuous cell lines of either tumoral (glioma, C6; neuroblastoma, M1; M1NN) or normal (NN;I6) origin actively accumulated [3H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.

  18. Carbon Availability Modifies Temperature Responses of Heterotrophic Microbial Respiration, Carbon Uptake Affinity, and Stable Carbon Isotope Discrimination

    PubMed Central

    Min, Kyungjin; Lehmeier, Christoph A.; Billings, Sharon A.

    2016-01-01

    Microbial transformations of organic carbon (OC) generate a large flux of CO2 into the atmosphere and influence the C balance of terrestrial and aquatic ecosystems. Yet, inherent heterogeneity in natural environments precludes direct quantification of multiple microbial C fluxes that underlie CO2 production. Here we used a continuous flow bioreactor coupled with a stable C isotope analyzer to determine the effects of temperature and C availability (cellobiose concentration) on C fluxes and 13C discrimination of a microbial population growing at steady-state in a homogeneous, well-mixed environment. We estimated C uptake affinity and C use efficiency (CUE) to characterize the physiological responses of microbes to changing environmental conditions. Temperature increased biomass-C specific respiration rate and C uptake affinity at lower C availability, but did not influence those parameters at higher C availability. CUE decreased non-linearly with increasing temperature. The non-linear, negative relationship between CUE and temperature was more pronounced under lower C availability than under relatively high C availability. We observed stable isotope fractionation between C substrate and microbial biomass C (7~12‰ depletion), and between microbial biomass and respired CO2 (4~10‰ depletion). Microbial discrimination against 13C-containing cellobiose during C uptake was influenced by temperature and C availability, while discrimination during respiration was only influenced by C availability. Shifts in C uptake affinity with temperature and C availability may have modified uptake-induced 13C fractionation. By stressing the importance of C availability on temperature responses of microbial C fluxes, C uptake affinity, CUE, and isotopic fractionation, this study contributes to a fundamental understanding of C flow through microbes. This will help guide parameterization of microbial responses to varying temperature and C availability within Earth-system models. PMID

  19. Carbon Availability Modifies Temperature Responses of Heterotrophic Microbial Respiration, Carbon Uptake Affinity, and Stable Carbon Isotope Discrimination.

    PubMed

    Min, Kyungjin; Lehmeier, Christoph A; Iv, Ford Ballantyne; Billings, Sharon A

    2016-01-01

    Microbial transformations of organic carbon (OC) generate a large flux of CO2 into the atmosphere and influence the C balance of terrestrial and aquatic ecosystems. Yet, inherent heterogeneity in natural environments precludes direct quantification of multiple microbial C fluxes that underlie CO2 production. Here we used a continuous flow bioreactor coupled with a stable C isotope analyzer to determine the effects of temperature and C availability (cellobiose concentration) on C fluxes and (13)C discrimination of a microbial population growing at steady-state in a homogeneous, well-mixed environment. We estimated C uptake affinity and C use efficiency (CUE) to characterize the physiological responses of microbes to changing environmental conditions. Temperature increased biomass-C specific respiration rate and C uptake affinity at lower C availability, but did not influence those parameters at higher C availability. CUE decreased non-linearly with increasing temperature. The non-linear, negative relationship between CUE and temperature was more pronounced under lower C availability than under relatively high C availability. We observed stable isotope fractionation between C substrate and microbial biomass C (7~12‰ depletion), and between microbial biomass and respired CO2 (4~10‰ depletion). Microbial discrimination against (13)C-containing cellobiose during C uptake was influenced by temperature and C availability, while discrimination during respiration was only influenced by C availability. Shifts in C uptake affinity with temperature and C availability may have modified uptake-induced (13)C fractionation. By stressing the importance of C availability on temperature responses of microbial C fluxes, C uptake affinity, CUE, and isotopic fractionation, this study contributes to a fundamental understanding of C flow through microbes. This will help guide parameterization of microbial responses to varying temperature and C availability within Earth-system models.

  20. Characterization of AMT-mediated high-affinity ammonium uptake in roots of maize (Zea mays L.).

    PubMed

    Gu, Riliang; Duan, Fengying; An, Xia; Zhang, Fusuo; von Wirén, Nicolaus; Yuan, Lixing

    2013-09-01

    High-affinity ammonium uptake in plant roots is mainly mediated by AMT1-type ammonium transporters, and their regulation varies depending on the plant species. In this study we aimed at characterizing AMT-mediated ammonium transport in maize, for which ammonium-based fertilizer is an important nitrogen (N) source. Two ammonium transporter genes, ZmAMT1;1a and ZmAMT1;3, were isolated from a maize root-specific cDNA library by functional complementation of an ammonium uptake-defective yeast mutant. Ectopic expression of both genes in an ammonium uptake-defective Arabidopsis mutant conferred high-affinity ammonium uptake capacities in roots with substrate affinities of 48 and 33 μM for ZmAMT1;1a and ZmAMT1;3, respectively. In situ hybridization revealed co-localization of both ZmAMT genes on the rhizodermis, suggesting an involvement in capturing ammonium from the rhizosphere. In N-deficient maize roots, influx increased significantly while ZmAMT expression did not. Ammonium resupply to N-deficient or nitrate-pre-cultured roots, however, rapidly enhanced both influx and ZmAMT transcript levels, revealing a substrate-inducible regulation of ammonium uptake. In conclusion, the two rhizodermis-localized transporters ZmAMT1;1a and ZmAMT1;3 are most probably the major components in the high-affinity transport system in maize roots. A particular regulatory feature is their persistent induction by ammonium rather than an up-regulation under N deficiency.

  1. The human organic cation transporter OCT1 mediates high affinity uptake of the anticancer drug daunorubicin

    PubMed Central

    Andreev, Emil; Brosseau, Nicolas; Carmona, Euridice; Mes-Masson, Anne-Marie; Ramotar, Dindial

    2016-01-01

    Anthracyclines such as daunorubicin are anticancer agents that are transported into cells, and exert cytotoxicity by blocking DNA metabolism. Although there is evidence for active uptake of anthracyclines into cells, the specific transporter involved in this process has not been identified. Using the high-grade serous ovarian cancer cell line TOV2223G, we show that OCT1 mediated the high affinity (Km ~ 5 μM) uptake of daunorubicin into the cells, and that micromolar amounts of choline completely abolished the drug entry. OCT1 downregulation by shRNA impaired daunorubicin uptake into the TOV2223G cells, and these cells were significantly more resistant to the drug in comparison to the control shRNA. Transfection of HEK293T cells, which accommodated the ectopic expression of OCT1, with a plasmid expressing OCT1-EYFP showed that the transporter was predominantly localized to the plasma membrane. These transfected cells exhibited an increase in the uptake of daunorubicin in comparison to control cells transfected with an empty EYFP vector. Furthermore, a variant of OCT1, OCT1-D474C-EYFP, failed to enhance daunorubicin uptake. This is the first report demonstrating that human OCT1 is involved in the high affinity transport of anthracyclines. We postulate that OCT1 defects may contribute to the resistance of cancer cells treated with anthracyclines. PMID:26861753

  2. Recombinant glucose uptake system

    DOEpatents

    Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

    1997-01-01

    Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

  3. The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots.

    PubMed

    Ragel, Paula; Ródenas, Reyes; García-Martín, Elena; Andrés, Zaida; Villalta, Irene; Nieves-Cordones, Manuel; Rivero, Rosa M; Martínez, Vicente; Pardo, Jose M; Quintero, Francisco J; Rubio, Francisco

    2015-12-01

    Plant growth and development requires efficient acquisition of essential elements. Potassium (K(+)) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K(+) uptake systems in the roots is essential to secure K(+) supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K(+) concentration is very low (<10 µm), K(+) nutrition depends exclusively on the high-affinity K(+) transporter5 (HAK5). Low-K(+)-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca(2+) sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K(+) transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K(+). Experiments of K(+) (Rb(+)) uptake and growth measurements in low-K(+) medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta.

  4. A low K+ signal is required for functional high-affinity K+ uptake through HAK5 transporters.

    PubMed

    Rubio, Francisco; Fon, Mario; Ródenas, Reyes; Nieves-Cordones, Manuel; Alemán, Fernando; Rivero, Rosa M; Martínez, Vicente

    2014-11-01

    The high-affinity K(+) transporter HAK5 is a key system for root K(+) uptake and, under very low external K(+), the only one capable of supplying K(+) to the plant. Functional HAK5-mediated K(+) uptake should be tightly regulated for plant adaptation to different environmental conditions. Thus, it has been described that the gene encoding the transporter is transcriptionally regulated, being highly induced under K(+) limitation. Here we show that environmental conditions, such as the lack of K(+), NO(3)(-) or P, that induced a hyperpolarization of the plasma membrane of root cells, induce HAK5 transcription. However, only the deprivation of K(+) produces functional HAK5-mediated K(+) uptake in the root. These results suggest on the one hand the existence of a posttranscriptional regulation of HAK5 elicited by the low K(+) signal and on the other that HAK5 may be involved in yet-unknown functions related to NO(3)(-) and P deficiencies. These results have been obtained here with Solanum lycopersicum (cv. Micro-Tom) as well as Arabidopsis thaliana plants, suggesting that the posttranscriptional regulation of high-affinity HAK transporters take place in all plant species.

  5. Stabilization of the Motion of Affine Systems

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

  6. Non-affine elasticity in jammed systems

    NASA Astrophysics Data System (ADS)

    Maloney, Craig

    2006-03-01

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

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

    PubMed

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

    1998-03-27

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

  8. Differential uptake of nanoparticles by endothelial cells through polyelectrolytes with affinity for caveolae

    PubMed Central

    Voigt, Julia; Christensen, Jon; Shastri, V. Prasad

    2014-01-01

    Nanoparticles (NPs) constitute an important medium for the targeted delivery of cancer therapeutics. Targeting of NPs to a specific cell type is traditionally achieved through the modification of the NP surface with peptides, aptamers, or other motifs that specifically recognize a cell-surface receptor, leading to internalization of NPs via clathrin and caveolae-mediated endocytosis. We have discovered that modifying the NP surface with anionic polyelectrolytes of varying lipophilicity can regulate the uptake of lipid NPs by endothelial and epithelial cells. Furthermore, we report the finding that synthetic polyelectrolytes composed of an aromatic sulfonic acid backbone exhibit specific affinity for caveolae of endothelial cells. By exploiting the higher expression of caveolae in endothelial cells in comparison with epithelial cells, a purely physiochemical approach to the targeted uptake of lipid NPs to endothelial cells is demonstrated. The ability to confer preferential affinity for NPs to cell surface domains by varying the charge and lipophilic characteristics of an NP surface offers a general means of achieving targeted delivery without the need for receptor–ligand-type targeting strategies. PMID:24516167

  9. Relative contribution of AtHAK5 and AtAKT1 to K+ uptake in the high-affinity range of concentrations.

    PubMed

    Rubio, Francisco; Nieves-Cordones, Manuel; Alemán, Fernando; Martínez, Vicente

    2008-12-01

    The relative contribution of the high-affinity K(+) transporter AtHAK5 and the inward rectifier K(+) channel AtAKT1 to K(+) uptake in the high-affinity range of concentrations was studied in Arabidopsis thaliana ecotype Columbia (Col-0). The results obtained with wild-type lines, with T-DNA insertion in both genes and specific uptake inhibitors, show that AtHAK5 and AtAKT1 mediate the NH4+-sensitive and the Ba(2+)-sensitive components of uptake, respectively, and that they are the two major contributors to uptake in the high-affinity range of Rb(+) concentrations. Using Rb(+) as a K(+) analogue, it was shown that AtHAK5 mediates absorption at lower Rb(+) concentrations than AtAKT1 and depletes external Rb(+) to values around 1 muM. Factors such as the presence of K(+) or NH4+ during plant growth determine the relative contribution of each system. The presence of NH4+ in the growth solution inhibits the induction of AtHAK5 by K(+) starvation. In K(+)-starved plants grown without NH4+, both systems are operative, but when NH4+ is present in the growth solution, AtAKT1 is probably the only system mediating Rb(+) absorption, and the capacity of the roots to deplete Rb(+) is reduced.

  10. Receptor regulation of the glutamate, GABA and taurine high-affinity uptake into astrocytes in primary culture.

    PubMed

    Hansson, E; Rönnbäck, L

    1991-05-10

    From experiments using dissociated primary astroglial cultures from newborn rat cerebral cortex, the stimulation of monoamine receptors (alpha, beta and 5HT) was shown to affect the high-affinity uptake kinetics of glutamate, GABA and taurine. In the presence of the alpha 1 agonist phenylephrine, there was an increased uptake (Vmax) of glutamate, while beta adrenoceptor activation slightly inhibited the glutamate uptake and stimulated the GABA and taurine uptakes. 5HT2 receptor stimulation caused a slight inhibition of the taurine uptake. The uptake rate of GABA was not affected by 5HT, alpha 1 or alpha 2 receptor agonists and the glutamate uptake was not affected by 5HT or alpha 2 receptor agonists. Nor was the taurine uptake affected by alpha 1 or alpha 2 receptor agonists. The active uptake of aspartate was unaffected by the presence of any of the monoamine receptor agonists used in this study. When the mechanisms behind these effects were studied, the GABA uptake seemed to be mediated via the G protein-adenylate cyclase complex in the receptor domain. Moreover, the K+ channels seemed to be involved. The taurine uptake, however, did not seem to be regulated by the same mechanism. It seems more probable that there is a direct interaction between the receptor and carrier of taurine at the membrane level. The mechanism underlying the receptor-regulated glutamate uptake is at present unclear, although it does not seem to involve protein kinase C.

  11. MKC-231, a choline uptake enhancer: (3) Mode of action of MKC-231 in the enhancement of high-affinity choline uptake.

    PubMed

    Takashina, Ken; Bessho, Tomoko; Mori, Reiko; Kawai, Kunji; Eguchi, Junichi; Saito, Ken-Ichi

    2008-07-01

    MKC-231, a putative cholinergic activity, is reported to improve learning and memory impaired in AF64A-treated animals. MKC-231 enhances high-affinity choline uptake (HACU) known as the rate-limiting step of acetylcholine (ACh) synthesis. We investigated the mode of action (MOA) of HACU enhancement by MKC-231. Intracerebroventricular (i.c.v.) injections of AF64A (3 nmol/brain) resulted in significant HACU reduction in hippocampal synaptosomes. Treatment with MKC-231 increased Vmax of HACU and Bmax of [3H]-HC-3 binding 1.6 and 1.7-fold, respectively. In studies of [3H]-MKC-231 binding and Biacore analysis, MKC-231 showed noticeable affinity for cloned high-affinity choline transporters (CHT1). The present study suggests that MKC-231 directly affects trafficking of CHT1 and increases the numbers of transporter, working for HACU, at the synaptic membrane.

  12. Uptake of (N-Me-3H)-choline by synaptosomes from the central nervous system of Locusta migratoria

    SciTech Connect

    Breer, H.

    1982-03-01

    The accumulation of 3H-choline by isolated synaptosomes from the central nervous system of locust was studied at concentrations varying from 0.05 to 40 microM. Kinetic analysis of the saturable process revealed a high-affinity and a low-affinity system. The high-affinity uptake was competitively inhibited by hemicholinium-3 and was absolutely dependent on external sodium. Elevated potassium concentrations inhibited choline uptake. The choline uptake by insect synaptosomes was found to be remarkably resistant to a variety of metabolic inhibitors. The reduced choline uptake under depolarizing conditions (high potassium concentration or veratridine) in the absence of calcium implies that electrochemical gradients are important for high-affinity choline uptake. Depolarization of preloaded synaptosomes under appropriate conditions resulted in a significant release of newly accumulated choline radioactivity.

  13. The CBL-Interacting Protein Kinase CIPK23 Regulates HAK5-Mediated High-Affinity K+ Uptake in Arabidopsis Roots1[OPEN

    PubMed Central

    Ragel, Paula; Ródenas, Reyes; García-Martín, Elena; Andrés, Zaida; Villalta, Irene; Nieves-Cordones, Manuel; Martínez, Vicente

    2015-01-01

    Plant growth and development requires efficient acquisition of essential elements. Potassium (K+) is an important macronutrient present in the soil solution at a wide range of concentrations. Regulation of the K+ uptake systems in the roots is essential to secure K+ supply. It has been shown in Arabidopsis (Arabidopsis thaliana) that when the external K+ concentration is very low (<10 µm), K+ nutrition depends exclusively on the high-affinity K+ transporter5 (HAK5). Low-K+-induced transcriptional activation of the gene encoding HAK5 has been previously reported. Here, we show the posttranscriptional regulation of HAK5 transport activity by phosphorylation. Expression in a heterologous system showed that the Ca2+ sensors calcineurin B-like (CBL1), CBL8, CBL9, and CBL10, together with CBL-interacting protein kinase23 (CIPK23), activated HAK5 in vivo. This activation produced an increase in the affinity and the Vmax of K+ transport. In vitro experiments show that the N terminus of HAK5 is phosphorylated by CIPK23. This supports the idea that phosphorylation of HAK5 induces a conformational change that increases its affinity for K+. Experiments of K+ (Rb+) uptake and growth measurements in low-K+ medium with Arabidopsis single mutants hak5, akt1, and cipk23, double mutants hak5 akt1, hak5 cipk23, and akt1 cipk23, and the triple mutant hak5 akt1 cipk23 confirmed the regulatory role of CIPK23 in planta. PMID:26474642

  14. The shoot is important for high-affinity nitrate uptake in Egeria densa, a submerged vascular plant.

    PubMed

    Takayanagi, Shu; Takagi, Yuma; Shimizu, Akifumi; Hasegawa, Hiroshi

    2012-09-01

    To understand the mechanisms of nitrate uptake by submerged vascular plants, a cDNA for a high-affinity nitrate transporter, NRT2, was isolated from Egeria densa, a submerged monocot. The deduced EdNRT2 protein was similar to the proteins of a conserved NRT2 group in higher plants. Real-time reverse transcription-PCR analysis revealed that after feeding whole plants with 0.2 mM nitrate, the EdNRT2 transcripts were induced in both shoots and roots within 0.5 h, reached the maximum by 1-3 h and then decreased. The EdNRT2 transcript levels in shoots were comparable to those in roots. When nitrate was applied separately to shoots and roots, the EdNRT2 transcripts were induced only in nitrate-treated organs and reached the maximum levels comparable to those in organs when nitrate was applied to whole plants. (15)N-nitrate feeding experiments demonstrated that both shoots and roots are responsible for nitrate uptake and that biomass and (15)N content in shoots was even higher than that in roots. We concluded that EdNRT2 is involved in high-affinity nitrate uptake by shoots and roots of E. densa, that nitrate is taken up independently by shoots and roots and that shoots play an important role in nitrate uptake from aquatic ecosystem.

  15. Specific cesium transport via the Escherichia coli Kup (TrkD) K+ uptake system.

    PubMed Central

    Bossemeyer, D; Schlösser, A; Bakker, E P

    1989-01-01

    Escherichia coli cells which contain a functional Kup (formerly TrkD) system took up Cs+ with a moderate rate and affinity. Kup is a separate K+ uptake system with relatively little discrimination in the transport of the cations K+, Rb+, and Cs+. Regardless of the presence or absence of Kup, K+-replete cells took up Cs+ primarily by a very low affinity mode, proportional to the ratio of the Cs+ and K+ concentrations in the medium. PMID:2649491

  16. Valproate is neuroprotective against malonate toxicity in rat striatum: an association with augmentation of high-affinity glutamate uptake.

    PubMed

    Morland, Cecilie; Boldingh, Karen Astrid; Iversen, Evy Grini; Hassel, Bjørnar

    2004-11-01

    The antiepileptic drug valproate (VPA) may be neuroprotective. We treated rats with VPA for 14 days (300 mg/kg twice daily) before intrastriatal injection of 1.5 micromol (1 M) of the succinate dehydrogenase inhibitor malonate. VPA-treated animals developed smaller lesions than control animals: 10 +/- 2 mm(3) versus 26 +/- 8 mm(3) (means +/- SD; P = 10(-4). Injection of NaCl that was equiosmolar with 1 M malonate caused lesions of only 1.2 +/- 0.4 mm(3) in control animals, whereas physiologic saline produced no lesion. VPA pretreatment reduced the malonate-induced extracellular accumulation of glutamate. This effect paralleled an increase in the striatal level of the glutamate transporter GLT, which augmented high-affinity glutamate uptake by 25%, as determined from the uptake of [(3)H] glutamate into striatal proteoliposomes. Malonate caused a 76% reduction in striatal adenosine triphosphate (ATP) content, but the glial, ATP-dependent formation of glutamine from radiolabeled glucose or glutamate was intact, indicating that glial ATP production supported uptake of glutamate. Striatal levels of HSP-70 and fos were reduced, and the levels of bcl-2 and phosphorylated extracellular signal-regulated kinase remained unaffected, but histone acetylation was increased by VPA treatment. The results suggest that augmentation of glutamate uptake may contribute importantly to VPA-mediated neuroprotection in striatum.

  17. Plasmid-Encoded Iron Uptake Systems.

    PubMed

    Di Lorenzo, Manuela; Stork, Michiel

    2014-12-01

    Plasmids confer genetic information that benefits the bacterial cells containing them. In pathogenic bacteria, plasmids often harbor virulence determinants that enhance the pathogenicity of the bacterium. The ability to acquire iron in environments where it is limited, for instance the eukaryotic host, is a critical factor for bacterial growth. To acquire iron, bacteria have evolved specific iron uptake mechanisms. These systems are often chromosomally encoded, while those that are plasmid-encoded are rare. Two main plasmid types, ColV and pJM1, have been shown to harbor determinants that increase virulence by providing the cell with essential iron for growth. It is clear that these two plasmid groups evolved independently from each other since they do not share similarities either in the plasmid backbones or in the iron uptake systems they harbor. The siderophores aerobactin and salmochelin that are found on ColV plasmids fall in the hydroxamate and catechol group, respectively, whereas both functional groups are present in the anguibactin siderophore, the only iron uptake system found on pJM1-type plasmids. Besides siderophore-mediated iron uptake, ColV plasmids carry additional genes involved in iron metabolism. These systems include ABC transporters, hemolysins, and a hemoglobin protease. ColV- and pJM1-like plasmids have been shown to confer virulence to their bacterial host, and this trait can be completely ascribed to their encoded iron uptake systems.

  18. Slight temperature changes affect protein affinity and cellular uptake/toxicity of nanoparticles

    NASA Astrophysics Data System (ADS)

    Mahmoudi, Morteza; Shokrgozar, Mohammad A.; Behzadi, Shahed

    2013-03-01

    It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular/organ temperature.It is known that what the cell actually ``sees'' at the nanoscale is an outer shell formed of `protein corona' on the surface of nanoparticles (NPs). The amount and composition of various proteins on the corona are strongly dependent on the biophysicochemical properties of NPs, which have been extensively studied. However, the effect of a small variation in temperature, due to the human circadian rhythm, on the composition of the protein corona and the affinity of various proteins to the surface of NPs, was ignored. Here, the effect of temperature on the composition of protein corona and the affinity of various proteins to the surface of NPs and, subsequently, cell responses to the protein coated NPs are probed. The results confirmed that cellular entrance, dispersion, and toxicity of NPs are strongly diverse with slight body temperature changes. This new finding can help scientists to maximise NP entrance to specific cells/organs with lower toxicity by adjusting the cellular

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

    PubMed

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

    2006-11-01

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

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

    PubMed Central

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

    2006-01-01

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

  1. Uptake of auxins into membrane vesicles isolated from pea stems: an in vitro auxin transport system

    SciTech Connect

    Slone, J.H.

    1985-01-01

    The objective of this research was to test the applicability of the chemiosmotic theory of auxin transport to a subcellular system. Membrane vesicles were isolated from the basal portion of the third internode of etiolated pea plants (Pisum sativum L. var. Alaska) by differential centrifugation. Uptake of auxin was determined by adding /sup 14/C-labeled indoleacetic acid (IAA) to vesicles. Nigericin, a monovalent cation ionophore, and the electrogenic protonophore, carbonyl-cyanide m-chlorophenylhydrazone (CCCP), at micromolar concentrations abolished saturable uptake. Bursting vesicles by sonication, osmotic shock and freeze/thawing also eliminated saturable uptake. As the temperature increased from 0 to 30/sup 0/C, saturable uptake decreased markedly. Nonsaturable auxin uptake was less affected by these treatments. The pH gradient-dependent uptake of auxin appeared to be a transmembrane uptake of auxin into the vesicles rather than surface binding. Unlabeled IAA, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-naphthaleneacetic acid (NAA) at low concentrations reduced the saturable accumulation of (/sup 14/C)IAA in vesicles, while phenylacetic acid, benzoic acid, and 1-NAA were effective only at high concentrations. Kinetic analysis revealed two types of sites: a high affinity site with an uptake capacity of 25 to 40 pmoles/g tissue, and a low affinity site with an uptake capacity of 260 to 600 pmole/g tissue, fresh wt. In conclusion, several principal elements of an auxin transport system, as specific by the chemiosmotic theory of polar auxin transport, were present in membrane vesicles isolated from relatively mature pea stem tissue. However, one important aspect of the theory was not demonstrated in this in vitro system - a TIBA/NPA-sensitive auxin efflux. The kinetics and specificity of auxin uptake strongly suggested that this system was physiologically significant.

  2. Modelling water uptake efficiency of root systems

    NASA Astrophysics Data System (ADS)

    Leitner, Daniel; Tron, Stefania; Schröder, Natalie; Bodner, Gernot; Javaux, Mathieu; Vanderborght, Jan; Vereecken, Harry; Schnepf, Andrea

    2016-04-01

    Water uptake is crucial for plant productivity. Trait based breeding for more water efficient crops will enable a sustainable agricultural management under specific pedoclimatic conditions, and can increase drought resistance of plants. Mathematical modelling can be used to find suitable root system traits for better water uptake efficiency defined as amount of water taken up per unit of root biomass. This approach requires large simulation times and large number of simulation runs, since we test different root systems under different pedoclimatic conditions. In this work, we model water movement by the 1-dimensional Richards equation with the soil hydraulic properties described according to the van Genuchten model. Climatic conditions serve as the upper boundary condition. The root system grows during the simulation period and water uptake is calculated via a sink term (after Tron et al. 2015). The goal of this work is to compare different free software tools based on different numerical schemes to solve the model. We compare implementations using DUMUX (based on finite volumes), Hydrus 1D (based on finite elements), and a Matlab implementation of Van Dam, J. C., & Feddes 2000 (based on finite differences). We analyse the methods for accuracy, speed and flexibility. Using this model case study, we can clearly show the impact of various root system traits on water uptake efficiency. Furthermore, we can quantify frequent simplifications that are introduced in the modelling step like considering a static root system instead of a growing one, or considering a sink term based on root density instead of considering the full root hydraulic model (Javaux et al. 2008). References Tron, S., Bodner, G., Laio, F., Ridolfi, L., & Leitner, D. (2015). Can diversity in root architecture explain plant water use efficiency? A modeling study. Ecological modelling, 312, 200-210. Van Dam, J. C., & Feddes, R. A. (2000). Numerical simulation of infiltration, evaporation and shallow

  3. High-Affinity Manganese Uptake by the Metal Transporter NRAMP1 Is Essential for Arabidopsis Growth in Low Manganese Conditions[C][W

    PubMed Central

    Cailliatte, Rémy; Schikora, Adam; Briat, Jean-François; Mari, Stéphane; Curie, Catherine

    2010-01-01

    In contrast with many other essential metals, the mechanisms of Mn acquisition in higher eukaryotes are seldom studied and poorly understood. We show here that Arabidopsis thaliana relies on a high-affinity uptake system to acquire Mn from the soil in conditions of low Mn availability and that this activity is catalyzed by the divalent metal transporter NRAMP1 (for Natural Resistance Associated Macrophage Protein 1). The nramp1-1 loss-of-function mutant grows poorly, contains less Mn than the wild type, and fails to take up Mn in conditions of Mn limitation, thus demonstrating that NRAMP1 is the major high-affinity Mn transporter in Arabidopsis. Based on confocal microscopy observation of an NRAMP1-green fluorescent protein fusion, we established that NRAMP1 is localized to the plasma membrane. Consistent with its function in Mn acquisition from the soil, NRAMP1 expression is restricted to the root and stimulated by Mn deficiency. Finally, we show that NRAMP1 restores the capacity of the iron-regulated transporter1 mutant to take up iron and cobalt, indicating that NRAMP1 has a broad selectivity in vivo. The role of transporters of the NRAMP family is well established in higher eukaryotes for iron but has been controversial for Mn. This study demonstrates that NRAMP1 is a physiological manganese transporter in Arabidopsis. PMID:20228245

  4. The Organization of High-Affinity Ammonium Uptake in Arabidopsis Roots Depends on the Spatial Arrangement and Biochemical Properties of AMT1-Type Transporters[W

    PubMed Central

    Yuan, Lixing; Loqué, Dominique; Kojima, Soichi; Rauch, Sabine; Ishiyama, Keiki; Inoue, Eri; Takahashi, Hideki; von Wirén, Nicolaus

    2007-01-01

    The AMMONIUM TRANSPORTER (AMT) family comprises six isoforms in Arabidopsis thaliana. Here, we describe the complete functional organization of root-expressed AMTs for high-affinity ammonium uptake. High-affinity influx of 15N-labeled ammonium in two transposon-tagged amt1;2 lines was reduced by 18 to 26% compared with wild-type plants. Enrichment of the AMT1;2 protein in the plasma membrane and localization of AMT1;2 promoter activity in the endodermis and root cortex indicated that AMT1;2 mediates the uptake of ammonium entering the root via the apoplasmic transport route. An amt1;1 amt1;2 amt1;3 amt2;1 quadruple mutant (qko) showed severe growth depression under ammonium supply and maintained only 5 to 10% of wild-type high-affinity ammonium uptake capacity. Transcriptional upregulation of AMT1;5 in nitrogen-deficient rhizodermal and root hair cells and the ability of AMT1;5 to transport ammonium in yeast suggested that AMT1;5 accounts for the remaining uptake capacity in qko. Triple and quadruple amt insertion lines revealed in vivo ammonium substrate affinities of 50, 234, 61, and 4.5 μM for AMT1;1, AMT1;2, AMT1;3, and AMT1;5, respectively, but no ammonium influx activity for AMT2;1. These data suggest that two principle means of achieving effective ammonium uptake in Arabidopsis roots are the spatial arrangement of AMT1-type ammonium transporters and the distribution of their transport capacities at different substrate affinities. PMID:17693533

  5. Modeling Nitrogen Uptake in Oilseed Rape cv Capitol during a Growth Cycle Using Influx Kinetics of Root Nitrate Transport Systems and Field Experimental Data

    PubMed Central

    Malagoli, Philippe; Lainé, Philippe; Le Deunff, Erwan; Rossato, Laurence; Ney, Bertrand; Ourry, Alain

    2004-01-01

    The use of kinetic equations of NO3- transport systems in oilseed rape (Brassica napus), determined by 15NO3- labeling under controlled conditions, combined with experimental field data from the INRA-Châlons rape database were used to model NO3- uptake during the plant growth cycle. The quantitative effects of different factors such as day/night cycle, ontogenetic stages, root temperature, photosynthetically active radiation, and soil nitrate availability on different components of the constitutive high-affinity transport systems, constitutive low-affinity transport systems, inducible low-affinity transport systems, and inducible high-affinity transport systems of nitrate were then determined to improve the model's predictions. Simulated uptake correlated well with measured values of nitrogen (N) uptake under field conditions for all N fertilization rates tested. Model outputs showed that the high-affinity transport system accounted for about 89% of total NO3- uptake (18% and 71% for constitutive high-affinity transport systems and inducible high-affinity transport systems, respectively) when no fertilizer was applied. The low-affinity transport system accounted for a minor proportion of total N uptake, and its activity was restricted to the early phase of the growth cycle. However, N fertilization in spring increased the duration of its contribution to total N uptake. Overall, data show that this mechanistic and environmentally regulated approach is a powerful means to simulate total N uptake in the field with the advantage of taking both physiologically regulated processes at the overall plant level and specific nitrate transport system characteristics into account. PMID:14671012

  6. K+ uptake in plant roots. The systems involved, their regulation and parallels in other organisms.

    PubMed

    Nieves-Cordones, Manuel; Alemán, Fernando; Martínez, Vicente; Rubio, Francisco

    2014-05-15

    Potassium (K(+)) is an essential macronutrient for plants. It is taken into the plant by the transport systems present in the plasma membranes of root epidermal and cortical cells. The identity of these systems and their regulation is beginning to be understood and the systems of K(+) transport in the model species Arabidopsis thaliana remain far better characterized than in any other plant species. Roots can activate different K(+) uptake systems to adapt to their environment, important to a sessile organism that needs to cope with a highly variable environment. The mechanisms of K(+) acquisition in the model species A. thaliana are the best characterized at the molecular level so far. According to the current model, non-selective channels are probably the main pathways for K(+) uptake at high concentrations (>10mM), while at intermediate concentrations (1mM), the inward rectifying channel AKT1 dominates K(+) uptake. Under lower concentrations of external K(+) (100μM), AKT1 channels, together with the high-affinity K(+) uptake system HAK5 contribute to K(+) acquisition, and at extremely low concentrations (<10μM) the only system capable of taking up K(+) is HAK5. Depending on the species the high-affinity system has been named HAK5 or HAK1, but in all cases it fulfills the same functions. The activation of these systems as a function of the K(+) availability is achieved by different mechanisms that include phosphorylation of AKT1 or induction of HAK5 transcription. Some of the characteristics of the systems for root K(+) uptake are shared by other organisms, whilst others are specific to plants. This indicates that some crucial properties of the ancestral of K(+) transport systems have been conserved through evolution while others have diverged among different kingdoms.

  7. High affinity binding of [3H]-tyramine in the central nervous system.

    PubMed Central

    Vaccari, A.

    1986-01-01

    Optimum assay conditions for the association of [3H]-para-tyramine [( 3H]-pTA) with rat brain membranes were characterized, and a saturable, reversible, drug-specific, and high affinity binding mechanism for this trace amine was revealed. The binding capacity (Bmax) for [3H]-pTA in the corpus striatum was approximately 30 times higher than that in the cerebellum, with similar dissociation constants (KD). The binding process of [3H]-pTA involved the dopamine system, inasmuch as (a) highest binding capacity was associated with dopamine-rich regions; (b) dopamine and pTA equally displaced specifically bound [3H]-pTA; (c) there was a severe loss in striatal binding capacity for [3H]-pTA and, reportedly, for [3H]-dopamine, following unilateral nigrostriatal lesion; (d) acute in vivo reserpine treatment markedly decreased the density of [3H]-pTA and, reportedly, of [3H]-dopamine binding sites. In competition experiments [3H]-pTA binding sites, though displaying nanomolar affinity for dopamine, revealed micromolar affinities for the dopamine agonists apomorphine and pergolide, and for several dopamine antagonists, while having very high affinity for reserpine, a marker for the catecholamine transporter in synaptic vesicles. The binding process of [3H]-pTA was both energy-dependent (ouabain-sensitive), and ATP-Mg2+-insensitive; furthermore, the potencies of various drugs in competing for [3H]-pTA binding to rat striatal membranes correlated well (r = 0.96) with their reported potencies in inhibiting [3H]-dopamine uptake into striatal synaptosomes. It is concluded that [3H]-pTA binds at a site located on/within synaptic vesicles where it is involved in the transport mechanism of dopamine. PMID:3801770

  8. Low uptake affinity cultivars with biochar to tackle Cd-tainted rice--A field study over four rice seasons in Hunan, China.

    PubMed

    Chen, De; Guo, Hu; Li, Ruiyue; Li, Lianqing; Pan, Genxing; Chang, Andrew; Joseph, Stephen

    2016-01-15

    Biochar is becoming an environmentally friendly material for remediation of heavy metal contaminated soils and improving food safety. A field trial over four rice seasons was conducted to investigate the use of biochar and low Cd accumulating cultivars on Cd uptake in a heavy metal contaminated soil. Wheat straw derived biochar was applied at 0, 20 and 40 t ha(-1). Two rice cultivars with differing Cd accumulation abilities were selected in each season. The results showed that both biochar and low Cd affinity cultivars significantly reduced rice grain Cd accumulation. Biochar had no significant effect the first season but thereafter consistently reduced rice grain Cd by a maximum of 61, 86 and 57% over the next three seasons. Zn accumulation in the rice grains was not decreased by biochar application, although available soil Zn was sharply reduced (35-91%). Indica conventional rice cultivars had much lower Cd, but higher Zn and lower Cd/Zn ratios in the grain than indica hybrid cultivars. Biochar was more effective for mitigating grain Cd accumulation in low Cd affinity cultivars than in high affinity cultivars. Soil pH was sustainably increased (up to nearly 1 unit) while available Cd significantly decreased by a maximum of 85% after biochar addition. The translocation of Cd from rice roots to shoots was reduced from 20 to 80% by biochar. Low uptake affinity cultivars combined with biochar reduced late rice grain Cd concentration and Cd/Zn ratios by 69-80% and 72-80%, respectively. It indicated that the management of combining biochar and low Cd affinity cultivars should be an efficient way to remediate Cd contaminated rice paddies and reduce health risk associated with consuming rice from these soils.

  9. Influence of affinity on antibody determination in microtiter ELISA systems

    SciTech Connect

    Peterman, J.H.; Voss, E.W. Jr.; Butler, J.E.

    1986-03-01

    Theoretically, all immunoassays are affinity (Ka) dependent when the product of the antibody (Ab) Ka and the free epitope concentration is less than 10. Thus, the degree of dependence on Ka depends on the concentration of available antigen in the system. The authors examined the binding of /sup 125/I-anti-fluorescein (a-FLU) monoclonal antibodies of different affinities to FLU-gelatin adsorbed on Immunlon 2 microtiter plates. Data obtained were in general agreement with our theoretical predictions; the percent of /sup 125/I-a-FLU which bound correlated with Ka, as did the shape of the titration curves. Measurement of 5 a-FLU monoclonals by the ELISA showed that the determination of Ab concentrations depends on the FLU-gelatin concentration, epitope density, and on the relationship between the Kas of test samples and the reference standard Ab preparation. Thus the ELISA is Ka dependent and should not be used routinely to estimate the absolute amount to Ab in unknown samples. However, the Ka dependency of the ELISA might provide a convenient assay for the estimation of the relative functional Ka (rfKa) of antibody preparations.

  10. Non-covalent interaction between dietary stilbenoids and human serum albumin: Structure-affinity relationship, and its influence on the stability, free radical scavenging activity and cell uptake of stilbenoids.

    PubMed

    Cao, Hui; Jia, Xueping; Shi, Jian; Xiao, Jianbo; Chen, Xiaoqing

    2016-07-01

    Dietary stilbenoids are associated with many benefits for human health, which depend on their bioavailability and bioaccessibility. The stilbenoid-human serum albumin (HSA) interactions are investigated to explore the structure-affinity relationship and influence on the stability, free radical scavenging activity and cell uptake of stilbenoids. The structure-affinity relationship of the stilbenoids-HSA interaction was found as: (1) the methoxylation enhanced the affinity, (2) an additional hydroxyl group increases the affinity and (3) the glycosylation significantly weakened the affinity. HSA obviously masked the free radical scavenging potential of stilbenoids. The stabilities of stilbenoids in different medium were determined as: HSA solution>human plasma>Dulbecco's modified Eagle's medium. It appears that the milk enhanced the cell uptake of stilbenoids with multi-hydroxyl groups and weakened the cell uptake of stilbenoids with methoxyl group on EA.hy 926 endothelial cells. The stilbenoids are hardly absorbed by human umbilical vein endothelial cells in the presence of milk.

  11. Anacardium occidentale bark lectin: purification, immobilization as an affinity model and influence in the uptake of technetium-99M by rat adipocytes.

    PubMed

    Maciel, Maria Inês Sucupira; de Mendonça Cavalcanti, Maria do Socorro; Napoleão, Thiago Henrique; Paiva, Patrícia Maria Guedes; de Almeida Catanho, Maria Teresa Jansem; Coelho, Luana Cassandra Breitenbach Barroso

    2012-10-01

    Lectins, proteins that recognize carbohydrates, have been immobilized on inert supports and used in the screening or purification of glycoproteins. Anacardium occidentale bark infusion has been used as a hypoglycemic agent in Brazil. The toxicity of natural products may be evaluated determining their capability to alter the biodistribution of technetium-99M ((99m)Tc). This work reports the isolation and characterization of a lectin from A. occidentale bark (AnocBL), its evaluation as an affinity support for glycoprotein isolation and lectin effect on the uptake of (99m)Tc by rat adipocytes. AnocBL was isolated from 80 % ammonium sulphate supernatant by affinity chromatography on fetuin-agarose. SDS-PAGE showed a single protein band of 47 kDa. The monossacharide L-arabinose and the glycoproteins fetuin, asialofetuin, ovomucoid, casein, thyroglobulin, peroxidase, fetal bovine serum and IgG inhibited the activity. The lectin activity was stable until 70 °C and at a pH range of 3.0-7.5. AnocBL-Sepharose column bound fetuin indicating that the lectin matrix may be used to obtain glycoconjugates of biotechnological interest. In vitro assay revealed that glucose and insulin increase (99m)Tc uptake by rat adipocytes. AnocBL decreases (99m)Tc uptake, and this effect was not detected in the presence of glucose. Fetuin inhibited AnocBL effect in all insulin concentrations.

  12. The ability of denbufylline to inhibit cyclic nucleotide phosphodiesterase and its affinity for adenosine receptors and the adenosine re-uptake site.

    PubMed Central

    Nicholson, C. D.; Jackman, S. A.; Wilke, R.

    1989-01-01

    1. Denbufylline has been examined for its ability to inhibit cyclic nucleotide phosphodiesterase isoenzymes from rat cardiac ventricle and cerebrum, as well as for its affinity for adenosine A1 and A2 receptors and the re-uptake site. For comparison, SK&F 94120, theophylline and 3-isobutyl-1-methyl-xanthine (IBMX) were examined as phosphodiesterase inhibitors whilst N6-cyclohexyladenosine, R(-)-N6-(2-phenylisopropyl)-adenosine, 5'-N-ethylcarboxamido-adenosine, 2-nitrobenzylthioinosine, theophylline and IBMX were examined for their affinity for adenosine binding sites. 2. This investigation confirmed the presence of four phosphodiesterase activities in rat cardiac ventricle; in rat cerebrum only three were present. 3. Denbufylline selective inhibited one form of Ca2+-independent, low Km cyclic AMP phosphodiesterase. The form inhibited was one of two present in cardiac ventricle and the sole one in cerebrum. This form was not inhibited by cyclic GMP. The inotropic agent SK&F 94120 selectively inhibited the form of cyclic AMP phosphodiesterase which was inhibited by cyclic GMP present in cardiac ventricle. Theophylline and IBMX were relatively non-selective phosphodiesterase inhibitors. 4. Denbufylline was a less potent inhibitor of ligand binding to adenosine receptors than of cyclic AMP phosphodiesterase. This contrasted with theophylline, which had a higher affinity for adenosine receptors, and IBMX which showed no marked selectivity. Denbufylline, theophylline and IBMX all had a low affinity for the adenosine re-uptake site. 5. Denbufylline is being developed as an agent for the therapy of multi-infarct dementia. The selective inhibition of a particular low Km cyclic AMP phosphodiesterase may account for the activity of this compound. PMID:2474352

  13. Robust adaptive control for a class of uncertain non-affine nonlinear systems using affine-type neural networks

    NASA Astrophysics Data System (ADS)

    Zhao, Shitie; Gao, Xianwen

    2016-08-01

    A robust adaptive control is proposed for a class of single-input single-output non-affine nonlinear systems. In order to approximate the unknown nonlinear function, a novel affine-type neural network is used, and then to compensate the approximation error and external disturbance a robust control term is employed. By Lyapunov stability analysis for the closed-loop system, it is proved that tracking errors asymptotically converge to zero. Moreover, an observer is designed to estimate the system states because all the states may not be available for measurements. Furthermore, the adaptation laws of neural networks and the robust controller are given out based on the Lyapunov stability theory. Finally, two simulation examples are presented to demonstrate the effectiveness of the proposed control method.

  14. Elevated sacroilac joint uptake ratios in systemic lupus erythematosus

    SciTech Connect

    De Smet, A.A.; Mahmood, T.; Robinson, R.G.; Lindsley, H.B.

    1984-08-01

    Sacroiliac joint radiographs and radionuclide sacroiliac joint uptake ratios were obtained on 14 patients with active systemic lupus erythematosus. Elevated joint ratios were found unilaterally in two patients and bilaterally in seven patients when their lupus was active. In patients whose disease became quiescent, the uptake ratios returned to normal. Two patients had persistently elevated ratios with continued clinical and laboratory evidence of active lupus. Mild sacroiliac joint sclerosis and erosions were detected on pelvic radiographs in these same two patients. Elevated quantitative sacroiliac joint uptake ratios may occur as a manifestation of active systemic lupus erythematosus.

  15. A phospholipid uptake system in the model plant Arabidopsis thaliana.

    PubMed

    Poulsen, Lisbeth R; López-Marqués, Rosa L; Pedas, Pai R; McDowell, Stephen C; Brown, Elizabeth; Kunze, Reinhard; Harper, Jeffrey F; Pomorski, Thomas G; Palmgren, Michael

    2015-07-27

    Plants use solar energy to produce lipids directly from inorganic elements and are not thought to require molecular systems for lipid uptake from the environment. Here we show that Arabidopsis thaliana Aminophospholipid ATPase10 (ALA10) is a P4-type ATPase flippase that internalizes exogenous phospholipids across the plasma membrane, after which they are rapidly metabolized. ALA10 expression and phospholipid uptake are high in the epidermal cells of the root tip and in guard cells, the latter of which regulate the size of stomatal apertures to modulate gas exchange. ALA10-knockout mutants exhibit reduced phospholipid uptake at the root tips and guard cells and are affected in growth and transpiration. The presence of a phospholipid uptake system in plants is surprising. Our results suggest that one possible physiological role of this system is to internalize lysophosphatidylcholine, a signalling lipid involved in root development and stomatal control.

  16. Compensatory Root Water Uptake of Overlapping Root Systems

    NASA Astrophysics Data System (ADS)

    Agee, E.; Ivanov, V. Y.; He, L.; Bisht, G.; Shahbaz, P.; Fatichi, S.; Gough, C. M.; Couvreur, V.; Matheny, A. M.; Bohrer, G.

    2015-12-01

    Land-surface models use simplified representations of root water uptake based on biomass distributions and empirical functions that constrain water uptake during unfavorable soil moisture conditions. These models fail to capture the observed hydraulic plasticity that allows plants to regulate root hydraulic conductivity and zones of active uptake based on local gradients. Recent developments in root water uptake modeling have sought to increase its mechanistic representation by bridging the gap between physically based microscopic models and computationally feasible macroscopic approaches. It remains to be demonstrated whether bulk parameterization of microscale characteristics (e.g., root system morphology and root conductivity) can improve process representation at the ecosystem scale. We employ the Couvreur method of microscopic uptake to yield macroscopic representation in a coupled soil-root model. Using a modified version of the PFLOTRAN model, which represents the 3-D physics of variably saturated soil, we model a one-hectare temperate forest stand under natural and synthetic climatic forcing. Our results show that as shallow soil layers dry, uptake at the tree and stand level shift to deeper soil layers, allowing the transpiration stream demanded by the atmosphere. We assess the potential capacity of the model to capture compensatory root water uptake. Further, the hydraulic plasticity of the root system is demonstrated by the quick response of uptake to rainfall pulses. These initial results indicate a promising direction for land surface models in which significant three-dimensional information from large root systems can be feasibly integrated into the forest scale simulations of root water uptake.

  17. Characterization of an AtCCX5 gene from Arabidopsis thaliana that involves in high-affinity K{sup +} uptake and Na{sup +} transport in yeast

    SciTech Connect

    Zhang, Xinxin; Zhang, Min; Takano, Tetsuo; Liu, Shenkui

    2011-10-14

    Highlights: {yields} The AtCCX5 protein coding a putative cation calcium exchanger was characterized. {yields} AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. {yields} AtCCX5 protein did not show the same transport properties as the CAXs. {yields} AtCCX5 protein involves in mediating high-affinity K{sup +} uptake in yeast. {yields} AtCCX5 protein also involves in Na{sup +} transport in yeast. -- Abstract: The gene for a putative cation calcium exchanger (CCX) from Arabidopsis thaliana, AtCCX5, was cloned and its function was analyzed in yeast. Green fluorescent protein-tagged AtCCX5 expressed in yeast was localized in the plasma membrane and nuclear periphery. The yeast transformants expressing AtCCX5 were created and their growth in the presence of various cations (K{sup +}, Na{sup +}, Ca{sup 2+}, Mg{sup 2+}, Fe{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Cd{sup 2+}, Mn{sup 2+}, Ba{sup 2+}, Ni{sup 2+}, Zn{sup 2+}, and Li{sup +}) were analyzed. AtCCX5 expression was found to affect the response to K{sup +} and Na{sup +} in yeast. The AtCCX5 transformant also showed a little better growth to Zn{sup 2+}. The yeast mutant 9.3 expressing AtCCX5 restored growth of the mutant on medium with low K{sup +} (0.5 mM), and also suppressed its Na{sup +} sensitivity. Ion uptake experiments showed that AtCCX5 mediated relatively high-affinity K{sup +} uptake and was also involved in Na{sup +} transport in yeast. Taken together, these findings suggest that the AtCCX5 is a novel transport protein involves in mediating high-affinity K{sup +} uptake and Na{sup +} transport in yeast.

  18. Fluoxetine, a selective inhibitor of serotonin uptake, potentiates morphine analgesia without altering its discriminative stimulus properties or affinity for opioid receptors

    SciTech Connect

    Hynes, M.D.; Lochner, M.A.; Bemis, K.G.; Hymson, D.L.

    1985-06-17

    The analgesic effect of morphine in the rat tail jerk assay was enhanced by the serotonin uptake inhibitor, fluoxetine. Tail jerk latency was not affected by fluoxetine alone. Morphine's affinity for opioid receptors labeled in vitro with /sup 3/H-naloxone or /sup 3/H-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin was not altered by fluoxetine, which has no affinity for these sites at concentrations as high as 1000 nM. In rats trained to discriminate morphine from saline, fluoxetine at doses of 5 or 10 mg/kg were recognized as saline. Increasing the fluoxetine dose to 20 mg/kg did not result in generalization to either saline or morphine. The dose response curve for morphine generalization was not significantly altered by fluoxetine doses of 5 or 10 mg/kg. Those rats treated with the combination of morphine and 20 mg/kg of fluoxetine did not exhibit saline or morphine appropriate responding. Fluoxetine potentiates the analgesic properties of morphine without enhancing its affinity for opioid receptors or its discriminative stimulus properties. 30 references, 2 figures, 2 tables.

  19. Distribution and ultrastructure of neurons in opossum piriform cortex displaying immunoreactivity to GABA and GAD and high-affinity tritiated GABA uptake

    SciTech Connect

    Haberly, L.B.; Hansen, D.J.; Feig, S.L.; Presto, S.

    1987-12-08

    GABAergic neurons have been identified in the piriform cortex of the opossum at light and electron microscopic levels by immunocytochemical localization of GABA and the GABA-synthesizing enzyme glutamic acid decarboxylase and by autoradiographic visualization of high-affinity /sup 3/H-GABA uptake. Four major neuron populations have been distinguished on the basis of soma size, shape, and segregation at specific depths and locations: large horizontal cells in layer Ia of the anterior piriform cortex, small globular cells with thin dendrites concentrated in layers Ib and II of the posterior piriform cortex, and multipolar and fusiform cells concentrated in the deep part of layer III in anterior and posterior parts of the piriform cortex and the subjacent endopiriform nucleus. All four populations were well visualized with both antisera, but the large layer Ia horizontal cells displayed only very light /sup 3/H-GABA uptake, thus suggesting a lack of local axon collaterals or lack of high-affinity GABA uptake sites. The large, ultrastructurally distinctive somata of layer Ia horizontal cells receive a very small number of symmetrical synapses; the thin, axonlike dendrites of small globular cells are exclusively postsynaptic and receive large numbers of both symmetrical and asymmetrical synapses, in contrast to somata which receive a small number of both types; and the deep multipolar and fusiform cells receive a highly variable number of symmetrical and asymmetrical synapses on somata and proximal dendrites. Labeled puncta of axon terminal dimensions were found in large numbers in the neuropil surrounding pyramidal cell somata in layer II and in the endopiriform nucleus. Moderately large numbers of labeled puncta were found in layer I at the depth of pyramidal cell apical dendrites with greater numbers in layer Ia at the depth of distal apical segments than in layer Ib.

  20. Cloning and functional characterization of the high-affinity K+ transporter HAK1 of pepper.

    PubMed

    Martínez-Cordero, M Angeles; Martínez, Vicente; Rubio, Francisco

    2004-10-01

    High-affinity K+ uptake in plants plays a crucial role in K+ nutrition and different systems have been postulated to contribute to the high-affinity K+ uptake. The results presented here with pepper (Capsicum annum) demonstrate that a HAK1-type transporter greatly contributes to the high-affinity K+ uptake observed in roots. Pepper plants starved of K+ for 3 d showed high-affinity K+ uptake (Km of 6 microM K+) that was very sensitive to NH and their roots expressed a high-affinity K+ transporter, CaHAK1, which clusters in group I of the KT/HAK/KUP family of transporters. When expressed in yeast ( Saccharomyces cerevisiae ), CaHAK1 mediated high-affinity K+ and Rb+ uptake with Km values of 3.3 and 1.9 microM, respectively. Rb+ uptake was competitively inhibited by micromolar concentrations of NH and Cs+, and by millimolar concentrations of Na+.

  1. 3H-GABA uptake selectively labels identifiable neurons in the leech central nervous system

    SciTech Connect

    Cline, H.T.

    1983-04-10

    Segmental ganglia of the leech ventral nerve cord synthesize the neurotransmitter gamma-aminobutyric acid (GABA) when incubated in the presence of the precursor glutamate, suggesting that there may be GABA-ergic neurons in the leech nerve cord. GABA-accumulating neurons of the two taxonomically distant leech species, Haementeria ghilianii and Hirudo medicinalis, have been labeled by taking advantage of their high-affinity uptake system for the neurotransmitter. Autoradiography of sectioned segmental ganglia previously exposed to 3H-GABA reveals a reproducible pattern of about thirty 3H-GABA-labeled neuronal cell bodies per ganglion. The majority of 3H-GABA-labeled neuronal cell bodies are bilaterally paired, although some apparently unpaired cell bodies also accumulate label. Neuronal processes were reproducibly labeled by GABA uptake and could be traced in the neuropil through commissures and fiber tracts into the segmental nerve roots and interganglionic connectives, respectively.

  2. An observing system simulation for Southern Ocean carbon dioxide uptake.

    PubMed

    Majkut, Joseph D; Carter, Brendan R; Frölicher, Thomas L; Dufour, Carolina O; Rodgers, Keith B; Sarmiento, Jorge L

    2014-07-13

    The Southern Ocean is critically important to the oceanic uptake of anthropogenic CO2. Up to half of the excess CO2 currently in the ocean entered through the Southern Ocean. That uptake helps to maintain the global carbon balance and buffers transient climate change from fossil fuel emissions. However, the future evolution of the uptake is uncertain, because our understanding of the dynamics that govern the Southern Ocean CO2 uptake is incomplete. Sparse observations and incomplete model formulations limit our ability to constrain the monthly and annual uptake, interannual variability and long-term trends. Float-based sampling of ocean biogeochemistry provides an opportunity for transforming our understanding of the Southern Ocean CO2 flux. In this work, we review current estimates of the CO2 uptake in the Southern Ocean and projections of its response to climate change. We then show, via an observational system simulation experiment, that float-based sampling provides a significant opportunity for measuring the mean fluxes and monitoring the mean uptake over decadal scales.

  3. High-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity in neuronal cultures for mechanistic and drug discovery studies.

    PubMed

    Ray, Balmiki; Bailey, Jason A; Simon, Jay R; Lahiri, Debomoy K

    2012-07-01

    Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory-related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzyme A by the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High-affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate-limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well-documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide-based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK-N-SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease.

  4. Effects of ammonia on high affinity glutamate uptake and glutamate transporter EAAT3 expression in cultured rat cerebellar granule cells.

    PubMed

    Chan, Helen; Zwingmann, Claudia; Pannunzio, Marc; Butterworth, Roger F

    2003-07-01

    Increased levels of extracellular glutamate are a consistent feature of hepatic encephalopathy (HE) associated with liver failure and other hyperammonemic pathologies. Reduction of glutamate uptake has been described in ammonia-exposed cultured astrocytes, synaptosomes, and in animal models of hyperammonemia. In the present study, we examine the effects of pathophysiological concentrations of ammonia on D-aspartate (a non-metabolizable analog of glutamate) uptake by cultured rat cerebellar granule neurons. Exposure of these cells to ammonia resulted in time-dependent (24% reduction at 24h and 60% reduction at 5 days, P<0.001) and dose-dependent (21, 37, and 57% reduction at 1, 2.5, and 5mM for 5 days, P<0.01) suppression of D-aspartate uptake. Kinetic analyses revealed significant decreases in the velocity of uptake (V(max)) (37% decrease at 2.5mM NH(4)Cl, P<0.05 and 52% decrease at 5mM NH(4)Cl, P<0.001) as well as significant reductions in K(m) values (25% reduction at 2.5mM NH(4)Cl, P<0.05 and 45% reduction at 5mM NH(4)Cl, P<0.001). Western blotting, on the other hand, showed no significant changes in the neuronal glutamate transporter EAAC1/EAAT3 protein, the only glutamate transporter currently known to be expressed by these cells. In addition, 1H combined with 13C-NMR spectroscopy studies using the stable isotope [1-13C]-glucose demonstrated a significant increase in intracellular glutamate levels derived from the oxidative metabolism of glucose, rather than from the deamidation of exogenous glutamine in cultured granule neurons exposed to ammonia. The present study provides evidence that the effects of ammonia on glutamate uptake are not solely an astrocytic phenomenon and that unlike the astrocytic glutamate transporter counterpart, EAAT3 protein expression in cultured cerebellar granule cells is not down-regulated when exposed to ammonia. Decrease of glutamate uptake in these cellular preparations may afford an additional regulatory mechanism aimed at

  5. Ferrous iron uptake in Cryptococcus neoformans.

    PubMed

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

    1998-09-01

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

  6. A high-affinity and specific carrier-mediated mechanism for uptake of thiamine pyrophosphate by human colonic epithelial cells.

    PubMed

    Nabokina, Svetlana M; Said, Hamid M

    2012-08-01

    All mammals require exogenous sources of thiamine (vitamin B1), as they lack the ability to synthesize the vitamin. These sources are dietary and bacterial (the latter is in reference to the vitamin, which is synthesized by the normal microflora of the large intestine). Bacterially generated thiamine exists in the free, as well as the pyrophosphorylated [thiamine pyrophosphate (TPP)], form. With no (or very little) phosphatase activity in the colon, we hypothesized that the bacterially generated TPP can also be taken up by colonocytes. To test this hypothesis, we examined [(3)H]TPP uptake in the human-derived, nontransformed colonic epithelial NCM460 cells and purified apical membrane vesicles isolated from the colon of human organ donors. Uptake of TPP by NCM460 cells occurred without metabolic alterations in the transported substrate and 1) was pH- and Na(+)-independent, but energy-dependent, 2) was saturable as a function of concentration (apparent K(m) = 0.157 ± 0.028 μM), 3) was highly specific for TPP and not affected by free thiamine (or its analogs) or by thiamine monophosphate and unrelated folate derivatives, 4) was adaptively regulated by extracellular substrate (TPP) level via what appears to be a transcriptionally mediated mechanism(s), and 5) appeared to be influenced by an intracellular Ca(2+)/calmodulin-mediated regulatory pathway. These findings suggest the involvement of a carrier-mediated mechanism for TPP uptake by colonic NCM460 cells, which was further confirmed by results from studies of native human colonic apical membrane vesicles. The results also suggest that the bacterially synthesized TPP in the large intestine is bioavailable and may contribute to overall body homeostasis of vitamin B1 and, especially, to the cellular nutrition of the local colonocytes.

  7. Variation for N Uptake System in Maize: Genotypic Response to N Supply

    PubMed Central

    Garnett, Trevor; Plett, Darren; Conn, Vanessa; Conn, Simon; Rabie, Huwaida; Rafalski, J. Antoni; Dhugga, Kanwarpal; Tester, Mark A.; Kaiser, Brent N.

    2015-01-01

    An understanding of the adaptations made by plants in their nitrogen (N) uptake systems in response to reduced N supply is important to the development of cereals with enhanced N uptake efficiency (NUpE). Twenty seven diverse genotypes of maize (Zea mays, L.) were grown in hydroponics for 3 weeks with limiting or adequate N supply. Genotypic response to N was assessed on the basis of biomass characteristics and the activities of the nitrate (NO3−) and ammonium (NH4+) high-affinity transport systems. Genotypes differed greatly for the ability to maintain biomass with reduced N. Although, the N response in underlying biomass and N transport related characteristics was less than that for biomass, there were clear relationships, most importantly, lines that maintained biomass at reduced N maintained net N uptake with no change in size of the root relative to the shoot. The root uptake capacity for both NO3− and NH4+ increased with reduced N. Transcript levels of putative NO3− and NH4+ transporter genes in the root tissue of a subset of the genotypes revealed that predominately ZmNRT2 transcript levels responded to N treatments. The correlation between the ratio of transcripts of ZmNRT2.2 between the two N levels and a genotype's ability to maintain biomass with reduced N suggests a role for these transporters in enhancing NUpE. The observed variation in the ability to capture N at low N provides scope for both improving NUpE in maize and also to better understand the N uptake system in cereals. PMID:26617612

  8. LjMOT1, a high-affinity molybdate transporter from Lotus japonicus, is essential for molybdate uptake, but not for the delivery to nodules.

    PubMed

    Duan, Guilan; Hakoyama, Tsuneo; Kamiya, Takehiro; Miwa, Hiroki; Lombardo, Fabien; Sato, Shusei; Tabata, Satoshi; Chen, Zheng; Watanabe, Toshihiro; Shinano, Takuro; Fujiwara, Toru

    2017-03-09

    Molybdenum (Mo) is an essential nutrient for plants, and is required for nitrogenase activity of legumes. However, the pathways of Mo uptake from soils and then delivery to the nodules have not been characterized in legumes. In this study, we characterized a high-affinity Mo transporter (LjMOT1) from Lotus japonicus. Mo concentrations in an ethyl methanesulfonate-mutagenized line (ljmot1) decreased by 70-95% compared with wild type (WT). By comparing the DNA sequences of four AtMOT1 homologs between mutant and WT lines, one point mutation was found in LjMOT1, which altered Trp(292) to a stop codon; no mutation was found in the other homologous genes. The phenotype of Mo concentrations in F2 progeny from ljmot1 and WT crosses were associated with genotypes of LjMOT1. Introduction of endogenous LjMOT1 to ljmot1 restored Mo accumulation to approximately 60-70% of the WT. Yeast expressing LjMOT1 exhibited high Mo uptake activity, and the Km was 182 nM. LjMOT1 was expressed mainly in roots, and its expression was not affected by Mo supply or rhizobium inoculation. Although Mo accumulation in the nodules of ljmot1 was significantly lower than that of WT, it was still high enough for normal nodulation and nitrogenase activity, even for cotyledons removed ljmot1 plants grown under low Mo conditions, in this case the plant growth was significantly inhibited by Mo deficiency. Our results suggest that LjMOT1 is an essential Mo transporter in L. japonicus for Mo uptake from the soil and growth, but is not Mo delivery to the nodules. This article is protected by copyright. All rights reserved.

  9. Mathematical modeling of the low and high affinity arabinose transport systems in Escherichia coli.

    PubMed

    Yildirim, Necmettin

    2012-04-01

    A mathematical model was developed for the low and high affinity arabinose transport systems in E. coli. The model is a system of three ordinary differential equations and takes the dynamics of mRNAs for the araE and araFGH proteins and the internal arabinose into account. Special attention was paid to estimate the model parameters from the literature. Our analysis and simulations suggest that the high affinity transport system helps the low affinity transport system to respond to high concentration of extracellular arabinose faster, whereas the high affinity transport system responds to a small amount of extracellular arabinose. Steady state analysis of the model also predicts that there is a regime for the extracellular concentration of arabinose where the arabinose system can show bistable behavior.

  10. Potassium uptake system Trk2 is crucial for yeast cell viability during anhydrobiosis.

    PubMed

    Borovikova, Diana; Herynkova, Pavla; Rapoport, Alexander; Sychrova, Hana

    2014-01-01

    Yeasts grow at very different potassium concentrations, adapting their intracellular cation levels to changes in the external environment. Potassium homeostasis is maintained with the help of several transporters mediating the uptake and efflux of potassium with various affinities and mechanisms. In the model yeast Saccharomyces cerevisiae, two uptake systems, Trk1 and Trk2, are responsible for the accumulation of a relatively high intracellular potassium content (200-300 mM) and the efflux of surplus potassium is mediated by the Tok1 channel and active exporters Ena ATPase and Nha1 cation/proton antiporter. Using a series of deletion mutants, we studied the role of individual potassium transporters in yeast cell resistance to dehydration. The Trk2 transporter (whose role in S. cerevisiae physiology was not clear) is important for cell viability in the stationary phase of growth and, moreover, it plays a crucial role in the yeast survival of dehydration/rehydration treatments. Mutants lacking the TRK2 gene accumulated significantly lower amounts of potassium ions in the stationary culture growth phase, and these lower amounts correlated with decreased resistance to dehydration/rehydration stress. Our results showed Trk2 to be the major potassium uptake system in stationary cells, and potassium content to be a crucial parameter for desiccation survival.

  11. Mechanism of the influence of EGTA on the affinity of the Ca-transporting and Ca-binding systems of the cell for calcium

    SciTech Connect

    Orolv, S.N.; Sitozhevskii, A.V.; Pokudin, N.I.; Agnaev, V.M.

    1986-05-20

    The activity of Ca-ATPases of erythrocyte ghosts and the sarcoplasmic reticulum, as the rate of ATP-dependent uptake of /sup 44/Ca by inside-out vesicles of erythrocyte membranes, vesicles of sarcoplasmic reticulum and mitochondria, was investigated. Evidently in all cases, the introduction of EGTA into the incubation medium leads to an increase in the affinity of the Ca-pumps for Ca/sup 2 +/ without any change in their maximum activity. An analogous effect of EGTA was detected in a determination of the affinity of Ca/sup 2 +/ of calmodulin, troponin S, and the fluorescent dye quin 2. It is suggested that the action of EGTA on the affinity of the Ca-transporting and Ca-binding systems of the cell for Ca/sup 2 +/ is associated with displacement of impurities of di- and trivalent cations.

  12. Plant-uptake of uranium: Hydroponic and soil system studies

    USGS Publications Warehouse

    Ramaswami, A.; Carr, P.; Burkhardt, M.

    2001-01-01

    Limited information is available on screening and selection of terrestrial plants for uptake and translocation of uranium from soil. This article evaluates the removal of uranium from water and soil by selected plants, comparing plant performance in hydroponic systems with that in two soil systems (a sandy-loam soil and an organic-rich soil). Plants selected for this study were Sunflower (Helianthus giganteus), Spring Vetch (Vicia sativa), Hairy Vetch (Vicia villosa), Juniper (Juniperus monosperma), Indian Mustard (Brassica juncea), and Bush Bean (Phaseolus nanus). Plant performance was evaluated both in terms of the percent uranium extracted from the three systems, as well as the biological absorption coefficient (BAC) that normalized uranium uptake to plant biomass. Study results indicate that uranium extraction efficiency decreased sharply across hydroponic, sandy and organic soil systems, indicating that soil organic matter sequestered uranium, rendering it largely unavailable for plant uptake. These results indicate that site-specific soils must be used to screen plants for uranium extraction capability; plant behavior in hydroponic systems does not correlate well with that in soil systems. One plant species, Juniper, exhibited consistent uranium extraction efficiencies and BACs in both sandy and organic soils, suggesting unique uranium extraction capabilities.

  13. Induction of nitrate uptake in maize roots: expression of a putative high-affinity nitrate transporter and plasma membrane H+-ATPase isoforms.

    PubMed

    Santi, Simonetta; Locci, Geraldine; Monte, Rossella; Pinton, Roberto; Varanini, Zeno

    2003-08-01

    An investigation was carried out to assess the effect of nitrate supply on the root plasma membrane (PM) H+-ATPase of etiolated maize (Zea mays L.) seedlings grown in hydroponics. The treatment induced higher uptake rates of the anion and the expression of a putative high-affinity nitrate transporter gene (ZmNRT2.1), the first to be identified in maize. Root PM H+-ATPase activity displayed a similar time-course pattern as that of net nitrate uptake and investigations were carried out to determine which of the two isoforms reported to date in maize, MHA1 and 2, responded to the treatment. MHA1 was not expressed under the conditions analysed. Genome analysis revealed that MHA2, described as the most abundant form in all maize tissues, was not present in the maize hybrid investigated, but a similar form was found instead and named MHA3. A second gene (named MHA4) was also identified and partially sequenced. Both genes, classified as members of the PM H+-ATPase subfamily II, responded to nitrate supply, although to different degrees: MHA4, in particular, proved more sensitive than MHA3, with a greater up- and down-regulation in response to the treatment. Increased expression of subfamily II genes resulted in higher steady-state levels of the enzyme in the root tissues and enhanced ATP-hydrolysing activity. The results support the idea that greater proton-pumping activity is required when nitrate inflow increases and suggest that nitrate may be the signal triggering the expression of the two members of PM H+-ATPase subfamily II.

  14. Fur-regulated iron uptake system of Edwardsiella ictaluri and its influence on pathogenesis and immunogenicity in the catfish host.

    PubMed

    Santander, Javier; Golden, Greg; Wanda, Soo-Young; Curtiss, Roy

    2012-08-01

    The ability of bacterial pathogens to take up iron from the host during infection is necessary for their multiplication within the host. However, host high-affinity iron binding proteins limit levels of free iron in fluids and tissues. To overcome this deficiency of iron during infection, bacterial pathogens have developed iron uptake systems that are upregulated in the absence of iron, typically tightly controlled by the ferric uptake regulator (Fur) protein. The iron uptake system of Edwardsiella ictaluri, a host-restricted pathogen of channel catfish (Ictalurus punctatus) and the main pathogen of this fish in aquaculture, is unknown. Here we describe the E. ictaluri Fur protein, the iron uptake machinery controlled by Fur, and the effects of fur gene deletion on virulence and immunogenicity in the fish host. Analysis of the E. ictaluri Fur protein shows that it lacks the N-terminal region found in the majority of pathogen-encoded Fur proteins. However, it is fully functional in regulated genes encoding iron uptake proteins. E. ictaluri grown under iron-limited conditions upregulates an outer membrane protein (HemR) that shows heme-hemoglobin transport activity and is tightly regulated by Fur. In vivo studies showed that an E. ictaluri Δfur mutant is attenuated and immune protective in zebrafish (Danio rerio) and catfish (Ictalurus punctatus), triggering systemic immunity. We conclude that an E. ictaluri Δfur mutant could be an effective component of an immersion-oral vaccine for the catfish industry.

  15. Fur-Regulated Iron Uptake System of Edwardsiella ictaluri and Its Influence on Pathogenesis and Immunogenicity in the Catfish Host

    PubMed Central

    Golden, Greg; Wanda, Soo-Young; Curtiss, Roy

    2012-01-01

    The ability of bacterial pathogens to take up iron from the host during infection is necessary for their multiplication within the host. However, host high-affinity iron binding proteins limit levels of free iron in fluids and tissues. To overcome this deficiency of iron during infection, bacterial pathogens have developed iron uptake systems that are upregulated in the absence of iron, typically tightly controlled by the ferric uptake regulator (Fur) protein. The iron uptake system of Edwardsiella ictaluri, a host-restricted pathogen of channel catfish (Ictalurus punctatus) and the main pathogen of this fish in aquaculture, is unknown. Here we describe the E. ictaluri Fur protein, the iron uptake machinery controlled by Fur, and the effects of fur gene deletion on virulence and immunogenicity in the fish host. Analysis of the E. ictaluri Fur protein shows that it lacks the N-terminal region found in the majority of pathogen-encoded Fur proteins. However, it is fully functional in regulated genes encoding iron uptake proteins. E. ictaluri grown under iron-limited conditions upregulates an outer membrane protein (HemR) that shows heme-hemoglobin transport activity and is tightly regulated by Fur. In vivo studies showed that an E. ictaluri Δfur mutant is attenuated and immune protective in zebrafish (Danio rerio) and catfish (Ictalurus punctatus), triggering systemic immunity. We conclude that an E. ictaluri Δfur mutant could be an effective component of an immersion-oral vaccine for the catfish industry. PMID:22615248

  16. Low- and high-affinity transport systems for citric acid in the yeast Candida utilis.

    PubMed Central

    Cássio, F; Leáo, C

    1991-01-01

    Citric acid-grown cells of the yeast Candida utilis induced two transport systems for citric acid, presumably a proton symport and a facilitated diffusion system for the charged and the undissociated forms of the acid, respectively. Both systems could be observed simultaneously when the transport was measured at 25 degrees C with labelled citric acid at pH 3.5 with the following kinetic parameters: for the low-affinity system, Vmax, 1.14 nmol of undissociated citric acid s-1 mg (dry weight) of cells-1, and Km, 0.59 mM undissociated acid; for the high-affinity system, Vmax, 0.38 nmol of citrate s-1 mg (dry weight) of cells-1, and Km, 0.056 mM citrate. At high pH values (above 5.0), the low-affinity system was absent or not measurable. The two transport systems exhibited different substrate specificities. Isocitric acid was a competitive inhibitor of citric acid for the high-affinity system, suggesting that these tricarboxylic acids used the same transport system, while aconitic, tricarballylic, trimesic, and hemimellitic acids were not competitive inhibitors. With respect to the low-affinity system, isocitric acid, L-lactic acid, and L-malic acid were competitive inhibitors, suggesting that all of these mono-, di-, and tricarboxylic acids used the same low-affinity transport system. The two transport systems were repressed by glucose, and as a consequence diauxic growth was observed. Both systems were inducible, and not only citric acid but also lactic acid and malic acid may induce those transport systems. The induction of both systems was not dependent on the relative concentration of the anionic form(s) and of undissociated citric acid in the culture medium.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1664712

  17. The Long and Winding Road: from the high affinity choline uptake site to clinical trials for malignant brain tumors

    PubMed Central

    Lowenstein, Pedro R.; Castro, Maria G.

    2016-01-01

    Malignant brain tumors are one of the most lethal cancers. They originate from glial cells and invade throughout the brain. Current standard of care involves surgical resection, radiotherapy and chemotherapy, and median survival is currently ~14–20 months post-diagnosis. Glioma tumors are highly infiltrative. Given that the brain immune system is deficient in priming systemic immune responses to glioma antigens present within the brain, we proposed to reconstitute the brain immune system to achieve immunological priming from within the brain. Two adenoviral vectors are injected into the resection cavity or remaining tumor. One adenoviral vector expresses the HSV-1 derived thymidine kinase which converts ganciclovir into a compound only cytotoxic to dividing glioma cells. The second adenovirus expresses the cytokine fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L differentiates precursors into dendritic cells and acts as a chemokine for dendritic cells. HSV-1/ganciclovir killing of tumor cells releases tumor antigens that are taken up by dendritic cells within the brain tumor microenvironment. Tumor killing also releases HMGB1, a TLR2 agonist that activates dendritic cells. HMGB1 activated dendritic cells, loaded with glioma antigens, migrate to cervical lymph nodes to stimulate a systemic CD8+ T cells cytotoxic immune response against glioma. This immune response is specific to glioma tumors, induces immunological memory, and does neither cause brain toxicity nor autoimmune responses. An IND was granted by the FDA on 4/7/2011. A Phase I, first in person, to test whether re-engineering the brain immune system is potentially therapeutic is ongoing. PMID:27288077

  18. Best approximation of functions in L{sub p} by polynomials on affine system

    SciTech Connect

    Terekhin, Pavel A

    2011-02-28

    Estimates of the best L{sub p}-approximation of functions by polynomials in an affine system (system of dilations and translations), which are similar to well-known estimates due to Ul'yanov and Golubov for approximations in the Haar system, are obtained. An analogue of A.F. Timan and M.F. Timan's inequality is shown to hold under certain conditions on the generating function of the affine system; this analogue fails for the Haar system for 1

  19. Dual-tagging system for the affinity purification of mammalian protein complexes

    SciTech Connect

    Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory {Greg} B; Huang, Ying; Wu, Jun; Liu, Yie; Wang, Yisong

    2007-01-01

    Although affinity purification coupled with mass spectrometry (MS) provides a powerful tool to study protein-protein interactions, this strategy has encountered numerous difficulties when adapted to mammalian cells. Here we describe a Gateway{reg_sign}-compatible dual-tag affinity purification system that integrates regulatable expression, tetracysteine motifs, and various combinations of affinity tags to facilitate the cloning, detection, and purification of bait proteins and their interacting partners. Utilizing the human telomere binding protein TRF2 as a benchmark, we demonstrate bait protein recoveries upwards of approximately 16% from as little as 1-7 x 10{sup 7} cells and successfully identify known TRF2 interacting proteins, suggesting that our dual-tag affinity purification approach is a capable new tool for expanding the capacity to explore mammalian proteomic networks.

  20. Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps.

    PubMed

    Scherzer, Sönke; Böhm, Jennifer; Krol, Elzbieta; Shabala, Lana; Kreuzer, Ines; Larisch, Christina; Bemm, Felix; Al-Rasheid, Khaled A S; Shabala, Sergey; Rennenberg, Heinz; Neher, Erwin; Hedrich, Rainer

    2015-06-09

    The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K(+) uptake system in the Venus flytrap. In search of K(+) transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K(+)-transporter genes into Xenopus oocytes, however, both putative K(+) transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K(+) transporter 1 (AKT1), we coexpressed the putative K(+) transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K(+) uptake. DmKT1 was found to be a K(+)-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around -120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K(+), reducing its concentration from millimolar levels down to trace levels.

  1. Calcium sensor kinase activates potassium uptake systems in gland cells of Venus flytraps

    PubMed Central

    Scherzer, Sönke; Böhm, Jennifer; Krol, Elzbieta; Shabala, Lana; Kreuzer, Ines; Larisch, Christina; Bemm, Felix; Al-Rasheid, Khaled A. S.; Shabala, Sergey; Rennenberg, Heinz; Neher, Erwin; Hedrich, Rainer

    2015-01-01

    The Darwin plant Dionaea muscipula is able to grow on mineral-poor soil, because it gains essential nutrients from captured animal prey. Given that no nutrients remain in the trap when it opens after the consumption of an animal meal, we here asked the question of how Dionaea sequesters prey-derived potassium. We show that prey capture triggers expression of a K+ uptake system in the Venus flytrap. In search of K+ transporters endowed with adequate properties for this role, we screened a Dionaea expressed sequence tag (EST) database and identified DmKT1 and DmHAK5 as candidates. On insect and touch hormone stimulation, the number of transcripts of these transporters increased in flytraps. After cRNA injection of K+-transporter genes into Xenopus oocytes, however, both putative K+ transporters remained silent. Assuming that calcium sensor kinases are regulating Arabidopsis K+ transporter 1 (AKT1), we coexpressed the putative K+ transporters with a large set of kinases and identified the CBL9-CIPK23 pair as the major activating complex for both transporters in Dionaea K+ uptake. DmKT1 was found to be a K+-selective channel of voltage-dependent high capacity and low affinity, whereas DmHAK5 was identified as the first, to our knowledge, proton-driven, high-affinity potassium transporter with weak selectivity. When the Venus flytrap is processing its prey, the gland cell membrane potential is maintained around −120 mV, and the apoplast is acidified to pH 3. These conditions in the green stomach formed by the closed flytrap allow DmKT1 and DmHAK5 to acquire prey-derived K+, reducing its concentration from millimolar levels down to trace levels. PMID:25997445

  2. Optimising root system hydraulic architectures for water uptake

    NASA Astrophysics Data System (ADS)

    Meunier, Félicien; Couvreur, Valentin; Draye, Xavier; Javaux, Mathieu

    2015-04-01

    In this study we started from local hydraulic analysis of idealized root systems to develop a mathematical framework necessary for the understanding of global root systems behaviors. The underlying assumption of this study was that the plant is naturally optimised for the water uptake. The root system is thus a pipe network dedicated to the capture and transport of water. The main objective of the present research is to explain the fitness of major types of root architectures to their environment. In a first step, we developed links between local hydraulic properties and macroscopic parameters of (un)branched roots. The outcome of such an approach were functions of apparent conductance of entire root system and uptake distribution along the roots. We compared our development with some allometric scaling laws for the root water uptake: under the same simplifying assumptions we were able to obtain the same results and even to expand them to more physiological cases. Using empirical data of measured root conductance, we were also able to fit extremely well the data-set with this model. In a second stage we used generic architecture parameters and an existent root growth model to generate various types of root systems (from fibrous to tap). We combined both sides (hydraulic and architecture) then to maximize under a volume constraint either apparent conductance of root systems or the soil volume explored by active roots during the plant growth period. This approach has led to the sensitive parameters of the macroscopic parameters (conductance and location of the water uptake) of each single plant selected for this study. Scientific questions such as: "What is the optimal sowing density of a given hydraulic architecture ?" or "Which plant traits can we change to better explore the soil domain ?" can be also addressed with this approach: some potential applications are illustrated. The next (and ultimate phase) will be to validate our conclusions with real architectures

  3. Spatiotemporal variation of nitrate uptake kinetics within the maize (Zea mays L.) root system is associated with greater nitrate uptake and interactions with architectural phenes.

    PubMed

    York, Larry M; Silberbush, Moshe; Lynch, Jonathan P

    2016-06-01

    Increasing maize nitrogen acquisition efficiency is a major goal for the 21st century. Nitrate uptake kinetics (NUK) are defined by I max and K m, which denote the maximum uptake rate and the affinity of transporters, respectively. Because NUK have been studied predominantly at the molecular and whole-root system levels, little is known about the functional importance of NUK variation within root systems. A novel method was created to measure NUK of root segments that demonstrated variation in NUK among root classes (seminal, lateral, crown, and brace). I max varied among root class, plant age, and nitrate deprivation combinations, but was most affected by plant age, which increased I max, and nitrate deprivation time, which decreased I max K m was greatest for crown roots. The functional-structural simulation SimRoot was used for sensitivity analysis of plant growth to root segment I max and K m, as well as to test interactions of I max with root system architectural phenes. Simulated plant growth was more sensitive to I max than K m, and reached an asymptote near the maximum I max observed in the empirical studies. Increasing the I max of lateral roots had the largest effect on shoot growth. Additive effects of I max and architectural phenes on nitrate uptake were observed. Empirically, only lateral root tips aged 20 d operated at the maximum I max, and simulations demonstrated that increasing all seminal and lateral classes to this maximum rate could increase plant growth by as much as 26%. Therefore, optimizing I max for all maize root classes merits attention as a promising breeding goal.

  4. A tetra-layer microfluidic system for peptide affinity screening through integrated sample injection.

    PubMed

    Wang, Weizhi; Huang, Yanyan; Jin, Yulong; Liu, Guoquan; Chen, Yi; Ma, Huimin; Zhao, Rui

    2013-05-21

    A novel integrated microfluidic system was designed and fabricated for affinity peptide screening with in situ detection. A tetra-layer microfluidic hybrid chip containing two top eccentric diffluent layers, an inter-layer and a bottom screening layer, was developed as the core device of the system. The eccentric diffluent layers were ingeniously invented for the vertical sample delivery from 2 top-inlets into 12 bottom-inlets, which eliminated the use of excessive accessories and complicated pipelines currently used in other systems. By using six pH gradient generators, the magnetic bead-based screening in 36 parallel chambers was simultaneously carried out under 6 different pH conditions from 5.4 to 8.2. This allowed simultaneous screening of 6 compounds and each under 6 different pH conditions. The fabricated chip system was applied to screening of affinity peptides towards β-endorphin antibody. The affinities of the peptide ligands to the antibody were assessed by on-chip confocal detection. The results from the screening study using this system indicated that the pentapeptide with the sequence of YGGFL had the highest affinity towards β-endorphin antibody at pH 7.1, which was further confirmed by the ELISA assay using a 96-well plate format. This microfluidic screening system is automatic, low-cost and reusable for not only peptide screening but also other bioactive compounds screening towards target molecules.

  5. Novel affinity chromatographic system for the single-step purification of glycosaminoglycans from complex systems using volatile buffers.

    PubMed

    Hodson, B A; Pepper, D S; Dawes, J

    1991-04-19

    A new system for the isolation and purification of glycosaminoglycans (GAGs) and related molecules from complex systems such as plasma is described. Affinity chromatography which exploits the very high affinity between the polymeric base Polybrene and sulphated polysaccharides was used. A novel volatile buffer system composed of ammonium formate and formic acid, which allows the complete recovery of samples, was developed, and elution conditions were optimised for the separation and purification of GAGs of different charge densities. Using this system the losses associated with dialysis and desalting, frequently necessary preliminaries to further analysis, are avoided.

  6. A Lyapunov method for stability analysis of piecewise-affine systems over non-invariant domains

    NASA Astrophysics Data System (ADS)

    Rubagotti, Matteo; Zaccarian, Luca; Bemporad, Alberto

    2016-05-01

    This paper analyses stability of discrete-time piecewise-affine systems, defined on possibly non-invariant domains, taking into account the possible presence of multiple dynamics in each of the polytopic regions of the system. An algorithm based on linear programming is proposed, in order to prove exponential stability of the origin and to find a positively invariant estimate of its region of attraction. The results are based on the definition of a piecewise-affine Lyapunov function, which is in general discontinuous on the boundaries of the regions. The proposed method is proven to lead to feasible solutions in a broader range of cases as compared to a previously proposed approach. Two numerical examples are shown, among which a case where the proposed method is applied to a closed-loop system, to which model predictive control was applied without a-priori guarantee of stability.

  7. Piecewise affine models of chaotic attractors: The Rössler and Lorenz systems

    NASA Astrophysics Data System (ADS)

    Amaral, Gleison F. V.; Letellier, Christophe; Aguirre, Luis Antonio

    2006-03-01

    This paper proposes a procedure by which it is possible to synthesize Rössler [Phys. Lett. A 57, 397-398 (1976)] and Lorenz [J. Atmos. Sci. 20, 130-141 (1963)] dynamics by means of only two affine linear systems and an abrupt switching law. Comparison of different (valid) switching laws suggests that parameters of such a law behave as codimension one bifurcation parameters that can be changed to produce various dynamical regimes equivalent to those observed with the original systems. Topological analysis is used to characterize the resulting attractors and to compare them with the original attractors. The paper provides guidelines that are helpful to synthesize other chaotic dynamics by means of switching affine linear systems.

  8. Identification of multiple cellular uptake pathways of polystyrene nanoparticles and factors affecting the uptake: relevance for drug delivery systems.

    PubMed

    Firdessa, Rebuma; Oelschlaeger, Tobias A; Moll, Heidrun

    2014-01-01

    Nanoparticles may address challenges by human diseases through improving diagnosis, vaccination and treatment. The uptake mechanism regulates the type of threat a particle poses on the host cells and how a cell responds to it. Hence, understanding the uptake mechanisms and cellular interactions of nanoparticles at the cellular and subcellular level is a prerequisite for their effective biomedical applications. The present study shows the uptake mechanisms of polystyrene nanoparticles and factors affecting their uptake in bone marrow-derived macrophages, 293T kidney epithelial cells and L929 fibroblasts. Labeling with the endocytic marker FM4-64 and transmission electron microscopy studies show that the nanoparticles were internalized rapidly via endocytosis and accumulated in intracellular vesicles. Soon after their internalizations, nanoparticles trafficked to organelles with acidic pH. Analysis of the ultrastructural morphology of the plasma membrane invaginations or extravasations provides clear evidence for the involvement of several uptake routes in parallel to internalize a given type of nanoparticles by mammalian cells, highlighting the complexity of the nanoparticle-cell interactions. Blocking the specific endocytic pathways by different pharmacological inhibitors shows similar outcomes. The potential to take up nanoparticles varies highly among different cell types in a particle sizes-, time- and energy-dependent manner. Furthermore, infection and the activation status of bone marrow-derived macrophages significantly affect the uptake potential of the cells, indicating the need to understand the diseases' pathogenesis to establish effective and rational drug-delivery systems. This study enhances our understanding of the application of nanotechnology in biomedical sciences.

  9. The competitor-introduced Ggamma recruitment system, a new approach for screening affinity-enhanced proteins.

    PubMed

    Fukuda, Nobuo; Ishii, Jun; Tanaka, Tsutomu; Kondo, Akihiko

    2010-04-01

    We have developed a new approach based on the Ggamma recruitment system to screen affinity-enhanced proteins by expressing a binding competitor. The previously established Ggamma recruitment system is a yeast two-hybrid (Y2H) system that utilizes G-protein signaling, and is based on the fact that membrane localization of the G-protein gamma subunit (Ggamma) is essential for signal transduction in yeast. In the original Y2H system, an engineered Ggamma that lacks membrane localization upon deletion of the lipid modification site (Ggamma(cyto)) is produced, and a candidate protein with an artificial lipidation site and its counterpart fused with Ggamma(cyto) are expressed. As protein-protein interactions bring Ggamma(cyto) towards the plasma membrane, G-protein signaling can be activated, and the interaction is detected by various cellular responses as the readout. In the current study, we expressed a third cytosolic protein that competes with the candidate protein to specifically isolate affinity-enhanced mutants from a mutation library of the candidate protein. Enhancing the affinity of the protein candidate guides the counterpart-Ggamma(cyto) fusion protein towards the plasma membrane and activates signaling. Using mutants of the Z domain derived from Staphylococcus aureus protein A as candidate proteins or competitors, and the Fc portion of human immunoglobulin G (IgG) as the counterpart, we demonstrate that affinity-enhanced proteins can be effectively screened from a library containing a 10 000-fold excess of non-enhanced proteins. This new approach, called the competitor-introduced Ggamma recruitment system, will be useful for efficient discovery of rare valuable candidates hidden among excess ordinary ones.

  10. 21 CFR 862.1715 - Triiodothyronine uptake test system.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... albumin of serum and plasma. The device provides an indirect measurement of thyrkoxine levels in serum and plasma. Measurements of triiodothyronine uptake are used in the diagnosis and treatment of...

  11. 21 CFR 862.1715 - Triiodothyronine uptake test system.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... albumin of serum and plasma. The device provides an indirect measurement of thyrkoxine levels in serum and plasma. Measurements of triiodothyronine uptake are used in the diagnosis and treatment of...

  12. 21 CFR 862.1715 - Triiodothyronine uptake test system.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... albumin of serum and plasma. The device provides an indirect measurement of thyrkoxine levels in serum and plasma. Measurements of triiodothyronine uptake are used in the diagnosis and treatment of...

  13. 21 CFR 862.1715 - Triiodothyronine uptake test system.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... albumin of serum and plasma. The device provides an indirect measurement of thyrkoxine levels in serum and plasma. Measurements of triiodothyronine uptake are used in the diagnosis and treatment of...

  14. 21 CFR 862.1715 - Triiodothyronine uptake test system.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... albumin of serum and plasma. The device provides an indirect measurement of thyrkoxine levels in serum and plasma. Measurements of triiodothyronine uptake are used in the diagnosis and treatment of...

  15. Identification of piecewise affine systems based on fuzzy PCA-guided robust clustering technique

    NASA Astrophysics Data System (ADS)

    Khanmirza, Esmaeel; Nazarahari, Milad; Mousavi, Alireza

    2016-12-01

    Hybrid systems are a class of dynamical systems whose behaviors are based on the interaction between discrete and continuous dynamical behaviors. Since a general method for the analysis of hybrid systems is not available, some researchers have focused on specific types of hybrid systems. Piecewise affine (PWA) systems are one of the subsets of hybrid systems. The identification of PWA systems includes the estimation of the parameters of affine subsystems and the coefficients of the hyperplanes defining the partition of the state-input domain. In this paper, we have proposed a PWA identification approach based on a modified clustering technique. By using a fuzzy PCA-guided robust k-means clustering algorithm along with neighborhood outlier detection, the two main drawbacks of the well-known clustering algorithms, i.e., the poor initialization and the presence of outliers, are eliminated. Furthermore, this modified clustering technique enables us to determine the number of subsystems without any prior knowledge about system. In addition, applying the structure of the state-input domain, that is, considering the time sequence of input-output pairs, provides a more efficient clustering algorithm, which is the other novelty of this work. Finally, the proposed algorithm has been evaluated by parameter identification of an IGV servo actuator. Simulation together with experiment analysis has proved the effectiveness of the proposed method.

  16. Characterization of the ferrioxamine uptake system of Nitrosomonas europaea.

    PubMed

    Wei, Xueming; Sayavedra-Soto, Luis A; Arp, Daniel J

    2007-12-01

    The chemolithoautotroph Nitrosomonas europaea has two genes predicted to encode outer-membrane (OM) ferrioxamine transporters. Expression of the ferrioxamine uptake system required induction, as shown by the shorter lag phase in ferrioxamine-containing cultures when ferrioxamine-exposed cells were used as an inoculum. The two OM ferrioxamine siderophore transporters encoded by foxA(1) (NE1097) and foxA(2) (NE1088) were produced only in cells grown in Fe-limited ferrioxamine-containing medium. The inactivation of foxA(1), singly or in combination with foxA(2), prevented growth in Fe-limited medium containing excess desferrioxamine (DFX). The foxA(2)-disrupted single mutant grew poorly in the regular Fe-limited (0.2 microM) medium with 10 microM DFX, but grew well when the Fe level was raised to 1.0 microM with 10 microM DFX. For efficient acquisition of Fe-loaded ferrioxamine, N. europaea needs both ferrioxamine transporters FoxA(1) and FoxA(2). FoxA(1) probably regulates its own production, and it controls the production of FoxA(2) as well.

  17. Strains of the Harmful Cyanobacterium Microcystis aeruginosa Differ in Gene Expression and Activity of Inorganic Carbon Uptake Systems at Elevated CO2 Levels.

    PubMed

    Sandrini, Giovanni; Jakupovic, Dennis; Matthijs, Hans C P; Huisman, Jef

    2015-11-01

    Cyanobacteria are generally assumed to be effective competitors at low CO2 levels because of their efficient CO2-concentrating mechanism (CCM), and yet how bloom-forming cyanobacteria respond to rising CO2 concentrations is less clear. Here, we investigate changes in CCM gene expression at ambient CO2 (400 ppm) and elevated CO2 (1,100 ppm) in six strains of the harmful cyanobacterium Microcystis. All strains downregulated cmpA encoding the high-affinity bicarbonate uptake system BCT1, whereas both the low- and high-affinity CO2 uptake genes were expressed constitutively. Four strains downregulated the bicarbonate uptake genes bicA and/or sbtA, whereas two strains showed constitutive expression of the bicA-sbtA operon. In one of the latter strains, a transposon insert in bicA caused low bicA and sbtA transcript levels, which made this strain solely dependent on BCT1 for bicarbonate uptake. Activity measurements of the inorganic carbon (Ci) uptake systems confirmed the CCM gene expression results. Interestingly, genes encoding the RuBisCO enzyme, structural carboxysome components, and carbonic anhydrases were not regulated. Hence, Microcystis mainly regulates the initial uptake of inorganic carbon, which might be an effective strategy for a species experiencing strongly fluctuating Ci concentrations. Our results show that CCM gene regulation of Microcystis varies among strains. The observed genetic and phenotypic variation in CCM responses may offer an important template for natural selection, leading to major changes in the genetic composition of harmful cyanobacterial blooms at elevated CO2.

  18. Strains of the Harmful Cyanobacterium Microcystis aeruginosa Differ in Gene Expression and Activity of Inorganic Carbon Uptake Systems at Elevated CO2 Levels

    PubMed Central

    Sandrini, Giovanni; Jakupovic, Dennis; Matthijs, Hans C. P.

    2015-01-01

    Cyanobacteria are generally assumed to be effective competitors at low CO2 levels because of their efficient CO2-concentrating mechanism (CCM), and yet how bloom-forming cyanobacteria respond to rising CO2 concentrations is less clear. Here, we investigate changes in CCM gene expression at ambient CO2 (400 ppm) and elevated CO2 (1,100 ppm) in six strains of the harmful cyanobacterium Microcystis. All strains downregulated cmpA encoding the high-affinity bicarbonate uptake system BCT1, whereas both the low- and high-affinity CO2 uptake genes were expressed constitutively. Four strains downregulated the bicarbonate uptake genes bicA and/or sbtA, whereas two strains showed constitutive expression of the bicA-sbtA operon. In one of the latter strains, a transposon insert in bicA caused low bicA and sbtA transcript levels, which made this strain solely dependent on BCT1 for bicarbonate uptake. Activity measurements of the inorganic carbon (Ci) uptake systems confirmed the CCM gene expression results. Interestingly, genes encoding the RuBisCO enzyme, structural carboxysome components, and carbonic anhydrases were not regulated. Hence, Microcystis mainly regulates the initial uptake of inorganic carbon, which might be an effective strategy for a species experiencing strongly fluctuating Ci concentrations. Our results show that CCM gene regulation of Microcystis varies among strains. The observed genetic and phenotypic variation in CCM responses may offer an important template for natural selection, leading to major changes in the genetic composition of harmful cyanobacterial blooms at elevated CO2. PMID:26319871

  19. Affine Maps of the Polarization Vector for Quantum Systems of Arbitrary Dimension

    NASA Astrophysics Data System (ADS)

    Byrd, Mark; Bishop, C. Allen; Ou, Yong-Cheng

    2011-03-01

    The operator-sum decomposition (OS) of a mapping from one density matrix to another has many applications in quantum information science. To this mapping there corresponds an affine map which provides a geometric description of the density matrix in terms of the polarization vector representation. This has been thoroughly explored for qubits since the components of the polarization vector are measurable quantities (corresponding to expectation values of Hermitian operators) and also because it enables the description of map domains geometrically. Here we extend the OS-affine map correspondence to qudits, briefly discuss general properties of the map, the form for particular important cases, and provide several explicit results for qutrit maps. We use the affine map and a singular-value-like decomposition, to find positivity constraints that provide a symmetry for small polarization vector magnitudes (states which are closer to the maximally mixed state) which is broken as the polarization vector increases in magnitude (a state becomes more pure). The dependence of this symmetry on the magnitude of the polarization vector implies the polar decomposition of the map can not be used as it can for the qubit case. However, it still leads us to a connection between positivity and purity for general d-state systems. This material is based upon work supported by NSF-Grant No. 0545798 to MSB.

  20. Functional analysis of [methyl-(3)H]choline uptake in glioblastoma cells: Influence of anti-cancer and central nervous system drugs.

    PubMed

    Taguchi, Chiaki; Inazu, Masato; Saiki, Iwao; Yara, Miki; Hara, Naomi; Yamanaka, Tsuyoshi; Uchino, Hiroyuki

    2014-04-01

    Positron emission tomography (PET) and PET/computed tomography (PET-CT) studies with (11)C- or (18)F-labeled choline derivatives are used for PET imaging in glioblastoma patients. However, the nature of the choline transport system in glioblastoma is poorly understood. In this study, we performed a functional characterization of [methyl-(3)H]choline uptake and sought to identify the transporters that mediate choline uptake in the human glioblastoma cell lines A-172 and U-251MG. In addition, we examined the influence of anti-cancer drugs and central nervous system drugs on the transport of [methyl-(3)H]choline. High- and low-affinity choline transport systems were present in A-172 cells, U-251MG cells and astrocytes, and these were Na(+)-independent and pH-dependent. Cell viability in A-172 cells was not affected by choline deficiency. However, cell viability in U-251MG cells was significantly inhibited by choline deficiency. Both A-172 and U-251MG cells have two different choline transporters, choline transporter-like protein 1 (CTL1) and CTL2. In A-172 cells, CTL1 is predominantly expressed, whereas in U-251MG cells, CTL2 is predominantly expressed. Treatment with anti-cancer drugs such as cisplatin, etoposide and vincristine influenced [methyl-(3)H]choline uptake in U-251MG cells, but not A-172 cells. Central nervous system drugs such as imipramine, fluvoxamine, paroxetine, reboxetine, citalopram and donepezil did not affect cell viability or [methyl-(3)H]choline uptake. The data presented here suggest that CTL1 and CTL2 are functionally expressed in A-172 and U-251MG cells and are responsible for [methyl-(3)H]choline uptake that relies on a directed H(+) gradient as a driving force. Furthermore, while anti-cancer drugs altered [methyl-(3)H]choline uptake, central nervous system drugs did not affect [methyl-(3)H]choline uptake.

  1. The high-affinity immunoglobulin E receptor (FcepsilonRI) regulates mitochondrial calcium uptake and a dihydropyridine receptor-mediated calcium influx in mast cells: Role of the FcepsilonRIbeta chain immunoreceptor tyrosine-based activation motif.

    PubMed

    Suzuki, Yoshihiro; Yoshimaru, Tetsuro; Inoue, Toshio; Nunomura, Satoshi; Ra, Chisei

    2008-04-01

    A growing body of evidence suggests that mitochondria take up calcium upon receptor (agonist) stimulation and that this contributes to the dynamics of spatiotemporal calcium signaling. We have previously shown that engagement of the high-affinity receptor for immunoglobulin E (FcepsilonRI) stimulates mitochondrial calcium ([Ca2+]m) uptake in mast cells. The present study was undertaken to investigate the mechanisms and biological significance of FcepsilonRI regulation of [Ca2+]m. Antigen stimulated [Ca2+]m uptake in a dose-dependent manner with a minimal effective dose of 0.03-3 ng/ml. This [Ca2+]m uptake took place immediately, reaching its peak within minutes and was inhibited by the src family kinase inhibitor PP1 and phosphatidylinositol-3-kinase inhibitor wortmannin. Analyses using mast cells expressing the wild-type or the mutated type of the FcepsilonRIbeta immunoreceptor tyrosine-based activation motif (ITAM) in which all tyrosine residues were replaced by phenylalanine revealed that the FcepsilonRIbeta ITAM is essential for a sustained [Ca2+]m uptake. The FcepsilonRIbeta ITAM was essential for overall calcium response upon weak FcepsilonRI stimulation (at low antigen concentration), while upon strong stimulation (at high antigen concentration) it appeared necessary selectively to an immediate calcium response that was sensitive to the dihydropyridine receptor (DHPR) antagonist nifedipine and wortmannin but not to the store-operated calcium entry (SOCE) antagonists such as 2-aminoethoxyphenyl borate and SK&F96365. These data demonstrate that the FcepsilonRIbeta regulates [Ca2+]m uptake in mast cells via the ITAM and suggest that this plays a key role in regulating calcium influx especially that induced via a DHPR-mediated calcium channel.

  2. Gallium-67 uptake by the thyroid associated with progressive systemic sclerosis

    SciTech Connect

    Sjoberg, R.J.; Blue, P.W.; Kidd, G.S.

    1989-01-01

    Although thyroidal uptake of gallium-67 has been described in several thyroid disorders, gallium-67 scanning is not commonly used in the evaluation of thyroid disease. Thyroidal gallium-67 uptake has been reported to occur frequently with subacute thyroiditis, anaplastic thyroid carcinoma, and thyroid lymphoma, and occasionally with Hashimoto's thyroiditis and follicular thyroid carcinoma. A patient is described with progressive systemic sclerosis who, while being scanned for possible active pulmonary involvement, was found incidentally to have abnormal gallium-67 uptake only in the thyroid gland. Fine needle aspiration cytology of the thyroid revealed Hashimoto's thyroiditis. Although Hashimoto's thyroiditis occurs with increased frequency in patients with progressive systemic sclerosis, thyroidal uptake of gallium-67 associated with progressive systemic sclerosis has not, to our knowledge, been previously described. Since aggressive thyroid malignancies frequently are imaged by gallium-67 scintigraphy, fine needle aspiration cytology of the thyroid often is essential in the evaluation of thyroidal gallium-67 uptake.

  3. Studies on Arabidopsis athak5, atakt1 double mutants disclose the range of concentrations at which AtHAK5, AtAKT1 and unknown systems mediate K uptake.

    PubMed

    Rubio, Francisco; Alemán, Fernando; Nieves-Cordones, Manuel; Martínez, Vicente

    2010-06-01

    The high-affinity K(+) transporter AtHAK5 and the inward-rectifier K(+) channel AtAKT1 have been described to contribute to K(+) uptake in Arabidopsis thaliana. Studies with T-DNA insertion lines showed that both systems participate in the high-affinity range of concentrations and only AtAKT1 in the low-affinity range. However the contribution of other systems could not be excluded with the information and plant material available. The results presented here with a double knock-out athak5, atakt1 mutant show that AtHAK5 is the only system mediating K(+) uptake at concentrations below 0.01 mM. In the range between 0.01 and 0.05 mM K(+) AtHAK5 and AtAKT1 are the only contributors to K(+) acquisition. At higher K(+) concentrations, unknown systems come into operation and participate together with AtAKT1 in low-affinity K(+) uptake. These systems can supply sufficient K(+) to promote plant growth even in the absence of AtAKT1 or in the presence of 10 mM K(+) where AtAKT1 is not essential.

  4. Enzyme- and affinity biomolecule-mediated polymerization systems for biological signal amplification and cell screening.

    PubMed

    Malinowska, Klara H; Nash, Michael A

    2016-06-01

    Enzyme-mediated polymerization and polymerization-based signal amplification have emerged as two closely related techniques that are broadly applicable in the nanobio sciences. We review recent progress on polymerization systems mediated by biological molecules (e.g., affinity molecules and enzymes), and highlight newly developed formats and configurations of these systems to perform such tasks as non-instrumented biodetection, synthesis of core-shell nanomaterials, isolation of rare cells, and high-throughput screening. We discuss useful features of biologically mediated polymerization systems, such as multiple mechanisms of amplification (e.g., enzymatic, radical chain propagation), and the ability to localize structures at interfaces and at cell surfaces with microscopic spatial confinement. We close with a perspective on desirable improvements that need to be addressed to adapt these molecular systems to future applications.

  5. Genetic diversity of inorganic carbon uptake systems causes variation in CO2 response of the cyanobacterium Microcystis.

    PubMed

    Sandrini, Giovanni; Matthijs, Hans C P; Verspagen, Jolanda M H; Muyzer, Gerard; Huisman, Jef

    2014-03-01

    Rising CO2 levels may act as an important selective factor on the CO2-concentrating mechanism (CCM) of cyanobacteria. We investigated genetic diversity in the CCM of Microcystis aeruginosa, a species producing harmful cyanobacterial blooms in many lakes worldwide. All 20 investigated Microcystis strains contained complete genes for two CO2 uptake systems, the ATP-dependent bicarbonate uptake system BCT1 and several carbonic anhydrases (CAs). However, 12 strains lacked either the high-flux bicarbonate transporter BicA or the high-affinity bicarbonate transporter SbtA. Both genes, bicA and sbtA, were located on the same operon, and the expression of this operon is most likely regulated by an additional LysR-type transcriptional regulator (CcmR2). Strains with only a small bicA fragment clustered together in the phylogenetic tree of sbtAB, and the bicA fragments were similar in strains isolated from different continents. This indicates that a common ancestor may first have lost most of its bicA gene and subsequently spread over the world. Growth experiments showed that strains with sbtA performed better at low inorganic carbon (Ci) conditions, whereas strains with bicA performed better at high Ci conditions. This offers an alternative explanation of previous competition experiments, as our results reveal that the competition at low CO2 levels was won by a specialist with only sbtA, whereas a generalist with both bicA and sbtA won at high CO2 levels. Hence, genetic and phenotypic variation in Ci uptake systems provide Microcystis with the potential for microevolutionary adaptation to changing CO2 conditions, with a selective advantage for bicA-containing strains in a high-CO2 world.

  6. Identification of high- and low-affinity NGF receptors during development of the chicken central nervous system

    SciTech Connect

    Escandon, E.; Chao, M.V. )

    1990-12-01

    In order to study regulation of the nerve growth factor (NGF) receptor during embryogenesis in chick brain, we have used affinity crosslinking of tissues with {sup 125}I-NGF. NGF interacts with high- and low-affinity receptors; high-affinity receptors are required for the majority of NGF's actions. Most measurements of receptor levels do not distinguish between high- and low-affinity forms of the receptor. We have used the lipophilic crosslinking agent HSAB to identify the high-affinity, functional receptor during development of the chicken central nervous system. A peak of expression during Embryonic Days 5-10 was detected in all regions of the chicken central nervous system, but, shortly after birth, only the cerebellar region displays significant levels of NGF receptor protein. The time course of expression confirms the dramatic regulation of the NGF receptor gene during defined embryonic periods. The detection of high-affinity NGF receptors in brain and neural retina provides strong evidence that NGF is involved in essential ontogenetic events in the development of the chicken central nervous system.

  7. Ferrioxamine B analogues: targeting the FoxA uptake system in the pathogenic Yersinia enterocolitica.

    PubMed

    Kornreich-Leshem, Hagit; Ziv, Carmit; Gumienna-Kontecka, Elzbieta; Arad-Yellin, Rina; Chen, Yona; Elhabiri, Mourad; Albrecht-Gary, Anne-Marie; Hadar, Yitzhak; Shanzer, Abraham

    2005-02-02

    A series of ferrioxamine B analogues that target the bacterium Yersinia enterocolitica were prepared. These iron carriers are composed of three hydroxamate-containing monomeric units. Two identical monomers consist of N-hydroxy-3-aminopropionic acid coupled with beta-alanine, and a third unit at the amino terminal is composed of N-hydroxy-3-aminopropionic acid and one of the following amino acids: beta-alanine (1a), phenylalanine (1b), cyclohexylalanine (1c), or glycine (1d). Thermodynamic results for representatives of the analogues have shown a strong destabilization (3-4 orders of magnitude) of the ferric complexes with respect to ferrioxamine B, probably due to shorter spacers and a more strained structure around the metal center. No significant effect of the variations at the N-terminal has been observed on the stability of the ferric complexes. By contrast, using in vivo radioactive uptake experiments, we have found that these modifications have a substantial effect on the mechanism of iron(III) uptake in the pathogenic bacteria Yersinia enterocolitica. Analogues 1a and 1d were utilized by the ferrioxamine B uptake system (FoxA), while 1b and 1c either used different uptake systems or were transported to the microbial cell nonspecifically by diffusion via the cell membrane. Transport via the FoxA system was also confirmed by uptake experiments with the FoxA deficient strain of Yersinia enterocolitica. A fluorescent marker, attached to 1a in a way that did not interfere with its biological activity, provided additional means to monitor the uptake mechanism by fluorescence techniques. Of particular interest is the observation that 1a was utilized by the uptake system of ferrioxamine B in Yersinia enterocolitica (FoxA) but failed to use the ferrioxamine uptake route in Pseudomonas putida. Here, we present a case in which biomimetic siderophore analogues deliberately designed for a particular bacterium can distinguish between related uptake systems of different

  8. Bacteria in an intense competition for iron: Key component of the Campylobacter jejuni iron uptake system scavenges enterobactin hydrolysis product

    PubMed Central

    Raines, Daniel J.; Moroz, Olga V.; Blagova, Elena V.; Turkenburg, Johan P.; Wilson, Keith S.

    2016-01-01

    To acquire essential Fe(III), bacteria produce and secrete siderophores with high affinity and selectivity for Fe(III) to mediate its uptake into the cell. Here, we show that the periplasmic binding protein CeuE of Campylobacter jejuni, which was previously thought to bind the Fe(III) complex of the hexadentate siderophore enterobactin (Kd ∼ 0.4 ± 0.1 µM), preferentially binds the Fe(III) complex of the tetradentate enterobactin hydrolysis product bis(2,3-dihydroxybenzoyl-l-Ser) (H5-bisDHBS) (Kd = 10.1 ± 3.8 nM). The protein selects Λ-configured [Fe(bisDHBS)]2− from a pool of diastereomeric Fe(III)-bisDHBS species that includes complexes with metal-to-ligand ratios of 1:1 and 2:3. Cocrystal structures show that, in addition to electrostatic interactions and hydrogen bonding, [Fe(bisDHBS)]2− binds through coordination of His227 and Tyr288 to the iron center. Similar binding is observed for the Fe(III) complex of the bidentate hydrolysis product 2,3-dihydroxybenzoyl-l-Ser, [Fe(monoDHBS)2]3−. The mutation of His227 and Tyr288 to noncoordinating residues (H227L/Y288F) resulted in a substantial loss of affinity for [Fe(bisDHBS)]2− (Kd ∼ 0.5 ± 0.2 µM). These results suggest a previously unidentified role for CeuE within the Fe(III) uptake system of C. jejuni, provide a molecular-level understanding of the underlying binding pocket adaptations, and rationalize reports on the use of enterobactin hydrolysis products by C. jejuni, Vibrio cholerae, and other bacteria with homologous periplasmic binding proteins. PMID:27162326

  9. Bacteria in an intense competition for iron: Key component of the Campylobacter jejuni iron uptake system scavenges enterobactin hydrolysis product.

    PubMed

    Raines, Daniel J; Moroz, Olga V; Blagova, Elena V; Turkenburg, Johan P; Wilson, Keith S; Duhme-Klair, Anne-K

    2016-05-24

    To acquire essential Fe(III), bacteria produce and secrete siderophores with high affinity and selectivity for Fe(III) to mediate its uptake into the cell. Here, we show that the periplasmic binding protein CeuE of Campylobacter jejuni, which was previously thought to bind the Fe(III) complex of the hexadentate siderophore enterobactin (Kd ∼ 0.4 ± 0.1 µM), preferentially binds the Fe(III) complex of the tetradentate enterobactin hydrolysis product bis(2,3-dihydroxybenzoyl-l-Ser) (H5-bisDHBS) (Kd = 10.1 ± 3.8 nM). The protein selects Λ-configured [Fe(bisDHBS)](2-) from a pool of diastereomeric Fe(III)-bisDHBS species that includes complexes with metal-to-ligand ratios of 1:1 and 2:3. Cocrystal structures show that, in addition to electrostatic interactions and hydrogen bonding, [Fe(bisDHBS)](2-) binds through coordination of His227 and Tyr288 to the iron center. Similar binding is observed for the Fe(III) complex of the bidentate hydrolysis product 2,3-dihydroxybenzoyl-l-Ser, [Fe(monoDHBS)2](3-) The mutation of His227 and Tyr288 to noncoordinating residues (H227L/Y288F) resulted in a substantial loss of affinity for [Fe(bisDHBS)](2-) (Kd ∼ 0.5 ± 0.2 µM). These results suggest a previously unidentified role for CeuE within the Fe(III) uptake system of C. jejuni, provide a molecular-level understanding of the underlying binding pocket adaptations, and rationalize reports on the use of enterobactin hydrolysis products by C. jejuni, Vibrio cholerae, and other bacteria with homologous periplasmic binding proteins.

  10. Comparison of monoclonal antibodies 17-1A and 323/A3: the influence of the affinity on tumour uptake and efficacy of radioimmunotherapy in human ovarian cancer xenografts.

    PubMed Central

    Kievit, E.; Pinedo, H. M.; Schlüper, H. M.; Haisma, H. J.; Boven, E.

    1996-01-01

    The low-affinity monoclonal antibody (MAb) chimeric 17-1A(c-17-1A) and the high-affinity MAb mouse 323/A3 (m-323/A3) were used to study the effect of the MAb affinity on the tumour uptake and efficacy of radioimmunotherapy in nude mice bearing subcutaneously the human ovarian cancer xenografts FMa, OVCAR-3 and Ov.Pe. Both MAbs are directed against the same pancarcinoma glycoprotein. In vitro, the number of binding sites on tumour cells at 4 degrees C was similar for both MAbs, but m-323/A3 had an approximately 5-fold higher affinity (1.3-3.0x10(9) M-1) than c-17-1A (3.0-5.4x10(8) M-1). This difference in affinity was more extreme at 37 degrees C, when no binding of c-17-1A could be observed. MAb m-323/A3 completely blocked binding of c-17-1A to tumour cells, whereas the reverse was not observed. Immunohistochemistry showed a similar but more intense staining pattern of m-323/A3 in human ovarian cancer xenografts than of c-17-1A. In vivo, the blood clearance in non-tumour-bearing nude mice was similar for both MAbs with terminal half-lives of 71.4 h for m-323/A3 and 62.7 h for c-17-1A. MAb m-323/A3 targeted better to tumour tissue, but was more heterogeneously distributed than c-17-1A. The cumulative absorbed radiation dose delivered by m-323/A3 to tumour tissue was 2.5- to 4.7-fold higher than that delivered by c-17-1A. When mice were treated with equivalent radiation doses of 131(I)m-323/A3 and 131(I)c-17-1A, based on a correction for the immunoreactivity of the radiolabelled MAbs, m-323/A3 induced a better growth inhibition in two of the three xenografts. When the radiation doses were adjusted to obtain a similar amount of radiation in the tumour c-17-1A was more effective in tumour growth inhibition in all three xenografts. Images Figure 3 Figure 4 PMID:8595159

  11. Optimality of affine control system of several species in competition on a sequential batch reactor

    NASA Astrophysics Data System (ADS)

    Rodríguez, J. C.; Ramírez, H.; Gajardo, P.; Rapaport, A.

    2014-09-01

    In this paper, we analyse the optimality of affine control system of several species in competition for a single substrate on a sequential batch reactor, with the objective being to reach a given (low) level of the substrate. We allow controls to be bounded measurable functions of time plus possible impulses. A suitable modification of the dynamics leads to a slightly different optimal control problem, without impulsive controls, for which we apply different optimality conditions derived from Pontryagin principle and the Hamilton-Jacobi-Bellman equation. We thus characterise the singular trajectories of our problem as the extremal trajectories keeping the substrate at a constant level. We also establish conditions for which an immediate one impulse (IOI) strategy is optimal. Some numerical experiences are then included in order to illustrate our study and show that those conditions are also necessary to ensure the optimality of the IOI strategy.

  12. Results of a combined model of root system growth and soil water uptake: evaluating the significance of root system architecture to plant water uptake

    NASA Astrophysics Data System (ADS)

    Bouda, M.; Saiers, J. E.

    2012-12-01

    Root system hydraulic architecture is a key determinant of plants' ability to withdraw water from the soil, satisfying transpirational demand. Presently, the representation of this component of the hydrological cycle in large-scale models is generally very simplistic, even though transpiration accounts for much of the terrestrial heat and water surface fluxes, and exercises control over photosynthetic uptake of CO2. In order to address this gap, we have developed a modelling approach that relies on several components. The first is RootGrow, original MATLAB code that simulates the stochastic growth of a root system as a function of an intrinsic set of parameters as well as its environment. We ran RootGrow coupled to the second component, a finite-element 3D simulation of the physics of water transport in the soil and root system using COMSOL, resulting in a combined model of root system development and water uptake. Model results show that root system architecture can affect water uptake by two separate mechanisms: (a) root system geometry determines the distribution of absorbing surface area throughout the soil domain, and (b) root system topology affects the water potential at the absorbing surfaces. In this study we sample the model's parameter space to demonstrate over what ranges of physically meaningful parameters (including hydraulic conductivity of plant tissues, soil type, and soil moisture level) these mechanisms significantly affect root systems' water withdrawal rate. The two mechanisms identified and our quantitative results will form the basis of a third component in this approach: developing simple analytical relationships characterising water uptake as a function of root system architecture that can be used in Ecosystem Demography Model v2.1 (ED2), a large-scale Dynamic Vegetation Model, based on a method of upscaling individual-based models of plant ecology.

  13. Integral reinforcement learning for continuous-time input-affine nonlinear systems with simultaneous invariant explorations.

    PubMed

    Lee, Jae Young; Park, Jin Bae; Choi, Yoon Ho

    2015-05-01

    This paper focuses on a class of reinforcement learning (RL) algorithms, named integral RL (I-RL), that solve continuous-time (CT) nonlinear optimal control problems with input-affine system dynamics. First, we extend the concepts of exploration, integral temporal difference, and invariant admissibility to the target CT nonlinear system that is governed by a control policy plus a probing signal called an exploration. Then, we show input-to-state stability (ISS) and invariant admissibility of the closed-loop systems with the policies generated by integral policy iteration (I-PI) or invariantly admissible PI (IA-PI) method. Based on these, three online I-RL algorithms named explorized I-PI and integral Q -learning I, II are proposed, all of which generate the same convergent sequences as I-PI and IA-PI under the required excitation condition on the exploration. All the proposed methods are partially or completely model free, and can simultaneously explore the state space in a stable manner during the online learning processes. ISS, invariant admissibility, and convergence properties of the proposed methods are also investigated, and related with these, we show the design principles of the exploration for safe learning. Neural-network-based implementation methods for the proposed schemes are also presented in this paper. Finally, several numerical simulations are carried out to verify the effectiveness of the proposed methods.

  14. Role of Iron Uptake Systems in Pseudomonas aeruginosa Virulence and Airway Infection

    PubMed Central

    Minandri, Fabrizia; Imperi, Francesco; Frangipani, Emanuela; Bonchi, Carlo; Visaggio, Daniela; Facchini, Marcella; Pasquali, Paolo; Bragonzi, Alessandra

    2016-01-01

    Pseudomonas aeruginosa is a leading cause of hospital-acquired pneumonia and chronic lung infections in cystic fibrosis patients. Iron is essential for bacterial growth, and P. aeruginosa expresses multiple iron uptake systems, whose role in lung infection deserves further investigation. P. aeruginosa Fe3+ uptake systems include the pyoverdine and pyochelin siderophores and two systems for heme uptake, all of which are dependent on the TonB energy transducer. P. aeruginosa also has the FeoB transporter for Fe2+ acquisition. To assess the roles of individual iron uptake systems in P. aeruginosa lung infection, single and double deletion mutants were generated in P. aeruginosa PAO1 and characterized in vitro, using iron-poor media and human serum, and in vivo, using a mouse model of lung infection. The iron uptake-null mutant (tonB1 feoB) and the Fe3+ transport mutant (tonB1) did not grow aerobically under low-iron conditions and were avirulent in the mouse model. Conversely, the wild type and the feoB, hasR phuR (heme uptake), and pchD (pyochelin) mutants grew in vitro and caused 60 to 90% mortality in mice. The pyoverdine mutant (pvdA) and the siderophore-null mutant (pvdA pchD) grew aerobically in iron-poor media but not in human serum, and they caused low mortality in mice (10 to 20%). To differentiate the roles of pyoverdine in iron uptake and virulence regulation, a pvdA fpvR double mutant defective in pyoverdine production but expressing wild-type levels of pyoverdine-regulated virulence factors was generated. Deletion of fpvR in the pvdA background partially restored the lethal phenotype, indicating that pyoverdine contributes to the pathogenesis of P. aeruginosa lung infection by combining iron transport and virulence-inducing capabilities. PMID:27271740

  15. Functional analysis of the ferric uptake requlator gene, fur, in Xanthomonas vesicatoria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Iron is essential for the growth and survival of many organisms. Intracellular iron homeostasis must be maintained for cell survival and protection against iron toxicity. The ferric uptake regulator protein (Fur) regulates the high-affinity ferric uptake system in many bacteria. To investigate the f...

  16. How to market an affiliation. St. Elizabeth Hospital and Mercy Medical Center affiliate to create Affinity Health System.

    PubMed

    1996-01-01

    When Wisconsin's St. Elizabeth Hospital and Mercy Medical Center affiliated to create Affinity Health System, Inc., strategic planning and a solid marketing plan carefully executed were instrumental in its success. A corporate identity campaign and product line identification were follow-up phases to the merger approval.

  17. An Inexpensive Electrode and Cell for Measurement of Oxygen Uptake in Chemical and Biochemical Systems.

    ERIC Educational Resources Information Center

    Brunet, Juan E.; And Others

    1983-01-01

    The continuous measurement of oxygen consumption in an enzymatic reaction is a frequent experimental fact and extremely important in the enzymatic activity of oxygenase. An electrochemical system, based on a polarographic method, has been developed to monitor the oxygen uptake. The system developed and electrode used are described. (JN)

  18. Cadmium uptake by cocoa trees in agroforestry and monoculture systems under conventional and organic management.

    PubMed

    Gramlich, A; Tandy, S; Andres, C; Chincheros Paniagua, J; Armengot, L; Schneider, M; Schulin, R

    2017-02-15

    Cadmium (Cd) uptake by cocoa has recently attracted attention, after the European Union (EU) decided to establish values for tolerable Cd concentrations in cocoa products. Bean Cd concentrations from some cocoa provenances, especially from Latin America, were found to exceed these values. Cadmium uptake by cocoa is expected not only to depend on a variety of soil factors, but also on plant and management factors. In this study, we investigated the influence of different production systems on Cd uptake by cocoa in a long-term field trial in the Alto Beni Region of Bolivia, where cocoa trees are grown in monocultures and in agroforestry systems, both under organic and conventional management. Leaf, fruits and roots of two cultivars were sampled from each production system along with soil samples collected around these trees. Leaf, pod husk and bean samples were analysed for Cd, iron (Fe) and zinc (Zn), the roots for mycorrhizal abundance and the soil samples for 'total' and 'available' Cd, Fe and Zn as well as DGT-available Cd and Zn, pH, organic matter, texture, 'available' phosphorus (P) and potassium (K). Only a small part of the variance in bean and pod husk Cd was explained by management, soil and plant factors. Furthermore, the production systems and cultivars alone had no significant influence on leaf Cd. However, we found lower Cd leaf contents in agroforestry systems than in monocultures when analysed in combination with DGT-available soil Cd, cocoa cultivar and soil organic matter. Overall, this model explained 60% of the variance of the leaf Cd concentrations. We explain lower leaf Cd concentrations in agroforestry systems by competition for Cd uptake with other plants. The cultivar effect may be explained by cultivar specific uptake capacities or by a growth effect translating into different uptake rates, as the cultivars were of different size.

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

    PubMed Central

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

    1996-01-01

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

  20. Alterations in dopamine and serotonin uptake systems in the striatum of the weaver mutant mouse.

    PubMed

    Stotz, E H; Palacios, J M; Landwehrmeyer, B; Norton, J; Ghetti, B; Simon, J R; Triarhou, L C

    1994-01-01

    In the striatum of the homozygous weaver mutant mouse (wv/wv), dopamine content, uptake and tyrosine hydroxylase activity are decreased compared to wild-type (+/+) mice. In mice heterozygous for the weaver gene (wv/+), these dopaminergic parameters exhibit only minor reductions compared to +/+ mice. The wv/wv striatum has recently been shown to have an increase in serotonin content. In the present study, the serotonin uptake system of the weaver striatum was investigated. Synaptosomal uptake of [3H] serotonin was determined in the dorsal portion of wv/wv and +/+ striatum, and serotonin uptake sites were examined by the binding of [3H] citalopram in the striatum of wv/wv, wv/+ and +/+ mice. The dopamine uptake system was also investigated in all three genotypes via the binding of [3H] mazindol. Synaptosomal uptake of [3H] serotonin was increased by 79% in the dorsal portion of the wv/wv striatum compared to that seen in the +/+ striatum. The binding of [3H] citalopram was increased by 62% in the dorsolateral and by 111% in the dorsomedial portions of the wv/wv striatum compared to +/+. [3H] Citalopram binding in the wv/+ striatum was also higher than +/+, but this increase did not reach statistical significance. Within the wv/wv striatum, [3H] mazindol binding was almost completely absent (88-89% reduction) in the dorsal portion and severely reduced in the other striatal areas. These data support the notion that the dorsal portion of the wv/wv striatum, which has the severest reduction in dopamine uptake, is hyperinnervated by serotonin fibers.

  1. Partial purification of the 5-hydroxytryptophan-reuptake system from human blood platelets using a citalopram-derived affinity resin

    SciTech Connect

    Biessen, E.A.L; Horn, A.S.; Robillard, G.T. )

    1990-04-03

    This paper describes a procedure for the synthesis and application of a citalopram-derived affinity resin in purifying the 5HT-reuptake system from human blood platelets. A two-step scheme has been developed for partial purification, based on wheat germ agglutinin-lectin (WGA) affinity and citalopram affinity chromatographies. Upon solubilization of the carrier with 1% digitonin, a 50-70-fold increase in specific ({sup 3}H) imipramine binding activity with a 70% recovery could be accomplished through WGA-lectin chromatography. The WGA pool was then subjected to affinity chromatography on citalopram-agarose. At least 90% of the binding capacity adsorbed to the column. Specific elution using 10 {mu}M citalopram resulted in a 22% recovery of binding activity. A 10,000-fold overall purification was obtained by using this two-step procedure. Analysis of the fractions on SDS-PAGE after {sup 125}I labeling revealed specific elution of 78- and 55-kDa proteins concomitant with the appearance of ({sup 3}H) imipramine binding activity. The pharmacological profile of the partially purified reuptake system correlated well with that derived from the crude membrane-bound reuptake system, suggesting a copurification of the 5HT binding activity and ({sup 3}H)imipramine binding activity.

  2. Determination of the kinetic rate constant of cyclodextrin supramolecular systems by high performance affinity chromatography.

    PubMed

    Li, Haiyan; Ge, Jingwen; Guo, Tao; Yang, Shuo; He, Zhonggui; York, Peter; Sun, Lixin; Xu, Xu; Zhang, Jiwen

    2013-08-30

    It is challenging and extremely difficult to measure the kinetics of supramolecular systems with extensive, weak binding (Ka<10(5)M(-1)), and fast dissociation, such as those composed of cyclodextrins and drugs. In this study, a modified peak profiling method based on high performance affinity chromatography (HPAC) was established to determine the dissociation rate constant of cyclodextrin supramolecular systems. The interactions of β-cyclodextrin with acetaminophen and sertraline were used to exemplify the method. The retention times, variances and the plate heights of the peaks for acetaminophen or sertraline, conventional non-retained substance (H2O) on the β-cyclodextrin bonded column and a control column were determined at four flow rates under linear elution conditions. Then, plate heights for the theoretical non-retained substance were estimated by the modified HPAC method, in consideration of the diffusion and stagnant mobile phase mass transfer. As a result, apparent dissociation rate constants of 1.82 (±0.01)s(-1) and 3.55 (±0.37)s(-1) were estimated for acetaminophen and sertraline respectively at pH 6.8 and 25°C with multiple flow rates. Following subtraction of the non-specific binding with the support, dissociation rate constants were estimated as 1.78 (±0.00) and 1.91 (±0.02)s(-1) for acetaminophen and sertraline, respectively. These results for acetaminophen and sertraline were in good agreement with the magnitude of the rate constants for other drugs determined by capillary electrophoresis reported in the literature and the peak fitting method we performed. The method described in this work is thought to be suitable for other supramolecules, with relatively weak, fast and extensive interactions.

  3. A low-cost affinity purification system using β-1,3-glucan recognition protein and curdlan beads.

    PubMed

    Horiuchi, Masataka; Takahasi, Kiyohiro; Kobashigawa, Yoshihiro; Ochiai, Masanori; Inagaki, Fuyuhiko

    2012-08-01

    Silkworm β-1,3-glucan recognition protein (βGRP) tightly and specifically associates with β-1,3-glucan. We report here an affinity purification system named the 'GRP system', which uses the association between the β-1,3-glucan recognition domain of βGRP (GRP-tag), as an affinity tag, and curdlan beads. Curdlan is a water-insoluble β-1,3-glucan reagent, the low cost of which (about 100 JPY/g) allows the economical preparation of beads. Curdlan beads can be readily prepared by solubilization in an alkaline solution, followed by neutralization, sonication and centrifugation. We applied the GRP system to preparation of several proteins and revealed that the expression levels of the GRP-tagged proteins in soluble fractions were two or three times higher than those of the glutathione S-transferase (GST)-tagged proteins. The purity of the GRP-tagged proteins on the curdlan beads was comparable to that of the GST-tagged proteins on glutathione beads. The chemical stability of the GRP system was more robust than conventional affinity systems under various conditions, including low pH (4-6). Biochemical and structural analyses revealed that proteins produced using the GRP system were structurally and functionally active. Thus, the GRP system is suitable for both the large- and small-scale preparation of recombinant proteins for functional and structural analyses.

  4. System A amino acid transporter SNAT2 shows subtype-specific affinity for betaine and hyperosmotic inducibility in placental trophoblasts.

    PubMed

    Nishimura, Tomohiro; Yagi, Risa; Usuda, Mariko; Oda, Kenji; Yamazaki, Mai; Suda, Sayaka; Takahashi, Yu; Okazaki, Fumiyasu; Sai, Yoshimichi; Higuchi, Kei; Maruyama, Tetsuo; Tomi, Masatoshi; Nakashima, Emi

    2014-05-01

    Betaine uptake is induced by hypertonic stress in a placental trophoblast cell line, and involvement of amino acid transport system A was proposed. Here, we aimed to identify the subtype(s) of system A that mediates hypertonicity-induced betaine uptake. Measurement of [(14)C]betaine uptake by HEK293 cells transiently transfected with human or rat sodium-coupled neutral amino acid transporters (SNATs), SNAT1, SNAT2 and SNAT4 revealed that only human and rat SNAT2 have betaine uptake activity. The Michaelis constants (Km) of betaine uptake by human and rat SNAT2 were estimated to be 5.3 mM and 4.6 mM, respectively. Betaine exclusively inhibited the uptake activity of SNAT2 among the rat system A subtypes. We found that rat SNAT1, SNAT2 and SNAT4 were expressed at the mRNA level under isotonic conditions, while expression of SNAT2 and SNAT4 was induced by hypertonicity in TR-TBT 18d-1 cells. Western blot analyses revealed that SNAT2 expression on plasma membrane of TR-TBT 18d-1 cells was more potently induced by hypertonicity than that in total cell lysate. Immunocytochemistry confirmed the induction of SNAT2 expression in TR-TBT 18d-1 cells exposed to hypertonic conditions and indicated that SNAT2 was localized on the plasma membrane in these cells. Our results indicate that SNAT2 transports betaine, and that tonicity-sensitive SNAT2 expression may be involved in regulation of betaine concentration in placental trophoblasts.

  5. In vitro uptake of bile acids by choroid plexus, kidney cortex and anterior uvea. I. The iodipamide-sensitive transport systems in the rabbit.

    PubMed

    Bárány, E H

    1975-02-01

    Renal cortex, anterior uvea, lateral choroid plexus and terminal ileum accumulate -14C-cholate, glycocholate, deoxycholate and chenodeoxycholate to considerable tissue/medium ratios. Iodipamide partly inhibits accumulation by kidney, uvea and plexus but not ileum. In renal cortex the sensitive part is similar to 10, 60 and 90 percent for dihydroxy acids, cholate and glycocholate respectively. Hippurate depresses uptake in kidney and uvea but hardly in plexus. Simultaneous uptake by renal cortex and uvea of -14C-cholate or glycocholate, -125I-iodipamide and -131I-o-iodohippurate was studied with unlabelled iodipamide and hippurate as inhibitors. The concentration-dependence of the inhibition required the assumption of 4 partly overlapping iodipamide-sensitive transport systems handling the 4 test substances: the hippurate (H)-system, one moderately (L(1)) and one very hippurate-resistant (L(2)) part of the liverlike L-system and a fourth system called BS, more evenly inhibitable by iodipamide and hippurate than the others. The L(2)-system carries iodipamide but very little bile acids. No iodipamide-sensitive system clearly specialized for bile acid transport was found. The systems have only moderate affinity for bile acids and probably treat them just as large organic anions. A new mathematical procedure to test the degree of complexity of composite transport systems without kinetic assumptions was used.

  6. Topical imiquimod yields systemic effects due to unintended oral uptake.

    PubMed

    Grine, Lynda; Steeland, Sophie; Van Ryckeghem, Sara; Ballegeer, Marlies; Lienenklaus, Stefan; Weiss, Siegfried; Sanders, Niek N; Vandenbroucke, Roosmarijn E; Libert, Claude

    2016-01-28

    Repetitive application of topical imiquimod is used as an experimental model for the induction of psoriasiform skin lesions in mice. The model is characterized by several inflammatory processes, including cytokine production both locally and systemically, cellular infiltration, and splenomegaly. To investigate the production of type I interferons in response to imiquimod-containing Aldara cream, IFNβ-luciferase reporter mice were imaged in vivo and ex vivo. Type I interferons were found to be produced in the skin, but also in the intestinal system caused by unintended ingestion of imiquimod by the mice. Through the use of Elizabethan collars to prevent ingestion, these effects, including psoriasiform lesions were nearly completely prevented. Our findings reveal that topical treatment with Aldara induces a psoriasiform skin inflammation, but that its mode of action depends on ingestion of the chemical, which leads to systemic responses and affects local inflammation. Therefore, potential ingestion of topical treatments during experimental procedures should be taken into account during assessment of cutaneous inflammatory parameters in skin disease models.

  7. Topical imiquimod yields systemic effects due to unintended oral uptake

    PubMed Central

    Grine, Lynda; Steeland, Sophie; Van Ryckeghem, Sara; Ballegeer, Marlies; Lienenklaus, Stefan; Weiss, Siegfried; Sanders, Niek N.; Vandenbroucke, Roosmarijn E.; Libert, Claude

    2016-01-01

    Repetitive application of topical imiquimod is used as an experimental model for the induction of psoriasiform skin lesions in mice. The model is characterized by several inflammatory processes, including cytokine production both locally and systemically, cellular infiltration, and splenomegaly. To investigate the production of type I interferons in response to imiquimod-containing Aldara cream, IFNβ-luciferase reporter mice were imaged in vivo and ex vivo. Type I interferons were found to be produced in the skin, but also in the intestinal system caused by unintended ingestion of imiquimod by the mice. Through the use of Elizabethan collars to prevent ingestion, these effects, including psoriasiform lesions were nearly completely prevented. Our findings reveal that topical treatment with Aldara induces a psoriasiform skin inflammation, but that its mode of action depends on ingestion of the chemical, which leads to systemic responses and affects local inflammation. Therefore, potential ingestion of topical treatments during experimental procedures should be taken into account during assessment of cutaneous inflammatory parameters in skin disease models. PMID:26818707

  8. AGIA Tag System Based on a High Affinity Rabbit Monoclonal Antibody against Human Dopamine Receptor D1 for Protein Analysis

    PubMed Central

    Yano, Tomoya; Takeda, Hiroyuki; Uematsu, Atsushi; Yamanaka, Satoshi; Nomura, Shunsuke; Nemoto, Keiichirou; Iwasaki, Takahiro; Takahashi, Hirotaka; Sawasaki, Tatsuya

    2016-01-01

    Polypeptide tag technology is widely used for protein detection and affinity purification. It consists of two fundamental elements: a peptide sequence and a binder which specifically binds to the peptide tag. In many tag systems, antibodies have been used as binder due to their high affinity and specificity. Recently, we obtained clone Ra48, a high-affinity rabbit monoclonal antibody (mAb) against dopamine receptor D1 (DRD1). Here, we report a novel tag system composed of Ra48 antibody and its epitope sequence. Using a deletion assay, we identified EEAAGIARP in the C-terminal region of DRD1 as the minimal epitope of Ra48 mAb, and we named this sequence the “AGIA” tag, based on its central sequence. The tag sequence does not include the four amino acids, Ser, Thr, Tyr, or Lys, which are susceptible to post-translational modification. We demonstrated performance of this new tag system in biochemical and cell biology applications. SPR analysis demonstrated that the affinity of the Ra48 mAb to the AGIA tag was 4.90 × 10−9 M. AGIA tag showed remarkably high sensitivity and specificity in immunoblotting. A number of AGIA-fused proteins overexpressed in animal and plant cells were detected by anti-AGIA antibody in immunoblotting and immunostaining with low background, and were immunoprecipitated efficiently. Furthermore, a single amino acid substitution of the second Glu to Asp (AGIA/E2D) enabled competitive dissociation of AGIA/E2D-tagged protein by adding wild-type AGIA peptide. It enabled one-step purification of AGIA/E2D-tagged recombinant proteins by peptide competition under physiological conditions. The sensitivity and specificity of the AGIA system makes it suitable for use in multiple methods for protein analysis. PMID:27271343

  9. Time-Resolved Investigation of Molecular Components Involved in the Induction of [Formula: see text] High Affinity Transport System in Maize Roots.

    PubMed

    Pii, Youry; Alessandrini, Massimiliano; Dall'Osto, Luca; Guardini, Katia; Prinsi, Bhakti; Espen, Luca; Zamboni, Anita; Varanini, Zeno

    2016-01-01

    The induction, i.e., the rapid increase of nitrate ([Formula: see text]) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to [Formula: see text] treatment were characterized in terms of changes in [Formula: see text] uptake rate and plasma membrane (PM) H(+)-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H(+)-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼150 kDa oligomer. The expression trend during the induction of the 11 identified PM H(+)-ATPase transcripts indicates that those mainly involved in the response to [Formula: see text] treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H(+)-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms.

  10. Time-Resolved Investigation of Molecular Components Involved in the Induction of NO3– High Affinity Transport System in Maize Roots

    PubMed Central

    Pii, Youry; Alessandrini, Massimiliano; Dall’Osto, Luca; Guardini, Katia; Prinsi, Bhakti; Espen, Luca; Zamboni, Anita; Varanini, Zeno

    2016-01-01

    The induction, i.e., the rapid increase of nitrate (NO3–) uptake following the exposure of roots to the anion, was studied integrating physiological and molecular levels in maize roots. Responses to NO3– treatment were characterized in terms of changes in NO3– uptake rate and plasma membrane (PM) H+-ATPase activity and related to transcriptional and protein profiles of NRT2, NRT3, and PM H+-ATPase gene families. The behavior of transcripts and proteins of ZmNRT2s and ZmNRT3s suggested that the regulation of the activity of inducible high-affinity transport system (iHATS) is mainly based on the transcriptional/translational modulation of the accessory protein ZmNRT3.1A. Furthermore, ZmNRT2.1 and ZmNRT3.1A appear to be associated in a ∼150 kDa oligomer. The expression trend during the induction of the 11 identified PM H+-ATPase transcripts indicates that those mainly involved in the response to NO3– treatment are ZmHA2 and ZmHA4. Yet, partial correlation between the gene expression, protein levels and enzyme activity suggests an involvement of post-transcriptional and post-translational mechanisms of regulation. A non-denaturing Deriphat-PAGE approach allowed demonstrating for the first time that PM H+-ATPase can occur in vivo as hexameric complex together with the already described monomeric and dimeric forms. PMID:27877183

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

    PubMed Central

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

    2011-01-01

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

  12. Effect of systemic infection induced by Pseudomonas aeruginosa on the brain uptake of colistin in mice.

    PubMed

    Jin, Liang; Li, Jian; Nation, Roger L; Nicolazzo, Joseph A

    2012-10-01

    In view of reports of colistin-induced neurotoxicity in infected patients, the aim of this study was to assess whether the integrity of the blood-brain barrier (BBB) and the brain uptake of colistin are altered in the presence of systemic Pseudomonas aeruginosa infection. Bacteremia was confirmed 8 h after intramuscular administration of P. aeruginosa ATCC 27853 to Swiss Outbred mice, at which time a single subcutaneous dose of colistin sulfate (40 mg/kg of body weight) or an intravenous dose of [(14)C]sucrose (2 μCi) was administered. Despite a substantial elevation in plasma levels of the proinflammatory cytokines tumor necrosis factor alpha, interleukin-1β, and interleukin-6 during bacterial infection, the brain uptake of colistin was similar between infected and noninfected mice with AUC(brain)/AUC(plasma) (where AUC(brain) is the area under the brain concentration-time curve and AUC(plasma) is the area under the plasma concentration-time curve) ratios of 0.023 and 0.024, respectively. Similarly, the brain-to-plasma ratios of [(14)C]sucrose were no different between infected and noninfected mice, consistent with a lack of effect of bacteremia on BBB integrity. To further correlate any relationship between BBB disruption and plasma levels of proinflammatory cytokines, BBB integrity, colistin brain uptake, and plasma proinflammatory cytokines were measured following the administration of Salmonella enterica lipopolysaccharide (LPS), an agent known to induce BBB disruption. Despite LPS inducing a 4-fold increase in colistin brain uptake and a significant (P < 0.05) 1.2-fold increase in [(14)C]sucrose BBB penetration, plasma cytokine levels were lower with LPS treatment relative to those obtained with bacterial infection with P. aeruginosa. This study demonstrates that the brain uptake of colistin is not increased in mice during P. aeruginosa-induced systemic bacteremia despite a significant increase in plasma levels of three proinflammatory cytokines.

  13. 27-Hydroxycholesterol impairs neuronal glucose uptake through an IRAP/GLUT4 system dysregulation.

    PubMed

    Ismail, Muhammad-Al-Mustafa; Mateos, Laura; Maioli, Silvia; Merino-Serrais, Paula; Ali, Zeina; Lodeiro, Maria; Westman, Eric; Leitersdorf, Eran; Gulyás, Balázs; Olof-Wahlund, Lars; Winblad, Bengt; Savitcheva, Irina; Björkhem, Ingemar; Cedazo-Mínguez, Angel

    2017-02-17

    Hypercholesterolemia is associated with cognitively deteriorated states. Here, we show that excess 27-hydroxycholesterol (27-OH), a cholesterol metabolite passing from the circulation into the brain, reduced in vivo brain glucose uptake, GLUT4 expression, and spatial memory. Furthermore, patients exhibiting higher 27-OH levels had reduced (18)F-fluorodeoxyglucose uptake. This interplay between 27-OH and glucose uptake revealed the engagement of the insulin-regulated aminopeptidase (IRAP). 27-OH increased the levels and activity of IRAP, countered the IRAP antagonist angiotensin IV (AngIV)-mediated glucose uptake, and enhanced the levels of the AngIV-degrading enzyme aminopeptidase N (AP-N). These effects were mediated by liver X receptors. Our results reveal a molecular link between cholesterol, brain glucose, and the brain renin-angiotensin system, all of which are affected in some neurodegenerative diseases. Thus, reducing 27-OH levels or inhibiting AP-N maybe a useful strategy in the prevention of the altered glucose metabolism and memory decline in these disorders.

  14. Minimization of the root of a quadratic functional under a system of affine equality constraints with application to portfolio management

    NASA Astrophysics Data System (ADS)

    Landsman, Zinoviy

    2008-10-01

    We present an explicit closed form solution of the problem of minimizing the root of a quadratic functional subject to a system of affine constraints. The result generalizes Z. Landsman, Minimization of the root of a quadratic functional under an affine equality constraint, J. Comput. Appl. Math. 2007, to appear, see , articles in press, where the optimization problem was solved under only one linear constraint. This is of interest for solving significant problems pertaining to financial economics as well as some classes of feasibility and optimization problems which frequently occur in tomography and other fields. The results are illustrated in the problem of optimal portfolio selection and the particular case when the expected return of finance portfolio is certain is discussed.

  15. An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

    DOE PAGES

    AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

    2015-11-19

    Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database inmore » which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the

  16. An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system

    SciTech Connect

    AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

    2015-11-19

    Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database in which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. Lastly, this database will facilitate the analysis of protein-DNA interactions and the

  17. Probing the influence of anomeric effects on the lithium ion affinity in 1,3-diaza systems: a computational study.

    PubMed

    Kesharwani, Manoj K; Thiel, Walter; Ganguly, Bishwajit

    2010-10-07

    Lithium ion affinities of methanediamine (MDA), N,N,N',N'-tetramethylmethanediamine (TMMDA), 1,3-diazacyclohexane (DAC), trans-3,5-diazabicyclo[4.4.0]decane (trans-3,5-DBD), trans-1,3-diazabicyclo[4.4.0]decane (trans-1,3-DBD), cis-1,3-diazabicyclo[4.4.0]decane (cis-1,3-DBD), 1,5-diazabicyclo[3.3.1]nonane (DBN), trans-decahydro-8a,9a-diazaanthracene (trans-DDA), cis-decahydro-8a,9a-diazaanthracene (cis-DDA), 1,3-diazetidine (DAT), 1,3-imidazolidine (IMD), and 1,3-diazepane (DAP) have been studied by using density functional theory (DFT) and correlated ab initio methods. Possible conformers of these compounds were optimized at the B3LYP/6-31+G* level, and relative energies were evaluated at the MP2/6-311+G**//B3LYP/6-31+G* level. The experimental lithium ion affinities for reference molecules (i.e., ammonia and trimethylamine) are well-reproduced at these levels of theory. NBO analysis shows the influence of anomeric effects (n(N) → σ*(C-N) hyperconjugative interactions) on the conformational stability of the title compounds; however, the electrostatic and steric contributions included in the NBO Lewis term also affect the stabilities in some cases. The influence of anomeric effect is apparent in cis-DDA, where the nitrogen involved in n(N) → σ*(C-N) hyperconjugative interaction (cis-DDA-Li2) has a lithium ion affinity 1.7 kcal/mol lower than the nitrogen not involved in n(N) → σ*(C-N) hyperconjugative interaction (cis-DDA-Li1). In general, the computed lithium ion affinities were found to be conformationally dependent. The NBO results showed that the lithium ion affinities are also governed by the interplay of n(N) → σ*(C-N) hyperconjugative interactions and the steric strain caused upon lithiation. Further, the ring size also influences the lithium ion affinities in the 1,3-diaza monocyclic systems. In some complexes multiple coordination of the lithium ion is possible by inversion of one of the nitrogen atoms.

  18. An exposure system for measuring nasal and lung uptake of vapors in rats

    SciTech Connect

    Dahl, A.R.; Brookins, L.K.; Gerde, P.

    1995-12-01

    Inhaled gases and vapors often produce biological damage in the nasal cavity and lower respiratory tract. The specific site within the respirator tract at which a gas or vapor is absorbed strongly influences the tissues at risk to potential toxic effects; to predict or to explain tissue or cell specific toxicity of inhaled gases or vapors, the sites at which they are absorbed must be known. The purpose of the work reported here was to develop a system for determining nose and lung absorption of vapors in rats, an animal commonly used in inhalation toxicity studies. In summary, the exposure system described allows us to measure in the rate: (1) nasal absorption and desorption of vapors; (2) net lung uptake of vapors; and (3) the effects of changed breathing parameters on vapor uptake.

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

    PubMed Central

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

    1992-01-01

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

  20. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    PubMed Central

    Carrick, Brian H.; Hao, Linxuan; Smaldino, Philip J.; Engelke, David R.

    2015-01-01

    Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies. PMID:26715090

  1. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

    PubMed

    Carrick, Brian H; Hao, Linxuan; Smaldino, Philip J; Engelke, David R

    2015-12-29

    Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant "CelTag" DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  2. An updated model for nitrate uptake modelling in plants. II. Assessment of active root involvement in nitrate uptake based on integrated root system age: measured versus modelled outputs

    PubMed Central

    Malagoli, Philippe; Le Deunff, Erwan

    2014-01-01

    Background and Aims An updated version of a mechanistic structural–functional model was developed to predict nitrogen (N) uptake throughout the growth cycle by a crop of winter oilseed rape, Brassica napus, grown under field conditions. Methods The functional component of the model derives from a revisited conceptual framework that combines the thermodynamic Flow–Force interpretation of nitrate uptake isotherms and environmental and in planta effects on nitrate influx. Estimation of the root biomass (structural component) is based upon a combination of root mapping along the soil depth profile in the field and a relationship between the specific root length and external nitrate concentration. The root biomass contributing actively to N uptake was determined by introduction of an integrated root system age that allows assignment of a root absorption capacity at a specific age of the root. Key Results Simulations were well matched to measured data of N taken up under field conditions for three levels of N fertilization. The model outputs indicated that the two topsoil layers (0–30 and 30–60 cm) contained 75–88 % of the total root length and biomass, and accounted for 90–95 % of N taken up at harvest. Conclusions This conceptual framework provides a model of nitrate uptake that is able to respond to external nitrate fluctuations at both functional and structural levels. PMID:24709791

  3. The yeast Aft2 transcription factor determines selenite toxicity by controlling the low affinity phosphate transport system

    PubMed Central

    Pérez-Sampietro, María; Serra-Cardona, Albert; Canadell, David; Casas, Celia; Ariño, Joaquín; Herrero, Enrique

    2016-01-01

    The yeast Saccharomyces cerevisiae is employed as a model to study the cellular mechanisms of toxicity and defense against selenite, the most frequent environmental selenium form. We show that yeast cells lacking Aft2, a transcription factor that together with Aft1 regulates iron homeostasis, are highly sensitive to selenite but, in contrast to aft1 mutants, this is not rescued by iron supplementation. The absence of Aft2 strongly potentiates the transcriptional responses to selenite, particularly for DNA damage- and oxidative stress-responsive genes, and results in intracellular hyperaccumulation of selenium. Overexpression of PHO4, the transcriptional activator of the PHO regulon under low phosphate conditions, partially reverses sensitivity and hyperaccumulation of selenite in a way that requires the presence of Spl2, a Pho4-controlled protein responsible for post-transcriptional downregulation of the low-affinity phosphate transporters Pho87 and Pho90. SPL2 expression is strongly downregulated in aft2 cells, especially upon selenite treatment. Selenite hypersensitivity of aft2 cells is fully rescued by deletion of PHO90, suggesting a major role for Pho90 in selenite uptake. We propose that the absence of Aft2 leads to enhanced Pho90 function, involving both Spl2-dependent and independent events and resulting in selenite hyperaccumulation and toxicity. PMID:27618952

  4. Role of potassium uptake systems in Sinorhizobium meliloti osmoadaptation and symbiotic performance.

    PubMed

    Domínguez-Ferreras, Ana; Muñoz, Socorro; Olivares, José; Soto, María J; Sanjuán, Juan

    2009-04-01

    Stimulation of potassium uptake is the most rapid response to an osmotic upshock in bacteria. This cation accumulates by a number of different transport systems whose importance has not been previously addressed for rhizobia. In silico analyses reveal the presence of genes encoding four possible potassium uptake systems in the genome of Sinorhizobium meliloti 1021: Kup1, Kup2, Trk, and Kdp. The study of the relevance of these systems under a number of different growth conditions and in symbiosis showed that the integrity of Kup1 or Trk is essential for growth under laboratory conditions even in osmotically balanced media and the absence of both systems leads to a reduced infectivity and competitiveness of the bacteria in alfalfa roots. Trk is the main system involved in the accumulation of potassium after an osmotic upshift and the most important system for growth of S. meliloti under hyperosmotic conditions. The other three systems, especially Kup1, are also relevant during the osmotic adaptation of the cell, and the relative importance of the Kdp system increases at low potassium concentrations.

  5. Tracking the diurnal signal of plant water uptake through the hydrologic system

    NASA Astrophysics Data System (ADS)

    Blume, Theresa; Hassler, Sibylle; Heidbüchel, Ingo; Weiler, Markus; Simard, Sonia; Güntner, Andreas; Heinrich, Ingo

    2015-04-01

    Plant water uptake during summer is characterized by strong diurnal fluctuations. As a result a diurnal sink term is imposed on catchment storage, affecting the unsaturated zone, sometimes the saturated zone and even streamflow. Detecting this signal and understanding its propagation through the hydrological system may help to better quantify eco-hydrological connectivity. The extent and strength of the propagation of this signal from plant to soil to ground- and stream water was investigated with a unique setup of 46 field sites in Luxemburg and 15 field sites in Germany. These sites cover a range of geologies, soils, topographies and types of vegetation. Vegetation types include grassland, pine forest (young and old) and different deciduous forest stands. Available data at all sites includes information at high temporal resolution from 3-5 soil moisture profiles, matrix potential, piezometers and sapflow sensors (as proxy for plant water uptake) as well as standard climate data. At sites with access to a stream, discharge or water level is also recorded. Signal strength (amplitude of diurnal fluctuations) can thus be traced through the system and gives an indication of the physical sphere of influence of plant water uptake i.e. the "eco-hydro-connectivity". Temporal dynamics of signal strength furthermore suggest a shifting spatial distribution of root water uptake with time. The analysis of time lags (or phase shifts) between daily fluctuations in temperature, radiation, sapflow, soil water, groundwater and streamflow gives further insights into the processes driving and propagating these signals and inter-site comparison allows for the investigation of local controls.

  6. Sequential peptide affinity purification system for the systematic isolation and identification of protein complexes from Escherichia coli.

    PubMed

    Babu, Mohan; Butland, Gareth; Pogoutse, Oxana; Li, Joyce; Greenblatt, Jack F; Emili, Andrew

    2009-01-01

    Biochemical purification of affinity-tagged proteins in combination with mass spectrometry methods is increasingly seen as a cornerstone of systems biology, as it allows for the systematic genome-scale characterization of macromolecular protein complexes, representing demarcated sets of stably interacting protein partners. Accurate and sensitive identification of both the specific and shared polypeptide components of distinct complexes requires purification to near homogeneity. To this end, a sequential peptide affinity (SPA) purification system was developed to enable the rapid and efficient isolation of native Escherichia coli protein complexes (J Proteome Res 3:463-468, 2004). SPA purification makes use of a dual-affinity tag, consisting of three modified FLAG sequences (3X FLAG) and a calmodulin binding peptide (CBP), spaced by a cleavage site for tobacco etch virus (TEV) protease (J Proteome Res 3:463-468, 2004). Using the lambda-phage Red homologous recombination system (PNAS 97:5978-5983, 2000), a DNA cassette, encoding the SPA-tag and a selectable marker flanked by gene-specific targeting sequences, is introduced into a selected locus in the E. coli chromosome so as to create a C-terminal fusion with the protein of interest. This procedure aims for near-endogenous levels of tagged protein production in the recombinant bacteria to avoid spurious, non-specific protein associations (J Proteome Res 3:463-468, 2004). In this chapter, we describe a detailed, optimized protocol for the tagging, purification, and subsequent mass spectrometry-based identification of the subunits of even low-abundance bacterial protein complexes isolated as part of an ongoing large-scale proteomic study in E. coli (Nature 433:531-537, 2005).

  7. Synthetic nanoparticles camouflaged with biomimetic erythrocyte membranes for reduced reticuloendothelial system uptake

    NASA Astrophysics Data System (ADS)

    Rao, Lang; Xu, Jun-Hua; Cai, Bo; Liu, Huiqin; Li, Ming; Jia, Yan; Xiao, Liang; Guo, Shi-Shang; Liu, Wei; Zhao, Xing-Zhong

    2016-02-01

    Suppression of the reticuloendothelial system (RES) uptake is one of the most challenging tasks in nanomedicine. Coating stratagems using polymers, such as poly(ethylene glycol) (PEG), have led to great success in this respect. Nevertheless, recent observations of immunological response toward these synthetic polymers have triggered a search for better alternatives. In this work, natural red blood cell (RBC) membranes are camouflaged on the surface of Fe3O4 nanoparticles for reducing the RES uptake. In vitro macrophage uptake, in vivo biodistribution and pharmacokinetic studies demonstrate that the RBC membrane is a superior alternative to the current gold standard PEG for nanoparticle ‘stealth’. Furthermore, we systematically investigate the in vivo potential toxicity of RBC membrane-coated nanoparticles by blood biochemistry, whole blood panel examination and histology analysis based on animal models. The combination of synthetic nanoparticles and natural cell membranes embodies a novel and biomimetic nanomaterial design strategy and presents a compelling property of functional materials for a broad range of biomedical applications.

  8. Purification of papain by metal affinity partitioning in aqueous two-phase polyethylene glycol/sodium sulfate systems.

    PubMed

    Jiang, Zhi-Guo; Zhang, Hai-De; Wang, Wei-Tao

    2015-05-01

    A simple and inexpensive aqueous two-phase affinity partitioning system using metal ligands was introduced to improve the selectivity of commercial papain extraction. Polyethylene glycol 4000 was first activated using epichlorohydrin, then it was covalently linked to iminodiacetic acid. Finally, the specific metal ligand Cu(2+) was attached to the polyethylene glycol-iminodiacetic acid. The chelated Cu(2+) content was measured by atomic absorption spectrometry as 0.88 mol/mol (polyethylene glycol). The effects on the purification at different conditions, including polyethylene glycol molecular weight (2000, 4000, and 6000), concentration of phase-forming components (polyethylene glycol 12-20% w/w and sodium sulfate 12-20%, w/w), metal ligand type, and concentration, system pH and the commercial papain loading on papain extraction, were systematically studied. Under optimum conditions of the system, i.e. 18% w/w sodium sulfate, 18% w/w polyethylene glycol 4000, 1% w/w polyethylene glycol-iminodiacetic acid-Cu(2+) and pH 7, a maximum yield of 90.3% and a degree of purification of 3.6-fold were obtained. Compared to aqueous two phase extraction without ligands, affinity partitioning was found to be an effective technique for the purification of commercial papain with higher extraction efficiency and degree of purification.

  9. C-X...H contacts in biomolecular systems: how they contribute to protein-ligand binding affinity.

    PubMed

    Lu, Yunxiang; Wang, Yong; Xu, Zhijian; Yan, Xiuhua; Luo, Xiaoming; Jiang, Hualiang; Zhu, Weiliang

    2009-09-17

    The hydrogen bond acceptor capability of halogens has long been underappreciated in the field of biology. In this work, we have surveyed structures of protein complexes with halogenated ligands to characterize geometrical preferences of C-X...H contacts and contributions of such interactions to protein-ligand binding affinity. Notably, F...H interactions in biomolecules exhibit a remarkably different behavior as compared to three other kinds of X...H (X = Cl, Br, I) interactions, which has been rationalized by means of ab initio calculations using simple model systems. The C-X...H contacts in biological systems are characterized as weak hydrogen bonding interactions. Furthermore, the electrophile "head on" and nucleophile "side on" interactions of halogens have been extensively investigated through the examination of interactions in protein structures and a two-layer ONIOM-based QM/MM method. In biomolecular systems, C-X...H contacts are recognized as secondary interaction contributions to C-X...O halogen bonds that play important roles in conferring specificity and affinity for halogenated ligands. The results presented here are within the context of their potential applications in drug design, including relevance to the development of accurate force fields for halogens.

  10. ℒ1 adaptive controller for a class of non-affine multi-input multi-output nonlinear systems

    NASA Astrophysics Data System (ADS)

    Luo, Jie; Cao, Chengyu; Yang, Qinmin

    2013-02-01

    In this article, an extension of the ℒ1 adaptive control design is introduced for a class of non-affine Multi-Input Multi-Output nonlinear systems with unknown dynamics and unmeasured states. The system dynamics is represented in the normal form with the bounded-input-bounded-output internal dynamics. At first, a stable virtual reference counterpart is constructed. Thereafter, a piece-wise continuous adaptive law is introduced to the actual system along with a low-pass filtered control signal that allows for achieving arbitrarily close tracking of the input and the output signals of the reference system. Rigorous mathematical proof is provided, and the theoretical results are verified with the simulation.

  11. FigA, a Putative Homolog of Low-Affinity Calcium System Member Fig1 in Saccharomyces cerevisiae, Is Involved in Growth and Asexual and Sexual Development in Aspergillus nidulans

    PubMed Central

    Zhang, Shizhu; Zheng, Hailin; Long, Nanbiao; Carbó, Natalia; Chen, Peiying; Aguilar, Pablo S.

    2014-01-01

    Calcium-mediated signaling pathways are widely employed in eukaryotes and are implicated in the regulation of diverse biological processes. In Saccharomyces cerevisiae, at least two different calcium uptake systems have been identified: the high-affinity calcium influx system (HACS) and the low-affinity calcium influx system (LACS). Compared to the HACS, the LACS in fungi is not well known. In this study, FigA, a homolog of the LACS member Fig1 from S. cerevisiae, was functionally characterized in the filamentous fungus Aspergillus nidulans. Loss of figA resulted in retardant hyphal growth and a sharp reduction of conidial production. Most importantly, FigA is essential for the homothallic mating (self-fertilization) process; further, FigA is required for heterothallic mating (outcrossing) in the absence of HACS midA. Interestingly, in a figA deletion mutant, adding extracellular Ca2+ rescued the hyphal growth defects but could not restore asexual and sexual reproduction. Furthermore, quantitative PCR results revealed that figA deletion sharply decreased the expression of brlA and nsdD, which are known as key regulators during asexual and sexual development, respectively. In addition, green fluorescent protein (GFP) tagging at the C terminus of FigA (FigA::GFP) showed that FigA localized to the center of the septum in mature hyphal cells, to the location between vesicles and metulae, and between the junctions of metulae and phialides in conidiophores. Thus, our findings suggest that FigA, apart from being a member of a calcium uptake system in A. nidulans, may play multiple unexplored roles during hyphal growth and asexual and sexual development. PMID:24376003

  12. Conservation of `Moving' Energy in Nonholonomic Systems with Affine Constraints and Integrability of Spheres on Rotating Surfaces

    NASA Astrophysics Data System (ADS)

    Fassò, Francesco; Sansonetto, Nicola

    2016-04-01

    Energy is in general not conserved for mechanical nonholonomic systems with affine constraints. In this article we point out that, nevertheless, in certain cases, there is a modification of the energy that is conserved. Such a function is the pull-back of the energy of the system written in a system of time-dependent coordinates in which the constraint is linear, and for this reason will be called a `moving' energy. After giving sufficient conditions for the existence of a conserved, time-independent moving energy, we point out the role of symmetry in this mechanism. Lastly, we apply these ideas to prove that the motions of a heavy homogeneous solid sphere that rolls inside a convex surface of revolution in uniform rotation about its vertical figure axis, are (at least for certain parameter values and in open regions of the phase space) quasi-periodic on tori of dimension up to three.

  13. Factors affecting phosphate uptake by Aerobacter aerogenes in a system relating cell numbers to 32P uptake.

    PubMed

    White, L A; MacLeod, R A

    1971-03-01

    The uptake of phosphate, from media limited in this ion, by resting cells of Aerobacter aerogenes has been investigated and shown to be dependent upon several factors. An incubation medium composed of 10(-2)m K(+), 5 x 10(-3)m Mg(2+), 1 mg of glucose per ml, and 1 muCi of (32)PO(4) (3-) per ml, buffered at pH 6.55 with 0.05 mN-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES), was found to stimulate optimum accumulation of (32)P-orthophosphate. The temperature of incubation, incubation time, the concentration of unlabeled orthophosphate, as well as arsenate, and several metabolic inhibitors were found to affect the accumulation. The labeled cells were collected on a membrane filter, which had been previously boiled in glass-distilled water, for measurement of the radioactivity accumulated. Under optimum conditions, as few as 20,000 cells were capable of accumulating detectable amounts of (32)P-orthophosphate in 1 hr of incubation.

  14. Characterization of cadmium uptake in Lactobacillus plantarum and isolation of cadmium and manganese uptake mutants

    SciTech Connect

    Hao, Z.; Reiske, H.R.; Wilson, D.B.

    1999-11-01

    Two different Cd{sup 2+} uptake systems were identified in Lactobacillus plantarum. One is a high-affinity, high-velocity Mn{sup 2+} uptake system which also takes up Cd{sup 2+} and is induced by Mn{sup 2+} starvation. The calculated K{sub m} and V{sub max} are 0.26 {mu}M and 3.6 {mu}mol g of dry cell{sup {minus}1} min{sup {minus}1}, respectively. Unlike Mn{sup 2+} uptake, which is facilitated by citrate and related tricarboxylic acids, Cd{sup 2+} uptake is weakly inhibited by citrate. Cd{sup 2+} and Mn{sup 2+} are competitive inhibitors of each other, and the affinity of the system for Cd{sup 2+} is higher than that for Mn{sup 2+}. The other Cd{sup 2+} uptake system is expressed in Mn{sup 2+}-sufficient cells, and no K{sub m} can be calculated for it because uptake is nonsaturable. Mn{sup 2+} does not compete for transport through this system, nor does any other tested cation, i.e., Zn{sup 2+}, Cu{sup 2+}, Co{sup 2+}, Mg{sup 2+}, Ca{sup 2+}, Fe{sup 2+}, or Ni{sup 2+}. Both systems require energy, since uncouplers completely inhibit their activities. Two Mn{sup 2+}-dependent L. plantarum mutants were isolated by chemical mutagenesis and ampicillin enrichment. They required more than 5,000 times as much Mn{sup 2+} for growth as the parental strain. Mn{sup 2+} starvation-induced Cd{sup 2+} uptake in both mutants was less than 5% the wild-type rate. The low level of long-term Mn{sup 2+} or Cd{sup 2+} accumulation by the mutant strains also shows that the mutations eliminate the high-affinity Mn{sup 2+} and Cd{sup 2+} uptake system.

  15. Cadmium Bioavailability, Uptake, Toxicity and Detoxification in Soil-Plant System.

    PubMed

    Shahid, Muhammad; Dumat, Camille; Khalid, Sana; Niazi, Nabeel Khan; Antunes, Paula M C

    This review summarizes the findings of the most recent studies, published from 2000 to 2016, which focus on the biogeochemical behavior of Cd in soil-plant systems and its impact on the ecosystem. For animals and people not subjected to a Cd-contaminated environment, consumption of Cd contaminated food (vegetables, cereals, pulses and legumes) is the main source of Cd exposure. As Cd does not have any known biological function, and can further cause serious deleterious effects both in plants and mammalian consumers, cycling of Cd within the soil-plant system is of high global relevance.The main source of Cd in soil is that which originates as emissions from various industrial processes. Within soil, Cd occurs in various chemical forms which differ greatly with respect to their lability and phytoavailability. Cadmium has a high phytoaccumulation index because of its low adsorption coefficient and high soil-plant mobility and thereby may enter the food chain. Plant uptake of Cd is believed to occur mainly via roots by specific and non-specific transporters of essential nutrients, as no Cd-specific transporter has yet been identified. Within plants, Cd causes phytotoxicity by decreasing nutrient uptake, inhibiting photosynthesis, plant growth and respiration, inducing lipid peroxidation and altering the antioxidant system and functioning of membranes. Plants tackle Cd toxicity via different defense strategies such as decreased Cd uptake or sequestration into vacuoles. In addition, various antioxidants combat Cd-induced overproduction of ROS. Other mechanisms involve the induction of phytochelatins, glutathione and salicylic acid.

  16. Albumin and Cr-EDTA uptake by systemic arteries, veins, and pulmonary artery of rabbit

    SciTech Connect

    Lever, M.J.; Jay, M.T. )

    1990-07-01

    Experiments have been performed both in vivo and in vitro to measure the steady-state uptake of labeled albumin and Cr-ethylenediaminetetraacetate by various blood vessels of the rabbit: the ascending and descending portions of the thoracic aorta, the carotid artery, the pulmonary artery, and the inferior vena cava. The in vitro experiments indicated that the wall tissues of the pulmonary artery and the vena cava have much greater distribution volumes for albumin than do the systemic arteries. This may in part explain the differences in wall tissue concentrations in vivo and, in turn, the differences between vessels in their susceptibility to atherosclerosis.

  17. Band gaps, ionization potentials, and electron affinities of periodic electron systems via the adiabatic-connection fluctuation-dissipation theorem

    NASA Astrophysics Data System (ADS)

    Trushin, Egor; Betzinger, Markus; Blügel, Stefan; Görling, Andreas

    2016-08-01

    An approach to calculate fundamental band gaps, ionization energies, and electron affinities of periodic electron systems is explored. Starting from total energies obtained with the help of the adiabatic-connection fluctuation-dissipation (ACFD) theorem, these physical observables are calculated according to their basic definition by differences of the total energies of the N -, (N -1 ) -, and (N +1 ) -electron system. The response functions entering the ACFD theorem are approximated here by the direct random phase approximation (dRPA). For a set of prototypical semiconductors and insulators it is shown that even with this quite drastic approximation the resulting band gaps are very close to experiment and of a similar quality to those from the computationally more involved G W approximation. By going beyond the dRPA in the future the accuracy of the calculated band gaps may be significantly improved further.

  18. Optimal allocation of building blocks between nutrient uptake systems in a microbe.

    PubMed

    van den Berg, Hugo A; Kiselev, Yuri N; Orlov, Michael V

    2002-03-01

    A bacterial cell must distribute its molecular building blocks among various types of nutrient uptake systems. If the microbe is to maximize its average growth rate, this allocation of building blocks must be adjusted to the environmental availabilities of the various nutrients. The adjustments can be found from growth balancing considerations. We give a full proof of optimality and uniqueness of the optimal allocation regime for a simple model of microbial growth and internal stores kinetics. This proof suggests likely candidates for optimal control regimes in the case of a more realistic model. These candidate regimes differ with respect to the information that the cell's control system must have access to. We pay particular attention to one of the three candidates, a feedback regime based on a cellular control system that monitors only internal reserve densities. We show that allocation converges rapidly to balanced growth under this control regime.

  19. Robust fuzzy output feedback controller for affine nonlinear systems via T-S fuzzy bilinear model: CSTR benchmark.

    PubMed

    Hamdy, M; Hamdan, I

    2015-07-01

    In this paper, a robust H∞ fuzzy output feedback controller is designed for a class of affine nonlinear systems with disturbance via Takagi-Sugeno (T-S) fuzzy bilinear model. The parallel distributed compensation (PDC) technique is utilized to design a fuzzy controller. The stability conditions of the overall closed loop T-S fuzzy bilinear model are formulated in terms of Lyapunov function via linear matrix inequality (LMI). The control law is robustified by H∞ sense to attenuate external disturbance. Moreover, the desired controller gains can be obtained by solving a set of LMI. A continuous stirred tank reactor (CSTR), which is a benchmark problem in nonlinear process control, is discussed in detail to verify the effectiveness of the proposed approach with a comparative study.

  20. Insights into the algebraic structure of Lorenz-like systems using feedback circuit analysis and piecewise affine models

    NASA Astrophysics Data System (ADS)

    Letellier, Christophe; Amaral, Gleison F. V.; Aguirre, Luis A.

    2007-06-01

    The characterization of chaotic attractors has been a widely addressed problem and there are now many different techniques to define their nature in a rather accurate way, at least in the case of a three-dimensional system. Nevertheless, the link between the structure of the ordinary differential equations and the topology of their solutions is still missing and only a few necessary conditions on the algebraic structure are known today. By using a feedback circuit analysis, it is shown that it is possible to identify the relevant terms of the equations, that is, the terms that really contribute to the structure of the phase portrait. Such analysis also provides some guidelines for constructing piecewise affine models. Moreover, equivalence classes can be defined on the basis of the active feedback circuits involved.

  1. A synthetic host-guest system achieves avidin-biotin affinity by overcoming enthalpy-entropy compensation.

    PubMed

    Rekharsky, Mikhail V; Mori, Tadashi; Yang, Cheng; Ko, Young Ho; Selvapalam, N; Kim, Hyunuk; Sobransingh, David; Kaifer, Angel E; Liu, Simin; Isaacs, Lyle; Chen, Wei; Moghaddam, Sarvin; Gilson, Michael K; Kim, Kimoon; Inoue, Yoshihisa

    2007-12-26

    The molecular host cucurbit[7]uril forms an extremely stable inclusion complex with the dicationic ferrocene derivative bis(trimethylammoniomethyl)ferrocene in aqueous solution. The equilibrium association constant for this host-guest pair is 3 x 10(15) M(-1) (K(d) = 3 x 10(-16) M), equivalent to that exhibited by the avidin-biotin pair. Although purely synthetic systems with larger association constants have been reported, the present one is unique because it does not rely on polyvalency. Instead, it achieves its extreme affinity by overcoming the compensatory enthalpy-entropy relationship usually observed in supramolecular complexes. Its disproportionately low entropic cost is traced to extensive host desolvation and to the rigidity of both the host and the guest.

  2. Testing a structural model of young driver willingness to uptake Smartphone Driver Support Systems.

    PubMed

    Kervick, Aoife A; Hogan, Michael J; O'Hora, Denis; Sarma, Kiran M

    2015-10-01

    There is growing interest in the potential value of using phone applications that can monitor driver behaviour (Smartphone Driver Support Systems, 'SDSSs') in mitigating risky driving by young people. However, their value in this regard will only be realised if young people are willing to use this technology. This paper reports the findings of a study in which a novel structural model of willingness to use SDSSs was tested. Grounded in the driver monitoring and Technology Acceptance (TA) research literature, the model incorporates the perceived risks and gains associated with potential SDSS usage and additional social cognitive factors, including perceived usability and social influences. A total of 333 smartphone users, aged 18-24, with full Irish driving licenses completed an online questionnaire examining willingness or Behavioural Intention (BI) to uptake a SDSS. Following exploratory and confirmatory factor analyses, structural equation modelling indicated that perceived gains and social influence factors had significant direct effects on BI. Perceived risks and social influence also had significant indirect effects on BI, as mediated by perceived gains. Overall, this model accounted for 72.5% of the variance in willingness to uptake SDSSs. Multi-group structural models highlighted invariance of effects across gender, high and low risk drivers, and those likely or unlikely to adopt novel phone app technologies. These findings have implications for our understanding of the willingness of young drivers to adopt and use SDSSs, and highlight potential factors that could be targeted in behavioural change interventions seeking to improve usage rates.

  3. Overlap of copper and iron uptake systems in mitochondria in Saccharomyces cerevisiae.

    PubMed

    Vest, Katherine E; Wang, Jing; Gammon, Micah G; Maynard, Margaret K; White, Olivia L; Cobine, Jai A; Mahone, Wilkerson K; Cobine, Paul A

    2016-01-01

    In Saccharomyces cerevisiae, the mitochondrial carrier family protein Pic2 imports copper into the matrix. Deletion of PIC2 causes defects in mitochondrial copper uptake and copper-dependent growth phenotypes owing to decreased cytochrome c oxidase activity. However, copper import is not completely eliminated in this mutant, so alternative transport systems must exist. Deletion of MRS3, a component of the iron import machinery, also causes a copper-dependent growth defect on non-fermentable carbon. Deletion of both PIC2 and MRS3 led to a more severe respiratory growth defect than either individual mutant. In addition, MRS3 expressed from a high copy number vector was able to suppress the oxygen consumption and copper uptake defects of a strain lacking PIC2. When expressed in Lactococcus lactis, Mrs3 mediated copper and iron import. Finally, a PIC2 and MRS3 double mutant prevented the copper-dependent activation of a heterologously expressed copper sensor in the mitochondrial intermembrane space. Taken together, these data support a role for the iron transporter Mrs3 in copper import into the mitochondrial matrix.

  4. Overlap of copper and iron uptake systems in mitochondria in Saccharomyces cerevisiae

    PubMed Central

    Wang, Jing; Gammon, Micah G.; Maynard, Margaret K.; White, Olivia L.; Cobine, Jai A.; Mahone, Wilkerson K.

    2016-01-01

    In Saccharomyces cerevisiae, the mitochondrial carrier family protein Pic2 imports copper into the matrix. Deletion of PIC2 causes defects in mitochondrial copper uptake and copper-dependent growth phenotypes owing to decreased cytochrome c oxidase activity. However, copper import is not completely eliminated in this mutant, so alternative transport systems must exist. Deletion of MRS3, a component of the iron import machinery, also causes a copper-dependent growth defect on non-fermentable carbon. Deletion of both PIC2 and MRS3 led to a more severe respiratory growth defect than either individual mutant. In addition, MRS3 expressed from a high copy number vector was able to suppress the oxygen consumption and copper uptake defects of a strain lacking PIC2. When expressed in Lactococcus lactis, Mrs3 mediated copper and iron import. Finally, a PIC2 and MRS3 double mutant prevented the copper-dependent activation of a heterologously expressed copper sensor in the mitochondrial intermembrane space. Taken together, these data support a role for the iron transporter Mrs3 in copper import into the mitochondrial matrix. PMID:26763345

  5. Differing patterns of striatal sup 18 F-dopa uptake in Parkinson's disease, multiple system atrophy, and progressive supranuclear palsy

    SciTech Connect

    Brooks, D.J.; Ibanez, V.; Sawle, G.V.; Quinn, N.; Lees, A.J.; Mathias, C.J.; Bannister, R.; Marsden, C.D.; Frackowiak, R.S. )

    1990-10-01

    Using positron emission tomography (PET), we studied regional striatal 18F-dopa uptake in 16 patients with L-dopa-responsive Parkinson's disease (PD), 18 patients with multiple system atrophy, and 10 patients with progressive supranuclear palsy. Results were compared with those of 30 age-matched normal volunteers. The patients with PD showed significantly reduced mean uptake of 18F-dopa in the caudate and putamen compared to controls, but while function in the posterior part of the putamen was severely impaired (45% of normal), function in the anterior part of the putamen and in the caudate was relatively spared (62% and 84% of normal). Mean 18F-dopa uptake in the posterior putamen was depressed to similar levels in all patients. Unlike patients with PD, the patients with progressive supranuclear palsy showed equally severe impairment of mean 18F-dopa uptake in the anterior and posterior putamen. Caudate 18F-dopa uptake was also significantly lower in patients with progressive supranuclear palsy than in patients with PD, being depressed to the same level as that in the putamen. Mean 18F-dopa uptake values in the anterior putamen and caudate in patients with multiple system atrophy lay between PD and progressive supranuclear palsy levels. Locomotor disability of individual patients with PD or multiple system atrophy correlated with decline in striatal 18F-dopa uptake, but this was not the case for the patients with progressive supranuclear palsy. We conclude that patients with PD have selective nigral pathological features with relative preservation of the dopaminergic function in the anterior putamen and caudate, whereas there is progressively more extensive nigral involvement in multiple system atrophy and progressive supranuclear palsy.

  6. Biochemical affinity sensing systems based on luminescence generation in the evanescent field of optical waveguides

    NASA Astrophysics Data System (ADS)

    Duveneck, Gert L.; Oroszlan, Peter; Abel, A. P.; Klee, B.; Steiner, V.; Ehrat, Markus; Gygax, D.; Widmer, H. M.

    1995-01-01

    We have developed a (bio)chemical analysis system based on luminescence generation and detection in the evanescent field associated with light guiding in an optical fiber. Our intention was directed towards optimization of not only the sensor, including the sensor handling and the immobilization of biochemical recognition elements, but also of the assay chemistry, with special emphasis on methods used for sensor regeneration, of the fluidic system, and of the experimental control software. Goals of this optimization process were not only to achieve high sensitivity, reproducibility and the related precision of the results, but also maximum regenerability of the sensors and system flexibility for a variety of different applications. Four examples of different bioaffinity assays, established on our sensor system, are presented: a competitive immunoassay for atrazine, a sandwich immunoassay for hirudin, a DNA hybridization assay, and first studies for the development of sensors based on membrane- bound receptors. In the atrazine assay, the sensor could be regenerated more than 300 times. In the hybridization assay, a detection limit of 7.5 multiplied by 10-14 M complementary fluorescein-labeled DNA was achieved. The performance of our system is compared with an established enzyme-linked immunosorbent assay (ELISA) on the example of the hirudin assay. In the concluding section of this paper, advantages and disadvantages of our fiberoptic, luminescence-based system, compared with commercialized systems, based on detection of changes of the effective refractive index, are discussed.

  7. Quantification of radiotracer uptake with a dedicated breast PET imaging system

    PubMed Central

    Raylman, Raymond R.; Smith, Mark F.; Kinahan, Paul E.; Majewski, Stan

    2008-01-01

    Tomographic breast imaging techniques can be used to quantify radiotracer uptake in breast and tumor tissue. However, physical processes common to PET imaging can confound accurate quantification. In this investigation, we assessed the effects of these phenomena and tested correction schemes for our new positron emission mammography–tomography system (PEM–PET). The PEM–PET scanner utilizes two sets of rotating planar detector heads. Each unit consists of a 4×3 array of Hamamatsu H8500 flat panel position sensitive photomultipliers coupled to a 96×72 array of 2×2×15 mm3 LYSO detector elements (pitch=2.1 mm). Image reconstruction is performed with a 3D-OSEM algorithm parallelized to run on a multiprocessor computer system. The reconstructed field-of-view is 15×15×15 cm3. Much of the testing procedures were based on NEMA-NU2∕2001 protocols. Count rate losses due to pulse pile-up, image contamination due to acceptance of random coincidences and Compton scatter, and image artifacts produced by photon attenuation were measured. It was found that the system was susceptible to count rate losses when moderate levels of radiation were present in the scanner due to the current design of the event trigger electronics. Application of corrections for Compton scattering, photon attenuation and dead time resulted in improved estimations of 18F concentration in simplified phantom studies. Results from these preliminary studies indicate that the PEM–PET scanner will be useful for the quantification of radiotracer uptake in breast tumors, possibly facilitating early assessment of cancer treatments. PMID:19070233

  8. Data-based fault-tolerant control for affine nonlinear systems with actuator faults.

    PubMed

    Xie, Chun-Hua; Yang, Guang-Hong

    2016-09-01

    This paper investigates the fault-tolerant control (FTC) problem for unknown nonlinear systems with actuator faults including stuck, outage, bias and loss of effectiveness. The upper bounds of stuck faults, bias faults and loss of effectiveness faults are unknown. A new data-based FTC scheme is proposed. It consists of the online estimations of the bounds and a state-dependent function. The estimations are adjusted online to compensate automatically the actuator faults. The state-dependent function solved by using real system data helps to stabilize the system. Furthermore, all signals in the resulting closed-loop system are uniformly bounded and the states converge asymptotically to zero. Compared with the existing results, the proposed approach is data-based. Finally, two simulation examples are provided to show the effectiveness of the proposed approach.

  9. Automated Immobilized Metal Affinity Chromatography System for Enrichment of Escherichia coli Phosphoproteome

    SciTech Connect

    Qu, Yi; Wu, Si; Zhao, Rui; Zink, Erika M.; Orton, Daniel J.; Moore, Ronald J.; Meng, Da; Clauss, Therese RW; Aldrich, Joshua T.; Lipton, Mary S.; Pasa-Tolic, Ljiljana

    2013-06-05

    Enrichment of bacterial phosphopeptides is an essential step prior to bottom-up mass spectrometry-based analysis of the phosphoproteome, which is fundamental to understanding the role of phosphoproteins in cell signaling and regulation of protein activity. We developed an automated IMAC system to enrich strong cation exchange-fractionated phosphopeptides from the soluble proteome of Escherichia coli MG1655 grown on minimal medium. Initial demonstration of the system resulted in identification of 75 phosphopeptides covering 52 phosphoproteins. Consistent with previous studies, many of these phosphoproteins are involved in the carbohydrate portion of central metabolism. The automated system utilizes a large capacity IMAC column that can effectively enrich phosphopeptides from a bacterial sample by increasing peptide loading and reducing the wash time. An additional benefit of the automated IMAC system is reduced labor and associated costs.

  10. Uptake Kinetics of Arsenic in Upland Rice Cultivar Zhonghan 221 Inoculated with Arbuscular Mycorrhizal Fungi.

    PubMed

    Chan, W F; Li, W C; Wong, M H

    2015-01-01

    Arbuscular mycorrhizal fungi (AMF) appear to be highly associated with arsenic (As) uptake in host plants because arsenate (As(V)) and phosphorus (P) share the same transporter, whereby AMF can enhance P uptake. A short-term experiment was conducted for low- (0 to 0.05 mM As) and high-affinity (0 to 2.5 mM As) uptake systems, to investigate the AMF role on As uptake mechanism in plants, which may explain As uptake kinetics in upland rice cultivar: Zhonghan 221. When concentration of As ranged from 0 to 0.05 mM, Funneliformis geosporum (Fg) significantly decreased arsenite (As(III)) and monomethylarsonicacid (MMA) uptake when (p < 0.05) compared to non-mycorrhizal (NM) treatment, since the major route for (As(III)) in rice roots-rice silicon transporter Lsi1 would be influenced by Fg inoculation at high As concentrations. Fg can also reduce As(V) uptake significantly (p < 0.05) under both uptake systems relative to NM treatment, whereas, Funneliformis mosseae (Fm) increased As(V) and MMA uptake in rice roots, with MMA uptake rate generally lower than As(III) and As(V). Using suitable AMF species inoculation with rice, As uptake and accumulation in rice grains can be reduced and the risk to human health, once consumed, can be minimized.

  11. Investigating the Uptake Mechanisms of Hydrogen Peroxide to Single and Polycrystalline Ice with a Novel Flow Tube System

    NASA Astrophysics Data System (ADS)

    Hong, Angela; Ammann, Markus; Bartels-Rausch, Thorsten

    2016-04-01

    Air-ice chemical interactions are important for describing the distribution and subsequent chemical fate of trace atmospheric gases within ice and snow and determining the oxidative capacities of these environments. The nature of this interaction is governed by a compound's physicochemical properties as well as ice microstructure. Hydrogen peroxide (H2O2), a reservoir of HOx radicals in the atmosphere and an important chromophore in snow and ice, is a trace gas that demonstrates complex uptake behaviour to frozen aqueous media by the reversible, fast adsorption to the air-ice interface, aggregation, and lateral interactions, and a slower process, ostensibly via uptake into the bulk. However, the exact mechanism and kinetics for the slow uptake of H2O2 and the size of this reservoir is unknown. It is important to describe and quantify this loss term, over environmentally-relevant timescales, accommodation of H2O2 into the bulk may be the dominant process which controls the composition and chemistry of the snow and overlying atmosphere. We hypothesize that the slow uptake of H2O2 occurs by diffusion into the grain boundaries of ice. To provide mechanistic insight to the macroscopic phenomenon of atmospheric gas uptake to ice, and discern various mechanisms including adsorption to air-ice interface and accommodation into the bulk through uptake into grain boundaries, we design, machine, and validate a novel flow reactor system featuring a Drilled Ice Flow Tube (DIFT). Our flow reactor system is uniquely suited to testing these uptake mechanisms: by controlling the degree of grain boundaries present in the DIFT (ie. monocrystalline or polycrystalline), we can directly observe the effect of the ice microstructure on the adsorptive and bulk uptake of trace atmospheric gases over long timescales (eg. on the order of hours). Here, we describe method development of the DIFT and demonstrate using polarised microscopy imagery that our experimental set-up allows for the direct

  12. Artificial hydrogenases: biohybrid and supramolecular systems for catalytic hydrogen production or uptake.

    PubMed

    Caserta, Giorgio; Roy, Souvik; Atta, Mohamed; Artero, Vincent; Fontecave, Marc

    2015-04-01

    There is an urgent need for cheap, abundant and efficient catalysts as an alternative to platinum for hydrogen production and oxidation in (photo)electrolyzers and fuel cells. Hydrogenases are attractive solutions. These enzymes use exclusively nickel and iron in their active sites and function with high catalytic rates at the thermodynamic equilibrium. As an alternative, a number of biomimetic and bioinspired catalysts for H2 production and/or uptake, based on Ni, Fe and Co, have been developed and shown to display encouraging performances. In this review we discuss specifically recent approaches aiming at incorporating these compounds within oligomeric and polymeric hosts. The latter are most often biological compounds (peptides, proteins, polysaccharides, etc.) but we also discuss non-biological scaffolds (synthetic polymers, Metal-organic-Frameworks, etc.) which can provide the appropriate environment to tune the activity and stability of the synthetic catalysts. These supramolecular catalytic systems thus define a class of original compounds so-called artificial hydrogenases.

  13. A novel sialic acid utilization and uptake system in the periodontal pathogen Tannerella forsythia.

    PubMed

    Roy, Sumita; Douglas, C W Ian; Stafford, Graham P

    2010-05-01

    Tannerella forsythia is a key contributor to periodontitis, but little is known of its virulence mechanisms. In this study we have investigated the role of sialic acid in biofilm growth of this periodontal pathogen. Our data show that biofilm growth of T. forsythia is stimulated by sialic acid, glycolyl sialic acid, and sialyllactose, all three of which are common sugar moieties on a range of important host glycoproteins. We have also established that growth on sialyllactose is dependent on the sialidase of T. forsythia since the sialidase inhibitor oseltamivir suppresses growth on sialyllactose. The genome of T. forsythia contains a sialic acid utilization locus, which also encodes a putative inner membrane sialic acid permease (NanT), and we have shown this is functional when it is expressed in Escherichia coli. This genomic locus also contains a putatively novel TonB-dependent outer membrane sialic acid transport system (TF0033-TF0034). In complementation studies using an Escherichia coli strain devoid of its outer membrane sialic acid transporters, the cloning and expression of the TF0033-TF0034 genes enabled an E. coli nanR nanC ompR strain to utilize sialic acid as the sole carbon and energy source. We have thus identified a novel sialic acid uptake system that couples an inner membrane permease with a TonB-dependent outer membrane transporter, and we propose to rename these novel sialic acid uptake genes nanO and nanU, respectively. Taken together, these data indicate that sialic acid is a key growth factor for this little-characterized oral pathogen and may be key to its physiology in vivo.

  14. Simultaneous nitrification and denitrification with anoxic phosphorus uptake in a membrane bioreactor system.

    PubMed

    Patel, Jignesh; Nakhla, George

    2006-10-01

    The performance of an innovative membrane bioreactor (MBR) process using anoxic phosphorus uptake with nitrification and denitrification for the treatment of municipal wastewater with respect to operational performance and effluent quality is addressed in this paper. The system was operated at steady-state conditions with a synthetic acetate-based wastewater at a hydraulic retention time (HRT) of 12 hours and on degritted municipal wastewater at a total system HRT of 6 hours. The MBR system was able to achieve 99% biochemical oxygen demand (BOD), chemical oxygen demand (COD), and ammonia-nitrogen (NH4(+)-N); 98% total Kjeldahl nitrogen (TKN); and 97% phosphorus removal, producing effluent BOD, COD, NH4+-N, TKN, nitrate-nitrogen, nitrite-nitrogen, and phosphate-phosphorus of <3, 14, 0.2, 0.26, 5.8, 0.21, and <0.01 mg/L, respectively, at the 6-hour HRT. The comparison of the synthetic and municipal wastewater run is presented in this paper. Steady-state mass balance on municipal wastewater was performed to reveal some key features of the modified MBR system.

  15. G196 epitope tag system: a novel monoclonal antibody, G196, recognizes the small, soluble peptide DLVPR with high affinity

    PubMed Central

    Tatsumi, Kasumi; Sakashita, Gyosuke; Nariai, Yuko; Okazaki, Kosuke; Kato, Hiroaki; Obayashi, Eiji; Yoshida, Hisashi; Sugiyama, Kanako; Park, Sam-Yong; Sekine, Joji; Urano, Takeshi

    2017-01-01

    The recognition specificity of monoclonal antibodies (mAbs) has made mAbs among the most frequently used tools in both basic science research and in clinical diagnosis and therapies. Precise determination of the epitope allows the development of epitope tag systems to be used with recombinant proteins for various purposes. Here we describe a new family of tag derived from the epitope recognized by a highly specific mAb G196. The minimal epitope was identified as the five amino acid sequence Asp-Leu-Val-Pro-Arg. Permutation analysis was used to characterize the binding requirements of mAb G196, and the variable regions of the mAb G196 were identified and structurally analyzed by X-ray crystallography. Isothermal titration calorimetry revealed the high affinity (Kd = 1.25 nM) of the mAb G196/G196-epitope peptide interaction, and G196-tag was used to detect several recombinant cytosolic and nuclear proteins in human and yeast cells. mAb G196 is valuable for developing a new peptide tagging system for cell biology and biochemistry research. PMID:28266535

  16. Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

    NASA Astrophysics Data System (ADS)

    Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

    2016-03-01

    Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer.

  17. Uptake of indolmycin in gram-positive bacteria.

    PubMed Central

    Werner, R G

    1980-01-01

    The antimicrobial activity of indolmycin correlates with the generation time of the investigated strains. Thus, in Staphylococcus aureus ATCC 13150 with a 37-min generation time, the minimal inhibitory concentration (MIC) was 0.6 microgram ml-1, and in Bacillus subtilis ATCC 27142 with a generation time of 23 min, the MIC reached 10.5 micrograms ml-1. Competition experiments in staphylococci and B. subtilis with aromatic amino acids demonstrated that indolmycin uses the uptake systems that are responsible for tryptophan. When the Ki values of indolmycin for the uptake of the aromatic amino acids in staphylococci were compared, there was a significantly higher influence on the uptake of tryptophan with respect to phenylalanine and tyrosine. In addition, indolmycin low resistant mutants of S. aureus ATCC 13150 showed a 10- to 100-fold decrease in Km value for the uptake of tryptophan and a 10-fold decrease for tyrosine uptake. The Km value for phenylalanine remained unchanged. A significant correlation existed between the Ki values of indolmycin for the uptake of tryptophan in the wild-type strains of S. aureus and B. subtilis and the MIC against the corresponding strain. Low Ki values corresponded to low MIC. These results imply that, in addition to improvement of the antibiotic structure for target affinity, the tryptophan uptake system can be used as a test model for the structural evaluation of indolmycin with respect to an increased transport activity into bacterial cells. PMID:7235673

  18. Nonrecirculating Hydroponic System Suitable for Uptake Studies at Very Low Nutrient Concentrations 1

    PubMed Central

    Gutschick, Vincent P.; Kay, Lou Ellen

    1991-01-01

    We describe the mechanical, electronic, hydraulic, and structural design of a nonrecirculating hydroponic system. The system is particularly suited to studies at very low nutrient concentrations, for which on-line concentration monitoring methods either do not exist or are costly and limited to monitoring relatively few individual plants. Solutions are mixed automatically to chosen concentrations, which can be set differently for every pump fed from a master supply of deionized water and nutrient concentrates. Pumping rates can be varied over a 50-fold range, up to 400 liters per day, which suffices to maintain a number of large, post-seedling plants in rapid growth at (sub)micromolar levels of N and P. The outflow of each pump is divided among as many as 12 separate root chambers. In each chamber one may monitor uptake by individual plant roots or segments thereof, by measuring nutrient depletion in batch samples of solution. The system is constructed from nontoxic materials that do not adsorb nutrient ions; no transient shifts of nitrate and phosphate concentrations are observable at the submicromolar level. Nonrecirculation of solutions limits problems of pH shifts, microbial contamination, and cumulative imbalances in unmonitored nutrients. We note several disadvantages, principally related to high consumption of deionized water and solutes. The reciprocating pumps can be constructed inexpensively, particularly by the researcher. We also report previously unattainable control of passive temperature rise of chambers exposed to full sunlight, by use of white epoxy paint. PMID:16668100

  19. Immobilized metal ion affinity partitioning, a method combining metal-protein interaction and partitioning of proteins in aqueous two-phase systems.

    PubMed

    Birkenmeier, G; Vijayalakshmi, M A; Stigbrand, T; Kopperschläger, G

    1991-02-22

    Immobilized metal ions were used for the affinity extraction of proteins in aqueous two-phase systems composed of polyethylene glycol (PEG) and dextran or PEG and salt. Soluble chelating polymers were prepared by covalent attachment of metal-chelating groups to PEG. The effect on the partitioning of proteins of such chelating PEG derivatives coordinated with different metal ions is demonstrated. The proteins studied were alpha 2-macroglobulin, tissue plasminogen activator, superoxide dismutase and monoclonal antibodies. The results indicate that immobilized metal ion affinity partitioning provides excellent potential for the extraction of proteins.

  20. The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus

    PubMed Central

    Tosi, Tommaso; Campeotto, Ivan; Corrigan, Rebecca M.; Freemont, Paul; Gründling, Angelika

    2017-01-01

    Staphylococcus aureus is an important opportunistic human pathogen that is highly resistant to osmotic stresses. In order to survive an increase in osmolarity, bacteria immediately take up potassium and small organic compounds, also referred to as compatible solutes. The second messenger c-di-AMP binds to several receptor proteins, most of which are involved in ion and potassium uptake, that help bacteria cope with osmotic stress. In this study, we identified OpuCA, the ATPase component of an uptake system for the compatible solute carnitine, as a c-di-AMP target protein in S. aureus and found that a strain overproducing c-di-AMP showed reduced carnitine uptake. The CBS domains of OpuCA bound to c-di-AMP, and a crystal structure revealed a putative binding pocket for c-di-AMP in the cleft between the two CBS domains. Thus, c-di-AMP is involved in regulating both branches of osmoprotection (potassium uptake and compatible solute uptake), suggesting that c-di-AMP is a general osmotic stress regulator. PMID:27531650

  1. Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing.

    PubMed

    Motejadded, Hassan; Altenbuchner, Josef

    2009-04-01

    An E. coli vector system was constructed which allows the expression of fusion genes via a L: -rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence for affinity purification, the Saccharomyces cerevisiae Smt3 protein for protein processing by proteolytic cleavage and the protein of interest. The Smt3 gene was codon-optimized for expression in E. coli. In a second rhamnose-inducible vector, the S. cerevisiae Ulp1 protease gene for processing Smt3 fusion proteins was fused in the same way to maltose-binding protein and His-tag sequence but without the Smt3 gene. The enhanced green fluorescent protein (eGFP) was used as reporter and protein of interest. Both fusion proteins (MalE-6xHis-Smt3-eGFP and MalE-6xHis-Ulp1) were efficiently produced in E. coli and separately purified by amylose resin. After proteolytic cleavage the products were applied to a Ni-NTA column to remove protease and tags. Pure eGFP protein was obtained in the flow-through of the column in a yield of around 35% of the crude cell extract.

  2. Bacillus subtilis MntR coordinates the transcriptional regulation of manganese uptake and efflux systems.

    PubMed

    Huang, Xiaojuan; Shin, Jung-Ho; Pinochet-Barros, Azul; Su, Tina T; Helmann, John D

    2017-01-01

    The Bacillus subtilis MntR metalloregulatory protein senses manganese, an essential element required for central metabolism, oxidative stress resistance and replication. An mntR null mutant is highly sensitive to Mn(II) intoxication, which is attributed in part to the constitutive expression of two importers: the proton-dependent NRAMP family transporter MntH and the ABC transporter MntABCD. Here, we show that an mntR null mutant is still sensitive to Mn(II) intoxication even if both of the import systems are absent. This Mn(II) sensitivity results from the requirement for MntR to activate the transcription of two genes encoding cation diffusion facilitator (CDF) family efflux pumps. Physiological studies indicate that MneP (formerly YdfM) serves as the primary Mn(II) efflux pump with MneS (formerly YeaB) playing a secondary role. Mutant strains lacking mneP are Mn(II) sensitive and accumulate elevated levels of Mn(II), and these effects are exacerbated in a mneP mneS double mutant. DNA-binding and in vitro transcription studies demonstrate that MntR binds to both the mneP and mneS regulatory regions and directly activates transcription in response to levels of Mn(II) several-fold higher than required for repression of import genes. These results highlight the delicate balance of Mn(II) uptake and efflux systems controlled by MntR.

  3. Quantification of stomatal uptake of ionic solutes using a new model system.

    PubMed

    Eichert, T; Burkhardt, J

    2001-04-01

    Evidence for stomatal uptake of solutes by leaves without the application of surfactants or pressure has recently been provided (Eichert et al., 1998). In the present study, experimental conditions were refined in that the water potential was held at <0 on the physiologically inner side of the epidermal strips (ES) by use of a ceramic plate or the proper mesophyll of the plant. The penetrated substances were immobilized on the inner side by ion exchange membranes. The influence of humidity, light, stomatal density, and re-wetting on the uptake of anions (fluorescein) and cations (Fe(3+)) was investigated, using leaves of Allium porrum, Commelina communis and Sedum telephium. Uptake increased with humidity, stomatal aperture and stomatal density. It was restricted to stomatal areas, and was especially high below the rims of drying droplets. Again, penetration of stomatal pores was observed. Uptake was strongly correlated with the number of penetrated stomata, although usually less than 10% of the stomata contributed to uptake. The number of stomata that had been penetrated was highly variable, increasing extremely significantly with the number of repeated drying/ wetting cycles. These results indicate that stomatal uptake can be a major pathway for the foliar uptake of ionic solutes. It is a dynamic process, depending on environmental conditions and history of the residues on the leaf, aspects that had been neglected in previous concepts.

  4. CONTINUOUS, AUTOMATED AND SIMULTANEOUS MEASUREMENT OF OXYGEN UPTAKE AND CARBON DIOXIDE EVOLUTION IN BIOLOGICAL SYSTEMS

    EPA Science Inventory

    Commercial respirometers are capable of continuously and automatically measuring oxygen uptake in bioreactors. A method for continuously and automatically measuring carbon dioxide evolution can be retrofitted to commercial respirometers. Continuous and automatic measurements of...

  5. The osmotolerant fructophilic yeast Zygosaccharomyces rouxii employs two plasma-membrane fructose uptake systems belonging to a new family of yeast sugar transporters.

    PubMed

    Leandro, Maria José; Sychrová, Hana; Prista, Catarina; Loureiro-Dias, Maria C

    2011-02-01

    Owing to its high resistance to weak-acid preservatives and extreme osmotolerance, Zygosaccharomyces rouxii is one of the main spoilage yeasts of sweet foods and beverages. In contrast with Saccharomyces cerevisiae, Z. rouxii is a fructophilic yeast; it consumes fructose faster than glucose. So far, to our knowledge, no specific Z. rouxii proteins responsible for this fructophilic behaviour have been characterized. We have identified two genes encoding putative fructose transporters in the Z. rouxii CBS 732 genome. Heterologous expression of these two Z. rouxii ORFs in a S. cerevisiae strain lacking its own hexose transporters (hxt-null) and subsequent kinetic analysis of sugar transport showed that both proteins are functionally expressed at the plasma membrane: ZrFfz1 is a high-capacity fructose-specific facilitator (K(m)∼400 mM and V(max)∼13 mmol h(-1) g(-1)) and ZrFfz2 is a facilitator transporting glucose and fructose with similar capacity and affinity (K(m)∼200 mM and V(max)∼4 mmol h(-1) g(-1)). These two proteins together with the Zygosaccharomyces bailii Ffz1 fructose-specific transporter belong to a new family of sugar transport systems mediating the uptake of hexoses via the facilitated diffusion mechanism, and are more homologous to drug/H(+) antiporters (regarding their primary protein structure) than to other yeast sugar transporters of the Sugar Porter family.

  6. Impacts of carbon source addition on denitrification and phosphorus uptake in enhanced biological phosphorus removal systems.

    PubMed

    Begum, Shamim A; Batista, Jacimaria R

    2013-01-01

    In this study, simultaneous denitrification and phosphorus (P) removal were investigated in batch tests using nitrified mixed liquor and secondary wastewater influent from a full-scale treatment plant and different levels of acetate and propionate as supplemental carbon sources. Without supplemental carbon source, denitrification occurred at low rate and P release and P uptake was negatively affected (i.e., P removal of only 59.7%). When acetate and propionate were supplied, denitrification and P release occurred simultaneously under anoxic conditions. For acetate and propionate at a C/N stoichiometric ratio of 7.6, P release was negatively affected by denitrification. For acetate, the percent P removal and denitrification were very similar for C/N ratios of 22 (5X stoichiometric) and 59 (10X stoichiometric). For propionate, both percent P removal and denitrification deteriorated for C/N ratios of 22 (5X stoichiometric) and 45 (10X stoichiometric). It was observed that carbon source added in excess to stoichiometric ratio was consumed in the aerobic zone, but P was not taken up. This implies that PAO bacteria may utilize the excess carbon source in the aerobic zone rather than their polyhydroxyalkanoate (PHA) reserves, thereby promoting deterioration of the system.

  7. Effects of dehydroepiandrosterone (DHEA) and lactate on glucose uptake in the central nervous system.

    PubMed

    de Souza, Danielle Kaiser; Ribeiro, Maria Flávia Marques; Kucharski, Luiz Carlos Rios

    2012-01-17

    Dehydroepiandrosterone (DHEA) prevents brain aging, enhances the cerebral metabolism and interacts with energy substrates. The interaction between lactate and DHEA on glucose uptake and lactate oxidation by various nervous structures was investigated and results demonstrate that the 2-(14)C-deoxiglucose (2-(14)C-Dglucose) uptake was stimulated by 10mM lactate in the hypothalamus and olfactory bulb, inhibited in the cerebral cortex and cerebellum, and unaffected in the hippocampus. We also show that, in both the cerebral cortex and hypothalamus, (14)C-lactate oxidation was higher than (14)C-glucose oxidation (p≤0.001), demonstrating a relevant role for lactate as energy substrate. The interaction of lactate and 10(-8)M DHEA was tested and, although DHEA had no significant effect on uptake in the cerebellum, hippocampus, or hypothalamus, 10(-8)M DHEA increased the 2-(14)C-Dglucose uptake in the cerebral cortex in the presence of lactate (p≤0.001), and in the olfactory bulb in the absence of lactate (p<0.05). However, DHEA had no significant effect on (14)C-lactate oxidation. We suggest that DHEA improves glucose uptake in specific conditions. Thus, DHEA may affect CNS metabolism and interact with lactate, which is the most important neuronal energy substrate, on glucose uptake.

  8. The nuclear uptake and retention of a synthetic progestin in the cardiovascular system of the baboon.

    PubMed

    Sheridan, P J; McGill, H C

    1984-06-01

    It has long been known that there is a sexual dimorphism in the incidence of coronary heart disease. This observation, together with more recent reports of increased cardiovascular disease associated with the use of oral contraceptives, led to a search for steroid receptors in the cardiovascular system. In this study we examined the nuclear uptake and retention of a synthetic progestin in the cardiovascular system of the baboons. Long term oophorectomized baboons were primed with estradiol benzoate for 3 days before the experiment (50 micrograms/kg, im) and adrenalectomized 2 days before the experiment. On the day of the experiment, the animals were injected under anesthesia with 2.5 micrograms/kg BW [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor-[6,7-3H]pregn-4-ene-3,20-dione) or with [3H] ORG 2058 plus a 1000-fold excess of unlabeled progesterone (control). One hour after the injection, the animals were rapidly exsanguinated, and parts of the cardiovascular system were removed and processed for autoradiography. Localization of the synthetic progestin was found in nuclei of between 25-75% of all smooth muscle cells of the media of all arteries examined and to a lesser extent in the nuclei of the fibroblasts and others cells of the adventitia. Localization of the synthetic progestin in the heart was limited to approximately 1% of the myocardial cells and less than 5% of interstitial cell nuclei. The pattern of localization found differs from that for estrogen and androgen and suggests the possible presence of estrogen-independent progesterone receptors in smooth muscle cells of the media of the aorta and coronary arteries.

  9. Nnuclear uptake and retention of a synthetic progestin in the cardiovascular system of the baboon

    SciTech Connect

    Sheridan, P.J.; McGill, H.C. Jr.

    1984-06-01

    It has long been known that there is a sexual dimorphism in the incidence of coronary heart disease. This observation, together with more recent reports of increased cardiovascular disease associated with the use of oral contraceptives, led to a search for steroid receptors in the cardiovascular system. In this study the nuclear uptake and retention of a synthetic progestin was examined in the cardiovascular system of the baboons. Long term oophorectomized baboons were primed with estradiol benzoate for 3 days before the experiment (50 micrograms/kg, im) and adrenalectomized 2 days before the experiment. On the day of the experiment, the animals were injected under anesthesia with 2.5 micrograms/kg BW (/sup 3/H)ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor-(6,7-/sup 3/H)pregn-4-ene-3,20-dione) or with (/sup 3/H) ORG 2058 plus a 1000-fold excess of unlabeled progesterone (control). One hour after the injection, the animals were rapidly exsanguinated, and parts of the cardiovascular system were removed and processed for autoradiography. Localization of the synthetic progestin was found in nuclei of between 25-75% of all smooth muscle cells of the media of all arteries examined and to a lesser extent in the nuclei of the fibroblasts and others cells of the adventitia. Localization of the synthetic progestin in the heart was limited to approximately 1% of the myocardial cells and less than 5% of interstitial cell nuclei. The pattern of localization found differs from that for estrogen and androgen and suggests the possible presence of estrogen-independent progesterone receptors in smooth muscle cells of the media of the aorta and coronary arteries.

  10. Hemoglobin Ypsilanti: a high-oxygen-affinity hemoglobin demonstrated by two automated high-pressure liquid chromatography systems.

    PubMed

    Mais, Daniel D; Boxer, Laurence A; Gulbranson, Ronald D; Keren, David F

    2007-11-01

    Hemoglobin (Hb) Ypsilanti is a rare high-oxygen-affinity hemoglobin. Like other high-oxygen-affinity hemoglobins, Hb Ypsilanti manifests as erythrocytosis. Because the migration of many high-oxygen-affinity variants on alkaline and acid gels does not differ from that of HbA, oxygen-hemoglobin dissociation studies are often used to document their presence. Hb Ypsilanti is a notable exception because its electrophoresis pattern on alkaline gel is highly characteristic, exemplifying the phenomenon of hybrid formation in variant hemoglobins. In the past few years, several laboratories have begun to use high-pressure liquid chromatography (HPLC) as a screen for hemoglobinopathies. We demonstrate the elution profile of Hb Ypsilanti on the 2 most widely used HPLC methods.

  11. Interplay between seven secondary metal uptake systems is required for full metal resistance of Cupriavidus metallidurans.

    PubMed

    Herzberg, M; Bauer, L; Kirsten, A; Nies, D H

    2016-03-01

    The beta-proteobacterium Cupriavidus metallidurans is able to grow in metal-contaminated environments due to having sophisticated metal efflux systems. Here, the contribution of all seven known secondary metal uptake systems (ZupT, PitA, CorA1, CorA2, CorA3, ZntB, HoxN) to metal resistance is characterized. In a strategic deletion approach, all ten double deletion mutants, a variety of triple and quadruple mutants, and from the Δ4 mutant (ΔzupT ΔcorA1 ΔcorA2 ΔcorA3) the mutants Δ5 (=Δ4 ΔpitA), Δ6 (=Δ4 ΔpitA ΔzntB), and finally Δ7 (ΔzupT ΔcorA1 ΔcorA2 ΔcorA3 ΔpitA ΔzntB ΔhoxN) were constructed. Metal resistance, metal content, and regulation of expression of these genes were characterized in these mutants. The ΔzupT single deletion strain exhibited an extended lag phase in Tris-buffered liquid mineral salts medium (TMM) compared to its parent strain AE104, indicating a decreased fitness level. Further deletions up to Δ6 did not influence growth in TMM without added metals but fitness of the Δ7 strain dropped to a lower level compared to Δ6, Δ5 and ΔzupT. The cells of the Δ7 multiple deletion strain still contained all essential metals, demonstrating that additional metal import systems must exist in C. metallidurans. PitA was an important contributor of metal:phosphate complexes to C. metallidurans. Up to Δ5 no evidence was found for increased expression of the transporter genes to recruit substitutes for the deleted importers. Only the hoxN-lacZ reporter gene fusion displayed a changed expression pattern in the Δ6 strain, indicating recruitment of HoxN. Metal resistance of the deletion strains decreased along the deletion series although all strains still contained metal efflux systems: up to the Δ6 mutant the overall fitness was kept at the ΔzupT mutant strain level at the cost of a diminished competence to handle μM concentrations of transition metals. Together, these data demonstrated an important contribution of the seven

  12. Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum

    SciTech Connect

    Hao, Z.; Chen, S.; Wilson, D.B.

    1999-11-01

    An Mn{sup 2+} and Cd{sup 2+} uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd{sup 2+} sensitivity as well as energy-dependent Cd{sup 2+} uptake activity. Both transcription and translation of mntA were induced by Mn{sup 2+} starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd{sup 2+} uptake systems have been identified in L. plantarum: one is a high-affinity Mn{sup 2+} and Cd{sup 2+} uptake system that is expressed in Mn{sup 2+}-starved cells, and the other is a nonsaturable Cd{sup 2+} uptake system that is expressed in Cd{sup 2+}-sufficient cells. MntA was not detected in an Mn{sup 2+}-dependent mutant of L. plantarum which had lost high-affinity Mn{sup 2+} and Cd{sup 2+} uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn{sup 2+} and Cd{sup 2+} transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn{sup 2+}- and Cd{sup 2+}-binding sites of MntA are discussed. A second clone containing a low-affinity Cd{sup 2+} transport system was also isolated.

  13. 4-Iodotomoxetine: a novel ligand for serotonin uptake sites.

    PubMed

    Kung, M P; Chumpradit, S; Billings, J; Kung, H

    1992-01-01

    The tomoxetine analog, R-4-iodotomoxetine, binds in vitro to a single site of rat cortical membranes with high affinity (Kd = 0.03 +/- 0.01 nM, n = 4) and can be blocked by a selective serotonin reuptake site inhibitor, paroxetine. The [125I]R-4-iodotomoxetine binding at equilibrium is saturable and is temperature- and Na(+)-dependent. The number of specific [125I]R-4-iodotomoxetine binding sites (Bmax = 356 +/- 20 fmol/mg protein) is similar to that of [3H]citalopram (329 +/- 30 fmol/mg protein), a known serotonin uptake inhibitor. The binding of [125I]R-4-iodotomoxetine is selectively inhibited by several serotonin uptake blockers, and a good correlation is demonstrated between the potency of various drugs to inhibit in vitro binding of [125I]R-4-iodotomoxetine and [3H]citalopram. In addition, lesions performed with the neurotoxin p-chloroamphetamine, which destroys monoamine neurons, including serotonergic neuronal system, result in a 90% reduction of [125I]R-4-iodotomoxetine binding when compared to sham controls. These results indicate that the binding sites labeled by [125I]R-4-iodotomoxetine are associated with the neuronal serotonin uptake sites. However, the in vivo and ex vivo results do not show regional localization corresponding to the distribution of serotonin uptake sites. The nonspecific uptake may be related to this compound's high lipophilicity (octanol-buffer partition coefficient = 1100 - 1400 at pH 7). Although the in vivo properties of [125I]R-4-iodotomoxetine make it an unlikely candidate for mapping serotonin uptake sites with SPECT, the high affinity and selectivity should make it a useful tool for in vitro studies of the serotonin uptake sites.

  14. UPTAKE OF HEAVY METALS IN BATCH SYSTEMS BY A RECYCLED IRON-BEARING MATERIAL

    EPA Science Inventory

    An iron-bearing material deriving from surface finishing operations in the manufacturing of cast-iron components demonstrates potential for removal of heavy metals from aqueous waste streams. Batch isotherm and rate experiments were conducted for uptake of cadmium, zinc, and lead...

  15. [Evaluation of the binding affinity and RNA interference of low-molecular-weight chitosan/siRNA complexes using an imaging system].

    PubMed

    Kawaguchi, Yasuhisa; Okuda, Tomoyuki; Ban, Tatsunori; Danjo, Kazumi; Okamoto, Hirokazu

    2009-04-01

    Chitosan is one of the attractive non-viral carriers for gene delivery including siRNA. However, common chitosan, which has a relatively high molecular weight, is insoluble in water, which might make it difficult to apply clinically. In this study, we investigated the efficacy of low-molecular-weight chitosan (LMWC), which is soluble in water, as a carrier for siRNA delivery. To evaluate the binding affinity and RNA interference (RNAi) of LMWC/siRNA complexes, a multi-well imaging system (IVIS) was adapted. CT26 cells stably expressing firefly luciferase (CT26/Luc cells) were established to evaluate RNAi. Evaluation of RNAi using lipofectamine(TM) 2000 was carried out by employing a luminometer with cell lysis and IVIS without cell lysis. The results were closely correlated, suggesting the advantages of the multi-well imaging system regarding screening, the visualization of results, and nondestructive evaluation. Fluorescence generated by ethidium bromide intercalated in the double strand of siRNA was markedly quenched at a higher ratio of LMWC to siRNA (N/P) and lower pH. Evaluation of the particle size and zeta potential of LMWC/siRNA complexes also indicated the higher binding affinity of LMWC with siRNA. At N/P=300 and pH 6.5, which satisfied the high-level binding affinity of LMWC with siRNA, significantly lower luminescence was detected in CT26/Luc cells treated with LMWC/siRNA compared with those treated with LMWC alone, suggesting the presence of RNAi. These results suggested that LMWC may be an effective carrier for siRNA delivery, and that the multi-well imaging system may be a powerful tool to evaluate the binding affinity and RNAi.

  16. Peri-implant and systemic effects of high-/low-affinity bisphosphonate-hydroxyapatite composite coatings in a rabbit model with peri-implant high bone turnover

    PubMed Central

    2012-01-01

    Background Hydroxyapatite (HA) coatings composed with bisphosphonates (BPs) which have high mineral-binding affinities have been confirmed to successfully enhance implant stability. However, few previous studies focused on HA coatings composed with low-affinity BPs or on systemic effects of locally released BPs. Methods In this long-term study, we developed two kinds of BP-HA composite coatings using either high-affinity BP (alendronate, ALN) or low-affinity BP (risedronate, RIS). Thirty-six rabbits were divided into three groups according to different coating applications (group I: HA, group II: ALN-HA, and group III: RIS-HA). Implants were inserted into the proximal region of the medullary cavity of the left tibiay. At insertion, 2 × 108 wear particles were injected around implants to induce a peri-implant high bone turnover environment. Both local (left tibias) and systemic (right tibias and lumbar vertebrae) inhibitory effect on bone resorption were compared, including bone-implant integration, bone architecture, bone mineral density (BMD), implant stability, and serum levels of bone turnover markers. Results The results indicated that ALN-HA composite coating, which could induce higher bone-implant contact (BIC) ratio, bone mass augmentation, BMD, and implant stability in the peri-implant region, was more potent on peri-implant bone, while RIS-HA composite coating, which had significant systemic effect, was more potent on non-peri-implant bone, especially lumbar vertebrae. Conclusions It is instructive and meaningful to further clinical studies that we could choose different BP-HA composite coatings according to the patient’s condition. PMID:22686414

  17. Nutrient Uptake by Microorganisms according to Kinetic Parameters from Theory as Related to Cytoarchitecture

    PubMed Central

    Button, D. K.

    1998-01-01

    The abilities of organisms to sequester substrate are described by the two kinetic constants specific affinity, a°, and maximal velocity Vmax. Specific affinity is derived from the frequency of substrate-molecule collisions with permease sites on the cell surface at subsaturating concentrations of substrates. Vmax is derived from the number of permeases and the effective residence time, τ, of the transported molecule on the permease. The results may be analyzed with affinity plots (v/S versus v, where v is the rate of substrate uptake), which extrapolate to the specific affinity and are usually concave up. A third derived parameter, the affinity constant KA, is similar to KM but is compared to the specific affinity rather than Vmax  and is defined as the concentration of substrate necessary to reduce the specific affinity by half. It can be determined in the absence of a maximal velocity measurement and is equal to the Michaelis constant for a system with hyperbolic kinetics. Both are taken as a measure of τ, with departure of KM from KA being affected by permease/enzyme ratios. Compilation of kinetic data indicates a 108-fold range in specific affinities and a smaller (103-fold) range in Vmax values. Data suggest that both specific affinities and maximal velocities can be underestimated by protocols which interrupt nutrient flow prior to kinetic analysis. A previously reported inverse relationship between specific affinity and saturation constants was confirmed. Comparisons of affinities with ambient concentrations of substrates indicated that only the largest a°S values are compatible with growth in natural systems. PMID:9729603

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

    SciTech Connect

    Suh, Uhi Rinn

    2015-01-15

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

  19. Oxygen uptake kinetics and energy system's contribution around maximal lactate steady state swimming intensity.

    PubMed

    Pelarigo, Jailton Gregório; Machado, Leandro; Fernandes, Ricardo Jorge; Greco, Camila Coelho; Vilas-Boas, João Paulo

    2017-01-01

    The purpose of this study was to examine the oxygen uptake ([Formula: see text]) kinetics and the energy systems' contribution at 97.5, 100 and 102.5% of the maximal lactate steady state (MLSS) swimming intensity. Ten elite female swimmers performed three-to-five 30 min submaximal constant swimming bouts at imposed paces for the determination of the swimming velocity (v) at 100%MLSS based on a 7 x 200 m intermittent incremental protocol until voluntary exhaustion to find the v associated at the individual anaerobic threshold. [Formula: see text] kinetics (cardiodynamic, primary and slow component phases) and the aerobic and anaerobic energy contributions were assessed during the continuous exercises, which the former was studied for the beginning and second phase of exercise. Subjects showed similar time delay (TD) (mean = 11.5-14.3 s) and time constant (τp) (mean = 13.8-16.3 s) as a function of v, but reduced amplitude of the primary component for 97.5% (35.7 ± 7.3 mL.kg.min-1) compared to 100 and 102.5%MLSS (41.0 ± 7.0 and 41.3 ± 5.4 mL.kg.min-1, respectively), and τp decreased (mean = 9.6-10.8 s) during the second phase of exercise. Despite the slow component did not occur for all swimmers at all swim intensities, when observed it tended to increase as a function of v. Moreover, the total energy contribution was almost exclusively aerobic (98-99%) at 97.5, 100 and 102.5%MLSS. We suggest that well-trained endurance swimmers with a fast TD and τp values may be able to adjust faster the physiological requirements to minimize the amplitude of the slow component appearance, parameter associated with the fatigue delay and increase in exhaustion time during performance, however, these fast adjustments were not able to control the progressive fatigue occurred slightly above MLSS, and most of swimmers reached exhaustion before 30min swam.

  20. Uptake and Disposition of Select Pharmaceuticals by Bluegill Exposed at Constant Concentrations in a Flow-Through Aquatic Exposure System.

    PubMed

    Zhao, Jian-Liang; Furlong, Edward T; Schoenfuss, Heiko L; Kolpin, Dana W; Bird, Kyle L; Feifarek, David J; Schwab, Eric A; Ying, Guang-Guo

    2017-03-29

    The increasing use of pharmaceuticals has led to their subsequent input into and release from wastewater treatment plants, with corresponding discharge into surface waters that may subsequently exert adverse effects upon aquatic organisms. Although the distribution of pharmaceuticals in surface water has been extensively studied, the details of uptake, internal distribution, and kinetic processing of pharmaceuticals in exposed fish have received less attention. For this research, we investigated the uptake, disposition, and toxicokinetics of five pharmaceuticals (diclofenac, methocarbamol, rosuvastatin, sulfamethoxazole, and temazepam) in bluegill sunfish (Lepomis macrochirus) exposed to environmentally relevant concentrations (1000-4000 ng L(-1)) in a flow-through exposure system. Temazepam and methocarbamol were consistently detected in bluegill biological samples with the highest concentrations in bile of 4, 940, and 180 ng g(-1), respectively, while sulfamethoxazole, diclofenac, and rosuvastatin were only infrequently detected. Over 30-day exposures, the relative magnitude of mean concentrations of temazepam and methocarbamol in biological samples generally followed the order: bile ≫ gut > liver and brain > muscle, plasma, and gill. Ranges of bioconcentration factors (BCFs) in different biological samples were 0.71-3960 and 0.13-48.6 for temazepam and methocarbamol, respectively. Log BCFs were statistically positively correlated to pH adjusted log Kow (that is, log Dow), with the strongest relations for liver and brain (r(2) = 0.92 and 0.99, respectively), implying that bioconcentration patterns of ionizable pharmaceuticals depend on molecular status, that is, whether a pharmaceutical is un-ionized or ionized at ambient tissue pH. Methocarbamol and temazepam underwent rapid uptake and elimination in bluegill biological compartments with uptake rate constants (Ku) and elimination rate constants (Ke) at 0.0066-0.0330 h(-1) and 0.0075-0.0384 h(-1), respectively

  1. Dendritic polymer imaging systems for the evaluation of conjugate uptake and cleavage.

    PubMed

    Krüger, Harald R; Nagel, Gregor; Wedepohl, Stefanie; Calderón, Marcelo

    2015-03-07

    Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening.

  2. Simultaneous monitoring of electrical capacitance and water uptake activity of plant root system

    NASA Astrophysics Data System (ADS)

    Cseresnyés, Imre; Takács, Tünde; Füzy, Anna; Rajkai, Kálmán

    2014-10-01

    Pot experiments were designed to test the applicability of root electrical capacitance measurement for in situ monitoring of root water uptake activity by growing cucumber and bean cultivars in a growth chamber. Half of the plants were inoculated with Funneliformis mosseae arbuscular mycorrhizal fungi, while the other half served as non-infected controls. Root electrical capacitance and daily transpiration were monitored during the whole plant ontogeny. Phenology-dependent changes of daily transpiration (related to root water uptake) and root electrical capacitance proved to be similar as they showed upward trends from seedling emergence to the beginning of flowering stage, and thereafter decreased continuously during fruit setting. A few days after arbuscular mycorrhizal fungi-colonization, daily transpiration and root electrical capacitance of infected plants became significantly higher than those of non-infected counterparts, and the relative increment of the measured parameters was greater for the more highly mycorrhizal-dependent bean cultivar compared to that of cucumber. Arbuscular mycorrhizal fungi colonization caused 29 and 69% relative increment in shoot dry mass for cucumbers and beans, respectively. Mycorrhization resulted in 37% increase in root dry mass for beans, but no significant difference was observed for cucumbers. Results indicate the potential of root electrical capacitance measurements for monitoring the changes and differences of root water uptake rate.

  3. Dendritic polymer imaging systems for the evaluation of conjugate uptake and cleavage

    NASA Astrophysics Data System (ADS)

    Krüger, Harald R.; Nagel, Gregor; Wedepohl, Stefanie; Calderón, Marcelo

    2015-02-01

    Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening.Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular

  4. Cloning of the trkAH gene cluster and characterization of the Trk K(+)-uptake system of Vibrio alginolyticus.

    PubMed

    Nakamura, T; Yamamuro, N; Stumpe, S; Unemoto, T; Bakker, E P

    1998-08-01

    K(+)-uptake genes of Vibrio alginolyticus were identified by cloning chromosomal DNA fragments of this organism into plasmids, followed by electroporation and selection for growth at low K+ concentrations of cells of an Escherichia coli strain defective in K+ uptake. A 4.1 kb DNA fragment contained a cluster of three ORFs on the same DNA strand: the previously identified trkA gene, a gene similar to E. coli trkH (V. alginolyticus trkH) and a new gene, orf1, whose function is not clear. Products of V. alginolyticus trkA and orf1 were detected in E. coli minicells. trkA and trkH from V. alginolyticus restored growth at low K+ concentrations of an E. coli delta trkA and an E. coli delta trkG delta trkH strain, respectively, suggesting that these V. alginolyticus genes can functionally replace their E. coli counterparts. In addition, a plasmid containing V. alginolyticus trkAH permitted growth of an E. coli delta sapABCDF (delta trkE) strain at low K+ concentrations. This effect was mainly due to V. alginolyticus trkH and was enhanced by trkA from this organism. Measurements of net K(+)-uptake rates indicated that the presence of these genes in E. coli renders the Trk systems independent of products from the E. coli sapABCDF (trkE) operon.

  5. Conformal field theory on affine Lie groups

    SciTech Connect

    Clubok, Kenneth Sherman

    1996-04-01

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

  6. High-Affinity Glucose Transport in Aspergillus nidulans Is Mediated by the Products of Two Related but Differentially Expressed Genes

    PubMed Central

    Ventura, Luisa; González, Ramón; Ramón, Daniel; MacCabe, Andrew P.

    2014-01-01

    Independent systems of high and low affinity effect glucose uptake in the filamentous fungus Aspergillus nidulans. Low-affinity uptake is known to be mediated by the product of the mstE gene. In the current work two genes, mstA and mstC, have been identified that encode high-affinity glucose transporter proteins. These proteins' primary structures share over 90% similarity, indicating that the corresponding genes share a common origin. Whilst the function of the paralogous proteins is little changed, they differ notably in their patterns of expression. The mstC gene is expressed during the early phases of germination and is subject to CreA-mediated carbon catabolite repression whereas mstA is expressed as a culture tends toward carbon starvation. In addition, various pieces of genetic evidence strongly support allelism of mstC and the previously described locus sorA. Overall, our data define MstC/SorA as a high-affinity glucose transporter expressed in germinating conidia, and MstA as a high-affinity glucose transporter that operates in vegetative hyphae under conditions of carbon limitation. PMID:24751997

  7. Discriminating the molecular pathways during uptake and release on nanoporous host systems

    NASA Astrophysics Data System (ADS)

    Heinke, L.; Kärger, J.

    2009-01-01

    The use of optical techniques, such as interference microscopy, has enabled the observation of transient concentration profiles generated by intracrystalline transport diffusion in nanoporous host materials. In this way, the relevant transport parameters become directly accessible by experiment. We demonstrate that this novel type of information allows one to determine the fraction of molecules which, during molecular uptake (or release), are entering (or leaving) the host crystal through its different faces. By means of numerical calculations of a large amount of different uptake processes, the fraction of the respective fluxes is found to be reasonably calculated by means of the analytical solution for constant (i.e., mean) transport parameters. A more straightforward procedure, based on the assumption that the fraction of the flux is inversely proportional to the time constant calculated with the mean transport parameters, also yields reasonable results. It is shown that even for anisotropic mass transfer with strong concentration dependencies, the divergence from the actual results is generally less than 8%. In most cases such deviations would be below the limits of the accuracy of the determined transport parameters.

  8. Discriminating the molecular pathways during uptake and release on nanoporous host systems.

    PubMed

    Heinke, L; Kärger, J

    2009-01-28

    The use of optical techniques, such as interference microscopy, has enabled the observation of transient concentration profiles generated by intracrystalline transport diffusion in nanoporous host materials. In this way, the relevant transport parameters become directly accessible by experiment. We demonstrate that this novel type of information allows one to determine the fraction of molecules which, during molecular uptake (or release), are entering (or leaving) the host crystal through its different faces. By means of numerical calculations of a large amount of different uptake processes, the fraction of the respective fluxes is found to be reasonably calculated by means of the analytical solution for constant (i.e., mean) transport parameters. A more straightforward procedure, based on the assumption that the fraction of the flux is inversely proportional to the time constant calculated with the mean transport parameters, also yields reasonable results. It is shown that even for anisotropic mass transfer with strong concentration dependencies, the divergence from the actual results is generally less than 8%. In most cases such deviations would be below the limits of the accuracy of the determined transport parameters.

  9. Androgen uptake in the female reproductive system of a nonhuman primate

    SciTech Connect

    Weaker, F.J.; Sheridan, P.J.

    1981-07-15

    The nuclear uptake and retention of /sup 3/H-dehydrotestosterone (/sup 3/H-DHT) were studied in the reproductive organs of the female rhesus monkey by autoradiography. Ovariectomized and adrenalectomized adult female monkeys were injected with 1 microgram of /sup 3/H-DHT (107 Ci/mmole) and were exsanguinated 1 hour later. The uterus, cervix, vagina, and fimbriae of the fallopian tubes were removed and processed for autoradiography. Localization of the androgen was observed in the nuclei of fibroblasts, but not in the nuclei of smooth muscle and tissues of the blood vessels, of all four organs. The uptake of androgen by the epithelium varied for each organ. The simple columnar epithelium of the fallopian tube demonstrated nuclear labeling, but the epithelium of the uterus did not. The cells of the germinative layer of the stratified squamous epithelium of both the cervix and vagina exhibited nuclear concentration of label; however, the simple columnar epithelium of the cervix contiguous with the uterus was free of label. The results suggest that androgens may have a physiologic role in the female reproductive organs, and that the therapeutic effect of drugs such as danazol may be mediated via an androgen receptor.

  10. Is nutrient uptake by plant roots sensitive to the rate of mass flow? Reappraisal of an old chestnut for spatially distributed root systems

    NASA Astrophysics Data System (ADS)

    McMurtrie, R. E.; Näsholm, T.

    2015-12-01

    Numerous modelling papers have considered the contribution of mass flow to nutrient uptake by a single plant root, but few have evaluated its contribution at the scale of an entire root system. We derive equations for nitrogen (N) influx per unit root surface area (J) and N uptake by a single root (U) as functions of soil nitrogen supply, root-length density (RLD) and the velocity of water at the root surface (vo). This model of N uptake by a single root can be used to evaluate N uptake by an entire root system if spatial distributions are known for soil N supply, root biomass and water-uptake velocity. In this paper we show that spatial distributions of RLD and vo can be estimated simultaneously under an optimisation hypothesis (MaxNup, McMurtrie et al. 2012), according to which total root mass and total water uptake are distributed vertically in order to maximise total N uptake. The MaxNup hypothesis leads to equations for optimal vertical profiles of RLD, vo, J and U, maximum rooting depth and the fraction of total available soil nitrogen taken up by the root system. Predicted values of vo are enhanced at depths where nitrogen influx per unit root surface area (J) is more sensitive to vo and diminished at depths where J is less sensitive to vo. Predicted vo is largest at the base of the root system where RLD is lowest, and is smallest in upper soil layers where RLD is highest. MaxNup thus predicts that water uptake will be distributed preferentially to soil depths where it will enhance nitrogen uptake U; this tendency will amplify the sensitivity of total N uptake to total water uptake, compared with strategies where vo is the same for all roots, or where vo is elevated for roots in upper soil layers. Reference McMurtrie RE, Iversen CM, Dewar RC, Medlyn BE, Näsholm T, Pepper DA, Norby RJ. 2012. Plant root distributions and nitrogen uptake predicted by a hypothesis of optimal root foraging. Ecology and Evolution 2: 1235-1250.

  11. An analytical fuzzy-based approach to ?-gain optimal control of input-affine nonlinear systems using Newton-type algorithm

    NASA Astrophysics Data System (ADS)

    Milic, Vladimir; Kasac, Josip; Novakovic, Branko

    2015-10-01

    This paper is concerned with ?-gain optimisation of input-affine nonlinear systems controlled by analytic fuzzy logic system. Unlike the conventional fuzzy-based strategies, the non-conventional analytic fuzzy control method does not require an explicit fuzzy rule base. As the first contribution of this paper, we prove, by using the Stone-Weierstrass theorem, that the proposed fuzzy system without rule base is universal approximator. The second contribution of this paper is an algorithm for solving a finite-horizon minimax problem for ?-gain optimisation. The proposed algorithm consists of recursive chain rule for first- and second-order derivatives, Newton's method, multi-step Adams method and automatic differentiation. Finally, the results of this paper are evaluated on a second-order nonlinear system.

  12. Central Nervous System Delivery of Intranasal Insulin: Mechanisms of Uptake and Effects on Cognition.

    PubMed

    Salameh, Therese S; Bullock, Kristin M; Hujoel, Isabel A; Niehoff, Michael L; Wolden-Hanson, Tami; Kim, Junghyun; Morley, John E; Farr, Susan A; Banks, William A

    2015-01-01

    Intranasal insulin has shown efficacy in patients with Alzheimer's disease (AD), but there are no preclinical studies determining whether or how it reaches the brain. Here, we showed that insulin applied at the level of the cribriform plate via the nasal route quickly distributed throughout the brain and reversed learning and memory deficits in an AD mouse model. Intranasal insulin entered the blood stream poorly and had no peripheral metabolic effects. Uptake into the brain from the cribriform plate was saturable, stimulated by PKC inhibition, and responded differently to cellular pathway inhibitors than did insulin transport at the blood-brain barrier. In summary, these results show intranasal delivery to be an effective way to deliver insulin to the brain.

  13. Hexachlorobenzene uptake by fathead minnows and macroinvertebrates in recirculating sediment/water systems

    SciTech Connect

    Schuytema, G.S.; Krawczyk, D.F.; Griffis, W.L.; Nebeker, A.V.; Robideaux, M.L.

    1990-01-01

    Fathead minnow (Pimephales promelas), the worm, Lumbriculus variegatus, and the amphipods Hyalella azteca and Gammarus lacustris were exposed to hexachlorobenzene (HCB) in water with and without a bed of HCB-spiked sediment. Water HCB concentrations were maintained by recirculation through HCB-packed columns. Recirculating HCB-bound particulates and possibly eroded HCB particulates were an added source of HCB in addition to the sediment bed. Significant bioaccumulation of HCB in animal tissues was observed in water-only and water-sediment exposures. The presence of the HCB-spiked sediment did not result in a significant increase in the uptake of HCB by the organisms, but there was a substantial increase in sediment HCB levels over time. Higher tissue HCB levels in aquaria without sediment suggest that the sediment was a more efficient sink for HCB than the organisms.

  14. Effect of the novel high affinity choline uptake enhancer 2-(2-oxopyrrolidin-1-yl)-N-(2,3-dimethyl-5,6,7,8-tetrahydrofuro[2,3-b] quinolin-4-yl)acetoamide on deficits of water maze learning in rats.

    PubMed

    Bessho, T; Takashina, K; Tabata, R; Ohshima, C; Chaki, H; Yamabe, H; Egawa, M; Tobe, A; Saito, K

    1996-04-01

    The pharmacological properties of MKC-231 (2-(2-oxopyrrolidin-1-yl)-N- (2,3-dimethyl-5,6,7,8-tetrahydrofuro[2,3-b]quinolin-4-yl) acetoamide, CAS 135463-81-9) in comparison with an acetylcholinesterase (AChE) inhibitor, tacrine (CAS 1684-40-8) were studied. MKC-231(10(-10)-10(-6) moll) significantly increased high affinity choline uptake (HACU) when it was incubated with the hippocampal synaptosomes of ethylcholine mustard aziridinium ion (AF64A) treated rats, but not of normal rats. MKC-231 did not affect the AChE activity, [3H]- quinuclidinyl benzilate binding, and [3H]-pirenzepine binding. Oral administration of MKC-231 (1-10 mg/kg) significantly improved the learning deficits in the Morris' water maze of AF64A-treated rats, but it did not produce any significant side effects, like tremor, salivation or hypothermia, which were observed in rats treated with high doses of tacrine. Tacrine (0.1-3 mg/kg p.o.) failed to ameliorate the learning deficits in AF64A-treated rats. These results suggest that MKC-231 is a novel and quite unique compound, which improves the memory impairment induced by AF64A through the enhancement of HACU without any side effects at the effective doses.

  15. A dielectric affinity microbiosensor

    NASA Astrophysics Data System (ADS)

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

    2010-01-01

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

  16. Affinity in electrophoresis.

    PubMed

    Heegaard, Niels H H

    2009-06-01

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

  17. NN-adaptive output feedback tracking control for a class of discrete-time non-affine systems with a dynamic compensator

    NASA Astrophysics Data System (ADS)

    Zhang, Lijun; Zhao, Jiemei; Qi, Xue; Jia, Heming

    2013-06-01

    The problem of tracking control for a class of uncertain non-affine discrete-time nonlinear systems with internal dynamics is addressed. The fixed point theorem is first employed to ensure the control problem in question is solvable and well-defined. Based on it, an adaptive output feedback control scheme based on neural network (NN) is presented. The proposed control algorithm consists of two parts: a dynamic compensator is introduced to stabilise the linear portion of the tracking error system; a single-hidden-layer neural network (SHL NN) approximation mechanism is introduced to cancel the uncertainties resulting from the non-affine function, where the recursive weight update rules of NN estimation are derived from the discrete-time version of Lyapunov control theory. Ultimate boundedness of the error signals is shown through Lyapunov's direct method and the discrete-time version of input-to-state stability (ISS) theory. Finally, a model of automatical underwater vehicle (AUV) is considered to show the effectiveness of the proposed control scheme.

  18. Induction of the high-affinity Na(+)-dependent glutamate transport system XAG- by hypertonic stress in the renal epithelial cell line NBL-1.

    PubMed Central

    Ferrer-Martinez, A; Felipe, A; Nicholson, B; Casado, J; Pastor-Anglada, M; McGivan, J

    1995-01-01

    The high-affinity Na(+)-dependent glutamate transport system XAG- is induced (threefold increase in Vmax. with no change in Km) by hypertonicity in the renal epithelial cell line NBL-1. This effect is dependent on protein synthesis and glycosylation and is accompanied by an increase in EAAC1 mRNA levels. Other Na(+)-dependent transport systems in this cell line do not respond to hypertonic stress. In contrast to recent findings [Ruiz-Montasell, Gomez-Angelats, Casado, Felipe, McGivan and Pastor-Anglada (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 9569-9573] showing that increased system A activity after hyperosmotic shock results from induction of a regulatory protein, this is the first demonstration that hypertonicity may increase the expression of the gene for an amino acid transport protein itself. Images Figure 4 PMID:7654212

  19. Functional supersensitivity of alpha 1-adrenergic system in spinal ventral horn is due to absence of an uptake system and not to postsynaptic change.

    PubMed

    Hirayama, T; Ono, H; Fukuda, H

    1991-01-25

    The excitatory effects of adrenoceptor agonists on ventral horn cells were compared using an extracellular recording technique in spinal cord slices isolated from non-treated and 6-hydroxydopamine (6-OHDA)-treated rats (intracisternally 14 days previously). In spinal cord slices isolated from 6-OHDA-treated rats, the concentration-response curves for the alpha 1-adrenoceptor-mediated facilitatory effects produced by noradrenaline and phenylephrine but not those produced by methoxamine and isoproterenol were shifted to the left. 6-OHDA pretreatment decreased the level and uptake of noradrenaline and increased the number of [3H]prazosin binding sites in the spinal cord. These results suggest that in 6-OHDA-induced denervation, functional supersensitivity of the alpha 1-adrenergic system in the spinal ventral horn is due to absence of an uptake system, and not to postsynaptic change.

  20. TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae.

    PubMed Central

    Gaber, R F; Styles, C A; Fink, G R

    1988-01-01

    We identified a 180-kilodalton plasma membrane protein in Saccharomyces cerevisiae required for high-affinity transport (uptake) of potassium. The gene that encodes this putative potassium transporter (TRK1) was cloned by its ability to relieve the potassium transport defect in trk1 cells. TRK1 encodes a protein 1,235 amino acids long that contains 12 potential membrane-spanning domains. Our results demonstrate the physical and functional independence of the yeast potassium and proton transport systems. TRK1 is nonessential in S. cerevisiae and maps to a locus unlinked to PMA1, the gene that encodes the plasma membrane ATPase. Haploid cells that contain a null allele of TRK1 (trk1 delta) rely on a low-affinity transporter for potassium uptake and, under certain conditions, exhibit energy-dependent loss of potassium, directly exposing the activity of a transporter responsible for the efflux of this ion. Images PMID:3043197

  1. Miniaturized reaction vessel system, method for performing site-specific biochemical reactions and affinity fractionation for use in DNA sequencing

    DOEpatents

    Mirzabekov, Andrei Darievich; Lysov, Yuri Petrovich; Dubley, Svetlana A.

    2000-01-01

    A method for fractionating and sequencing DNA via affinity interaction is provided comprising contacting cleaved DNA to a first array of oligonucleotide molecules to facilitate hybridization between said cleaved DNA and the molecules; extracting the hybridized DNA from the molecules; contacting said extracted hybridized DNA with a second array of oligonucleotide molecules, wherein the oligonucleotide molecules in the second array have specified base sequences that are complementary to said extracted hybridized DNA; and attaching labeled DNA to the second array of oligonucleotide molecules, wherein the labeled re-hybridized DNA have sequences that are complementary to the oligomers. The invention further provides a method for performing multi-step conversions of the chemical structure of compounds comprising supplying an array of polyacrylamide vessels separated by hydrophobic surfaces; immobilizing a plurality of reactants, such as enzymes, in the vessels so that each vessel contains one reactant; contacting the compounds to each of the vessels in a predetermined sequence and for a sufficient time to convert the compounds to a desired state; and isolating the converted compounds from said array.

  2. The role of hysteresis in modeling root water uptake, both for single root and root system models.

    NASA Astrophysics Data System (ADS)

    de Willigen, P.; Heinen, M.

    2009-04-01

    The water retention curve obtained by progressive extraction of water from an initially saturated soil (desorption) differs from that obtained by gradual addition of water to air-dry soil (absorption). This phenomenon is called hysteresis (Koorevaar et al., 1983). Common as its occurrence is, it is often neglected in the modeling of root water uptake. We will present here a model for the transport of water to a single root. The model solves Richard's equation in cylindrical coordinates where the water uptake rate is a function of the root water potential. The occurrence of hysteresis is accounted for by application of the modified dependent domain model developed by Mualem (1984) and used by Kool and Parker (1987). We will discuss the differences in results due to the inclusion of the hysteresis subroutine, when alternate wetting and drying cycles occur. The influence of soil type and transpiration reduction function will be discussed. The findings obtained for the single root model were used to upscale root water uptake to a root system. This is a part of the FUSSIM2 model of Heinen and de Willigen (1998) and Heinen (2001), where water transport in a soil profile is calculated. We will use an example for a soil profile where the root length density decreases exponentially with depth, and where again wetting and drying cycles alternate. References Heinen M., 2001. FUSSIM2: brief description of the simulation model and application to fertigation scenarios. Agronomie 21: 285-296. Heinen, M., and P. de Willigen, 1998. FUSSIM2 A two-dimensional simulation model for water flow, solute transport and root uptake of water and nutrients in partly unsaturated porous media, QASA No. 20, AB-DLO, Wageningen, The Netherlands, 140 p. Kool J.B. and J.C. Parker, 1987. Development and evaluation of closed form expressions for hysteretic soil hydraulic properties. Water Resour. Res. 23: 105 114. Koorevaar P., G. Menelik and C. Dirksen, 1983. Elements of soil physics. Elsevier

  3. Stimulatory effect of taurine on calcium ion uptake in rod outer segments of the rat retina is independent of taurine uptake.

    PubMed

    Militante, J D; Lombardini, J B

    1999-10-01

    Taurine stimulates ATP-dependent Ca(2+) uptake in the rat rod outer segments (ROS). This stimulation has been linked to the function of the cyclic nucleotide-gated cation channel, implying an important physiologic role for taurine in visual signal transduction. Calmodulin (CaM) has been reported to affect taurine transport in the choroid plexus and also to inhibit the cyclic nucleotide-gated channel; thus, the effects of the competitive CaM inhibitors trifluoperazine (TFP) and N-(8-aminooctyl)-5-iodonaphthalene-1-sulfonamide (J-8) were studied on Ca(2+) and taurine uptake in the rat ROS. Pretreatment of the ROS preparation with TFP and J-8 for 5 min before measurement of Ca(2+)-uptake activity produced inhibition of the effects of taurine on ATP-dependent Ca(2+) uptake. Both TFP and J-8 also were effective in inhibiting high-affinity taurine uptake. In both uptake systems, inhibition by TFP was noncompetitive. These data initially suggested that the stimulatory effects of taurine on ATP-dependent Ca(2+) uptake are dependent on taurine uptake. However, competitive inhibition of taurine uptake by guanidinoethane sulfonate did not produce any effect on the stimulatory effects of taurine. Previous studies have proposed that taurine binds directly to the plasma membrane, and our study demonstrated that TFP inhibits taurine binding to the ROS. In addition, our study demonstrated that taurine uptake is unaffected by varying the concentration of Ca(2+) and that the effects of TFP are independent of Ca(2+), suggesting that TFP acts through a CaM-independent mechanism.

  4. Affine dynamics with torsion

    NASA Astrophysics Data System (ADS)

    Gültekin, Kemal

    2016-03-01

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

  5. Lectin affinity electrophoresis.

    PubMed

    Kobayashi, Yuka

    2014-01-01

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

  6. Heterologous expression of BetL, a betaine uptake system, enhances the stress tolerance of Lactobacillus salivarius UCC118.

    PubMed

    Sheehan, Vivien M; Sleator, Roy D; Fitzgerald, Gerald F; Hill, Colin

    2006-03-01

    Given the increasing commercial and clinical relevance of probiotic cultures, improving the technological robustness of what are often process-sensitive cultures is an important biological goal. The nisin-controlled expression system was used to direct the heterologous expression of the listerial betaine uptake system BetL in the probiotic strain Lactobacillus salivarius UCC118. Following nisin induction, strains expressing betL exhibited a significant increase in resistance to several stresses, including elevated osmo-, cryo-, baro-, and chill tolerance, as well as increased resistance to spray- and freeze-drying. The ability to confer additional stress tolerance on a probiotic culture may be an important step in delivering viable cultures for maximal efficacy.

  7. Root controls on water redistribution and carbon uptake in the soil-plant system under current and future climate

    NASA Astrophysics Data System (ADS)

    Volpe, V.; Marani, M.; Albertson, J. D.; Katul, G.

    2013-10-01

    Understanding photosynthesis and plant water management as a coupled process remains an open scientific problem. Current eco-hydrologic models characteristically describe plant photosynthetic and hydraulic processes through ad hoc empirical parameterizations with no explicit accounting for the main pathways over which carbon and water uptake interact. Here, a soil-plant-atmosphere continuum model is proposed that mechanistically couples photosynthesis and transpiration rates, including the main leaf physiological controls exerted by stomata. The proposed approach links the soil-to-leaf hydraulic transport to stomatal regulation, and closes the coupled photosynthesis-transpiration problem by maximizing leaf carbon gain subject to a water loss constraint. The approach is evaluated against field data from a grass site and is shown to reproduce the main features of soil moisture dynamics and hydraulic redistribution. In particular, it is shown that the differential soil drying produced by diurnal root water uptake drives a significant upward redistribution of moisture both through a conventional Darcian flow and through the root system, consistent with observations. In a numerical soil drying experiment, it is demonstrated that more than 50% of diurnal transpiration is supplied by nocturnal upward water redistribution, and some 12% is provided directly through root hydraulic redistribution. For a prescribed leaf area density, the model is then used to diagnose how elevated atmospheric CO2 concentration and increased air temperature jointly impact soil moisture, transpiration, photosynthesis, and whole-plant water use efficiency, along with compensatory mechanisms such as hydraulic lift using several canonical forms of root-density distribution.

  8. Two glycerol uptake systems contribute to the high osmotolerance of Zygosaccharomyces rouxii.

    PubMed

    Dušková, Michala; Ferreira, Célia; Lucas, Cândida; Sychrová, Hana

    2015-08-01

    The accumulation of glycerol is essential for yeast viability upon hyperosmotic stress. Here we show that the osmotolerant yeast Zygosaccharomyces rouxii has two genes, ZrSTL1 and ZrSTL2, encoding transporters mediating the active uptake of glycerol in symport with protons, contributing to cell osmotolerance and intracellular pH homeostasis. The growth of mutants lacking one or both transporters is affected depending on the growth medium, carbon source, strain auxotrophies, osmotic conditions and the presence of external glycerol. These transporters are localised in the plasma membrane, they transport glycerol with similar kinetic parameters and besides their expected involvement in the cell survival of hyperosmotic stress, they surprisingly both contribute to an efficient survival of hypoosmotic shock and to the maintenance of intracellular pH homeostasis under non-stressed conditions. Unlike STL1 in Sa. cerevisiae, the two Z. rouxii STL genes are not repressed by glucose, but their expression and activity are downregulated by fructose and upregulated by non-fermentable carbon sources, with ZrSTL1 being more influenced than ZrSTL2. In summary, both transporters are highly important, though Z. rouxii CBS 732(T) cells do not use external glycerol as a source of carbon.

  9. Uptake and accumulation of antimicrobials, triclocarban and triclosan, by food crops in a hydroponic system.

    PubMed

    Mathews, Shiny; Henderson, Shannon; Reinhold, Dawn

    2014-05-01

    Commonly used in personal care products, triclocarban (TCC) and triclosan (TCS) are two chemicals with antimicrobial properties that have recently been recognized as environmental contaminants with the potential to adversely affect human health. The objective of the study described herein was to evaluate the potential of food crops to uptake TCC and TCS. Eleven food crops, grown in hydroponic nutrient media, were exposed to a mixture of 500 μg L(-1) TCC and TCS. After 4 weeks of exposure, roots accumulated 86-1,350 mg kg(-1) of antimicrobials and shoots had accumulated 0.33-5.35 mg kg(-1) of antimicrobials. Translocation from roots to shoots was less than 1.9 % for TCC and 3.7 % for TCS, with the greatest translocation for TCC observed for pepper, celery, and asparagus and for TCS observed for cabbage, broccoli, and asparagus. For edible tuber- or bulb-producing crops, the concentrations of both TCC and TCS were lower in the tubers than in the roots. Exposure calculations using national consumption data indicated that the average exposure to TCC and TCS from eating contaminated crops was substantially less than the exposure expected to cause adverse effects, but exceeded the predicted exposure from drinking water. Exposure to antimicrobials through food crops would be substantially reduced through limiting consumption of beets and onions.

  10. Affinity partitioning of albumin and alpha-fetoprotein in an aqueous two-phase system using poly(ethylene glycol)-bound triazine dyes.

    PubMed

    Birkenmeier, G; Usbeck, E; Kopperschläger, G

    1984-01-01

    Human albumin and alpha-fetoprotein are partitioned in an aqueous two-phase system composed of 10% (w/w) Dextran and 7.5% (w/w) poly(ethylene glycol). When a small amount of poly(ethylene glycol) is replaced by Cibacron Blue F3G-A-liganded poly(ethylene glycol) the partition coefficient, K, of albumin increases by the factor of about 4000 whereas the K value of alpha-fetoprotein undergoes only a small change. The change of the partition coefficient in a logarithmic scale induced by increasing dye-polymer concentrations turned out as a useful measure for the affinity of albumin and alpha-fetoprotein to the dyes. The effect of pH and salt concentration on the affinity partition of albumin and alpha-fetoprotein is demonstrated. The partition of the two proteins in presence of Cibacron Blue F3G-A-liganded poly(ethylene glycol) is compared with seven other triazine dye-poly(ethylene glycol) derivatives.

  11. Protein Delivery System Containing a Nickel-Immobilized Polymer for Multimerization of Affinity-Purified His-Tagged Proteins Enhances Cytosolic Transfer.

    PubMed

    Postupalenko, Viktoriia; Desplancq, Dominique; Orlov, Igor; Arntz, Youri; Spehner, Danièle; Mely, Yves; Klaholz, Bruno P; Schultz, Patrick; Weiss, Etienne; Zuber, Guy

    2015-09-01

    Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.

  12. Identification and functional assay of the interaction motifs in the partner protein OsNAR2.1 of the two-component system for high-affinity nitrate transport.

    PubMed

    Liu, Xiaoqin; Huang, Daimin; Tao, Jinyuan; Miller, Anthony J; Fan, Xiaorong; Xu, Guohua

    2014-10-01

    A partner protein, NAR2, is essential for high-affinity nitrate transport of the NRT2 protein in plants. However, the NAR2 motifs that interact with NRT2s for their plasma membrane (PM) localization and nitrate transporter activity have not been functionally characterized. In this study, OsNAR2.1 mutations with different carbon (C)-terminal deletions and nine different point mutations in the conserved regions of NAR2 homologs in plants were generated to explore the essential motifs involved in the interaction with OsNRT2.3a. Screening using the membrane yeast two-hybrid system and Xenopus oocytes for nitrogen-15 ((15)N) uptake demonstrated that either R100G or D109N point mutations impaired the OsNAR2.1 interaction with OsNRT2.3a. Western blotting and visualization using green fluorescent protein fused to either the N- or C-terminus of OsNAR2.1 indicated that OsNAR2.1 is expressed in both the PM and cytoplasm. The split-yellow fluorescent protein (YFP)/BiFC analyses indicated that OsNRT2.3a was targeted to the PM in the presence of OsNAR2.1, while either R100G or D109N mutation resulted in the loss of OsNRT2.3a-YFP signal in the PM. Based on these results, arginine 100 and aspartic acid 109 of the OsNAR2.1 protein are key amino acids in the interaction with OsNRT2.3a, and their interaction occurs in the PM but not cytoplasm.

  13. Novel peptide VIP-TAT with higher affinity for PAC1 inhibited scopolamine induced amnesia.

    PubMed

    Yu, Rongjie; Yang, Yanxu; Cui, Zekai; Zheng, Lijun; Zeng, Zhixing; Zhang, Huahua

    2014-10-01

    A novel peptide VIP-TAT with a cell penetrating peptide TAT at the C-terminus of VIP was constructed and prepared using intein mediated purification with an affinity chitin-binding tag (IMPACT) system to enhance the brain uptake efficiency for the medical application in central nervous system. It was found by labeling VIP-TAT and VIP with fluorescein isothiocyanate (FITC) that the extension with TAT increased the brain uptake efficiency of VIP-TAT significantly. Then short-term and long-term treatment with scopolamine (Scop) was used to evaluate the effect of VIP-TAT or VIP on Scop induced amnesia. Both short-term and long-term administration of VIP-TAT inhibited the latent time reduction in step-through test induced by Scop significantly, but long-term administration of VIP aggravated the Scop induced amnesia. Long-term i.p. injection of VIP-TAT was shown to have positive effect by inhibiting the oxidative damage, apoptosis and the cholinergic system activity reduction that induced by Scop, while VIP exerted negative effect in brain opposite to that in periphery system. The in vitro data showed that VIP-TAT had not only protective but also proliferative effect on Neuro2a cells which was inhibited by PAC1 antagonist PACAP(6-38). Competition binding assay and cAMP assay confirmed that VIP-TAT had higher affinity and activation for PAC1 than VIP. So it was concluded that the significantly stronger protective effect of VIP-TAT against Scop induced amnesia than VIP was due to (1) the enhanced brain uptake efficiency of VIP-TAT and (2) the increased affinity and activation of VIP-TAT for receptor PAC1.

  14. Genetically designed biomolecular capping system for mesoporous silica nanoparticles enables receptor-mediated cell uptake and controlled drug release

    NASA Astrophysics Data System (ADS)

    Datz, Stefan; Argyo, Christian; Gattner, Michael; Weiss, Veronika; Brunner, Korbinian; Bretzler, Johanna; von Schirnding, Constantin; Torrano, Adriano A.; Spada, Fabio; Vrabel, Milan; Engelke, Hanna; Bräuchle, Christoph; Carell, Thomas; Bein, Thomas

    2016-04-01

    Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the development of precisely controllable and highly modular theranostic systems.Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the

  15. Maltose uptake by the novel ABC transport system MusEFGK2I causes increased expression of ptsG in Corynebacterium glutamicum.

    PubMed

    Henrich, Alexander; Kuhlmann, Nora; Eck, Alexander W; Krämer, Reinhard; Seibold, Gerd M

    2013-06-01

    The Gram-positive Corynebacterium glutamicum efficiently metabolizes maltose by a pathway involving maltodextrin and glucose formation by 4-α-glucanotransferase, glucose phosphorylation by glucose kinases, and maltodextrin degradation via maltodextrin phosphorylase and α-phosphoglucomutase. However, maltose uptake in C. glutamicum has not been investigated. Interestingly, the presence of maltose in the medium causes increased expression of ptsG in C. glutamicum by an unknown mechanism, although the ptsG-encoded glucose-specific EII permease of the phosphotransferase system itself is not required for maltose utilization. We identified the maltose uptake system as an ABC transporter encoded by musK (cg2708; ATPase subunit), musE (cg2705; substrate binding protein), musF (cg2704; permease), and musG (cg2703; permease) by combination of data obtained from characterization of maltose uptake and reanalyses of transcriptome data. Deletion of the mus gene cluster in C. glutamicum Δmus abolished maltose uptake and utilization. Northern blotting and reverse transcription-PCR experiments revealed that musK and musE are transcribed monocistronically, whereas musF and musG are part of an operon together with cg2701 (musI), which encodes a membrane protein of unknown function with no homologies to characterized proteins. Characterization of growth and [(14)C]maltose uptake in the musI insertion strain C. glutamicum IMcg2701 showed that musI encodes a novel essential component of the maltose ABC transporter of C. glutamicum. Finally, ptsG expression during cultivation on different carbon sources was analyzed in the maltose uptake-deficient strain C. glutamicum Δmus. Indeed, maltose uptake by the novel ABC transport system MusEFGK2I is required for the positive effect of maltose on ptsG expression in C. glutamicum.

  16. The E. coli pET expression system revisited-mechanistic correlation between glucose and lactose uptake.

    PubMed

    Wurm, David Johannes; Veiter, Lukas; Ulonska, Sophia; Eggenreich, Britta; Herwig, Christoph; Spadiut, Oliver

    2016-10-01

    Therapeutic monoclonal antibodies are mainly produced in mammalian cells to date. However, unglycosylated antibody fragments can also be produced in the bacterium Escherichia coli which brings several advantages, like growth on cheap media and high productivity. One of the most popular E. coli strains for recombinant protein production is E. coli BL21(DE3) which is usually used in combination with the pET expression system. However, it is well known that induction by isopropyl β-D-1-thiogalactopyranoside (IPTG) stresses the cells and can lead to the formation of insoluble inclusion bodies. In this study, we revisited the pET expression system for the production of a novel antibody single-chain variable fragment (scFv) with the goal of maximizing the amount of soluble product. Thus, we (1) investigated whether lactose favors the recombinant production of soluble scFv compared to IPTG, (2) investigated whether the formation of soluble product can be influenced by the specific glucose uptake rate (q s,glu) during lactose induction, and (3) determined the mechanistic correlation between the specific lactose uptake rate (q s,lac) and q s,glu. We found that lactose induction gave a much greater amount of soluble scFv compared to IPTG, even when the growth rate was increased. Furthermore, we showed that the production of soluble protein could be tuned by varying q s,glu during lactose induction. Finally, we established a simple model describing the mechanistic correlation between q s,lac and q s,glu allowing tailored feeding and prevention of sugar accumulation. We believe that this mechanistic model might serve as platform knowledge for E. coli.

  17. Comparison between Arabidopsis and Rice for Main Pathways of K(+) and Na(+) Uptake by Roots.

    PubMed

    Nieves-Cordones, Manuel; Martínez, Vicente; Benito, Begoña; Rubio, Francisco

    2016-01-01

    K(+) is an essential macronutrient for plants. It is acquired by specific uptake systems located in roots. Although the concentrations of K(+) in the soil solution are widely variable, K(+) nutrition is secured by uptake systems that exhibit different affinities for K(+). Two main systems have been described for root K(+) uptake in several species: the high-affinity HAK5-like transporter and the inward-rectifier AKT1-like channel. Other unidentified systems may be also involved in root K(+) uptake, although they only seem to operate when K(+) is not limiting. The use of knock-out lines has allowed demonstrating their role in root K(+) uptake in Arabidopsis and rice. Plant adaptation to the different K(+) supplies relies on the finely tuned regulation of these systems. Low K(+)-induced transcriptional up-regulation of the genes encoding HAK5-like transporters occurs through a signal cascade that includes changes in the membrane potential of root cells and increases in ethylene and reactive oxygen species concentrations. Activation of AKT1 channels occurs through phosphorylation by the CIPK23/CBL1 complex. Recently, activation of the Arabidopsis HAK5 by the same complex has been reported, pointing to CIPK23/CBL as a central regulator of the plant's adaptation to low K(+). Na(+) is not an essential plant nutrient but it may be beneficial for some plants. At low concentrations, Na(+) improves growth, especially under K(+) deficiency. Thus, high-affinity Na(+) uptake systems have been described that belong to the HKT and HAK families of transporters. At high concentrations, typical of saline environments, Na(+) accumulates in plant tissues at high concentrations, producing alterations that include toxicity, water deficit and K(+) deficiency. Data concerning pathways for Na(+) uptake into roots under saline conditions are still scarce, although several possibilities have been proposed. The apoplast is a significant pathway for Na(+) uptake in rice grown under salinity

  18. Comparison between Arabidopsis and Rice for Main Pathways of K+ and Na+ Uptake by Roots

    PubMed Central

    Nieves-Cordones, Manuel; Martínez, Vicente; Benito, Begoña; Rubio, Francisco

    2016-01-01

    K+ is an essential macronutrient for plants. It is acquired by specific uptake systems located in roots. Although the concentrations of K+ in the soil solution are widely variable, K+ nutrition is secured by uptake systems that exhibit different affinities for K+. Two main systems have been described for root K+ uptake in several species: the high-affinity HAK5-like transporter and the inward-rectifier AKT1-like channel. Other unidentified systems may be also involved in root K+ uptake, although they only seem to operate when K+ is not limiting. The use of knock-out lines has allowed demonstrating their role in root K+ uptake in Arabidopsis and rice. Plant adaptation to the different K+ supplies relies on the finely tuned regulation of these systems. Low K+-induced transcriptional up-regulation of the genes encoding HAK5-like transporters occurs through a signal cascade that includes changes in the membrane potential of root cells and increases in ethylene and reactive oxygen species concentrations. Activation of AKT1 channels occurs through phosphorylation by the CIPK23/CBL1 complex. Recently, activation of the Arabidopsis HAK5 by the same complex has been reported, pointing to CIPK23/CBL as a central regulator of the plant’s adaptation to low K+. Na+ is not an essential plant nutrient but it may be beneficial for some plants. At low concentrations, Na+ improves growth, especially under K+ deficiency. Thus, high-affinity Na+ uptake systems have been described that belong to the HKT and HAK families of transporters. At high concentrations, typical of saline environments, Na+ accumulates in plant tissues at high concentrations, producing alterations that include toxicity, water deficit and K+ deficiency. Data concerning pathways for Na+ uptake into roots under saline conditions are still scarce, although several possibilities have been proposed. The apoplast is a significant pathway for Na+ uptake in rice grown under salinity conditions, but in other plant species

  19. Stabilization of affinity-tagged recombinant protein during/after its production in a cell-free system using wheat-germ extract.

    PubMed

    Kawarasaki, Yasuaki; Yamada, Yasuhiro; Ichimori, Maki; Shinbata, Tomoya; Kohda, Katsunori; Nakano, Hideo; Yamane, Tsuneo

    2003-01-01

    We found that the affinity tag fused to the carboxyl (C-) terminal of a single-chain Fv (scFv) antibody was proteolytically degraded in a wheat germ cell-free protein synthesis system. The addition of two extra residues of glycine to the tail of the cMyc tag significantly increased the stability of the tag, suggesting that wheat endogenous carboxypeptidase(s) play a primary role in the C-terminal tag-specific degradation. In addition to the modification of the tag sequence, addition of diisopropyl fluorophosphate, which is known as an inhibitor of carboxypeptidases, prevented the cMyc tag sequence degradation. The effects of other protease inhibitors on the translation reaction and stability of the synthesized protein are also reported.

  20. GD-aided IOL (input-output linearisation) controller for handling affine-form nonlinear system with loose condition on relative degree

    NASA Astrophysics Data System (ADS)

    Zhang, Yunong; Chen, Dechao; Jin, Long; Zhang, Yinyan; Yin, Yonghua

    2016-04-01

    Input-output linearisation (IOL) may encounter a singularity problem when applied to the tracking control of affine-form nonlinear system (AFNS), which may not have a well-defined relative degree. The singularity problem has occurred in the area of control for decades. In this paper, we incorporate the gradient dynamics (GD) into IOL, which leads to the GD-aided IOL method to solve the singularity problem, with the proposition of the loose condition on relative degree. Moreover, detailed theoretical analyses on tracking-error bound and convergence performance of the corresponding GD-aided IOL controller are presented. Simulations and comparisons substantiate that the proposed GD-aided IOL method is capable of completing the tracking-control task and conquering the singularity encountered in the AFNS.

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

    PubMed

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

    2010-12-23

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

  2. A method to obtain correct standard uptake values in Pinnacle treatment planning system for target volume delineation

    PubMed Central

    Salehzahi, Farshid; Tse, Jason; Lee, Jonathan; Selvaraj, Jothybasu

    2016-01-01

    Standardized uptake value (SUV) is an advanced tool for quantitative tumor identification and metabolic target volume delineation (TVD) in diagnostic and therapeutic settings. It is thus important to establish a quality assured process to maintain the traceability of data correctly by positron emission tomography (PET) systems. Patient administration of 18fluoro-deoxy-glucose is increasingly delivered by automated infusion systems (AISs). Whenever AIS is used, its accuracy and traceability measurement need verification. In addition, it was observed that the unreproducible SUV displayed in PET and the treatment planning system (TPS) may cause grave concerns for radiation oncologists for TVD. This concern may complicate the correlation of TVD on PET and TPS and their clinical reporting. The SUV traceability was established from the PET system to AIS. Its accuracy was verified by cross-referencing to the reference dose calibrator traceable to a primary standard. The SUV values were converted in TPS using the in-house “clinical tool” to be identical as in PET, to allow radiation oncologists to use SUV confidently. The outcome of this study enables the clinical groups to rely on the correct SUV values displayed on the TPS and to improve the quality of care for patients in clinical procedures. PMID:28144116

  3. An in situ optical imaging system for measuring lipid uptake, vessel contraction, and lymph flow in small animal lymphatic vessels

    NASA Astrophysics Data System (ADS)

    Kassis, Timothy; Weiler, Michael J.; Dixon, J. Brandon

    2012-03-01

    All dietary lipids are transported to venous circulation through the lymphatic system, yet the underlying mechanisms that regulate this process remain unclear. Understanding how the lymphatics functionally respond to changes in lipid load is important in the diagnosis and treatment of lipid and lymphatic related diseases such as obesity, hypercholesterolemia, and lymphedema. Therefore, we sought to develop an in situ imaging system to quantify and correlate lymphatic function as it relates to lipid transport. A custom-built optical set-up provides us with the capability of dual-channel imaging of both high-speed bright-field video and fluorescence simultaneously. This is achieved by dividing the light path into two optical bands. Utilizing high-speed and back-illuminated CCD cameras and post-acquisition image processing algorithms, we have the potential quantify correlations between vessel contraction, lymph flow and lipid concentration of mesenteric lymphatic vessels in situ. Local flow velocity is measured through lymphocyte tracking, vessel contraction through measurements of the vessel walls and lipid uptake through fluorescence intensity tracking of a fluorescent long chain fatty acid analogue, Bodipy FL C16. This system will prove to be an invaluable tool for both scientists studying lymphatic function in health and disease, and those investigating strategies for targeting the lymphatic system with orally delivered drugs.

  4. Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum.

    PubMed Central

    Farwick, M; Siewe, R M; Krämer, R

    1995-01-01

    Osmoregulatory uptake of glycine betaine in whole cells of Corynebacterium glutamicum ATCC 13032 (wild type) was studied. The cells actively take up glycine betaine when they are osmotically shocked. The total accumulation and uptake rate were dependent on the osmotic strength of the medium. Kinetic analysis revealed a high-affinity transport system (Km, 8.6 +/- 0.4 microM) with high maximum velocity (110 nmol.min-1.mg [dry weight]-1). Glycine betaine functioned as a compatible solute when added to the medium and allowed growth at an otherwise inhibitory osmotic strength of 1.5 M NaCl. Proline and ectoine could also be used as osmoprotectants. Glycine betaine is neither synthesized nor metabolized by C. glutamicum. The glycine betaine transport system is constitutively expressed at a basal level of activity. It can be induced up to eightfold by osmotic stress and is strongly regulated at the level of activity. The transport system is highly specific and has its pH optimum in the slightly alkaline range at about pH 8. The uptake of the zwitterionic glycine betaine is mediated by a secondary symport system coupled to cotransport of at least two Na+ ions. It is thus driven both by the membrane potential and the Na+ gradient. An extremely high accumulation (internal/external) ratio of up to 4 x 10(6) was measured, which represents the highest accumulation ratio observed for any transport system. PMID:7642496

  5. Dual-channel in-situ optical imaging system for quantifying lipid uptake and lymphatic pump function

    NASA Astrophysics Data System (ADS)

    Kassis, Timothy; Kohan, Alison B.; Weiler, Michael J.; Nipper, Matthew E.; Cornelius, Rachel; Tso, Patrick; Brandon Dixon, J.

    2012-08-01

    Nearly all dietary lipids are transported from the intestine to venous circulation through the lymphatic system, yet the mechanisms that regulate this process remain unclear. Elucidating the mechanisms involved in the functional response of lymphatics to changes in lipid load would provide valuable insight into recent implications of lymphatic dysfunction in lipid related diseases. Therefore, we sought to develop an in situ imaging system to quantify and correlate lymphatic function as it relates to lipid transport. The imaging platform provides the capability of dual-channel imaging of both high-speed bright-field video and fluorescence simultaneously. Utilizing post-acquisition image processing algorithms, we can quantify correlations between vessel pump function, lymph flow, and lipid concentration of mesenteric lymphatic vessels in situ. All image analysis is automated with customized LabVIEW virtual instruments; local flow is measured through lymphocyte velocity tracking, vessel contraction through measurements of the vessel wall displacement, and lipid uptake through fluorescence intensity tracking of an orally administered fluorescently labelled fatty acid analogue, BODIPY FL C16. This system will prove to be an invaluable tool for scientists studying intestinal lymphatic function in health and disease, and those investigating strategies for targeting the lymphatics with orally delivered drugs to avoid first pass metabolism.

  6. Disease activity in systemic lupus erythematosus is associated with an altered expression of low-affinity Fcγ receptors and costimulatory molecules on dendritic cells

    PubMed Central

    Carreño, Leandro J; Pacheco, Rodrigo; Gutierrez, Miguel A; Jacobelli, Sergio; Kalergis, Alexis M

    2009-01-01

    Dendritic cells (DCs) play a pivotal role in the interface between immunity and maintenance of peripheral tolerance. The capture of immunoglobulin G (IgG)-containing immune complexes (ICs) by low-affinity Fcγ receptors (FcγRs) expressed on DCs may influence the immunogenicity/tolerogenicity of these cells, depending on the activating/inhibitory potential of FcγRs. Because of the key role that low-affinity FcγRs play in determining the magnitude of the response in IC-driven inflammation, these receptors are likely to play a role in autoimmune diseases, such as systemic lupus erythematosus (SLE). To evaluate if an altered expression of costimulatory molecules and/or FcγRs could account for disease severity, we evaluated the expression of these molecules on immature and mature DCs derived from peripheral blood monocytes of SLE patients and healthy donors. Our results show an increased expression of the costimulatory molecules CD40 and CD86. Furthermore, the ratio of CD86/CD80 is higher in SLE patients compared with healthy donors. Conversely, while the expression of activating FcγRs was higher on DCs from SLE patients, expression of inhibitory FcγRs was lower, compared with DCs obtained from healthy donors. As a result, the activating to inhibitory FcγR ratio was significantly higher in DCs from SLE patients. The altered ratio of activating/inhibitory FcγRs on mature DCs showed a significant correlation with the activity of SLE, as determined by the SLE Disease Activity Index (SLEDAI) score. We postulate that the increased ratio of activating/inhibitory FcγRs expressed on DCs from SLE patients can contribute to the failure of peripheral tolerance in the IC-mediated phase of autoimmune pathogenesis. PMID:20067533

  7. Affine Sphere Relativity

    NASA Astrophysics Data System (ADS)

    Minguzzi, E.

    2017-03-01

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

  8. Measuring the serotonin uptake site using (/sup 3/H)paroxetine--a new serotonin uptake inhibitor

    SciTech Connect

    Gleiter, C.H.; Nutt, D.J.

    1988-01-01

    Serotonin is an important neurotransmitter that may be involved in ethanol preference and dependence. It is possible to label the serotonin uptake site in brain using the tricyclic antidepressant imipramine, but this also binds to other sites. We have used the new high-affinity uptake blocker paroxetine to define binding to this site and report it to have advantages over imipramine as a ligand.

  9. /sup 3/H-imipramine uptake into rat striatal slices and imipramine-induced /sup 3/H-dopamine efflux

    SciTech Connect

    Saito, R.; Kawasaki, K.; Ono, N.; Kamiya, H.

    1983-04-01

    The effect of imipramine on spontaneous efflux of radiolabelled dopamine (DA) from slices of rat striatum was examined by a superfusion method. Imipramine at concentrations of 10 - 100 microM enhanced the efflux of DA accumulated in a high-affinity uptake system in a concentration-dependent manner. This efflux of /sup 3/H-DA was not affected by conditions (Ca/sup 2 +/-free medium, 100 microM bretylium and 30 microM tetrodotoxin) which inhibited the release of /sup 3/H-DA by electrical stimulation. Furthermore, this imipramine-induced /sup 3/H-DA efflux was temperature-dependent. The uptake of /sup 3/H-imipramine into striatal slices was determined. This uptake was concentration- and temperature-dependent and increased linearly. These results are discussed in relation to the hypothesis that /sup 3/H-DA efflux by imipramine is connected with uptake of imipramine.

  10. Uptake of stormwater nitrogen in bioretention systems demonstrated from 15N tracer techniques

    NASA Astrophysics Data System (ADS)

    Houdeshel, D.; Hultine, K. R.; Pomeroy, C. A.

    2012-12-01

    Bioretention stormwater management systems are engineered ecosystems that capture urban stormwater in order to reduce the harmful effects of stormwater pollution on receiving waters. Bioretention systems have been shown to be effective at reducing the volume of runoff, and thereby reduce the nutrient loading to receiving waters from urban areas. However, little work has been done to evaluate the treatment processes that are responsible for reductions in effluent nitrogen (N). We hypothesize that the pulses of inorganic nitrogen associated with urban runoff events are captured in the plat tissues within these systems and not adsorbed to the soil media, thus creating a long-term, sustainable treatment approach to reducing the total nutrient loading to receiving waters. Nitrogen treatment performance was tested on two bioretention systems in Salt Lake City, UT: 1) an upland native community that does not require irrigation in semi-arid climates, and 2) a wetland community that requires 250 l of daily irrigation to offset the relatively high evaporative demand in the region. Each cell is sized to treat a 2.5 cm storm from a 140 m2 impervious surface: the area of the bioretention system is 10 m2. To test the N removal performance of each system, runoff events were simulated to represent an average precipitation regime using a synthetic stormwater blend starting in January, 2012. Effluent was collected from an underdrain and analyzed for total nitrogen (TN); mass removal was calculated for each month by subtracting the TN mass added to the garden minus the TN mass that flowed out of the garden. To test the hypothesis that plants assimilate stormwater N, 4 g of 100 atom% 15N NH4NO3 tracer was used as the N source in the synthetic stormwater during the first 2,000 l synthetic storm event in May. This isotopic label was calculated to enrich the total N pool of each garden to 100‰ 15N/14Nair. New growth was harvested from each plant in both cells and analyzed for 15N

  11. Characterization of cooperative bicarbonate uptake into chloroplast stroma in the green alga Chlamydomonas reinhardtii

    PubMed Central

    Yamano, Takashi; Sato, Emi; Iguchi, Hiro; Fukuda, Yuri; Fukuzawa, Hideya

    2015-01-01

    The supply of inorganic carbon (Ci; CO2 and HCO3–) is an environmental rate-limiting factor in aquatic photosynthetic organisms. To overcome the difficulty in acquiring Ci in limiting-CO2 conditions, an active Ci uptake system called the CO2-concentrating mechanism (CCM) is induced to increase CO2 concentrations in the chloroplast stroma. An ATP-binding cassette transporter, HLA3, and a formate/nitrite transporter homolog, LCIA, are reported to be associated with HCO3– uptake [Wang and Spalding (2014) Plant Physiol 166(4):2040–2050]. However, direct evidence of the route of HCO3– uptake from the outside of cells to the chloroplast stroma remains elusive owing to a lack of information on HLA3 localization and comparative analyses of the contribution of HLA3 and LCIA to the CCM. In this study, we revealed that HLA3 and LCIA are localized to the plasma membrane and chloroplast envelope, respectively. Insertion mutants of HLA3 and/or LCIA showed decreased Ci affinities/accumulation, especially in alkaline conditions where HCO3– is the predominant form of Ci. HLA3 and LCIA formed protein complexes independently, and the absence of LCIA decreased HLA3 mRNA accumulation, suggesting the presence of unidentified retrograde signals from the chloroplast to the nucleus to maintain HLA3 mRNA expression. Furthermore, although single overexpression of HLA3 or LCIA in high CO2 conditions did not affect Ci affinity, simultaneous overexpression of HLA3 with LCIA significantly increased Ci affinity/accumulation. These results highlight the HLA3/LCIA-driven cooperative uptake of HCO3– and a key role of LCIA in the maintenance of HLA3 stability as well as Ci affinity/accumulation in the CCM. PMID:26015566

  12. Ethanol intake and sup 3 H-serotonin uptake I: A study in Fawn-Hooded rats

    SciTech Connect

    Daoust, M.; Compagnon, P.; Legrand, E.; Boucly, P. )

    1991-01-01

    Ethanol intake and synaptosomal {sup 3}H-serotonin uptake were studied in male Fawn-Hooded and Sprague-Dawley rats. Fawn-Hooded rats consumed more alcohol and more water than Sprague-Dawley rats. Plasma alcohol levels of Sprague-Dawley rats were not detectable but were about 5 mg/dl in Fawn-Hooded rats. Ethanol intake increased the Vmax of serotonin uptake in Fawn-Hooded rats in hippocampus and cortex, but not in thalamus. In Fawn-Hooded rats, serotonin uptake (Vmax) was higher than in Sprague-Dawley rats cortex. Ethanol intake reduced the Vmax of serotonin uptake in Fawn-Hooded rats in hippocampus and cortex. In cortex, the carrier affinity for serotonin was increased in alcoholized Fawn-Hooded rats. These results indicate that synaptosomal {sup 3}H-serotonin uptake is affected by ethanol intake. In Fawn-Hooded rats, high ethanol consumption is associated with high serotonin uptake. In rats presenting high serotonin uptake, alcoholization reduces {sup 3}H-serotonin internalization in synaptosomes, indicating a specific sensitivity to alcohol intake of serotonin uptake system.

  13. Uptake and effectiveness of systemic insecticides as influenced by application technique

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of systemic neonicotinoid insecticides such as Imidacloprid and Thiamethoxam have been shown to be effective against different types of insects including sucking insect like aphids, whiteflies, scales and mealybugs. The most common forms of application of these neonicotinoid insecticides ha...

  14. Determination of foliar uptake of water droplets on waxy leaves in controlled environmental system

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Pertinent techniques for determination of plant cuticle permeability are needed to select proper doses of active ingredients and spray additives to improve pesticide application efficacy. A controlled environmental system with 100% relative humidity was developed for direct measurements of foliar up...

  15. Understanding Arsenic Dynamics in Agronomic Systems to Predict and Prevent Uptake by Crop Plants

    EPA Science Inventory

    This review is on arsenic in agronomic systems, and covers processes that influence the entry of arsenic into the human food supply. The scope is from sources of arsenic (natural and anthropogenic) in soils, biogeochemical and rhizosphere processes that control arsenic speciatio...

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-11-16

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

  18. Diatoms Si uptake capacity drives carbon export in coastal upwelling systems

    NASA Astrophysics Data System (ADS)

    Abrantes, Fatima; Cermeno, Pedro; Lopes, Cristina; Romero, Oscar; Matos, Lélia; Van Iperen, Jolanda; Rufino, Marta; Magalhães, Vitor

    2016-07-01

    Coastal upwelling systems account for approximately half of global ocean primary production and contribute disproportionately to biologically driven carbon sequestration. Diatoms, silica-precipitating microalgae, constitute the dominant phytoplankton in these productive regions, and their abundance and assemblage composition in the sedimentary record is considered one of the best proxies for primary production. The study of the sedimentary diatom abundance (SDA) and total organic carbon content (TOC) in the five most important coastal upwelling systems of the modern ocean (Iberia-Canary, Benguela, Peru-Humboldt, California, and Somalia-Oman) reveals a global-scale positive relationship between diatom production and organic carbon burial. The analysis of SDA in conjunction with environmental variables of coastal upwelling systems such as upwelling strength, satellite-derived net primary production, and surface water nutrient concentrations shows different relations between SDA and primary production on the regional scale. On the global scale, SDA appears modulated by the capacity of diatoms to take up silicic acid, which ultimately sets an upper limit to global export production in these ocean regions.

  19. 2-Methylisoborneol and geosmin uptake by organic sludge derived from a recirculating aquaculture system.

    PubMed

    Guttman, Lior; van Rijn, Jaap

    2009-02-01

    In a previous study on a recirculating fish culture system, levels of geosmin and 2-methylisoborneol were found to decrease when culture water was recirculated through the anaerobic sludge digestion treatment stage of the system. This finding led us to the present study in which the geosmin and 2-methylisoborneol removal capacity of the sludge derived from this treatment stage was examined in vitro. It was found that reduction of off-flavor compounds by the sludge was mediated by both chemical/physical sorption and biological degradation. At geosmin and 2-methylisoborneol concentrations within the range of those experienced in fish culture systems, chemical/physical sorption by the sludge was found to account for a 93% reduction in geosmin and a 79% reduction in 2-methylisoborneol from the overlying water within 48h of incubation. Combined with the biological degradation taking place in the sludge, a complete removal of these compounds from the water phase occurred within 9 days of incubation. By means of repeated washing of the geosmin and 2-methylisoborneol contaminate sludge with clean water, relatively small amounts of these compounds were released from the sludge, a possible indication for the fact that absorption, rather than adsorption, underlies the chemical/physical removal process.

  20. Phenolsulfonphthalein transport by potential-sensitive urate transport system.

    PubMed

    Itagaki, Shirou; Shimamoto, Soji; Sugawara, Mitsuru; Kobayashi, Michiya; Miyazaki, Katsumi; Hirano, Takeshi; Iseki, Ken

    2005-08-22

    The purpose of this study was to elucidate the transporter-mediated secretion systems for phenolsulfonphthalein in brush-border membranes. In human and rat renal brush-border membranes, a potential-sensitive transport system has been shown to be involved in the efflux of organic anions. The uptake of phenolsulfonphthalein into rat renal brush-border membrane vesicles was stimulated by an inside-positive membrane potential. This potential-sensitive uptake of phenolsulfonphthalein was inhibited by probenecid, pyrazinoate and urate. p-Aminohippurate had no effect on the potential-sensitive uptake of phenolsulfonphthalein. Moreover, urate competitively inhibited the uptake of phenolsulfonphthalein. On the other hand, the uptake of phenolsulfonphthalein was slightly increased in the presence of an outward Cl- gradient. These results suggest that phenolsulfonphthalein has high affinity for the potential-sensitive urate transport system but has low affinity for an anion exchanger.

  1. New aerobactin-mediated iron uptake system in a septicemia-causing strain of Enterobacter cloacae.

    PubMed Central

    Crosa, L M; Wolf, M K; Actis, L A; Sanders-Loehr, J; Crosa, J H

    1988-01-01

    Unlike the great majority of the aerobactin-producing enteric bacteria documented in the literature, Enterobacter cloacae EK33, isolated from a case of human neonatal meningitis, did not show any homology at the DNA level with the prototype aerobactin system encoded by the ColV-K30 plasmid. However, both the nuclear magnetic resonance spectrum and fast-atom bombardment mass spectrometry of the siderophore purified from EK33 confirmed its identity with aerobactin. Bioassay screening of a gene library of total DNA of EK33 led to the isolation of several aerobactin-positive clones. Under conditions of iron limitation, these clones expressed in Escherichia coli a protein of 72 kilodaltons that reacted with antiserum raised against the pColV-K30 74-kilodalton aerobactin receptor, while the original E. cloacae strain synthesized an 85-kilodalton protein which also cross-reacted with the antiserum. Restriction endonuclease analysis of the cloned DNA confirmed the structural differences between the two aerobactin genetic systems. Images PMID:3192510

  2. Cystine and dibasic amino acid uptake by opossum kidney cells

    SciTech Connect

    States, B.; Segal, S. )

    1990-06-01

    The characteristics of the uptake of L-cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which is found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystine/glutamate transport system present in LLC-PK1 cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high-affinity saturable process which is independent of medium sodium concentration. The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cystine with a ratio greater than 100:1 for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in human cystinuria.

  3. Studies with an immobilized metal affinity chromatography cassette system involving binuclear triazacyclononane-derived ligands: automation of batch adsorption measurements with tagged recombinant proteins.

    PubMed

    Petzold, Martin; Coghlan, Campbell J; Hearn, Milton T W

    2014-07-18

    This study describes the determination of the adsorption isotherms and binding kinetics of tagged recombinant proteins using a recently developed IMAC cassette system and employing automated robotic liquid handling procedures for IMAC resin screening. These results confirm that these new IMAC resins, generated from a variety of different metal-charged binuclear 1,4,7-triaza-cyclononane (tacn) ligands, interact with recombinant proteins containing a novel N-terminal metal binding tag, NT1A, with static binding capacities similar to those obtained with conventional hexa-His tagged proteins, but with significantly increased association constants. In addition, higher kinetic binding rates were observed with these new IMAC systems, an attribute that can be positively exploited to increase process productivity. The results from this investigation demonstrate that enhancements in binding capacities and affinities were achieved with these new IMAC resins and chosen NT1A tagged protein. Further, differences in the binding performances of the bis(tacn) xylenyl-bridged ligands were consistent with the distance between the metal binding centres of the two tacn moieties, the flexibility of the ligand and the potential contribution from the aromatic ring of the xylenyl group to undergo π/π stacking interactions with the tagged proteins.

  4. Enhancement of zidovudine uptake by dehydroepiandrosterone sulfate in rat syncytiotrophoblast cell line TR-TBT 18d-1.

    PubMed

    Nishimura, Tomohiro; Seki, Yoshiaki; Sato, Kazuko; Chishu, Takuya; Kose, Noriko; Terasaki, Tetsuya; Kang, Young-Sook; Sai, Yoshimichi; Nakashima, Emi

    2008-10-01

    AZT (3'-azido-3'-deoxythymidine; zidovudine), which is used for the prevention of mother-to-child transmission of HIV-1, is transplacentally transferred to the fetus across the blood-placenta barrier, which is composed of syncytiotrophoblasts. We recently showed that apical uptake of AZT by syncytiotrophoblasts is mediated by saturable transport system(s) in the TR-TBT 18d-1 cell line, and the cellular accumulation of AZT was increased in the presence of dehydroepiandrosterone sulfate (DHEAS). Here, we aimed to clarify the mechanism of this effect of DHEAS. Inhibitors of efflux transporters, including breast cancer resistance protein, P-glycoprotein, and multidrug resistance proteins, had little effect on the cellular accumulation of AZT in TR-TBT 18d-1. Kinetic study revealed that the rate constant for AZT uptake was greatly increased in the presence of 1 mM DHEAS. These results suggested that the effect of DHEAS was because of enhancement of the uptake process(es), rather than inhibition of efflux. When AZT uptake was analyzed according to the Michaelis-Menten equation, the estimated Michaelis constant, Km, for AZT uptake in the presence of 1 mM DHEAS was lower than that in its absence, whereas maximum uptake velocity, Vmax, and nonsaturable uptake clearance, kns, were similar in the presence and absence of DHEAS, indicating that DHEAS may change the recognition characteristics of the transporter for AZT in TR-TBT 18d-1. Thus, the increase of AZT uptake in TR-TBT 18d-1 cells in the presence of DHEAS was concluded to be because of a DHEAS-induced change in the affinity of AZT uptake system, although the transporter responsible for AZT uptake has not been identified.

  5. Effects of cultivation conditions on the uptake of arsenite and arsenic chemical species accumulated by Pteris vittata in hydroponics.

    PubMed

    Hatayama, Masayoshi; Sato, Takahiko; Shinoda, Kozo; Inoue, Chihiro

    2011-03-01

    The physiological responses of the arsenic-hyperaccumulator, Pteris vittata, such as arsenic uptake and chemical transformation in the fern, have been investigated. However, a few questions remain regarding arsenic treatment in hydroponics. Incubation conditions such as aeration, arsenic concentration, and incubation period might affect those responses of P. vittata in hydroponics. Arsenite uptake was low under anaerobic conditions, as previously reported. However, in an arsenite uptake experiment, phosphorous (P) starvation-dependent uptake of arsenate was observed under aerobic conditions. Time course-dependent analysis of arsenite oxidation showed that arsenite was gradually oxidized to arsenate during incubation. Arsenite oxidation was not observed in any of the control conditions, such as exposure to a nutrient solution or to culture medium only, or with the use of dried root; arsenite oxidation was only observed when live root was used. This result suggests that sufficient aeration allows the rhizosphere system to oxidize arsenite and enables the fern to efficiently take up arsenite as arsenate. X-ray absorption near edge structure (XANES) analyses showed that long-duration exposure to arsenic using a hydroponic system led to the accumulation of arsenate as the dominant species in the root tips, but not in the whole roots, partly because up-regulation of arsenate uptake by P starvation of the fern was caused and retained by long-time incubation. Analysis of concentration-dependent arsenate uptake by P. vittata showed that the uptake switched from a high-affinity transport system to a low-affinity system at high arsenate concentrations, which partially explains the increased arsenate abundance in the whole root.

  6. Multifunctional Delivery Systems for Advanced oral Uptake of Peptide/Protein Drugs.

    PubMed

    Park, Jin Woo; Kim, Sun Jin; Kwag, Dong Sup; Kim, Sol; Park, Jeyoung; Youn, Yu Seok; Bae, You Han; Lee, Eun Seong

    2015-01-01

    In recent years, advances in biotechnology and protein engineering have enabled the production of large quantities of proteins and peptides as important therapeutic agents. Various researchers have used biocompatible functional polymers to prepare oral dosage forms of proteins and peptides for chronic use and for easier administration to enhance patient compliance. However, there is a need to enhance their safety and effectiveness further. Most macromolecules undergo severe denaturation at low pH and enzymatic degradation in the gastrointestinal tract. The macromolecules' large molecular size and low lipophilicity cause low permeation through the intestinal membrane. The major strategies that have been used to overcome these challenges (in oral drug carrier systems) can be classified as follows: enteric coating or encapsulation with pH-sensitive polymers or mucoadhesive polymers, co-administration of protease inhibitors, incorporation of absorption enhancers, modification of the physicochemical properties of the macromolecules, and site-specific delivery to the colon. This review attempts to summarize the various advanced oral delivery carriers, including nanoparticles, lipid carriers, such as liposomes, nano-aggregates using amphiphilic polymers, complex coacervation of oppositely charged polyelectrolytes, and inorganic porous particles. The particles were formulated and/or surface modified with functional polysaccharides or synthetic polymers to improve oral bioavailability of proteins and peptides. We also discuss formulation strategies to overcome barriers, therapeutic efficacies in vivo, and potential benefits and issues for successful oral dosage forms of the proteins and peptides.

  7. Oral Uptake of Chlamydia psittaci by Ducklings Results in Systemic Dissemination

    PubMed Central

    Rossignol, Christelle; Scharf, Sabine; Sachse, Konrad; Berthon, Patricia; Durand, Benoit; Virlogeux-Payant, Isabelle; Borel, Nicole; Laroucau, Karine

    2016-01-01

    Enteric infections caused by Chlamydia (C.) psittaci are frequent in ducks, but mostly remain subclinical under field conditions. To emulate natural infection, we investigated the pathogenic potential of a C. psittaci field strain in orally inoculated 4-day-old ducklings. Three different challenge doses were tested and seven contact animals were also mock-inoculated with buffer in each group. Over the course of ten days, the birds were monitored for clinical symptoms and chlamydial dissemination before final examination of tissues using histopathology and immunohistochemistry. While the challenge strain disseminated systemically to all internal organs, mild signs of diarrhea were confined to ducklings inoculated with the highest dose (4.3 x 108 IFU/mL, Group 1). No other clinical symptoms or histopathological lesions were seen. The chlamydial load in internal organs as measured by PCR depended on the challenge dose and was unevenly distributed, i.e. high loads in spleen, liver, and distal small and large intestinal tract (ileum, cecum and rectum) vs. ten times lower values in lungs and proximal small intestinal tract (duodenum and jejunum). Notably, the C. psittaci infection of contact birds became evident on day 10 post-infection, with bacterial loads comparable to those of experimentally-infected animals, thus suggesting rapid bird-to-bird transmission of the challenge strain. PMID:27168325

  8. Examination of zinc uptake in a combined system using sludge, minerals and ultrafiltration membranes.

    PubMed

    Katsou, Evina; Malamis, Simos; Haralambous, Katherine

    2010-10-15

    This work investigates the feasibility of zinc removal from wastewater with the use of ultrafiltration (UF) membranes combined with natural minerals and sludge. Activated sludge obtained from a membrane bioreactor (MBR) was enriched with initial zinc concentration of 320 mg/L and specific concentrations of zeolite, bentonite and vermiculite. The mixture was agitated and placed inside a batch ultrafiltration unit where the filtration process took place. The effect of several parameters on zinc removal was investigated including the mineral type, quantity and grain size, the metal-mineral contact time and the associated kinetics, the pH value, the zinc initial concentration and sludge mixed liquor suspended solids (MLSS) concentration. The ultrafiltration membranes without any mineral addition were able to remove 38-78% of zinc ions due to biosorption on sludge flocs. The addition of minerals increased the Zn(II) removal efficiencies reaching in some cases more than 90%. Bentonite was the most effective mineral in zinc removal followed by vermiculite. Alkaline pH values favoured zinc removal due to enhanced chemical precipitation. A three-stage adsorption process was identified where the boundary layer diffusion process was followed by a two-stage intraparticle diffusion process. Powder size vermiculite was more effective than granular vermiculite in zinc removal. Minerals also resulted in membrane fouling mitigation since the membrane permeability drop was reduced. The combined sludge-mineral-ultrafiltration system can be effectively employed for the treatment of industrial wastewater.

  9. Nevirapine uptake into the central nervous system of the Guinea pig: an in situ brain perfusion study.

    PubMed

    Gibbs, J E; Gaffen, Z; Thomas, S A

    2006-05-01

    The presence of human immunodeficiency virus (HIV) in the central nervous system (CNS) is associated with the development of HIV-1-associated dementia (HAD), a major cause of HIV-related mortality. To eradicate HIV in the CNS, anti-HIV drugs need to reach the brain and cerebrospinal fluid (CSF) in therapeutic concentrations. This involves passage through the blood-brain and blood-CSF barriers. Using a well established guinea pig in situ brain perfusion model, this study investigated whether nevirapine [6H-dipyrido(3,2-b:2',3'-e)(1,4)diazepin-6-one,11-cyclopropyl-5,11-dihydro-4-methyl], a non-nucleoside reverse transcriptase inhibitor (NNRTI), could effectively accumulate in the CNS. [(3)H]Nevirapine was coperfused with [(14)C]mannitol (a vascular/paracellular permeability marker) through the carotid arteries for up to 30 min, and accumulation in the brain, CSF, and choroid plexus was measured. [(3)H]Nevirapine uptake into the cerebrum was greater than uptake of [(14)C]mannitol, indicating significant passage across the blood-brain barrier and accumulation into the brain (this was further confirmed with capillary depletion and high-performance liquid chromatography analyses). Likewise, [(3)H]nevirapine showed a great ability to cross the blood-CSF barrier and accumulate in the CSF, compared with [(14)C]mannitol. The CNS accumulation of [(3)H]nevirapine was unaffected by 100 muM nevirapine, suggesting that passage across the blood-brain barrier can occur by diffusion. Furthermore, coperfusion with 100 muM efavirenz [2H-3,1-benzoxazin-2-one, 6-chloro-4-(cyclopropylethynyl)-1,4-dihydro-4-(trifluoromethyl)-, (4S)-; another NNRTI] did not significantly alter CNS accumulation of [(3)H]nevirapine, indicating that the efficacy of nevirapine in the CNS would not be altered by the addition of this drug to a combination therapy. Together, these data indicate that this anti-HIV drug should be beneficial in the eradication of HIV within the CNS and the subsequent treatment of

  10. Nitrogen regulator GlnR controls uptake and utilization of non-phosphotransferase-system carbon sources in actinomycetes

    PubMed Central

    Liao, Cheng-Heng; Yao, Lili; Xu, Ya; Liu, Wei-Bing; Zhou, Ying; Ye, Bang-Ce

    2015-01-01

    The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producing Saccharopolyspora erythraea. Deletion of the glnR gene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen- and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals. PMID:26644570

  11. Nitrogen regulator GlnR controls uptake and utilization of non-phosphotransferase-system carbon sources in actinomycetes.

    PubMed

    Liao, Cheng-Heng; Yao, Lili; Xu, Ya; Liu, Wei-Bing; Zhou, Ying; Ye, Bang-Ce

    2015-12-22

    The regulatory mechanisms underlying the uptake and utilization of multiple types of carbohydrates in actinomycetes remain poorly understood. In this study, we show that GlnR (central regulator of nitrogen metabolism) serves as a universal regulator of nitrogen metabolism and plays an important, previously unknown role in controlling the transport of non-phosphotransferase-system (PTS) carbon sources in actinomycetes. It was observed that GlnR can directly interact with the promoters of most (13 of 20) carbohydrate ATP-binding cassette (ABC) transporter loci and can activate the transcription of these genes in response to nitrogen availability in industrial, erythromycin-producing Saccharopolyspora erythraea. Deletion of the glnR gene resulted in severe growth retardation under the culture conditions used, with select ABC-transported carbohydrates (maltose, sorbitol, mannitol, cellobiose, trehalose, or mannose) used as the sole carbon source. Furthermore, we found that GlnR-mediated regulation of carbohydrate transport was highly conserved in actinomycetes. These results demonstrate that GlnR serves a role beyond nitrogen metabolism, mediating critical functions in carbon metabolism and crosstalk of nitrogen- and carbon-metabolism pathways in response to the nutritional states of cells. These findings provide insights into the molecular regulation of transport and metabolism of non-PTS carbohydrates and reveal potential applications for the cofermentation of biomass-derived sugars in the production of biofuels and bio-based chemicals.

  12. Root system architecture in Arabidopsis grown in culture is regulated by sucrose uptake in the aerial tissues.

    PubMed

    Macgregor, Dana R; Deak, Karen I; Ingram, Paul A; Malamy, Jocelyn E

    2008-10-01

    This article presents a detailed model for the regulation of lateral root formation in Arabidopsis thaliana seedlings grown in culture. We demonstrate that direct contact between the aerial tissues and sucrose in the growth media is necessary and sufficient to promote emergence of lateral root primordia from the parent root. Mild osmotic stress is perceived by the root, which then sends an abscisic acid-dependent signal that causes a decrease in the permeability of aerial tissues; this reduces uptake of sucrose from the culture media, which leads to a repression of lateral root formation. Osmotic repression of lateral root formation in culture can be overcome by mutations that cause the cuticle of a plant's aerial tissues to become more permeable. Indeed, we report here that the previously described lateral root development2 mutant overcomes osmotic repression of lateral root formation because of a point mutation in Long Chain Acyl-CoA Synthetase2, a gene essential for cutin biosynthesis. Together, our findings (1) impact the interpretation of experiments that use Arabidopsis grown in culture to study root system architecture; (2) identify sucrose as an unexpected regulator of lateral root formation; (3) demonstrate mechanisms by which roots communicate information to aerial tissues and receive information in turn; and (4) provide insights into the regulatory pathways that allow plants to be developmentally plastic while preserving the essential balance between aboveground and belowground organs.

  13. Predicting binding affinities of host-guest systems in the SAMPL3 blind challenge: the performance of relative free energy calculations

    NASA Astrophysics Data System (ADS)

    König, Gerhard; Brooks, Bernard R.

    2012-05-01

    Relative free energy calculations based on molecular dynamics simulations are combined with available experimental binding free energies to predict unknown binding affinities of acyclic Cucurbituril complexes in the blind SAMPL3 competition. The predictions yield root mean square errors between 2.6 and 3.2 kcal/mol for seven host-guest systems. Those deviations are comparable to results for solvation free energies of small organic molecules. However, the standard deviations found in our simulations range from 0.4 to 2.4 kcal/mol, which indicates the need for better sampling. Three different approaches are compared. Bennett's Acceptance Ratio Method and thermodynamic integration based on the trapezoidal rule with 12 λ-points exhibit a root mean square error of 2.6 kcal/mol, while thermodynamic integration with Simpson's rule and 11 λ-points leads to a root mean square error of 3.2 kcal/mol. In terms of absolute median errors, Bennett's Acceptance Ratio Method performs better than thermodynamic integration with the trapezoidal rule (1.7 vs. 2.9 kcal/mol). Simulations of the deprotonated forms of the guest molecules exhibit a poorer correspondence to experimental results with a root mean square error of 5.2 kcal/mol. In addition, a decrease of the buffer concentration by approximately 20 mM in the simulations raises the root mean square error to 3.8 kcal/mol.

  14. Predicting binding affinities of host-guest systems in the SAMPL3 blind challenge: the performance of relative free energy calculations.

    PubMed

    König, Gerhard; Brooks, Bernard R

    2012-05-01

    Relative free energy calculations based on molecular dynamics simulations are combined with available experimental binding free energies to predict unknown binding affinities of acyclic Cucurbituril complexes in the blind SAMPL3 competition. The predictions yield root mean square errors between 2.6 and 3.2 kcal/mol for seven host-guest systems. Those deviations are comparable to results for solvation free energies of small organic molecules. However, the standard deviations found in our simulations range from 0.4 to 2.4 kcal/mol, which indicates the need for better sampling. Three different approaches are compared. Bennett's Acceptance Ratio Method and thermodynamic integration based on the trapezoidal rule with 12 λ-points exhibit a root mean square error of 2.6 kcal/mol, while thermodynamic integration with Simpson's rule and 11 λ-points leads to a root mean square error of 3.2 kcal/mol. In terms of absolute median errors, Bennett's Acceptance Ratio Method performs better than thermodynamic integration with the trapezoidal rule (1.7 vs. 2.9 kcal/mol). Simulations of the deprotonated forms of the guest molecules exhibit a poorer correspondence to experimental results with a root mean square error of 5.2 kcal/mol. In addition, a decrease of the buffer concentration by approximately 20 mM in the simulations raises the root mean square error to 3.8 kcal/mol.

  15. UPTAKE OF RADIONUCLIDE METALS BY SPME FIBERS

    SciTech Connect

    Duff, M; S Crump, S; Robert02 Ray, R; Keisha Martin, K; Donna Beals, D

    2006-08-28

    The Federal Bureau of Investigation (FBI) Laboratory currently does not have on site facilities for handling radioactive evidentiary materials and there are no established FBI methods or procedures for decontaminating high explosive (HE) and fire debris (FD) evidence while maintaining evidentiary value. One experimental method for the isolation of HE and FD residue involves using solid phase microextraction or SPME fibers to remove residue of interest. Due to their high affinity for organics, SPME fibers should have little affinity for most metals. However, no studies have measured the affinity of radionuclides for SPME fibers. The focus of this research was to examine the affinity of dissolved radionuclide ({sup 239/240}Pu, {sup 238}U, {sup 237}Np, {sup 85}Sr, {sup 133}Ba, {sup 137}Cs, {sup 60}Co and {sup 226}Ra) and stable radionuclide surrogate metals (Sr, Co, Ir, Re, Ni, Ba, Cs, Nb, Zr, Ru, and Nd) for SPME fibers at the exposure conditions that favor the uptake of HE and FD residues. Our results from radiochemical and mass spectrometric analyses indicate these metals have little measurable affinity for these SPME fibers during conditions that are conducive to HE and FD residue uptake with subsequent analysis by liquid or gas phase chromatography with mass spectrometric detection.

  16. Application of affinity aqueous two-phase systems for the fractionation of CD133(+) stem cells from human umbilical cord blood.

    PubMed

    González-González, Mirna; Rito-Palomares, Marco

    2015-03-01

    In a further attempt to establish a novel stem cell primary recovery strategy, the use of aqueous two-phase systems (ATPS) complemented with the use of antibodies (known as immunoaffinity ATPS) is explored in this work. This type of liquid-liquid extraction systems exploits antigen-antibody affinity and represents a novel and selective approach for the purification of stem cells. The proposed bioengineering strategies include the implementation of traditional [polyethylene glycol (PEG), dextran (DEX) and ficoll] and novel (Ucon) immunoaffinity ATPS to prove the viability of cluster of differentiation 133 (CD133(+) ) stem cells from human umbilical cord blood. Furthermore, the addition of the antibody is implemented to identify conditions under which contaminants and stem cells of interest concentrate in opposite phases. The objective of this work is to establish the initial basis for the development of a novel and scalable purification bioprocess for the selective recovery of CD133(+) stem cells employing immunoaffinity ATPS. The reported methodology allows a partitioning of 62% CD133(+) stem cells to the top phase of the ficoll 400,000-DEX 70,000 immunoaffinity ATPS. In PEG 8,000-DEX 500,000 and Ucon-DEX 75,000 systems, no difference was observed when compared with the conventional ATPS (without antibody addition), as the CD133 antibody does not have preference for the desired clean top phase. In all experiments, cell viability was at least 98% after ATPS recovery. This research highlights the challenges that must be addressed to allow the potential establishment of a separation process using immunoaffinity ATPS for the recovery and purification of stem cells.

  17. Absence of Rapid Exchange Component in a Low-Affinity Carrier Transport

    PubMed Central

    LeFevre, Paul G.

    1963-01-01

    A previous study showed that human red blood cells equilibrate much less rapidly with D-glucose at moderately high concentrations than with C14-glucose added after the net movement is completed. This had been predicted from a simple reversible mobile-carrier mediated-transport model system suggested by the net monosaccharide transport kinetics in these cells, but is also consistent with the more complex models proposed for certain active transport systems to account for elevation of tracer fluxes of even low-affinity "substrates" when their trans-concentration is raised. The simple model predicts, however, that with any sugar showing a much lower apparent affinity for the reactive sites, such as D-ribose, this phenomenon would not be observed, and tracer equilibration should proceed at approximately the same rate as net uptake. The latter expectation was confirmed experimentally by analyses of the ribose, or radioactivity, content of washed red cells sampled serially during incubation with ribose or C14-ribose in the appropriate mixtures. The tracer ribose movement showed no evidence of a relatively rapid exchange component. The relative rapidity of glucose tracer uptake into cells preloaded with ordinary glucose may therefore more readily be attributed simply to depression of tracer efflux by competition for the saturated reactive sites, than to any action of the trans-concentration on the influx by way of a coupled exchange process. PMID:13929247

  18. [Effects of reduced N application rate on yield and nutrient uptake and utilization in maize-soybean relay strip intercropping system].

    PubMed

    Yong, Tai-Wen; Liu, Xiao-Ming; Wen-Yu, Liu; Su, Ben-Ying; Song, Chun; Yang, Feng; Wang, Xiao-Chun; Yang, Wen-Yu

    2014-02-01

    A field experiment with three N application rates (0, 180, 240 N kg x hm(-2), representing zero, reduced and conventional N application, respectively) and three planting patterns (maize monoculture, soybean monoculture and maize-soybean relay strip intercropping) was conducted to reveal the effects of cropping patterns and N application rates on yield, nutrient uptake and nitrogen use efficiency of maize and soybean. The results showed that the grain yield, N, P and K uptake and harvest index of the intercropped maize reduced slightly compared with the monoculture maize, however these indices of the intercropped soybean increased significantly compared with the monoculture. With the increase in nitrogen fertilizer application, the excellence of relay strip intercropping was weakened in the maize-soybean intercropping system. The grain yield, economic coefficient, N, P and K uptake, harvest index, N agronomy efficiency and N uptake efficiency of maize and soybean increased significantly at the reduced nitrogen rate (180 N kg x hm(-2)), but the rate of soil N contribution declined, compared with the conventional rate of N application by local farmers (240 N kg x hm(-2)). In the reduced nitrogen rate treatment, total soil N and P contents of the maize strip reduced, whereas the total soil N, P and K contents of soybean strip and the total K content of maize strip increased compared with the zero N application treatment. With the reduced N application, the annual total grain yield, N, P and K uptake of above-ground biomass in the maize-soybean relay strip intercropping system were higher than in the monoculture, and the land equivalent ratio (LER) was 2.28. N uptake efficiency of maize in the relay strip intercropping system was 20.2% higher than in the maize monoculture, and the index of soybean was 30.5% lower than in the monoculture. The rate of soil N contribution in the relay strip intercropping system was 20.0% and 8.8% lower than in the maize and soybean

  19. Genome-wide assessment of the carriers involved in the cellular uptake of drugs: a model system in yeast

    PubMed Central

    2011-01-01

    Background The uptake of drugs into cells has traditionally been considered to be predominantly via passive diffusion through the bilayer portion of the cell membrane. The recent recognition that drug uptake is mostly carrier-mediated raises the question of which drugs use which carriers. Results To answer this, we have constructed a chemical genomics platform built upon the yeast gene deletion collection, using competition experiments in batch fermenters and robotic automation of cytotoxicity screens, including protection by 'natural' substrates. Using these, we tested 26 different drugs and identified the carriers required for 18 of the drugs to gain entry into yeast cells. Conclusions As well as providing a useful platform technology, these results further substantiate the notion that the cellular uptake of pharmaceutical drugs normally occurs via carrier-mediated transport and indicates that establishing the identity and tissue distribution of such carriers should be a major consideration in the design of safe and effective drugs. PMID:22023736

  20. Effect of monocular deprivation on uptake and binding of [3H]glutamate in the visual system of rat brain.

    PubMed

    Schliebs, R; Kunert, E; Bigl, V

    1984-11-01

    [3H]Glutamate uptake and binding studies were performed in the visual cortices, lateral geniculate nuclei (LGN), and superior colliculi of 3-month-old rats with one eyelid surgically closed from postnatal day 10 (monocular deprivation). Uptake and binding were highest in the lateral geniculate nucleus followed by the visual cortex (69% and 15%, respectively compared to LGN values) and the superior colliculus (32% and 59% of LGN values). Monocular deprivation did not affect [3H]glutamate uptake in any of the visual regions examined. However, a 46% decrease in [3H]glutamate binding in the lateral geniculate nucleus ipsilateral to the sutured eye was detected. Binding levels in other regions were not affected.

  1. Effects of nitrogen fertilization on soil nutrient concentration and phosphatase activity and forage nutrient uptake from a grazed pasture system.

    PubMed

    Dillard, Sandra Leanne; Wood, Charles Wesley; Wood, Brenda Hall; Feng, Yucheng; Owsley, Walter Frank; Muntifering, Russell Brian

    2015-05-01

    Over a 3-year period, the effect of differing N-application regimes on soil extractable-P concentration, soil phosphatase activity, and forage P uptake in a P-enriched grazed-pasture system was investigated. In the fall of each year, six 0.28-ha plots were overseeded with triticale ( × Triticosecale rimpaui Wittm.) and crimson clover (Trifolium incarnatum) into a tall fescue (Lolium arundinacea)/bermudagrass (Cynodon dactylon) sod and assigned to 1 of 3 N-fertilizer treatments (n = 2): 100% of N recommendation in a split application (100N), 50% in a single application (50N), and 0% of N recommendation (0N) for triticale. Cattle commenced grazing the following spring and grazed until May. In the summer, plots were overseeded with cowpea (Vigna unguiculata), fertilized at the same rates by reference to N recommendations for bermudagrass, and grazed by cattle until September. There were no effects of N fertilization on soil phosphatase activity, electrical conductivity, or concentrations of water-soluble P. Concentrations of extractable P decreased in plots receiving 50N, but increasing N fertilization to 100N resulted in no further reduction in extractable P. Forage biomass, foliar P concentrations, and forage P mass were not affected by N fertilization rates at the plant-community level, but responses were observed within individual forage species. Results are interpreted to mean that N fertilization at 50% of the agronomic recommendation for the grass component can increase forage P mass of specific forages and decrease soil extractable P, thus providing opportunity for decreasing P losses from grazed pasture.

  2. Analytical performance of a versatile laboratory microscopic X-ray fluorescence system for metal uptake studies on argillaceous rocks

    NASA Astrophysics Data System (ADS)

    Gergely, Felicián; Osán, János; Szabó, B. Katalin; Török, Szabina

    2016-02-01

    Laboratory-scale microscopic X-ray fluorescence (micro-XRF) plays an increasingly important role in various fields where multielemental investigations of samples are indispensable. In case of geological samples, the reasonable detection limits (LOD) and spatial resolutions are necessary to identify the trace element content in microcrystalline level. The present study focuses on the analytical performance of a versatile laboratory-scale micro-XRF system with various options of X-ray sources and detectors to find the optimal experimental configuration in terms of sensitivities and LOD for selected elements in loaded petrographic thin sections. The method was tested for sorption studies involving thin sections prepared from cores of Boda Claystone Formation, which is a potential site for a high-level radioactive waste repository. Loaded ions in the sorption measurements were Cs(I) and Ni(II) chemically representing fission and corrosion products. Based on the collected elemental maps, the correlation between the elements representative of main rock components and the selected loaded ion was studied. For the elements of interest, Cs(I) and Ni(II) low-power iMOXS source with polycapillary and silicon drift detector was found to be the best configuration to reach the optimal LOD values. Laboratory micro-XRF was excellent to identify the responsible key minerals for the uptake of Cs(I). In case of nickel, careful corrections were needed because of the relatively high Ca content of the rock samples. The results were compared to synchrotron radiation micro-XRF.

  3. Ten-fold augmentation of endothelial uptake of vascular endothelial growth factor with ultrasound after systemic administration

    NASA Technical Reports Server (NTRS)

    Mukherjee, D.; Wong, J.; Griffin, B.; Ellis, S. G.; Porter, T.; Sen, S.; Thomas, J. D.

    2000-01-01

    OBJECTIVES: In this study, the feasibility of delivering and enhancing the uptake of vascular endothelial growth factor (VEGF) into the intact endothelium by using ultrasound (US) facilitation was determined. BACKGROUND: A limitation of tissue-targeted drug delivery is the need for direct arterial cannulation. We postulate a mechanism by which agents injected intravenously may be targeted to a tissue using US and ultrasonic contrast agents. METHODS: We used a rat model to test the ability of US and an ultrasonic contrast agent perflurocarbon exposed sonicated dextrose albumin (PESDA) to increase uptake of VEGF in the myocardium. Continuous wave Doppler US (0.6 W/cm2 at 1 MHz for 15 min) was applied to the chest wall overlying the myocardium during intravenous injection with either VEGF (100 microg/kg) alone or a combination of VEGF and PESDA (0.1%). Control rats had VEGF infused without US or PESDA. The VEGF uptake was measured quantitatively in the heart, lung, liver and kidneys by enzyme-linked immunosorbent assay (ng/g of tissue) and morphologically by fluorescence microscopy. RESULTS: There was an eight-fold increase in VEGF uptake in the heart by US alone (16.86 +/- 1.56 vs. 2.11 +/- 0.953 ng/g of tissue, p < 0.0001) and a 13-fold increase with US + PESDA (26.78 +/- 2.88 vs. 2.11 +/- 0.953 ng/g of tissue, p < 0.0001) compared with control rats. Fluorescence microscopy revealed deposition of VEGF in the endothelium of small intramyocardial arterioles. CONCLUSIONS: These results show a marked increase in endothelial VEGF uptake with US and US + PESDA. Thus, US may be used to augment endothelial VEGF uptake 10-fold to 13-fold.

  4. Effect of Uptake-one inhibitors on the uptake of norepinephrine and metaiodobenzylguanidine

    SciTech Connect

    Tobes, M.C.; Jaques, S. Jr.; Wieland, D.M.; Sisson, J.C.

    1985-08-01

    The mechanisms underlying the uptake of the radiopharmaceutical metaiodobenzylguanidine (MIBG) and the catecholamine norepinephrine (NE) were studied using cultured bovine adrenomedullary cells as an in vitro model system. Sodium-dependent and sodium-independent uptake systems have been identified and characterized for both MIBG and NE. The sodium-dependent uptake of Ne and MIBG was inhibited by the selective Uptake-one inhibitors, desmethylimipramine (DMI) and cocaine, whereas the sodium-independent uptake for NE and MIBG was much less sensitive to inhibition by these agents. The sodium-dependent uptake system fulfills the criteria for the neuronal Uptake-one system, and the sodium-independent uptake system fulfills the criteria for a passive diffusion mechanism. Arterial concentrations proximal to the dog adrenal were very small suggesting that the sodium-dependent (Uptake-one) system is predominant in vivo. Consistent with the in vitro observations, the in vivo uptake of MIBG and NE into dog adrenal medullae was effectively blocked by pretreatment with DMI or cocaine. Therefore, iodine-131 MIBG scintigraphy of the adrenal appears to reflect uptake by way of the Uptake-one system.

  5. Crystallization and preliminary crystallographic studies of PotA, a membrane-associated ATPase of the spermidine-preferential uptake system in Thermotoga maritima

    PubMed Central

    Sugiyama, Shigeru; Kashiwagi, Keiko; Kakinouchi, Keisuke; Tomitori, Hideyuki; Kanai, Ken; Murata, Michio; Adachi, Hiroaki; Matsumura, Hiroyoshi; Takano, Kazufumi; Murakami, Satoshi; Inoue, Tsuyoshi; Mori, Yusuke; Igarashi, Kazuei

    2014-01-01

    A membrane-associated ATPase, PotA, is a component of the spermidine-preferential uptake system in prokaryotes that plays an important role in normal cell growth by regulating the cellular polyamine concentration. No three-dimensional structures of membrane-associated ATPases in polyamine-uptake systems have been determined to date. Here, the crystallization and preliminary X-ray diffraction analysis of PotA from Thermotoga maritima are reported. Diffraction data were collected and processed to 2.7 Å resolution from both native and selenomethionine-labelled crystals. Preliminary crystallographic analysis revealed that the crystals belonged to the hexagonal space group P3112 (or P3212), with unit-cell parameters a = b = 88.9, c = 221.2 Å, α = 90, β = 90, γ = 120°, indicating that a dimer was present in the asymmetric unit. PMID:24915082

  6. ‘And then there were three’: highly efficient uptake of potassium by foliar trichomes of epiphytic bromeliads

    PubMed Central

    Winkler, Uwe; Zotz, Gerhard

    2010-01-01

    Background and Aims Vascular epiphytes have to acquire nutrients from atmospheric wash out, stem-flow, canopy soils and trapped litter. Physiological studies on the adaptations to nutrient acquisition and plant utilization of nutrients have focused on phosphorus and nitrogen; potassium, as a third highly abundant nutrient element, has received minor attention. In the present study, potassium uptake kinetics by leaves, within-plant distribution and nutrient accumulation were analysed to gain an improved understanding of physiological adaptations to non-terrestrial nutrient supply of plants. Methods Radioactively labelled 86RbCl was used as an analogue to study uptake kinetics of potassium absorbed from tanks of epiphytes, its plant distribution and the correlation between uptake efficiency and abundance of trichomes, functioning as uptake organs of leaves. Potassium in leaves was additionally analysed by atomic absorption spectroscopy to assess plant responses to potassium deficiency. Key Results Labelled rubidium was taken up from tanks over a wide range of concentrations, 0·01–90 mm, which was achieved by two uptake systems. In four tank epiphytes, the high-affinity transporters had average Km values of 41·2 µm, and the low-affinity transporters average Km values of 44·8 mm. Further analysis in Vriesea splenriet showed that high-affinity uptake of rubidium was an ATP-dependent process, while low-affinity uptake was mediated by a K+-channel. The kinetic properties of both types of transporters are comparable with those of potassium transporters in roots of terrestrial plants. Specific differences in uptake velocities of epiphytes are correlated with the abundance of trichomes on their leaf surfaces. The main sinks for potassium were fully grown leaves. These leaves thus function as internal potassium sources, which allow growth to be maintained during periods of low external potassium availability. Conclusions Vascular epiphytes possess effective mechanisms

  7. Multiple biosynthetic and uptake systems mediate siderophore-dependent iron acquisition in Streptomyces coelicolor A3(2) and Streptomyces ambofaciens ATCC 23877.

    PubMed

    Barona-Gómez, Francisco; Lautru, Sylvie; Francou, Francois-Xavier; Leblond, Pierre; Pernodet, Jean-Luc; Challis, Gregory L

    2006-11-01

    Siderophore-mediated iron acquisition has been well studied in many bacterial pathogens because it contributes to virulence. In contrast, siderophore-mediated iron acquisition by saprophytic bacteria has received relatively little attention. The independent identification of the des and cch gene clusters that direct production of the tris-hydroxamate ferric iron-chelators desferrioxamine E and coelichelin, respectively, which could potentially act as siderophores in the saprophyte Streptomyces coelicolor A3(2), has recently been reported. Here it is shown that the des cluster also directs production of desferrioxamine B in S. coelicolor and that very similar des and cch clusters direct production of desferrioxamines E and B, and coelichelin, respectively, in Streptomyces ambofaciens ATCC 23877. Sequence analyses of the des and cch clusters suggest that components of ferric-siderophore uptake systems are also encoded within each cluster. The construction and analysis of a series of mutants of S. coelicolor lacking just biosynthetic genes or both the biosynthetic and siderophore uptake genes from the des and cch clusters demonstrated that coelichelin and desferrioxamines E and B all function as siderophores in this organism and that at least one of these metabolites is required for growth under defined conditions even in the presence of significant quantities of ferric iron. These experiments also demonstrated that a third siderophore uptake system must be present in S. coelicolor, in addition to the two encoded within the cch and des clusters, which show selectivity for coelichelin and desferrioxamine E, respectively. The ability of the S. coelicolor mutants to utilize a range of exogenous xenosiderophores for iron acquisition was also examined, showing that the third siderophore-iron transport system has broad specificity for tris-hydroxamate-containing siderophores. Together, these results define a complex system of multiple biosynthetic and uptake pathways for

  8. Dynamic model of flexible phytoplankton nutrient uptake

    PubMed Central

    Bonachela, Juan A.; Raghib, Michael; Levin, Simon A.

    2011-01-01

    The metabolic machinery of marine microbes can be remarkably plastic, allowing organisms to persist under extreme nutrient limitation. With some exceptions, most theoretical approaches to nutrient uptake in phytoplankton are largely dominated by the classic Michaelis–Menten (MM) uptake functional form, whose constant parameters cannot account for the observed plasticity in the uptake apparatus. Following seminal ideas by earlier researchers, we propose a simple cell-level model based on a dynamic view of the uptake process whereby the cell can regulate the synthesis of uptake proteins in response to changes in both internal and external nutrient concentrations. In our flexible approach, the maximum uptake rate and nutrient affinity increase monotonically as the external nutrient concentration decreases. For low to medium nutrient availability, our model predicts uptake and growth rates larger than the classic MM counterparts, while matching the classic MM results for large nutrient concentrations. These results have important consequences for global coupled models of ocean circulation and biogeochemistry, which lack this regulatory mechanism and are thus likely to underestimate phytoplankton abundances and growth rates in oligotrophic regions of the ocean. PMID:22143781

  9. One-step affinity tag purification of full-length recombinant human AP-1 complexes from bacterial inclusion bodies using a polycistronic expression system.

    PubMed

    Wang, Wei-Ming; Lee, A-Young; Chiang, Cheng-Ming

    2008-05-01

    The AP-1 transcription factor is a dimeric protein complex formed primarily between Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) family members. These distinct AP-1 complexes are expressed in many cell types and modulate target gene expression implicated in cell proliferation, differentiation, and stress responses. Although the importance of AP-1 has long been recognized, the biochemical characterization of AP-1 remains limited in part due to the difficulty in purifying full-length, reconstituted dimers with active DNA-binding and transcriptional activity. Using a combination of bacterial coexpression and epitope-tagging methods, we successfully purified all 12 heterodimers (3 Junx4 Fos) of full-length human AP-1 complexes as well as c-Jun/c-Jun, JunD/JunD, and c-Jun/JunD dimers from bacterial inclusion bodies using one-step nickel-NTA affinity tag purification following denaturation and renaturation of coexpressed AP-1 subunits. Coexpression of two constitutive components in a dimeric AP-1 complex helps stabilize the proteins when compared with individual protein expression in bacteria. Purified dimeric AP-1 complexes are functional in sequence-specific DNA binding, as illustrated by electrophoretic mobility shift assays and DNase I footprinting, and are also active in transcription with in vitro-reconstituted human papillomavirus (HPV) chromatin containing AP-1-binding sites in the native configuration of HPV nucleosomes. The availability of these recombinant full-length human AP-1 complexes has greatly facilitated mechanistic studies of AP-1-regulated gene transcription in many biological systems.

  10. Substrate-induced internalization of the high-affinity choline transporter.

    PubMed

    Okuda, Takashi; Konishi, Asami; Misawa, Hidemi; Haga, Tatsuya

    2011-10-19

    Cholinergic neurons are endowed with a high-affinity choline uptake system for efficient synthesis of acetylcholine at the presynaptic terminals. The high-affinity choline transporter CHT1 is responsible for choline uptake, the rate-limiting step in acetylcholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylcholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylcholine synthesis at the cholinergic presynaptic terminals.

  11. The influence of secretion elicitors and external pH on the kinetics of D-alanine uptake by the trap lobes of Dionaea muscipula Ellis (Venus's Flytrap).

    PubMed

    Rea, P A; Whatley, F R

    1983-08-01

    Simple kinetic techniques were used to examine the mechanism of D-alanine uptake by the adaxial surfaces of the trap lobes of Dionaea muscipula Ellis (Venus's Flytrap.) On the basis of these analyses, the uptake of D-alanine was found to depend on the time during which the trap lobes were inoculated with elicitors of secretion before excision and measurement of uptake. Disks taken from traps that had not been subjected to a preceding period of inoculation with secretion elicitors showed a low basal rate of uptake which was neither pH-dependent nor exhibited saturation with respect to external D-alanine concentration. Disks from preinoculated traps, on the other hand, displayed an enhanced rate of uptake which showed both pH-dependence and saturation with respect to external D-alanine concentration. The capacity for enhanced uptake was lost upon prolonged inoculation or when inoculation was stopped. Of the compounds tested, only elicitors of secretion caused an enhancement of uptake. The enhanced rate of D-alanine uptake is temperature-sensitive with a Q10 characteristic of a mediated process. Uncouplers cause an instantaneous abolition of uptake whereas the effects of terminal-oxidase inhibitors are time-dependent. The pH-dependence of uptake is inferred to result from an increased affinity of the carrier system for D-alanine at low pH values. Although the ionic state of D-alanine is relatively unaffected over the pH range examined, a decrease in the external pH from 6.0 to 3.8 decreases the apparent K m for uptake by four-fold but increases V max by only 30%. It is concluded that the acid secreted by the digestive glands of Dionaea plays a direct role in facilitating the uptake of amino acids from the trap cavity.

  12. Kernel Affine Projection Algorithms

    NASA Astrophysics Data System (ADS)

    Liu, Weifeng; Príncipe, José C.

    2008-12-01

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

  13. Dual Mechanisms of Ion Uptake in Relation to Vacuolation in Corn Roots

    PubMed Central

    Torii, Kenji; Laties, George G.

    1966-01-01

    Absorption isotherms for chloride and rubidium ions have been determined through a wide concentration range for nonvacuolate root tips, and for vacuolate subapical sections of corn root. In the range 0 to 0.5 mm, chloride absorption is hyperbolic with concentration in both tips and proximal sections. At high concentrations, 1 to 50 mm, a second multiple-hyperbolic isotherm for chloride is noted in vacuolate tissue, while the isotherm for nonvacuolate tips rises exponentially. A linear to exponentially rising isotherm is taken to signify diffusive permeation. The same distinction between tip and subapical tissue characterizes Rb absorption. Rb uptake is indifferent to the nature of the counterion at all concentrations in the tip, while the counterion exerts a predictable influence on Rb absorption in proximal tissue. The effect of a poorly absorbable anion on Rb uptake is greater in the high concentration range. Evidence is presented for the metabolic nature of ion transport into nonvacuolate root tips. Verification is offered that ion uptake is mediated by dual mechanisms, and the thesis is developed that the high-affinity (low Ks) system mediates ion passage through the plasma membrane while the low-affinity (high Ks) system implements transport through the tonoplast. PMID:16656332

  14. Dual mechanisms of ion uptake in relation to vacuolation in corn roots.

    PubMed

    Torii, K; Laties, G G

    1966-05-01

    Absorption isotherms for chloride and rubidium ions have been determined through a wide concentration range for nonvacuolate root tips, and for vacuolate subapical sections of corn root. In the range 0 to 0.5 mm, chloride absorption is hyperbolic with concentration in both tips and proximal sections. At high concentrations, 1 to 50 mm, a second multiple-hyperbolic isotherm for chloride is noted in vacuolate tissue, while the isotherm for nonvacuolate tips rises exponentially. A linear to exponentially rising isotherm is taken to signify diffusive permeation.The same distinction between tip and subapical tissue characterizes Rb absorption. Rb uptake is indifferent to the nature of the counterion at all concentrations in the tip, while the counterion exerts a predictable influence on Rb absorption in proximal tissue. The effect of a poorly absorbable anion on Rb uptake is greater in the high concentration range. Evidence is presented for the metabolic nature of ion transport into nonvacuolate root tips. Verification is offered that ion uptake is mediated by dual mechanisms, and the thesis is developed that the high-affinity (low K(s)) system mediates ion passage through the plasma membrane while the low-affinity (high K(s)) system implements transport through the tonoplast.

  15. Adjoint affine fusion and tadpoles

    NASA Astrophysics Data System (ADS)

    Urichuk, Andrew; Walton, Mark A.

    2016-06-01

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

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

    PubMed

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

    2017-05-01

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

  17. Uptake and distribution of nitrogen from acidic fog within a ponderosa pine (Pinus ponderosa Laws.)/litter/soil system

    SciTech Connect

    Fenn, M.E.; Leininger, T.D.

    1995-11-01

    The magnitude and importance of wet deposition of N in forests of the South Coast (Los Angeles) Air Basin have not been well characterized. We exposed 3-yr-old ponderosa pine (Pinus ponderosa Laws.) seedlings growing in native forest soil to acidic fog treatments (pH 3.1) simulating fog chemistry from a pine forest near Los Angeles, California. Fog solutions contained either {sup 15}NH{sub 4}{sup +}, {sup 15}NO{sub 3}{sup {minus}}, or unlabeled N. The fog treatments were applied in open-top chambers in six 5-hr exposures. Soil treatments within each of the fog exposures were bare soil, soil overlain with L- and F-litter, and soil covered with plastic during the fog events to prevent fogwater from contacting soil. Seedlings were harvested and samples were collected 15 wk after the fog treatments. Uptake of {sup 15}N by roots was by far the dominant pathway for plant assimilation of fog-deposited {sup 15}N. Deposition of N in fog supplied 9.4% and 8.7% of the total N in current-year crown biomass in the litter-overlay and bare-soil treatments, respectively. Total N concentrations in every plant fraction except current-year stems were significantly higher in the bare-soil treatment than in the plastic-covered soil treatment. Less than 5% of the {sup 15}N deposited directly to the seedling crowns was retained by the plants in the covered-soil treatment, whereas 57% of the {sup 15}N deposited to the seedling/litter/soil systems was incorporated into plant biomass. The litter layers retained {sup 15}NH{sub 4}{sup +} more effectively than {sup 15}NH{sub 4}{sup +} more effectively than {sup 15}NO{sub 3}. Data from this study suggest that N deposited from fog may be an important source of N for plant growth in forests of the SCAB where fog occurrence and pollution exposure coincide. 5 refs., 5 figs., 3 tabs.

  18. Uncoupler-Resistant Glucose Uptake by the Thermophilic Glycolytic Anaerobe Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum)

    PubMed Central

    Cook, Gregory M.; Janssen, Peter H.; Morgan, Hugh W.

    1993-01-01

    The transport of glucose across the bacterial cell membrane of Thermoanaerobacter thermosulfuricus (Clostridium thermohydrosulfuricum) Rt8.B1 was governed by a permease which did not catalyze concomitant substrate transport and phosphorylation and thus was not a phosphoenolpyruvate-dependent phosphotransferase. Glucose uptake was carrier mediated, could not be driven by an artificial membrane potential (Δψ) in the presence or absence of sodium, and was not sensitive to inhibitors which dissipate the proton motive force (Δp; tetrachlorosalicylanilide, N,N-dicyclohexylcarboiimide, and 2,4-dinitrophenol), and no uptake of the nonmetabolizable analog 2-deoxyglucose could be demonstrated. The glucokinase apparent Km for glucose (0.21 mM) was similar to the Kt (affinity constant) for glucose uptake (0.15 mM), suggesting that glucokinase controls the rate of glucose uptake. Inhibitors of ATP synthesis (iodoacetate and sodium fluoride) also inhibited glucose uptake, and this effect was due to a reduction in the level of ATP available to glucokinase for glucose phosphorylation. These results indicated that T. thermosulfuricus Rt8.B1 lacks a concentrative uptake system for glucose and that uptake is via facilitated diffusion, followed by ATP-dependent phosphorylation by glucokinase. In T. thermosulfuricus Rt8.B1, glucose is metabolized by the Embden-Meyerhof-Parnas pathway, which yields 2 mol of ATP (G. M. Cook, unpublished data). Since only 1 mol of ATP is used to transport 1 mol of glucose, the energetics of this system are therefore similar to those found in bacteria which possess a phosphotransferase. PMID:16349043

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

    PubMed Central

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

    2005-01-01

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

  20. Impact of assimilable nitrogen availability in glucose uptake kinetics in Saccharomyces cerevisiae during alcoholic fermentation

    PubMed Central

    2012-01-01

    Background The expression and activity of the different Saccharomyces cerevisiae hexose uptake systems (Hxt) and the kinetics of glucose uptake are considered essential to industrial alcoholic fermentation performance. However, the dynamics of glucose uptake kinetics during the different stages of fermentation, depending on glucose and nitrogen availability, is very poorly characterized. The objective of the present work was to examine thoroughly the alterations occurring in glucose uptake kinetics during alcoholic fermentation, by the wine strain S. cerevisiae PYCC 4072, of a synthetic grape juice basal medium with either a limiting or non-limiting initial nitrogen concentration and following nitrogen supplementation of the nitrogen-depleted sluggish fermentation. Results Independently of the initial concentration of the nitrogen source, glucose transport capacity is maximal during the early stages of fermentation and presumably sustained by the low-affinity and high-capacity glucose transporter Hxt1p. During nitrogen-limited sluggish fermentation, glucose uptake capacity was reduced to approximately 20% of its initial values (Vmax = 4.9 ± 0.8 compared to 21.9 ± 1.2 μmol h-1 10-8 cells), being presumably sustained by the low-affinity glucose transporter Hxt3p (considering the calculated Km = 39.2 ± 8.6 mM). The supplementation of the sluggish fermentation broth with ammonium led to the increase of glucose transport capacity associated to the expression of different glucose uptake systems with low and high affinities for glucose (Km = 58.2 ± 9.1 and 2.7 ± 0.4 mM). A biclustering analysis carried out using microarray data, previously obtained for this yeast strain transcriptional response to equivalent fermentation conditions, indicates that the activation of the expression of genes encoding the glucose transporters Hxt2p (during the transition period to active fermentation) and Hxt3p, Hxt4p, Hxt6p and Hxt7p (during the

  1. Glycinergic-Fipronil Uptake Is Mediated by an Amino Acid Carrier System and Induces the Expression of Amino Acid Transporter Genes in Ricinus communis Seedlings.

    PubMed

    Xie, Yun; Zhao, Jun-Long; Wang, Chuan-Wei; Yu, Ai-Xin; Liu, Niu; Chen, Li; Lin, Fei; Xu, Han-Hong

    2016-05-18

    Phloem-mobile insecticides are efficient for piercing and sucking insect control. Introduction of sugar or amino acid groups to the parent compound can improve the phloem mobility of insecticides, so a glycinergic-fipronil conjugate (GlyF), 2-(3-(3-cyano-1-(2,6-dichloro-4-(trifluoromethyl)phenyl)-4-((trifluoromethyl)sulfinyl)-1H-pyrazole-5-yl)ureido) acetic acid, was designed and synthesized. Although the "Kleier model" predicted that this conjugate is not phloem mobile, GlyF can be continually detected during a 5 h collection of Ricinus communis phloem sap. Furthermore, an R. communis seedling cotyledon disk uptake experiment demonstrates that the uptake of GlyF is sensitive to pH, carbonyl cyanide m-chlorophenylhydrazone (CCCP), temperature, and p-chloromercuribenzenesulfonic acid (pCMBS) and is likely mediated by amino acid carrier system. To explore the roles of amino acid transporters (AATs) in GlyF uptake, a total of 62 AAT genes were identified from the R. communis genome in silico. Phylogenetic analysis revealed that AATs in R. communis were organized into the ATF (amino acid transporter) and APC (amino acid, polyaminem and choline transporter) superfamilies, with five subfamilies in ATF and two in APC. Furthermore, the expression profiles of 20 abundantly expressed AATs (cycle threshold (Ct) values <27) were analyzed at 1, 3, and 6 h after GlyF treatment by RT-qPCR. The results demonstrated that expression levels of four AAT genes, RcLHT6, RcANT15, RcProT2, and RcCAT2, were induced by the GlyF treatment in R. communis seedlings. On the basis of the observation that the expression profile of the four candidate genes is similar to the time course observation for GlyF foliar disk uptake, it is suggested that those four genes are possible candidates involved in the uptake of GlyF. These results contribute to a better understanding of the mechanism of GlyF uptake as well as phloem loading from a molecular biology perspective and facilitate functional

  2. Organic cation uptake in vitro by the rabbit iris-ciliary body, renal cortex, and choroid plexus.

    PubMed

    Bárány, E H

    1976-05-01

    The uptake in vitro of radioactively labeled test substances was studied in tissues from albino rabbits. Choroid plexus, slices of outer renal cortex, and iris-ciliary body were incubated in a K-rich medium containing one of the cations 14C-Emepronium (Cetiprin), 14C-tetraethylammonium, 14C-choline, or 125I-o-iodobenzyltrimethylammonium and sometimes the anions 131I-o-iodohippurate and 125I-iodipamide. Choroid plexus and renal cortex accumulated all test substances, some to very high tissue-medium ratios. The iris-ciliary body preparation accumulated the anions well but the organic cations only weakly. The only convincing uptake was that of Emepronium. The affinity of this uptake system seemed to be similar to that in the kidney, half-saturating around 10(-4)M Emepronium.

  3. Development of a cell-based nonradioactive glucose uptake assay system for SGLT1 and SGLT2.

    PubMed

    Kanwal, Abhinav; Singh, Shailendra Pratap; Grover, Paramjit; Banerjee, Sanjay Kumar

    2012-10-01

    Sodium-dependent glucose cotransporters (SGLT1 and SGLT2), which have a key role in the absorption of glucose in the kidney and/or gastrointestinal tract, have been proposed as a novel therapeutic strategy for diabetes and cardiomyopathy. Here we developed a simple cell-based, nonradioactive method for functional screening of SGLT1 and SGLT2 inhibitors. Stable cell lines expressing human SGLT1 and SGLT2 were established by transfecting HEK293 cells with vectors (pCMV6-Neo) having full-length human SGLT1 and SGLT2 and selecting the positive clones following neomycin treatment. We confirmed the gene expression of SGLT1 and SGLT2 by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting. Furthermore, to analyze the function of SGLTs, we incubated stable cell lines with 2-deoxyglucose or fluorescent d-glucose analog (2-NBDG) and performed glucose uptake assay. A significant (P<0.001) increase in glucose uptake was observed in both cell lines. The increased glucose uptake in both cell lines was completely inhibited when treated with nonspecific SGLT1/SGLT2 inhibitors and phlorizin (100μM), but not when treated with nonspecific sodium-independent facilitative glucose transporter (GLUT) inhibitors (100μM). Taken together, our data suggest that cell-based methods developed for screening SGLT1/SGLT2 inhibitors are phlorizin sensitive and specific for respective glucose transporters. This assay provides a simple and rapid method for identifying novel and selective SGLT inhibitors.

  4. Smooth affine shear tight frames: digitization and applications

    NASA Astrophysics Data System (ADS)

    Zhuang, Xiaosheng

    2015-08-01

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

  5. Effects of warming on uptake and translocation of cadmium (Cd) and copper (Cu) in a contaminated soil-rice system under Free Air Temperature Increase (FATI).

    PubMed

    Ge, Li-Qiang; Cang, Long; Liu, Hui; Zhou, Dong-Mei

    2016-07-01

    Global warming has received growing attentions about its potential threats to human in recent, however little is known about its effects on transfer of heavy metals in agro-ecosystem, especially for Cd in rice. Pot experiments were conducted to evaluate Cd/Cu translocation in a contaminated soil-rice system under Free Air Temperature Increase (FATI). The results showed that warming gradually decreased soil porewater pH and increased water-soluble Cd/Cu concentration, reduced formation of iron plaque on root surface, and thus significantly increased total uptake of Cd/Cu by rice. Subsequently, warming significantly promoted Cd translocation from root to shoot, and increased Cd distribution percentage in shoot, while Cu was not significantly affected. Enhanced Cd uptake and translocation synergistically resulted in higher rice grain contamination with increasing concentration from 0.27 to 0.65 and 0.14-0.40 mg kg(-1) for Indica and Japonica rice, respectively. However increase of Cu in brown grain was only attributed to its uptake enhancement under warming. Our study provides a new understanding about the food production insecurity of heavy metal contaminated soil under the future global warming.

  6. Electron Affinity Calculations for Thioethers

    NASA Technical Reports Server (NTRS)

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

    1997-01-01

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

  7. Effect of IDA and TREN chelating agents and buffer systems on the purification of human IgG with immobilized nickel affinity membranes.

    PubMed

    Ribeiro, Mariana Borsoi; Vijayalakshmi, Mookambesvaran; Todorova-Balvay, Daniele; Bueno, Sonia Maria Alves

    2008-01-01

    The purification of IgG from human plasma was studied by comparing two affinity membranes complexed with Ni(II), prepared by coupling iminodiacetic acid (IDA) and Tris(2-aminoethyl)amine (TREN) to poly(ethylenevinyl alcohol), PEVA, hollow fiber membranes. The Ni(II)-TREN-PEVA hollow fiber membrane had lower capacity for human IgG than the complex Ni(II)-IDA-PEVA, but with similar selectivity. The IgG in peak fractions eluted from the Ni(II)-IDA-PEVA with a stepwise concentration gradient of Tris-HCl pH 7.0 (100-700 mM) reached a purity of 98% (based on IgG, IgM, IgA, albumin, and transferrin nephelometric analysis). Adsorption IgG data at different temperatures (4-37 degrees C) were analyzed using Langmuir model resulting in a calculated maximum capacity at 25 degrees C of 204.6 mg of IgG/g of dry membrane. Decrease in Kd with increasing temperature (1.7x10(-5) to 5.3x10(-6) M) indicated an increase in affinity with increased temperature. The positive value of enthalpy change (26.2 kJ/mol) indicated that the adsorption of IgG in affinity membrane is endothermic. Therefore, lower temperature induces adsorption as verified experimentally.

  8. The electron affinity of gallium nitride (GaN) and digallium nitride (GaNGa): the importance of the basis set superposition error in strongly bound systems.

    PubMed

    Tzeli, Demeter; Tsekouras, Athanassios A

    2008-04-14

    The electron affinity of GaN and Ga2N as well as the geometries and the dissociation energies of the ground states of gallium nitrides GaN, GaN(-), Ga2N, and Ga2N(-) were systematically studied by employing the coupled cluster method, RCCSD(T), in conjunction with a series of basis sets, (aug-)cc-pVxZ(-PP), x=D, T, Q, and 5 and cc-pwCVxZ(-PP), x=D, T, and Q. The calculated dissociation energy and the electron affinity of GaN are 2.12 and 1.84 eV, respectively, and those of Ga2N are 6.31 and 2.53 eV. The last value is in excellent agreement with a recent experimental value for the electron affinity of Ga2N of 2.506+/-0.008 eV. For such quality in the results to be achieved, the Ga 3d electrons had to be included in the correlation space. Moreover, when a basis set is used, which has not been developed for the number of the electrons which are correlated in a calculation, the quantities calculated need to be corrected for the basis set superposition error.

  9. Bromocriptine tablet of self-microemulsifying system adsorbed onto porous carrier to stimulate lipoproteins secretion for brain cellular uptake.

    PubMed

    Thongrangsalit, Sirigul; Phaechamud, Thawatchai; Lipipun, Vimolmas; Ritthidej, Garnpimol C

    2015-07-01

    Both low solubility and high hepatic metabolism cause low oral bioavailability of bromocriptine mesylate (BM) leading to very low drug amount in brain. Self-microemulsion (SME) tablets were developed to improve solubility, stimulate lipoprotein synthesis to promote lymphatic transport, avoid hepatic metabolism and target drug to brain. SME liquid containing castor oil, Tween(®) 80 and Cremophor(®) EL was prepared and then adsorbed onto solid carries, Aerosil(®)200, Aeroperl(®)300 or NeusilinUS2(®), yielding SME powders. The optimal ratios of SME liquid to carriers determined from flowability and scanning electron photomicrographs before tableting were 1.5:1, 2:1 and 2.5:1 for Aerosil(®)200, Aeroperl(®)300 and NeusilinUS2(®), respectively. Only Aeroperl(®)300 SME tablet had comparable dissolution to BM commercial tablet. From in vitro study in Caco-2 cells, fluorescein loaded SME tablet showed higher uptake than fluorescein loaded in either oil or surfactant. Although significantly lower amount of drug was permeated from SME tablet than from commercial tablet, higher drug uptake was obviously observed (P<0.05). In addition, higher lipoprotein synthesis expressing as content of apolipoprotein B (apo-B) found in secreted chylomicron resulted in higher drug uptake in co-culture of brain endothelial cells (bEnd.3) and astrocytes (CTX TNA2) from drug loaded SME tablet when compared to commercial tablet (P<0.05) due to binding of apo-B to LDL receptors expressed on the surface of endothelial cells. Therefore, tablet of SME adsorbed onto porous carrier potentially delivered BM to brain via lymphatic transport by increasing the lipoprotein synthesis.

  10. Norepinephrine uptake by rat jejunum: Modulation by angiotensin II

    SciTech Connect

    Suvannapura, A.; Levens, N.R. )

    1988-02-01

    Angiotensin II (ANG II) is believed to stimulate sodium and water absorption from the small intestine by enhancing sympathetic nerve transmission. This study is designed to determine whether ANG II can enhance sympathetic neurotransmission within the small intestine by inhibition norepinephrine (NE) uptake. Intracellular NE accumulation by rat jejunum was concentration dependent and resolved into high- and low-affinity components. The high-affinity component (uptake 1) exhibited a Michaelis constant (K{sub m}) of 1.72 {mu}M and a maximum velocity (V{sub max}) of 1.19 nmol {center dot} g{sup {minus}1} {center dot} 10 min{sup {minus}1}. The low-affinity component (uptake 2) exhibited a K{sub m} of 111.1 {mu}M and a V{sub max} of 37.1 nmol {center dot} g{sup {minus}1} {center dot} 10 min{sup {minus}1}. Cocaine, an inhibitor of neuronal uptake, inhibited the intracellular accumulation of label by 80%. Treatment of animals with 6-hydroxydopamine, which depletes norepinephrine from sympathetic terminals, also attenuated NE uptake by 60%. Thus accumulation within sympathetic nerves constitutes the major form of ({sup 3}H)NE uptake into rat jejunum. ANG II inhibited intracellular ({sup 3}H)NE uptake in a concentration-dependent manner. At a dose of 1 mM, ANG II inhibited intracellular ({sup 3}H)NE accumulation by 60%. Cocaine failed to potentiate the inhibition of ({sup 3}H)NE uptake produced by ANG II. Thus ANG II appears to prevent ({sup 3}H)NE accumulation within rat jejunum by inhibiting neuronal uptake.

  11. Functional Analysis of the Ferric Uptake Regulator Gene fur in Xanthomonas vesicatoria.

    PubMed

    Liu, Huiqin; Dong, Chunling; Zhao, Tingchang; Han, Jucai; Wang, Tieling; Wen, Xiangzhen; Huang, Qi

    2016-01-01

    Iron is essential for the growth and survival of many organisms. Intracellular iron homeostasis must be maintained for cell survival and protection against iron toxicity. The ferric uptake regulator protein (Fur) regulates the high-affinity ferric uptake system in many bacteria. To investigate the function of the fur gene in Xanthomonas vesicatoria (Xv), we generated a fur mutant strain, fur-m, by site-directed mutagenesis. Whereas siderophore production increased in the Xv fur mutant, extracellular polysaccharide production, biofilm formation, swimming ability and quorum sensing signals were all significantly decreased. The fur mutant also had significantly reduced virulence in tomato leaves. The above-mentioned phenotypes significantly recovered when the Xv fur mutation allele was complemented with a wild-type fur gene. Thus, Fur either negatively or positively regulates multiple important physiological functions in Xv.

  12. Functional Analysis of the Ferric Uptake Regulator Gene fur in Xanthomonas vesicatoria

    PubMed Central

    Liu, Huiqin; Dong, Chunling; Zhao, Tingchang; Han, Jucai; Wang, Tieling; Wen, Xiangzhen; Huang, Qi

    2016-01-01

    Iron is essential for the growth and survival of many organisms. Intracellular iron homeostasis must be maintained for cell survival and protection against iron toxicity. The ferric uptake regulator protein (Fur) regulates the high-affinity ferric uptake system in many bacteria. To investigate the function of the fur gene in Xanthomonas vesicatoria (Xv), we generated a fur mutant strain, fur-m, by site-directed mutagenesis. Whereas siderophore production increased in the Xv fur mutant, extracellular polysaccharide production, biofilm formation, swimming ability and quorum sensing signals were all significantly decreased. The fur mutant also had significantly reduced virulence in tomato leaves. The above-mentioned phenotypes significantly recovered when the Xv fur mutation allele was complemented with a wild-type fur gene. Thus, Fur either negatively or positively regulates multiple important physiological functions in Xv. PMID:26910324

  13. Atopic donor status does not influence the uptake of the major grass pollen allergen, Phl p 5, by dendritic cells.

    PubMed

    Ashjaei, Kazem; Palmberger, Dieter; Bublin, Merima; Bajna, Erika; Breiteneder, Heimo; Grabherr, Reingard; Ellinger, Isabella; Hoffmann-Sommergruber, Karin

    2015-09-01

    Dendritic cells (DCs) are sentinels of the immune system for antigen recognition and uptake, as well as presentation to naïve T cells for stimulation or priming. Internalization and endocytic degradation of allergens by DCs are important steps required for T cell priming. In the current study we investigated binding and internalization of purified recombinant non-glycosylated grass pollen allergen, Phl p 5, and natural non-specific lipid transfer protein from sunflower, SF-nsLTP to human monocyte derived dendritic cells (MoDCs). Colocalization of Phl p 5 with low affinity (CD23) or high affinity receptor (FcεRI) was investigated by immunofluorescence staining. Likewise, localization of the allergens in early (EE) and late endosomes (LE) was detected by co-staining for early endosome antigen (EEA1) and lysosomal-associated membrane protein 1 (LAMP1). In our experimental setting we could demonstrate that Phl p 5 as well as SF-nsLTP bound to MoDCs from both, grass pollen allergic and non-allergic individuals. Competitive allergen uptake experiments demonstrated non-preferential and simultaneous uptake of Phl p 5 and SF-nsLTP by MoDCs. No overlap of signals from Phl p 5 and CD23 or FcεRI was detectable, excluding IgE-mediated uptake for this allergen. Both allergens, Phl p 5 and SF-nsLTP, were localized in early and late endosomes. The present study applied a set of methods to assess the allergen uptake by MoDCs in an in vitro model. No qualitative and quantitative differences in the allergen uptake of both, Phl p 5 and SF-nsLTP were detected in single and competitive assays.

  14. Water uptake patterns and root system architecture of Zea mays in a natural soil under influence of drought stress monitored by MRI

    NASA Astrophysics Data System (ADS)

    Merz, Steffen; Pohlmeier, Andreas; Seidler, Christina; van Dusschoten, Dagmar; Vereecken, Harry

    2012-04-01

    The interface between roots and soil plays a key role in water transport in the Soil-Plant-Atmosphere-Continuum (SPAC). The transport which changes with the degree of dehydration is influenced by both the hydraulic conductivity of roots and the soil. One important factor in plant growth is the amount of available water in the soil, which correlates directly with soil texture. Water uptake of plant roots and water uptake patterns in soil can be monitored using non-invasive 1H Nuclear Magnetic Resonance Imaging (MRI). In a preceding study the effect of root water uptake and uniform desiccation patterns under drought conditions were observed for Ricinus communis grown in a model medium (Pohlmeier et al. 2008). Continuing these studies, the new aspect is the determination of water uptake patterns and root system architecture in a natural soil. The general challenge of MRI in soils are the inherent fast relaxation times T2* and T2 of the soil matrix. With the use of conventional sequences only water in macropores can be determined. The loss of sensitivity can be overcome by MRI sequences with sufficiently short detection times. In this work we employed and assessed two methods: SPI (Single Point Imaging) detects the T2* relaxation with a dead time of < 0.05 ms and SE3D (Spin Echo 3D) probes T2,eff with an echo time of about 0.8 ms. Zea mays, planted in a cylindrical container filled with a natural soil was completely sealed after 4 weeks of growth to avoid evaporation, so water loss took place via transpiration only. The water content of the soil was determined gravimetrically and by means of MRI each 2nd day over a period of 14 days. Furthermore a SEMS (Spin Echo Multi Slice) sequence was used to visualize the growth of root system architecture. This study shows that SPI3D and SE3D are feasible for the determination of water content in a natural soil up to a certain detection limit. We observed quite uniform water uptake patterns during drying of the soil until water

  15. Characterization of Na+ uptake in the endangered desert pupfish, Cyprinodon macularius (Baird and Girard)

    PubMed Central

    Brix, Kevin V.; Grosell, Martin

    2013-01-01

    This study provided an initial characterization of Na+ uptake in saline freshwater by the endangered pupfish, Cyprinodon macularius. This species occurs only in several saline water systems in the southwestern USA and northern Mexico, where salinity is largely controlled by water-management practices. Consequently, understanding the osmoregulatory capacity of this species is important for their conservation. The lower acclimation limit of C. macularius in freshwater was found to be 2 mM Na+. Fish acclimated to 2 or 7 mM Na+ displayed similar Na+ uptake kinetics, with Km values of 4321 and 3672 μM and Vmax values of 4771 and 3602 nmol g−1 h−1, respectively. A series of experiments using pharmacological inhibitors indicated that Na+ uptake in C. macularius was not sensitive to bumetanide, metolazone, or phenamil. These results indicate the Na+–K+–2Cl− cotransporter, Na+–Cl− cotransporter, and the Na+ channel–H+-ATPase system are likely not to be involved in Na+ uptake at the apical membrane of fish gill ionocytes in fish acclimated to 2 or 7 mM Na+. However, Na+ uptake was sensitive to 1 × 10−3 M amiloride (not 1 × 10−4 or 1 × 10−5 M), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and ethoxzolamide. These data suggest that C. macularius relies on a low-affinity Na+–H+ exchanger for apical Na+ uptake and that H+ ions generated via carbonic anhydrase-mediated CO2 hydration are important for the function of this protein. PMID:27293589

  16. Characterization of Na(+) uptake in the endangered desert pupfish, Cyprinodon macularius (Baird and Girard).

    PubMed

    Brix, Kevin V; Grosell, Martin

    2013-01-01

    This study provided an initial characterization of Na(+) uptake in saline freshwater by the endangered pupfish, Cyprinodon macularius. This species occurs only in several saline water systems in the southwestern USA and northern Mexico, where salinity is largely controlled by water-management practices. Consequently, understanding the osmoregulatory capacity of this species is important for their conservation. The lower acclimation limit of C. macularius in freshwater was found to be 2 mM Na(+). Fish acclimated to 2 or 7 mM Na(+) displayed similar Na(+) uptake kinetics, with K m values of 4321 and 3672 μM and V max values of 4771 and 3602 nmol g(-1) h(-1), respectively. A series of experiments using pharmacological inhibitors indicated that Na(+) uptake in C. macularius was not sensitive to bumetanide, metolazone, or phenamil. These results indicate the Na(+)-K(+)-2Cl(-) cotransporter, Na(+)-Cl(-) cotransporter, and the Na(+) channel-H(+)-ATPase system are likely not to be involved in Na(+) uptake at the apical membrane of fish gill ionocytes in fish acclimated to 2 or 7 mM Na(+). However, Na(+) uptake was sensitive to 1 × 10(-3) M amiloride (not 1 × 10(-4) or 1 × 10(-5) M), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and ethoxzolamide. These data suggest that C. macularius relies on a low-affinity Na(+)-H(+) exchanger for apical Na(+) uptake and that H(+) ions generated via carbonic anhydrase-mediated CO2 hydration are important for the function of this protein.

  17. Contractions of affine spherical varieties

    SciTech Connect

    Arzhantsev, I V

    1999-08-31

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

  18. Method development and application to determine potential plant uptake of antibiotics and other drugs in irrigated crop production systems.

    PubMed

    Jones-Lepp, Tammy L; Sanchez, Charles A; Moy, Thomas; Kazemi, Reza

    2010-11-24

    Studies have shown the detection of emerging contaminants (ECs), of which pharmaceuticals are a subset, in surface waters across the United States. The objective of this study was to develop methods, and apply them, to evaluate the potential for food chain transfer when EC-containing waters are used for crop irrigation. Greenhouse experiments were performed in which select food crops were irrigated with water spiked with three antibiotics. Field experiments, at two different sites, were conducted. Select crops were irrigated with wastewater effluent known to contain ECs, EC-free well water, and Colorado River water containing trace-level ECs. The results of the greenhouse studies show the potential for uptake of one or more of the antibiotics evaluated, albeit at very low levels. In those food crops watered with wastewater effluent, only an industrial flavoring agent, N,N'-dimethylphenethylamine (DMPEA), was consistently found. None of the evaluated contaminants were found in crops irrigated with Colorado River water.

  19. Characterization of cellular uptake and toxicity of aminosilane-coated iron oxide nanoparticles with different charges in central nervous system-relevant cell culture models

    PubMed Central

    Sun, Zhizhi; Yathindranath, Vinith; Worden, Matthew; Thliveris, James A; Chu, Stephanie; Parkinson, Fiona E; Hegmann, Torsten; Miller, Donald W

    2013-01-01

    Background Aminosilane-coated iron oxide nanoparticles (AmS-IONPs) have been widely used in constructing complex and multifunctional drug delivery systems. However, the biocompatibility and uptake characteristics of AmS-IONPs in central nervous system (CNS)-relevant cells are unknown. The purpose of this study was to determine the effect of surface charge and magnetic field on toxicity and uptake of AmS-IONPs in CNS-relevant cell types. Methods The toxicity and uptake profile of positively charged AmS-IONPs and negatively charged COOH-AmS-IONPs of similar size were examined using a mouse brain microvessel endothelial cell line (bEnd.3) and primary cultured mouse astrocytes and neurons. Cell accumulation of IONPs was examined using the ferrozine assay, and cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results No toxicity was observed in bEnd.3 cells at concentrations up to 200 μg/mL for either AmS-IONPs or COOH-AmS-IONPs. AmS-IONPs at concentrations above 200 μg/mL reduced neuron viability by 50% in the presence or absence of a magnetic field, while only 20% reductions in viability were observed with COOH-AmS-IONPs. Similar concentrations of AmS-IONPs in astrocyte cultures reduced viability to 75% but only in the presence of a magnetic field, while exposure to COOH-AmS-IONPs reduced viability to 65% and 35% in the absence and presence of a magnetic field, respectively. Cellular accumulation of AmS-IONPs was greater in all cell types examined compared to COOH-AmS-IONPs. Rank order of cellular uptake for AmS-IONPs was astrocytes > bEnd.3 > neurons. Accumulation of COOH-AmS-IONPs was minimal and similar in magnitude in different cell types. Magnetic field exposure enhanced cellular accumulation of both AmS- and COOH-AmS-IONPs. Conclusion Both IONP compositions were nontoxic at concentrations below 100 μg/mL in all cell types examined. At doses above 100 μg/mL, neurons were more sensitive to Am

  20. Kinetic properties of a micronutrient transporter from Pisum sativum indicate a primary function in Fe uptake from the soil.

    PubMed

    Cohen, Clara K; Garvin, David F; Kochian, Leon V

    2004-03-01

    Fe uptake in dicotyledonous plants is mediated by a root plasma membrane-bound ferric reductase that reduces extracellular Fe(III)-chelates, releasing Fe(2+) ions, which are then absorbed via a metal ion transporter. We previously showed that Fe deficiency induces an increased capacity to absorb Fe and other micronutrient and heavy metals such as Zn(2+) and Cd(2+) into pea ( Pisum sativum L.) roots [Cohen et al. (1998) Plant Physiol 116:1063-1072). To investigate the molecular basis for this phenomenon, an Fe-regulated transporter that is a homologue of the Arabidopsis IRT1 micronutrient transporter was isolated from pea seedlings. This cDNA clone, designated RIT1 for root iron transporter, encodes a 348 amino acid polypeptide with eight putative membrane-spanning domains that is induced under Fe deficiency and can functionally complement yeast mutants defective in high- and low-affinity Fe transport. Chelate buffer techniques were used to control Fe(2+) in the uptake solution at nanomolar activities representative of those found in the rhizosphere, and radiotracer methodologies were employed to show that RIT1 is a very high-affinity (59)Fe(2+) uptake system ( K(m) =54-93 nM). Additionally, radiotracer ((65)Zn, (109)Cd) flux techniques were used to show that RIT can also mediate a lower affinity Zn and Cd influx ( K(m) of 4 and 100 microM, for Zn(2+) and Cd(2+), respectively). These findings suggest that, in typical agricultural soils, RIT1 functions primarily as a high-affinity Fe(2+) transporter that mediates root Fe acquisition. This is consistent with recent findings with Arabidopsis IRT1 knockout mutants that strongly suggest that this transporter plays a key role in root Fe uptake and nutrition. However, the ability of RIT1 to facilitate Zn and Cd uptake when these metals are present at elevated concentrations suggests that RIT1 may be one pathway for the entry of toxic metals into the food chain. Furthermore, the finding that plant Fe deficiency status may

  1. Comparison of the uptake of methacrylate-based nanoparticles in static and dynamic in vitro systems as well as in vivo.

    PubMed

    Rinkenauer, Alexandra C; Press, Adrian T; Raasch, Martin; Pietsch, Christian; Schweizer, Simon; Schwörer, Simon; Rudolph, Karl L; Mosig, Alexander; Bauer, Michael; Traeger, Anja; Schubert, Ulrich S

    2015-10-28

    Polymer-based nanoparticles are promising drug delivery systems allowing the development of new drug and treatment strategies with reduced side effects. However, it remains a challenge to screen for new and effective nanoparticle-based systems in vitro. Important factors influencing the behavior of nanoparticles in vivo cannot be simulated in screening assays in vitro, which still represent the main tools in academic research and pharmaceutical industry. These systems have serious drawbacks in the development of nanoparticle-based drug delivery systems, since they do not consider the highly complex processes influencing nanoparticle clearance, distribution, and uptake in vivo. In particular, the transfer of in vitro nanoparticle performance to in vivo models often fails, demonstrating the urgent need for novel in vitro tools that can imitate aspects of the in vivo situation more accurate. Dynamic cell culture, where cells are cultured and incubated in the presence of shear stress has the potential to bridge this gap by mimicking key-features of organs and vessels. Our approach implements and compares a chip-based dynamic cell culture model to the common static cell culture and mouse model to assess its capability to predict the in vivo success more accurately, by using a well-defined poly((methyl methacrylate)-co-(methacrylic acid)) and poly((methyl methacrylate)-co-(2-dimethylamino ethylmethacrylate)) based nanoparticle library. After characterization in static and dynamic in vitro cell culture we were able to show that physiological conditions such as cell-cell communication of co-cultured endothelial cells and macrophages as well as mechanotransductive signaling through shear stress significantly alter cellular nanoparticle uptake. In addition, it could be demonstrated by using dynamic cell cultures that the in vivo situation is simulated more accurately and thereby can be applied as a novel system to investigate the performance of nanoparticle systems in vivo

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

    PubMed

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

    2016-01-01

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

  3. Enhancement of CO2 Affinity in a Polymer of Intrinsic Microporosity by Amine Modification

    PubMed Central

    2014-01-01

    Nitrile groups in the polymer of intrinsic microporosity PIM-1 were reduced to primary amines using borane complexes. In adsorption experiments, the novel amine–PIM-1 showed higher CO2 uptake and higher CO2/N2 sorption selectivity than the parent polymer, with very evident dual-mode sorption behavior. In gas permeation with six light gases, the individual contributions of solubility and diffusion to the overall permeability was determined via time-lag analysis. The high CO2 affinity drastically restricts diffusion at low pressures and lowers CO2 permeability compared to the parent PIM-1. Furthermore, the size-sieving properties of the polymer are increased, which can be attributed to a higher stiffness of the system arising from hydrogen bonding of the amine groups. Thus, for the H2/CO2 gas pair, whereas PIM-1 favors CO2, amine–PIM-1 shows permselectivity toward H2, breaking the Robeson 2008 upper bound. PMID:24860196

  4. Na sup + uptake into colonic enterocyte membrane vesicles

    SciTech Connect

    Bridges, R.J.; Garty, H.; Benos, D.J.; Rummel, W. Weizmann Institute of Science, Rehovot Univ. of Alabama, Birmingham )

    1988-04-01

    Na{sup +} uptake was studied in colonic enterocyte membrane vesicles prepared from normal and dexamethasone-treated rats. Vesicles from rats treated with dexamethasone demonstrated a fivefold greater {sup 22}Na{sup +} uptake compared with vesicles from normal rats. Most of the tracer uptake in membranes derived from treated rats occurred through a conductive, amiloride-blockable pathway located in vesicles with low native K{sup +} permeability and high Cl{sup {minus}} permeability. Kinetic analysis of the amiloride inhibition curve revealed the presence of two amiloride-blockable pathways, one with a high affinity accounting for 85% of the uptake, and one with a low affinity accounting for only 12% of the uptake. Only the low-affinity pathway was detected with vesicles from normal rats. The high sensitivity to amiloride, the dependence on dexamethasone pretreatment, and the relative permeabilities to K{sup +} and Cl{sup {minus}} indicate that most of the {sup 22}Na{sup +} uptake in membranes derived from treated rats is through a Na{sup +}-specific channel located in apical membrane vesicles. Preincubation of the isolated cells from dexamethasone-treated rats at 37{degree}C in Ca{sup 2+}-free solutions before homogenization and membrane vesicle purification caused a 5- to 10-fold increase in amiloride-blockable {sup 22}Na{sup +} uptake compared with vesicles derived from cells maintained at 0{degree}C. The addition of Ca{sup 2+}, but not of Mg{sup 2+}, to the incubation solution markedly reduced this temperature-dependent enhancement in {sup 22}Na{sup +} uptake. These results suggest that Na{sup +} transport in colonic enterocytes from dexamethasone-treated rats is regulated by a Ca{sup 2+}-dependent, temperature-sensitive process which causes a sustained change in the apical membrane.

  5. Electrogenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells

    NASA Astrophysics Data System (ADS)

    Brew, Helen; Attwell, David

    1987-06-01

    Glutamate is taken up avidly by glial cells in the central nervous system1. Glutamate uptake may terminate the transmitter action of glutamate released from neurons1, and keep extracellular glutamate at concentrations below those which are neurotoxic. We report here that glutamate evokes a large inward current in retinal glial cells which have their membrane potential and intracellular ion concentrations controlled by the whole-cell patch-clamp technique2. This current seems to be due to an electrogenic glutamate uptake carrier, which transports at least two sodium ions with every glutamate anion carried into the cell. Glutamate uptake is strongly voltage-dependent, decreasing at depolarized potentials: when fully activated, it contributes almost half of the conductance in the part of the glial cell membrane facing the retinal neurons. The spatial localization, glutamate affinity and magnitude of the uptake are appropriate for terminating the synaptic action of glutamate released from photoreceptors and bipolar cells. These data challenge present explanations of how the b-wave of the electroretinogram is generated, and suggest a mechanism for non-vesicular voltage-dependent release of glutamate from neurons.

  6. Tracing source, mixing and uptaking processes of carbon in an epikarst spring-pond system in southeastern Guizhou of China by carbon isotopes (13C-14C)

    NASA Astrophysics Data System (ADS)

    Zhao, M.; Chen, B.; Liu, Z.; Li, H. C.; Yang, R.

    2015-12-01

    δ13C and Δ14C of dissolved inorganic carbon (DIC), particulate organic carbon (POC) and aquatic plants from a karst spring and two spring-fed ponds in Laqiao, Maolan County, Guizhou Province in January, July and October of 2013 were measured to understand the roles of aquatic photosynthesis through DIC uptake in karst surface waters. The mean Δ14C and δ13C values of DIC for the spring pool, midstream and downstream ponds are -60.6±26.3‰ and -13.53±1.97‰, -62.8±62.9‰ and -11.72±2.72‰, and -54.2±56.5‰ and -9.40±2.03‰, respectively. Both Δ14C and δ13C show seasonal variations, with lower Δ14C values but heavier δ13C values in dry season and vice versa in summer rainy season. This observation indicates that (1) the main carbon source of the spring DIC is from limestone bedrock dissolution and soil CO2 with higher contribution in summer due to higher productivity; and (2) 13C and 14C have different behaviors during DIC uptake by aquatic plants and during CO2 exchange between DIC and the atmospheric CO2. Biological uptake of CO2 will not affect the Δ14C of DIC, but lead to δ13CDIC enrichment. CO2 exchange between DIC and the atmospheric CO2 should elevate both the Δ14C and δ13C of DIC. In Laqiao spring-pond system, it seems that the effect of biological uptake on the Δ14C and δ13C of DIC is much stronger than that of CO2 exchange with the atmosphere. The mean Δ14C values of POC from the spring pool, midstream and downstream ponds are -308.1 ±64.3‰, -164.4±84.4‰ and -195.1±108.5‰, respectively, indicating mixture of aquatic algae and detrital particle (clay and dust). More aquatic algae were formed in the stream ponds especially in the summer. SEM results of the POC samples support this conclusion. Furthermore, the Δ14C values of the submerged aquatic plants range from -200.0‰ to -51.3 ‰ and were similar to those of the DIC, indicating that the aquatic plants used DIC for photosynthesis. The Δ14C value of an emergent plant

  7. The uptake machinery of clostridial actin ADP-ribosylating toxins--a cell delivery system for fusion proteins and polypeptide drugs.

    PubMed

    Barth, Holger; Blöcker, Dagmar; Aktories, Klaus

    2002-12-01

    Several bacterial protein toxins, including Clostridium botulinum C2 toxin, Clostridum perfringens iota toxin, Clostridium difficile ADP-ribosyltransferase, and the Bacillus-produced vegetative insecticidal proteins, target the cytoskeleton by ADP-ribosylation of actin. All these toxins are binary in structure and consist of an enzyme component, possessing ADP-ribosyltransferase activity and a separated binding and translocation component, which is involved in the delivery of the enzyme component into the cell. The toxins are not only important virulence factors but also cell biological tools to study the function of the actin cytoskeleton. Moreover, the binary toxins turned out to be effective transporter systems for the delivery of specific fusion toxins (e.g., Rho-ADP-ribosylating C3 exoenzyme) into cells. The present review describes the biological functions of the toxins, focuses on recent studies on the uptake and delivery mechanism and discusses the usage as a drug delivery system.

  8. Development, qualification, validation and application of the neutral red uptake assay in Chinese Hamster Ovary (CHO) cells using a VITROCELL® VC10® smoke exposure system.

    PubMed

    Fields, Wanda; Fowler, Kathy; Hargreaves, Victoria; Reeve, Lesley; Bombick, Betsy

    2017-04-01

    Cytotoxicity assessment of combustible tobacco products by neutral red uptake (NRU) has historically used total particulate matter (TPM) or solvent captured gas vapor phase (GVP), rather than fresh whole smoke. Here, the development, validation and application of the NRU assay in Chinese Hamster Ovary (CHO) cells, following exposure to fresh whole smoke generated with the VITROCELL® VC10® system is described. Whole smoke exposure is particularly important as both particulate and vapor phases of tobacco smoke show cytotoxicity in vitro. The VITROCELL® VC10® system provides exposure at the air liquid interface (ALI) to mimic in vivo conditions for assessing the toxicological impact of smoke in vitro. Instrument and assay validations are crucial for comparative analyses.

  9. Effects of PEG-Induced Water Deficit in Solanum nigrum on Zn and Ni Uptake and Translocation in Split Root Systems

    PubMed Central

    Feller, Urs; Anders, Iwona; Wei, Shuhe

    2015-01-01

    Drought strongly influences root activities in crop plants and weeds. This paper is focused on the performance of the heavy metal accumulator Solanum nigrum, a plant which might be helpful for phytoremediation. The water potential in a split root system was decreased by the addition of polyethylene glycol (PEG 6000). Rubidium, strontium and radionuclides of heavy metals were used as markers to investigate the uptake into roots, the release to the shoot via the xylem, and finally the basipetal transport via the phloem to unlabeled roots. The uptake into the roots (total contents in the plant) was for most makers more severely decreased than the transport to the shoot or the export from the shoot to the unlabeled roots via the phloem. Regardless of the water potential in the labeling solution, 63Ni and 65Zn were selectively redistributed within the plant. From autoradiographs, it became evident that 65Zn accumulated in root tips, in the apical shoot meristem and in axillary buds, while 63Ni accumulated in young expanded leaves and roots but not in the meristems. Since both radionuclides are mobile in the phloem and are, therefore, well redistributed within the plant, the unequal transfer to shoot and root apical meristems is most likely caused by differences in the cell-to-cell transport in differentiation zones without functional phloem (immature sieve tubes). PMID:27135329

  10. Effects of PEG-Induced Water Deficit in Solanum nigrum on Zn and Ni Uptake and Translocation in Split Root Systems.

    PubMed

    Feller, Urs; Anders, Iwona; Wei, Shuhe

    2015-06-05

    Drought strongly influences root activities in crop plants and weeds. This paper is focused on the performance of the heavy metal accumulator Solanum nigrum, a plant which might be helpful for phytoremediation. The water potential in a split root system was decreased by the addition of polyethylene glycol (PEG 6000). Rubidium, strontium and radionuclides of heavy metals were used as markers to investigate the uptake into roots, the release to the shoot via the xylem, and finally the basipetal transport via the phloem to unlabeled roots. The uptake into the roots (total contents in the plant) was for most makers more severely decreased than the transport to the shoot or the export from the shoot to the unlabeled roots via the phloem. Regardless of the water potential in the labeling solution, (63)Ni and (65)Zn were selectively redistributed within the plant. From autoradiographs, it became evident that (65)Zn accumulated in root tips, in the apical shoot meristem and in axillary buds, while (63)Ni accumulated in young expanded leaves and roots but not in the meristems. Since both radionuclides are mobile in the phloem and are, therefore, well redistributed within the plant, the unequal transfer to shoot and root apical meristems is most likely caused by differences in the cell-to-cell transport in differentiation zones without functional phloem (immature sieve tubes).

  11. Linear algorithms of affine synthesis in the Lebesgue space L^1 \\lbrack 0,1 \\rbrack

    NASA Astrophysics Data System (ADS)

    Terekhin, Pavel A.

    2010-10-01

    We prove that there are no linear algorithms of affine synthesis for affine systems in the Lebesgue space L^1 \\lbrack 0,1 \\rbrack with respect to the model space \\ell^1, although the corresponding affine synthesis problem has a positive solution under the most general assumptions. At the same time, by imposing additional conditions on the generating function of the affine system, we can give an explicit linear algorithm of affine synthesis in the Lebesgue space when the model space is that of the coefficients of the system. This linear algorithm generalizes the Fourier-Haar expansion into orthogonal series.

  12. Optimal Affine-Invariant Point Matching

    NASA Astrophysics Data System (ADS)

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

    1989-03-01

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

  13. High-Affinity Transport of Choline-O-Sulfate and Its Use as a Compatible Solute in Bacillus subtilis

    PubMed Central

    Nau-Wagner, Gabriele; Boch, Jens; Le Good, J. Ann; Bremer, Erhard

    1999-01-01

    We report here that the naturally occurring choline ester choline-O-sulfate serves as an effective compatible solute for Bacillus subtilis, and we have identified a high-affinity ATP-binding cassette (ABC) transport system responsible for its uptake. The osmoprotective effect of this trimethylammonium compound closely matches that of the potent and widely employed osmoprotectant glycine betaine. Growth experiments with a set of B. subtilis strains carrying defined mutations in the glycine betaine uptake systems OpuA, OpuC, and OpuD and in the high-affinity choline transporter OpuB revealed that choline-O-sulfate was specifically acquired from the environment via OpuC. Competition experiments demonstrated that choline-O-sulfate functioned as an effective competitive inhibitor for OpuC-mediated glycine betaine uptake, with a Ki of approximately 4 μM. Uptake studies with [1,2-dimethyl-14C]choline-O-sulfate showed that its transport was stimulated by high osmolality, and kinetic analysis revealed that OpuC has high affinity for choline-O-sulfate, with a Km value of 4 ± 1 μM and a maximum rate of transport (Vmax) of 54 ± 3 nmol/min · mg of protein in cells grown in minimal medium with 0.4 M NaCl. Growth studies utilizing a B. subtilis mutant defective in the choline to glycine betaine synthesis pathway and natural abundance 13C nuclear magnetic resonance spectroscopy of whole-cell extracts from the wild-type strain demonstrated that choline-O-sulfate was accumulated in the cytoplasm and was not hydrolyzed to choline by B. subtilis. In contrast, the osmoprotective effect of acetylcholine for B. subtilis is dependent on its biotransformation into glycine betaine. Choline-O-sulfate was not used as the sole carbon, nitrogen, or sulfur source, and our findings thus characterize this choline ester as an effective compatible solute and metabolically inert stress compound for B. subtilis. OpuC mediates the efficient transport not only of glycine betaine and choline

  14. Effect of the inhibition of dopamine uptake on the dopamine- and dimethyldopamine-induced-inhibition of the potassium-evoked release of [3H]acetylcholine from striatal slices.

    PubMed

    Farooqui, T; Wallace, R A; Miller, D D; Uretsky, N J

    1989-01-01

    1. Dimethyldopamine was eight times more potent than dopamine in activating the D2 receptor that inhibits the potassium-evoked release of [3H]acetylcholine from striatal slices. 2. Cocaine and mazindol produced an eight-fold shift in the concentration-response curve for dopamine, but not for dimethyldopamine. 3. The IC50 of dimethyldopamine for the inhibition of [3H]dopamine uptake was thirty times greater than that for dopamine. 4. Dopamine may be less potent than dimethyldopamine at the D2 receptor because dopamine has a higher affinity for the dopamine uptake system, resulting in its rapid removal from the vicinity of the receptor.

  15. On the physics of the symbol--matter problem in biological systems and the origin of life: affine Hilbert spaces model of the robustness of the internal quantum dynamics of biological systems.

    PubMed

    Balázs, András

    2003-06-01

    In the present paper, some physical considerations of the biological symbol-matter problem is exposed. First of all, the physical concept of quantum dynamical internal measuremental robustness is discussed. In this context, the significance of introducing affine molecular Hilbert spaces, the original (primordeal) internal quantum measurement, and the global constraining nature of time-inversion symmetry restoring, as a special restoration force, is discussed at some length. It is pointed out, as a summary, that global robustness of the internal dynamics of quantum measurements is due to two basic factors: on one hand, the global constraining nature of the chosen specific (symmetry-) restoring force, and on the other, the individual robustness of the discrete local internal measuremental interactions. The second condition is supposed to follow from a system-internalised ("objective") Bohr-type Copenhagen interpretation of quantum mechanics, corresponding, in an external context, to the Generalized Complementarity Principle of Bohr and Elsasser. It is not claimed, however, that this latter problem has been, as yet, satisfactorily settled physically. In fact, if it were, it would amount to a specifically biological quantum theory of internal measurement, which had to be rooted in the original primordeal global internal measurement, amounting to the origin of the genetic code.

  16. Affinity+: Semi-Structured Brainstorming on Large Displays

    SciTech Connect

    Burtner, Edwin R.; May, Richard A.; Scarberry, Randall E.; LaMothe, Ryan R.; Endert, Alexander

    2013-04-27

    Affinity diagraming is a powerful method for encouraging and capturing lateral thinking in a group environment. The Affinity+ Concept was designed to improve the collaborative brainstorm process through the use of large display surfaces in conjunction with mobile devices like smart phones and tablets. The system works by capturing the ideas digitally and allowing users to sort and group them on a large touch screen manually. Additionally, Affinity+ incorporates theme detection, topic clustering, and other processing algorithms that help bring structured analytic techniques to the process without requiring explicit leadership roles and other overhead typically involved in these activities.

  17. Identification of major zinc-binding proteins from a marine cyanobacterium: insight into metal uptake in oligotrophic environments.

    PubMed

    Barnett, James Paul; Scanlan, David John; Blindauer, Claudia Andrea

    2014-07-01

    Marine cyanobacteria make a significant contribution to primary production whilst occupying some of the most nutrient poor regions of the world's oceans. The low bioavailability of trace metals can limit the growth of phytoplankton in ocean waters, but only scarce data are available on the requirements of marine microbes for zinc. Recent genome mining studies suggest that marine cyanobacteria have both uptake systems for zinc and proteins that utilize zinc as a cofactor. In this study, the oligotrophic strain Synechococcus sp. WH8102 was grown at different zinc concentrations. Using metalloproteomics approaches, we demonstrate that even though this organism's growth was not affected by extremely low zinc levels, cells accumulated significant quantities of zinc, which was shown to be protein-associated by 2D liquid chromatography and ICP-MS. This indicates that the mechanisms for zinc uptake in Synechococcus sp. WH8102 are extremely efficient. Significantly, expression of SYNW2224, a putative porin, was up-regulated during growth in zinc-depleted conditions. Furthermore, along with 30 other proteins, SYNW2224 was captured by immobilised zinc affinity chromatography, indicating the presence of surface-exposed site(s) with metal-binding capacity. It is proposed that this porin plays a role in high-affinity zinc uptake in this and other cyanobacteria.

  18. Cloning of chrysanthemum high-affinity nitrate transporter family (CmNRT2) and characterization of CmNRT2.1

    PubMed Central

    Gu, Chunsun; Song, Aiping; Zhang, Xiaoxue; Wang, Haibin; Li, Ting; Chen, Yu; Jiang, Jiafu; Chen, Fadi; Chen, Sumei

    2016-01-01

    The family of NITRATE TRANSPORTER 2 (NRT2) proteins belongs to the high affinity transport system (HATS) proteins which acts at low nitrate concentrations. The relevant gene content of the chrysanthemum genome was explored here by isolating the full length sequences of six distinct CmNRT2 genes. One of these (CmNRT2.1) was investigated at the functional level. Its transcription level was inducible by low concentrations of both nitrate and ammonium. A yeast two hybrid assay showed that CmNRT2.1 interacts with CmNAR2, while a BiFC assay demonstrated that the interaction occurs at the plasma membrane. Arabidopsis thaliana plants heterologously expressing CmNRT2.1 displayed an enhanced rate of labeled nitrogen uptake, suggesting that CmNRT2.1 represents a high affinity root nitrate transporter. PMID:27004464

  19. Chemical binding affinity estimation using MSB

    NASA Astrophysics Data System (ADS)

    Weaver, John B.; Rauwerdink, Adam M.

    2011-03-01

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

  20. Revision, uptake and coding issues related to the open access Orchard Sports Injury Classification System (OSICS) versions 8, 9 and 10.1

    PubMed Central

    Orchard, John; Rae, Katherine; Brooks, John; Hägglund, Martin; Til, Lluis; Wales, David; Wood, Tim

    2010-01-01

    The Orchard Sports Injury Classification System (OSICS) is one of the world’s most commonly used systems for coding injury diagnoses in sports injury surveillance systems. Its major strengths are that it has wide usage, has codes specific to sports medicine and that it is free to use. Literature searches and stakeholder consultations were made to assess the uptake of OSICS and to develop new versions. OSICS was commonly used in the sports of football (soccer), Australian football, rugby union, cricket and tennis. It is referenced in international papers in three sports and used in four commercially available computerised injury management systems. Suggested injury categories for the major sports are presented. New versions OSICS 9 (three digit codes) and OSICS 10.1 (four digit codes) are presented. OSICS is a potentially helpful component of a comprehensive sports injury surveillance system, but many other components are required. Choices made in developing these components should ideally be agreed upon by groups of researchers in consensus statements. PMID:24198559

  1. The increased binding affinity of curcumin with human serum albumin in the presence of rutin and baicalin: A potential for drug delivery system

    NASA Astrophysics Data System (ADS)

    Liu, Bing-Mi; Zhang, Jun; Hao, Ai-Jun; Xu, Liang; Wang, Dan; Ji, Hui; Sun, Shi-Jie; Chen, Bo-Qi; Liu, Bin

    2016-02-01

    The impacts of rutin and baicalin on the interaction of curcumin (CU) with human serum albumin (HSA) were investigated by fluorescence and circular dichroism (CD) spectroscopies under imitated physiological conditions. The results showed that the fluorescence quenching of HSA by CU was a simultaneous static and dynamic quenching process, irrespective of the presence or absence of flavonoids. The binding constants between CU and HSA in the absence and presence of rutin and baicalin were 2.268 × 105 M- 1, 3.062 × 105 M- 1, and 3.271 × 105 M- 1, indicating that the binding affinity was increased in the case of two flavonoids. Furthermore, the binding distance determined according to Förster's theory was decreased in the presence of flavonoids. Combined with the fact that flavonoids and CU have the same binding site (site I), it can be concluded that they may simultaneously bind in different regions in site I, and formed a ternary complex of flavonoid-HSA-CU. Meanwhile, the results of fluorescence quenching, CD and three-dimensional fluorescence spectra revealed that flavonoids further strengthened the microenvironmental and conformational changes of HSA induced by CU binding. Therefore, it is possible to develop a novel complex involving CU, flavonoid and HSA for CU delivery. The work may provide some valuable information in terms of improving the poor bioavailabiliy of CU.

  2. Discovery of Compounds that Positively Modulate the High Affinity Choline Transporter

    PubMed Central

    Choudhary, Parul; Armstrong, Emma J.; Jorgensen, Csilla C.; Piotrowski, Mary; Barthmes, Maria; Torella, Rubben; Johnston, Sarah E.; Maruyama, Yuya; Janiszewski, John S.; Storer, R. Ian; Skerratt, Sarah E.; Benn, Caroline L.

    2017-01-01

    Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the SLC5A7 gene). This transporter is comparatively well-characterized but otherwise unexplored as a potential drug target. We therefore sought to identify small molecules that would enable testing of the hypothesis that positive modulation of CHT mediated transport would enhance activity-dependent cholinergic signaling. We utilized existing and novel screening techniques for their ability to reveal both positive and negative modulation of CHT using literature tools. A screening campaign was initiated with a bespoke compound library comprising both the Pfizer Chemogenomic Library (CGL) of 2,753 molecules designed specifically to help enable the elucidation of new mechanisms in phenotypic screens and 887 compounds from a virtual screening campaign to select molecules with field-based similarities to reported negative and positive allosteric modulators. We identified a number of previously unknown active and structurally distinct molecules that could be used as tools to further explore CHT biology or as a starting point for further medicinal chemistry. PMID:28289374

  3. A current perspective on applications of macrocyclic‐peptide‐based high‐affinity ligands

    PubMed Central

    Leenheer, Daniël; ten Dijke, Peter

    2016-01-01

    Abstract Monoclonal antibodies can bind with high affinity and high selectivity to their targets. As a tool in therapeutics or diagnostics, however, their large size (∼150 kDa) reduces penetration into tissue and prevents passive cellular uptake. To overcome these and other problems, minimized protein scaffolds have been chosen or engineered, with care taken to not compromise binding affinity or specificity. An alternate approach is to begin with a minimal non‐antibody scaffold and select functional ligands from a de novo library. We will discuss the structure, production, applications, strengths, and weaknesses of several classes of antibody‐derived ligands, that is, antibodies, intrabodies, and nanobodies, and nonantibody‐derived ligands, that is, monobodies, affibodies, and macrocyclic peptides. In particular, this review is focussed on macrocyclic peptides produced by the Random non‐standard Peptides Integrated Discovery (RaPID) system that are small in size (typically ∼2 kDa), but are able to perform tasks typically handled by larger proteinaceous ligands. PMID:27352774

  4. Uptake of intact nucleoside monophosphates by Bdellovibrio bacteriovorus 109J.

    PubMed Central

    Ruby, E G; McCabe, J B; Barke, J I

    1985-01-01

    The degraded nucleic acids and ribosomes of its prey cell provide Bdellovibrio bacteriovorus 109J with a source of ribonucleoside monophosphates and deoxyribonucleoside monophosphates for biosynthesis and respiration. We demonstrate that bdellovibrios, in contrast to almost all other bacteria, take up these nucleoside monophosphates into the cell in an intact, phosphorylated form. In this way they are able to assimilate more effectively the cellular contents of their prey. Studies with UMP and dTMP demonstrate that they are transported and accumulated against a concentration gradient, achieving internal levels at least 10 times the external levels. Treatment of the bdellovibrios with azide or carbonyl cyanide m-chlorophenylhydrazone eliminates their ability to either transport or maintain accumulated UMP and suggests the presence of a freely reversible exchange mechanism. There are at least two separate classes of transport systems for nucleoside monophosphates, each exhibiting partial specificity for either ribonucleoside monophosphates or deoxyribonucleoside monophosphates. Kinetic analyses of UMP transport in different developmental stages of strain 109J indicate that each stage expresses a single, saturable uptake system with a distinct apparent substrate affinity constant (Kt) of 104 microM in attack phase cells and 35 microM in prematurely released growth phase filaments. The capacity for transport of UMP by the growth phase filaments was 2.4 times that of the attack phase cells. These data, in addition to the apparent lack of environmental control of UMP transport capacity in attack phase cells, suggest that there are two transport systems for UMP in bdellovibrios and that the high-affinity, high-capacity growth phase system is developmentally regulated. PMID:4030692

  5. Affine Contractions on the Plane

    ERIC Educational Resources Information Center

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

    2007-01-01

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

  6. Affinity-aware checkpoint restart

    SciTech Connect

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

    2014-12-08

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

  7. Affinity-aware checkpoint restart

    DOE PAGES

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

    2014-12-08

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

  8. ELECTRON AFFINITIES OF INORGANIC RADICALS.

    DTIC Science & Technology

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

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

    PubMed Central

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

    2015-01-01

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

  10. The mechanism of zinc uptake by cultured rat liver cells.

    PubMed Central

    Taylor, J A; Simons, T J

    1994-01-01

    1. The initial rate of 65Zn uptake into cultured rat hepatocytes has been measured over a range of Zn2+ concentrations from 3 x 10(-10) M to 5 x 10(-6) M. Histidine and albumin were used to buffer Zn2+ ions at concentrations below 1 x 10(-6) M. 2. The results suggest there are two mechanisms for Zn2+ uptake; a high-affinity, saturable pathway, with a maximum velocity (Vmax) of 20-30 pmol (mg protein)-1 min-1 and a Michaelis-Menten constant (Km) of about 2 x 10(-9) M Zn2+ (with histidine), and a low-affinity, linear pathway, that only makes a significant contribution to Zn2+ uptake at Zn2+ concentrations above 1 x 10(-6) M. 3. Transport via the high-affinity pathway is dependent on the concentration of Zn2+ ions and not on the concentrations of Zn(2+)-ligand complexes, suggesting that Zn2+ is the transported species. 4. The affinity of the saturable pathway for Zn2+ is slightly lower in the presence of albumin, with a Km of about 1.3 x 10(-8) M. The reason for this is uncertain. PMID:8014898

  11. Heme uptake across the outer membrane as revealed by crystal structures of the receptor–hemophore complex

    PubMed Central

    Krieg, Stefanie; Huché, Frédéric; Diederichs, Kay; Izadi-Pruneyre, Nadia; Lecroisey, Anne; Wandersman, Cécile; Delepelaire, Philippe; Welte, Wolfram

    2009-01-01

    Gram-negative bacteria use specific heme uptake systems, relying on outer membrane receptors and excreted heme-binding proteins (hemophores) to scavenge and actively transport heme. To unravel the unknown molecular details involved, we present 3 structures of the Serratia marcescens receptor HasR in complex with its hemophore HasA. The transfer of heme over a distance of 9 Å from its high-affinity site in HasA into a site of lower affinity in HasR is coupled with the exergonic complex formation of the 2 proteins. Upon docking to the receptor, 1 of the 2 axial heme coordinations of the hemophore is initially broken, but the position and orientation of the heme is preserved. Subsequently, steric displacement of heme by a receptor residue ruptures the other axial coordination, leading to heme transfer into the receptor. PMID:19144921

  12. Competition behaviour of metal uptake in cementitious systems: An XRD and EXAFS investigation of Nd- and Zn-loaded 11 Å tobermorite

    NASA Astrophysics Data System (ADS)

    Vespa, M.; Dähn, R.; Wieland, E.

    Cement-based materials play an important role in multi-barrier concepts developed worldwide for the safe disposal of hazardous and radioactive wastes. Cement is used to condition and stabilize the waste materials and to construct the engineered barrier systems (container, backfill and liner materials) of repositories for radioactive waste. In this study, bulk-X-ray absorption spectroscopy (XAS) was used to investigate the uptake mechanism of Nd on the crystalline C-S-H phase 11 Å tobermorite in the presence of Zn (co-absorbing metal), and vice versa, as potential competitor under strongly alkaline conditions (pH = 12.5-13.3). The Zn and Nd concentration in all samples was 50,000 ppm, whereas the reaction times varied from 1 to 6 months. Extended X-ray absorption fine structure (EXAFS) data of the Nd LIII-edge indicate that the local structural environment of Nd consists of ∼7-8 O atoms at 2.42 Å, ∼7-8 Si at ∼3.67 Å and ∼5-6 Ca at ∼3.8 Å, and that this environment remains unchanged in the presence and absence of Zn. In contrary, Zn K-edge EXAFS data exhibit distinct differences in the presence and absence of Nd as co-absorbing element. Data analysis indicates that Zn is tetrahedrally coordinated (∼4 O at ∼1.96 Å) and the obtained structural data in the simultaneous presence of Nd and Zn are consistent with the formation of mixed Zn surface complexes and Zn bound in the interlayer remaining in these positions also with prolonged reaction times (up to 6 months). However, without the co-absorbing element Nd, strong structural changes in the uptake mechanisms of Zn are observable, e.g., after 3 month reaction time Zn-Zn backscattering pairs can be observed. These findings suggest that Nd has an influence on the incorporation of Zn in the tobermorite structure. In addition, the results of this study indicate that competitive uptake of metal cations with similar sorption behaviour by C-S-H phases can take place, deserving further attention in future

  13. Mechanism of glutamate uptake in Zymomonas mobilis.

    PubMed Central

    Ruhrmann, J; Krämer, R

    1992-01-01

    The energetics of the anaerobic gram-negative bacterium Zymomonas mobilis, a well-known ethanol-producing organism, is based solely on synthesis of 1 mol of ATP per mol of glucose by the Entner-Doudoroff pathway. When grown in the presence of glucose as a carbon and energy source, Z. mobilis had a cytosolic ATP content of 3.5 to 4 mM. Because of effective pH homeostasis, the components of the proton motive force strongly depended on the external pH. At pH 5.5, i.e., around the optimal pH for growth, the proton motive force was about -135 mV and was composed of a pH gradient of 0.6 pH units (internal pH 6.1) and a membrane potential of about -100 mV. Measurement of these parameters was complicated since ionophores and lipophilic probes were ineffective in this organism. So far, only glucose transport by facilitated diffusion is well characterized for Z. mobilis. We investigated a constitutive secondary glutamate uptake system. Glutamate can be used as a nitrogen source for Z. mobilis. Transport of glutamate at pH 5.5 shows a relatively high Vmax of 40 mumol.min-1.g (dry mass) of cells-1 and a low affinity (Km = 1.05 mM). Glutamate is taken up by a symport with two H+ ions, leading to substantial accumulation in the cytosol at low pH values. PMID:1332937

  14. P2X7 receptor activation downmodulates Na(+)-dependent high-affinity GABA and glutamate transport into rat brain cortex synaptosomes.

    PubMed

    Barros-Barbosa, A R; Lobo, M G; Ferreirinha, F; Correia-de-Sá, P; Cordeiro, J M

    2015-10-15

    Sodium-dependent high-affinity amino-acid transporters play crucial roles in terminating synaptic transmission in the central nervous system (CNS). However, there is lack of information about the mechanisms underlying the regulation of amino-acid transport by fast-acting neuromodulators, like ATP. Here, we investigated whether activation of the ATP-sensitive P2X7 receptor modulates Na(+)-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake into nerve terminals (synaptosomes) of the rat cerebral cortex. Radiolabeled neurotransmitter accumulation was evaluated by liquid scintillation spectrometry. The cell-permeant sodium-selective fluorescent indicator, SBFI-AM, was used to estimate Na(+) influx across plasma membrane. 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-300 μM), a prototypic P2X7 receptor agonist, concentration-dependently decreased [(3)H]GABA (14%) and [(14)C]glutamate (24%) uptake; BzATP decreased transport maximum velocity (Vmax) without affecting the Michaelis constant (Km) values. The selective P2X7 receptor antagonist, A-438079 (3 μM), prevented inhibition of [(3)H]GABA and [(14)C]glutamate uptake by BzATP (100 μM). The inhibitory effect of BzATP coincided with its ability to increase intracellular Na(+) and was mimicked by Na(+) ionophores, like gramicidin and monensin. Increases in intracellular Na(+) (with veratridine or ouabain) or substitution of extracellular Na(+) by N-methyl-D-glucamine (NMDG)(+) all decreased [(3)H]GABA and [(14)C]glutamate uptake and attenuated BzATP effects. Uptake inhibition by BzATP (100 μM) was also attenuated by calmidazolium, which selectively inhibits Na(+) currents through the P2X7 receptor pore. In conclusion, disruption of the Na(+) gradient by P2X7 receptor activation downmodulates high-affinity GABA and glutamate uptake into rat cortical synaptosomes. Interference with amino-acid transport efficacy may constitute a novel target for therapeutic management of cortical excitability.

  15. Transport of the aromatic amino acids into isolated rat liver cells. Properties of uptake by two distinct systems.

    PubMed Central

    Salter, M; Knowles, R G; Pogson, C I

    1986-01-01

    The transport of the aromatic amino acids into isolated rat liver cells was studied. There was a rapid and substantial binding of the aromatic amino acids, L-alanine and L-leucine to the plasma membrane. This has important consequences for the determination of rates of transport and intracellular concentrations of the amino acids. Inhibition studies with a variety of substrates of various transport systems gave results consistent with aromatic amino acid transport being catalysed by two systems: a 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH)-insensitive aromatic D- and L-amino acid-specific system, and the L-type system (BCH-sensitive). The BCH-insensitive component of transport was Na+-independent and facilitated non-concentrative transport of the aromatic amino acids; it was unaffected by culture of liver cells for 24 h, by 48 h starvation, dexamethasone phosphate or glucagon. Kinetic properties of the BCH-inhibitable component were similar to those previously reported for the L2-system in liver cells. The BCH-insensitive component was a comparatively low-Km low-Vmax. transport system that we suggest is similar to the T-transport system previously seen only in human red blood cells. The results are discussed with reference to the importance of the T- and L-systems in the control of aromatic L-amino acid degradation in the liver. PMID:3954748

  16. Solid lipid nanoparticles (SLN) of Efavirenz as lymph targeting drug delivery system: Elucidation of mechanism of uptake using chylomicron flow blocking approach.

    PubMed

    Makwana, Vivek; Jain, Rashmi; Patel, Komal; Nivsarkar, Manish; Joshi, Amita

    2015-11-10

    The aim of the present work was to develop a lymph targeted SLN formulation of antiretroviral (ARV) drug and to have an understanding of its underlying mechanism of uptake by the lymphatics. The lymphatics are the inaccessible reservoirs of HIV in human body. Efavirenz (EFV) is a BCS class II, ARV drug that undergoes extensive first pass metabolism. The EFV SLN formulation was prepared using Gelucire 44/14, Compritol 888 ATO, Lipoid S 75 and Poloxamer 188 by hot homogenization technique followed by ultrasonication method, with mean particle size of 168 nm, polydispersity index (PDI) <0.220, and mean zeta potential of -35.55 mV. DSC and XRPD studies revealed change in crystallinity index of drug when incorporated into SLN. In vitro drug release was found to be prolonged and biphasic in PBS pH 6.8. There was no significant change in the mean particle size, PDI, zeta potential and entrapment efficiency of EFV SLN after storage at 30 ± 2°C/60 ± 5%RH for two months. The results from lymphatic transport and tissue distribution study indicate that a significant part of the EFV had by-passed portal system and was recovered in the lymph via chylomicron uptake mechanism. Reduction in the amount (44.70%) of the EFV reaching to liver indicates that major amount of EFV bypasses the liver and thereby, enhances the oral bioavailability of the EFV. A significant amount of EFV was found in spleen, a major lymphatic organ. EFV SLN seems to have potential to target the ARV to lymphatics for the better management of HIV.

  17. ( sup 14 C)-Sucrose uptake by guard cell protoplasts of pisum sativum, argenteum mutant

    SciTech Connect

    Rohrig, K.; Raschke, K. )

    1991-05-01

    Guard cells rely on import for their supply with reduced carbon. The authors tested by silicone oil centrifugation the ability of guard cell protoplasts to accumulated ({sup 14}C)-sucrose. Uptake rates were corrected after measurement of {sup 14}C-sorbitol and {sup 3}H{sub 2}O spaces. Sucrose uptake followed biphasic kinetics, with a high-affinity component below 1 mM external sucrose (apparent K{sub m} 0.8 mM at 25C) and a low-affinity nonsaturable component above. Uptake depended on pH (optimum at pH 5.0). Variations in the concentrations of external KCl, CCCP, and valinomycin indicated that about one-half of the sucrose uptake rate could be related to an electrochemical gradient across the plasmalemma. Total uptake rates measured at 5 mM external sucrose seem to be sufficient to replenish emptied plastids with starch within a few hours.

  18. Uptake of glycerol 3-phosphate and some of its analogs by the hexose phosphate transport system of Escherichia coli.

    PubMed Central

    Guth, A; Engel, R; Tropp, B E

    1980-01-01

    The hexose phosphate transport system transported glycerol 3-phosphate and its analogs 3,4-dihydroxybutyl-1-phosphonate, glyceraldehyde 3-phosphate, and 3-hydroxy-4-oxobutyl-1-phosphonate. PMID:6995450

  19. High-affinity binding of fibronectin to cultured Kupffer cells

    SciTech Connect

    Cardarelli, P.M.; Blumenstock, F.A.; McKeown-Longo, P.J.; Saba, T.M.; Mazurkiewicz, J.E.; Dias, J.A. )

    1990-11-01

    Hepatic Kupffer cells are a major component of the reticuloendothelial or macrophage system. They were the first phagocytic cell type whose phagocytosis was shown to be influenced by plasma fibronectin, a dimeric opsonic glycoprotein. In the current study, the binding of soluble radioiodinated fibronectin purified from rat serum to isolated rat hepatic Kupffer cells was investigated using a cultured Kupffer cell monolayer technique. Binding was specific, since unlabeled purified fibronectin competed in a dose-dependent manner with the 125I-fibronectin for binding to the Kupffer cells. Addition of gelatin enhanced the binding of 125I-fibronectin to Kupffer cells. The phagocytosis of gelatinized-coated red cells by Kupffer cells was increased either by preopsonizing the target particles with purified fibronectin or by the addition of purified fibronectin to the culture medium. In contrast, exposure of the Kupffer cells to medium containing purified fibronectin followed by wash-removal of the fibronectin did not increase the uptake of gelatin-coated red blood cells, even though fibronectin was detected on the surface of the Kupffer cells by immunofluorescence. Trypsinized monolayers expressed decreased capacity to bind 125I-fibronectin as well as fibronectin-coated sheep erythrocytes. The binding of 125I-fibronectin-gelatin complexes was inhibited by excess unlabeled fibronectin. We calculated that specific high-affinity (Kd = 7.46 x 10(-9) M) binding sites for fibronectin exist on Kupffer cells. There are approximately 2,800-3,500 binding sites or putative fibronectin receptors per Kupffer cell. These sites appear to mediate the enhanced phagocytosis of gelatin-coated particles opsonized by fibronectin.

  20. Calcium Mitigates Arsenic Toxicity in Rice Seedlings by Reducing Arsenic Uptake and Modulating the Antioxidant Defense and Glyoxalase Systems and Stress Markers.

    PubMed

    Rahman, Anisur; Mostofa, Mohammad Golam; Alam, Md Mahabub; Nahar, Kamrun; Hasanuzzaman, Mirza; Fujita, Masayuki

    2015-01-01

    The effect of exogenous calcium (Ca) on hydroponically grown rice seedlings was studied under arsenic (As) stress by investigating the antioxidant and glyoxalase systems. Fourteen-day-old rice (Oryza sativa L. cv. BRRI dhan29) seedlings were exposed to 0.5 and 1 mM Na2HAsO4 alone and in combination with 10 mM CaCl2 (Ca) for 5 days. Both levels of As caused growth inhibition, chlorosis, reduced leaf RWC, and increased As accumulation in the rice seedlings. Both doses of As in growth medium induced oxidative stress through overproduction of reactive oxygen species (ROS) by disrupting the antioxidant defense and glyoxalase systems. Exogenous application of Ca along with both levels of As significantly decreased As accumulation and restored plant growth and water loss. Calcium supplementation in the As-exposed rice seedlings reduced ROS production, increased ascorbate (AsA) content, and increased the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD), and the glyoxalase I (Gly I) and glyoxalase II (Gly II) enzymes compared with seedlings exposed to As only. These results suggest that Ca supplementation improves rice seedlings tolerance to As-induced oxidative stress by reducing As uptake, enhancing their antioxidant defense and glyoxalase systems, and also improving growth and physiological condition.

  1. The uptake of NO3-, NO2-, and NH4+ by intact wheat (Triticum aestivum) seedlings. I. Induction and kinetics of transport systems

    NASA Technical Reports Server (NTRS)

    Goyal, S. S.; Huffaker, R. C.

    1986-01-01

    The inducibility and kinetics of the NO3-, NO2-, and NH4+ transporters in roots of wheat seedlings (Triticum aestivum cv Yercora Rojo) were characterized using precise methods approaching constant analysis of the substrate solutions. A microcomputer-controlled automated high performance liquid chromatography system was used to determine the depletion of each N species (initially at 1 millimolar) from complete nutrient solutions. Uptake rate analyses were performed using computerized curve-fitting techniques. More precise estimates were obtained for the time required for the extent of the induction of each transporter. Up to 10 and 6 hours, respectively, were required to achieve apparent full induction of the NO3- and NO2- transporters. Evidence for substrate inducibility of the NH4+ transporters requiring 5 hours is presented. The transport of NO3- was mediated by a dual system (or dual phasic), whereas only single systems were found for transport of NO2- and NH4+. The Km values for NO3-, NO2-, and NH4+ were, respectively, 0.027, 0.054, and 0.05 millimolar. The Km for mechanism II of NO3- transport could not be defined in this study as it exhibited only apparent first order kinetics up to 1 millimolar.

  2. Calcium Mitigates Arsenic Toxicity in Rice Seedlings by Reducing Arsenic Uptake and Modulating the Antioxidant Defense and Glyoxalase Systems and Stress Markers

    PubMed Central

    Rahman, Anisur; Mostofa, Mohammad Golam; Alam, Md. Mahabub; Nahar, Kamrun; Hasanuzzaman, Mirza; Fujita, Masayuki

    2015-01-01

    The effect of exogenous calcium (Ca) on hydroponically grown rice seedlings was studied under arsenic (As) stress by investigating the antioxidant and glyoxalase systems. Fourteen-day-old rice (Oryza sativa L. cv. BRRI dhan29) seedlings were exposed to 0.5 and 1 mM Na2HAsO4 alone and in combination with 10 mM CaCl2 (Ca) for 5 days. Both levels of As caused growth inhibition, chlorosis, reduced leaf RWC, and increased As accumulation in the rice seedlings. Both doses of As in growth medium induced oxidative stress through overproduction of reactive oxygen species (ROS) by disrupting the antioxidant defense and glyoxalase systems. Exogenous application of Ca along with both levels of As significantly decreased As accumulation and restored plant growth and water loss. Calcium supplementation in the As-exposed rice seedlings reduced ROS production, increased ascorbate (AsA) content, and increased the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glutathione peroxidase (GPX), superoxide dismutase (SOD), and the glyoxalase I (Gly I) and glyoxalase II (Gly II) enzymes compared with seedlings exposed to As only. These results suggest that Ca supplementation improves rice seedlings tolerance to As-induced oxidative stress by reducing As uptake, enhancing their antioxidant defense and glyoxalase systems, and also improving growth and physiological condition. PMID:26798635

  3. Thyroid Scan and Uptake

    MedlinePlus

    ... procedures within the last two months that used iodine-based contrast material. Your doctor will instruct you ... a type of nuclear medicine imaging. The radioactive iodine uptake test (RAIU) is also known as a ...

  4. Thyroid Scan and Uptake

    MedlinePlus Videos and Cool Tools

    ... A thyroid scan is a type of nuclear medicine imaging. The radioactive iodine uptake test (RAIU) is ... thyroid function, but does not involve imaging. Nuclear medicine is a branch of medical imaging that uses ...

  5. PHARMACEUTICAL AND BIOMEDICAL APPLICATIONS OF AFFINITY CHROMATOGRAPHY: RECENT TRENDS AND DEVELOPMENTS

    PubMed Central

    Hage, David S.; Anguizola, Jeanethe A.; Bi, Cong; Li, Rong; Matsuda, Ryan; Papastavros, Efthimia; Pfaunmiller, Erika; Vargas, John; Zheng, Xiwei

    2012-01-01

    Affinity chromatography is a separation technique that has become increasingly important in work with biological samples and pharmaceutical agents. This method is based on the use of a biologically-related agent as a stationary phase to selectively retain analytes or to study biological interactions. This review discusses the basic principles behind affinity chromatography and examines recent developments that have occurred in the use of this method for biomedical and pharmaceutical analysis. Techniques based on traditional affinity supports are discussed, but an emphasis is placed on methods in which affinity columns are used as part of HPLC systems or in combination with other analytical methods. General formats for affinity chromatography that are considered include step elution schemes, weak affinity chromatography, affinity extraction and affinity depletion. Specific separation techniques that are examined include lectin affinity chromatography, boronate affinity chromatography, immunoaffinity chromatography, and immobilized metal ion affinity chromatography. Approaches for the study of biological interactions by affinity chromatography are also presented, such as the measurement of equilibrium constants, rate constants, or competition and displacement effects. In addition, related developments in the use of immobilized enzyme reactors, molecularly imprinted polymers, dye ligands and aptamers are briefly considered. PMID:22305083

  6. Assessment of root uptake and systemic vine-transport of Salmonella enterica sv. Typhimurium by melon (Cucumis melo) during field production.

    PubMed

    Lopez-Velasco, Gabriela; Sbodio, Adrian; Tomás-Callejas, Alejandro; Wei, Polly; Tan, Kin Hup; Suslow, Trevor V

    2012-08-01

    Among melons, cantaloupes are most frequently implicated in outbreaks and surveillance-based recalls due to Salmonella enterica. There is limited but compelling evidence that associates irrigation water quality as a significant risk of preharvest contamination of melons. However, the potential for root uptake from water and soil and subsequent systemic transport of Salmonella into melon fruit is uncharacterized. The aim of this work was to determine whether root uptake of S. enterica results in systemic transport to fruit at high doses of applied inoculum through sub-surface drip and furrow irrigation during field production of melons. Cantaloupe and honeydew were grown under field conditions, in a silt clay loam soil using standard agronomic practices for California. An attenuated S. enterica sv. Typhimurium strain was applied during furrow irrigation and, in separate plots, buried drip-emitter lines delivered the inoculum directly into the established root zone. Contamination of the water resulted in soil contamination within furrows however Salmonella was not detected on top of the beds or around melon roots of furrow-irrigated rows demonstrating absence of detectable lateral transfer across the soil profile. In contrast, positive detection of the applied isolate occurred in soil and the rhizosphere in drip injected plots; survival of Salmonella was at least 41 days. Despite high populations of the applied bacteria in the rhizosphere, after surface disinfection, internalized Salmonella was not detected in mature melon fruit (n=485). Contamination of the applied Salmonella was detected on the rind surface of melons if fruit developed in contact with soil on the sides of the inoculated furrows. Following an unusual and heavy rain event during fruit maturation, melons collected from the central area of the beds, were shown to harbor the furrow-applied Salmonella. Delivery of Salmonella directly into the peduncle, after minor puncture wounding, resulted in detection

  7. FUNCTIONAL OVERLAP OF ROOT SYSTEMS IN AN OLD-GROWTH FOREST INFERRED FROM TRACER 15N UPTAKE

    EPA Science Inventory

    Belowground competition for nutrients and water is considered a key factor affecting spatial organization and productivity of individual stems within forest stands, yet there are few data describing the lateral extent and overlap of competing root systems. We quantified the func...

  8. Development and application of an arabinose-inducible expression system by facilitating inducer uptake in Corynebacterium glutamicum.

    PubMed

    Zhang, Yun; Shang, Xiuling; Lai, Shujuan; Zhang, Guoqiang; Liang, Yong; Wen, Tingyi

    2012-08-01

    Corynebacterium glutamicum is currently used for the industrial production of a variety of biological materials. Many available inducible expression systems in this species use lac-derived promoters from Escherichia coli that exhibit much lower levels of inducible expression and leaky basal expression. We developed an arabinose-inducible expression system that contains the L-arabinose regulator AraC, the P(BAD) promoter from the araBAD operon, and the L-arabinose transporter AraE, all of which are derived from E. coli. The level of inducible P(BAD)-based expression could be modulated over a wide concentration range from 0.001 to 0.4% L-arabinose. This system tightly controlled the expression of the uracil phosphoribosyltransferase without leaky expression. When the gene encoding green fluorescent protein (GFP) was under the control of P(BAD) promoter, flow cytometry analysis showed that GFP was expressed in a highly homogeneous profile throughout the cell population. In contrast to the case in E. coli, P(BAD) induction was not significantly affected in the presence of different carbon sources in C. glutamicum, which makes it useful in fermentation applications. We used this system to regulate the expression of the odhI gene from C. glutamicum, which encodes an inhibitor of α-oxoglutarate dehydrogenase, resulting in high levels of glutamate production (up to 13.7 mM) under biotin nonlimiting conditions. This system provides an efficient tool available for molecular biology and metabolic engineering of C. glutamicum.

  9. Comparative Metabolism of Carbon Tetrachloride in Rats, Mice and Hamsters Using Gas Uptake and PBPK Modeling

    SciTech Connect

    Thrall, Karla D. ); Vucelick, Mark E.; Gies, Richard A. ); Zangar, Richard C. ); Weitz, Karl K. ); Poet, Torka S. ); Springer, David L. ); Grant, Donna M. ); Benson, Janet M.

    2000-08-25

    No study has comprehensively compared the rate of metabolism of carbon tetrachloride (CCl4) across species. Therefore, the in vivo metabolism of CCl4 was evaluated using groups of male animals (F344 rats, B6C3F1 mice, and Syrian hamsters) exposed to 40-1800 ppm CCl4 in a closed, recirculating gas-uptake system. For each species, an optimal fit of the family of uptake curves was obtained by adjusting Michaelis-Menten metabolic constants Km (affinity) and Vmax (capacity) using a physiologically based pharmacokinetic (PBPK) model. The results show that the mouse has a slightly higher capacity and lower affinity for metabolizing CCl4 compared to the rat, while the hamster has a higher capacity and lower affinity than either rat or mouse. A comparison of the Vmax to Km ratio, normalized for mg of liver protein (L/hr/mg) across species indicates that hamsters metabolize more CCl4 than either rats or mice, and should be more susceptible to CCl4-induced hepatotoxicity. These species comparisons were evaluated against toxicokinetic studies conducted in animals exposed by nose-only inhalation to 20 ppm 14C-labeled CCl4 for 4 hours. The toxicokinetic study results are consistent with the in vivo rates of metabolism, with rats eliminating less radioactivity associated with metabolism (14CO2 and urine/feces) and more radioactivity associated with the parent compound (radioactivity trapped on charcoal) compared to either hamsters or mice. The in vivo metabolic constants determined here, together with in vitro constants determined using rat, mouse, hamster and human liver microsomes, were used to estimate human in vivo metabolic rates of 1.49 mg/hr/kg body weight and 0.25 mg/L for Vmax and Km, respectively. Normalizing the rate of metabolism (Vmax/Km) by mg liver protein, the rate of metabolism of CCl4 differs across species, with hamster > mouse& > rat > human.

  10. Traits and selection strategies to improve root systems and water uptake in water-limited wheat crops.

    PubMed

    Wasson, A P; Richards, R A; Chatrath, R; Misra, S C; Prasad, S V Sai; Rebetzke, G J; Kirkegaard, J A; Christopher, J; Watt, M

    2012-05-01

    Wheat yields globally will depend increasingly on good management to conserve rainfall and new varieties that use water efficiently for grain production. Here we propose an approach for developing new varieties to make better use of deep stored water. We focus on water-limited wheat production in the summer-dominant rainfall regions of India and Australia, but the approach is generally applicable to other environments and root-based constraints. Use of stored deep water is valuable because it is more predictable than variable in-season rainfall and can be measured prior to sowing. Further, this moisture is converted into grain with twice the efficiently of in-season rainfall since it is taken up later in crop growth during the grain-filling period when the roots reach deeper layers. We propose that wheat varieties with a deeper root system, a redistribution of branch root density from the surface to depth, and with greater radial hydraulic conductivity at depth would have higher yields in rainfed systems where crops rely on deep water for grain fill. Developing selection systems for mature root system traits is challenging as there are limited high-throughput phenotyping methods for roots in the field, and there is a risk that traits selected in the lab on young plants will not translate into mature root system traits in the field. We give an example of a breeding programme that combines laboratory and field phenotyping with proof of concept evaluation of the trait at the beginning of the selection programme. This would greatly enhance confidence in a high-throughput laboratory or field screen, and avoid investment in screens without yield value. This approach requires careful selection of field sites and years that allow expression of deep roots and increased yield. It also requires careful selection and crossing of germplasm to allow comparison of root expression among genotypes that are similar for other traits, especially flowering time and disease and toxicity

  11. Micromolar-affinity benzodiazepine receptors regulate voltage-sensitive calcium channels in nerve terminal preparations.

    PubMed Central

    Taft, W C; DeLorenzo, R J

    1984-01-01

    Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance. PMID:6328498

  12. Micromolar-Affinity Benzodiazepine Receptors Regulate Voltage-Sensitive Calcium Channels in Nerve Terminal Preparations

    NASA Astrophysics Data System (ADS)

    Taft, William C.; Delorenzo, Robert J.

    1984-05-01

    Benzodiazepines in micromolar concentrations significantly inhibit depolarization-sensitive Ca2+ uptake in intact nerve-terminal preparations. Benzodiazepine inhibition of Ca2+ uptake is concentration dependent and stereospecific. Micromolar-affinity benzodiazepine receptors have been identified and characterized in brain membrane and shown to be distinct from nanomolar-affinity benzodiazepine receptors. Evidence is presented that micromolar, and not nanomolar, benzodiazepine binding sites mediate benzodiazepine inhibition of Ca2+ uptake. Irreversible binding to micromolar benzodiazepine binding sites also irreversibly blocked depolarization-dependent Ca2+ uptake in synaptosomes, indicating that these compounds may represent a useful marker for identifying the molecular components of Ca2+ channels in brain. Characterization of benzodiazepine inhibition of Ca2+ uptake demonstrates that these drugs function as Ca2+ channel antagonists, because benzodiazepines effectively blocked voltage-sensitive Ca2+ uptake inhibited by Mn2+, Co2+, verapamil, nitrendipine, and nimodipine. These results indicate that micromolar benzodiazepine binding sites regulate voltage-sensitive Ca2+ channels in brain membrane and suggest that some of the neuronal stabilizing effects of micromolar benzodiazepine receptors may be mediated by the regulation of Ca2+ conductance.

  13. Cyclic RGD peptide-modified liposomal drug delivery system for targeted oral apatinib administration: enhanced cellular uptake and improved therapeutic effects

    PubMed Central

    Song, Zhiwang; Lin, Yun; Zhang, Xia; Feng, Chan; Lu, Yonglin; Gao, Yong; Dong, Chunyan

    2017-01-01

    Apatinib is an oral tyrosine kinase inhibitor, which selectively targets vascular endothelial growth factor receptor 2 and has the potential to treat many tumors therapeutically. Cyclic arginylglycylaspartic acid (cRGD)- and polyethylene glycol (PEG)-modified liposomes (cRGD-Lipo-PEG) were constructed to act as a targeted delivery system for the delivery of apatinib to the human colonic cancer cell line, HCT116. These cRGD-modified liposomes specifically recognized integrin αvβ3 and exhibited greater uptake efficiency with respect to delivering liposomes into HCT116 cells when compared to nontargeted liposomes (Lipo-PEG), as well as greater death of tumor cells and apoptosis. The mechanism by which cRGD-Lipo-PEG targets cells was elucidated further with competition assays. To determine the anticancer efficacy in vivo, nude mice were implanted with HCT116 xenografts and treated with apatinib-loaded liposomes or free apatinib intravenously or via intragastric administration. The active and passive targeting of cRGD-Lipo-PEG led to significant tumor treatment targeting ability, better inhibition of tumor growth, and less toxicity when compared with treatments using uncombined apatinib. The results presented strongly support the case for cRGD-Lipo-PEG representing a targeted delivery system for apatinib in the treatment of colonic cancer. PMID:28331317

  14. Functional components of the bacterial CzcCBA efflux system reduce cadmium uptake and accumulation in transgenic tobacco plants.

    PubMed

    Nesler, Andrea; DalCorso, Giovanni; Fasani, Elisa; Manara, Anna; Di Sansebastiano, Gian Pietro; Argese, Emanuele; Furini, Antonella

    2017-03-25

    Cadmium (Cd) is a toxic trace element released into the environment by industrial and agricultural practices, threatening the health of plants and contaminating the food/feed chain. Biotechnology can be used to develop plant varieties with a higher capacity for Cd accumulation (for use in phytoremediation programs) or a lower capacity for Cd accumulation (to reduce Cd levels in food and feed). Here we generated transgenic tobacco plants expressing components of the Pseudomonas putida CzcCBA efflux system. Plants were transformed with combinations of the CzcC, CzcB and CzcA genes, and the impact on Cd mobilization was analysed. Plants expressing PpCzcC showed no differences in Cd accumulation, whereas those expressing PpCzcB or PpCzcA accumulated less Cd in the shoots, but more Cd in the roots. Plants expressing both PpCzcB and PpCzcA accumulated less Cd in the shoots and roots compared to controls, whereas plants expressing all three genes showed a significant reduction in Cd levels only in shoots. These results show that components of the CzcCBA system can be expressed in plants and may be useful for developing plants with a reduced capacity to accumulate Cd in the shoots, potentially reducing the toxicity of food/feed crops cultivated in Cd-contaminated soils.

  15. Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots.

    PubMed Central

    Gazzarrini, S; Lejay, L; Gojon, A; Ninnemann, O; Frommer, W B; von Wirén, N

    1999-01-01

    Ammonium and nitrate are the prevalent nitrogen sources for growth and development of higher plants. 15N-uptake studies demonstrated that ammonium is preferred up to 20-fold over nitrate by Arabidopsis plants. To study the regulation and complex kinetics of ammonium uptake, we isolated two new ammonium transporter (AMT) genes and showed that they functionally complemented an ammonium uptake-deficient yeast mutant. Uptake studies with 14C-methylammonium and inhibition by ammonium yielded distinct substrate affinities between uptake into plant roots showed that nitrogen supply and time of day differentially regulated the individual carriers. Transcript levels of AtAMT1;1, which possesses an affinity in the nanomolar range, steeply increased with ammonium uptake in roots when nitrogen nutrition became limiting, whereas those of AtAMT1;3 increased slightly, with AtAMT1;2 being more constitutively expressed. All three ammonium transporters showed diurnal variation in expression, but AtAMT1;3 transcript levels peaked with ammonium uptake at the end of the light period, suggesting that AtAMT1;3 provides a link between nitrogen assimilation and carbon provision in roots. Our results show that high-affinity ammonium uptake in roots is regulated in relation to the physiological status of the plant at the transcriptional level and by substrate affinities of individual members of the AMT1 gene family. PMID:10330477

  16. Assessing the impact of geogenic chromium uptake by carrots (Daucus carota) grown in Asopos river basin.

    PubMed

    Lilli, Maria A; Syranidou, Evdokia; Palliou, Andriana; Nikolaidis, Nikolaos P; Karatzas, George; Kalogerakis, Nicolas

    2017-01-01

    A methodology was developed to assess the impact of geogenic origin hexavalent chromium uptake by carrots, and the risk of human consumption of carrots grown in Asopos River basin in Greece. A field scale experiment was conducted with carrots cultivated in treatment plots, with and without compost amendment, in order to assess the impact of carbon in the mobility and uptake of chromium by plants. The results suggested that there is a trend for chromium mobilization and uptake in the surface and the leaves of the carrots cultivated in the treatment plot with the higher carbon addition, but not in the core of the carrots. Limited mobility of hexavalent chromium in the soil-plant-water system is presented due to the affinity of chromium to be retained in the solid phase and be uptaken by plants. Hexavalent chromium tolerant bacterial strains were isolated from the carrots. These endophytic bacteria, present in all parts of the plant, were able to reduce hexavalent chromium to trivalent form to levels below the detection limit. Finally, a site-specific risk assessment analysis suggested no adverse effects to human health due to the consumption of carrots. These findings are of particular importance since they confirm that carrots grown in soils with geogenic origin chromium does not pose any adverse risk for human consumption, but could also have the beneficial effect of the micronutrient trivalent chromium.

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

    PubMed

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

    2008-02-08

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

  18. Nutritional status and ion uptake response of Gynura bicolor DC. between Porous-tube and traditional hydroponic growth systems

    NASA Astrophysics Data System (ADS)

    Wang, Minjuan; Fu, Yuming; Liu, Hong

    2015-08-01

    Hydroponic culture has traditionally been used for Bioregenerative Life Support Systems (BLSS) because the optimal environment for roots supports high growth rates. Recent developments in Porous-tube Nutrient Delivery System (PTNDS) also offer high control of the root environment which is designed to provide a means for accurate environmental control and to allow for two-phase flow separation in microgravity. This study compared the effects of PTNDS and traditional hydroponic cultures on biomass yield, nutritional composition and antioxidant defense system (T-AOC, GSH, H2O2 and MDA) of G. bicolor, and ionic concentration (NH4+, K+, Mg2+, Ca2+, NO3-, H2 PO4-, SO42-) of nutrient solution during planting period in controlled environment chambers. The results indicated that the biomass production and yield of G. bicolor grown in PTNDS were higher than in hydroponic culture, although Relative water content (RWC), leaf length and shoot height were not significantly different. PTNDS cultivation enhanced calories from 139.5 to 182.3 kJ/100 g dry matter, and carbohydrate from 4.8 to 7.3 g/100 g dry matter and reduced the amount of protein from 7.3 to 4.8 g/100 g dry matter and ash from 1.4 to1.0 g/100 g dry matter, compared with hydroponic culture. PTNDS cultivation accumulated the nutrition elements of Ca, Cu, Fe and Zn, and reduced Na concentration. T-AOC and GSH contents were significantly lower in PTNDS than in hydroponic culture in the first harvest. After the first harvest, the contents of MDA and H2O2 were significantly higher in PTNDS than in hydroponic culture. However, the activity of T-AOC and GSH and H2O2 and MDA contents had no significant differences under both cultures after the second and third harvest. Higher concentrations of K+, Mg2+ and Ca2+ were found in nutrient solution of plants grown in hydroponics culture compared to PTNDS, wherein lower concentrations of NO3-, H2 PO4- and SO42- occurred. Our results demonstrate that PTNDS culture has more

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

    PubMed

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

    2016-03-21

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

  20. Cyanide uptake from wastewater by modified natrolite zeolite-iron oxyhydroxide system: application of isotherm and kinetic models.

    PubMed

    Noroozifar, Meissam; Khorasani-Motlagh, Mozhgan; Fard, Parisa Ahmadzadeh

    2009-07-30

    A method for the removal of cyanides from wastewater is described. The method involves the adsorption of cyanides by a modified natural zeolite (natrolite) using batch technique. A new iron oxyhydroxide-natrolite system was used in this study. A combination of XRD, XRF and FTIR spectroscopies, as well as TG/DSC thermal analyses was used for characterization of zeolitic materials. Effects of parameters such as pH, amount of adsorbent and contact time on the cyanide removing yield are studied. It was observed that the yield increases by increasing dosage of adsorbent and contact time at a fixed pH 7.5. A yield of 82% was achieved at optimum conditions for removing cyanide from industrial wastewaters. The experimental data obtained for optimum conditions were selected for modeling the adsorption behavior of the materials using six isotherm equations (Freundlich, Langmuir, Langmuir-Freundlich, Dubinin-Radushkevich, Redlich-Peterson and Toth). The obtained modeling results indicated that, although the three-parameter models, taking into account the surface heterogeneity, provided the closest approach to the measurement data, the parameters estimates could be highly biased. The kinetic studies proved that the second-order kinetic was the applicable model.

  1. Novel ISCOMs from Quillaja brasiliensis saponins induce mucosal and systemic antibody production, T-cell responses and improved antigen uptake.

    PubMed

    Cibulski, Samuel Paulo; Mourglia-Ettlin, Gustavo; Teixeira, Thais Fumaco; Quirici, Lenora; Roehe, Paulo Michel; Ferreira, Fernando; Silveira, Fernando

    2016-02-24

    In the last decades, significant efforts have been dedicated to the search for novel vaccine adjuvants. In this regard, saponins and its formulations as "immunostimulating complexes" (ISCOMs) have shown to be capable of stimulating potent humoral and cellular immune responses, enhanced cytokine production and activation of cytotoxic T cells. The immunological activity of ISCOMs formulated with a saponin fraction extracted from Quillaja brasiliensis (QB-90 fraction) as an alternative to classical ISCOMs based on Quil A(®) (IQA) is presented here. The ISCOMs prepared with QB-90, named IQB-90, typically consist of 40-50 nm, spherical, cage-like particles, built up by QB-90, cholesterol, phospholipids and antigen (ovalbumin, OVA). These nanoparticles were efficiently uptaken in vitro by murine bone marrow-derived dendritic cells. Subcutaneously inoculated IQB-90 induced strong serum antibody responses encompassing specific IgG1 and IgG2a, robust DTH reactions, significant T cell proliferation and increases in Th1 (IFN-γ and IL-2) cytokine responses. Intranasally delivered IQB-90 elicited serum IgG and IgG1, and mucosal IgA responses at distal systemic sites (nasal passages, large intestine and vaginal lumen). These results indicate that IQB-90 is a promising alternative to classic ISCOMs as vaccine adjuvants, capable of enhancing humoral and cellular immunity to levels comparable to those induced by ISCOMs manufactured with Quillaja saponaria saponins.

  2. In silico survey of the mitochondrial protein uptake and maturation systems in the brown alga Ectocarpus siliculosus.

    PubMed

    Delage, Ludovic; Leblanc, Catherine; Nyvall Collén, Pi; Gschloessl, Bernhard; Oudot, Marie-Pierre; Sterck, Lieven; Poulain, Julie; Aury, Jean-Marc; Cock, J Mark

    2011-01-01

    The acquisition of mitochondria was a key event in eukaryote evolution. The aim of this study was to identify homologues of the components of the mitochondrial protein import machinery in the brown alga Ectocarpus and to use this information to investigate the evolutionary history of this fundamental cellular process. Detailed searches were carried out both for components of the protein import system and for related peptidases. Comparative and phylogenetic analyses were used to investigate the evolution of mitochondrial proteins during eukaryote diversification. Key observations include phylogenetic evidence for very ancient origins for many protein import components (Tim21, Tim50, for example) and indications of differences between the outer membrane receptors that recognize the mitochondrial targeting signals, suggesting replacement, rearrangement and/or emergence of new components across the major eukaryotic lineages. Overall, the mitochondrial protein import components analysed in this study confirmed a high level of conservation during evolution, indicating that most are derived from very ancient, ancestral proteins. Several of the protein import components identified in Ectocarpus, such as Tim21, Tim50 and metaxin, have also been found in other stramenopiles and this study suggests an early origin during the evolution of the eukaryotes.

  3. Inhibited muscle amino acid uptake in sepsis.

    PubMed Central

    Hasselgren, P O; James, J H; Fischer, J E

    1986-01-01

    Amino acid uptake in vivo was determined in soleus (SOL) muscle, diaphragm, heart, and liver following intravenous injection of [3H]-alpha-amino-isobutyric acid ([3H]-AIB) in rats made septic by cecal ligation and puncture (CLP) and in sham-operated controls. Muscle amino acid transport was also measured in vitro by determining uptake of [3H]-AIB in incubated extensor digitorum longus (EDL) and SOL muscles. Results were expressed as distribution ratio between [3H]-AIB in intracellular and extracellular fluid. AIB uptake in vivo was reduced by 90% in SOL and cardiac muscle and by 45% in diaphragm 16 hours after CLP. In contrast, AIB uptake by liver was almost four times higher in septic than in control animals. AIB uptake in vitro was reduced by 18% in EDL 8 hours after CLP but was not significantly altered in SOL at the same time point. Sixteen hours after CLP, AIB uptake was significantly reduced in both muscles, i.e., by 17% in EDL and by 65% in SOL. When muscles from untreated rats were incubated in the presence of plasma from septic animals (16 hours CLP) or from animals injected with endotoxin (2 mg/kg body weight), AIB uptake was reduced. Addition of endotoxin in vitro (2-200 micrograms/ml) to incubated muscles did not affect AIB uptake. The results suggest that sepsis leads to marked impairment of amino acid transport system A in muscle and that this impairment is mediated by a circulating factor that is not endotoxin. Reduced uptake of amino acids by skeletal muscle during sepsis may divert amino acids to the liver for increased gluconeogenesis and protein synthesis. PMID:3963895

  4. Uptake and metabolism of 10:2 fluorotelomer alcohol in soil-earthworm (Eisenia fetida) and soil-wheat (Triticum aestivum L.) systems.

    PubMed

    Zhao, Shuyan; Zhu, Lingyan

    2017-01-01

    The behavior of 10:2 fluorotelomer alcohol (10:2 FTOH) in the systems of soil-earthworm (Eisenia fetida), soil-wheat (Triticum aestivum L.) and soil-earthworm-wheat, including degradation in soil, uptake and metabolism in wheat and earthworms were investigated. Several perfluorocarboxylic acids (PFCAs) as degradation products of 10:2 FTOH were identified in the soil, plant and earthworms. 10:2 FTOH could be biodegraded to perfluorooctanoate (PFOA), perfluorononanate (PFNA) and perfluorodecanoate (PFDA) in soil in the absence or presence of wheat/earthworms, and PFDA was the predominant metabolite. Accumulation of initial 10:2 FTOH and its metabolites were observed in the wheat and earthworms, suggesting that 10:2 FTOH could be bioaccumulated in wheat and earthworms and biotransformed to the highly stable PFCAs. Perfluoropentanoic acid (PFPeA), perfluorohexanoic (PFHxA) and PFDA were detected in wheat root, while PFDA and perfluoroundecanoic acid (PFUnDA) were detected in shoot. PFNA and PFDA were determined in earthworms and the concentration of PFDA was much higher. The presence of earthworms and/or plant stimulated the microbial degradation of 10:2 FTOH in soil. The results supplied important evidence that degradation of 10:2 FTOH was an important potential source of PFCAs in the environment and in biota.

  5. Kinetics of caesium and potassium absorption by roots of three grass pastures and competitive effects of potassium on caesium uptake in Cynodon sp

    SciTech Connect

    Ayub, J. Juri; Velasco, R. H.; Valverde, L. Rubio; Garcia-Sanchez, M. J.; Fernandez, J. A.

    2008-08-07

    Caesium uptake by plant roots has been normally associated with the uptake of potassium as the potassium transport systems present in plants have also the capacity to transport caesium. Three grass species (Eragrostis curvula, Cynodon sp and Distichlis spicata) growing in seminatural grassland of central Argentina were selected to study their capability to incorporate Cs{sup +} (and K{sup +}) using electrophysiological techniques. Although the {sup 137}Cs soil inventory ranged between 328-730 Bq m{sup -2} in this region, no {sup 137}Cs activity was detected in these plants. However, all the species, submitted previously to K{sup +} starvation, showed the uptake of both Cs{sup +} and K{sup +} when micromolar concentrations of these cations were present in the medium. The uptake showed saturation kinetics for both cations that could be fitted to the Michelis-Menten model. K{sub M} values were smaller for K{sup +} than for Cs{sup +}, indicating a higher affinity for the first cation. The presence of increasing K{sup +} concentrations in the assay medium inhibited Cs{sup +} uptake in Cynodon sp., as expected if both cations are transported by the same transport systems. This effect is due to the competition of both ions for the union sites of the high affinity potassium transporters. In field situation, where soil concentration of Cs{sup +} is smaller than K{sup +} concentration, is then expectable that caesium activity in plants is not detectable. Nevertheless, the studied plants would have the capacity to incorporate caesium if its availability in soil solution increases. In addition, studies of Cs/K interaction can help us to understand the variability in transfer factors.

  6. Kinetics of caesium and potassium absorption by roots of three grass pastures and competitive effects of potassium on caesium uptake in Cynodon sp.

    NASA Astrophysics Data System (ADS)

    Ayub, J. Juri; Valverde, L. Rubio; Garcia-Sanchez, M. J.; Fernandez, J. A.; Velasco, R. H.

    2008-08-01

    Caesium uptake by plant roots has been normally associated with the uptake of potassium as the potassium transport systems present in plants have also the capacity to transport caesium. Three grass species (Eragrostis curvula, Cynodon sp and Distichlis spicata) growing in seminatural grassland of central Argentina were selected to study their capability to incorporate Cs+ (and K+) using electrophysiological techniques. Although the 137Cs soil inventory ranged between 328-730 Bq m-2 in this region, no 137Cs activity was detected in these plants. However, all the species, submitted previously to K+ starvation, showed the uptake of both Cs+ and K+ when micromolar concentrations of these cations were present in the medium. The uptake showed saturation kinetics for both cations that could be fitted to the Michelis-Menten model. KM values were smaller for K+ than for Cs+, indicating a higher affinity for the first cation. The presence of increasing K+ concentrations in the assay medium inhibited Cs+ uptake in Cynodon sp., as expected if both cations are transported by the same transport systems. This effect is due to the competition of both ions for the union sites of the high affinity potassium transporters. In field situation, where soil concentration of Cs+ is smaller than K+ concentration, is then expectable that caesium activity in plants is not detectable. Nevertheless, the studied plants would have the capacity to incorporate caesium if its availability in soil solution increases. In addition, studies of Cs/K interaction can help us to understand the variability in transfer factors.

  7. Behavior of decabromodiphenyl ether (BDE-209) in the soil-plant system: uptake, translocation, and metabolism in plants and dissipation in soil.

    PubMed

    Huang, Honglin; Zhang, Shuzhen; Christie, Peter; Wang, Sen; Xie, Mei

    2010-01-15

    Deca-bromodiphenyl ether (BDE-209) is the major component of the commercial deca-BDE flame retardant. There is increasing concern over BDE-209 due to its increasing occurrence in the environment and in humans. In this study the behavior of BDE-209 in the soil-plant system was investigated. Accumulation of BDE-209 was observed in the roots and shoots of all the six plant species examined, namely ryegrass, alfalfa, pumpkin, summer squash, maize, and radish. Root uptake of BDE-209 was positively correlated with root lipid content (P < 0.001, R(2) = 0.81). The translocation factor (TF, C(shoot)/C(root)) of BDE-209 was inversely related to its concentration in roots. Nineteen lower brominated (di- to nona-) PBDEs were detected in the soil and plant samples and five hydroxylated congeners were detected in the plant samples, indicating debromination and hydroxylation of BDE-209 in the soil-plant system. Evidence of a relatively higher proportion of penta- through di-BDE congeners in plant tissues than in the soil indicates that there is further debromination of PBDEs within plants or low brominated PBDEs are more readily taken up by plants. A significant negative correlation between the residual BDE-209 concentration in soil and the soil microbial biomass measured as the total phospholipid fatty acids (PLFAs) (P < 0.05, R(2) = 0.74) suggests that microbial metabolism and degradation contribute to BDE-209 dissipation in soil. These results provide important information about the behavior of BDE-209 in the soil-plant system.

  8. Nitrogen uptake and utilization by intact plants

    NASA Technical Reports Server (NTRS)

    Raper, C. D., Jr.; Tolley-Henry, L. C.

    1986-01-01

    The results of experiments support the proposed conceptual model that relates nitrogen uptake activity by plants as a balanced interdependence between the carbon-supplying function of the shoot and the nitrogen-supplying function of the roots. The data are being used to modify a dynamic simulation of plant growth, which presently describes carbon flows through the plant, to describe nitrogen uptake and assimilation within the plant system. Although several models have been proposed to predict nitrogen uptake and partitioning, they emphasize root characteristics affecting nutrient uptake and relay on empirical methods to describe the relationship between nitrogen and carbon flows within the plant. Researchers, on the other hand, propose to continue to attempt a mechanistic solution in which the effects of environment on nitrogen (as well as carbon) assimilation are incorporated through their direct effects on photosynthesis, respiration, and aging processes.

  9. Computer simulation of uranyl uptake by the rough lipopolysaccharide membrane of Pseudomonas aeruginosa.

    PubMed

    Lins, Roberto D; Vorpagel, Erich R; Guglielmi, Matteo; Straatsma, T P

    2008-01-01

    Heavy metal environmental contaminants cannot be destroyed but require containment, preferably in concentrated form, in a solid or immobile form for recycling or final disposal. Microorganisms are able to take up and deposit high levels of contaminant metals, including radioactive metals such as uranium and plutonium, into their cell wall. Consequently, these microbial systems are of great interest as the basis for potential environmental bioremediation technologies. The outer membranes of Gram-negative microbes are highly nonsymmetric and exhibit a significant electrostatic potential gradient across the membrane. This gradient has a significant effect on the uptake and transport of charged and dipolar compounds. However, the effectiveness of microbial systems for environmental remediation will depend strongly on specific properties that determine the uptake of targeted contaminants by a particular cell wall. To aid in the design of microbial remediation technologies, knowledge of the factors that determine the affinity of a particular bacterial outer membrane for the most common ionic species found in contaminated soils and groundwater is of great importance. Using our previously developed model for the lipopolysaccharide (LPS) membrane of Pseudomonas aeruginosa, this work presents the potentials of mean force as the estimate of the free energy profile for uptake of sodium, calcium, chloride, uranyl ions, and a water molecule by the bacterial LPS membrane. A compatible classical parameter set for uranyl has been developed and validated. Results show that the uptake of uranyl is energetically a favorable process relative to the other ions studied. At neutral pH, this nuclide is shown to be retained on the surface of the LPS membrane through chelation with the carboxyl and hydroxyl groups located in the outer core.

  10. Development of a BALB/c 3T3 neutral red uptake cytotoxicity test using a mainstream cigarette smoke exposure system

    PubMed Central

    2014-01-01

    Background Tobacco smoke toxicity has traditionally been assessed using the particulate fraction under submerged culture conditions which omits the vapour phase elements from any subsequent analysis. Therefore, methodologies that assess the full interactions and complexities of tobacco smoke are required. Here we describe the adaption of a modified BALB/c 3T3 neutral red uptake (NRU) cytotoxicity test methodology, which is based on the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) protocol for in vitro acute toxicity testing. The methodology described takes into account the synergies of both the particulate and vapour phase of tobacco smoke. This is of particular importance as both phases have been independently shown to induce in vitro cellular cytotoxicity. Findings The findings from this study indicate that mainstream tobacco smoke and the gas vapour phase (GVP), generated using the Vitrocell® VC 10 smoke exposure system, have distinct and significantly different toxicity profiles. Within the system tested, mainstream tobacco smoke produced a dilution IC50 (dilution (L/min) at which 50% cytotoxicity is observed) of 6.02 L/min, whereas the GVP produced a dilution IC50 of 3.20 L/min. In addition, we also demonstrated significant dose-for-dose differences between mainstream cigarette smoke and the GVP fraction (P < 0.05). This demonstrates the importance of testing the entire tobacco smoke aerosol and not just the particulate fraction, as has been the historical preference. Conclusions We have adapted the NRU methodology based on the ICCVAM protocol to capture the full interactions and complexities of tobacco smoke. This methodology could also be used to assess the performance of traditional cigarettes, blend and filter technologies, tobacco smoke fractions and individual test aerosols. PMID:24935030

  11. Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake.

    PubMed

    Zimnicka, Adriana M; Ivy, Kristin; Kaplan, Jack H

    2011-03-01

    Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ∼90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60-70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in

  12. Fatigue damage prognosis using affine arithmetic

    NASA Astrophysics Data System (ADS)

    Gbaguidi, Audrey; Kim, Daewon

    2014-02-01

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

  13. Biphasic Affinity Chromatographic Approach for Deep Tyrosine Phosphoproteome Analysis.

    PubMed

    Deng, Zhenzhen; Dong, Mingming; Wang, Yan; Dong, Jing; Li, Shawn S-C; Zou, Hanfa; Ye, Mingliang

    2017-02-21

    Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.

  14. An inducible expression system of histidine-tagged proteins in Streptomyces lividans for one-step purification by Ni2+ affinity chromatography.

    PubMed

    Enguita, F J; de la Fuente, J L; Martín, J F; Liras, P

    1996-04-01

    An expression and purification cassette containing the aminoglycoside phosphotransferase gene (aph) as selective marker has been constructed in the Escherichia coli vector pULHis2. DNA fragments inserted in the cassette can be easily subcloned in pIJ699 to give vectors for overexpression of genes in Streptomyces and purification of proteins by a one-step procedure. The expression system uses the thiostrepton-inducible promoter tipA for expression and a six histidine coding nucleotide sequence that is fused in frame to the foreign gene inserted in the polylinker. The pULHis2-derived expression vector has been used satisfactorily to express and to purify the P7 and P8 proteins of Nocardia lactamdurans which carry out the methoxylation of cephalosporin C to 7-methoxycephalosporin C.

  15. Catecholamine uptake sites: characterization, localization, and a role in the production of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism

    SciTech Connect

    Javitch, J.A.

    1985-01-01

    Dopamine and norepinephrine are inactivated by specific high affinity transport systems which mediate the recapture of the amines into presynaptic nerve terminals. (/sup 3/H)Maxindol labels neuronal dopamine uptake sites in corpus striatum membranes and neuronal norepinephrine uptake sites in cerebral cortex and submaxillary/sublingual gland membranes. The potencies of various inhibitors of biogenic amine uptake in reducing (/sup 3/H)mazindol binding in striatal membranes correlate with their potencies for inhibition of neurona (/sup 3/H)dopamine accumulation, whereas their potencies in reducing (/sup 3/H)mazindol binding to cortical and salivary gland membranes correlate with their potencies for inhibition of neuronal (/sup 3/H)norepinephrine accumulation. The association of (/sup 3/H)mazindol binding sites with neuronal dopamine uptake sites in the corpus striatum is further supported by the reduction of (/sup 3/H)mazindol binding sites in striatal membranes following destruction of dopaminergic neurons by 6-hydroxydopamine. Similarly, destruction of noradrenergic neurons by N-(2-chloro-ethyl)-N-ethyl-2-bromobenzylamine(DSP-4) decreases (/sup 3/H)mazindol binding to cortical membranes. Dopamine and norepinephrine uptake sites in rat brain have been differentially visualized using (/sup 3/H)mazindol autoradiography. N-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces neuropathologic and clinical abnormalities in humans and animals that closely resemble idiopathic Parkinson disease. (/sup 3/H)MPTP binds with high affinity to brain membranes. The chemical specificity of the binding sites corresponds to structure-activity requirements for neurotoxicity.

  16. Capacity and Plasticity of Potassium Channels and High-Affinity Transporters in Roots of Barley and Arabidopsis1[C][W

    PubMed Central

    Coskun, Devrim; Britto, Dev T.; Li, Mingyuan; Oh, Saehong; Kronzucker, Herbert J.

    2013-01-01

    The role of potassium (K+) transporters in high- and low-affinity K+ uptake was examined in roots of intact barley (Hordeum vulgare) and Arabidopsis (Arabidopsis thaliana) plants by use of 42K radiotracing, electrophysiology, pharmacology, and mutant analysis. Comparisons were made between results from barley and five genotypes of Arabidopsis, including single and double knockout mutants for the high-affinity transporter, AtHAK5, and the Shaker-type channel, AtAKT1. In Arabidopsis, steady-state K+ influx at low external K+ concentration ([K+]ext = 22.5 µm) was predominantly mediated by AtAKT1 when high-affinity transport was inhibited by ammonium, whereas in barley, by contrast, K+ channels could not operate below 100 µm. Withdrawal of ammonium resulted in an immediate and dramatic stimulation of K+ influx in barley, indicating a shift from active to passive K+ uptake at low [K+]ext and yielding fluxes as high as 36 µmol g (root fresh weight)−1 h−1 at 5 mm [K+]ext, among the highest transporter-mediated K+ fluxes hitherto reported. This ammonium-withdrawal effect was also established in all Arabidopsis lines (the wild types, atakt1, athak5, and athak5 atakt1) at low [K+]ext, revealing the concerted involvement of several transport systems. The ammonium-withdrawal effect coincided with a suppression of K+ efflux and a significant hyperpolarization of the plasma membrane in all genotypes except athak5 atakt1, could be sustained over 24 h, and resulted in increased tissue K+ accumulation. We discuss key differences and similarities in K+ acquisition between two important model systems and reveal novel aspects of K+ transport in planta. PMID:23553635

  17. Affinity membrane introduction mass spectrometry

    SciTech Connect

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

    1995-02-15

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

  18. Effects of nitrification inhibitors (DCD and DMPP) on nitrous oxide emission, crop yield and nitrogen uptake in a wheat-maize cropping system

    NASA Astrophysics Data System (ADS)

    Liu, C.; Wang, K.; Zheng, X.

    2013-04-01

    The application of nitrification inhibitors together with ammonium-based fertilizers is proposed as a potent method to decrease nitrous oxide (N2O) emission while promoting crop yield and nitrogen use efficiency in fertilized agricultural fields. To evaluate the effects of nitrification inhibitors, we conducted year-round measurements of N2O fluxes, yield, aboveground biomass, plant carbon and nitrogen contents, soil inorganic nitrogen and dissolved organic carbon contents and the main environmental factors for urea (U), urea + dicyandiamide (DCD) and urea + 3,4-dimethylpyrazol phosphate (DMPP) treatments in a wheat-maize rotation field. The cumulative N2O emissions were calculated to be 4.49 ± 0.21, 2.93 ± 0.06 and 2.78 ± 0.16 kg N ha-1 yr-1 for the U, DCD and DMPP treatments, respectively. Therefore, the DCD and DMPP treatments significantly decreased the annual emissions by 35% and 38%, respectively (p < 0.01). The variations of soil temperature, moisture and inorganic nitrogen content regulated the seasonal fluctuation of N2O emissions. When the emissions presented clearly temporal variations, high-frequency measurements or optimized sampling schedule for intermittent measurements would likely provide more accurate estimations of annual cumulative emission and treatment effect. The application of nitrification inhibitors significantly increased the soil inorganic nitrogen content (p < 0.01); shifted the main soil inorganic nitrogen form from nitrate to ammonium; and tended to increase the dissolved organic carbon content, crop yield, aboveground biomass and nitrogen uptake by aboveground plant. The results demonstrate the roles the nitrification inhibitors play in enhancing yield and nitrogen use efficiency and reducing N2O emission from the wheat-maize cropping system.

  19. Effects of nitrification inhibitors (DCD and DMPP) on nitrous oxide emission, crop yield and nitrogen uptake in a wheat-maize cropping system

    NASA Astrophysics Data System (ADS)

    Liu, C.; Wang, K.; Zheng, X.

    2013-01-01

    The application of nitrification inhibitors together with ammonium-based fertilizers is proposed as a potent method to decrease nitrous oxide (N2O) emission while promoting yield and nitrogen use efficiency in fertilized agricultural fields. To evaluate the effects of nitrification inhibitors, we conducted year-round measurements of N2O fluxes, yield, aboveground biomass, plant carbon and nitrogen contents, soil inorganic nitrogen and dissolved organic carbon contents and the main environmental factors for urea (U), urea + dicyandiamide (DCD) and urea + 3,4-dimethylpyrazol-phosphate (DMPP) treatments in a wheat-maize rotation field. The cumulative N2O emissions were calculated to be 4.49 ± 0.21, 2.93 ± 0.06 and 2.78 ± 0.16 kg N ha-1 yr-1 for the U, DCD and DMPP treatments, respectively. Therefore, the DCD and DMPP treatments decreased the annual emissions by 35% and 38%, respectively. The variations of soil temperature, moisture and inorganic nitrogen content regulated the seasonal fluctuation of N2O emissions. When the emissions presented clearly temporal variations, year-round and high-frequency measurements should be adopted to estimate annual cumulative emissions and treatment effects. The application of nitrification inhibitors increased the soil inorganic nitrogen and dissolved organic carbon availability and shifted the main soil inorganic nitrogen form from nitrate to ammonium. The annual yield, aboveground biomass and nitrogen uptake by aboveground plants increased by 8.5-9.1%, 8.6-9.7% and 10.9-13.2%, respectively, for the DCD and DMPP treatments compared with the U treatment. The results demonstrate the roles the nitrification inhibitors play in enhancing yield and nitrogen use efficiency and reducing N2O emission from the wheat-maize cropping system.

  20. Crystal structure of the heme-IsdC complex, the central conduit of the Isd iron/heme uptake system in Staphylococcus aureus.

    PubMed

    Sharp, Katherine H; Schneider, Sabine; Cockayne, Alan; Paoli, Max

    2007-04-06

    Pathogens such as Staphylococcus aureus require iron to survive and have evolved specialized proteins to steal heme from their host. IsdC is the central conduit of the Isd (iron-regulated surface determinant) multicomponent heme uptake machinery; staphylococcal cell-surface proteins such as IsdA, IsdB, and IsdH are thought to funnel their molecular cargo to IsdC, which then mediates the transfer of the iron-containing nutrient to the membrane translocation system IsdDEF. The structure of the heme-IsdC complex reveals a novel heme site within an immunoglobulin-like domain and sheds light on its binding mechanism. The folding topology is reminiscent of the architecture of cytochrome f, cellobiose dehydrogenase, and ethylbenzene dehydrogenase; in these three proteins, the heme is bound in an equivalent position, but interestingly, IsdC features a distinct binding pocket with the ligand located next to the hydrophobic core of the beta-sandwich. The iron is coordinated with a tyrosine surrounded by several non-polar side chains that cluster into a tightly packed proximal side. On the other hand, the distal side is relatively exposed with a short helical peptide segment that acts as a lip clasping onto almost half of the porphyrin plane. This structural feature is argued to play a role in the mechanism of binding and release by switching to an open conformation and thus loosening the interactions holding the heme. The structure of the heme-IsdC complex provides a template for the understanding of other proteins, such as IsdA, IsdB, and IsdH, that contain the same heme-binding module as IsdC, known as the NEAT (near transporter) domain.

  1. Describing high-dimensional dynamics with low-dimensional piecewise affine models: applications to renewable energy.

    PubMed

    Hirata, Yoshito; Aihara, Kazuyuki

    2012-06-01

    We introduce a low-dimensional description for a high-dimensional system, which is a piecewise affine model whose state space is divided by permutations. We show that the proposed model tends to predict wind speeds and photovoltaic outputs for the time scales from seconds to 100 s better than by global affine models. In addition, computations using the piecewise affine model are much faster than those of usual nonlinear models such as radial basis function models.

  2. Antisymmetric tensor generalizations of affine vector fields

    PubMed Central

    Morisawa, Yoshiyuki; Tomoda, Kentaro

    2016-01-01

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

  3. An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client.

    PubMed

    Bigenzahn, Johannes W; Fauster, Astrid; Rebsamen, Manuele; Kandasamy, Richard K; Scorzoni, Stefania; Vladimer, Gregory I; Müller, André C; Gstaiger, Matthias; Zuber, Johannes; Bennett, Keiryn L; Superti-Furga, Giulio

    2016-03-01

    Tandem affinity purification-mass spectrometry (TAP-MS) is a popular strategy for the identification of protein-protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression.

  4. An Inducible Retroviral Expression System for Tandem Affinity Purification Mass-Spectrometry-Based Proteomics Identifies Mixed Lineage Kinase Domain-like Protein (MLKL) as an Heat Shock Protein 90 (HSP90) Client*

    PubMed Central

    Bigenzahn, Johannes W.; Fauster, Astrid; Rebsamen, Manuele; Kandasamy, Richard K.; Scorzoni, Stefania; Vladimer, Gregory I.; Müller, André C.; Gstaiger, Matthias; Zuber, Johannes; Bennett, Keiryn L.; Superti-Furga, Giulio

    2016-01-01

    Tandem affinity purification–mass spectrometry (TAP-MS) is a popular strategy for the identification of protein–protein interactions, characterization of protein complexes, and entire networks. Its employment in cellular settings best fitting the relevant physiology is limited by convenient expression vector systems. We developed an easy-to-handle, inducible, dually selectable retroviral expression vector allowing dose- and time-dependent control of bait proteins bearing the efficient streptavidin-hemagglutinin (SH)-tag at their N- or C termini. Concomitant expression of a reporter fluorophore allows to monitor bait-expressing cells by flow cytometry or microscopy and enables high-throughput phenotypic assays. We used the system to successfully characterize the interactome of the neuroblastoma RAS viral oncogene homolog (NRAS) Gly12Asp (G12D) mutant and exploited the advantage of reporter fluorophore expression by tracking cytokine-independent cell growth using flow cytometry. Moreover, we tested the feasibility of studying cytotoxicity-mediating proteins with the vector system on the cell death-inducing mixed lineage kinase domain-like protein (MLKL) Ser358Asp (S358D) mutant. Interaction proteomics analysis of MLKL Ser358Asp (S358D) identified heat shock protein 90 (HSP90) as a high-confidence interacting protein. Further phenotypic characterization established MLKL as a novel HSP90 client. In summary, this novel inducible expression system enables SH-tag-based interaction studies in the cell line proficient for the respective phenotypic or signaling context and constitutes a valuable tool for experimental approaches requiring inducible or traceable protein expression. PMID:26933192

  5. Supramolecular polymers as dynamic multicomponent cellular uptake carriers.

    PubMed

    Petkau-Milroy, Katja; Sonntag, Michael H; van Onzen, Arthur H A M; Brunsveld, Luc

    2012-05-16

    Supramolecular synthesis represents a flexible approach to the generation of dynamic multicomponent materials with tunable properties. Here, cellular uptake systems based on dynamic supramolecular copolymers have been developed using a combination of differently functionalized discotic molecules. Discotics featuring peripheral amine functionalities that endow the supramolecular polymer with cellular uptake capabilities were readily synthesized. This enabled the uptake of otherwise cell-impermeable discotics via cotransport as a function of supramolecular coassembly. Dynamic multicomponent and multifunctional supramolecular polymers represent a novel and unique platform for modular cellular uptake systems.

  6. A Novel Vertex Affinity for Community Detection

    SciTech Connect

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

    2015-10-05

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

  7. Structural determinants of sigma receptor affinity

    SciTech Connect

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

    1987-12-01

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

  8. Selenium Uptake and Volatilization by Marine Algae

    NASA Astrophysics Data System (ADS)

    Luxem, Katja E.; Vriens, Bas; Wagner, Bettina; Behra, Renata; Winkel, Lenny H. E.

    2015-04-01

    Selenium (Se) is an essential trace nutrient for humans. An estimated one half to one billion people worldwide suffer from Se deficiency, which is due to low concentrations and bioavailability of Se in soils where crops are grown. It has been hypothesized that more than half of the atmospheric Se deposition to soils is derived from the marine system, where microorganisms methylate and volatilize Se. Based on model results from the late 1980s, the atmospheric flux of these biogenic volatile Se compounds is around 9 Gt/year, with two thirds coming from the marine biosphere. Algae, fungi, and bacteria are known to methylate Se. Although algal Se uptake, metabolism, and methylation influence the speciation and bioavailability of Se in the oceans, these processes have not been quantified under environmentally relevant conditions and are likely to differ among organisms. Therefore, we are investigating the uptake and methylation of the two main inorganic Se species (selenate and selenite) by three globally relevant microalgae: Phaeocystis globosa, the coccolithophorid Emiliania huxleyi, and the diatom Thalassiosira oceanica. Selenium uptake and methylation were quantified in a batch experiment, where parallel gas-tight microcosms in a climate chamber were coupled to a gas-trapping system. For E. huxleyi, selenite uptake was strongly dependent on aqueous phosphate concentrations, which agrees with prior evidence that selenite uptake by phosphate transporters is a significant Se source for marine algae. Selenate uptake was much lower than selenite uptake. The most important volatile Se compounds produced were dimethyl selenide, dimethyl diselenide, and dimethyl selenyl sulfide. Production rates of volatile Se species were larger with increasing intracellular Se concentration and in the decline phase of the alga. Similar experiments are being carried out with P. globosa and T. oceanica. Our results indicate that marine algae are important for the global cycling of Se

  9. Plant Water Uptake in Drying Soils1

    PubMed Central

    Lobet, Guillaume; Couvreur, Valentin; Meunier, Félicien; Javaux, Mathieu; Draye, Xavier

    2014-01-01

    Over the last decade, investigations on root water uptake have evolved toward a deeper integration of the soil and roots compartment properties, with the goal of improving our understanding of water acquisition from drying soils. This evolution parallels the increasing attention of agronomists to suboptimal crop production environments. Recent results have led to the description of root system architectures that might contribute to deep-water extraction or to water-saving strategies. In addition, the manipulation of root hydraulic properties would provide further opportunities to improve water uptake. However, modeling studies highlight the role of soil hydraulics in the control of water uptake in drying soil and call for integrative soil-plant system approaches. PMID:24515834

  10. Third system for neutral amino acid transport in a marine pseudomonad.

    PubMed Central

    Pearce, S M; Hildebrandt, V A; Lee, T

    1977-01-01

    Uptake of leucine by the marine pseudomonad B-16 is an energy-dependent, concentrative process. Respiratory inhibitors, uncouplers, and sulfhydryl reagents block transport. The uptake of leucine is Na+ dependent, although the relationship between the rate of leucine uptake and Na+ concentration depends, to some extent, on the ionic strength of the suspending assay medium and the manner in which cells are washed prior to assay. Leucine transport can be separated into at least two systems: a low-affinity system with an apparent Km of 1.3 X 10(-5) M, and a high-affinity system with an apparent Km of 1.9 X 10(-7) M. The high-affinity system shows a specificity unusual for bacterial systems in that both aromatic and aliphatic amino acids inhibit leucine transport, provided that they have hydrophobic side chains of a length greater than that of two carbon atoms. The system exhibits strict stereospecificity for the L form. Phenylalanine inhibition was investigated in more detail. The Ki for inhibition of leucine transport by phenylalanine is about 1.4 X 10(-7) M. Phenylalanine itself is transported by an energy-dependent process whose specificity is the same as the high-affinity leucine transport system, as is expected if both amino acids share the same transport system. Studies with protoplasts indicate that a periplasmic binding protein is not an essential part of this transport system. Fein and MacLeod (J. Bacteriol. 124:1177-1190, 1975) reported two neutral amino acid transport systems in strain B-16: the DAG system, serving glycine, D-alanine, D-serine, and alpha-aminoisobutyric acid; and the LIV system, serving L-leucine, L-isoleucine, L-valine, and L-alanine. The high-affinity system reported here is a third neutral amino acid transport system in this marine pseudomonad. We propose the name "LIV-II" system. PMID:856786

  11. Affinity proteomics to study endogenous protein complexes: Pointers, pitfalls, preferences and perspectives

    PubMed Central

    LaCava, John; Molloy, Kelly R.; Taylor, Martin S.; Domanski, Michal; Chait, Brian T.; Rout, Michael P.

    2015-01-01

    Dissecting and studying cellular systems requires the ability to specifically isolate distinct proteins along with the co-assembled constituents of their associated complexes. Affinity capture techniques leverage high affinity, high specificity reagents to target and capture proteins of interest along with specifically associated proteins from cell extracts. Affinity capture coupled to mass spectrometry (MS)-based proteomic analyses has enabled the isolation and characterization of a wide range of endogenous protein complexes. Here, we outline effective procedures for the affinity capture of protein complexes, highlighting best practices and common pitfalls. PMID:25757543

  12. Synthesis, Biodistribution and In vitro Evaluation of Brain Permeable High Affinity Type 2 Cannabinoid Receptor Agonists [11C]MA2 and [18F]MA3

    PubMed Central

    Ahamed, Muneer; van Veghel, Daisy; Ullmer, Christoph; Van Laere, Koen; Verbruggen, Alfons; Bormans, Guy M.

    2016-01-01

    The type 2 cannabinoid receptor (CB2) is a member of the endocannabinoid system and is known for its important role in (neuro)inflammation. A PET-imaging agent that allows in vivo visualization of CB2 expression may thus allow quantification of neuroinflammation. In this paper, we report the synthesis, radiosynthesis, biodistribution and in vitro evaluation of a carbon-11 ([11C]MA2) and a fluorine-18 ([18F]MA3) labeled analog of a highly potent N-arylamide oxadiazole CB2 agonist (EC50 = 0.015 nM). MA2 and MA3 behaved as potent CB2 agonist (EC50: 3 nM and 0.1 nM, respectively) and their in vitro binding affinity for hCB2 was found to be 87 nM and 0.8 nM, respectively. Also MA3 (substituted with a fluoro ethyl group) was found to have higher binding affinity and EC50 values when compared to the originally reported trifluoromethyl analog 12. [11C]MA2 and [18F]MA3 were successfully synthesized with good radiochemical yield, high radiochemical purity and high specific activity. In mice, both tracers were efficiently cleared from blood and all major organs by the hepatobiliary pathway and importantly these compounds showed high brain uptake. In conclusion, [11C]MA2 and [18F]MA3 are shown to be high potent CB2 agonists with good brain uptake, these favorable characteristics makes them potential PET probes for in vivo imaging of brain CB2 receptors. However, in view of its higher affinity and selectivity, further detailed evaluation of MA3 as a PET tracer for CB2 is warranted. PMID:27713686

  13. Relationship of sup(99m)Tc-MDP uptake to regional osseous circulation in skeletally immature and mature dogs

    SciTech Connect

    McKinstry, P.; Schnitzer, J.E.; Light, T.R.; Ogden, J.A.; Hoffer, P.

    1982-05-01

    Uptake of intravenously injected sup(99m)Tc-MDP in multiple regions of healthy skeletally immature and mature dogs was correlated with regional chondro-osseous blood flow determined by radioactively labeled microspheres. Compared to the microsphere distribution, the distribution of sup(99m)Tc-MDP indicated that blood flow is an important, but not exclusive, factor in the uptake of bone-seeking tracers. Other factors such as the affinity of the tracer for the various types of chondro-osseous tissues must also affect tracer uptake. A measure of the relative affinity of sup(99m)Tc-MDP for bone was derived by the ratio of the percentage of tracer uptake to the percentage of blood flow. The juxta-ephyseal region demonstrated the greatest affinity for the tracer, followed in decreasing affinity, by the cortical bone, epiphyseal cartilage, trabecular bone of the metaphysis and secondary ossification center, and marrow space. Within the spongiosa, affinity generally increased as the proportion of osseous trabeculae relative to marrow space increased. sup(99m)Tc-MDP uptake is disproportionately increased in areas of active bone growth and remodeling where the surface area of hydroxyapatite crystals available for adsorption is probably highest.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-05-01

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

  16. Etiology of amyloidosis determines myocardial 99mTc-DPD uptake in amyloidotic cardiomyopathy.

    PubMed

    Longhi, Simone; Bonfiglioli, Rachele; Obici, Laura; Gagliardi, Christian; Milandri, Agnese; Lorenzini, Massimiliano; Guidalotti, Pier Luigi; Merlini, Giampaolo; Rapezzi, Claudio

    2015-05-01

    Tc-DPD (Tc-3,3-diphosphono-1,2-propanodicarboxylic acid) has a high affinity for transthyretin (TTR)-infiltrated myocardium, allowing a differential diagnosis with light chain cardiac amyloidosis and other nonamyloidotic cardiomyopathies with a hypertrophic phenotype, in which myocardial tracer uptake is low or absent. Myocardial bone tracer uptake in the rarer forms of amyloidosis (eg, apolipoprotein-related) has been rarely studied. We present 4 cases of cardiac amyloidosis that underwent Tc-DPD scintigraphy; myocardial DPD uptake was present in patients with ATTR, wtTTR and apolipoprotein AI and negative in cases with AL and apolipoprotein AII-related disease.

  17. STP10 encodes a high-affinity monosaccharide transporter and is induced under low-glucose conditions in pollen tubes of Arabidopsis

    PubMed Central

    Rottmann, Theresa; Zierer, Wolfgang; Subert, Christa; Sauer, Norbert; Stadler, Ruth

    2016-01-01

    Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose–H+ symporter. Analyses of STP10 mRNA and STP10 promoter–reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10. qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10 T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway. PMID:26893494

  18. STP10 encodes a high-affinity monosaccharide transporter and is induced under low-glucose conditions in pollen tubes of Arabidopsis.

    PubMed

    Rottmann, Theresa; Zierer, Wolfgang; Subert, Christa; Sauer, Norbert; Stadler, Ruth

    2016-04-01

    Pollen tubes are fast growing, photosynthetically inactive cells. Their energy demand is covered by specific transport proteins in the plasma membrane that mediate the uptake of sugars. Here we report on the functional characterization of AtSTP10, a previously uncharacterized member of the SUGAR TRANSPORT PROTEIN family. Heterologous expression of STP10 cDNA in yeast revealed that the encoded protein catalyses the high-affinity uptake of glucose, galactose and mannose. The transporter is sensitive to uncouplers of transmembrane proton gradients, indicating that the protein acts as a hexose-H(+)symporter. Analyses of STP10 mRNA and STP10 promoter-reporter gene studies revealed a sink-specific expression pattern of STP10 in primordia of lateral roots and in pollen tubes. This restriction to sink organs is mediated by intragenic regions of STP10 qPCR analyses with cDNA of in vitro grown pollen tubes showed that STP10 expression was down-regulated in the presence of 50mM glucose. However, in pollen tubes of glucose-insensitive plants, which lack the glucose sensor hexokinase1 (HXK1), no glucose-induced down-regulation of STP10 expression was detected. A stp10T-DNA insertion line developed normally, which may point towards functional redundancy. The data presented in this paper indicate that a high-affinity glucose uptake system is induced in growing pollen tubes under low glucose conditions and that this regulation may occur through the hexokinase pathway.

  19. Ocean uptake of carbon dioxide

    SciTech Connect

    Peng, Tsung-Hung; Takahashi, Taro

    1993-06-01

    Factors controlling the capacity of the ocean for taking up anthropogenic C0{sup 2} include carbon chemistry, distribution of alkalinity, pCO{sup 2} and total concentration of dissolved C0{sup 2}, sea-air pCO{sup 2} difference, gas exchange rate across the sea-air interface, biological carbon pump, ocean water circulation and mixing, and dissolution of carbonate in deep sea sediments. A general review of these processes is given and models of ocean-atmosphere system based on our understanding of these regulating processes axe used to estimate the magnitude of C0{sup 2} uptake by the ocean. We conclude that the ocean can absorb up to 35% of the fossil fuel emission. Direct measurements show that 55% Of C0{sup 2} from fossil fuel burning remains in the atmosphere. The remaining 10% is not accounted for by atmospheric increases and ocean uptake. In addition, it is estimated that an amount equivalent to 30% of recent annual fossil fuel emissions is released into the atmosphere as a result of deforestation and farming. To balance global carbon budget, a sizable carbon sink besides the ocean is needed. Storage of carbon in terrestrial biosphere as a result of C0{sup 2} fertilization is a potential candidate for such missing carbon sinks.

  20. Ocean uptake of carbon dioxide

    SciTech Connect

    Peng, Tsung-Hung ); Takahashi, Taro . Lamont-Doherty Earth Observatory)

    1993-01-01

    Factors controlling the capacity of the ocean for taking up anthropogenic C0[sup 2] include carbon chemistry, distribution of alkalinity, pCO[sup 2] and total concentration of dissolved C0[sup 2], sea-air pCO[sup 2] difference, gas exchange rate across the sea-air interface, biological carbon pump, ocean water circulation and mixing, and dissolution of carbonate in deep sea sediments. A general review of these processes is given and models of ocean-atmosphere system based on our understanding of these regulating processes axe used to estimate the magnitude of C0[sup 2] uptake by the ocean. We conclude that the ocean can absorb up to 35% of the fossil fuel emission. Direct measurements show that 55% Of C0[sup 2] from fossil fuel burning remains in the atmosphere. The remaining 10% is not accounted for by atmospheric increases and ocean uptake. In addition, it is estimated that an amount equivalent to 30% of recent annual fossil fuel emissions is released into the atmosphere as a result of deforestation and farming. To balance global carbon budget, a sizable carbon sink besides the ocean is needed. Storage of carbon in terrestrial biosphere as a result of C0[sup 2] fertilization is a potential candidate for such missing carbon sinks.

  1. Improving image segmentation by learning region affinities

    SciTech Connect

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

    2010-11-03

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

  2. Nitrogen Uptake in Spinach

    NASA Astrophysics Data System (ADS)

    Ramirez, J.; VanBenthem, P.

    2013-12-01

    A plant's absorption of nitrogen can be encouraged by a variety of environmental factors, especially the application of fertilizers. As a common limiting factor in plant growth, not up taking enough nitrogen can be a result of an unhealthy plant. Moreover, as farmers seek out methods to increase growth of plants, fertilizers are used as a solution to the issue of nitrogen deficiency to incorporate additional nitrogen from chemical or organic sources, by not using the right fertilizer can greatly affect the plats. The point of this research project is to determine the effect of various fertilizers on the plant growth, and to correlate the measured nitrogen, water and chlorophyll content in spinach leaves. Spinach leaves were used because they are known to quickly uptake chemicals in the environment. The spinach plants were exposed to four different growing parameters, which are referred to as control, ammonium nitrate, MiracleGro , and organic. The spinach was originally placed in nitrogen deficient soil with only 2.2x10 4 weight percent (wt. %) nitrogen. The leaves in the control group were grown in this nitrogen deficient soil without any fertilizer added. Ammomium nitrate and MiracleGro were added to the spinach in the A and MG groups, respectively, and organic chicken stool was used for the O group. By using a spectral imaging system and flame combustion techniques, the chlorophyll content can be related to the nitrogen content in the spinach leaves. In these spinach leaves, nitrogen and chlorophyll content were measured, chlorophyll is a green pigment that plays a crucial role in producing nutrients for green plants. The lack of chlorophyll will allow the plant to become susceptible to diseases, so it is extremely important that the plants have a high content of chlorophyll. The role of nitrogen in chlorophyll is very important and helps in the creation of chlorophyll; therefore it is necessary that an appropriate amount of nitrogen is added for optimal growth

  3. Effects of a 60 Hz magnetic field on central cholinergic systems of the rat

    SciTech Connect

    Lai, H.; Carino, M.A.; Horita, A.; Guy, A.W. )

    1993-03-15

    The authors studied the effects of an acute exposure to a 60 Hz magnetic field on sodium-dependent, high-affinity choline uptake in the brain of the rat. Decreases in uptake were observed in the frontal cortex and hippocampus after the animals were exposed to a magnetic field at flux densities [>=] 0.75 mT. These effects of the magnetic field were blocked by pretreating the animals with the narcotic antagonist naltrexone, but not by the peripheral opioid antagonist, naloxone methiodide. These data indicate that the magnetic-field-induced decreases in high-affinity choline uptake in the rat brain were mediated by endogenous opioids in the central nervous systems.

  4. The Impact of the Affinity Learning Authoring Tool on Student Learning

    ERIC Educational Resources Information Center

    Soh, Leen-Kiat; Fowler, David; Zygielbaum, Art I.

    2008-01-01

    Affinity Learning is a system that allows the user to build a lesson on a subject matter by breaking it down into concepts, misconceptions, assessments, and remediation steps. Examples and questions can also used in these components. Affinity Learning has been found to be effective and can offer critical insights to student learning strategies.…

  5. Platelet serotonin content and uptake in spontaneously hypertensive rats

    SciTech Connect

    Guicheney, P.; Legros, M.; Marcel, D.; Kamal, L.; Meyer, P.

    1985-02-18

    Platelet serotonin (5-HT) content and uptake were studied in male SHR and WKY at various ages. Blood was withdrawn from the carotid artery under anesthesia and 5-HT levels determined from platelet rich plasma (PRP) using a HPLC technique coupled with an electrochemical detection method. Platelet 5-HT uptake was studied by incubating PRP at 37/sup 0/C for 10 sec with increasing concentrations of /sup 3/H-5HT. Lineweaver-Burk plots of /sup 3/H-5HT uptake were linear suggesting simple Michaelis-Menten uptake kinetics. The SHR had more platelets than age-matched controls and consequently a higher blood circulating pool of 5-HT. Nevertheless, the 5-HT platelet levels were similar to those of their age-matched rats. The 5 week-old SHR and WKY had greater numbers of platelets and higher 5-HT platelet levels than the older rats of both strains. The affinity constants (Km) and the maximal velocities (Vmax) of platelet 5-HT uptake did not differ significantly between the 12 week- and the 6 month-old SHR and WKY. These data suggest that the SHR do not show the same impairment in platelet 5-HT metabolism as observed in essential hypertension in man.

  6. EFFECT OF HUMIC ACID ON UPTAKE AND TRANSFER OF COPPER FROM MICROBES TO CILIATES TO COPEPODS

    EPA Science Inventory

    This research is part of an ongoing project designed to determine the effect of humic acid on the uptake and transfer of metals by marine organisms at the lower end of the food chain. Binding affinities for Cu, Cd, Zn, and Cr to Suwannee River humic acid were determined at variou...

  7. Blood flow and oxygen uptake during exercise

    NASA Technical Reports Server (NTRS)

    Mitchell, J. W.; Stolwijk, J. A. J.; Nadel, E. R.

    1973-01-01

    A model is developed for predicting oxygen uptake, muscle blood flow, and blood chemistry changes under exercise conditions. In this model, the working muscle mass system is analyzed. The conservation of matter principle is applied to the oxygen in a unit mass of working muscle under transient exercise conditions. This principle is used to relate the inflow of oxygen carried with the blood to the outflow carried with blood, the rate of change of oxygen stored in the muscle myoglobin, and the uptake by the muscle. Standard blood chemistry relations are incorporated to evaluate venous levels of oxygen, pH, and carbon dioxide.

  8. Methionine uptake in Corynebacterium glutamicum by MetQNI and by MetPS, a novel methionine and alanine importer of the NSS neurotransmitter transporter family.

    PubMed

    Trötschel, Christian; Follmann, Martin; Nettekoven, Jeannine A; Mohrbach, Tobias; Forrest, Lucy R; Burkovski, Andreas; Marin, Kay; Krämer, Reinhard

    2008-12-02

    The soil bacterium Corynebacterium glutamicum is a model organism in amino acid biotechnology. Here we present the identification of two different L-methionine uptake systems including the first characterization of a bacterial secondary methionine carrier. The primary carrier MetQNI is a high affinity ABC-type transporter specific for l-methionine. Its expression is under the control of the transcription factor McbR, the global regulator of sulfur metabolism in C. glutamicum. Besides MetQNI, a novel secondary methionine uptake system of the NSS (neurotransmitter:sodium symporter) family was identified and named MetP. The MetP system is characterized by a lower affinity for methionine and uses Na(+) ions for energetic coupling. It is also the main alanine transporter in C. glutamicum and is expressed constitutively. These observations are consistent with models of methionine, alanine, and leucine bound to MetP, derived from the X-ray crystal structure of the LeuT transporter from Aquifex aeolicus. Complementation studies show that MetP consists of two components, a large subunit with 12 predicted transmembrane segments and, surprisingly, an additional subunit with one predicted transmembrane segment only. Thus, this new member of the NSS transporter family adds a novel feature to this class of carriers, namely, the functional dependence on an additional small subunit.

  9. Heavy metal uptake of Geosiphon pyriforme

    NASA Astrophysics Data System (ADS)

    Scheloske, Stefan; Maetz, Mischa; Schüßler, Arthur

    2001-07-01

    Geosiphon pyriforme represents the only known endosymbiosis between a fungus, belonging to the arbuscular mycorrhizal (AM) fungi, and cyanobacteria (blue-green algae). Therefore we use Geosiphon as a model system for the widespread AM symbiosis and try to answer some basic questions regarding heavy metal uptake or resistance of AM fungi. We present quantitative micro-PIXE measurements of a set of heavy metals (Cu, Cd, Tl, Pb) taken up by Geosiphon-cells. The uptake is studied as a function of the metal concentration in the nutrient solution and of the time Geosiphon spent in the heavy metal enriched medium. The measured heavy metal concentrations range from several ppm to some hundred ppm. Also the influence of the heavy metal uptake on the nutrition transfer of other elements will be discussed.

  10. Extraosseous myocardial uptake incidentally detected during bone scan: report of three cases and a systematic literature review of extraosseous uptake.

    PubMed

    Caobelli, Federico; Paghera, Barbara; Pizzocaro, Claudio; Guerra, Ugo Paolo

    2013-01-01

    Bone scintigraphy is widely considered as an important technique able to investigate various pathological conditions of the skeletal system. Many unexpected extraosseous uptakes have been reported in literature. We present here three cases of unexpected 99mTc-oxidronate (HDP) myocardial extraosseous uptakes in patients undergoing bone scan for staging purposes. In particular, we present the first reported case ofa myocardial uptake in a patient with IgM-related amyloidosis. Subsequently, we perform a review of the existing literature about extraosseous uptakes.

  11. Inositol uptake in rat aorta

    SciTech Connect

    Rapoport, R.M.; Van Gorp, C.; Chang, Ki-Churl )

    1990-01-01

    {sup 3}H-inositol uptake into deendothelialized aorta was linear for at least 2 h and was composed of both a saturable, Na{sup +}-dependent, and a nonsaturable, Na{sup +}-independent component. The Na{sup +}-dependent component of inositol uptake had a K{sub m} of 50 {mu}M and a V{sub max} of 289 pmol/mg prot/h. Exposure to LiCl, ouabain, or Ca{sup 2+} - free Krebs-Ringer bicarbonate solution inhibited uptake. Metabolic poisoning with dinitrophenol, as well as incubation with phloretin, an inhibitor of carrier-mediated hexose transport, also inhibited uptake. Exposure to norepinephrine decreased inositol uptake, while phorbol myristate acetate was without effect. Isobutylmethylxanthine significantly increased inositol uptake, while the increased uptake due to dibutyryl cyclic AMP and forskolin were not statistically significant. Sodium nitroprusside, and activator of guanylate cyclase, and 8-bromo cyclic GMP, were without effect on uptake, as was methylene blue, an inhibitor of guanylate cyclase. Inositol uptake into the aorta was increased when the endothelium was allowed to remain intact, although this effect was likely due to uptake in both the endothelial and smooth muscle cells.

  12. Different components of /sup 3/H-imipramine binding in rat brain membranes: relation to serotonin uptake sites

    SciTech Connect

    Gobbi, M.; Taddei, C.; Mennini, T.

    1988-01-01

    In the present paper, the authors confirm and extend previous studies showing heterogeneous /sup 3/H-imipramine (/sup 3/H-IMI) binding sites. Inhibition curves of various drugs (serotonin, imipramine, desmethyl-imipramine, d-fenfluramine, d-norfenfluramine and indalpine, a potent serotonin uptake inhibitor) obtained using 2 nM /sup 3/H-IMI and in presence of 120 mM NaCl, confirmed the presence of at least three /sup 3/H-IMI binding sites: two of these were serotonin-insensitive while the third one was selectively inhibited by serotonin and indalpine with nanomolar affinities. Moreover this last component was found to be selectively modulated by chronic imipramine treatment thus suggesting a close relation to serontonin uptake mechanism. These data indicate that the use of a more selective inhibitors of the serotonin-sensitive component (like indalpine or serotonin itself) to define non specific /sup 3/H-IMI, may be of help in understanding its relation with serotonin uptake system. 22 references, 2 figures, 2 tables.

  13. Elongation changes of exploratory and root hair systems induced by aminocyclopropane carboxylic acid and aminoethoxyvinylglycine affect nitrate uptake and BnNrt2.1 and BnNrt1.1 transporter gene expression in oilseed rape.

    PubMed

    Leblanc, Antonin; Renault, Hugues; Lecourt, Julien; Etienne, Philippe; Deleu, Carole; Le Deunff, Erwan

    2008-04-01

    Ethylene is a plant hormone that plays a major role in the elongation of both exploratory and root hair systems. Here, we demonstrate in Brassica napus seedlings that treatments with the ethylene precursor, aminocyclopropane carboxylic acid (ACC) and the ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG), cause modification of the dynamic processes of primary root and root hair elongation in a dose-dependent way. Moreover, restoration of root elongation in AVG-treated seedlings by 1 mm l-glutamate suggested that high concentrations of AVG affect root elongation through nonoverlapping ethylene metabolic pathway involving pyridoxal 5'-P-dependent enzymes of nitrate (N) metabolism. In this respect, treatments with high concentrations of ACC and AVG (10 mum) over 5 d revealed significant differences in relationships between root growth architecture and N uptake capacities. Indeed, if these treatments decreased severely the elongation of the exploratory root system (primary root and lateral roots) they had opposing effects on the root hair system. Although ACC increased the length and number of root hairs, the rate of N uptake and the transcript level of the N transporter BnNrt2.1 were markedly reduced. In contrast, the decrease in root hair length and number in AVG-treated seedlings was overcompensated by an increase of N uptake and BnNrt2.1 gene expression. These root architectural changes demonstrated that BnNrt2.1 expression levels were more correlated to the changes of the exploratory root system than the changes of the root hair system. The difference between treatments in N transporters BnNrt1.1 and BnNrt2.1 gene expression is discussed with regard to presumed transport functions of BnNrt1.1 in relation to root elongation.

  14. Elongation Changes of Exploratory and Root Hair Systems Induced by Aminocyclopropane Carboxylic Acid and Aminoethoxyvinylglycine Affect Nitrate Uptake and BnNrt2.1 and BnNrt1.1 Transporter Gene Expression in Oilseed Rape[W

    PubMed Central

    Leblanc, Antonin; Renault, Hugues; Lecourt, Julien; Etienne, Philippe; Deleu, Carole; Le Deunff, Erwan

    2008-01-01

    Ethylene is a plant hormone that plays a major role in the elongation of both exploratory and root hair systems. Here, we demonstrate in Brassica napus seedlings that treatments with the ethylene precursor, aminocyclopropane carboxylic acid (ACC) and the ethylene biosynthesis inhibitor, aminoethoxyvinylglycine (AVG), cause modification of the dynamic processes of primary root and root hair elongation in a dose-dependent way. Moreover, restoration of root elongation in AVG-treated seedlings by 1 mm l-glutamate suggested that high concentrations of AVG affect root elongation through nonoverlapping ethylene metabolic pathway involving pyridoxal 5′-P-dependent enzymes of nitrate (N) metabolism. In this respect, treatments with high concentrations of ACC and AVG (10 μm) over 5 d revealed significant differences in relationships between root growth architecture and N uptake capacities. Indeed, if these treatments decreased severely the elongation of the exploratory root system (primary root and lateral roots) they had opposing effects on the root hair system. Although ACC increased the length and number of root hairs, the rate of N uptake and the transcript level of the N transporter BnNrt2.1 were markedly reduced. In contrast, the decrease in root hair length and number in AVG-treated seedlings was overcompensated by an increase of N uptake and BnNrt2.1 gene expression. These root architectural changes demonstrated that BnNrt2.1 expression levels were more correlated to the changes of the exploratory root system than the changes of the root hair system. The difference between treatments in N transporters BnNrt1.1 and BnNrt2.1 gene expression is discussed with regard to presumed transport functions of BnNrt1.1 in relation to root elongation. PMID:18287493

  15. The transcriptional regulator Np20 is the zinc uptake regulator in Pseudomonas aeruginosa.

    PubMed

    Ellison, Matthew L; Farrow, John M; Farrow, John Matthew; Parrish, Whitney; Danell, Allison S; Pesci, Everett C

    2013-01-01

    Zinc is essential for all bacteria, but excess amounts of the metal can have toxic effects. To address this, bacteria have developed tightly regulated zinc uptake systems, such as the ZnuABC zinc transporter which is regulated by the Fur-like zinc uptake regulator (Zur). In Pseudomonas aeruginosa, a Zur protein has yet to be identified experimentally, however, sequence alignment revealed that the zinc-responsive transcriptional regulator Np20, encoded by np20 (PA5499), shares high sequence identity with Zur found in other bacteria. In this study, we set out to determine whether Np20 was functioning as Zur in P. aeruginosa. Using RT-PCR, we determined that np20 (hereafter known as zur) formed a polycistronic operon with znuC and znuB. Mutant strains, lacking the putative znuA, znuB, or znuC genes were found to grow poorly in zinc deplete conditions as compared to wild-type strain PAO1. Intracellular zinc concentrations in strain PAO-Zur (Δzur) were found to be higher than those for strain PAO1, further implicating the zur as the zinc uptake regulator. Reporter gene fusions and real time RT-PCR revealed that transcription of znuA was repressed in a zinc-dependent manner in strain PAO1, however zinc-dependent transcriptional repression was alleviated in strain PAO-Zur, suggesting that the P. aeruginosa Zur homolog (ZurPA) directly regulates expression of znuA. Electrophoretic mobility shift assays also revealed that recombinant ZurPA specifically binds to the promoter region of znuA and does not bind in the presence of the zinc chelator N,N',N-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN). Taken together, these data support the notion that Np20 is the P. aeruginosa Zur, which regulates the transcription of the genes encoding the high affinity ZnuABC zinc transport system.

  16. The Cutting Edge of Affinity Electrophoresis Technology

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  18. Biotin transport in the rat central nervous system.

    PubMed

    Lo, W; Kadlecek, T; Packman, S

    1991-12-01

    Previous studies in the biotin-deficient rat have shown that brain biotin concentrations and the activity of biotin-dependent carboxylases are relatively preserved in the face of biotin starvation and systemic biotin deficiency. These data suggested the existence of a concentration mechanism for biotin in brain, and the present studies were undertaken to further characterize brain biotin transport. We presently show that rat cerebrospinal fluid biotin concentrations are 2.5 times higher than serum concentrations, consistent with the existence of a concentrative mechanism for biotin. Further, we demonstrate uptake of 3H-biotin into rat brain from blood at physiologic biotin concentrations, using single pass clearance measurements of a brain uptake index. The calculated brain uptake indices for biotin, and the inhibition kinetics, are consistent with the possible existence of a low affinity mediated uptake mechanism. The results have implications for the pathophysiology of human biotin-responsive multiple carboxylase deficiency.

  19. Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants

    PubMed Central

    Hafke, Jens B.; Höll, Sabina-Roxana; Kühn, Christina; van Bel, Aart J. E.

    2013-01-01

    Apart from cut aphid stylets in combination with electrophysiology, no attempts have been made thus far to measure in vivo sucrose-uptake properties of sieve elements. We investigated the kinetics of sucrose uptake by single sieve elements and phloem parenchyma cells in Vicia faba plants. To this end, microelectrodes were inserted into free-lying phloem cells in the main vein of the youngest fully-expanded leaf, half-way along the stem, in the transition zone between the autotrophic and heterotrophic part of the stem, and in the root axis. A top-to-bottom membrane potential gradient of sieve elements was observed along the stem (−130 mV to −110 mV), while the membrane potential of the phloem parenchyma cells was stable (approx. −100 mV). In roots, the membrane potential of sieve elements dropped abruptly to −55 mV. Bathing solutions having various sucrose concentrations were administered and sucrose/H+-induced depolarizations were recorded. Data analysis by non-linear least-square data fittings as well as by linear Eadie–Hofstee (EH) -transformations pointed at biphasic Michaelis–Menten kinetics (2 MM, EH: Km1 1.2–1.8 mM, Km2 6.6–9.0 mM) of sucrose uptake by sieve elements. However, Akaike's Information Criterion (AIC) favored single MM kinetics. Using single MM as the best-fitting model, Km values for sucrose uptake by sieve elements decreased along the plant axis from 1 to 7 mM. For phloem parenchyma cells, higher Km values (EH: Km1 10 mM, Km2 70 mM) as compared to sieve elements were found. In preliminary patch-clamp experiments with sieve-element protoplasts, small sucrose-coupled proton currents (−0.1 to −0.3 pA/pF) were detected in the whole-cell mode. In conclusion (a) Km values for sucrose uptake measured by electrophysiology are similar to those obtained with heterologous systems, (b) electrophysiology provides a useful tool for in situ determination of Km values, (c) As yet, it remains unclear if one or two uptake systems are involved

  20. Electrophysiological approach to determine kinetic parameters of sucrose uptake by single sieve elements or phloem parenchyma cells in intact Vicia faba plants.

    PubMed

    Hafke, Jens B; Höll, Sabina-Roxana; Kühn, Christina; van Bel, Aart J E

    2013-01-01

    Apart from cut aphid stylets in combination with electrophysiology, no attempts have been made thus far to measure in vivo sucrose-uptake properties of sieve elements. We investigated the kinetics of sucrose uptake by single sieve elements and phloem parenchyma cells in Vicia faba plants. To this end, microelectrodes were inserted into free-lying phloem cells in the main vein of the youngest fully-expanded leaf, half-way along the stem, in the transition zone between the autotrophic and heterotrophic part of the stem, and in the root axis. A top-to-bottom membrane potential gradient of sieve elements was observed along the stem (-130 mV to -110 mV), while the membrane potential of the phloem parenchyma cells was stable (approx. -100 mV). In roots, the membrane potential of sieve elements dropped abruptly to -55 mV. Bathing solutions having various sucrose concentrations were administered and sucrose/H(+)-induced depolarizations were recorded. Data analysis by non-linear least-square data fittings as well as by linear Eadie-Hofstee (EH) -transformations pointed at biphasic Michaelis-Menten kinetics (2 MM, EH: K m1 1.2-1.8 mM, K m2 6.6-9.0 mM) of sucrose uptake by sieve elements. However, Akaike's Information Criterion (AIC) favored single MM kinetics. Using single MM as the best-fitting model, K m values for sucrose uptake by sieve elements decreased along the plant axis from 1 to 7 mM. For phloem parenchyma cells, higher K m values (EH: K m1 10 mM, K m2 70 mM) as compared to sieve elements were found. In preliminary patch-clamp experiments with sieve-element protoplasts, small sucrose-coupled proton currents (-0.1 to -0.3 pA/pF) were detected in the whole-cell mode. In conclusion (a) K m values for sucrose uptake measured by electrophysiology are similar to those obtained with heterologous systems, (b) electrophysiology provides a useful tool for in situ determination of K m values, (c) As yet, it remains unclear if one or two uptake systems are involved in sucrose

  1. Overexpression of BetS, a Sinorhizobium meliloti high-affinity betaine transporter, in bacteroids from Medicago sativa nodules sustains nitrogen fixation during early salt stress adaptation.

    PubMed

    Boscari, Alexandre; Van de Sype, Ghislaine; Le Rudulier, Daniel; Mandon, Karine

    2006-08-01

    Sinorhizobium meliloti possesses several betaine transporters to cope with salt stress, and BetS represents a crucial high-affinity glycine and proline betaine uptake system involved in the rapid acquisition of betaines by cells subjected to osmotic upshock. Using a transcriptional lacZ (beta-galactosidase) fusion, we showed that betS is expressed during the establishment of the symbiosis and in mature nitrogen-fixing nodules. However, neither Nod nor Fix phenotypes were impaired in a betS mutant. BetS is functional in isolated bacteroids, and its activity is strongly activated by high osmolarity. In bacteroids from a betS mutant, glycine betaine and proline betaine uptake was reduced by 85 to 65%, indicating that BetS is a major component of the overall betaine uptake activity in bacteroids in response to osmotic stress. Upon betS overexpression (strain UNA349) in free-living cells, glycine betaine transport was 2.3-fold higher than in the wild-type strain. Interestingly, the accumulation of proline betaine, the endogenous betaine synthesized by alfalfa plants, was 41% higher in UNA349 bacteroids from alfalfa plants subjected to 1 week of salinization (0.3 M NaCl) than in wild-type bacteroids. In parallel, a much better maintenance of nitrogen fixation activity was observed in 7-day-salinized plants nodulated with the overexpressing strain than in wild-type nodulated plants. Taken altogether, these results are consistent with the major role of BetS as an emergency system involved in the rapid uptake of betaines in isolated and in planta osmotically stressed bacteroids of S. meliloti.

  2. Siderophore uptake in bacteria and the battle for iron with the host; a bird's eye view.

    PubMed

    Chu, Byron C; Garcia-Herrero, Alicia; Johanson, Ted H; Krewulak, Karla D; Lau, Cheryl K; Peacock, R Sean; Slavinskaya, Zoya; Vogel, Hans J

    2010-08-01

    Siderophores are biosynthetically produced and secreted by many bacteria, yeasts, fungi and plants, to scavenge for ferric iron (Fe(3+)). They are selective iron-chelators that have an extremely high affinity for binding this trivalent metal ion. The ferric ion is poorly soluble but it is the form of iron that is predominantly found in oxygenated environments. Siderophore uptake in bacteria has been extensively studied and over the last decade, detailed structural information for many of the proteins that are involved in their transport has become available. Specifically, numerous crystal structures for outer membrane siderophore transporters, as well as for soluble periplasmic siderophore-binding proteins, have been reported. Moreover, unique siderophore-binding proteins have recently been serendipitously discovered in humans, and the structures of some of their siderophore-complexes have been characterized. The binding pockets for different ferric-siderophores in these proteins have been described in great molecular detail. In addition to highlighting this structural information, in this review paper we will also briefly discuss the relevant chemical properties of iron, and provide a perspective on our current understanding of the human and bacterial iron uptake pathways. Potential clinical uses of siderophores will also be discussed. The emerging overall picture is that iron metabolism plays an extremely important role during bacterial infections. Because levels of free ferric iron in biological systems are always extremely low, there is serious competition for iron and for ferric-siderophores between pathogenic bacteria and the human or animal host.

  3. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    SciTech Connect

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    )/sup 3/H)cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 3/H)cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of (/sup 3/H)cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  4. Positron-labeled dopamine agonists for probing the high affinity states of dopamine subtype 2 receptors.

    PubMed

    Hwang, Dah-Ren; Narendran, Raj; Laruelle, Marc

    2005-01-01

    It is well documented that guanidine nucleotide-coupled dopamine subtype 2 receptors (D2) are configured in high and low affinity states for the dopamine agonist in vitro. However, it is still unclear whether these functional states exist in vivo. We hypothesized that positron-labeled D2 agonist and Positron Emission Tomography can be used to probe these functional states noninvasively. Recently, we demonstrated in nonhuman primates that N-[11C]propyl-norapomorphine (NPA), a full D2 agonist, is a suitable tracer for imaging the high affinity states of D2 receptors in vivo. We also developed kinetic modeling method to derive receptor parameters, such as binding potential (BP) and specific uptake ratios (V3''). When coupled with a dopamine releasing drug, amphetamine, NPA was found to be more sensitive than antagonist tracers, such as [11C]raclopride (RAC), to endogenous dopamine concentration changes (by about 42%). This finding suggests that NPA is a superior tracer for reporting endogenous DA concentration. In addition, the difference of the BP or V3'' of NPA and RAC under control and amphetamine challenge conditions could be used to estimate the functional states of D2 receptors in vivo. On the basis of our findings and the assumptions that NPA binds only to the high affinity states and RAC binds equally to both affinity states, we proposed that about 70% of the D2 receptors are configured in the high affinity states in vivo.

  5. Novel trends in affinity biosensors: current challenges and perspectives

    NASA Astrophysics Data System (ADS)

    Arugula, Mary A.; Simonian, Aleksandr

    2014-03-01

    Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ‘aptamers’ and ‘phages’ which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives.

  6. Kinetic Studies of Biological Interactions By Affinity Chromatography

    PubMed Central

    Schiel, John E.; Hage, David S.

    2009-01-01

    The rates at which biological interactions occur can provide important information on the mechanism and behavior of such processes in living systems. This review will discuss how affinity chromatography can be used as a tool to examine the kinetics of biological interactions. This approach, referred to here as biointeraction chromatography, uses a column with an immobilized binding agent to examine the association or dissociation of this agent with other compounds. The use of HPLC-based affinity columns in kinetic studies has received particular attention in recent years. Advantages of using HPLC with affinity chromatography for this purpose include the ability to reuse the same ligand within a column for a large number of experiments, and the good precision and accuracy of this approach. A number of techniques are available for kinetic studies through the use of affinity columns and biointeraction chromatography. These approaches include plate height measurements, peak profiling, peak fitting, split-peak measurements, and peak decay analysis. The general principles for each of these methods are discussed in this review and some recent applications of these techniques are presented. The advantages and potential limitations of each approach are also considered. PMID:19391173

  7. AGE-RELATED EFFECTS OF CHLORPYRIFOS ON HIGH AFFINITY CHOLINE UPTAKE IN RAT BRAIN. (R825811)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  8. A putative role for the plasma membrane potential in the control of the expression of the gene encoding the tomato high-affinity potassium transporter HAK5.

    PubMed

    Nieves-Cordones, Manuel; Miller, Anthony J; Alemán, Fernando; Martínez, Vicente; Rubio, Francisco

    2008-12-01

    A chimeric CaHAK1-LeHAK5 transporter with only 15 amino acids of CaHAK1 in the N-terminus mediates high-affinity K(+) uptake in yeast cells. Kinetic and expression analyses strongly suggest that LeHAK5 mediates a significant proportion of the high-affinity K(+) uptake shown by K(+)-starved tomato (Solanum lycopersicum) plants. The development of high-affinity K(+) uptake, putatively mediated by LeHAK5, was correlated with increased LeHAK5 mRNA levels and a more negative electrical potential difference across the plasma membrane of root epidermal and cortical cells. However, this increase in high-affinity K(+) uptake was not correlated with the root K(+) content. Thus, (i) growth conditions that result in a hyperpolarized root plasma membrane potential, such as K(+) starvation or growth in the presence of NH(4) (+), but which do not decrease the K(+) content, lead to increased LeHAK5 expression; (ii) the presence of NaCl in the growth solution, which prevents the hyperpolarization induced by K(+) starvation, also prevents LeHAK5 expression. Moreover, once the gene is induced, depolarization of the plasma membrane potential then produces a decrease in the LeHAK5 mRNA. On the basis of these results, we propose that the plant membrane electrical potential plays a role in the regulation of the expression of this gene encoding a high-affinity K(+) transporter.

  9. Uptake and transport of a novel anticancer drug-delivery system: lactosyl-norcantharidin-associated N-trimethyl chitosan nanoparticles across intestinal Caco-2 cell monolayers.

    PubMed

    Guan, Min; Zhu, Qiao-Ling; Liu, Yang; Bei, Yong-Yan; Gu, Zong-Lin; Zhang, Xue-Nong; Zhang, Qiang

    2012-01-01

    In this paper, novel liver-targeting nanoparticles (NPs), lactosyl-norcantharidin (Lac-NCTD)-associated N-trimethyl chitosan (TMC) NPs (Lac-NCTD-TMC-NPs), were prepared using ionic cross-linkage. The physical properties, particle size, and encapsulation efficiency of the nanoparticles were then investigated. The continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells (Caco-2) cell monolayer model was used to study the transport mechanism of Lac-NCTD, and the effects of factors such as time, temperature, pH level, drug concentration, enhancers, and inhibitors. This model was also used to indicate the differences among Lac-NCTD, Lac-NCTD-associated chitosan NPs (Lac-NCTD-CS-NPs), and Lac-NCTD-TMC- NPs in the absorption and transportation of membranes. Drug concentration levels were measured using high-performance liquid chromatography. Active transport and paracellular transport were suggested to be both the primary and secondary mechanisms for Lac-NCTD absorption, respectively. Lac-NCTD uptake and absorption were not controlled by pH levels, but were positively correlated to uptake time, and negatively correlated to temperature. The basolateral to apical apparent permeability coefficients (Papps) were higher than those of the apical to basolateral values. The inhibitor of P-glycoprotein and the multidrug resistance-associated protein 2 significantly enhanced the uptake amount of Lac-NCTD. Compared with Lac-NCTD, Lac-NCTD-CS-NPs and Lac-NCTD-TMC-NPs significantly enhanced drug absorption. Additionally, the latter exhibited stronger action. Lac-NCTD-NPs could penetrate the plasma membrane of Caco-2 cells and translocate into the cytoplasm and even into the nucleus. Nanoparticles were uptaken into Caco-2 cells through the endocytosis pathway.

  10. Potassium Uptake Modulates Staphylococcus aureus Metabolism

    PubMed Central

    Gries, Casey M.; Sadykov, Marat R.; Bulock, Logan L.; Chaudhari, Sujata S.; Thomas, Vinai C.; Bose, Jeffrey L.

    2016-01-01

    ABSTRACT As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K+) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K+ uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K+ deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K+ uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization. PMID:27340697

  11. Visualizing Antibody Affinity Maturation in Germinal Centers

    PubMed Central

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

    2016-01-01

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

  12. PRINCIPLES OF AFFINITY-BASED BIOSENSORS

    EPA Science Inventory

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

  13. Protein purification using PDZ affinity chromatography.

    PubMed

    Walkup, Ward G; Kennedy, Mary B

    2015-04-01

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

  14. Affinity Electrophoresis Using Ligands Attached To Polymers

    NASA Technical Reports Server (NTRS)

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

    1990-01-01

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

  15. Experimental allergic encephalomyelitis (EAE) in mice selectively bred to produce high affinity (HA) or low affinity (LA) antibody responses.

    PubMed Central

    Devey, M E; Major, P J; Bleasdale-Barr, K M; Holland, G P; Dal Canto, M C; Paterson, P Y

    1990-01-01

    Induction of experimental allergic encephalomyelitis (EAE) in mice genetically selected to produce either high affinity (HA) or low affinity (LA) antibody responses has revealed significant differences in disease susceptibility between the two lines. HA mice were highly susceptible to EAE following subcutaneous sensitization to mouse central nervous system (CNS) tissue emulsified in Freund's complete adjuvant (FCA). Furthermore, of HA mice surviving acute EAE, up to 93% subsequently developed chronic relapsing disease (CREAE) characterized by variable demyelinating inflammatory changes within the spinal cord. In contrast, LA mice, despite having a major histocompatability complex (MHC) haplotype associated with susceptibility to EAE, were highly resistant to the disease and showed no signs of CREAE when observed for up to 100 days post